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7788903
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Role of endogenous endothelin-1 in experimental renal hypertension in dogs.
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Endothelin-1, a vasoconstrictive peptide released by endothelium, may be involved in the pathophysiology of hypertension. The goal of the present study was to evaluate the role of endogenous endothelin-1 in renal hypertension in dogs. The model of hypertension consisted of silk tissue wrapping of the left kidney, which produced hypertension associated with perinephritis after 6 to 8 weeks. Thirty-two anesthetized open chest dogs were studied randomly: 8 dogs with perinephritic hypertension received the nonpeptidic ETA-ETB receptor antagonist bosentan (group 1); 8 other hypertensive dogs received the vehicle solution (group 2); 8 healthy dogs received bosentan (group 3); and 8 healthy dogs received the vehicle solution (group 4). Bosentan was injected as an intravenous bolus (3 mg/kg) followed by a 1-hour infusion at a rate of 7 mg.kg-1.h-1. In hypertensive dogs, bosentan produced a similar decrease (P = .0001) of both left ventricular systolic and mean aortic pressures, which averaged 38 mm Hg (-22% and -24%, respectively). These parameters remained unchanged with the vehicle solution. Left ventricular end-diastolic and left atrial pressures also declined significantly with bosentan (P = .0005 and P < .05, respectively). Left ventricular lengths tended to decrease. The other cardiovascular parameters (heart rate, peak [+]dP/dt, time constant of relaxation, and coronary vascular resistance) did not change significantly. In healthy dogs, bosentan decreased mean aortic pressure by 19 mm Hg (P = .004). Vehicle solution had no effect. Plasma endothelin-1 levels, similar under basal conditions in healthy and hypertensive dogs, increased 30-fold with bosentan (P = .0001). Specific endothelin-1 receptor antagonism markedly lowers blood pressure in experimental hypertension but is less effective on blood pressure of healthy animals. This suggests that endothelin-1 plays a role in the pathophysiology of hypertension but contributes to a lesser extent to the maintenance of normal blood pressure. This role of endothelin-1 is unrelated to its plasma levels. The increase of plasma endothelin-1 with bosentan, due either to a displacement of endothelin-1 from its receptor or to a feedback mechanism, does not prevent this blood pressure reduction.
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7788901
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[Current malaria stratification in China].
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During the past 30 years since 1958 when malaria stratification was first made, a great change in malaria situation has occurred. The original stratification can not represent the current status of malaria distribution. Therefore, it needs to be redivided so as to provide basis for future malaria control work. Taking species of mosquito vector and malaria incidence as the main indices and making reference to the natural geographical division, the current malaria endemic area in China can be divided into four regions, i.e. (1) Western region: it is mostly a natural non-endemic area, except some limited areas with sporadic cases in Xinjiang Autonomous Region; (2) Northern region: the whole north-eastern and partial northern China belong to the areas where malaria has been basically eliminated; but sporadic occurrence still exist in most part of the northern China; (3) Central region: it is mostly a hypo-endemic area, only a small part has meso-endemicity; (4) Southern region: nearly 50% has meso-endemicity and the other 50% has hypo-endemicity. The demarcation line of natural non-endemic areas and the areas where malaria has been basically eliminated has been defined.
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7788900
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[Effects of different fixatives for Trichomonas vaginalis in indirect fluorescent antibody test].
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This paper dealt with the effects of different fixatives for Trichomonas vaginalis in indirect fluorescent antibody test. T. vaginalis fixed by formalin, methanol or alcohol displayed clear flagella. The parasites fixed by methanol and alcohol showed specific ring-like fluorescence with 1-2 bright dots. Antigen slides prepared from different Trichomonas strains, different number of parasites and anti-Trichomonas vaginalis McAb strains in IFA presented different results. Antigen slides showed the best results when using parasites at concentrations of 1.2 x 10(6) cells/ml or 2.4 x 10(5) cells/ml. Antigen preparations stored at 4 degrees C or -20 degrees C remained reactive throughout the experimental period of three months.
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7788899
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[A clinicopathological study of eighteen autopsy cases with acquired toxoplasmosis].
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Eighteen autopsy cases of acquired toxoplasmosis in New York City were studied. Seventeen cases were with acquired immunodeficiency syndrome (AIDS) and one patient with Hodgkin's disease. All 18 cases involved the brain and nine of them disseminated to the heart (8 cases), lung (4 cases), pancreas (3 cases), alimentary tract (2 cases) and urogenital organs (3 cases). The authors divided the acquired toxoplasmosis into (1) immunocompetent, (2) immunocompromised and (3) immunodeficient types. The autopsy findings showed that the brain, heart and lung were the most susceptible organs. Pseudocysts were also found in lungs and alimentary tract, suggesting an autoinfection by swallowing sputum containing Toxoplasma. Toxoplasma in the urogenital organs might become a source for sexual transmitted toxoplasmosis.
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7788898
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[Efficacy of praziquantel combined with albendazole in the treatment of clonorchiasis].
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This paper reported on the clinical efficacy of praziquantel-albendazole combination in the treatment of clonorchiasis in Guangxi. Two hundred and two cases were divided into three groups. The first group comprised 66 cases who were treated with praziquantel at 180 mg/kg bwt for three days. The second group consisted of 62 cases who were treated with albendazole at 90 mg/kg bwt for three days. In the third group, 74 cases were treated with 90 mg/kg bwt praziquantel plus 45 mg/kg albendazole for three days. The results showed that the negative conversion rates were 98.5%, 61.3% and 87.8% in groups one, two and three, respectively, one month after treatment. In some cases who continued to be egg positive, the reduction rates in faecal egg counts were 99.7%, 65.0% and 97.3%, respectively. Although the cure rate in group three was not as high as in group one, the side effects were more mild and transient and the cost of treatment could be reduced by 60% as compared with group one. In cases with light and moderate infections, the praziquantel-albendazole combination proved more effective. In addition, the praziquantel-albendazole combination was also highly effective for treating those cases who were infected with Ascaris, Trichuris or hookworm.
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7788897
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[Observation on antibody levels of rabbits infected with single sex cercariae of Schistosoma japonicum].
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Nine rabbits infected with male S. japonicum cercariae (worm recovery 15-133, mean 53.1) and 9 rabbits infected with female cercariae (worm recovery 1-102, mean 36.1) were bled periodically until 1 year post-infection. The sera were tested by ELISA, LA and CHR for antibody detection. Results showed that the positive rates were 88.9%, 55.6% and 83.3%, respectively. The duration of positive reaction was 3-51 wk post-infection for ELISA, 4-17 wk for LA and 2-35 wk for CHR and the fluctuated results were obtained in ELISA while positive CHR and LA results usually remained stable. The above-mentioned results suggest that antibody detection methods should not be neglected due to its sensitivity although antigen detection methods are considered more favorable. For the surveillance of schistosomiasis in endemic areas where schistosomiasis is under control, antibody detection methods are still worthy of recommending for case finding of new infections.
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7788896
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[Pathological changes in the livers of rabbits infected with schistosome cercariae and treated with artemether or praziquantel in the early stage of infection].
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When rabbits infected with Schistosoma japonicum cercariae for 7 d were treated ig with artemether (Art) 30 mg.kg-1, and repeated at 1-wk intervals (i.e. on d 14, 21, 28 after infection) for 4 times, dead worms were found in the liver sections, but no egg or egg granuloma was detected. In infected rabbits treated repeatedly with the above-mentioned dosage of Art at 2-wk intervals (i.e. on d 21, 35, 49) after the first dosing on d 7 after infection, few eggs and egg granulomas were seen occasionally. In another experiment, praziquantel (Pra) 40 mg.kg-1 was given ig to rabbits on d 21 after infection and the same dosage was administered repeatedly at 1- or 2-wk intervals for 2 times, the numbers of eggs and egg granulomas detected in the liver sections of these 2 groups were much less as compared with the control. In the latter group treated at 2-wk intervals, only degenerated eggs and egg granulomas were detected occasionally in the liver sections. The results indicate that if Art or Pra was given to rabbits in the early stage after infection, no apparent or only slight damage of the liver induced by the eggs was seen due to the fact that most or even all of the female worms were killed.
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7788895
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Scanning electron microscope observation on tegumental alteration of Schistosoma japonicum induced by levo- and dextro-praziquantel.
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When mice infected with Schistosoma japonicum for 35 d were treated ig with levo-praziquantel (L-Pra) 150 mg.kg-1 or dextro-praziquantel (D-Pra) 150-600 mg.kg-1, the alterations of the worm tegument were examined by scanning electron microscopy within 1-24 h after administration. The results showed that L-Pra induced apparent and extensive damage on the worm surface including severe swelling, fusion, erosion and peeling of tegument accompanied by attachment of the host leukocytes. The discoid-like sensory structures usually showed swelling, deformation and collapse. With D-Pra 150 mg.kg-1, only slight damage of tegument was seen. When the dose was increased to 600 mg.kg-1, the damage of worm tegument was similar to that induced by L-Pra 150 mg.kg-1 but the intensity of the damage was slighter as compared to that induced by L-Pra. The results suggested that the actions of the 2 optical enantiomers of praziquantel were different in quantity but not in quality.
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7788894
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[Preparation and characterization of monoclonal antibodies against esterase of malathion-resistant Culex quinquefasciatus].
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Five clones of monoclonal antibodies (McAbs) were generated from BALB/c mice which were activized with PPD and immunized with esterases extracted from malathion-resistant Culex quinquefasciatus. All 5 clones of McAbs belonged to the IgG1 subtype, additive index (A.I.) of every two McAbs did not exceeded 50%. After a 1 h of incubation, the McAbs at the titer of 1:1,000 completely inhibited the activity of the amplified esterases. Of 8 strains of adult mosquitoes studied by Western-blot assay, all exhibited the 64 kDa esterase band recognized by the McAbs, six (SP, FS, SH, CD, RD, RM) showed more bands of different MW (43-60 kDa), the higher the organophosphorus resistant level, the more and the denser the bands. The above McAbs had little or no effect against the esterases of dimethrin-resistant strain and the larva stage of malathion-resistant Culex quinquefasciatus.
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7788893
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[Studies on amino acid and chemical element in five species of filariae].
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Five species of human and domestic animal filariae (B. m, S. d, S. l, S. e, D. i) after lyophilization and hydrolysis were resolved using HPLC and type WFX-IB AAS to determine amino acid (AA) and microelement (ME) content, respectively. The results showed that there were 16 and 17 AA in B. malayi and the animal filariae, respectively. Among AA, the total amount (microgram/mg dry wt.) was markedly higher in animal filariae than that in B. malayi in which the content of acidic and aliphatic AA was significantly higher than those of basic and aromatic AA, respectively. Among five ME (Zn, Cu, Fe, Mn, Cd) and two microelements (Ca, Mg) found in all these filariae. Zn Content was the predominant ME and Ca was much more than Mg.
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7788891
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[In vitro effect of metronidazole ester on Trichomonas vaginalis and Entamoeba histolytica].
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Metronidazole ester, a new nitroimidazole drug, was synthesized by the Department of Physical Chemistry, School of Pharmacy, Shanghai Medical University. The trichomonadicidal and amoebicidal activity of metronidazole ester was evaluated in vitro by the cultivation of Trichomonas vaginalis and Entamoeba histolytica at different drug concentrations. The results indicated that the 100% lethal concentration for T. vaginalis and E. histolytica was 2.6 micrograms/ml and 40 micrograms/ml, respectively after incubation with metronidazole ester for 48 h. In comparison with metronidazole, the effect of metronidazole ester for both protozoa was similar.
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7788892
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[Dynamics of IL-2 and IFN-gamma levels induced by sea or Con A in spleen cells of Schistosoma japonicum-infected mice].
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The capacity of mitogen Con A and SEA-stimulated spleen cells to produce cytokines IFN-gamma and IL-2 was studied in S. japonicum-infected mice every two weeks from 0 to 14 wk after infection. The results showed that the levels of these two cytokines began to rise at the 4th wk after infection and reached a peak level at 6-8 wk, then declined to the levels similar to those pre-infection at 12-14 wk after infection. The IFN-gamma level reached the peak earlier than the IL-2 level. The dynamics of cytokine level in both mitogen and antigen-stimulated group was similar. The results suggest that IL-2 and IFN-gamma might be the essential cytokines involved in egg granuloma formation in schistosomiasis japonica.
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7788890
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Purification of 31/32 kDa proteins of adult Schistosoma japonicum as antigens (Sj 31/32) for ELISA and IHA.
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Major diagnostic proteins of 31/32 kDa were purified from soluble adult worm homogenate of Schistosoma japonicum using AcA ultragel chromatography and additional treatments. The purity of the isolated antigenic proteins (Sj 31/32) was demonstrated by SDS-PAGE and Western blotting technique. The antigens showed a negative reaction when stained by PAS and remained active after treatment with sodium periodate, moving toward anode in the electrophoretic field. These purified antigens were used for detecting the specific antibody in sera from patients with schistosomiasis japonica by ELISA and IHA. As compared with SEA, Sj 31/32 kDa was found as sensitive as but much more specific than SEA in immunodiagnosis.
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7788889
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[Research on the factors influencing the sporogonic multiplication of Plasmodium vivax in the mosquito vector].
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Volunteers with no previous malaria history infected with northern Guangdong isolate of Plasmodium vivax and mosquito vector, Anopheles stephensi, were the objects and materials used in the research. Totally there were 16 volunteers, half of them were infected by mosquito-bite and another half by blood-inoculation. Blood was drawn when parasitemia reached a certain level and the erythrocytic forms developed into large trophozoites. Mosquitoes fed on infected blood from 5 and 4 donors of the two groups gave high gland infection rate and high gland indices respectively, but the highest gland indices were got from volunteers infected by mosquito-bite. The time of blood-feeding is important, only those blood samples taken from the people 3 to 8 days after the first onset gave good sporozoite harvest and the gland infection was greatly reduced on the 9th day. During this period (d3-8), the density of gametocyte had obvious influence on the intensity of mosquito infection. When the sex ratio of gametocyte (female:male) was 4:1 or less, the result would be good. For the purpose of reflecting more perfectly the circumstance of the sporogonic multiplication of plasmodium in the mosquito vector, we suggest the concept of gut/gland infection intensity which equals the product of the infection rate and the index of oocyst/positive gland.
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7788886
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Functional coupling between glycolysis and sarcoplasmic reticulum Ca2+ transport.
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To investigate whether the energy derived from glycolysis is functionally coupled to Ca2+ active transport in sarcoplasmic reticulum (SR), we determined whether glycolytic enzymes were associated with SR membranes and whether metabolism through these enzymes was capable of supporting 45Ca transport. Sealed right-side-out SR vesicles were isolated by step sucrose gradient from rabbit skeletal and cardiac muscle. Intravesicular 45Ca transport was measured after the addition of glycolytic substrates and cofactors specific for each of the glycolytic reactions being studied or after the addition of exogenous ATP and was expressed as transport sensitive to the specific Ca(2+)-ATPase inhibitor thapsigargin. We found that the entire chain of glycolytic enzymes from aldolase onward, including aldolase, GAPDH, phosphoglycerate kinase (PGK), phosphoglyceromutase, enolase, and pyruvate kinase (PK), was associated with SR vesicles from both cardiac and skeletal muscle. Iodoacetic acid, an inhibitor of GAPDH, eliminated 45Ca transport supported by fructose-1,6-diphosphate, the substrate for aldolase, but transport was completely restored by phosphoenolpyruvate (the substrate for PK), indicating that both of the ATP-producing glycolytic enzymes, GAPDH/PGK and PK, were associated with the SR and functionally capable of providing ATP for the Ca2+ pump. Addition of a soluble hexokinase ATP trap eliminated 45Ca transport fueled by exogenous ATP but had markedly less effect on 45Ca transport supported by endogenously produced ATP (via glycolysis). Similarly, at very low concentrations of ATP and ADP (10 to 50 nmol/L), ATP that was produced endogenously from ADP and phosphoenolpyruvate supported 15-fold more 45Ca transport than ATP that was supplied exogenously at the same concentration. These results are consistent with functional coupling of glycolytic ATP to Ca2+ transport and support the hypothesis that ATP generated by SR-associated glycolytic enzymes may play an important role in cellular Ca2+ homeostasis by driving the SR Ca2+ pump.
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7788888
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[Nationwide survey of the distribution of human parasites in China--infection with parasite species in human population].
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The infection rate of main species of parasites and their character by first nationwide survey of human parasites was made. The overall infection rate of human intestinal parasite and the infection rate of most species of parasites were higher in females than that in males. The infection rate according to the age group, the highest infection rate was found in the group aged 5-14 years. With regard to the relation between parasitic infections and occupations of the infected persons, the highest infection rates of Ascaris lumbricoides were exhibited in school children, of hookworm and Clonorchis sinensis were in halfpeasants and halfmerchant and vegetable grower; of Trichuris trichiura were in fishers, of Giardia lamblia, Entamoeba histolytica and Taenia were in herdsmen and halfherdsmen and halfpeasants. In this survey it is also shown that each nationality has their main species of parasites. The family clustering of some main parasites were proved by some province/autonomous region/municipality.
