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7787210
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Orthotopic liver transplantation in a patient with severe hemophilia A: a life-saving treatment for the first Italian case.
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Clinical cure of hemophilia A by orthotopic liver transplantation has been reported in 11 cases. We describe the first successful Italian case. A 27-year-old man had cirrhosis caused by previous infections with the hepatitis B, C and D viruses following life-long treatment with factor VIII concentrates made from large plasma pools. He was, however, seronegative for the human immunodeficiency virus. In the year before transplantation, life-threatening gastrointestinal bleeding due to severe esophageal varices required a large transfusion regimen (on average, 13 bags of red cell concentrates and 35,000 U of factor VIII/week). To perform orthotopic liver transplantation 8,000 U of factor VIII were given during surgery together with 10 bags of red cells and 11 of fresh-frozen plasma. Intraoperative bleeding was not different from that of non-hemophilic patients undergoing orthotopic liver transplantation. No additional factor VIII was used after transplantation and factor VIII levels in plasma were always above 50 U/dl, reaching the highest value of 184 U/dl on day 4 post transplantation. He was discharged from hospital 10 weeks after transplantation with factor VIII levels of 68 U/dl. All virological markers are currently negative, except anti-hepatitis C virus antibodies. In this patient orthotopic liver transplantation was a life-saving treatment for end-stage cirrhosis and a cure for hemophilia A.
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7787209
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Insulin-like growth factor-I, interleukin-1 alpha and beta in pancreatic cancer: role in tumor invasiveness and associated diabetes.
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We evaluated levels of insulin-like growth factor-I and interleukin-1 alpha and beta in patients with pancreatic cancer; the role of these substances in tumor spread and in hyperglycemia was also investigated. Thirty pancreatic cancer patients (21 with hyperglycemia) were compared with others with diseases causing hyperglycemia [liver cirrhosis (14 cases, 12 with hyperglycemia), chronic pancreatitis (20 cases, 12 with hyperglycemia), type I diabetes mellitus (13 cases, all hyperglycemic)]. Insulin-like growth factor-I was significantly reduced in patients with liver cirrhosis, probably due to a reduced hepatic capacity for synthesis. It was increased in 6 of 30 pancreatic cancer patients; in these subjects it was correlated with alanine aminotransferase and C-peptide, but not with tumor diameter or the presence of metastases. Interleukin-1 alpha and beta were both elevated in pancreatic cancer patients. The former was high, while the latter was low when liver metastases were present. Neither was related to glucose or C-peptide levels. In summary, insulin-like growth factor-I levels are increased in some pancreatic cancer patients but this does not seem to favor tumor spread; however IGF-I could be involved influencing glucose homeostasis. Interleukin-1 alpha increased, while interleukin-1 beta decreased in pancreatic cancer patients with metastases, suggesting a different involvement of these two substances in pancreatic cancer spread.
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7787208
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Detection of hepatitis D virus RNA in serum by a reverse transcription, polymerase chain reaction-based assay.
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We designed a reverse transcription, polymerase chain reaction-based assay for serum hepatitis D virus RNA. Amplified hepatitis D virus cDNA was revealed by ethidium bromide staining, followed by blotting onto a nylon membrane and hybridization with a 32phosphorus-labelled oligonucleotide, or by a DNA enzyme immunoassay (DEIA) using a double stranded DNA-specific monoclonal antibody. The absolute sensitivity was expressed as number of hepatitis D virus RNA molecules, using a serum of known viral RNA concentration. Three sets of primers were used, encompassing the base positions 66-686 (variable rod-stabilizing region), 701-962 (conserved, viroid-like domain) and 886-1,333 (portion of the open reading frame 5 encoding for the carboxyterminus of the hepatitis D antigen) of the viral genome. The lower detection limits, after amplification of the three RNA portions, as assessed by ethidium bromide staining, were 7.5 x 10(6), 7.5 x 10(4) and 7.5 x 10(2) molecules of hepatitis D virus RNA per assay, respectively. The region encompassing bases 886-1,333 was chosen for blotting and hybridization to a radiolabelled oligonucleotide probe or for a capture-based DNA enzyme immunoassay, where the microplate was coated with this same probe. The two procedures showed comparable sensitivity, i.e., about 10 molecules of viral RNA per assay. The specificity of the assay was further on a panel of both anti-hepatitis D-positive and -negative sera. Amplification of serum hepatitis D virus RNA by reverse transcription/polymerase chain reaction followed by detection of the amplified cDNA by DNA enzyme immunoassay is a promising and feasible routine assay for detecting low amounts of circulating virions.
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7787207
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The role of cytokines in the acquired immunodeficiency syndrome.
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HIV replication in vitro is regulated by many factors, including various exogeneous stimuli and proteins encoded by either virus or cellular genomes. During the asymptomatic period, cells latently or chronically infected with HIV gradually express virus, leading to immunosuppression and opportunistic infection. These conditions would result in the increased secretion of cytokines, especially TNF, from infected and uninfected cells, which can induce HIV and killing of infected cells. A vicious circle is then set in motion in which heterologous microbial infections directly or indirectly activate HIV and the production of cytokines, thereby accelerating lymphocyte depletion and immunodeficiency. AIDS is a disorder of the immune network caused by a unique retrovirus HIV. However, if the whole story described above is true, this disease can also be termed a "cytokine disease". Immunity resembles a "double-edged sword", with aspects not only protective, but also deleterious to the host. Therefore, it is essential to more extensively investigate the mechanism of cytokine regulation of HIV expression in vivo, not only to understand the complex pathophysiology of AIDS, but also to design a therapeutic strategy to halt this deadly disease.
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7787206
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Cytokines as adjuvants in immunocompromised hosts.
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Vaccination of immunocompromised subjects may be ineffective due to the poor immune responses induced. In addition, vaccination with live attenuated organisms may also be dangerous due to the possible lack of control of the infection. This review describes the protection of cytokines in the vaccination of immunocompromised individuals. Cytokines have possible roles as immunological adjuvants, enhancing immune responses to vaccination, and can also have effects on the growth of live vaccines or vaccine vectors.
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7787205
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Lymphotropism of hepatitis B and C viruses: an update and a newcomer.
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The mechanisms of viral persistence are complex and include infection of the lymphoid cells. In the case of hepatitis B virus, early observations have suggested that HBV may infect peripheral blood mononuclear cells (PBMC). In animal models studies in chronic hepatitis B patients have further confirmed that viral DNA replicative intermediates, as well as viral transcripts and proteins, can be detected in PBMC under certain conditions. The consequences of this lymphotropism are not fully understood, but it seems likely that PBMC represent an extrahepatic reservoir of virus. The ability of hepatitis C virus to infect PBMC has been demonstrated in vivo and in vitro. The link between HCV lymphotropism and both the natural history of the viral infection and the immunological disorders frequently observed in HCV infections still needs to be established. In both cases, the infection of PBMC by HBV or HCV may represent the source of infection of the liver graft in patients transplanted for end-stage liver disease associated with HBV or HCV.
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7787204
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Revision of the ICIDH: mental health aspects. WHO/MNH Disability Working Group.
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This article reviews the key issues arising in the revision of the International Classification of Impairments, Disabilities, and Handicaps (ICIDH) from a mental health perspective, and describes the work of the Disability Working Group of the WHO's Division of Mental Health. The ICIDH, which describes the consequences of disorders at three levels as impairments, disabilities, and handicaps, is generally applicable and useful for mental health purposes. While some impairments are mainly a consequence of 'mental' disorders (e.g. cognitive impairment), there should be no differences between mental and physical disorders in the classification scheme, to avoid a dichotomy between mind and body. There is also a need to improve the ways in which interference with the performance of social roles is described, since this is often the most obvious consequence of mental disorders. This article presents the potentials of the ICIDH in the field of mental health, and gives recommendations for the development of the revision process of the ICIDH. To stimulate the process of producing a 'common language' in the ICIDH related to mental health issues, former and potential users of the ICIDH are invited to give comments and suggestions.
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7787203
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Application of the ICIDH in survey research on rehabilitation: the emergence of the functional diagnosis.
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The International Classification of Impairments, Disabilities, and Handicaps (ICIDH) has been hypothesized to be an excellent conceptual framework for the functional diagnosis, i.e. the evaluation of health problems by rehabilitation specialists. The ICIDH-based functional diagnosis was assessed in a series of survey studies on physical therapy, exercise therapy, occupational therapy, podiatry, and speech therapy. The results show that the functional diagnosis tends to be reliable; that treatment goals, derived from the diagnosis, offer a sensible characterization of care given by rehabilitation specialists; and that treatment goals validity predict the selection of interventions by rehabilitation specialists. It is concluded that the application of the ICIDH in research on the functional diagnosis is highly appropriate. In future studies the concept of the functional diagnosis and the diagnostic procedures should be further developed.
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7787202
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The need for revision of the ICIDH: an example--problems in gait.
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In this article a proposal is formulated for adjustment of the Classification of Impairments and the Classification of Disabilities of the International Classification of Impairments, Disabilities, and Handicaps (ICIDH). This proposal is a product of a project from the Dutch National Institute of Research and Postgraduate Education in Physical Therapy. This project is conducted in close cooperation with the professional national organizations of five health professions. To give an indication of the kind of changes proposed, the disabilities and impairments necessary for classifying the complaints, the examination findings, the treatment goals and the treatment results in patients with gait problems are discussed.
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7787201
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Using the handicap code of the ICIDH for classifying patients by intensity of nursing care requirements.
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An 11-class patient classification system (PCS) has been built on a recode of two dimensions of the handicap code of the ICIDH: physical independence and mobility handicaps. The proposed system, called MAC XI, explains 78% of the variance of nursing care hours required by nursing-home residents and extended-care hospital patients. This percentage of variation is higher than the one explained by traditional dependency grids such as the Exton-Smith, Murphy, Kuntzmann and SMAF. MAC XI, based on two dimensions of the handicap code, is thus a powerful tool for predicting intensity of nursing care for staffing and budgeting purposes in long-term care institutions.
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7787200
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Rehabilitation Activities Profile: the ICIDH as a framework for a problem-oriented assessment method in rehabilitation medicine.
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The Rehabilitation Activities Profile (RAP) is an ICIDH-based assessment method that covers the domains of communication, mobility, personal care, occupation, and relationships. Disabilities and handicaps in these domains are assessed on four-point Likert scales for severity. Problems perceived by the patient associated with these disabilities or handicaps are also assessed on four-point Likert scales for severity. High scores on perceived problems represent a patient's priorities. Information is gathered through a semi-structured interview with the patient; proxies and observations can be used as additional sources of information. Assessment can be performed at two levels. The first level is a global one, serving as a screening device. If disabilities or handicaps are identified, the second level provides for an in-depth assessment of those specific disabilities and handicaps as well as the related perceived problems. The method is designed to assist screening, goal-setting, and outcome evaluation of individual patients.
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7787199
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Disability, resources, role demands and mobility handicap.
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Research on disablement highlights a wide variability in the impact of disabling conditions on individuals' lives. However, in most of this work, the relationships between impairment/disability and features of individuals' social and physical environments are not specified conceptually. Recent conceptual work in the context of the International Classification of Impairments, Disabilities, and Handicaps (ICIDH) suggests that the impact of impairment/disability on individuals' lives is contingent on levels of resources and other aspects of social context. The research question addressed in this paper is whether selected social factors affect the impact of impairment/disability on mobility handicap, defined as 'the individual's ability to move about effectively in his/her surroundings'. Two types of social factors are considered: resources such as help from others or having a car available; and social role obligations such as having a job or visiting relatives. Data are derived from a 1986 probability sample of 570 individuals with disabilities living in communities in Calderdale, Yorkshire, England. Multiple-regression models indicate that the impact of walking disability on mobility handicap was reduced by availability of a car in the household and school or job obligations. Other impairments/disabilities, resources and social role demands examined did not act on mobility outcomes in this manner. Implications for conceptualizing and testing relationships between impairment, disability, handicap and social and physical environments are discussed. A critical task for future research is the investigation of personal and social resources and barriers that may moderate the impact of disability on individuals' lives.
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7787198
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The ICIDH in the USA: applications and relevance to ADA goals.
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The measurements of disability and environmental barriers have received growing nationwide attention and dissemination in the United States, particularly since the 1990 passage of the Americans with Disabilities Act (ADA). This article presents a brief overview of selected ICIDH uses and applications in the USA. Potential contributions of the ICIDH for ADA implementation are discussed, and parallel definitions of disability in the ICIDH and the ADA are cited.
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7787197
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Documenting environmental factors for preventing the handicap creation process: Quebec contributions relating to ICIDH and social participation of people with functional differences.
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This article presents the conceptual origins and the usefulness of an holistic model, stressing the role of environmental determinants in the performance of day-to-day activities and the fulfilment of social roles by persons with disabilities. The Quebec Committee on ICIDH contribution to the development of knowledge on the clarification of the relationship of disabled individual-environment and the understanding of the handicap creation process in relation with ICIDH experimentations is presented. Finally, on the basis of anthropological social research conducted in Quebec, the article illustrates a methodology for environmental analysis of the handicap situations of disabled clients in rehabilitation and social integration support programmes.
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7787196
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Human experience in disablement: the imperative of the ICIDH.