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7788887
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Expression of a mutation causing hypertrophic cardiomyopathy disrupts sarcomere assembly in adult feline cardiac myocytes.
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Mutations in the beta-myosin heavy chain (beta MyHC) induce hypertrophic cardiomyopathy (HCM), cardiac hypertrophy, and sarcomere disarray, with the latter being the characteristic hallmark. Thus, we sought to determine whether expression of mutant beta MyHC in adult feline cardiac myocytes, a species known to develop HCM with a phenotype identical to that in humans, induces sarcomere disarray. A full-length beta MyHC cDNA was cloned from a human heart cDNA library, and an HCM-causing mutation (Arg403Gln) was induced in the beta MyHC cDNA by site-directed mutagenesis using polymerase chain reaction (PCR). The normal and mutant beta MyHC cDNAs were cloned into p delta E1spIB shuttle vector, downstream from a cytomegalovirus (CMV) promoter. Replication-deficient recombinant adenoviral constructs (Ad5/CMV/beta MyHC-N and Ad5/CMV/beta MyHC-403) were generated through homologous recombination of p delta E1spIB/CMV/beta MyHC-N or Ad5/CMV/beta MyHC-403 and pBHG10 after cotransfection in 293 host cells. Infection of COS-1 cells with the beta MyHC construct resulted in the expression of a full-length myosin protein. Efficiency of infection of isolated adult cardiac myocytes was > 95%. Expression of the beta MyHC constructs into mRNA at 48 hours after infection of feline cardiac myocytes was confirmed by reverse transcription-PCR. The net total protein and beta-myosin synthesis were determined by using the amount of incorporation of [3H]phenylalanine into total protein and beta-myosin, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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7788885
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L-methionine augments mammalian myocardial contraction by sensitizing the myofilament to Ca2+.
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L-Methionine is an essential amino acid that has been reported to have a potent positive inotropic effect on the mammalian myocardium. We studied the mechanisms of the inotropic effect in ventricular myocardium from the rabbit. In the isolated coronary-perfused whole heart, L-methionine in a millimolar range exerted concentration-dependent positive inotropic effects on the isovolumic left ventricle, which were associated with negative lusitropic effects (prolonged time course of relaxation). The chronotropic state and the coronary perfusion pressure were not affected. These complex effects on the isolated whole heart were not blocked by pretreatment with (mumol/L) propranolol 1, prazosin 1, carbachol 3, staurosporine 1, or [Ser1,Ile8]angiotensin II 0.1. To further study the subcellular mechanisms, isolated ventricular papillary muscles from the same species were loaded with a bioluminescent indicator, aequorin, to monitor [Ca2+]i. In the presence of 3 mmol/L L-methionine, the isometric tension showed a similar combination of the positive inotropic and negative lusitropic effects as observed in the whole heart. In contrast, the simultaneously recorded intracellular Ca2+ signals did not increase in amplitude but instead decreased. The [Ca2+]i-tension relation shifted to the left compared with that obtained in response to [Ca2+]o. In saponin (250 micrograms/mL)-treated skinned preparations, 3 mmol/L L-methionine also shifted the force-pCa curve to the left by 0.16 pCa units. This is the first demonstration that an essential amino acid directly acts on the myofilaments and modulates their responsiveness to Ca2+, thereby producing a positive inotropic effect.
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7788884
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Regulation of aldosterone biosynthesis by adrenal renin is mediated through AT1 receptors in renin transgenic rats.
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The transgenic (TG) rat (mREN2)27 is characterized by overexpression of the additional mouse Ren-2d gene in the adrenal cortex with marked suppression of renal renin. We have previously shown that in salt-depleted TG rats enhanced activation of mineralocorticoid biosynthesis is associated with selective stimulation of adrenal renin. To investigate whether the local renin-angiotensin system regulates aldosterone biosynthesis in the adrenal cortex of TG rats, we studied the effects of the AT1-angiotensin subtype receptor antagonist DuP 753 on aldosterone production in 5-week-old TG rats during salt restriction. All the rats (n = 56) were shifted from regular chow to a diet containing only 0.04% NaCl for 1 week. The AT1-receptor antagonist DuP 753 (10 mg/kg per day in drinking water) was administered to 27 of these rats during low-salt diet. Subgroups of rats were killed at 0,4, and 7 days. Low-salt diet increased both adrenal renin activity (from 31 +/- 3 to 77 +/- 4 and 85 +/- 2 ng angiotensin I.h-1.mg protein-1 at 4 and 7 days, respectively; P < .001) and mRNA (by 68.4 +/- 10% and 80 +/- 17% from baseline, P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)
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7788883
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Localization of atrial natriuretic factor receptors in the mesenteric arterial bed. Comparison with angiotensin II and endothelin receptors.
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Although receptors for atrial natriuretic factor (ANF) and angiotensin II (Ang II) have been reported in rat mesenteric arteries, both peptides induce weak biological responses. Endothelin-1 (ET-1) evokes a potent vasoconstriction in the mesenteric artery. To identify the tissue localization of ANF, Ang II, and ET-1 receptors, radioligand binding experiments with 125I-ANF, 125I-[Sar1,Ile8]Ang II, and 125I-ET-1 were performed in defatted mesenteric arteries and in the surrounding adipose tissue. 125I-ANF binding assays in adipose tissue showed a single class of high-affinity binding sites (Bmax, 420 +/- 16 fmol/mg protein; Kd, 343 +/- 16 pmol/L). In vascular membranes, most 125I-ANF binding was nonspecific. The majority of receptors present in adipose tissue recognized ANF, C-type natriuretic peptide (CNP), and des-[Gln18,Ser19,Gly20,Leu21,Gly22]ANF-(4- 23) (C-ANF) with close affinities, with C-ANF competing for > 98% of the binding sites. In adipocytes, ANF and CNP stimulated cGMP generation. cGMP production by mesenteric arteries was stimulated by sodium nitroprusside but not by ANF or CNP. Autoradiographic localization of 125I-ANF and 125I-ET-1 showed that in the case of ANF, most specific binding occurred in adipocytes, whereas for ET-1, specific binding was present in both adipose tissue and mesenteric arteries. Cross-linking of 125I-ANF followed by SDS-PAGE revealed two receptor species of 130 and 70 kD in adipose membranes and none in vascular tissue. Both were completely displaced by ANF, CNP, and C-ANF. 125I-[Sar1,Ile8]Ang II binding assays in adipose tissue exhibited a single class of binding sites (Bmax, 211 +/- 4 fmol/mg protein; Kd, 520 +/- 10 pmol/L.(ABSTRACT TRUNCATED AT 250 WORDS)
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7788882
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Measurement of platelet-activating factor in a canine model of coronary thrombosis and in endarterectomy samples from patients with advanced coronary artery disease.
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Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator of numerous inflammatory and thrombotic responses. The purpose of this study was to determine if PAF synthesis is elevated in damaged coronary arteries after a sustained period of cyclic flow variation (CFV), a phenomenon caused by alternating periods of thrombosis and reperfusion at sites of endothelial injury. Cyclic flow was established and maintained in the left anterior descending coronary arteries (LADs) of 10 dogs. After 8 hours of CFV, the section of damaged LAD containing the thrombus and control sections of the circumflex artery, carotid artery, and saphenous vein was excised, and the total lipids were extracted. The PAF was then purified by silica column chromatography and high-performance liquid chromatography and assayed by both a rabbit platelet bioassay and a PAF radioimmunoassay. With the platelet bioassay, PAF levels of 8.9 +/- 4.0 (range, 4.8 to 15.5) pg/mg wet wt were found in the damaged LADs from the 10 dogs. This PAF bioactivity was completely inhibited by a PAF receptor antagonist. When the radioimmunoassay was used, slightly higher PAF levels of 16.3 +/- 12.9 (range, 4.5 to 41.8) pg/mg wet wt were observed in the LADs. Overall, these PAF levels were 3- to 64-fold higher than in the control vessels when either assay method was used. Although increases in PAF were observed in the damaged LADs, measurements of PAF in blood samples taken from the LAD and the aorta (control) failed to demonstrate any site-specific increase of PAF in the blood. In related experiments, PAF was also measured in 23 endarterectomy samples taken from the coronary arteries of 16 patients with severe atherosclerosis. The PAF levels in these samples were highly variable (2.9 +/- 2.2 [range, 0.3 to 8.5] pg/mg wet wt) and showed no correlation with tissue mass, suggesting that PAF is affected by factors other than the simple presence of atherosclerotic tissue in the vessel. These findings provide direct evidence that PAF is synthesized locally at the site of endothelial injury during thrombosis and that PAF accumulates in the atherosclerotic plaque of some patients with advanced coronary artery disease.
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7788881
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Endothelial cell interactions with synthetic peptides from the carboxyl-terminal heparin-binding domains of fibronectin.
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Fibronectin (FN) plays an important role in endothelial cell adhesion, spreading, and motility. Within FN, a number of functional domains have been identified, including the 33/66-kD carboxyl-terminal heparin-binding fragments, which support the adhesion of vascular endothelial cells. A number of synthetic peptides representing amino acid sequences within the 33/66-kD fragments have been shown to promote the adhesion, spreading, and migration of a variety of cell types. Our working hypothesis is that one or more of these sequences may also mediate vascular endothelial cell adhesion, spreading, and migration to the 33/66-kD fragments. In support of this hypothesis, we have demonstrated that endothelial cells from various sources adhered in a concentration-dependent manner to surfaces coated with FN, the 33/66-kD fragments, and synthetic peptides derived from the 33/66-kD fragments of FN. FN and the 33/66-kD fragments also promoted endothelial cell spreading and migration. Although each of the six synthetic peptides tested supported endothelial cell adhesion, only one of these peptides within the carboxyl-terminal heparin-binding domain (FN-C/H-V) promoted endothelial cell spreading and migration. Cell spreading on FN-C/H-V, as well as on FN and the 33/66-kD fragments, was associated with the formation of a well-developed actin cytoskeleton and the formation of focal contacts. FN-C/H-V (but not scrambled FN-C/H-V) inhibited cell spreading on FN and the 33/66-kD fragments in a concentration-dependent manner. FN-C/H-V had a modest effect on the adhesion of a clonal population of rat heart endothelial cells (RHE-1A) to the 33/66-kD fragments of FN and no effect on RHE-1A cell adhesion to FN. These findings suggest that peptide FN-C/H-V is unique among this group of peptides derived from the 33/66-kD heparin-binding fragments of FN in its ability to promote the adhesion, spreading, and migration of vascular endothelial cells and further suggest that the sequence defined by this peptide plays an important role in vascular endothelial cell interactions with the 33/66-kD fragments of FN.
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7788879
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Functional identification of histamine H3-receptors in the human heart.
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Norepinephrine release contributes to ischemic cardiac dysfunction and arrhythmias. Because activation of histamine H3-receptors inhibits norepinephrine release, we searched for the presence of H3-receptors directly in sympathetic nerve endings (cardiac synaptosomes) isolated from surgical specimens of human atria. Norepinephrine was released by depolarization with K+. The presence of H3-receptors was ascertained because the selective H3-receptor agonists (R) alpha-methylhistamine and imetit reduced norepinephrine release, and the specific H3-receptor antagonist thioperamide blocked this effect. Norepinephrine release was exocytotic, since it was inhibited by the N-type Ca(2+)-channel blocker omega-conotoxin and the protein kinase C inhibitor Ro31-8220. Functional relevance of these H3-receptors was obtained by showing that transmural electrical stimulation of sympathetic nerve endings in human atrial tissue increased contractility, an effect blocked by propranolol and attenuated in a concentration-dependent manner by (R) alpha-methylhistamine. Also, thioperamide antagonized the effect of (R) alpha-methylhistamine. Our findings are the first demonstration that H3-receptors are present in sympathetic nerve endings in the human heart, where they modulate adrenergic responses by inhibiting norepinephrine release. Since myocardial ischemia causes intracardiac histamine release, H3-receptor-induced attenuation of sympathetic neurotransmission may be clinically relevant.
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7788880
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Acetylcholine-sensitive intracellular Ca2+ store in fresh endothelial cells and evidence for ryanodine receptors.
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In a freshly isolated endothelial cell preparation from rabbit aorta, the regulation of the acetylcholine (ACh)-sensitive intracellular Ca2+ store and the effects of the Ca(2+)-induced Ca2+ release agonists ryanodine and caffeine were studied using fura 2 imaging fluorescence microscopy. ACh (10 mumol/L) caused a transient release of Ca2+ from an intracellular store, presumably via an inositol tris-phosphate-sensitive mechanism. This ACh response could be repeated in the presence of extracellular Ca2+ but was obtained only once in Ca(2+)-free bathing solution, which shows that a depleted intracellular Ca2+ store can be rapidly refilled from the extracellular space. Refilling can be prevented by the endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 mumol/L), implying that Ca2+ enters the cytoplasm before accumulation in the endoplasmic reticulum. Ionomycin (10 mumol/L) caused a large Ca2+ release even after the ACh-releasable store had been emptied, indicating the existence of other ACh-insensitive stores, perhaps including the mitochondria. In one third of the cells studied, ACh induced oscillations in [Ca2+]i that were dependent on extracellular Ca2+. Also investigated were the effects of caffeine and ryanodine. In this cell preparation neither caffeine nor ryanodine induced a Ca2+ transient but instead slowly increased [Ca2+]i. It was observed that both caffeine and ryanodine were able to slowly deplete the ACh-sensitive store. These results indicate the presence of functional ryanodine receptors in native endothelial cells and demonstrate overlap between the caffeine and agonist-sensitive Ca2+ stores. We also found that caffeine was able to directly inhibit the process of ACh-induced Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)
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7788878
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Osmotic compression of single cardiac myocytes eliminates the reduction in Ca2+ sensitivity of tension at short sarcomere length.
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According to the Frank-Starling relation, cardiac output varies as a function of end-diastolic volume of the ventricle. The cellular basis of the relation is thought to involve length-dependent variations in Ca2+ sensitivity of tension; ie, as sarcomere length is increased in cardiac muscle, Ca2+ sensitivity of tension also increases. One possible explanation for this effect is that the decrease in myocyte diameter as muscle length is increased reduces the lateral spacing between thick and thin filaments, thereby increasing the likelihood of cross-bridge interaction with actin. To examine this idea, we measured the effects of osmotic compression of single skinned cardiac myocytes on Ca2+ sensitivity of tension. Single myocytes from rat enzymatically digested ventricles were attached to a force transducer and piezoelectric translator, and tension-pCa relations were subsequently characterized at short sarcomere length (SL), at the same short SL in the presence of 2.5% dextran, and at long SL. The pCa (-log[Ca2+]) for half-maximal tension (ie, pCa50) increased from 5.54 +/- 0.09 to 5.65 +/- 0.10 (n = 7, mean +/- SD, P < .001) as SL was increased from approximately 1.85 to approximately 2.25 microns. Osmotic compression of myocytes at short length also increased Ca2+ sensitivity of tension, shifting tension-pCa relations to [Ca2+] levels similar to those observed at long length (pCa50, 5.68 +/- 0.11). These results support the idea that the length dependence of Ca2+ sensitivity of tension in cardiac muscle arises in large part from the changes in interfilament lattice spacing that accompany changes in SL.
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7788877
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Binding of A1 adenosine receptor ligand [3H]8-cyclopentyl-1,3-dipropylxanthine in coronary smooth muscle.
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Vascular smooth muscle has been reported to contain the A1 subtype of adenosine receptors, but the existence of such receptor(s) in coronary smooth muscle has not been established. In the present study, the 3H-labeled A1-selective antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) was used to demonstrate the specific binding in porcine coronary artery smooth muscle membranes. The binding was saturable with a Bmax of 6.43 +/- 1.02 fmol/mg protein. Scatchard analysis of the binding data provided a single binding site with a Kd of 0.21 +/- 0.025 nmol/L. In the competition experiments, adenosine receptor agonists and antagonists showed the following order of potency (nmol/L): S-N6-(2-endonorbornyl)adenosine (S-ENBA) 0.11 = R(-)-N6-phenylisopropyladenosine 0.32 > DPCPX 3.2 = xanthine amine congener 2.4 = N6-cyclopentyladenosine 2.67 > 5'-(N-ethylcarboxamido)-adenosine 7.35 >> 2-[p-(2-carboxyethyl)-phenethyl-amino]-5'-(N-ethylcarboxamido)- adenosine 1000 > theophylline 83,000. This order of potency fits the criteria for the A1 adenosine receptor. S-ENBA, a highly selective A1 receptor agonist, was used to investigate the effect on isoproterenol-mediated vasorelaxation and cAMP accumulation. S-ENBA (0.1 to 10 nmol/L) dose-dependently shifted the isoproterenol-mediated (10(-8) to 10(-5) mol/L) vasorelaxation to the right in vascular rings. S-ENBA (10 nmol/L) inhibited the basal cAMP levels by 36% and attenuated the isoproterenol (10(-5) mol/L)-stimulated cAMP by 25% in the coronary rings. These inhibitory effects of S-ENBA on isoproterenol-mediated cAMP-accumulation and vasorelaxation were abolished by pertussis toxin (100 ng/mL, overnight) treatment of the arteries.(ABSTRACT TRUNCATED AT 250 WORDS)
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7788876
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Transverse shear along myocardial cleavage planes provides a mechanism for normal systolic wall thickening.