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The ICIDH recognizes that the perspectives of people whose lives are personally shaped by disablement are critical to improving our understanding of disease consequences. However, this 'insider's perspective' has received comparatively little attention in medical research. This article suggests that the weight of medical history mitigates against human experience as a focus of investigation, traces the history of the International Classification of Diseases (ICD) from this perspective, and suggests that these historical influences are still operative in disablement studies. The ICIDH's inclusion of the insider's perspective represents a significant departure from traditional ways of thinking in medical science, and allows for important changes in the discourse about disablement. This article uses phenomenological theory to show how the insider's perspective might be more fully integrated into clinical research and theory development. Finally, this article suggests modifications to the ICIDH that would help to include more explicitly the insider's perspective in its structure.
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7787191
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Molecular cloning of an ozone-induced 1-aminocyclopropane-1-carboxylate synthase cDNA and its relationship with a loss of rbcS in potato (Solanum tuberosum L.) plants.
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Acute or chronic exposure of potato plants to ozone (O3) induces ethylene production. We isolated a 1586 bp cDNA (pOIP-1) encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase from a cDNA library constructed with mRNA extracted from O3-treated leaves. The clone has a 1365 bp open reading frame and a 221 bp trailing sequence. The active site found in all ACC synthases and 11 of the 12 amino acid residues conserved in aminotransferases are found in pOIP-1. Northern analysis showed that the mRNA encoding ACC synthase was detectable 1 h after the onset of O3 exposure, and the message increased over time as did ethylene production. Concurrent with the increased ACC synthase mRNA was a decrease in the message for the Rubisco small subunit (rbcS) with no change in the large subunit (rbcL). When the plants were treated with aminooxyacetic acid (AOA), both ethylene production and level of ACC synthase transcript were inhibited. The decline in rbcS was also inhibited by AOA suggesting a correlation between ethylene production and loss of rbcS. Based on nuclear run-on studies it appears that the increase in ACC synthase mRNA may result from O3-induced transcriptional activity.
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7787195
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The ICIDH: health problems in a medical and social perspective.
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The ICIDH is a classification of health problems. Impairments, disabilities, and handicaps are to be considered as three different interpretations of a person's health state. Use of the ICIDH is indicated when a person's health problems need to be described at a particular point in time, both in medical and in social terms, i.e. including their significance for the daily life functioning of the patient. It is argued that the ICIDH is relevant to health problems other than just those for disabled and for chronically ill. The ICIDH subtitle and the model explaining the relations between disease and impairment-disability-handicap do not reflect a clear notion of the perspective of the ICIDH on health problems. An overview of ICIDH applications shows that the ICIDH is being used not so much for the evaluation of the health-care system but for many other purposes, among these the understanding of therapeutic activities.
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7787194
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Reflection on the definition of impairment and disability as defined by the World Health Organization.
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The International Classification of Impairments, Disabilities, and Handicaps (ICIDH) is gaining wide acceptance inside and outside the field of rehabilitation medicine. Impairment and disability are concepts which are often used interchangeably or defined differently. The World Health Organization (WHO) has defined impairment and disability in the ICIDH. In this article an analysis is made of these definitions and the characteristics that are given for the definitions. Based on this analysis, modifications for the definitions and characteristics of impairment and disability are suggested.
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7787193
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The ICIDH: evolution, status, and prospects.
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The assessment of disability prevalence in populations is a long-standing concern. In the mid-1970s the World Health Organization (WHO) introduced a scheme for the measurement of the consequences of disease. The classification of the long-term non-fatal consequences of disease is structured on three axes, corresponding roughly to experiences at the level of organ or function (impairment--1009 items), individual action (disability--338 items) and societal interaction (handicap/disadvantage--72 items). The International Classification of Impairments, Disabilities, and Handicaps (ICIDH) is now well established. This paper describes developments in the use of the ICIDH since 1980, in assessing the prevalence of disability in populations, in formulating policy decisions, in management at institution level, and in the care of individuals. It lists problems identified in the use of the ICIDH, such as the need to clarify the role and interrelationship of environmental factors in the definition and development of the different planes addressed by the ICIDH, problems of overlap between disabilities and handicaps, and between impairments and disabilities. Suggestions for improvement include a greater emphasis on presenting handicap as a description of the interrelation between impairments or disabilities and their physical and social environment. It is anticipated that a revised proposal will be finalized for 1998 and formally issued in 1999.
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7787190
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Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco.
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The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.
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7787187
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Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae).
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Recent studies on chalcone synthase (CHS) and the related stilbene synthase (STS) suggest that the structure of chs-like genes in plants has evolved into different forms, whose members have both different regulation and capacity to code for different but related enzymatic activities. We have studied the diversity of chs-like genes by analysing the structure, expression patterns and catalytic properties of the corresponding enzymes of three genes that are active during corolla development in Gerbera hybrida. The expression patterns demonstrate that chs-like genes are representatives of three distinct genetic programmes that are active during organ differentiation in gerbera. Gchs1 and gchs3 code for typical CHS enzymes, and their gene expression pattern temporally correlates with flavonol (gchs1, gchs3) and anthocyanin (gchs1) synthesis during corolla development. Gchs2 is different. The expression pattern does not correlate with the pigmentation pattern, the amino acid sequence deviates considerably from the consensus of typical CHSs, and the catalytic properties are different. The data indicate that it represents a new member in the large superfamily of chs and chs-related genes.
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7787189
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Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plants.
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The heat shock (hs) response during plant growth and development was analyzed in tobacco and Arabidopsis using chimaeric beta-glucuronidase reporter genes (hs-Gus) driven by a soybean hs promoter. Fluorimetric measurements and histochemical staining revealed high Gus activities in leaves, roots, and flowers exclusively after heat stress. The highest levels of heat-inducible expression were found in the vascular tissues. Without heat stress, a developmental induction of hs-Gus was indicated by the accumulation of high levels of Gus in transgenic tobacco seeds. There was no developmental induction of hs-Gus in Arabidopsis seeds. In situ hybridization to the RNA of the small heat shock protein gene Athsp17.6 in tissue sections revealed an expression in heat-shocked leaves but no expression in control leaves of Arabidopsis. However, a high level of constitutive expression of hs genes was detected in meristematic and provascular tissues of the Arabidopsis embryo. The developmental and tissue-specific regulation of the hs response is discussed.
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7787188
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Narbonin, a novel 2S protein from Vicia narbonensis L. seeds: cDNA, gene structure and developmentally regulated formation.
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cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M(r) ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.
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7787186
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Isolation of a carrot gene expressed specifically during early-stage somatic embryogenesis.
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We report the first successful isolation by subtractive hybridization of a gene expressed specifically during somatic embryogenesis. Embryogenic cell clusters, 32-50 microns in diameter, were isolated by sieving and density-gradient centrifugation. The cDNA library was constructed from proglobulars which were formed from embryogenic cell clusters 3 days after transfer to auxin-free modified Lin and Staba's medium. For use as probe in screening, the same cDNA used for library construction was enriched for specific sequences using subtractive hybridization. The cDNA used for subtraction was prepared from suspension cultures 5 days after subculturing in auxin-containing medium. Nine independent differentially expressed cDNA clones were obtained from a screen of 150,000 recombinant phages. Northern analysis indicated one of these, CEM6, to be expressed specifically during somatic embryogenesis. In addition, one hybridizing transcript was detected in plantlet cotyledons, and two transcripts were detected in hypocotyls. Two separate and distinct hybridizing transcripts are expressed specifically in hypocotyl tissue. The amino acid sequence deduced from the nucleotide sequence of the CEM6 cDNA indicates that it encodes a glycine-rich protein containing a hydrophobic signal-sequence like domain. Its early embryo-specific expression and sequence characteristics suggest an important role as a cell wall protein in embryogenesis.
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7787185
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Identification and characterization of genes with unstable transcripts (GUTs) in tobacco.
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Plants and other higher eukaryotes have the ability to recognize and target specific transcripts for rapid decay from among the majority of relatively stable mRNAs present within cells. However, little is known about the nature of unstable transcripts in plants, or the mechanisms that facilitate their rapid degradation. As a first step toward understanding how plants distinguish between unstable and stable transcripts, a novel differential screen was used to identify cDNAs for genes with unstable transcripts (GUTs), solely on the basis of the instability of their mRNAs. cDNA probes were prepared from tobacco cells that had been depleted of highly unstable mRNAs by treatment for 90 min with a transcriptional inhibitor, and from control, untreated cells. GUT clones were selected on the basis of weak hybridization to the former probe relative to the latter probe. Half-life measurements performed on the mRNAs hybridizing to eight GUT clones indicated that each was unstable, with a half-life on the order of about an hour or less. All eight of the cDNAs corresponded to new tobacco genes, and four showed sequence similarity with genes from other species, including the eukaryotic family of DNAJ homologs, a tomato wound-inducible protein, and histone H3. In addition to providing information about the types of transcripts that are inherently unstable in plants, the GUT clones should provide excellent tools for the identification of cis- and trans-acting determinants of mRNA instability.
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7787182
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The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene.
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The gdhA gene of Synechocystis PCC 6803, which encodes an NADP-dependent glutamate dehydrogenase (NADP-GDH), has been cloned by complementation of an Escherichia coli glutamate auxotroph. This gene was found to code for a polypeptide of 428 amino acid residues, whose sequence shows high identity with those of archaebacteria (42-47%), some Gram-positive bacteria (40-44%) and mammals (37%). The minimal fragment of Synechocystis DNA required for complementation (2kb) carries the gdhA gene preceded by an open reading frame (ORF2) encoding a polypeptide of 130 amino acids. ORF2 and gdhA are co-transcribed as a 1.9 kb mRNA, but shorter transcripts including only gdhA were also detected. Two promoter regions were identified upon transcriptional fusion to the cat reporter gene of a promoter probe plasmid. Transcription from the promoter upstream of ORF2 was found to be regulated depending on the growth phase of Synechocystis, in parallel to NADP-GDH activity. This promoter is expressed in Escherichia coli too, in contrast to the second promoter, located between ORF2 and gdhA, which was silent in E. coli and did not respond to the stage of growth in Synechocystis. Disruption of the cyanobacterial gdhA gene with a chloramphenicol resistance cassette yielded a mutant strain totally lacking NADP-GDH activity, demonstrating that this gene is not essential to Synechocystis 6803 under our laboratory conditions.
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7787183
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Structural organization and differential expression of carrot beta-fructofuranosidase genes: identification of a gene coding for a flower bud-specific isozyme.
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Three genomic clones (Inv*Dc1, Inv*Dc2 and Inv*Dc3) were isolated by using the cDNA for carrot cell wall beta-fructofuranosidase as a probe. The expression patterns of the three genes differed markedly. High levels of Inv*Dc1 transcripts were found in leaves and roots of young carrot, whereas in plants with developing tap roots no transcripts were detected. A high level of mRNA of Inv*Dc1 was also present in suspension-cultured cells. In developing reproductive organs, only low levels of transcripts of Inv*Dc1 were found in flower buds and flowers and none at later stages of development. In contrast, Inv*Dc2 and Inv*Dc3 were not expressed in vegetative plant organs. Invb1*Dc1 was exclusively and strongly expressed in flower buds, and Inv*Dc3 at a very low level in suspension-cultured cells.
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7787184
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The location of untranscribed DNA sequences within ras genes essential for eliciting plant growth suppression.
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Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition. All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect. The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect. This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein. Small deletions at either the 5' or 3' end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect. A 36 bp region at the 5' end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences. Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not. These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region.
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7787180
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Structural and functional characterization of two wheat histone H2B promoters.
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Two wheat histone H2B genes (TH123 and TH153) were isolated. Nucleotide sequence analysis revealed that some characteristic sequence motifs were conserved in both the 5'- and 3'-flanking regions. A canonical TATA box and several CCAAT sequences were present in the presumed promoter regions. Motifs similar or identical to the hexamer (ACGTCA) and octamer (CGCGGATC) motifs that are positive cis-acting elements of the wheat H3 (TH012) promoter were also observed in both the H2B promoters. A gel mobility shift assay indicated that the hexamer and hexamer-like motifs bound the wheat bZIP proteins HBP-1a and/or HBP-1b in vitro. A novel sequence motif, (A/T)(G/A)AAAT(A/G), was found downstream of a translational stop codon as observed in several plant histone H2B cDNAs. Promoter activity was analyzed with H2B promoter-GUS fusion genes in the transient system using tobacco protoplasts. Studies of the promoter function in transgenic tobacco plants showed that the H2B promoters were preferentially active in meristematic tissues. Taken together, our data indicate that the H2B genes are regulated, in part, by the same mechanism as found in H3 and H4 gene transcription.
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7787181
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Expression of three osmotin-like protein genes in response to osmotic stress and fungal infection in potato.
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We have characterized three cDNAs encoding osmotin-like proteins from potato (Solanum commersonii) cell cultures. These cDNAs (pA13, pA35, and pA81) have extensive nucleotide identity in the coding regions but low homology in the 3' non-coding sequences, and may encode three isoforms of potato pathogenesis-related (PR) type 5 proteins. Using gene-specific probes, RNA gel blot analyses showed constitutive accumulation of osmotin-like protein mRNAs in cell cultures, leaves, stems, roots and flowers, with high abundance in the roots and mature flowers. Treatments with abscisic acid (ABA), low temperature, and NaCl increased the accumulation of all three mRNAs in S. commersonii cell cultures and plants grown in vitro. Salicylic acid (SA), and wounding resulted in a moderate increase in the levels of pA13 and pA81 but not pA35 mRNAs. Infection with the fungus Phytophthora infestans activated strong and non-systemic expression of all three osmotin-like protein genes. The accumulation of osmotin-like proteins, however, was detected only in P. infestans-infected tissues but not in plants treated with ABA, SA, NaCl, low temperature, or wounding.