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Recent studies in humans and other species show that there is substantial transverse shear strain in the left ventricular myocardium, and others have shown transverse myocardial laminae separated by cleavage planes. We proposed that cellular rearrangement based on shearing along myocardial cleavage planes could account for > 50% of normal systolic wall thickening, since < 50% can be explained by increases in myocyte diameter. To test this hypothesis, we measured strains at two sites with different cleavage-plane anatomy in eight open-chest dogs. Columns of radiopaque markers were implanted in the left ventricular anterior free wall and septum. Markers were tracked with biplane cineradiography, and strains were quantified by using finite deformation techniques. Hearts were perfusion-fixed with glutaraldehyde, and cleavage-plane orientations at the bead sites were measured in three orthogonal planes. At subendocardial sites of the anterior left ventricular wall, where the cleavage planes approach the endocardium obliquely from the apical side of the surface normal in the longitudinal-radial plane (-67 +/- 11 degrees), systolic longitudinal-radial transverse shear (E23) was positive (0.14 +/- 0.08). At the septal sites where the subendocardial cleavage planes approach the endocardium obliquely from above the surface normal (44 +/- 12 degrees), E23 was negative (-0.12 +/- 0.08). The differences in cleavage-plane angle and E23 at the two sites were each highly significant (P < .0005). At both sites, the transverse shear strain accompanied substantial systolic wall thickening at the subendocardium (anterior, E33 = 0.44 +/- 0.16; septum, E33 = 0.22 +/- 0.14). These data are not representative of the behavior in midwall and outer wall sites, where cleavage-plane orientation was not consistently different between anterior left ventricle and septum. Our data indicate that rearrangement of myocytes by slippage along myocardial cleavage planes is in the correct direction and of sufficient magnitude in the subendocardium (inner third) to account for a substantial proportion (> 50%) of systolic wall thickening. Furthermore, three-dimensional reconstruction of the myocardial laminae and local comparison with maximum strain vectors indicate that for the inner third of the ventricular wall the maximum shear deformation is a result of relative sliding between myocardial laminae.
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7788875
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Dithionite increases radical formation and decreases vasoconstriction in the lung. Evidence that dithionite does not mimic alveolar hypoxia.
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Dithionite is a powerful reducing agent used to deoxygenate hemoglobin and create anaerobic conditions in vitro. Recently, dithionite has been used as a convenient means of creating "hypoxia" in experiments studying the O2 sensor in the pulmonary circulation and carotid body. We evaluated the hypothesis that hypoxia created by hypoxic ventilation and that created by dithionite have different effects on the pulmonary circulation. In vitro, dithionite (10(-5) to 10(-3) mol/L), added to oxygenated Krebs' solution, rapidly created superoxide anion in a dose-dependent manner. Dithionite consumed O2 in parallel with the generation of superoxide radical, with both processes peaking within seconds. Anoxia was sustained only if resupply of O2 was prevented. In isolated rat lungs (whether perfused with autologous blood or Krebs' solution), hypoxic ventilation alone lowered perfusate PO2 from approximately 140 to 40 mm Hg and decreased lung levels of activated oxygen species (AOS), measured by luminol-enhanced chemiluminescence, before the onset of hypoxic pulmonary vasoconstriction. Constrictor responses to angiotensin II and KCl were not impaired by intermittent hypoxic challenges, and lung weight did not increase. In contrast, dithionite impaired constrictor responses of the Krebs' solution-perfused lungs to all vasoconstrictors tested and increased lung weight. When given as a bolus (5 x 10(-3) mol/L) into the pulmonary artery during normoxic ventilation, dithionite caused no vasoconstriction and only briefly lowered PO2 (because of constant resupply of O2 from the alveoli). When superimposed on hypoxic ventilation, dithionite further lowered PO2 from approximately 40 to approximately 0 mm Hg and caused additional constriction. Unlike hypoxic ventilation, dithionite increased AOS production. Antioxidant enzymes diminished dithionite-induced radical production and diminished the loss of vascular reactivity and lung edema. In conclusion, unlike hypoxic ventilation, dithionite causes edema and loss of vascular reactivity in the lung by generating superoxide anion and hydrogen peroxide. Hypoxia elicited by dithionite is not equivalent to authentic hypoxia because of the obligatory associated generation of AOS. Dithionite usage should not be substituted for authentic hypoxia in studies of O2 sensing.
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7788874
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Role of atrial contraction in diastolic pressure elevation induced by rapid pacing of hypertrophied canine ventricle.
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The mechanism of diastolic pressure elevation induced by acute rapid pacing in pressure-load hypertrophied left ventricles (LVs) remains incompletely understood. It has been ascribed to abnormalities of coronary flow, metabolism, and calcium cycling. However, rapid pacing also alters the timing of atrial and ventricular stimulation relative to the diastolic filling period, and this could also influence diastolic pressures. To test the role of such mechanical factors, LV pressure-volume hemodynamics were measured in closed-chested anesthetized dogs during and after abrupt cessation of rapid atrial pacing. Twenty-one dogs were studied: 6 dogs with LV hypertrophy (LVH) induced by perinephritic hypertension, 5 sham-operated normotensive dogs, and 10 acute normotensive control dogs. In LVH dogs, but not in sham-operated or control dogs, end-diastolic pressure rose progressively with increasing heart rate from 5.6 +/- 3.1 mm Hg at baseline to 22.6 +/- 8.1 mm Hg at 220 beats per minute. In all hearts, rapid pacing shifted the timing of left atrial contraction so that it occurred near the onset of LV filling rather than at end diastole. However, in LVH hearts, early LV diastolic pressure and peak atrial pressure were also markedly elevated. Most striking, immediately after terminating the pacing, diastolic pressure declined to near baseline. This rapid pressure decline occurred just when atrial systole would have ensued and before ventricular activation would have followed had pacing continued. Thus, diastolic pressure elevation resolved before a change in ventricular pacing rate. The role of atrial contraction was further explored by simultaneous atrioventricular pacing. This shifted the time of atrial systole so that it occurred during LV isovolumic contraction, while maintaining the identical LV pacing rate. This change eliminated the diastolic pressure elevation found previously. Further analysis revealed that the pressure increase during rapid pacing was not due simply to partial LV filling imposed on a relaxing ventricle or to hypertension or an intact pericardium. These data indicate that mechanical effects of atrioventricular interaction play an important role in tachycardia-induced diastolic dysfunction in this model of LVH and can be more causative than ischemia or metabolic factors in this setting.
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7788873
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Arrhythmia and delayed recovery of cardiac action potential during reperfusion after ischemia. Role of oxygen radical-induced no-reflow phenomenon.
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The role of reactive metabolites of oxygen, oxygen radicals (O-Rs), as mediators of potentially arrhythmogenic alterations in cellular electrical properties and contractile dysfunction of cardiac muscle during reperfusion after ischemia was investigated. Electrical and mechanical activities of arterially perfused guinea pig right ventricular walls were recorded simultaneously with intracellular microelectrodes and a force transducer. Preparations were maintained in Krebs-Henseleit solution (perfusion rate, 1.5 mL/min) and subjected to 30 minutes of no-flow ischemia followed by 60 minutes of reperfusion or pretreated with O-R scavengers (superoxide dismutase, 50 U/mL; catalase, 600 U/mL; and mannitol, 2 mmol/L) for 10 to 20 minutes, followed by 30 minutes of ischemia and 60 minutes of reperfusion. Reperfusion in untreated preparations caused (1) depolarization of resting membrane potential by 8 to 10 mV and slow recovery of action potential duration requiring 60 minutes to attain the preischemic duration, (2) tachyarrhythmias and premature action potentials, (3) postischemic contractile dysfunction, and (4) increased coronary perfusion pressure in untreated preparations. Pretreatment with scavenger cocktail affected neither electrical nor contractile activity before or during no-flow ischemia, but it (1) accelerated recovery of resting membrane potential and action potential duration, (2) reduced the incidence of tachyarrhythmia, (3) improved contractile function, and (4) inhibited the rise in perfusion pressure on reflow. Reperfusion with an exogenous O-R-generating system containing xanthine/xanthine oxidase (X/XO, 2 mmol/L:10 mU/mL) inhibited recovery of action potential duration and contractility. Treatment of normoxic arterially perfused right ventricular walls with X/XO caused a decline in action potential duration by approximately 20% within 30 minutes. In contrast, X/XO caused a 30% increase in the duration of action potentials in superfused papillary muscles or small strips of right ventricular walls over the same time period. Pretreatment with sodium nitroprusside (10 mumol/L) inhibited the decline in duration induced by X/XO in normoxic right ventricular walls but was without effect on prolongation due to X/XO in papillary muscles. Reperfusion with nitroprusside after no-flow ischemia caused (1) accelerated recovery of preischemic action potential configuration, (2) a significant decline in the incidence of reperfusion arrhythmias, (3) improved postischemic contractile performance, and (4) inhibition of the increase in perfusion pressure associated with reflow. The data indicate that slow recovery of the action potential duration caused by O-Rs in reperfusion cannot be explained by the direct effects of O-Rs on cardiac myocytes.(ABSTRACT TRUNCATED AT 400 WORDS)
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7788872
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Two components of the delayed rectifier K+ current in ventricular myocytes of the guinea pig type. Theoretical formulation and their role in repolarization.
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Two distinct delayed rectifier K+ currents, IKr and IKs, were found recently in ventricular cells. We formulated these currents theoretically and investigated their roles in action potential repolarization and the restitution of action potential duration (APD). The Luo-Rudy (L-R) model of the ventricular action potential was used in the simulations. The single delayed rectifier K+ current in the model was replaced by IKr and IKs. Our results show that IKs is the major outward current during the plateau repolarization. A specific block of either IKr or IKs can effectively prolong APD to the same degree. Therefore, either channel provides a target for class III antiarrhythmic drugs. In the simulated guinea pig ventricular cell, complete block of IKr does not result in early afterdepolarizations (EADs). In contrast, > 80% block of IKs results in abnormal repolarization and EADs. This behavior reflects the high IKs-to-IKr density ratio (approximately 8:1) in this cell and can be reversed (ie, IKr block can cause EADs) by reducing the ratio of IKs to IKr. The computed APD restitution curve is consistent with the experimental behavior, displaying fast APD variation at short diastolic intervals (DIs) and downward shift at longer DIs with the decrease of basic drive cycle length (BCL). Examining the ionic currents and their underlying kinetic processes, we found that activation of both IKr and IKs is the primary determinant of the APD restitution at shorter DIs, with Ca2+ current through L-type channels (ICa) playing a minor role. The rate of APD change depends on the relative densities of IKr and IKs; it increases when the IKr-to-IKs density ratio is large. The BCL-dependent shift of restitution at longer DIs is primarily attributed to long-lasting changes in [Ca2+]i. This in turn causes different degrees of Ca(2+)-dependent inactivation of ICa and different degrees of Ca(2+)-dependent conductance of IKs at very long DIs (> 5 s) for different BCLs. This BCL dependence of ICa and IKs that is secondary to long-lasting changes in [Ca2+]i is responsible for APD changes at long DIs and can be viewed as a "memory property" of cardiac cells.
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7788871
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[Ca2+]i inhibition of K+ channels in canine pulmonary artery. Novel mechanism for hypoxia-induced membrane depolarization.
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Experiments were performed on smooth muscle cells isolated from canine pulmonary artery to identify the type of K+ channel modulated by hypoxia and examine the possible role of [Ca2+]i in hypoxic K+ channel inhibition. Whole-cell patch-clamp experiments revealed that hypoxia (induced by the O2 scavenger, sodium dithionite) reduced macroscopic K+ currents, an effect that could be prevented by strong intracellular buffering of [Ca2+]i. The inhibitory effects of hypoxia were mimicked by acute exposure of cells to caffeine and could be prevented by caffeine pretreatment, suggesting an important obligatory role of [Ca2+]i in hypoxic inhibition of K+ currents. Exposure of cells to low concentrations of 4-aminopyridine (4-AP, 1 mmol/L) prevented hypoxic inhibition of macroscopic K+ currents, whereas low concentrations of tetraethylammonium were without effect, suggesting that the target K+ channel inhibited by hypoxia is a voltage-dependent delayed rectifier K+ channel, which is inhibited by [Ca2+]i. Hypoxia failed to consistently modify the activity of large-conductance (118 picosiemens [pS] in physiological K+) Ca(2+)-activated K+ channels in inside-out membrane patches but reduced open probability of smaller-conductance (25-pS) delayed rectifier K+ channels in cell-attached membrane patches. In inside-out membrane patches, 1 mumol/L Ca2+ added to the cytoplasmic surface significantly reduced open probability of small-conductance (25-pS) 4-AP-sensitive delayed rectifier K+ channels. Whole-cell current measurements using symmetrical K+ to increase driving force for small currents active near the cell's resting membrane potential revealed the presence of a 4-AP-sensitive K+ current that activated near -65 mV and was inhibited by hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)
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7788870
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[Ca2+]i inhibition of K+ channels in canine renal artery. Novel mechanism for agonist-induced membrane depolarization.
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The patch-clamp technique was used to examine the inhibition of delayed rectifier K+ channels by agents that release intracellular Ca2+. During voltage-clamp experiments on isolated myocytes with 4-aminopyridine (4-AP, 10 mmol/L) and niflumic acid (100 mumol/L) present to inhibit delayed rectifier K+ current (IK(dr)) and Ca(2+)-activated Cl- current (ICl(Ca)), angiotensin II (Ang II) and caffeine increased Ca(2+)-activated K+ current (IK(Ca)) between -25 and 80 mV (n = 5). Conversely, with charybdotoxin (ChTX, 100 nmol/L) and niflumic acid (100 mumol/L) present to inhibit IK(Ca) and ICl(Ca), Ang II and caffeine only caused inhibition of IK(dr). Block was achieved within 15 seconds of drug application and was reversible upon washout (n = 5). The effects of Ang II on IK(Ca) and IK(dr) were inhibited by the specific Ang II receptor antagonist losartan (1 mmol/L, n = 3). Intracellular BAPTA (10 mmol/L) also abolished the effects of Ang II and caffeine on both IK(Ca) and IK(dr). In current-clamp experiments, the application of ChTX (100 nmol/L) and niflumic acid (100 mumol/L) caused little change in resting membrane potential; however, subsequent application of caffeine (10 mmol/L) caused a 26 +/- 2.9 mV depolarization from -54 +/- 3.1 to -28 +/- 1.7 mV (n = 6). 4-AP (10 mmol/L) blocked the caffeine-induced depolarization. When isolated cells were loaded with the Ca2+ indicator indo 1 (100 mumol/L), Ang II, caffeine, and 4-AP increased [Ca2+]i and depolarized the cells. Both Ang II and caffeine caused an increase in [Ca2+]i that preceded membrane depolarization, whereas 4-AP depolarized the cell first and then caused an increase in [Ca2+]i (n = 4). In inside-out patches, with 200 nmol/L ChTX in the patch pipette to block large-conductance Ca(2+)-activated K+ channels, a 45 +/- 7-picosiemen 4-AP-sensitive K+ channel was identified that was sensitive to cytoplasmic Ca2+ (n = 6). Increasing intracellular Ca2+ decreased channel opening probability [NxP(open), where N is the number of functional channels in a patch and P(open) is the opening probability] at all membrane potentials examined. At 0 mV, increasing Ca2+ from < 5 to 200 and 600 nmol/L free Ca2+ decreased NxP(open) by 52 +/- 3% and 73 +/- 7%, respectively (n = 6). The decrease in opening probability of the delayed rectifier K+ channel resulted from a concentration- and voltage-dependent decrease in mean open time. The decrease in mean open time reflected significant decreases and increases in open and closed time constants, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)
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7788869
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Alpha 1-adrenergic receptor stimulation decreases maximum shortening velocity of skinned single ventricular myocytes from rats.