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7787179
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A GCC element and a G-box motif participate in ethylene-induced expression of the PRB-1b gene.
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The PRB-1b gene codes for a basic-type pathogenesis-related protein and is activated at the transcriptional level by the plant hormone ethylene. To identify cis-acting DNA elements essential for ethylene induction, deleted and mutant forms of the PRB-1b promoter, fused to the beta-glucuronidase (GUS) coding region, were introduced in transgenic tobacco plants. A 73 bp fragment (X1 region) of the PRB-1b promoter, located between positions -213 and -141, was sufficient to confer ethylene responsiveness to the reporter gene. The X1 region contains a TAAGAGCCGCC motif (GCC-box) well conserved in several ethylene-inducible genes. A substitution mutation in this sequence, in the context of a 213 bp PRB-1b promoter, completely abolished ethylene induction in transgenic tobacco, defining this conserved motif as part of a cis-acting element responsive to ethylene. Three other mutations in the X1 region caused a pronounced decrease in the PRB-1b promoter activity in transgenic plants, but did not affect ethylene inducibility. One of them, localized in a G-box like motif (CACGTG), disrupted the binding site for a nuclear factor, as observed in gel-shift analysis. Interestingly, the mobility of the complex formed on the G-box element was dependent on its phosphorylation state. These results suggest that a cis-acting element involved in the perception of the ethylene signal resides in a GCC motif and acts in concert with additional elements in the regulation of ethylene-induced PRB-1b expression.
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7787178
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A corm-specific gene encodes tarin, a major globulin of taro (Colocasia esculenta L. Schott).
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A gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (G1, ca. 28 kDa) was isolated from a lambda Charon 35 library, using a cDNA derived from a highly abundant corm-specific mRNA, as probe. The gene, named tar1, and the corresponding cDNA were characterized and compared. No introns were found. The major transcription start site was determined by primer extension analysis. The gene has an open reading frame (ORF) of 765 bp, and the deduced amino acid sequence indicated a precursor polypeptide of 255 residues that is post-translationally processed into two subunits of about 12.5 kDa each. The deduced protein is 45% homologous to curculin, a sweet-tasting protein found in the fruit pulp of Curculigo latifolia and 40% homologous to a mannose-binding lectin from Galanthus nivalis. Significant similarity was also found at the nucleic acid sequence level with genes encoding lectins from plant species of the Amaryllidaceae and Lilliaceae families.
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7787177
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Expression of Agrobacterium rhizogenes auxin biosynthesis genes in transgenic tobacco plants.
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Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells. The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements. This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes. Neither wound nor hormone induction could be detected on transgenic leaf discs. However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts. The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli. Histological analysis showed that the expression was located in cells reactivated by in vitro culture. Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the beta-glucuronidase activity of the chimaeric genes. These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.
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7787176
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Characterization of cDNA clones for differentially expressed genes in embryos of dormant and nondormant Avena fatua L. caryopses.
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The molecular regulation of seed dormancy was investigated using differential display to visualize and isolate cDNAs representing differentially expressed genes during early imbibition of dormant and nondormant Avena fatua L. embryos. Of about 3000 cDNA bands examined, 5 cDNAs hybridized with mRNAs exhibiting dormancy-associated expression patterns during the first 48 h of inhibition, while many more nondormancy-associated cDNAs were observed. Dormancy-associated clone AFD1 hybridized with a 1.5 kb mRNA barely detectable in dry dormant and nondormant embryos that became more abundant in dormant embryos after 24 h of imbibition. Clone AFD2 hybridized with two mRNAs, a 1.3 kb message constitutively expressed in dormant and nondormant embryos and a 0.9 kb message present at higher levels in dormant embryos after 3 h of imbibition. Nondormancy-associated clones AFN1, AFN2 and AFN3 hybridized with 1.5 kb, 1.7 kb and 1.1 kb mRNAs, respectively, that were more abundant in nondormant embryos during imbibition. Expression patterns of some mRNAs in dormant embryos induced to germinate by GA3 treatment were different than water controls, but were not identical to those observed in nondormant embryos. DNA sequence analysis revealed 76% sequence identity between clone AFN3 and a Citrus sinensis glutathione peroxidase-like cDNA, while significant sequence similarities with known genes were not found for other clones. Southern hybridization analyses showed that all clones represent low (1 to 4) copy number genes.
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7787174
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Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato.
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3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the beta-glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R2 pollen of R1 progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.
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7787175
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Molecular cloning and characterization of a pea chitinase gene expressed in response to wounding, fungal infection and the elicitor chitosan.
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The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chi1. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.
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7787173
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Successful in vitro fertilization and pregnancy in a patient with autoimmune chronic active hepatitis and cirrhosis.
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Fertility is reduced in women with chronic active autoimmune hepatitis (AIH) and pregnancy is hazardous. This report describes a 33 year old woman with AIH and cirrhosis in whom a successful pregnancy was achieved following in vitro fertilization/embryo transfer. Disease exacerbation during pregnancy was controlled by azathioprine and an increased dose of prednisone, and a healthy child was delivered by Caesarean section at 36 weeks gestation. Since the perinatal care of preterm infants and the obstetric care available to women with complicated medical problems has improved markedly in recent years and since active disease can be controlled by adequate immunosuppressive therapy, we propose that it is justified to allow these patients access to in vitro fertilization programmes.
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7787172
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The role of gastrin and cholecystokinin in normal and neoplastic gastrointestinal growth.
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Gastrin and cholecystokinin (CCK) act as growth factors for the gastric mucosa and the pancreas, respectively. CCK is also responsible, via the CCK-A receptor, for the pancreatic hyperplasia observed following the feeding of protease inhibitors or pancreaticobiliary diversion. Hypergastrinaemia does not increase the incidence of spontaneous gastrointestinal carcinoma, but does stimulate the proliferation of gastric enterochromaffin-like cells via the gastrin/CCK-B receptor, with a consequent increase in the incidence of gastric carcinoids. Whether gastrin influences mutagen-induced gastrointestinal carcinogenesis is still controversial, but CCK clearly enhances the induction by carcinogens of acinar tumours in the pancreas. While gastrin increases xenograft growth of 50% of gastrointestinal tumours tested, effects on the proliferation of gastrointestinal tumour cell lines in vitro have been more difficult to demonstrate, perhaps because many cell lines are already maximally stimulated by autocrine gastrin. Gastrin mRNA and progastrin, but not mature amidated gastrin, have been detected in all gastrointestinal cell lines tested. Although cell proliferation is inhibited by gastrin/CCK receptor antagonists, the spectrum of antagonist affinities is not consistent with binding to either CCK-A or gastrin/CCK-B receptors. Definition of the molecular structure of the receptor involved in the autocrine loop may lead to novel therapies for gastrointestinal cancer.
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7787167
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Effects of nifedipine on hepatic venous pressure gradient and portal vein blood flow in patients with cirrhosis.
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We investigated the effects of nifedipine on splanchnic haemodynamics in 13 patients with cirrhosis and portal hypertension, and in 10 control subjects using hepatic venous catheterization and pulsed Doppler ultrasound. There were no significant changes in systemic or splanchnic haemodynamics in control patients. In contrast, systemic vasodilatation, evidenced by significant decreases in mean arterial pressure and systemic vascular resistance, was observed in patients 20 min after sublingual application of 10 mg nifedipine. Moreover, hepatic venous pressure gradient and portal vein blood flow significantly increased after nifedipine administration. There was a significant correlation between the percentage increases in portal vein blood flow and in hepatic venous pressure gradient. However, no correlation was found between the percentage change in cardiac output and that in portal vein blood flow. Thus the increase in portal vein blood flow appears to be related to splanchnic arterial vasodilatation by nifedipine. Consequently, nifedipine has deleterious effects on portal haemodynamics in patients with cirrhosis. As nifedipine may potentially increase the risk of variceal haemorrhage in patients with less advanced varices, this drug should be used with caution in patients with chronic liver disease.
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7787165
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Influence of ascites on the chemotaxis of granulocytes in patients with cirrhosis.
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Spontaneous bacterial peritonitis is a specific infectious complication in liver cirrhosis. The reasons for the preferred location of infection on the peritoneum are not clear. The aims of the present study were to ascertain whether hepatogenic ascites fluid is chemotactically effective, what part is played by complement factor C3 and whether there are inhibitors of chemotaxis in ascites. Chemotaxis of granulocytes in serum and ascites fluid was measured in 18 patients with cirrhosis and ascites and in 18 healthy individuals using the Boyden chamber method. In the patients, the chemotactic effect of serum was reduced significantly. Ascites fluid had lower chemotactic activity than autologous serum (P < 0.01), directly correlated to C3 levels (P < 0.025). There was a significant correlation between chemotaxis in serum and in ascites fluid (P < 0.005). Adding ascites fluid to serum led to reduction of chemotactic activity only in the patients (P < 0.025). In conclusion, the chemotactic effect of ascites fluid is considerably lower than that of serum and is proportional to local concentrations of C3. Chemotaxis-inhibiting factors can also be identified in ascites fluid, their pathogenetic relevance being limited.
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7787166
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Hepatitis B vaccination alone is not adequate for the categorizing of adult subjects with isolated anti-HBc.
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To evaluate the meaning of isolated antibody to hepatitis B core antigen (anti-HBc), 88 Chinese subjects with isolated anti-HBc received rescreening of hepatitis B virus (HBV) markers. Eighty (90.9%) of them were still positive for this antibody and 29 were also found to be positive for antibody to hepatitis B surface antigen (anti-HBs). The remaining 51 subjects (58.0%) were positive for anti-HBc alone; 50 of them received a four-dose schedule of hepatitis B (HB) vaccine. After the initial dose, only one vaccinee disclosed an amnestic anti-HBs response, that is, anti-HBs titre > 1000 miu/mL. Forty-five vaccinees completed the vaccination schedule and 44 (97.8%) had anti-HBs response. The anti-HBs responses in 25 of these vaccinees were compared with 25 age- and sex-matched normal susceptible vaccinees. The anti-HBs response rates in both groups were the same (96 vs 96%). However, the geometric mean titre was significantly lower in the vaccinees with isolated anti-HBc (512 mIU/mL vs 4688 mIU/mL, P < 0.001). Prevaccinated sera were available in 49 vaccinees with isolated anti-HBc for detection of antibody to hepatitis B e antigen (anti-HBe) and HBV DNA; 37 (75.5%) of them had one or two of these markers. As we regarded the rescreening of HBV markers, response to hepatitis B vaccination and presence or absence of anti-HBe and/or HBV DNA together for categorizing the 88 subjects with isolated anti-HBc, at least three-quarters of them had past infection of HBV. The subjects with false positive anti-HBc test were a minor group.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787163
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A non-invasive method for evaluating cirrhotic portal hypertension by administration of 99mTc-MIBI per rectum.
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A study was performed to evaluate radio-isotopic imaging using technetium-99m hexakis 2-methoxyisobutyl isonitrile administered per rectum to assess portal collateral circulation. The heart-liver ratios (H/L; mean +/- standard deviation) in 15 controls, 13 cases of histologically confirmed viral hepatitis and 57 cirrhosis patients were 0.27 +/- 0.11, 0.43 +/- 0.14 and 1.00 +/- 0.28, respectively (P < 0.001). Among the cirrhosis patients those with the Child-Pugh classification A, B and C had H/L of 0.56 +/- 0.14, 1.00 +/- 0.20 and 1.19 +/- 0.26, respectively (P < 0.001). A high value of H/L was associated with a high risk of hepatic encephalopathy (1.25 +/- 0.17, P < 0.01) and oesophageal varices (1.02 +/- 0.20, P < 0.01). There were associations between H/L and serum bilirubin (P < 0.01), albumin (P < 0.05) and prothrombin time (P < 0.05). The results also showed a good correlation between H/L and portal vein pressure measured during operation in 13 patients (P < 0.001, r = 0.87). The regression equation: y = 6.77 + 32.5 H/L, allowed portal vein pressure to be estimated. The prognostic value of the test was supported by the fact that good correlations were observed between the H/L ratio and widely accepted prognostic classification (Child-Pugh). It is suggested that this new method could be a reliable non-invasive way to give an indication of the degree of portasystemic shunting to evaluate the prognosis and to follow up the effects of medications for reducing portal hypertension in patients with cirrhotic portal hypertension.
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7787164
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Carbohydrate-deficient transferrin in alcoholics with liver disease.
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To assess the relationship between carbohydrate-deficient transferrin (CDT) and alcoholic liver disease, we measured the ratio of carbohydrate-deficient transferrin to total transferrin (rCDT) in 32 male alcoholics with liver disease (Child-Pugh class A, 8; B, 11; C, 13) and 14 male alcoholics without clinically evident liver disease. Twenty of 32 with liver disease and six of 14 without clinically apparent liver disease had recent abstinence. The 32 patients with liver disease were assessed, in addition to the Child-Pugh class, using a linear prognostic score, the Combined Clinical and Laboratory Index (CCLI). Transferrin and CDT were measured by isocratic anion exchange chromatography and a radio-immunoassay. When the total group (n = 46) was divided into those with recent abstinence (n = 26) and those without (n = 20), the rCDT was lower in the abstainers than non-abstainers (0.7 +/- 0.6 vs 2.9 +/- 2.4, P < 0.005). Similarly, abstainers with liver disease (n = 20) had a significantly lower rCDT than non-abstainers (n = 12) with liver disease (0.7 +/- 0.7 vs 3.5 +/- 2.8, P < 0.005). The rCDT in the 20 abstaining patients with liver disease did not differ significantly between Child-Pugh classes. Furthermore, there was no correlation between the CCLI and rCDT (r = 0.05). We conclude that the relationship between rCDT and alcohol abuse is not appreciably altered by the presence of clinically severe liver disease in male alcoholics.