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alpha 1-Adrenergic agonists have negative inotropic effects on mammalian myocardium under some conditions, and biochemical experiments measuring the Ca(2+)-activated actomyosin ATPase activity of myofibrillar preparations suggest that this may result from a decrease in cross-bridge cycling rate caused by phosphorylation of myofilament proteins. Experiments with intact ventricular preparations, however, have failed to demonstrate a mechanical manifestation of a decrease in cycling rate. The present study examined the effect of alpha 1-adrenergic receptor stimulation on maximum shortening velocity in skinned single ventricular myocytes from rats. Enzymatically isolated myocytes were incubated with the beta-receptor antagonist propranolol in the presence or absence of the alpha 1-adrenergic receptor agonist phenylephrine and were then rapidly skinned to preserve the phosphorylation state of myofilament proteins. The velocity of unloaded shortening (Vo) was determined by use of the slack-test method and compared between skinned control and phenylephrine-treated cells. The relationship between isometric tension and [Ca2+] was also assessed for each myocyte. Vo was significantly lower in the alpha 1-adrenergic receptor agonist-treated cells than in the control cells, but there was no effect on Ca2+ sensitivity of isometric tension. In addition, the myosin heavy chain isoform composition accounted for a significant amount of the variation in Vo within the treatment groups. On the basis of these and previous results we propose that alpha 1-adrenergic receptor stimulation inhibits cross-bridge cycling rate at the level of myofilament proteins by a mechanism that may involve phosphorylation of troponin I by protein kinase C.
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7788868
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Effects of Levosimendan, a cardiotonic agent targeted to troponin C, on cardiac function and on phosphorylation and Ca2+ sensitivity of cardiac myofibrils and sarcoplasmic reticulum in guinea pig heart.
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A new cardiotonic agent, (R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)-phenyl] hydrazono]propanedinitrile (Levosimendan), has been developed and screened for its ability to bind to cardiac troponin C. In perfused hearts, low concentrations of 0.03 or 0.1 mumol/L Levosimendan increased +dP/dt, but did not affect the speed of relaxation and produced only a slight increase in spontaneous heart rate in the hearts perfused with 0.1 mumol/L of the drug. In these same hearts, perfusion with 0.03 mumol/L Levosimendan did not alter the 32P incorporation into troponin I or C protein, whereas a slight but significant increase was noted for phospholamban, with no detectable change in tissue cAMP levels. Administration of 0.1 or 0.3 mumol/L Levosimendan significantly increased myocardial cAMP levels as well as the phosphorylation of phospholamban, troponin I, and C protein. Levosimendan (0.03 to 10 mumol/L) reversibly increased force generated by detergent-extracted fiber bundles over a range of submaximally activating free Ca2+ concentrations with no significant effect on maximum force or on Ca2+ binding to myofilament troponin C. There was no direct effect of Levosimendan on Ca2+ uptake by vesicles of sarcoplasmic reticulum (SR). In contrast, under conditions optimal for cAMP-dependent phosphorylation, Levosimendan slightly but significantly lowered the concentration of Ca2+, yielding half-maximal uptake rates by the SR vesicles. Our results indicate that at low concentrations Levosimendan acts preferably as a Ca2+ sensitizer, whereas at higher concentrations its action as a phosphodiesterase inhibitor contributes to the positive inotropic effect.
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7788867
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Molecular regulation of atrioventricular valvuloseptal morphogenesis.
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The majority of congenital heart defects arise from abnormal development of valvuloseptal tissue. The primordia of the valve leaflets and membranous septa of the heart are the cardiac cushions. Remodeling of the cushions is associated with a transitional extracellular matrix that includes sulfated proteoglycans and the microfibrillar proteins fibulin and fibrillin. Cushion formation is restricted to the AV canal and ventricular outflow tract regions of the primary heart tube. The proper placement of the cushions may be the result of the development of the primary heart tube as a segmented organ, as well as the subsequent looping of the heart. Segmentation of the heart tube may be demonstrated by the alternating molecular expression pattern along the longitudinal axis. In support of this hypothesis is the restricted expression of BMP-4 and msx-2 to the AV canal and ventricular outflow tract. The importance of looping for cushion positioning may imply that the iv and inv genes and retinoic acid are important for the proper patterning of the heart. The cells of the cushions evolve from endocardial cells that undergo an epithelial-to-mesenchymal transformation. This developmental event is regulated by the myocardium and is probably due to the production of protein complexes, present within the cardiac jelly of the cushion-forming regions, that consist of fibronectin and the ES proteins. Both the cushion mesenchyme and its endocardial cell antecedents express JB3, an ECM protein. JB3 expression is also featured within the heart-forming fields of the primary mesoderm, from which the endocardial progenitors of the cushion cells originate.(ABSTRACT TRUNCATED AT 250 WORDS)
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7788866
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Non-specific inhibition of cytochrome P450 activities by chlorophyllin in human and rat liver microsomes.
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Chlorophyllin, a copper/sodium salt of chlorophyll used in the treatment of geriatric patients, inhibits the mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1-methyl-5H- pyrido[4,3-b]indole (Trp-P-2), aflatoxin B1 and benzo[a]pyrene (B[a]P). Recent in vitro and in vivo studies have shown that a molecular complex is formed between IQ and chlorophyllin, suggesting that this complex formation might be responsible for the antigenotoxic effect of chlorophyllin observed. Cytochrome P450 (P450) enzymes appear to be the major catalysts in the bioactivation of these carcinogens. We have investigated the in vitro effects of chlorophyllin on several P450 activities including ethoxyresorufin O-deethylation, benzyloxyresorufin O-debenzylation, coumarin 7-hydroxylation, 7-ethoxycourmarin O-deethylation, B[a]P 3-hydroxylation, and chlorzoxazone 6-hydroxylation. Chlorophyllin non-specifically inhibited all of P450 activities observed. Spectrally detectable P450 was also destroyed in microsomes and purified P450 in a reconstituted system in the presence of chlorophyllin and an NADPH-generating system. These results suggest that the antigenotoxic effect of chlorophyllin might be due to inhibition of P450 enzymes involving bioactivation of carcinogens in addition to molecular complex formation between carcinogens and chlorophyllin. Comparison of the apparent Ki for P450 inactivation with previously estimated constants for chlorophyllin-IQ complexation suggest that P450 inhibition should be the dominant mechanism of inhibition.
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7788865
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Mammary carcinogenicity in female CD rats of a diol epoxide metabolite of fluoranthene, a commonly occurring environmental pollutant.
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We examined the mammary carcinogenicity in CD rats of anti-2,3-dihydroxy-1,10b-epoxy-10b,1,2,3-tetrahydro-fluoranthene (FDE), a genotoxic metabolite of the environmental pollutant fluoranthene. FDE (2 mumol or 10 mumol) in 0.1 ml dimethyl sulfoxide (DMSO) was injected beneath each of the three left thoracic nipples of groups of 20 rats each, with 0.1 ml DMSO alone being injected under the right nipples. On the next day, the procedure was repeated for the three inguinal nipples on each side. anti-3,4-Dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BcPDE, 2 mumol per nipple) was used as a positive control and DMSO alone as a negative control. Tumor development was assessed weekly by palpation and the experiment was terminated after 41 weeks. Eighty five percent of the rats in each of the FDE treated groups developed histologically confirmed mammary tumors, compared to 11% in the DMSO treated animals (P < 0.01). Most tumors were on the left side. The lower dose of FDE induced a significant number of adenomas while the higher dose induced significant incidences of both adenomas and adenocarcinomas compared to controls. BcPDE was a powerful mammary carcinogen, confirming our previous observation. The results of this study demonstrate the carcinogenicity of FDE to the CD rat mammary gland. Since FDE is a potentially transportable human metabolite of fluoranthene, its possible role as an etiologic factor in breast cancer deserves further study.
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7788864
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Site specific reduction of colon cancer incidence, without a concomitant reduction in cryptal cell proliferation, in 1,2-dimethylhydrazine treated rats by diets containing 10% pectin with 5% or 20% corn oil.
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The effects of specific dietary interventions on incidence of carcinogen-induced cancer and on cryptal cell proliferation in areas of the colon located either over aggregates of lymphoid nodules (ALN) or away from ALN was investigated. Groups of dimethylhydrazine (DMH) treated rats or non-DMH-treated rats were fed a basal AIN-76 diet less fiber of any type, or the basal fiber free diet supplemented with 10% pectin and with 5%, 10%, or 20% corn oil. The adenocarcinoma (AC) incidence was determined in regions of the colon, i.e. ascending, descending, descending over the ALN and descending away from the ALN. The results indicate that: (i) factors associated with ALN promote AC formation, (ii) dietary modifications (addition of pectin and of 20% corn oil to the diet) each cause significant site specific suppression of AC incidence, (iii) DMH-treatment rendered crypts non-responsive to the suppression of cryptal cell proliferation which occurred in the rats not treated with DMH (suggestive of a DMH-induced loss in the regulation of cell proliferation) and (iv) reduction of AC incidence was not always accompanied by reduction in crypt cell proliferation. Studies of intervention procedures designed to prevent colon cancer should take into account the colon site specific tumorigenic response to the preventive agent and should not rely on a single biomarker to predict the efficacy of the intervention.
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7788863
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DNA adduct formation in B6C3F1 mice and Fischer-344 rats exposed to 1,2,3-trichloropropane.
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1,2,3-Trichloropropane (TCP) is a multispecies, multisite carcinogen which has been found to be an environmental contaminant. In this study, we have characterized and measured DNA adducts formed in vivo following exposure to TCP. [14C]TCP was administered to male B6C3F1 mice and Fischer-344 rats by gavage at doses used in the NTP carcinogenesis bioassay. Both target and nontarget organs were examined for the formation of DNA adducts. Adducts were hydrolyzed from DNA by neutral thermal or mild acid hydrolysis, isolated by HPLC, and detected and quantitated by measurement of radioactivity. The HPLC elution profile of radioactivity suggested that one major DNA adduct was formed. To characterize this adduct, larger yields were induced in rats by intraperitoneal administration of TCP (300 mg/kg). The DNA adduct was isolated by HPLC based on coelution with the radiolabeled adduct, and compared to previously identified adducts. The isolated adduct coeluted with S-[1-(hydroxymethyl)-2-(N7-guanyl)-ethyl]glutathione, an adduct derived from the structurally related carcinogen 1,2-dibromo-3-chloropropane (DBCP). Analysis by electrospray mass spectrometry suggested that the TCP-induced adduct and the DBCP-derived adduct were identical. The 14C-labeled DNA adduct was distributed widely among the organs examined. Adduct levels varied depending on species, organ, and dose. In rat organs, adduct concentrations for the low dose ranged from 0.8 to 6.6 mumol per mol guanine and from 7.1 to 47.6 mumol per mol guanine for the high dose. In the mouse, adduct yields ranged from 0.32 to 28.1 mumol per mol guanine for the low dose and from 12.2 to 208.1 mumol per mol guanine for the high dose. The relationship between DNA adduct formation and organ-specific tumorigenesis was unclear. Although relatively high concentrations of DNA adducts were detected in target organs, several nontarget sites also contained high adduct levels. Our data suggest that factors in addition to adduct formation may be important in TCP-induced carcinogenesis.
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7788862
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Metabolism of aflatoxin B1 by rabbit and rat nasal mucosa microsomes and purified cytochrome P450, including isoforms 2A10 and 2A11.
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The nasal mucosa of some mammalian species are susceptible to the toxicity of aflatoxin B1 (AFB1), a potent hepatocarcinogen, but little is known about the nasal enzymes involved in the metabolic activation of AFB1 or the metabolites produced. In the present study, the metabolism of AFB1 was studied with nasal microsomes from rats and rabbits and with several purified isozymes of rabbit P450 in a reconstituted enzyme system. The rates of AFB1-N7-guanine DNA adduct formation with rabbit and rat nasal microsomes are over 3- and 10-fold higher, respectively, than with liver microsomes from the same species. On the other hand, the rates of formation of AFM1 (9a-hydroxy-AFB1) and AFQ1 (3-hydroxy-AFB1) products known to be less toxic, are lower with nasal than with liver microsomes. Of particular interest, nasal microsomes produce high levels of six unidentified polar metabolites that are not formed by microsomes from liver or several other tissues. These same products are also generated by P450 NMa purified from rabbit nasal microsomes in a reconstituted system, but not by five other isozymes of cytochrome P450 (1A2, 2B4, 2E1, 2G1, 3A6) that are known to be present in nasal microsomes. AFB1-DNA adducts are formed by P450 NMa at a rate 3-fold higher than that by nasal microsomes. The DNA adducts are formed at much slower rates by P450s 2G1, 2B4, and 1A2, and adducts are not formed at measurable rates by P450s 2E1 and 3A6. Moreover, AFB1-DNA adduct formation is also catalyzed by cDNA-derived, heterologously expressed P450s 2A10 and 2A11, both of which are known to be present in the purified P450 NMa preparation. The Km and Vmax values of the two isozymes for DNA adduct formation are comparable to those for nasal microsomes. Furthermore, the formation of AFB1-DNA adducts by nasal microsomes is decreased by nicotine, a known inhibitor of P450 NMa. These data indicate that members of the P450 2A gene subfamily play an important role in the metabolic activation of AFB1 in rabbit and rat nasal mucosa and suggest a molecular basis for assessing the health risk associated with inhalation exposure to this procarcinogen in humans.
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7788860
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Expression of cytochrome P450 and microsomal epoxide hydrolase in cervical and oral epithelial cells immortalized by human papillomavirus type 16 E6/E7 genes.
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Epidemiological evidence suggests that the presence of human papillomaviruses (HPV), when combined with smoking behaviors, considerably enhances the risk of developing oral, cervical, vulvar, and/or anal carcinomas. It is well established that the cytochrome P450 (CYP), microsomal epoxide hydrolase (mEH), and other biotransformation enzymes are important modulators of the bioactivation and detoxification of many environmental chemicals, including constituents of tobacco smoke such as certain nitrosamines and polycyclic aromatic hydrocarbons (PAH). Since there is little information regarding oral and cervical epithelial-specific expression of these genes, established primary and HPV-immortalized oral and cervical epithelial cell lines were analyzed for morphology, mRNA and protein expression patterns of specific CYPs and mEH. Primary human oral and cervical epithelial cells were immortalized using retroviral infection with HPV-16 E6/E7 genes. Primary human keratinocyte cells were immortalized by transfection of HPV-18 and made tumorigenic with nitrosomethylurea treatment. Expression profiles for mEH, CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP3A, and CYP2E1 were evaluated in these cultures in the presence or absence of a PAH inducer, using reverse transcriptase-coupled polymerase chain reaction analysis. mEH gene expression was evident in all cultures, while CYP2A6 mRNA was not detected in any of the cell lines, regardless of culture conditions. CYP2E1 mRNA expression was greatest in the oral epithelial cultures and detectable in all other epithelial cultures except for the HPV-18 immortalized keratinocyte cell line. Elevated levels of CYP2D6 mRNA existed in both oral epithelial cell lines and the HPV-16 immortalized cervical epithelial cells when compared to the other cell lines examined. CYP1A1 and CYP1A2 mRNAs were detected in all the cells and several cultures were inducible by PAH exposure. To corroborate the RT/PCR data, Western immunoblotting experiments were conducted on selected samples. Using these methods, CYP1A1 and CYP2E1 proteins were detected in primary and HPV-immortalized oral and cervical epithelial cultures. These data indicate that both primary and HPV immortalized cells appear to express certain biotransformation enzymes necessary for the activation of tobacco-specific nitrosamines and PAHs. Although the overall impact of HPV gene infection on expression of these systems remains to be fully elucidated, as in vitro system is characterized which should prove useful in examining interactive mechanisms of HPV with xenobiotic activation in the etiology of squamous cell carcinomas.
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7788861
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Expression of Ah receptor (TCDD receptor) during human monocytic differentiation.
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We have previously found a high expression of human Ah receptor (TCDD receptor) mRNA in peripheral blood cells of individuals. In this paper, the expression of this gene in blood cells was first investigated in fractions of nucleated cells, revealing predominant expression of the Ah receptor gene in the monocyte fraction. Then the expression levels of AhR mRNA in various hematopoietic cell lines were examined together with those of Arnt and P450IA1. AhR was expressed at high levels in monocytoid U937, THP1, and HEL/S cells, and at moderate levels in promyelocytic HL60 cells and erythroblastic HEL cells. However, it was not detected in lymphoid cells MOLT4 (T cell) and BALL1 (B cell), nor in K562 erythroblasts. Furthermore, a specific induction of AhR during monocytic differentiation was investigated in HL60 and HEL cells. HL60 cells were induced to differentiate toward monocytes-macrophages by incubation with phorbol ester, showing a 5- to 2-fold increase of AhR mRNA. The incubation with transforming growth factor beta 1 and 1 alpha,25-dihydroxyvitamin D3 resulted in a 5- to 7-fold increase of AhR mRNA. The HEL cells also exhibited a similar elevation of AhR mRNA level, when they had differentiated toward monocyte-macrophage cells by these combined inducers, but little change in the mRNA level was observed when the cells were induced to differentiate into other cell types. Treatment of the differentiated HL60 cells with 3-methylcholanthrene, a ligand of AhR, induced the expression of the P450IA1 gene. These results indicated that expression of AhR mRNA was significantly induced during monocytic differentiation and that the differentiated cells were responsive to xenobiotics. Our results suggest that AhR may play an important role in the function of monocytes and also in the eventual activation of environmental carcinogens.
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7788859
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Localization of methylation sites in the human O6-methylguanine-DNA methyltransferase promoter: correlation with gene suppression.