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7787160
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Correlation between serum pepsinogen concentration and gastric acidity measured by 24 h pH monitoring.
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The relationship between the intragastric pH measured by 24 h pH monitoring system and the serum pepsinogen I/II ratio was studied in 68 cases. When pepsinogen I/II ratio was compared with pH 3.0 holding time (the percentage time during which the gastric pH is above 3.0), there was a negative correlation between these two parameters (correlation coefficient r = -0.62, P < 0.001). Furthermore, there was also a strong negative correlation between the early morning (from 03.00 to 06.00 h) gastric pH and pepsinogen I/II ratio (r = -0.76, P < 0.001). Accordingly, by simply measuring serum pepsinogen I and II, it may be possible to infer gastric acidity and to obtain the information concerning the early morning intragastric pH.
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7787162
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Plasma kallikrein clearance by the liver of chronic carbon tetrachloride-treated rats.
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We have previously reported that the endocytosis of rat plasma kallikrein (RPK) by hepatocytes is a calcium-independent and beta-galactoside-dependent mechanism. We now report the clearance of RPK by the liver of four groups of rats: normal, inflamed (48 h ex-turpentine) and two groups chronically treated with CCl4 (52 mg/kg per week, intragastrically, for 9-12 weeks). Each liver was isolated, exsanguinated and perfused at 37 degrees C with 30 mL of BSA-Krebs-Henseleit-bicarbonate medium containing 10 nmol/L RPK. Although all rats received the same mild CCl4 treatment, the liver histology showed that they evolved either to severe hepatitis (serum alanine aminotransferase [ALT] 4852 +/- 885 U/L, parenchymatous necrosis in the perivenous region) or to compensated cirrhosis (serum ALT 209 +/- 42 U/L, vigorous fibrous encircling regeneration nodules); neither jaundice nor ascites was noted. The results show that serum albumin was not altered among the groups and that: the acute-phase response by itself (inflamed group) increased RPK clearance rate (3.01 +/- 0.59 mL/min) as compared with the normal group (1.85 +/- 0.14 mL/min); the CCl4 treatment, although induced an acute-phase response, decreased (P < 0.01) RPK clearance rates (0.80 +/- 0.11 mL/min hepatitis group and 0.98 +/- 0.10 mL/min cirrhosis group). These findings suggest that the hepatic clearance rate of plasma kallikrein is an early indicator of liver injury.
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7787161
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Immunoglobulin G subclass distribution of human anticolon antibodies in ulcerative colitis.
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Immunoglobulin G (IgG) subclasses of anticolon antibodies were studied in patients with ulcerative colitis (UC) using enzyme-linked immunosorbent assay (ELISA). The concentrations of total serum IgG subclasses were also measured by ELISA. The values for total serum IgG subclasses in patients with UC were not significantly different from those in normal controls, while the ratio of IgG1 to IgG2 in the patients was significantly higher than that in normal controls. All four IgG subclasses of autoantibodies were demonstrated in the sera of the patients. IgG4 anticolon antibodies were detected most frequently (15 out of 18 patients, 83%). IgG2 was the next most prevalent (9 of 18 patients, 50%). The activity of anticolon antibodies in each subclass did not correlate with the concentration of the corresponding serum IgG subclass. Seven cell lines producing anticolon antibodies were obtained from the colonic mucosa of the patients by Epstein-Barr virus (EBV) transformation. IgG subclasses of anticolon antibodies secreted by these cell lines were also varied. IgG4 subclass was secreted by three EBV transformed cell lines, all of which produced IgG4 anticolon antibodies. These results suggest that all four different IgG subclasses could respond to the colon antigens and that various antigens in colonic mucosa or lumen may contribute to the induction of those autoantibodies. In addition, the prominence of IgG4 anticolon antibodies may support the pathogenic role of this subclass in UC as in other autoimmune diseases.
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7787158
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Colonic mucosal antioxidant enzymes and lipid peroxide levels in normal subjects and patients with ulcerative colitis.
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Oxygen-derived free radicals have been implicated in the pathogenesis of ulcerative colitis. Mammalian tissues contain antioxidant systems that offer protection from the damaging effect of these active species. In the present study, the activity of the antioxidant enzymes catalase, glutathione peroxidase, glutathione transferase and glutathione reductase were measured in rectal biopsies from patients with ulcerative colitis and compared with that obtained from normal subjects. A significant decrease in the activity of glutathione transferase was observed in ulcerative colitis (48.32 +/- 6.73 units/mg protein, mean +/- s.e.) compared to normal (68.20 +/- 6.83; P = 0.015). There was no difference in the activity of other antioxidant enzymes between controls and ulcerative colitis. Myeloperoxidase, a marker for neutrophil infiltration, was considerably increased in ulcerative colitis while malonaldehyde, the end product of lipid peroxidation, was not increased. The reduced activity of glutathione transferase in ulcerative colitis may be an additional factor in the pathogenesis of mucosal damage in this disease.
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7787159
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Sulphated macromolecules produced by in vivo labelling in the rat gastric mucosa.
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The aim of this study was to investigate the nature and distribution of sulphated macromolecules of the extracellular matrix in rat gastric mucosa. This was achieved by developing an in vivo labelling system. An intraperitoneal injection of 1 mCi [35S]-sulphate was given for either 4 h (0.01% incorporation into macromolecular fraction) or 8 h (0.13% incorporation). At the end of the labelling period the stomach was removed and the mucosa and submucosa was either taken as a single combined sample or separated into four layers by blunt dissection. Each sample was papain digested and analysed by ion-exchange chromatography. This analysis revealed sulphated species of differing charge existing in differing proportions throughout the mucosa. These sulphated species eluted at NaCl concentrations of approximately 0 (A), 0.19 (B), 0.34 (C) and 0.78 mol/L (D) from a Q-Sepharose ion exchange column. Further analysis by size exclusion chromatography and chemical and enzymatic digestion showed that peaks B and C had molecular weights of 2.4 x 10(5) and 2.8 x 10(5), respectively and were resistant to chondroitinase ABC, heparitinase and nitrous acid digestion. Peak D was found to contain a polydisperse population of molecules with a molecular weight range of approximately 1 x 10(4) to 6 x 10(4). This sample was susceptible to nitrous acid and chondroitinase ABC digestion and was found predominantly in the sample isolated from deeper in the tissue. We have thus developed an in vivo labelling technique for sulphated macromolecules that can be used in the further study of injury to the gastric mucosa.
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7787157
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A single standard nocturnal dose of nizatidine enhances the healing of active duodenal ulcers among Chinese.
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Chinese people have a lower maximal acid output and gastrin response to meals compared to Western people. The aim of the present study was to assess the efficacy of a half-single nocturnal dose of nizatidine (150 mg at bedtime [h.s.], n = 40) with a standard full dose given once nightly (300 mg h.s., n = 38) or twice daily (150 mg twice a day [b.i.d.], n = 43) in the treatment of Chinese patients with active duodenal ulcers. An endoscopy was performed upon entry and at 4 week intervals until the ulcer healed (up to 8 weeks). There is no statistical difference in healing rates after 4 weeks of treatment (52.5, 52 and 47% in nizatidine 150 mg h.s., 150 mg b.i.d. and nizatidine 300 mg h.s., respectively) whereas nizatidine 300 h.s. had a significantly higher healing rate compared to nizatidine 150 mg h.s. and b.i.d. after 8 weeks of treatment (89 vs 70 and 67%, P < 0.05) by uni- and multivariate analysis of clinical and endoscopic characteristics. Symptomatic response was not significantly different in these three treated groups. Our study suggested that a half-single nocturnal dose of nizatidine is not ideal for the treatment of duodenal ulcer in Chinese patients, whereas a single standard nocturnal dose appears more effective than a twice-daily regimen.
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7787156
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Familial juvenile polyposis with adenomatous-carcinomatous change.
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A family tree of 118 members with five members found to have juvenile polyposis-adenomatous change and four juvenile polyposis-adenomatous-carcinomatous change is presented. All the patients developed bleeding per rectum between 10 and 17 years of age. Four members died of colonic malignancy between 30 and 55 years of age. Colonoscopy in five living members revealed typical juvenile polyps throughout the whole length of the colon and atypical large lobulated polyps containing adenomatous change in juvenile polyps in the rectosigmoid area. An autosomal dominant mode of transmission was evident on analysis of the pedigree. Gastric hyperplastic polyps were present in three of the five living members. Familial juvenile polyposis may have the potential to progress into adenoma-carcinoma sequence and is not always a benign disorder. Colonoscopic surveillance should be done to detect adenomatous change if any member of the juvenile polyposis family develops colonic malignancy.
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7787151
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Behavioral compliance with dialysis prescription in hemodialysis patients.
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The relationship between compliance and outcome is poorly understood, partially because there has been no gold standard for measuring compliance in hemodialysis patients. To investigate interrelationships between psychological, medical, and compliance factors, hemodialysis (HD) patients were studied with the Beck Depression Inventory, and a subset, the Cognitive Depression Index, the Perception of Illness Effects scale, and the Multidimensional Scale of Perceived Social Support. Behavioral compliance was measured in three ways: (1) percent time compliance (signifying "shortening behavior"); (2) percent attendance (signifying "skipping behavior) (3) percent total time compliance, assessing patients' time on dialysis normalized for prescribed time, including all shortenings and absences. Standard compliance indicators (predialysis serum potassium and phosphorus concentrations and interdialytic weight gain) were also analyzed. The patients' mean Beck Depression Inventory was in the range of mild depression. The prevalence of depression was 25.5%. Both depression indices correlated with Perception of Illness Effects scale scores. In general, social support was related to both measures of depression and perception of illness effects. Total time compliance was 95.8 +/- 5.0%. Younger patients were more likely to skip treatments compared with older patients. Time compliance comprised a wide spectrum, with most patients relatively compliant, whereas a small proportion received far less than their prescribed dialysis. Skipping and shortening behaviors did not correlate, suggesting that these constitute two separate types of noncompliant behaviors. Time compliance parameters did not correlate with potassium levels or interdialytic weight gain, but did correlate with phosphorus levels. Interrelationships between behavioral compliance measures and other parameters varied between units and patients of different gender. Finally, behavioral compliance patterns were stable over months in patients.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787155
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The effects of abdominal irradiation for seminoma of the testis on gastrointestinal function.
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To evaluate the long-term effects of abdominal irradiation for the treatment of seminoma of the testis on gastrointestinal function, 15 randomly selected patients who had been treated for stage I seminoma of the testis 2-10 years previously had the following parameters of gastrointestinal function evaluated: gastrointestinal symptoms; absorption of bile acid; vitamin B12; lactose and fat; gastric emptying; small intestinal and total gut transit; and intestinal permeability. Results were compared to those obtained in 18 normal volunteers. There was an increased prevalence of gastrointestinal symptoms (P < 0.01) in the patients and stool frequency was above the control range in two of them. Gastric emptying was faster (P < 0.01) in the patients. There were no significant differences in vitamin B12, bile acid, lactose or fat absorption, small intestinal transit or whole gut transit between the two groups, although faecal fat excretion was greater than the control range in three of the patients. At least one parameter of gastrointestinal function was abnormal in 11 of the 15 patients. Patients with right-sided seminoma had a greater bowel frequency when compared to those with left-sided seminoma (P < 0.05). We conclude that mild abnormalities in gastrointestinal function occur frequently when abdominal irradiation is used to treat stage I seminoma.
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7787154
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Ras oncogene and p53 gene hotspot mutations in colorectal cancers.
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Ras oncogene and p53 gene mutations are frequently observed in colorectal cancers. The role of co-operation between these two genes in the tumorigenesis of colorectal cancer was evaluated. Point mutations in K-ras oncogene and hotspot codons of p53 gene of colorectal cancers were evaluated by naturally created or amplified created restriction site method. Nine of 42 cases (21.4%) of colorectal cancer showed K-ras oncogene mutations. Six of 42 cases (14.3%) of colorectal cancer showed p53 gene hotspot point mutations. The low frequency of p53 gene mutation in this series may be due to racial difference or different hotspot codons. When six cases with mutated p53 gene were examined, only one (16.7%) showed concurrent K-ras oncogene codon 12 and p53 gene codon 248 mutations. We concluded that the co-operation between ras oncogene and p53 gene hotspot point mutations in the tumorigenesis of colorectal cancer in Chinese was not common. Other factors such as adenomatous polyposis coli gene mutations, oncogene activation or tumour suppression gene inactivation may be involved.
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7787152
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Comparison of continuous ambulatory peritoneal dialysis-related infections with different "Y-tubing" exchange systems.
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Despite several modifications of the continuous ambulatory peritoneal dialysis (CAPD) technique over the last decade, peritonitis remains a major source of morbidity and is the leading cause of dropout for patients maintained on CAPD therapy. Recently, Baxter Healthcare introduced the Ultra Twin bag system, which uses drainage and infusion bags both secured to Y connecting tubing. Previous nonrandomized studies comparing the Ultra Twin bag system with other systems have indicated an improvement in the peritonitis rate with the Ultra Twin bag system. In this study, 82 patients were randomized to use the Ultra Twin bag system or the Ultra Y-set system, which uses only the drainage bag already attached to the Y connecting tubing. Peritonitis rates were significantly lower with the Ultra Twin bag system, one episode per 33.9 patient months, compared with the Ultra Y-set system, one episode per 11.7 patient months (P < 0.05). Furthermore, the 1-yr infection-free survival rates with the Ultra Twin bag system and the Ultra Y-set system were 71 and 40%, respectively. Exit-site infections were lower with the Ultra Twin bag system, one episode per 12.5 patient months, compared with the Ultra Y-set system, one episode per 28.3 patient months, although this difference was not statistically significant (P = 0.084). The effect of the reduction in the infection rate on patient dropout with the Ultra Twin bag system remains to be addressed.
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7787150
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Aggressive, long-term cyclosporine therapy for steroid-resistant focal segmental glomerulosclerosis.
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Short-term cyclosporine (CsA) has been shown to reduce the proteinuria in refractory nephrotic syndrome, but the effect on disease progression has not been evaluated. This study was undertaken to evaluate whether maintenance CsA therapy in steroid-resistant focal segmental glomerulosclerosis (FSGS) will prevent progression to ESRD. Twenty-one black and Hispanic children (mean age, 8.4 +/- 4.5 yr) with biopsy-proven, steroid/cyclophosphamide-resistant FSGS were treated with CsA (initiated at 6 mg/kg per day and titrated to the serum cholesterol level to achieve a response). The mean CsA dose was 7 (4 to 20) mg/kg per day, the duration of CsA therapy was 27.5 (3 to 97) months, and the duration of follow-up was 8.5 +/- 4.7 yr. At the end of CsA therapy, the mean (+/- SE) proteinuria fell from 6.2 +/- 0.2 to 2.0 +/- 0.1 g/24 h (P < 0.001), the mean albumin rose from 1.95 +/- 0.04 to 3.41 +/- 0.04 g/dL (P < 0.001), the mean cholesterol decreased from 472 +/- 12.7 to 257 +/- 5.3 mg/dL (P < 0.005), and the mean creatinine rose from 0.79 +/- 0.02 to 1.16 +/- 0.03 mg/dL (P < 0.005). Seven children continue to receive maintenance CsA therapy, and 14 patients have had CsA stopped: 6 for an increase in serum creatinine and/or continued proteinuria, 5 for sustained remission, 2 for noncompliance, and 1 for pregnancy. Five (24%) of the 21 patients progressed to ESRD.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787149
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Dietary vitamin E supplementation ameliorates renal injury in chronic puromycin aminonucleoside nephropathy.
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Chronic puromycin aminonucleoside nephropathy (PAN) is an experimental analog of focal segmental glomerulosclerosis. Progressive renal damage in this model is partly mediated by excessive production of oxidant species. Whether dietary supplementation with vitamin E, an endogenous lipophilic antioxidant, ameliorates the severity of chronic PAN was tested. PAN was induced by seven serial injections of the glomerular epithelial cell toxin puromycin aminonucleoside, 2 mg/100 g body wt per dose, over a 12-wk period. Experimental animals (N = 8) received vitamin E-enriched chow (100 IU/kg), whereas control PAN rats (N = 10) were fed standard rodent diet containing vitamin E (30 IU/kg of chow). The administration of vitamin E had no effect on somatic growth or blood pressure; however, rats with PAN fed the vitamin E-enriched diet had an increased hematocrit. In addition, the experimental diet resulted in a 50% reduction in urinary total protein and albumin excretion and stabilization of the serum albumin, cholesterol, and triglyceride concentrations (P < 0.01). The inulin clearance was 69% higher in the vitamin E-supplemented animals (P < 0.001). Tubular function, namely, phosphate reabsorption and beta 2-microglobulin excretion, was improved in rats with chronic PAN treated with the vitamin E-enriched diet. There was a significant decrease in glomerulosclerosis and glomerular planar area, and tubulointerstitial scarring was diminished in vitamin E-treated animals with chronic PAN (P < 0.01). These beneficial effects on renal structure and function were associated with reduced malondialdehyde content in the kidney and liver.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787148
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Familial risk, age at onset, and cause of end-stage renal disease in white Americans.
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A strong familial clustering of ESRD has been reported among African Americans, suggesting that factors predisposing to renal failure, whether genetic, environmental, or both, may disproportionately affect certain families. A case-control study was undertaken to determine if a familial risk of ESRD was present among white Americans, if this risk differed among causes of ESRD, and if variability in age at onset was attributed to familial factors. Data were obtained from 103 white American patients (cases) with ESRD receiving dialysis treatments at the Bowman Gray School of Medicine's affiliated dialysis facility in Winston-Salem, NC. One hundred three age-, sex- and race-matched non-ESRD controls were consecutively selected from the Wake Forest University Physicians internal medicine clinic. Odds ratios (OR) and associated 95% confidence intervals (CI) were calculated to signify the prevalence of a relative with ESRD among cases versus controls. The presence of either a first- or second-degree relative increased a white American's risk for developing ESRD nearly threefold (OR = 2.7, 95% CI 1.1 to 7.2; P = 0.038), whereas the presence of either a first-, second- or third-degree relative with ESRD increased the risk nearly fourfold (OR = 3.5, 95% CI 1.5 to 8.4; P = 0.004). Cases with chronic glomerulonephritis and Type II diabetic nephropathy as the cause of ESRD had relatives with ESRD more often than cases with Type I diabetic nephropathy, interstitial nephritis, or renal artery stenosis. The average correlation (f) of ages at onset of ESRD among individuals in a single family (cases and their relatives) was 55%.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787147
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Modifications in glomerular polyanion distribution in adriamycin nephrosis.
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Previous reports have suggested that, in proteinuria induced by adriamycin (ADR), the functional size barrier of the glomerular basement membrane (GBM) is altered as the result of a sieving defect, whereas the functional charge barrier of the glomeruli remains intact. The aim of this study was to reevaluate the effect of ADR on anionic constituents in the glomerular capillary wall (GCW). Kidneys of nephrotic rats, induced by the injection of 7.5 mg/kg ADR, and controls were resected, and cortices were isolated 24 h and 10 days postinjection, fixed with formaldehyde, and embedded in paraffin. For the histochemical evaluation of sialyl residues, deparafinized sections were treated with biotin-labeled peanut agglutinin (PNA), before or after neuraminidase treatment. PNA binding was visualized by the avidin-biotin-peroxidase complex and interacted with hydrogen peroxide and diaminobenzidine. For electron microscopy, kidney cortices were fixed with glutaraldehyde and embedded in araldite or LR-white. The postembedding localization of anionic sites was carried out by cationic colloidal gold (CCG), directly applied on thin LR-white sections. Although in the 24-h ADR group, kidney functions and glomerular morphology were generally unaltered, the 10-day ADR group exhibited severe proteinuria, hypoalbuminemia, and massive fusion of intercalated foot processes of the podocytes. Intense PNA binding was observed after neuraminidase treatment in the GCW of the controls. This was gradually decreased in the 24-h ADR kidneys and further decreased in the 10-day ADR, indicating a gradual decrease in glomerular sialic acid content.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787146
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Prostaglandin F2 alpha- and 12-O-tetradecanoylphorbol-13-acetate-induced alterations in the pathways of renal ammoniagenesis.
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The mechanisms whereby prostaglandin F2 alpha (PGF2 alpha) and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit ammoniagenesis and the reason why they behave differently at pH 7.4, were examined with (15N)glutamine to assess the metabolic pathways and 2'-7'-bis(2-carboxyethyl)-5-(and-6)-carboxylfluorescein, acetoxymethylester (BCECF-AM) to evaluate Na+/H+ antiporter activity. LLC-PK1 cultures were incubated for 1 h in a Krebs-Hensleit bicarbonate buffer of pH 7.4 and pH 6.8 supplemented either with 5-15N- or 2-15N-labeled glutamine, followed by the assessment of (15N)ammonia and (15N)amino acid formation. Exposure of cells to either PGF2 alpha or TPA completely inhibited the low pH-induced increases in (15N)ammonia formation from incubations with 5-15N, reflecting reduced flux through the mitochondrial phosphate-dependent glutaminase, and from (2-15N)glutamine, reflecting reduced flux through the mitochondrial glutamate dehydrogenase pathway. They also qualitatively reversed the acute acidosis-induced changes in (15N)alanine formation and (15N)glutamate accumulation in the media. By contrast only TPA, but not PGF2 alpha, modified glutamine metabolism at pH 7.4. Na+/H+ antiporter activity was assessed under both acidified and basal (pH 7.4) conditions by measuring changes in intracellular pH in cells loaded with BCECF. TPA and PGF2 alpha both stimulated Na+/H+ antiporter activity comparably under acidified conditions. When cells were studied at pH 7.4, TPA but not PGF2 alpha stimulated the Na+/H+ antiporter and increased steady-state intracellular pH.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787145
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The study of the effect of intensity of blood pressure management on the progression of type 1 diabetic nephropathy: study design and baseline patient characteristics. Collaborative Study Group.
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A randomized, prospective, clinical trial has been initiated to continue follow-up in a subset of the patients previously enrolled in the recently completed Study of Angiotensin-Converting Enzyme Inhibition (ACEi) in Type 1 Diabetic Nephropathy. In that study, the use of captopril was associated with a 48% reduction in the risk of doubling the serum creatinine and a 50% reduction in the risk of experiencing dialysis, transplantation, or death, compared with the use of placebo. These effects were independent of captopril's effect on the blood pressure. This study is designed to determine whether the level of mean arterial blood pressure (MAP), using the ACE inhibitor ramipril as the primary therapy, is associated with an improved prognosis of diabetic nephropathy with respect to (1) the rate of decline in renal function; (2) the rate of progression to end-stage renal failure; (3) the clinical course of proteinuria; (4) morbidity; and (5) mortality. Patients are randomized into one of two distinct blood pressure control groups, an Intensive Group #1, MAP < or = 92 mm Hg; and a Moderate Group #2, MAP 100 to 107 mm Hg. Patients previously enrolled in the "Study of ACEi in Type 1 Diabetic Nephropathy" whose serum creatinine was less than 4.0 mg/dL (354 mumol/L) were eligible for randomization into this study. All patients will receive ramipril (2.5 to 10.0 mg/day) as the primary therapy, with the addition or removal of other antihypertensive agents as needed to achieve the assigned blood pressure goal.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787143
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Renal hemodynamics and plasma and kidney angiotensin II in established diabetes mellitus in rats: effect of sodium and salt restriction.
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Six weeks after the onset of insulin-treated streptozotocin diabetes (STZ) in Munich-Wistar rats, the effect of a low-sodium (LNa) and a low-salt (LNaCl) diet on renal function and on plasma and kidney tissue angiotensin II (AIIp, AIIk) was tested. Clearance experiments were performed in anesthetized rats 7 days after starting on LNa or LNaCl. On a control diet, STZ exhibited an increase in GFR, RBF, and kidney weight (KW) and a reduction in renal vascular resistance (RVR) and AIIk, but no change in AIIp, compared with nondiabetic normal rats (CON). Although sodium restriction reduced and salt restriction increased AIIk in CON, both diets increased AIIp without affecting renal hemodynamics or KW. In diabetic rats, both salt and sodium restriction further increased GFR and RBF by reducing RVR, increased KW, and changed AIIk and AIIp in a similar pattern, but at significantly lower values compared with CON. Daily treatment of STZ-LNa with the AII-receptor blocker losartan (20 mg/L, in drinking water) did not affect the reduction in RVR and the increase in KW but slightly reduced RBF because of a decrease in mean arterial blood pressure and further increased GFR. It was concluded that (1) AIIk but not AIIp is affected differently by LNa compared with LNaCl in both CON and STZ; (2) LNaCl and LNa change AIIp and AIIk in a similar pattern but at significant lower values in STZ compared with CON; and (3) with regard to renal hemodynamics and KW, the response to LNa and LNaCl is different in CON compared with rats 6 wk after the onset of diabetes mellitus, the latter exhibiting a further increase in renal hyperfiltration and KW by a mechanism that is not directly AII receptor dependent.
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7787144
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Impaired renal autoregulatory ability in dogs with reduced renal mass.
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In normal dogs, the renal autoregulatory mechanism limits the effect of changes in renal arterial pressure (RAP) on RBF, renal outer cortical blood flow (OCBF), and GFR by adjusting the vascular tone of the preglomerular arterioles. To determine the extent to which autoregulatory ability was impaired in remnant renal tissue in dogs, the effects of variations in RAP on RBF, OCBF, and GFR were studied after sham-operation (Group 1; N = 5), 3/4 nephrectomy (Group 2; N = 5), or 7/8 nephrectomy (Group 3; N = 5). In Group 1, the RBF, OCBF, and GFR were not significantly affected by variations in RAP between 75 and 125 mm Hg, indicating intact renal autoregulatory ability. In contrast, both groups of partially nephrectomized dogs exhibited a loss of renal autoregulatory ability below 100 mm Hg (P < 0.05). As RAP rose above 100 mm Hg, dogs with 7/8 nephrectomy exhibited a greater increase than control dogs in RBF (0.586 +/- 0.211 versus -0.080 +/- 0.030 percent change in RBF/mm Hg change in RAP; P < 0.05), OCBF (0.408 +/- 0.157 versus -0.059 +/- 0.054 percent change in RBF/mm Hg change in RAP; P < 0.05), and GFR (0.784 +/- 0.230 versus 0.134 +/- 0.049 percent change in RBF/mm Hg change in RAP; P < 0.05). The ability of the renal vasculature to maintain renal function stable above 100 mm Hg was intermediate in Group 2 and not significantly different from corresponding values for Group 1.(ABSTRACT TRUNCATED AT 250 WORDS)
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7787142
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Renal hemodynamics and sodium handling in moderate renal insufficiency: the role of insulin resistance and dyslipidemia.