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Adducts of O6-alkylguanine in DNA that are induced by cytotoxic, carcinogenic or mutagenic alkylating agents can be removed by the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). Human tumor cell lines that do not express this enzyme (Mer-) are hypersensitive to the effects of such alkylating agents, although the molecular basis of MGMT gene suppression is not yet understood. Previous studies suggested that Mer- cells deficient in this enzyme lack neither the gene nor the trans-acting factors necessary for normal transcription. Methylation of CpG dinucleotides is an attractive mechanism to account for suppression of the MGMT gene; however, there have been reports of both direct and inverse correlations between methylation and MGMT expression. We previously demonstrated an inverse correlation between methylation at a single SmaI site in the human MGMT promoter and gene expression. To substantiate this observation, we examined additional CpGs in the promoters of three Mer+ and three Mer- cell lines, using rare methylation-sensitive restriction sites, and then sought to identify the region where methylation correlated with gene expression. Six CpGs in the region from -245 bp to +225 bp (relative to the transcription start site) were completely unmethylated in all Mer+ cells, whereas in Mer- cells were at least partially methylated. The methylation status of CpGs further upstream did not correlate with MGMT expression. We conclude, therefore, that the association between CpG methylation and suppressed MGMT gene activity extends to sites other than SmaI but is limited to a core region of the promoter.
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7788858
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Metabolism of the food-derived carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in nonhuman primates.
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The metabolism and disposition of the food mutagen and rodent carcinogen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was investigated in cynomolgus monkeys. Monkeys were administered a single dose of radiolabeled [14C]MeIQx (2.2 or 50 mumol/kg). Peak blood levels of radioactivity were observed within 1-3 h after dosing and declined rapidly thereafter. By 72 h after dosing, approximately 50% and 70% of the 2.2 mumol/kg, and 50 mumol/kg dose, respectively, was excreted in the urine. Approximately 15-20% of either dose was recovered in the feces. Eight metabolites and the parent compound were detected in urine by HPLC. The parent compound accounted for approximately 15-25% of the dose excreted in the urine. Seven MeIQx urinary metabolites were identified. Five metabolites were identical to MeIQx metabolites previously found in rats: MeIQx-N2-glucuronide, MeIQx-N2-sulfamate, MeIQx-5-sulfate, MeIQx-5-O-glucuronide, and 8-CH2OH-MeIQx-5-sulfate. Cynomolgus monkeys, however, metabolized MeIQx to a novel glucuronide conjugate of MeIQx not found in rats. Based upon mass spectroscopy and proton NMR analyses, the structure of this metabolite was consistent with an N1-glucuronide of MeIQx. This metabolite was the major urinary metabolite found in monkeys, accounting for 31-37% of the dose excreted in the urine over a 24 h period. One additional metabolite identified in urine and feces of MeIQx treated cynomolgus monkeys, that has not been found previously in any other animal model, was 7-oxo-MeIQx, a likely enteric bacterial metabolite of MeIQx. 7-Oxo-MeIQx accounted for 20-25% of the dose of MeIQx found in the urine and was the major fecal metabolite. The N2-glucuronide conjugate of the carcinogenic metabolite 2-hydroxyamino-3,8-dimethylimidazo[4,5-f]quinoxaline (NHOH-MeIQx) was not detected in urine or bile of monkeys, even after 10 daily doses of MeIQx (100 mumol/kg) were given. The results indicate that MeIQx is metabolically processed in monkeys via multiple pathways of detoxification. However, MeIQx is poorly metabolically activated via cytochrome P450 mediated N-oxidation. The in vivo metabolism of MeIQx in cynomolgus monkeys is different from that of the structurally related food-derived mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), which is readily metabolically activated by this species and in contrast to MeIQx, has been shown to be a powerful hepatic carcinogen.
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7788857
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Anti-benzo[a]pyrene diolepoxide--DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking.
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The level of (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) bound to DNA of lymphocytes plus monocytes in 39 coke oven workers exposed to polycyclic aromatic hydrocarbons (PAH) and 39 non-exposed persons (controls) were investigated, each of the groups consisting of smokers and non-smokers. The adduct level was measured by an improved HPLC/fluorescence method (Rojas, M., Alexandrov, K., van Schooten, F. J., Hillebrand, M., Kriek, E. and Bartsch, H., Carcinogenesis, 15, 557-560, 1994) through the release of the corresponding benzo[a]pyrene (B[a]P) tetrols. The anti-BPDE-DNA adduct was detected in 51% of coke oven workers exposed to PAH and in 18% of the non-exposed (control) subjects. The mean level of anti-BPDE-DNA adducts/10(8) nucleotides in coke oven workers (15.7 +/- 37.8) was approximately 8 times higher than in non-exposed subjects (2.0 +/- 8.7). The interindividual variation of adduct levels was approximately 100-fold in coke oven workers and approximately 50-fold in controls respectively. Smokers in the exposed group had 3.5 times more DNA adducts than non-smokers. With the exception of one non-smoker with very high adduct levels (52.8 adducts/10(8)), the control subjects showed the presence of barely detectable adducts in only 16% of the samples examined. The increased in vivo formation in some smokers and high variability of anti-BPDE-DNA adducts in coke oven workers suggests variations in genetically controlled activation/inactivation reactions of PAH metabolism.
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7788856
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Effect of three tumour promoters on the stability of hepatocyte cultures and apoptosis after transforming growth factor-beta 1.
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Tumour promoters like the anti-androgen cyproterone acetate (CPA), the peroxisome proliferator nafenopin (NAF) and phenobarbital (PB) stimulate liver growth in rodents. Transforming growth factor-beta 1 (TGF-beta 1) is expressed in livers after treatment with CPA (Oberhammer et al., submitted) and some peroxisome proliferators. In this paper we describe the influence of CPA, NAF and PB on the stability of hepatocyte cultures and induction of apoptosis by TGF-beta 1. All three tumour promoters had a stabilizing effect on confluent monolayers of hepatocytes, partially preventing the usually occurring dedifferentiation and detachment processes. CPA on its own was able to induce apoptosis at the high dose of 10 microM. No induction of apoptosis could be observed after PB and NAF. At any dose above 0.01 microM CPA enhanced TGF-beta 1-induced apoptosis (5.8-fold increase with 10 microM CPA). Thus the combination of 10 microM CPA and 1 ng/ml TGF-beta 1 induced apoptosis in 90% of the plated hepatocytes. At a high dose (10 microM) NAF produced a 35% reduction in apoptosis induced by TGF-beta 1, in parallel with a stabilizing effect on cell number. PB did not affect the rate of apoptosis induced by TGF-beta 1. As demonstrated by immunohistochemical detection of PCNA, TGF-beta 1 prevented induction of PCNA by epidermal growth factor (EGF). No induction of PCNA was observable in CPA-treated cultures. In untreated and EGF-treated cultures TGF-beta 1 was able to induce apoptosis to the same extent within 30 h. In CPA-treated cultures this period was shortened to 12 h. Thus CPA shortens the lag phase of induction of apoptosis by shifting hepatocytes to a point before S phase, where they are highly susceptible to TGF-beta 1-induced apoptosis.
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7788855
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Mutagenicity and specific mutation spectrum induced by 8-methoxypsoralen plus a low dose of UVA in the hprt gene in diploid human fibroblasts.
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Exposure of cells to 8-methoxypsoralen plus a low dosage of UVA (365 nm) generates mainly monoadducts (PUVA-I treatment), while further irradiation of PUVA-I treated cells after removal of 8-methoxypsoralen (PUVA-II treatment) converts a high frequency of monoadducts to crosslinks. In this study, a comparison was made of the cytotoxicity and mutagenicity of PUVA-I-treated cells obtained here with those induced by PUVA-II treatment in our previous report. PUVA-I treatment slightly affected the colony-forming ability of cells. However, the 6-thioguanine-resistant cells were markedly increased from 3/10(6) clonable cells in UVA-irradiated populations to 47/10(6) clonable cells in PUVA-I-treated populations. Those results indicated that PUVA-I was more mutagenic than PUVA-II at equal cytotoxic doses, implying that psoralen monoadducts are less cytotoxic and as mutagenic as crosslinks. Mutations in the hypoxanthine (guanine) phosphoribosyltransferase gene of independent PUVA-I mutants were characterized by direct sequencing of cDNA and/or genomic DNA that were amplified by polymerase chain reaction. All the 30 sequenced mutants had single base substitutions. Of those mutations, 21 occurred in the coding region and the others were in the consensus sequences at exon-intron boundaries, thereby resulting in aberrant cDNA. The majority of base substitutions were T to A transversions (23/30); 22 were located at the thymine of 5'TA sites. All of the 24 T.A base pair substitutions (including one T to C) had thymine located on the non-transcribed strand. Five of the six G.C base substitutions were located at the 5' TG or 5' CA sites on the non-transcribed strand. The frequencies of mutations at 5'TA and 5'TG/5'CA sites were similar in PUVA-I- and PUVA-II-induced mutants. However, the specific kind of T.A base pair substitutions induced by PUVA-I is strikingly different from that induced by PUVA-II. While the transient misalignment-realignment model could account for PUVA-II-induced T.A base substitutions, the low cytotoxic effect and the specific T to A substitutions of PUVA-I treatment might be a result of rapid incorporation of nucleotides after insertion of an adenine or a thymine opposite the psoralen monoadducts on the template by DNA polymerases.
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7788854
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Dose dependence of diethylnitrosamine-induced nuclear enlargement in embryonal turkey liver.
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Avian embryos (turkey) were exposed to diethylnitrosamine in ovo. On the first day of incubation doses of 0.5-5.0 mg/egg were injected into the white of the fertilized egg. The experiment was terminated 4 days before hatching. Livers were removed and prepared for subsequent histological examination. In haematoxylin and eosin stained sections the areas of hepatocyte nuclear profiles were measured by semi-automatic image analysis. In liver samples of diethylnitrosamine-exposed embryos hepatocyte nuclei of more than twice the size of normal hepatocyte nuclei were found. The incidence of the enlarged nuclei was clearly dose dependent. An increase in the size of hepatocyte nuclei was observed after low doses of diethylnitrosamine that did not induce common signs of non-specific toxic effects, e.g. cell death, fat vacuoles or loss of glycogen. The slope of the dose-response curve was rather steep. A 10-fold increase in the dose of the carcinogen resulted in a 100-fold increase in the incidence of enlarged hepatocyte nuclei. In combination with preneoplastic foci of altered hepatocytes, the quantification of nuclear enlargement can provide a valuable complementary parameter for the evaluation of carcinogen-induced effects in ovo.
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7788853
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Promotion of N-nitrosodimethylamine-initiated mouse lung tumors following single or multiple low dose exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.
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The environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is highly toxic to several rodent species and may have adverse health effects in exposed human populations. Further, TCDD has been shown to be a potent liver tumor promoter in the rat after repeated administration. These studies were conducted to determine the tumor promoting capability of TCDD in the Swiss mouse following single or multiple exposures. Following tumor initiation with N-nitrosodimethylamine (NDMA; 25 mg/kg), animals were given either a single dose (1.6, 16 or 48 micrograms/kg) or repeated injections (0.05 microgram/kg/week for 20 weeks) of TCDD and sacrificed at 52 weeks of age. Neither NDMA nor TCDD caused an increase in incidence of liver tumors. NDMA induced lung tumors in 100% of animals, with 12 +/- 0.1 tumors/mouse. The multiplicity of lung tumors was significantly increased by low dose TCDD treatment, with 20 +/- 2.6 tumors/mouse following a single 1.6 micrograms/kg dose (P = 0.016) and 18 +/- 1.7 (P = 0.031) following repeated 0.05 microgram/kg doses (x 20). Higher doses of TCDD did not increase multiplicity of lung tumors and, in fact, may have been toxic to the lungs of NDMA-treated mice, as evidenced by the infiltration of pigmented macrophages. These data demonstrate the potent tumor promoting capability of TCDD in mouse lung.
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7788852
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Activation of phosphorylation of plasma membrane insulin-like growth factor-I receptors in the kidney of Syrian hamsters by diethylstilbestrol.
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In the present work we have investigated activation of phosphorylation of plasma membrane insulin-like growth factor-I receptors (IGF-IR) by diethylstilbestrol (DES). Insulin-like growth factor-I (IGF-I) stimulated the activity of membrane protein tyrosine kinase(s) (PTK) in both normal and DES-treated hamster kidneys. The level of IGF-I-stimulated PTK(s) almost doubled after 15 days of DES treatment. Autophosphorylation experiments revealed that phosphorylation of a 95 kDa band (presumably the beta subunit of IGF-IR) was 2-fold higher in the membranes of kidney from DES-treated animals compared with controls. To understand the mechanism of activation of IGF-I-dependent PTK by DES, we investigated the relationship between the binding capacity of IGF-I to membrane proteins and the level of IGF-IR. The binding of [125I]IGF-I to membranes from the DES-treated group was 30% higher than that of age-matched normal kidney (P < 0.001). Scatchard analysis of the binding data for both normal and DES-treated hamster kidney revealed a single class binding site for IGF-I with a dissociation constant (Kd) of 4.1 and 4.6 nM and a maximum binding capacity (Bmax) of 1786 and 2086 fmol/200 micrograms protein respectively. Therefore, the difference observed in [125I]IGF-I binding between DES-treated and normal kidney membranes may be partially due to an increase in the number of IGF-I binding sites, with no change in the affinity of the receptors for IGF-I. An enhanced level of IGF-IRs in membranes from DES-treated animals was visualized by autoradiography following affinity labeling of membrane proteins subjected to SDS-PAGE. Under reducing conditions a molecular band of 132 kDa was evident. The 132 kDa band represents the alpha-subunit of IGF-IRs. Northern blot analyses revealed that DES treatment increased the level of IGF-IR mRNA 2-fold compared with that of controls. These findings suggest that an enhanced level of IGF-IR coupled with qualitative changes may be responsible for the activation of IGF-I-dependent PTK on DES exposure. Whether the stimulation of IGF-IR phosphorylation by exposure to a carcinogenic dose of DES may be a factor in the induction of renal cancer in Syrian hamsters is not clear.
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7788851
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Cells transfected with transferrin receptor cDNA lacking the iron regulatory domain become more sensitive to the DNA-damaging action of oxidative stress.
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Chinese hamster V79 cells were transfected with human transferrin receptor cDNA, lacking the 3' untranslatable regulatory sequence. One of the clones obtained was investigated completely. It exhibited the cDNA sequences recombined into the genome, transcribed the corresponding mRNA and became partially constitutive in iron uptake from transferrin. The rate of iron uptake was significantly higher in these cells than in the parental ones, as was the intracellular iron content, assessed indirectly by measuring the activation of the regulatory protein responsible for iron homeostasis control. The transfected cells were more sensitive to the DNA-damaging action of H2O2. This corroborates the important role that iron plays as a mediator of DNA damage by reactive oxygen species. It also points to the possibility that mutations at the regulatory sequences of transferrin receptors, leading to partial disturbance in iron homeostasis, might render the cells more prone to further mutations and malignant transformations by reactive oxygen species.
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7788850
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Effects of testosterone, testosterone propionate, 17 beta-trenbolone and progesterone on cell transformation and mutagenesis in Syrian hamster embryo cells.
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Testosterone, testosterone propionate, 17 beta-trenbolone and progesterone, which represent the main endogenous and synthetic androgens and a progestin, were evaluated for possible cell transformation and genetic effects in Syrian hamster embryo (SHE) cells. Cell growth was reduced by treatment with the steroids at 10-30 micrograms/ml in a dose-related manner. Testosterone and testosterone propionate were less toxic than the other two steroids. Testosterone, testosterone propionate and progesterone induced morphological transformation of SHE cells with similar transformation frequencies. The most potent effects were observed with testosterone propionate, which induced cell transformation at 1-30 micrograms/ml in a dose-related manner. Testosterone and progesterone transformed cells only at the highest dose (30 micrograms/ml). 17 beta-Trenbolone did not induce a statistically significant level of cell transformations at any dose tested (up to 30 micrograms/ml). The transformation frequencies induced by testosterone, testosterone propionate and progesterone were less than one-half that induced by benzo[a]pyrene at 1 microgram/ml. None of these steroids induced significant increases in frequencies of chromosome aberrations or aneuploidy. Gene mutations were not observed for testosterone at the HPRT or Na+/K+ ATPase locus. Because these steroids are also associated with carcinogenic activity in vivo, these in vitro findings provide a model and new insights into the study of the mechanisms of androgen- and progestin-induced cell transformation.
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7788849
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Rat CYP1B1: an adrenal cytochrome P450 that exhibits sex-dependent expression in livers and kidneys of TCDD-treated animals.
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The broad spectrum of biological responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is believed to be due to the alteration in expression of TCDD-inducible genes. The aim of this study was to investigate the effects of TCDD on the in vivo tissue-specific expression of the recently identified TCDD-inducible cytochrome P450 CYP1B1 [Sutter et al. (1994) J. Biol. Chem., 269, 13092-13099] in Sprague-Dawley rats. We cloned the 5.0 kb rat homolog of CYP1B1 from a TCDD-treated rat liver cDNA library and showed that the rat and human CYP1B1 predicted amino acid sequences are 80% identical. RNA hybridization analysis showed that CYP1B1 is constitutively expressed in the adrenal glands and also in the testes of untreated rats. This tissue distribution suggests that CYP1B1 may be a physiological steroid hydroxylase. Seventy-two hours post-administration of 25 micrograms/kg body wt TCDD by gavage, steady-state levels of the 5.1 kb CYP1B1 RNA were increased > 50-fold in liver, and to a lesser extent in kidneys, lung, heart and ovaries. Average CYP1B1 RNA levels were significantly higher in the kidneys and livers of TCDD-treated females than in those from similarly treated males. In contrast, no significant sex-difference was observed in the levels of CYP1A1 in these tissues in TCDD-treated animals. In Sprague-Dawley rats, TCDD is a more potent hepatocarcinogen in females than in males. The induction of CYP1B1 in TCDD rat liver may be a contributing factor to the carcinogenic action of this persistent environmental pollutant.
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7788848
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Induction at high incidence of ductal prostate adenocarcinomas in NBL/Cr and Sprague-Dawley Hsd:SD rats treated with a combination of testosterone and estradiol-17 beta or diethylstilbestrol.
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This study determined the incidence of prostate adenocarcinoma following long-term treatment of NBL and Sprague-Dawley rats with estradiol-17 beta or diethylstilbestrol (DES) plus testosterone and it defined the origin of these tumors. NBL and Sprague-Dawley rats were treated with two Silastic tubing implants (i.d. 1.6 mm, o.d. 3.2 mm) containing a 2 cm long filling of testosterone and one implant containing a 1 cm long filling of estradiol-17 beta or DES. Control animals received empty implants. Treated animals were killed when moribund and controls were killed at 91 (NBL) or 75 (Sprague-Dawley) weeks after initiation of treatment and accessory sex glands were sampled for histopathological examination of multiple step sections. Prostatic adenocarcinoma occurred in 100% of NBL rats after treatment with estradiol-17 beta or DES plus testosterone for 44 and 59 weeks (group means) respectively. Adenocarcinoma incidences were lower in Sprague-Dawley rats. The adenocarcinomas were small, microscopic, invasive tumors and they were spatially closely associated with the periurethral ducts of the dorsal, lateral and/or anterior (= coagulating gland) prostate, but never with the ducts of the ventral lobe and seminal vesicles. One adenocarcinoma was of uncertain origin. Duct-acinar dysplastic lesions occurred in the periphery of the dorsal and lateral prostate of all hormone-treated NBL and many Sprague-Dawley rats, but did not appear to give rise to carcinoma. Although some adenocarcinomas were contiguous with dysplastic ducts of the peripheral dorsolateral prostate, the main mass of these neoplasms was located in the periurethral area. Also, most adenocarcinomas were only connected with the periurethral ducts, in which atypical hyperplasia occurred following hormone treatment for 36 weeks or longer. Thus atypical hyperplasia of the periurethral prostate ducts, but not peripheral duct-acinar dysplasia, appeared to be the likely precursor of the induced carcinomas. Testosterone plus DES, but not estradiol-17 beta, induced marked dysplasia-like lesions in the acini of the ventral prostate of all NBL and many Sprague-Dawley rats. These lesions had progressed to carcinoma in situ (or adenoma) in 46% of NBL rats.
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7788847
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Transplacental transfer of environmental genotoxins: polycyclic aromatic hydrocarbon-albumin in non-smoking women, and the effect of maternal GSTM1 genotype.
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Transplacental transfer of genotoxic material has been determined by measuring the polycyclic aromatic hydrocarbon-albumin adduct level in serum isolated from the mother and the umbilical cord using a competitive ELISA assay and the antibody (8E11) against benzo[a]pyrene (B[a]P) tetrols. Smoking women (median 5.54 fmol B[a]P equiv/microgram albumin; 21 cases) and non-smoking women living in rural areas (median 4.99; 30) had higher adduct levels than non-smoking women living in suburbia (median 4.09; 37), whereas non-smoking women living in the city of Aarhus had an intermediate level (median 4.82; 40). Exposure to passive smoking did not modify the adduct levels. When all non-smoking cases were combined, the transport time to/from the home became a major contributing factor to the adduct level. The median adduct level in umbilical cord blood was significantly lower than in maternal blood, the maternal/fetal ratio being approximately 1.3, and a positive association between the adduct levels in the mother and umbilical cord blood was observed. The frequency of the GSTM1 null genotype in the study population, females aged 19-44, was 55.4%, but the GSTM1 genotype did not significantly alter the serum albumin adduct level. This study indicates that the competitive ELISA to detect B[a]P bound to serum albumin is sensitive enough to detect differences in the burden of genotoxic compounds in non-occupational exposed individuals. The lower adduct level in people living in suburbia suggests that local production of incomplete combustion products, like vehicle exhaust or heat generation, is the major contributing factor to genotoxic compounds in the general environment.
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7788846
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DNA damage as assessed by 32P-postlabelling in three rat strains exposed to dietary tamoxifen: the relationship between cell proliferation and liver tumour formation.
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Tamoxifen was administered in the diet (420 p.p.m.) to female F344 (Fischer), Wistar (LAC-P) and LEW (Lewis) rats to determine for each strain the early morphological and biochemical changes associated with the subsequent development of liver cancer. Hepatic DNA damage, as determined by 32P-postlabelling, showed a cumulative increase with time from 500 adducts/10(8) nucleotides at 30 days to almost 3000 adducts/10(8) nucleotides after 180 days, with little difference between strains at this time point. A significant strain difference was found in the number of adducts present in the Fischer rats at 90 days, compared to the Wistar and Lewis strains. There was a marked strain differences in the time to development of liver tumours. After 6 months treatment, both Wistar and Lewis rats had tumours while none were seen in the Fischer animals. After 11 months, all of the Wistar and Lewis rats had developed liver carcinoma, while the Fischer rats developed liver carcinoma by 20 months. Depression in cell proliferation, relative to age-matched controls, was seen in the livers of Fischer rats after six months of exposure to tamoxifen, in contrast to an increase in the Wistar and Lewis rats. This observation is consistent with the promotion of foci to tumours and the subsequent progression of tumours to carcinomas in the latter two strains. These data may assist in establishing the possible risk factors, such as extent of DNA damage and increased liver cell proliferation, to women with long-term prophylactic exposure to tamoxifen.
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7788845
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Aberrant expression of gap junction proteins (connexins) is associated with tumor progression during multistage mouse skin carcinogenesis in vivo.
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To elucidate what changes in the expression of gap junction proteins (connexins) occur at what stages during multistage mouse skin carcinogenesis in vivo, we immunohistochemically and morphometrically analyzed the expression of connexin 26 (Cx26) and connexin 43 (Cx43) in papillomas, well-, moderately- and poorly-differentiated squamous cell carcinomas, as well as in squamous cell carcinomas at invasion sites and those metastasized into lymph nodes in female CD-1 mice as a result of treatment with dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. In papillomas, no clear reduction of the two connexins was observed; however, Cx26 and Cx43 were frequently co-localized in the same gap junction plaques, whereas the two kinds of Cxs were differentially expressed in normal and surrounding non-tumorous epidermis. In squamous cell carcinomas, the expression of both Cx26 and Cx43 significantly decreased compared with surrounding non-tumorous epidermis and papillomas. The Western blot analysis confirmed that both Cx26 and Cx43 proteins were reduced in squamous cell carcinomas compared with papillomas. Furthermore, the expression of Cx26 was reduced as cancer cells became morphologically less differentiated, while that of Cx43 did not change. Squamous cell carcinomas at invasive sites showed clear reduction of Cx26 and Cx43. In squamous cell carcinomas metastasized into lymph nodes, Cx26 was expressed, but few carcinoma cells expressed Cx43. The localization of E-cadherin on the plasma membrane between cancer cells was maintained even at invasive and metastatic sites. Our data suggest that quantitative and qualitative changes in connexin expression are associated with tumor progression, including the loss of differentiation, and invasion and metastasis, during multistage mouse skin carcinogenesis.
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7788844
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Spectrum of mutations induced by methyl and ethyl methanesulfonate at the hprt locus of normal and tag expressing Chinese hamster fibroblasts.
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This work describes the isolation and characterization of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) induced 6-thioguanine-resistant mutants in normal and Escherichia coli tag gene expressing Chinese hamster fibroblast, RJKO, cells. It was previously shown that increased removal of 3-alkylated adenine, effected by 3-methyladenine DNA glycosylase I (Tag), reduces the frequencies of hprt mutations induced by alkylating agents which produce mostly N-alkylation (MMS and EMS) to half the normal rate. In order to identify which type of mutation is suppressed by increased 3-alkyladenine repair we have determined the DNA base sequence changes of the hprt cDNA in 61 independent MMS- and EMS-induced mutant clones. For both cell types and irrespective of the agent used, the majority of mutations were GC to AT transitions originating in the non-transcribed strand. Only 6/55 base substitutions occurred at AT base pairs: five AT to GC transitions and one AT to CG transversion. Six mutations were found to be deletions. These results indicate that 3-alkylated adenines in DNA are not directly premutagenic. The fact that the mutation frequency is reduced by increased 3-alkyladenine removal might be explained by postulating the existence in mammalian cells of an SOS-like response turned on by cytotoxic lesions like 3-alkyladenine, or, alternatively, that increased removal of 3-alkyladenine increases the number of single-strand breaks in DNA, which stalls DNA replication and allows a prolonged time for DNA repair by the alkyltransferase.
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7788843
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Detection of O6-methylguanine, O4-methylthymine and O4-ethylthymine in human liver and peripheral blood leukocyte DNA.
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O6-Methylguanine, O4-methylthymine and O4-ethylthymine were determined by a recently developed, highly sensitive and specific method (PREPI, pre-fractionation, 32P-post-labeling and immunoprecipitation) in human liver DNA obtained from 15 autopsy specimens and in 15 peripheral blood leukocyte DNA samples obtained from healthy volunteers. All the cases had no obvious history of recent occupational or therapeutic exposure to alkylating agents. In the human liver DNA O6-methylguanine was detected in 13 cases at levels of 1.1-6.7 adducts/10(7) guanine; two cases were below the detection limit of approximately 1.1 x 10(-8). O4-Methylthymine and O4-ethylthymine were detected in all the liver samples at levels of 0.1-14 adducts and 0.5-140 adducts/10(7) thymine respectively. The mean value of the ratio of O6-methylguanine to O4-methylthymine was about 6. Among the three DNA adducts measured there was no significant correlation between any two combinations of adducts. In peripheral leukocytes of healthy volunteers O6-methylguanine was detected at 0.7-4.6 adducts/10(8) guanine, this being approximately 3-6% of that in livers. Neither O4-methylthymine nor O4-ethylthymine was above the detection limits. These results suggest that humans are exposed to exogenous and/or endogenous methylating and ethylating agents in daily life.
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7788842
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Inhibition of apoptosis during 2,3,7,8-tetrachlorodibenzo-p-dioxin-mediated tumour promotion in rat liver.
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The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on cell division and cell death (apoptosis) in glutathione S-transferase (GST-P)-positive liver foci were analyzed in diethylnitrosamine-initiated female Wistar rats that were treated with TCDD, either acutely for 3 days or chronically for 115 days. Apoptotic bodies were quantitated in liver sections simultaneously stained for GST-P expression and H&E using a novel fluorescence microscopic detection method which greatly facilitates recognition of apoptotic bodies due to their high level of eosin fluorescence. While TCDD treatment only marginally affected cell division in GST-P-positive liver foci, as estimated by 5-bromo-2'-deoxyuridine-labelling, apoptotic indices were decreased to approximately 60% and approximately 10% of control values after acute and chronic TCDD treatment, respectively. In normal liver tissue, apoptotic indices were only slightly reduced by TCDD treatment, suggesting selective inhibition of apoptosis in the enzyme-altered cell population by the dioxin. Since inhibition of apoptosis in GST-P-positive liver foci was by far more pronounced than changes in cell division, our data suggest that the promoting activity of TCDD is preferentially mediated by a decrease of apoptosis in enzyme-altered liver foci.
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7788840
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Role of GSTT1 and GSTM1 genotypes in determining individual sensitivity to sister chromatid exchange induction by diepoxybutane in cultured human lymphocytes.
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The individual genotoxic response of cultured human lymphocytes to diepoxybutane (DEB), an epoxide metabolite of 1,3-butadiene, shows a bimodal distribution. Blood donors can be classified as either DEB-sensitive or DEB-resistant on the basis of the frequency of sister chromatid exchanges (SCEs) induced by DEB in whole-blood lymphocyte cultures. The genetic basis of this phenomenon has thusfar been unknown. To investigate if differences in the ability of individuals to detoxify DEB could explain the bimodal response, sister chromatid exchanges (SCEs) induced by a 48-h treatment with DEB (2 and 5 microM) were analyzed in whole-blood lymphocyte cultures of 20 human donors with known genotypes of two polymorphic glutathione S-transferases (GSTs), GSTT1 and GSTM1. Both polymorphisms include a homozygous null genotype lacking the respective GST gene and isozyme. The mean frequency of SCEs/cell was 1.6 times higher among GSTT1 null donors (n = 8) than GSTT1 positive donors (n = 12) at both 2 microM DEB (mean 67.3 versus 40.9) and 5 microM DEB (mean 123.2 versus 77.5), with no overlapping in DEB-induced individual SCE frequencies between the two genotypes. Thus, all DEB-sensitive individuals were of the GSTT1 null genotype, while all DEB-resistant persons had a detectable GSTT1 gene. A significant (P < 0.05) negative correlation (r = -0.65 at 5 microM, r = -0.56 at 2 microM) was obtained in the GSTT1 positive donors between DEB-induced individual SCE frequency and RBC GSTT1 activity, measured by formaldehyde formation from dichloromethane; the GSTT1 null individuals showed no GSTT1 activity. At 5 microM DEB, the lymphocyte cultures of the GSTT1 null donors also had a significantly decreased replication index, indicating an impact of GSTT1 genotype on the cytotoxicity of DEB. No influence on DEB-induced SCEs or cytotoxic effects was observed for GSTM1 genotype. It is concluded that sensitivity to in vitro SCE induction by DEB is explained by the lack of GSTT1.
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7788841
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DNA repair synthesis and DNA fragmentation in primary cultures of human and rat hepatocytes exposed to cyproterone acetate.
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Cyproterone acetate (CPA), a synthetic steroid used in human therapy, has recently been shown to cause DNA damage in cultured rat hepatocytes and in rat liver. In the present study we have investigated whether CPA also induces genotoxic effects in human hepatocytes. Genotoxicity of CPA was determined by measuring the capability of the compound for inducing DNA repair synthesis and for causing the formation of DNA single-strand breaks. Autoradiography and alkaline elution were used to quantitate DNA repair and DNA fragmentation, respectively. Exposure of hepatocytes to CPA for 20 h induced DNA repair synthesis in two hepatocyte preparations derived from males and in four of the five preparations derived from females. In cultures from some donors, induction of repair was detected at 1 microM CPA, the lowest concentration tested. The maximum effect generally occurred at 10-20 microM. Only a very slight increase in the frequency of DNA single-strand breaks was found following exposure of the hepatocytes to 50 microM CPA for 20 h. For comparative purposes, the effects of CPA on DNA repair and DNA fragmentation were also determined in cultured rat hepatocytes. A strong induction of DNA repair synthesis, but only a slight enhancement in DNA fragmentation was observed in CPA-treated hepatocytes derived from female rats. These results indicate that the measurement of repair is a more sensitive indicator for the genotoxicity of CPA than the measurement of DNA fragmentation. No genotoxic effects of CPA were detectable in hepatocyte cultures derived from male rats. The present findings show that CPA is genotoxic in human hepatocytes and that the striking sex difference in the genotoxicity of CPA in rat cells is not observed with human cells.
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7788834
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Canadian anaesthesia physician resource planning--is it possible?
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This study was undertaken with the objective of assessing current sources of information for anaesthesia Physician Resource Planning (PRP). Four major data bases, the annual reports of Health and Welfare Canada (H&W), the education statistics from the Canadian Post-M.D. Education Registry (CAPER), the Royal College of Physicians and Surgeons of Canada (RCPSC) and the Physician Resource Data System of the Canadian Medical Association (PRDS), were examined for the period 1982 to 1991. The ratio of the number of surgical (S) to anaesthesia (A) clinicians decreased over this period despite an increase in the S:A ratios for trainees and certificants. The number of female anaesthetists has progressively increased. A steady decline in the number of rural anaesthetists has occurred. Age distribution of active certified anaesthetists revealed marked inter-regional differences. Little change was noted in the total mean hours worked per week. Each database provided valuable, but limited, data. The PRDS data is useful in assessing trends (age, sex and practice activity). Information provided by H&W tends to underestimate anaesthesia resource information by at least 10%. While information obtained from RCPSC and CAPER is accurate, the current mode of presentation of data limits their usefulness. Integrating data from all the databases appears to provide a meaningful assessment for PRP rather than assessing each database in isolation. Interpretation of the information and its value must take into account the limitations of the data being provided. Assessing present and planning future needs based on the current information structure will prove extremely difficult.