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Insulin infusion during euglycemia causes antinatriuresis and renal vasodilation in healthy humans, whereas the effects of acute insulin infusion on tubular sodium handling and renal hemodynamics in chronic renal disease are unknown. The response to euglycemic insulin infusion was investigated in two homogeneous patient groups with a slight renal impairment-one with nephrotic syndrome (GFR, 64 mL/min; N = 9) and one with non-nephrotic immunoglobulin A nephropathy (GFR, 70 mL/min; N = 8). In addition, nine renal transplant recipients (GFR, 44 +/- 6 mL/min) were investigated. The results were compared with those of 12 healthy controls (GFR, 105 +/- 4 mL/min). Renal hemodynamics and renal tubular sodium handling were estimated with inulin, p-aminohippurate, sodium, and lithium clearances. The results showed that patients with nephrotic syndrome (5.0 +/- 0.4 mg/kg per minute) and renal transplant recipients (4.8 +/- 0.6 mg/kg per minute) had a significant lower metabolic clearance of glucose as compared with control subjects (7.9 +/- 0.4 mg/kg per minute), whereas patients with immunoglobulin A nephropathy (6.7 +/- 0.6 mg/kg per minute) had a metabolic clearance of glucose that was similar to that of the controls. Despite insulin resistance to carbohydrate metabolism, insulin infusion still induced hypokalemia and antinatriuresis in patients with nephrotic syndrome and renal transplant recipients. Insulin infusion caused a significant 13% increase in RPF and lithium clearance in control subjects, and a positive Spearman rank correlation (Rs = 0.41; P < 0.05) was observed between the changes in p-aminohippurate and lithium clearances during insulin infusion in the combined patient group, suggesting that impaired renal vasodilation may contribute to abnormal proximal tubular sodium handling and sodium retention. The results also suggest that hypertriglyceridemia could be a factor contributing to abnormal proximal tubular sodium handling in chronic renal disease.
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7787140
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Rapidly progressive glomerulonephritis after immunotherapy for cancer.
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Cytokines have been used in experimental and standard protocols for immune enhancement for cancer. The combination of interleukin-2 and interferon-alpha 2 beta has been used in experimental protocols for metastatic renal cell carcinoma. A man who developed rapidly progressive renal failure after receiving this combination therapy is reported. A renal biopsy revealed a pauci-immune crescentic glomerulonephritis. Antineutrophil cytoplasmic antibodies and antiglomerular basement membrane antibodies were absent. The spectrum of renal disease and potentially related extrarenal manifestations associated with interleukin-2 and inteferon-alpha are reviewed. A pathogenesis of altered cell-mediated immunity, consistent with abnormalities in extrarenal organs after immune enhancement, is proposed.
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7787141
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Intravenous immune globulin in the treatment of patients with systemic lupus erythematosus and end-stage renal disease.
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Intravenous immune globulin (IVIg) has been advocated as efficacious therapy for a variety of disorders including idiopathic thrombocytopenic purpura and Kawasaki disease. Several reports have also documented the effectiveness of IVIg in systemic lupus erythematosus (SLE). Two patients with symptomatic SLE and ESRD were treated with IVIg. Both patients tolerated IVIg administration well and demonstrated clinical and serologic improvement. Both individuals also experienced a transient decline in serum albumin concentration with IVIg treatment. The mechanisms by which IVIg might have effected improvement in these patients are varied and are likely related to the immunomodulatory actions of IVIg. The reversible change in albumin concentration seen in these individuals may be secondary to abrupt alterations in oncotic homeostasis. Despite this unusual effect, the documented improvement in these patients suggests that IVIg therapy may be of benefit in patients with active SLE and ESRD. Further studies are warranted to examine the mechanisms by which IVIg may exert its therapeutic effect.
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7787139
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Structure-stabilizing forces in the glomerular tuft.
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The glomerular tuft is constantly exposed to considerable expansile forces resulting from high capillary pressures. Counterforces must be generated in order to maintain structural stability. This review analyzes those structures of the glomerular tuft capable of developing such stabilizing forces. Two systems are described. A basic system consists of the glomerular basement membrane (GBM) and the mesangium. The GBM represents the main skeletal element of the glomerular tuft. In general, opposing portions of the GBM are bridged by contractile mesangial cell processes, generating inwardly directed forces that balance the expansile forces resulting from pressure gradients across the GBM. A second structure-stabilizing role of the podocytes appears to be superimposed on this system. Podocytes are attached to the GBM by numerous foot processes that contain a contractile system. The foot process attachments probably stabilize small patches of the underlying GBM, counteracting local elastic distension. In addition, podocytes may contribute to the stabilization of the folding pattern of the tuft by linking neighboring capillary loops to each other.
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7787135
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The influence of pyruvic acid on the pharmacokinetics of sulphadiazine in rabbits.
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During the past few years, acetylation polymorphism has been shown to be a proven, established fact, and N-acetyltransferase, an enzyme that transfers an acetyl group to the substrate, has been recognized as the main factor in acetylation polymorphism. In a recent study, a significant difference between the acetylation phenotype and plasma pyruvic acid (PA) concentration in rabbits was found. In this report, the influence of PA on the pharmacokinetics of sulphadiazine (SDZ), a drug that has been used in pharmacogenetic studies of acetylation, was studied. By using a loading dose of 300 mg kg-1, and an infusion rate of 7.5 mg min-1 kg-1 of PA, the concentration of PA reached a steady state (Css approximately equal to 100 micrograms mL-1) in 30 min. During PA infusion in rapid-acetylation rabbits, no significant changes were found in any of the pharmacokinetic parameters for SDZ. However, differences were found in the beta half-life, AUC, clearance, and k10 of SDZ in slow acetylators: the beta half-life decreased from 115.74 +/- 12.47 min to 62.96 +/- 4.36 min (p < 0.001); AUC decreased from 10,617.38 +/- 1179.81 micrograms min mL-1 to 6217.14 +/- 391.32 micrograms min mL-1 (p < 0.001); clearance increased from 0.0044 +/- 0.0008 L min-1 kg-1 to 0.0068 +/- 0.0007 L min-1 kg-1 (p < 0.001); and k10 increased from 0.0090 +/- 0.0009 min-1 to 0.0193 +/- 0.0028 min-1 (p < 0.005). The reason for this may be that PA influences the elimination of SDZ in slow-acetylation rabbits.
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7787134
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The influence of food intake on the effect of two controlled release formulations of furosemide.
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Differences in the urinary excretion rate of furosemide may explain discrepancies observed between the bioavailability and the total diuretic effect of different formulations of this drug. Furosemide was given at a dose of 60 mg as two oral controlled release (CR) formulations (FR and LR), with and without breakfast, in a randomized, four-treatment, four-period, crossover design to 28 healthy volunteers. Urinary volume, and contents of furosemide and sodium, were measured in samples taken over 24 h. The extent and rate of absorption of furosemide from FR were decreased after breakfast as compared to fasting: the mean (SD) of total furosemide excreted decreased from 11.38 (3.12) to 7.73 (1.67) mg, p < 0.0001, and the median (range) mean residence time increased from 6.3 (4.1-9.3) to 9.5 (5.9-11.8) h, p < 0.001. On the other hand, the extent of absorption of LR was increased after breakfast, from 8.04 (3.32) to 9.45 (1.83) mg, p < 0.05, without a significant change in MRT. FR had a higher extent and rate of absorption than LR during fasting, but its extent of absorption was lower than that of LR in the postprandial state. Interestingly, the total fraction of furosemide absorbed, as estimated from total furosemide excretion, was not correlated with the total diuresis (r2 = 0.079) and the differences in drug response compared among the four periods were much smaller than would be expected from the differences in amount absorbed. This discrepancy may be explained by differences in urinary excretion rate of furosemide and, related to this, differences in efficiency profiles between the four treatments. Therefore, the urinary excretion profile of a formulation of furosemide may be more important for the cumulated drug effect than the amount absorbed.
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7787133
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Disposition of [3H]fluvastatin following single oral doses in beagle dogs and rhesus monkeys with bile fistulae.
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The disposition of [3H]fluvastatin was examined following single oral doses in dogs (12.4 mg kg-1) and monkeys (0.48 and 45.5 mg kg-1) with bile fistulae. Serial plasma and complete urine, feces, and bile were collected at designated intervals for 3 or 4 d, and were analyzed for total radioactivity and unchanged fluvastatin. In the dog, peak radioactivity concentrations (Cmax) averaged 7260 ng equiv. mL-1 and the mean time to peak (tmax) was approximately 9 h. In the monkey, the mean radioactivity tmax values were approximately 5 and 13 h following the low and high doses, the respective Cmax values being 116 and 10,400 ng equiv. mL-1. The mean AUC of total radioactivity was proportional to the dose while that of fluvastatin was overproportional to dose, suggesting dose independent absorption but saturable first-pass effect. The AUC ratio of unchanged fluvastatin versus total radioactivity was approximately 63% in the dog, and 9% and 13% for the low and high doses, respectively in the monkey. The bile was the major excretory route of radioactivity (dog, 56%; low-dose monkey, 73%; high-dose monkey, 69%) whereas the renal pathway accounted for < 5% of the dose in both species. Approximately 12% of the biliary radioactivity in the dog was due to intact fluvastatin, compared with 0% and 7.5% after the low and high doses in the monkey. These results showed a smaller extent of fluvastatin metabolism in the dog than in the monkey, and suggested that metabolism in the monkey was saturable in the dose range studied.
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7787131
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The effect of food on the absorption of 14C-SDZ FOX 988, an antidiabetic agent, in healthy human volunteers.
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The objective of this study is to examine the effect of food on oral absorption of SDZ FOX 988 (FOX 988), an antidiabetic agent, and circulating levels of its active metabolite, SDZ 53-450 (53-450). Sixteen normal volunteers received a single 10 mg dose of 14C-FOX 988, either as gelatin capsules or in a suspension (0.5% CMC). For subjects receiving each formulation, four subjects received a meal, consisting of 50% fat by calories, immediately following dosing, while the other four received the same meal at 2 h post-dose. Serial blood, urine, and fecal samples were collected for 120 h and analyzed for total radioactivity. Blood concentrations of 53-450 were analyzed using an HPLC-UV method. Concomitant administration with food increased the extent of FOX 988 absorption from either suspension or capsule, as shown by an increase in AUC and in urinary recovery of radioactivity. Blood concentrations of 53-450 were only detected in subjects receiving food at dosing. No difference in absorption was observed between the capsule and the suspension. Results from this study showed that oral absorption of FOX 988 is enhanced by co-administration of food in normal volunteers.
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7787132
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Pharmacokinetic analysis of diethylcarbonate prodrugs of ibuprofen and naproxen.
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The pharmacokinetics of ibuprofen diethylcarbonate (ibudice) and naproxen diethythylcarbonate (napdice), two new diethylcarbonate prodrugs of ibuprofen and naproxen, was investigated in dogs. The rationale for the development of ibudice and napdice was that esterification of the carboxylic moiety of the parent compounds would suppress gastrotoxicity without adversely affecting their anti-inflammatory activity. In addition the biotransformation of the prodrugs to the parent compounds may be utilized to achieve rate and time controlled drug delivery of the active entities. Following oral administration the diethylcarbonate esters were rapidly biotransformed to the parent compounds and no ibudice or napdice was detected in the plasma. The relative bioavailability of ibuprofen and naproxen, following oral administration of ibudice and napdice, was 96% and 74%, respectively, and the rate of absorption was not significantly different from that obtained following oral dosing of the parent compound. Stability studies in gastric and intestinal juice showed that, unlike napdice, ibudice was stable in gastric juice, with both prodrugs undergoing rapid biotransformation to their parent compounds in intestinal juice. This rapid conversion led to the lack of sustained release performance following oral administration of ibudice or napidice.
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7787130
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Pharmacokinetics of dolasetron following single- and multiple-dose intravenous administration to normal male subjects.
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Dolasetron, Anzemet, a 5-hydroxytryptamine receptor antagonist, is under investigation as an antiemetic agent. The keto-reduced metabolite of dolasetron has been identified in human plasma and is probably responsible for the majority of the antiemetic activity. This study evaluated the pharmacokinetics of dolasetron and the reduced metabolite following single and multiple intravenous (IV) infusions of dolasetron mesylate in healthy male subjects. Four groups of subjects (six active/two placebo) received either dolasetron mesylate or placebo in single IV doses ranging from 0.30 to 0.60 mg kg-1 on day 1 and multiple IV doses ranging from 0.60 to 1.20 mg kg-1 d-1 on days 2-9. Dolasetron could be detected for less than 1 h, while the reduced metabolite appeared rapidly in the plasma, reaching a maximal plasma concentration in less than 1 h. Reduced metabolic maximal plasma concentration was proportional to the dose and the area under plasma concentration curve was linear based on regression analysis. The half-life of reduced metabolite ranged from 3.82 to 7.46 h. The mean renal clearance of reduced metabolite was 2.20-4.43 mL min-1 kg-1 and was dose independent. All of the evidence supports dose independent pharmacokinetics for the reduced metabolite. Upon multiple dosing, the reduced metabolite AUC can be predicted from the single-dose pharmacokinetics of this metabolite.
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7787129
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Pharmacokinetics of amoxicillin coadministered with a saline-polyethylene glycol solution in healthy volunteers.
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The pharmacokinetics of orally administered amoxicillin were investigated in 12 healthy volunteers in a crossover design. They received either a placebo or a saline-polyethylene glycol solution (SPG) for 4 d, the last dose being given simultaneously with 1 g amoxicillin; blood samples were drawn for the next 12 h. Amoxicillin kinetics were similar in the two treatments but small differences in some pharmacokinetic parameters reached significance. The mean +/- SD area under the curve was lower with SPG (43.8 +/- 6.8 against 47.8 +/- 8.2 mg h L-1, p < 0.05) but the treatments were equivalent according to Westlake's test (95% confidence interval = 14.95%). Analysis of SPG against placebo amoxicillin absorption kinetics after fitting the data to a Weibull model revealed a longer duration of the absorption, a slower rate of absorption, and a different shape of the curve. No clinical consequences are expected from these minor variations but possible mechanisms could be relevant to other drugs.
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7787128
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Nutritional supplementation and prevention of congenital abnormalities.
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Recent intervention studies have shown a reduction of occurrence and recurrence of neural tube defects caused by the periconceptional supplementation of folic-acid-containing multivitamins or pharmacological doses of folic acid alone. This new primary preventive method can also reduce the occurrence of other major congenital abnormalities, mainly the reduction of conotruncal cardiovascular malformations, defects of the urinary tract, congenital hypertrophic pyloric stenosis and congenital limb deficiencies. The rate of cleft lip, palate, or both, was not lowered by periconceptional multivitamin supplementation, however. The underlying biologic mechanisms of this protective effect are not still understood, but naturally occurring folates (polyglutamates) or synthetic folic acid (monoglutamate) have a key role. The debate over supplementation concerns which vitamins (folic-acid-containing multivitamins, or folic acid alone); what doses (0.4 mg, 0.8 mg or 4 mg folic acid) and to whom (whether it is worthwhile differentiating between women at high and low risk). At present there are three possibilities: folate- and other vitamin-rich diet, supplementation, and food fortification to provide appropriate multivitamin and folic acid consumption for all women of childbearing age who are capable of becoming pregnant.
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7787122
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Congenital infection.
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The past year has shed much new light on congenital infection. A key development has been the application of polymerase chain reaction technology to the diagnosis of intrauterine infection. This technique appears to be the diagnostic tool of choice for toxoplasmosis and cytomegalovirus. Pharmacologic treatment appears to reduce the sequellae of toxoplasmosis when treated either in utero or postnatally. Obstetric interventions may reduce vertical transmission. Education has been shown to reduce the incidence of seroconversion for toxoplasmosis, and HIV-positive women treated with zidovudine have a dramatically reduced rate of transmission to their offspring.
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7787125
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Antepartum fetal surveillance.
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In recent years the advances in prenatal diagnosis of fetal asphyxia have been dramatic and increasing at a remarkable pace. Now that a solid base of multivariable testing has been accepted as the method of choice for assessment of fetal health, the focus of clinical research has shifted towards identification of factors and signs that might predict not only risk of death and immediate morbid outcome but also the quality of life after delivery. The papers reviewed here outline the scope of the problem of cerebral palsy and offer clues as to how fetal disease may become manifest as child or adult sustained neurological injury. Concurrently, the advances in refinement of the testing methods are reviewed. Clearly a better understanding of the etiology, physiology, and range of fetal responses to asphyxia will be the key to effecting not only better survival rates but also a higher quality of survival.
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7787123
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Low-dose aspirin therapy in obstetrics.
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The results of four large controlled clinical trials, encompassing over 13,000 pregnant women, indicate that low-dose aspirin is of benefit in the prevention of pre-eclampsia in women at high risk. The preventive effect on fetal growth retardation seems to be small, and no therapeutic benefits could be established. Low-dose aspirin appears to be safe for mother, fetus, and neonate.
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7787124
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The pathophysiology of premature rupture of the membranes.
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Premature rupture of the membranes (PROM) is the condition in which the chorioamnion is disrupted before the onset of labor. This condition creates a dilemma for the practicing obstetrician, because once the membranes have broken the risk of fetal or maternal infection, or both, increases. Preterm PROM adds to this management challenge, mainly because of the added problem of prematurity. Although the epidemiology of PROM has been well defined, the exact etiology has yet to be understood. However, using the associated clinical risk factors of PROM, researchers in this field have contributed to our understanding of the causes. Various mechanisms have been proposed, including mechanical, as well as infectious or inflammatory processes. The purpose of this article is to review the various proposed mechanisms of PROM. Maternal risk factors for PROM are presented, mainly to place into context the current literature involving both in-vitro and in-vivo research. It is apparent that a single pathophysiologic mechanism is not responsible for all cases of PROM, but rather a combination of processes is in operation.
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7787120
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Early detection of structural anomalies and markers of chromosomal aberrations by transvaginal ultrasonography.
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Transvaginal ultrasonography is emerging as the newest method for the early detection of structural malformations and markers of chromosomal aberrations. According to a multicenter registry on the onset of fetal anomalies, many defects are potentially detectable at an early transvaginal scan. Moreover, cystic hygroma and other nuchal signs thought to indicate a very significant risk for a genetic problem detected during the first trimester may resolve and not be detected at the later abdominal scan. Current research presented in this review indicates a possible transition from an abdominal scan at 18-22 weeks to two scans: an early transvaginal scan at 12-14 weeks for the detection of gross congenital anomalies and markers of chromosomal aberrations, and then an abdominal scan at 22 weeks for cardiac and other anomalies.
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7787121
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Multifetal pregnancy reduction and selective termination.
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Multifetal pregnancy reduction has had a major impact upon perinatal morbidity and mortality following iatrogenic multiple pregnancies. There are still residual risks, however, that are higher for higher order multiples than for pregnancies that started out at lower numbers. There are many, but not complete, parallels between reductions for number per se, and selective termination because of diagnosed abnormalities.
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7787116
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X-ray evidence that in contracting live frog muscles there exist two distinct populations of myosin heads.
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Using synchrotron radiation and whole muscles, 2 ms time-resolved x-ray diffraction patterns were recorded at 8 degrees C. The results show that in both isotonic and isometric contractions, as well as in length changes imposed at maximum tension [Po], the meridional third myosin layer line consists of two distinct reflections with different intensities and spacings that measure approximately 14,623 and 14,412 nm at Po. Although the intensity behavior of the two reflections is strikingly different during quick releases, it is very similar during stretches. Study of the time courses indicates that myosin heads diffracting at Po with the approximately 14.623 nm periodicity are actively involved in tension production. Those diffracting at Po with the periodicity of approximately 14.412 nm appear not be associated with tension production during isometric contraction and releases, but the results suggest that they are recruited during stretches and here contribute to tension production. Our most important conclusion is that under all conditions of contraction we have investigated there exist two populations of myosin heads, each with a well defined axial disposition and configuration.
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7787115
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Changes in the x-ray diffraction pattern from single, intact muscle fibers produced by rapid shortening and stretch.
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Changes in the x-ray diffraction patterns produced by 100-microseconds-length steps imposed during tetanic stimulation were recorded from single intact fibers of frog tibialis anterior muscle. For shortening steps, a staircase length change was applied, with a 20-ms interval between steps. For stretches, each 20-ms cycle started with a stretch, followed after 4 ms by shortening to the original length. Each shortening step in the staircase and each stretch in the stretch/shortening protocol produced a response similar to that of a single step from the isometric steady state. The intensity of the 14.5-nm x-ray reflection arising from the axial repeat of the myosin heads along their filaments decreased after both shortening and stretch; this decrease was not accompanied by broadening along or across the meridian. The relationship between the intensity after the length step and step amplitude was approximately linear for both stretches and shortening steps, extrapolating to zero intensity for 11-nm stretches and 13-nm shortening steps, but there was no significant intensity change for the first approximately 2 nm of shortening. These results are broadly consistent with conventional models of muscle contraction in which myosin heads move through about 10 nm during the working stroke in the shortening direction, but an additional distortion of the myosin heads may be produced by a stretch.
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7787119
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Early amniocentesis.
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Amniocentesis before 15 weeks has been introduced in many prenatal diagnosis centres. There are, however, major anatomical differences between the appearance of fetus at 15-16 weeks, when amniocentesis has normally been performed, and in the first and early second trimester. This is mainly because of the presence of extraembryonic coelome and the relatively small amount of amniotic fluid in early pregnancy. Several small cohort studies have indicated no increased fetal loss rate with early amniocentesis. Others have suggested an increased risk of respiratory distress among the newborn. The only randomized study published, however, found a significantly increased rate of fetal losses after early amniocentesis. This incomplete study indicates the urgency of further randomized studies of early amniocentesis. A randomized study comparing early amniocentesis with transabdominal chorionic villus sampling is being undertaken at our centre in Copenhagen using a filter method that reduces the volume of fluid needed for karyotyping.
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7787118
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Chorionic villus sampling.
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Chorionic villus sampling (CVS) has the great advantage over mid-trimester amniocentesis of producing early results. Moreover, rapid analytic techniques have significantly reduced the waiting time between sampling and diagnosis, whereas progress in recombinant DNA technology and human gene mapping has led to an increase in the range of conditions it can detect. The role of CVS in twin pregnancy has been investigated and compared with amniocentesis. Although these techniques are equally safe, CVS should be considered the approach of choice because of a number of technical advantages, and in relation to selective fetal reduction in discordant twins. Confined placental mosaicism has been investigated and a list of chromosomes related to adverse pregnancy outcome has been compiled. Recent reports have substantially contributed to solving the controversy on the hypothetical relationship between limb reduction defects and CVS. Analysis of limb reduction defects among more than 130,000 cases reported to the World Health Organization CVS registry has been unable to find any relationship between sampling and fetal malformations, including limb reduction defects.
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7787117
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Fetal cells in maternal blood.
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Fetal cells have been successfully detected in maternal blood in all three trimesters of gestation in a substantial proportion of normal pregnancies. Various enrichment techniques have been developed for fetal trophoblast cells, leucocytes and nucleated red blood cells. Nucleated red blood cells are considered to be best suited for noninvasive prenatal diagnosis. Fluorescence in-situ hybridization detected the first cases of fetal trisomy in maternal blood after enrichment of fetal nucleated red blood cells. Despite recent encouraging results, accurate and reproducible diagnoses of fetal anomalies by polymerase chain reaction or fluorescence in-situ hybridization require further optimization of enrichment devices and detection protocols.
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7787114
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Millisecond time resolution electron cryo-microscopy of the M-ATP transient kinetic state of the acto-myosin ATPase.
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The structure of the AM-ATP transient kinetic state of the acto-myosin ATPase cycle has been examined by electron microscopy using frozen-hydrated specimens prepared in low ionic strength. By spraying grids layered with the acto-S1 complex with ATP immediately before freezing, it was possible to examine the structure of the ternary complex with a time resolution of 10 ms. Disordered binding of the S1 was observed, suggesting more than one attachment geometry. This could be due to the presence of more than one biochemical intermediate, or to a single intermediate binding in more than one conformation.
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7787113
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Transients of fluorescence polarization in skeletal muscle fibers labeled with rhodamine on the regulatory light chain.
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Structural changes of the myosin heads were correlated with mechanical events in the cross-bridge cycle by measuring fluorescence polarization signals at high time resolution from rhodamine probes bound to myosin regulatory light chains in skeletal muscle fibers. Motions of the cross-bridges were partially synchronized either by applying quick length changes to the fibers during active contractions or by activating the fibers from rigor by photolysis of caged ATP in the presence of Ca2+. With fibers in rigor, the fluorescence polarization values indicate that the probe dipoles are quite well ordered and are directed away from the muscle fiber axis. After photorelease of ATP from caged ATP, changes in polarization signals are consistent with broadening of the distribution of probe orientations. The signal deflections occur when ATP binds to actomyosin or when the cross-bridges detach, but the orientational distribution changes surprisingly little during active force development. In contrast, when staircases of quick releases are applied to labeled fibers during active contractions, the fluorescence polarization signals suggest a concerted rotation of the probes. The results indicate that the light chain region of myosin tilts during the quick release and/or during the tension recovery phase within the next few ms.
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7787112
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Three-dimensional structure of the acrosomal filament of Limulus sperm by 400 kV electron cryomicroscopy.
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The acrosomal bundle of Limulus sperm was imaged by electron cryomicroscopy, and the three-dimensional structure of a filament computationally isolated from the bundle was determined by helical image reconstruction. The actin model of Holmes was fit into the map, and its interactions with scruin, the actin-binding and cross-linking protein, were studied. Scruin binds to two consecutive actins along the helix via subdomains 1 and 3. These interactions involve helix-loop-beta motifs that are present in both actin subdomains (in different monomers) in positions available for binding by the same scruin molecule as it wraps around the actin. Taking first the structural motif homology and then looking for sequence pattern similarities, a stretch of 12 out of 20 matches in hydrophobicity is found. Scruin itself has been found to have an internal tandem homology.
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7787111
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Fluorescence polarization from isomers of tetramethylrhodamine at SH-1 in rabbit psoas muscle fibers.
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We have used fluorescence polarization to examine orientational changes of the 5- and 6-isomers of acetamidotetramethylrhodamine (ATR) covalently bound to SH-1 (Cys-707 of the myosin heavy chain) in single, skinned fibers from rabbit psoas muscle after rapid length steps or photolysis of caged nucleotides. Similar results were obtained with both the 5- and 6-isomers of ATR. After the photolysis of caged ATP, large and rapid changes in the fluorescence polarization signals were observed and were complete well before appreciable force had been generated. Changes in the fluorescence polarization signals after the photolysis of caged ADP were similar to those after the photolysis of caged ATP, despite an almost negligible change in force. The fluorescence polarization signals remained almost constant after rapid length steps in both rigor and active muscle fibers. These results suggest that structural changes at SH-1 monitored by 5- or 6-ATR are not associated directly with the force-generating event of muscle contraction, but may be involved in the communication pathway between the nucleotide and actin-binding sites of myosin.