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7788833
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Upper airway involvement in Parkinson's disease resulting in postoperative respiratory failure.
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A 71-yr-old man with a six-year history of Parkinson's disease (PD), Type II diabetes mellitus, myocardial infarction, and remote 20 pack-year smoking history, underwent an anterior resection of the rectum for carcinoma. Sixty hours later, the patient suffered a respiratory arrest; his antiparkinsonian medications had not been resumed. Preoperative flow-volume loops showed the characteristic saw-tooth pattern of PD indicating dysfunction of the striated muscle of the upper airway. Although postoperative respiratory distress was managed as lower airway obstruction, at the time of intubation there were no signs of lower airway pathology. Upper airway dysfunction and obstruction secondary to PD is thought to have been a contributing factor to the postoperative respiratory distress and failure. This case is presented to draw attention to the risk of upper airway dysfunction in Parkinson's Disease, especially with the withdrawal of antiparkinsonian medications.
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7788832
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Pressure support ventilation with the laryngeal mask airway: a method to manage severe reactive airway disease postoperatively.
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The use of a laryngeal mask airway (LMA) and a bi-level positive airway pressure (BiPAP) machine is described in a post-operative thoracotomy patient with reactive airway disease. The LMA was placed to avoid reintubation of the trachea after a double lumen tube was no longer necessary. Placement in an awakening patient and positive-pressure ventilatory support were well tolerated and did not trigger a bronchospastic response. The patient was able to cough and breathe deeply with the LMA while receiving ventilatory assistance in the post-anaesthesia care unit (PACU). The LMA is a therapeutic option to tracheal reintubation in patients who need postoperative ventilatory support after one-lung anaesthesia.
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7788831
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Placental transfer of lidocaine hydrochloride after prolonged continuous maternal intravenous administration.
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We treated a patient with arrhythmia during pregnancy with prolonged intravenous administration of lidocaine hydrochloride. This was a case of twin-to-twin transfusion syndrome and the arrhythmia was caused by ritodrine therapy. In total, 14.1 g lidocaine (50 mg.hr-1 for 282 hr) were used. Since there are no descriptions of human placental transfer of lidocaine after such a prolonged continuous intravenous administration, we measured lidocaine concentrations in maternal and fetal serum, and in the amniotic fluid (AF) at delivery. Fetal serum lidocaine concentrations (donor: 0.83 microgram.ml-1; recipient: 0.82 microgram.ml-1) were lower than in the maternal serum (1.6 micrograms.ml-1), while the AF lidocaine concentrations (donor: 1.05 micrograms.ml-1; recipient: 1.04 micrograms.ml-1) were higher than those of the fetal sera. The fetal/maternal concentration ratios of lidocaine were 0.52 for the donor and 0.51 for the recipient, which were similar to those described previously after administration of lidocaine in labour.
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7788828
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Paediatric cardiac catheterization: innovations.
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In recent years interventional procedures have been introduced to the field of paediatric cardiac catheterization. These procedures continue to develop in complexity and increasingly are being applied to patients with reduced cardiovascular reserve, as an alternative to cardiac surgery or when cardiac surgery with cardiopulmonary bypass is contraindicated. More frequently anaesthetists are being called upon to provide support in sedating, anaesthetizing or/and resuscitating these patients. The purpose of this review is to give a comprehensive update of the interventional procedures and to review the anaesthetic management techniques as they apply to the catheterization laboratory. We will discuss possible complications and management strategies from our own experience and the experience of others. We have observed that as more complicated procedures are performed the anaesthetist plays a pivotal role in the management of the patient from arrival to departure from the cardiac catheterization laboratory, and in preventing mortality and major morbidity. Although the economic consequences of interventional cardiological techniques remain unclear, the field continues to expand and more complex procedures are continually being introduced.
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7788830
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Alternating nitroglycerin and syntocinon to facilitate uterine exploration and removal of an adherent placenta.
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Nitroglycerin (NTG) has been demonstrated to provide uterine relaxation in the management of various obstetric complications. A 32-yr-old woman presented 40 min postpartum for manual removal of a retained placenta. Repeated, alternating doses of NTG 250 micrograms and syntocinon (SYN) 10U iv were used over 15 min to produce periods of uterine relaxation and contraction respectively for uterine exploration. Multiple attempts to extract the placenta failed and a diagnosis of placenta accreta was made. There were no major side effects from this combination of drugs apart from a transient 20% decrease in blood pressure after NTG, which responded to ephedrine 10-15 mg iv. The rapid change in uterine tone was believed to be due not only to the short duration of action of NTG and SYN, but also to the possible physiological antagonism between the two drugs. The mechanism of interaction may involve calcium mobilization and myosin light chain phosphorylation. We conclude that NTG and SYN can be used to produce alternating periods of uterine relaxation and contraction rapidly and consistently with little sustained effects from either agents.
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7788829
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Maternal death following epidural anaesthesia for caesarean section delivery in a patient with unsuspected sepsis.
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Sepsis in the parturient may be difficult to recognize in light of the physiological changes associated with pregnancy. The purposes of this report are to highlight the signs and symptoms which indicate an underlying septic process and the management of these patients in the peripartum period. This 32-yr-old GII PI woman with twin gestation presented at 36 wk in labour. Her temperature was 35.3 degrees C, she was normotensive and had a normal white blood cell count. After epidural anaesthesia was administered for Caesarean section, she became apnoeic, pulseless and unresponsive. Despite aggressive cardiopulmonary resuscitation, neither she nor her twin babies survived. Post mortem blood work revealed a considerable left shift of her white blood count (> 60% bands) and an anion gap acidosis. Autopsy revealed evidence of widespread Group A beta-haemolytic streptococcal sepsis. Diagnosis of sepsis in the parturient involves assessment of the patient's temperature, WBC and differential and acid-base status. Evaluation of the intravascular volume must precede anaesthetic intervention. Epidural anaesthesia may be considered in the labouring and Caesarean section patient who has been fluid-resuscitated. Emergency operative delivery may result in cardiovascular compromise in the patient with severe sepsis.
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7788827
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Modification of the haemodynamic responses to induction of anaesthesia and tracheal intubation with alfentanil, esmolol and their combination.
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The purpose of this double-blind randomized work was to study the effect of alfentanil and esmolol and their half-dose combination on the increases of heart rate and arterial pressure and on the prolongation of the QTc interval of the ECG occurring during anaesthetic induction. Sixty ASA class I-II patient with mean age ranging from 26 to 32 yr among the groups. Patients were allocated to one of four equal groups to receive saline, esmolol 2 mg.kg-1, alfentanil 0.03 mg.kg-1 and alfentanil 0.015 mg.kg-1+esmolol 1 mg.kg-1. Anaesthesia was induced with thiopentone. Succinylcholine was used to facilitate tracheal intubation. Haemodynamic variables were measured non-invasively and the QTc interval with the aid of a microcomputer. Comparisons between the groups were performed using two-way analysis of variance with repeated measures. Both alfentanil and alfentanil-esmolol prevented the increase of heart rate and arterial pressure caused by intubation whereas esmolol prevented only the increase of the heart rate. None of the treatments prevented prolongation of the QTc interval after intubation and only alfentanil prevented that after succinylcholine. The present results suggest that in the prevention of the haemodynamic responses to tracheal intubation, the half-dose combination of alfentanil and esmolol is as effective as alfentanil and superior to esmolol. The combination is preferable to relatively large doses of either drug in circumstances where side effects, such as respiratory depression due to alfentanil or bradycardia due to both drugs should be minimized.
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7788826
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Thoracic epidural clonidine and morphine for postoperative pain relief.
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This study was undertaken to evaluate the potentiation of the postoperative analgesic effect of thoracic epidural morphine by coadministration of thoracic epidural clonidine in a randomized double-blinded design. Twenty patients underwent radical gastrectomy under combined general anaesthesia (enflurane and nitrous oxide/oxygen) and epidural anaesthesia with local anaesthetics. They received a thoracic epidural bolus injection of either 0.05 mg.kg-1 morphine plus 3 micrograms.kg-1 clonidine (M+C group; n = 10) or 0.05 mg.kg-1 morphine alone (M group; n = 10) immediately before completion of surgery. All patients received iv morphine via patient-controlled analgesia (PCA) equipment for 24 hr postoperative period, and the PCA iv consumption of morphine was the primary variable of efficacy of the analgesic regimen. In addition, data analyses included mean arterial blood pressure, heart rate, respiratory rate, arterial blood gas measurement, sedation score, and visual analogue pain scale score (VAS). The cumulative number of iv morphine injections via PCA was less in the M+C group than in the M group at each hour for 24 hr postoperative period (P < 0.05), while the numbers of PCA morphine injections per hour beyond nine hours after surgery were higher in the M group than in the M+C group (P < 0.05). Sedation score was higher, and VAS and mean blood pressure were lower in the M+C group only at one hour after surgery compared with the M group. We conclude that the combined single thoracic epidural administration of morphine plus clonidine produces a more potent and longer lasting analgesia than does morphine alone.
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7788825
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Self-administered intranasal meperidine for postoperative pain management.
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Recent studies have demonstrated that intranasal is comparable to intravenous opioid titration in its pain-relieving effect. In these studies, however, the intranasal opioid titration was performed by the investigator, and the treatment period was two hours or less. The purpose of this randomized, prospective study was to investigate whether intranasal opioid administration by the patients themselves for a prolonged postoperative period may be regarded as a therapeutic alternative for postoperative pain management. Forty-four orthopaedic patients were studied over a 12-hr period on the first day after surgery. Twenty-two had free access to intranasal meperidine (nasal group) and were allowed to administer six intranasal puffs (27 mg per dose). The next self-administration was only permitted after a delay of at least ten minutes. Another 22 patients received intermittent subcutaneous meperidine injections (25 or 50 mg) on request (sc group). Pain intensity was recorded at 30-min intervals with the aid of the 101-point numerical rating scale. The pain score was lower in the nasal than in the sc group at the 30, 150 to 330, 420 to 480 and 540 to 600 min measuring points (P = < 0.05). The meperidine requirement was 112.9 +/- 81.3 mg in the nasal and 103.4 +/- 41.5 mg in the sc group (NS). Two patients in each group complained of nausea and vomiting. Thirteen of the 21 nasal and nine of the 15 sc patients who completed the final questionnaire rated the pain management as excellent or good (NS).(ABSTRACT TRUNCATED AT 250 WORDS)
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7788824
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Incidence of malignant hyperthermia reactions in 2,214 patients undergoing muscle biopsy.
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To determine the incidence of malignant hyperthermia (MH) reactions after trigger-free anaesthesia in a large population of MH-susceptible (MHS) patients, the charts of 2,214 patients who underwent elective muscle biopsy for malignant hyperthermia were reviewed. Either general or regional anaesthesia with non-triggering drugs was used. For general anaesthesia, the trachea was intubated in the absence of muscle relaxants. The halothane/caffeine contracture test was performed on the biopsied muscle. Suspected MH reactions were identified according to their site of occurrence (in the operating room, recovery room or ward). Ninety-seven percent of patients received a general anaesthetic; 3% received a regional anaesthetic or field block. Of the 2214 patients whose muscles were biopsied, 1082 patients were biopsy-positive for MH. Five patients in whom MH reactions were diagnosed in the recovery room were all subsequently proved to be biopsy-positive for MH. Four of the five received intravenous dantrolene as part of their therapy; the fifth received only symptomatic therapy as parenteral dantrolene was not commercially available. All patients recovered completely from their reactions. We conclude that the incidence of MH reactions in biopsy-positive patients who receive a trigger-free anaesthetic for minor surgery is small (0.46%, (0.15-1.07%, 95% CI)). These reactions occur in the immediate postoperative period.
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7788823
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Oral ondansetron decreases vomiting after tonsillectomy in children.
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Vomiting is a common, unpleasant aftermath of tonsillectomy in children. Intraoperative intravenous ondansetron (OND) reduces vomiting after this operation. Our double-blind, placebo-controlled, randomized investigation studied the effect of the oral form of OND on vomiting after outpatient tonsillectomy in children. We studied 233 healthy children age 2-14 yr undergoing elective tonsillectomy. Subjects were given placebo (PLAC) or OND 0.1 mg.kg-1 rounded off to the nearest 2 mg one hr before surgery. Anaesthesia was induced with either propofol or halothane/N2O. Vecuronium 0.1 mg.kg-1 was administered at the discretion of the anaesthetist. Anaesthesia was maintained with halothane/N2O, 50 micrograms.kg-1 midazolam iv and 1-1.5 mg.kg-1 codeine im. At the end of surgery, residual neuromuscular blockade was reversed with neostigmine and atropine. All episodes of in-hospital emesis were recorded by nursing staff. Rescue antiemetics in the hospital were 1 mg.kg-1 dimenhydrinate iv for vomiting x 2 and 50 micrograms.kg-1 droperidol iv for vomiting x 4. Parents kept a diary of emesis after discharge. Postoperative pain was treated with morphine, codeine and/or acetaminophen. The two groups were similar with respect to demographic data, induction technique and anaesthesia time. Oral OND (n = 109) reduced postoperative emesis from 54% to 39%, P < 0.05. This effect was most dramatic in-hospital, where 10% of the OND-patients and 30% of the PLAC-group vomited, P < 0.05. The OND-subjects required fewer rescue antiemetics, 7% vs 17%, P < 0.05. In conclusion, oral ondansetron decreased the incidence of vomiting after outpatient tonsillectomy in children.
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7788822
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Nitrous oxide does not increase vomiting in children after myringotomy.
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The aim of this study was to establish whether nitrous oxide has a clinically important effect on postoperative vomiting in children after myringotomy. We studied 320 healthy children of ages 0.5-13 yr undergoing elective myringotomy and tube insertion. Induction and maintenance of anaesthesia were randomized to inhalation with either 70% N2O/30% O2/halothane or 100%O2/halothane. Surgical technique and postoperative management were not influenced by this study. Vomiting in the recovery room (PAR) and Day Care Surgical Unit (DCSU) was recorded by nurses unaware of the anaesthetic technique. Parents were contacted 24-48 hr after surgery to ascertain the incidence of vomiting after discharge. The groups were similar with respect to demographic data, except that the anaesthesia time was greater among the 158 patients in the N2O-treated group (11 +/- 4 vs 12 +/- 4 min, mean +/- SD). The incidence of vomiting was 13% in both groups. Most of the 42 patients that had emesis only vomited once or twice. The incidence of vomiting was not altered by sex (13% vs 13%) or duration of anaesthesia. The incidence of vomiting increased with increasing age. The children aged less than 3 yr vomited 4% of the time, those aged 3-5 vomited 11% of the time, those aged 6-8 yr vomited 17% of the time, while the incidence of vomiting among those aged 9-13 yr was 31%. Vomiting prolonged the postoperative hospital stay from 75 to 92 min, P < 0.001, ANOVA. In summary, we have been unable to demonstrate that N2O induces vomiting by children after a brief general anaesthetic for myringotomy.
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7788818
|
Early diagnosis of colorectal cancer.
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It is reasonable to suggest that survival in colorectal cancer is closely linked to the anatomical stage of the tumour, implying that detection and removal of less advanced cancers will lead to a prolongation of survival. Early diagnosis of colorectal cancer therefore should be an important goal to strive for. An effort to achieve early diagnosis may be based on identification of high risk patients, and screening of groups at intermediate risk of developing colorectal cancer. Patients with well defined precancerous conditions should be included in surveillance programme. Prophylactic surgical treatment should be considered in longstanding ulcerative colitis and familial polyposis syndrome. Improved methods of genetic mapping and identification of the genetic defects that predisposed to cancer will probably be of great importance in the future. To reach groups of the population for early diagnosis of colorectal cancer large-scale screening is probably necessary. A change towards less advanced cancers is likely to be the result of mass screening. Improved survival with acceptable cost-benefit ratio remains to be shown.
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7788816
|
[Nuclear DNA in neoplastic pathology of the thyroid gland].
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We have analysed the nuclear DNA content in a series of 33 thyroid tumours (18 carcinomas and 15 adenomas) and, for comparison, in 189 nodular strumas and 17 tissue samples of normal thyroids, calculating cell cycle parameters and presence of aneuploid DNA. Cell suspensions were prepared for each of three tissue fragments taken for different areas of the surgical specimens. Cell subpopulations with aneuploid DNA content were present in 39% of carcinomas, 27% of adenomas, 20% of strumas, but were absent in the samples from normal thyroids. The presence of hyperdiploid DNA was much more frequent than that of hypodiploid DNA in all pathologic tissues. The mean proliferation index was 14.1 in the carcinomas, 6.6 in the adenomas, 7.1 in the strumas and 6.1 in normal thyroid tissues. The percent of histograms with a coefficient of variation of the G0-G1 peak greater than 5 was highest in the carcinomas (26%). The significance and implications of the reported data are discussed for the interpretation of thyroid neoplastic pathologies.