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7787109
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Single kinesin molecules stressed with optical tweezers.
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Using the optical tweezers to pull on microtubules, we have stretched and twisted single kinesin molecules adsorbed to glass surfaces. Preliminary measurements suggest that the mechanical system is very compliant, with an apparent stretch of 120 nm with < 2 pN of force. Although measurements of the series compliance of the bead-microtubule structure are still in progress, the kinesin attachment site does not slip with stretch. However, under torsional stress, kinesin appears to slip. With torques < 2 pN-microns approximately 1 Hz in 2 mM AMP-PNP, there is no apparent limit to the number of revolutions that the microtubule can rotate around the kinesin attachment site (n = 44). Preliminary data from other nucleotide conditions are similar. Although there are rare instances of torsional elasticity where the attachment site unwinds, the restoring forces are not constant with angular position, also indicating slippage. Mechanisms of mechanochemical transduction must account for linear force generation in the presence of angular "slippage."
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7787107
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Motility assays using myosin attached to surfaces through specific binding to monoclonal antibodies.
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We have analyzed the dependence of actin filament movement on the mode of myosin attachment to surfaces. Monoclonal antibodies that bind to three distinct sites were used to tether myosin to nitrocellulose-coated glass. One antibody reacts with an epitope on the regulatory light chain located at the head-rod junction. The other two react with sites in the rod domain, one in the S2 region near the S2-LMM hinge, and the other at the C terminus of the myosin rod. These monoclonal antibodies were used to provide increasing flexibility in the mode of attachment. Fast skeletal muscle myosin monomers were bound to the surfaces through the specific interaction with these monoclonal antibodies and the sliding movement of fluorescently labeled actin filaments analyzed by video microscopy. Each of these antibodies produced stable, myosin-coated surfaces that supported uniform movement of actin over the course of several hours. Attachment of myosin through the anti-S2 and anti-LMM monoclonal antibodies yielded a maximum velocity of 10 microns/s at 30 degrees C, whereas attachment through anti-LC2 produced a lower velocity of 4-5 microns/s. Each antibody showed a characteristic minimum myosin density below which sliding movement was no longer supported and an exponential dependence of actin filament velocity on myosin surface density below Vmax. Maximum sliding velocity was achieved over a range of myosin surface densities. Thus, the specific mode of attachment can influence the characteristic velocity of actin filament movement and the surface density needed to support movement. These data are being used to analyze the dynamics of sliding filament assays and evaluate estimates of the average number of motor molecules per unit length of actin required to support movement.
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7787102
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Solution structure of two molecular motor domains: nonclaret disjunctional and kinesin.
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The effects of selected ligands on the structure of the truncated heavy-chain chemomechanical motor domains of Drosophila ncd and human kinesin were compared using the technique of transient electric birefringence. The 366-amino acid C-terminal motor domain of Drosophila nonclaret disjunctional, ncd(335-700), and the 349-amino acid N-terminal motor domain of human kinesin, kinesin(349), were studied at 4 degrees C in neutral buffers with ionic strength of 100 mM to form complexes with either MgADP or MgADP.Vi. The rotational diffusion time adjusted to 20 degrees C and water, tau 20,W, for ncd(335-700).MgADP is 32.8 ns, and for ncd(335-700).MgADP.Vi is 34.8 ns, suggesting prolate ellipsoids with dimensions 9.40 x 3.77 nm and 9.73 x 3.70 nm, respectively. The specific Kerr constant, Ksp, of ncd is -1.65 x 10(-12) cm2V-2 for the MgADP complex and -1.15 x 10(-12) cm2V-2 for the MgADP.Vi complex. The large negative Ksp for a prolate protein suggests an unusual charge distribution with two long surfaces with opposite charge. The tau 20,W for kinesin(349).MgADP is longer than the corresponding ncd motor and shows a decrease with increased electric field. The kinesin(349).MgADP.Vi complex has a longer tau 20,W. The Ksp for kinesin(349) is 0.36 x 10(-12) cm2V-2 for each complex.
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7787101
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The working stroke of myosin crossbridges.
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Recent x-ray diffraction measurements of the axial periodicities of the actin and myosin filaments in contracting muscles show that they are stretched by small but significant amounts by the developed tension, so that at least one half, possibly more, of the observed compliance of a sarcomere must reside in the filaments themselves. This implies that the movement steps of a crossbridge deduced from quick-release experiments may be shorter than some previous estimates, necessitating at least two steps to account for current in vitro single-molecule measurements. Intensity measurements of the wider angle x-ray reflections also show some unexpected features.
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7787100
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Actin's view of actomyosin interface.
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Actomyosin interactions were examined by using yeast actin mutants with alanines replacing charged amino acid pairs D24/D25, E99/E100, D80/D81, and E83/K84. In the in vitro motility experiments, actin filaments of D24A/D25A or E99A/E100A mutants moved in the presence of 0.7% methylcellulose at the velocities of wild-type actin. Without methylcellulose, these mutant filaments, but not the D80/D81 or E83/K84 filaments, dissociated from the assay surface upon addition of ATP. Measurements of myosin subfragment-1 (S1) binding to D24A/D25A- and E99A/E100A-polymerized actins in the presence of ATP revealed a three- and twofold decrease in their binding constant, respectively, compared with wild-type actin. In contrast to this, all monomeric actins had the same binding affinity for S1. The rates and extents of polymerization of D24A/D25A and E99A/E100A actins by S1 were reduced in comparison to wild-type actin. The local structure of subdomain-2 on actin, as probed by subtilisin cleavage, was not altered for either mutant. A twofold decrease in nucleotide exchange was detected for the D24A/D25A mutant actin. These results demonstrate the involvement of the D24/D25 and E99/E100 residues in the weak binding of myosin to actin and reveal that residues D80/D81 and E83/K84 do not modulate actomyosin interactions.
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7787099
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Is myosin a "back door" enzyme?
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ATP has been modeled into the active site of chicken skeletal myosin subfragment-1 using the adenylate kinase.Ap5A structure as a starting reference. The resulting docked ATP.S1 structure is justified in that it rationalizes the photolabeling data from several ATP analogs. The gamma-phosphate of ATP sits at the bottom of the active site pocket and is partially visible via a view along the prominent 50-kDa cleft of S1 but not when viewed from above the active site. It is postulated that actin binding promotes the movement of the P-loop and Arg-245 to allow Pi from ATP to leave via a "back-door" in the 50-kDa fragment while ADP is still bound at the active site. Such a mechanism can explain a number of experimental observations, including the kinetics of ATP hydrolysis, the nucleotide dependence of Pi exchange into ATP, and the formation of stable myosin.ADP.vanadate complexes in muscle fibers.
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7787098
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A single myosin head can be cross-linked to the N termini of two adjacent actin monomers.
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Myosin subfragment-1 (S1) can be cross-linked to two actin monomers by 1-ethyl-3-[3-(dimethylamino)-propyl]-carbodiimide only when F-actin is in excess over S1. Electron micrographs of the covalent actin2-S1 complex showed that S1 was cross-linked to two adjacent monomers of the same actin filament. Cross-linking experiments with pre-proteolyzed S1 derivatives in combination with a proteolytic dissection of the intact covalent actin2-S1 adduct (m = 265 kDa), revealed that two N-terminal segments of actin (residues 1-28) were covalently attached to a single S1 molecule. One was cross-linked to either the 20-kDa or the 50-kDa heavy chain fragments of S1, and the other only to the 50-kDa region. The doubly cross-linked product was formed under physiological ionic strength with S1 or with reconstituted myosin filaments, regardless of the presence of ADP or the regulatory proteins, tropomyosin and troponin. Finally, we found that this cross-linking could also take place within myofibrils in the rigor state. These results demonstrate that under nonsaturating conditions, the actin-S1 interface encompasses a much larger region than that recently proposed for the nonphysiological, fully saturated actin filaments.
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7787095
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Single-molecule mechanics of heavy meromyosin and S1 interacting with rabbit or Drosophila actins using optical tweezers.
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Single-molecule mechanical interactions between rabbit heavy meromyosin (HMM) or subfragment 1 (S1) and rabbit actin were measured with an optical tweezers piconewton, nanometer transducer. Similar intermittent interactions were observed with HMM and S1. The mean magnitude of the single interaction isotonic displacements was 20 nm for HMM and 15 nm with S1. The mean value of the force of single-molecule interactions was 1.8 pN for HMM and 1.7 pN with S1. The stiffness of myosin S1 was determined by applying a sinusoidal length change to the thin filament and measuring the corresponding force; the mean stiffness was 0.13 pN nm-1. By moving an actin filament over a long distance past an isolated S1 head, we found that cross-bridge attachment occurred preferentially at a periodicity of about 40 nm, similar to that of the actin helical repeat. Rate constants for the probability of detachment of HMM from actin were determined from histograms of the lifetime of the attached state. This gave a value of 8 s-1 or 0.8 x 10(6) M-1 s-1 for binding of ATP to the rigor complex. We conclude (1) that our HMM-actin interactions involve just one head, (2) that compliance of the cross-bridge is not in myosin subfragment 2, although we cannot say to what extent contributions arise from myosin S1 or actin, and (3) that the elemental movement can be caused by a change of shape of the S1 head, but that this would have to be much greater than the movements suggested from structural studies of S1 (Rayment et al., 1993).
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7787096
|
Are actin filaments moving under unloaded conditions in the in vitro motility assay?
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With sliding actin-filament motility assays, filament velocity should be independent of changes in the level of actomyosin activation under unloaded conditions. Using a simple modification of the motility assay to measure relative changes in isometric force (activation), we determined that isometric force increased 200-fold with thiophosphorylation of the myosin regulatory light chain, and that with thiophosphorylated myosin, isometric force was further increased by the addition of saturating smooth-muscle tropomyosin (100%) or tropomyosin plus calponin (500%), and decreased with the addition of saturating caldesmon (-100%). Under "reducing conditions," filament velocity (2.0 microns/s) was constant for mixtures of dephosphorylated and thiophosphorylated myosin containing > 5% thiophosphorylated myosin, and was unaffected by the addition of saturating concentrations of tropomyosin or caldesmon. In contrast, "standard assay conditions" were found to be nonreducing. With fully thiophosphorylated smooth-muscle myosin, saturating smooth-muscle tropomyosin increased velocity to 150% of control, and caldesmon halted all filament motion; with fully dephosphorylated myosin (< 0.002 mol/mol) filaments only moved when tropomyosin or tropomyosin plus calponin was added. Taken together, these observations suggest that under "standard conditions" a mechanical load is present that is eliminated by "reducing conditions." Filament velocity was insensitive to changes in cross-bridge density, under all conditions, suggesting that noncycling cross-bridges, generated by photochemical oxidation of myosin, is a likely source of mechanical loading.
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7787094
|
Characterization of single actin-myosin interactions.
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The feedback-enhanced laser trap assay (Finer et al., 1994) allows the measurement of force and displacement produced by single myosin molecules interacting with an actin filament suspended in solution by two laser traps. The average displacement of 11 nm at low load and the average force of 4 pN near isometric conditions are consistent with the conventional swinging cross-bridge model of muscle contraction (Huxley, 1969). The durations of single actin-myosin interactions at low load, 3-7 ms, suggest a relatively small duty ratio. Event durations can be increased either by reducing the ATP concentration until ATP binding is rate-limiting or by lowering the temperature. For sufficiently long interactions near isometric conditions, low frequency force fluctuations were observed within the time frame of a single event. Single myosin events can be measured at ionic strengths that disrupt weak binding actomyosin interactions, supporting the postulate of distinct weak and strong binding states. Myosin-generated force and displacement were measured simultaneously against several different loads to generate a force-displacement curve. The linear appearance of this curve suggests that the myosin powerstroke is driven by the release of a strained linear elastic element with a stiffness of approximately 0.4 pN nm-1.
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7787093
|
Conformational changes of the myosin heads during hydrolysis of ATP as analyzed by x-ray solution scattering.
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We have shown for the first time that the myosin head (subfragment-1, S1), the energy-transducing component in the actomyosin motor system undergoes a distinct shape change during hydrolysis of ATP using x-ray solution scattering techniques. Among various analogs for intermediate states of the S1 ATPase cycle, the complexes with MgADP and vanadate (S1.ADP.Vi), MgADP and beryllium fluoride (S1.ADP.BeF3), or MgADP and aluminum fluoride (S1.ADP.AIF4) showed a shape change similar to that in the presence of MgATP, but the complexes with ATP gamma S (S1.ADP gamma S) and MgADP trapped by cross-linking with pPDM (S1.ADP-pPDM) seemed to have a shape similar to that of nucleotide-free S1. These results indicate that the shape of an S1**.ADP.Pi state is more rounded or bent than in other intermediate states of the S1 ATPase cycle. Such changes occur in light chain 2-deficient S1 and also in smooth muscle S1. However, MgADP-fluoride complexes with smooth muscle S1 (without phosphorylation of a regulatory light chain) seemed to have a structure similar to that of nucleotide-free S1. Analysis of x-ray scattering data indicated that a conformational change of S1 in the presence of MgATP might be caused by a hinge-like bending movement between the catalytic and regulatory domains. The global change of S1 is correlated with some specific changes of a nucleotide-binding moiety.
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