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7788815
|
[Dysplasia and colorectal neoplasms in a group of patients with ulcerative rectocolitis (URC)].
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In our Medical Department we examined 74 patients with ulcerative colitis between January 1987 and December 1992. All the patients have been controlled by colonoscopy and multiple rectal byopsies. As started by different studies, the ulcerative colitis is predictive of mucosal dysplasia alterations and colo-rectal carcinoma. Our experience confirms that the occurrence is not so high as reported by some preliminary studies. These results could probably attributed to better diagnostic and therapeutic strategies now available.
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7788814
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[Tumor markers in malignant anorectal pathology].
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The Authors note the limits and the more rational directions of using the several tumour markers that they utilized in the study of the neoplastic pathology of the anus-rectum. They particularly point out the benefit as "dynamic indices" of variation of the tumor mass during the neoplasm's progress and treatment. For these neoplasies any new marker offers advantages of diagnostic accuracy and a greater specificity more CEA. Nevertheless other markers used appear interesting as biochemical parameters for tumour growth. The study and the tumour marker's development supported, in recent years, the introduction of new diagnostic methods that include the utilization of monoclonal localization of the tumour mass.
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7788813
|
[Primary non-Hodgkin's lymphomas of the colon: apropos of a case with double localization].
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A case of non-Hodgkin's lymphoma involving the caecum and the rectum was resected. The disease occurs in 1-4% of gastrointestinal neoplasms and comprises 0.2-0.65% of colorectal tumours. The caecum is frequently involved while primary malignant lymphoma localized to the caecum and the rectum is more uncommon. The most representative histological types are histiocytic or lymphocytic tumours. The disease commonly presents as a painful abdominal mass and is associated with weight loss and diarrhoea. Barium enema and pancolonoscopy are sufficient for diagnosis, but echotomography and Tc scan are useful for staging. Surgery is the treatment of choice and overall 5-years survival is 30-40%. The role of adjuvant therapy has not yet been clearly defined, although data from some studies indicate increased survival in those patients given adjuvant therapy compared with patients treated with surgical resection alone.
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7788812
|
[Ogilvie syndrome: a case report].
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The Authors report a case of Ogilvie's syndrome observed directly. Remembering this disease is extremely rare, they review the pathogenic hypotheses and clinical picture, describing particularly therapeutic, conservative and surgical management.
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7788811
|
[Current trends in the treatment of perforated colonic diverticulitis].
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The authors, after having examined the cases of complicated diverticulitis operated between 1985 and 1993, confirm that the surgical treatment for this particular pathology is strictly dependent on the local or general seriousness. In the light of this they consider an operation for resection and anastomosis 1st stage justifiable only in selected cases; colostomy and drainage in particularly serious cases, preferring Hartmann's method of operation for the 3rd and 4th stages of Chappuis and Chon. The results obtained agree in a satisfactory manner with the data in the literature.
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7788810
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[Malignant tumors of the small intestine. Clinical and therapeutic considerations on 23 observed cases].
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Small bowel tumours observed by the Authors at the IInd Surgical pathology of Pavia University between 1974 and 1992 were reviewed. There were found 23 cases; istopathologic findings, clinical presentation, diagnostic aspects and therapeutic options are discussed. The primary role of surgery in the treatment of these cancers is emphasized, but this can be curative only if diagnosis has been timely.
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7788809
|
[Continence in low resections].
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Anorectal function is an important problem after low anterior resection procedure. This paper reports the results from 14 patients undergoing to low resection at Surgical Pathology Institute of the University of Verona. In 12 cases the restoration of the bowel continuity has been obtained by colon-anal anastomosis (Parks Operation) and in 3 cases by colo-rectal anastomosis at the anorectal ring level. The patients have been examined in the preoperative and in the postoperative period by clinical and manometric study. The results confirm that low resection does not involve faecal continence.
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7788808
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[Local excision of rectal tumors: Y. Mason's operation].
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Twenty-nine patients with cancer of the low rectum were treated by local excision performed by a transsphincteric approach (Mason's operation). Patient's selection requires a careful digital examination, biopsy, CT or MR and intraluminal ultrasound. In our series an accurate and strictly selection provides low recurrence rates with no mortality and low postoperative complications. Mason's operation, when criteria for appropriate patients selection are followed, is a valid alternative to mayor surgical procedures as APR.
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7788807
|
Colorectal cancer emergencies.
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From the variety of operative choices for colorectal cancer emergencies, it is difficult to select the best one for the individual patient, who is often old and frail. Appropriate handling of the emergency situation is essential. Treatment of the patient's malignant disease is secondary. The choice of surgical procedure in colorectal cancer emergencies must be based on sound clinical judgement and should be in keeping with the technical skill and experience of the individual surgeon. Surgical failures have major negative consequences not only for the immediate postoperative course, but also for long-term survival and quality of life of these patients.
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7788806
|
The leaking colorectal anastomosis; why does it happen and how do we prevent it?
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The ingredients predisposing for an anastomotic leak can be attributed both to the patient and to the surgeon. Some causative elements can be remedied; others cannot be changed very much. In cancer surgery the quest for cure involves ligation of the inferior mesenteric artery in the treatment of left-sided tumours. This obviously creates a conflict with the goal of constructing a well circulated anastomosis. In the treatment of rectal cancers total mesorectal excision has in recent years been put forward as the crucial step in avoiding local recurrences. The use of drains is a matter of controversy. The rationale for using them is to avoid pelvic collections with subsequent infection. The anastomosis is the point of least resistance so this is where a pelvic abscess is liable to break through, thus creating a leak. The choice of anastomosis, single layer or double layer, has been extensively dealt with and is beyond the scope of this presentation. My opinion is that you should stick to the type of anastomosis you are familiar with and try to refine your technique as much as possible. Continuous surgical audit and improved surgical training are likely the most important measures to lower the complication rates in colorectal surgery.
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7788805
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[Conservative surgery in rectal cancer].
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The Authors analyse their experience about 387 cases operated for rectal cancer. The choice of the operation was not related to tumour's features, but was related to the location of the neoplasia. The safety margin of rectal resection was reduced up to a minimum of 2 cm. in lower tumours, where it was not possible to meet this condition an abdomino-perineal resection was performed. A lumbo-aortic lymphadenectomy with high ligature of inferior mesenteric artery was always performed; in 17 cases it was extended to the pelvic nodes. In all the patients the whole mesorectum was removed and a wide pelvic dissection was performed to avoid the so called cone effect. Since 1991 all the B2-C patients undergo preoperative radiotherapy. Recurrence rate and survival are similar both after abdomino-perineal and sphincter-saving resections. Among the different risk factors, the Authors have found a statistically significant relation only with the stage. The Authors, therefore, conclude that sphincter-saving resection is the best surgical procedure because of the god quality of life. They stress the importance of a lumboarotic lymphadenectomy with high ligature of inferior mesenteric artery. As far as the complementary therapy is concerned, the discussion is still open, especially about the timing and the choice of the adjuvant treatment.
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7788804
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Sphincter preserving techniques in rectal cancer and their limitations.
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For a patient with a tumour in the anal canal or with the inferior margin 5 cm or less from the anal verge an abdominoperineal rectal excision with permanent sigmoid colostomy is the only available treatment. The Turnbull-Cutait pull-through technique involved full mobilization of the rectum, complete eversion of the anorectal stump and pull-through of the colon, the excess of which was resected in the second stage of the operation, followed by suture of the colonic and rectal mucosa. Abdomino sacral resection has been practised by a few surgeons only. Abdominotransanal resection with sutured colo-anal sleeve anastomosis was the operation developed by Sir Alan Parks. In this operation the anorectal stump is not everted through the anus as in the Turnbull-Cutait operation. There has been a great revival of interest in low anterior resections with the introduction of the EEA instrument for stapling the colorectal anastomosis and there is still today a great enthusiasm for this technique. In an attempt to improve the results trial are going on at present to evaluate the importance of a J-configured neo-rectum constructed from the sigmoid or descending colon and stapled to the top of the anal canal or handsutured to the pectinate line after endoanal mucosectomy. Many patients with rectal carcinoma are old and often suffer from intercurrent diseases such as hypertension, diabetes etc. There is considerable postoperative complication. An abdominoperineal resection with a colostomy is by no means outdated for an ultralow or low sited rectal cancer.
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7788803
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Phase behavior of mixtures of cholesterol and saturated phosphatidylglycerols.
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The interaction of cholesterol with a series of saturated phosphatidylglycerols was investigated using differential scanning calorimetry and X-ray diffraction. We find that the miscibility of cholesterol in phosphatidylglycerol bilayers is lower than in the corresponding phosphatidylcholine bilayers and decreases with increasing acyl chain length of the phospholipid. The influence of the negative charge of the phosphatidylglycerol on cholesterol miscibility is discussed.
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7788802
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Sterol carrier protein-2 stimulates intermembrane sterol transfer by direct membrane interaction.
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It is unclear how the cytosolic sterol carrier protein-2 (SCP-2) binds sterols and enhances sterol transfer between membranes. Therefore, human recombinant SCP-2 was used in conjunction with phase fluorometry, dialysis, and chemical labeling techniques to show if a direct membrane effect accounted for this activity. SCP-2 directly interacted with L-cell fibroblast plasma membrane vesicles as determined by increased fluorescence anisotropy of coumarin-labeled protein (CPM-SCP-2). Furthermore, a new fluorescence lifetime component due to plasma membrane-bound CPM-SCP-2 was observed. Dialysis studies with 3H- cholesterol loaded plasma membranes indicated that SCP-2, added to the donor compartment, stimulated sterol transfer whether or not the dialysis membrane was permeable to SCP-2. Nevertheless, ligand-binding experiments indicated that chemically blocking the SCP-2 sterol binding site inhibited the ability of SCP-2 to enhance sterol transfer between plasma membrane vesicles. SCP-2 did not stimulate plasma membrane fusion. Addition of SCP-2 to plasma membranes increased the anisotropy plasma membrane proteins covalently reacted with CPM, but not that of lipids labeled with the fatty acid analogue octadecyl rhodamine B. In conclusion, the data are consistent with SCP-2 stimulating intermembrane sterol transfer by direct interaction with sterol in the membrane and enhancing its desorption from the membrane.
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7788801
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Kinetics and thermodynamics of emulsion delivery of lipophilic antioxidants to cells in culture.
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Oil-in-water emulsions are being used increasingly for the delivery of lipophilic drugs, but the fundamental physicochemical principles governing such delivery have not been explored. We determined the kinetics and thermodynamics of delivery from emulsions to cells in culture for two lipophilic compounds, U74006 and U74500. Two fundamental properties dominate the delivery, (a) the concentration of the compound in the lipid phase of the emulsion is directly proportional to the concentration of the compound in cells at equilibrium, and (b) the rate of transfer is directly proportional to the concentration of particles in contact with the cells. Thus, the transfer is consistent with direct partitioning from the lipid phase of the emulsion to cells and occurs by the direct collision of emulsion particles with cells. The details of the mechanism of delivery differ between the two compounds. Specifically, delivery of U74006 is first-order with respect to the drug accumulating in the cells. The transfer of U74500 is best described as a sum of two simultaneous pseudo first-order processes consistent with delivery from a single donor compartment to two receiver compartments. Furthermore, two molecules of U74500 appear to be involved in each transfer event. Our results show that relatively simple principles govern the delivery of compounds from oil-in-water emulsions to cells.
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7788800
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Monolayer study of mixtures of diacetylenic phosphatidylcholine and phospholipids containing metal-chelating iminodiacetic acid headgroup.
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The mixing behavior of polymerizable diacetylenic phosphatidylcholine (1) with two diacetylenic phospholipids containing the iminodiacetic acid (IDA) functionality in the headgroup region was studied at the air-water interface. All three phospholipids contained tricosa-10,12-diynoyl acyl chains. In phospholipid 2, the choline group (-CH2-CH2-NMe3) was replaced by an -CH2-CH2-N-(CH2-COOH)2 functionality. In phospholipid 3, the IDA unit was linked to the phosphate headgroup via a sulfonate linker, -CH2-CH2-OS(O2)-O-CH2-CH2-. Monolayers were prepared by mixing polymerizable 1,2 bis(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine (1) with metal-chelating IDA phospholipids (2, 3) on a 10 mM CuCl2 subphase. Studies of monolayer properties of mixtures of 2 and 3 with 1 demonstrated that mixtures of 2 with 1 had better mixing behavior than mixtures of phospholipids 1 and 3.
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7788799
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Microdomain formation in phosphatidylethanolamine bilayers detected by 2H-NMR.
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In deuterium NMR spectra of phosphatidylethanolamine bilayers with an extremely high content of saturated fatty acids, each C1 deuteron of the glycerol backbone gave rise to a doublet [Yoshikawa et al., (1988) Biochim. Biophys. Acta 944, 321-328]. This suggests the presence of two backbone conformations, the exchange between which is slow on an NMR time-scale. The origin of the two conformations has been investigated in this work using saturated 1,2-diacyl-sn-glycero-3-phosphoethanolamine specifically deuterated in the glycerol backbone. The results showed that the two conformations originate from different domains, which have different fatty acid compositions. The differential scanning calorimetry of the bilayers suggested that the size of the domain is not large enough to show an independent phase transition. Thus, the formation of microdomains in the phosphatidylethanolamine bilayers has been concluded. Conformational difference in different domains was shown to be restricted to the C1 position of the glycerol backbone. The microdomains of phosphatidylethanolamine were retained even in the presence of other phospholipids.
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7788797
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Phase behaviour of novel phospholipid analogues.
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The phase behaviour of phospholipid analogues containing a phosphoramide moiety was investigated using polarized light microscopy. A complex liquid-crystalline polymorphism consisting of lamellar, and normal topology hexagonal and intermediate phases was observed and found to depend on the structure of the phosphoramide headgroup and its methylation. In addition, a lower consolute phase boundary in the micellar solution part of the phase diagram was observed in some systems. The aggregation behaviour of these novel amphiphiles was compared with that of quaternary ammonium and oligo-ethylene oxide amphiphiles.
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7788798
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Photo-effect in phospholipid liposome containing riboflavin.
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The photovoltage developed in a photoelectrochemical cell consisting of riboflavin (RFN), a neutral dye and phospholipid (PL) liposome in aqueous solution has been found to correlate with the Stern-Volmer constant (KSV) or quenching-rate constant (Kq) of RFN-PL studied by fluorescence spectra. A possible mechanisms of photovoltage generation suggests the photo-induced electron transfer from PL to RFN in liposome through the excited state molecular interaction.
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7788796
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Exclusion of SP-C, but not SP-B, by gel phase palmitoyl lipids.
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The interactions of the hydrophobic pulmonary surfactant proteins, SP-C and SP-B, with lipid bilayers were assessed by fluorescence energy transfer. SP-C and SP-B were labeled with the fluorescent probe, succinimidyl nitrobenzoxadiazolyl amino hexanoate (NBD). Fluorescence energy transfer from NBD-SP-C and NBD-SP-B to four distinct indocarbocyanine probes (CnDiI) was utilized to determine the association of the surfactant proteins with various lipid acyl chains. In lipid mixtures including DPPC and DPPG, SP-C was associated with shorter chain and unsaturated lipids below the bulk lipid phase transition. Longer chain saturated CnDiI were excluded from SP-C aggregates. In contrast, SP-B demonstrated little acyl chain preference. The association of SP-C with shorter chain and unsaturated lipids below the bulk phase transition is interpreted to arise from a mismatch in the length of the hydrophobic region of the SP-C alpha-helix relative to the length of the hydrophobic region of dipalmitoyl lipids in the gel phase.
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7788795
|
Cholesteryl hemisuccinate-cholesterol interaction: miscibility properties of the sterols.
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Miscibility of cholesterol and cholesteryl hemisuccinate was investigated by differential scanning calorimetry and small-angle X-ray scattering. In an excess of cholesterol, above 2:1 mol ratio, phase separation takes place into a mixed phase and an almost pure cholesterol phase.
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7788794
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Ganglioside GD1a generates domains of high curvature in phosphatidylethanolamine liposomes as determined by solid state 31P-NMR spectroscopy.
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We have studied the effects of gangliosides on the polymorphic behaviour of phosphatidylethanolamines. The ganglioside GD1a promotes the formation of phases which give rise to isotropic 31P-NMR resonance lines, particularly in the temperature range of the L alpha to HII transition. In addition, higher mol fractions of ganglioside raise the L alpha to HII phase transition temperature. Our results demonstrate that small mol fractions of gangliosides can have profound effects on the molecular organization of phosphatidylethanolamines.
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