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9450985

Free fatty acids (FFA), a link between obesity and insulin resistance.

Evidence, gained from human studies, is reviewed showing that elevation of plasma FFA levels produce peripheral and probably also hepatic insulin resistance in obese healthy and diabetic subjects. First, plasma FFA levels are elevated in most obese subjects. Second, physiological elevations of plasma FFA inhibit acutely as well as chronically insulin stimulated glucose uptake in a dose dependent fashion. Responsible for this inhibition is a FFA induced defect in insulin stimulated glucose transport and/or phosphorylation which develops after 3-4 hours of raising plasma FFA and a second defect, consisting of inhibition of glycogen synthase, the rate limiting enzyme of glycogen synthesis, which develops after 4-6 hours. FFA induced inhibition of fatty acid oxidation (Randle effect) does not affect insulin stimulated glucose uptake or glycogen synthesis and thus does not cause insulin resistance. Elevated plasma FFA levels also modestly increase insulin suppressed endogenous glucose production (EGP) although this effect has not been found by all investigators. The reasons why it has been difficult to demonstrate unequivocal effects of FFA on EGP include 1) the fact that FFA promote insulin secretion which counteracts its effect on EGP (FFA increase, while insulin decreases EGP); 2) the recognition that FFA induced increase in gluconeogenesis may pensated by intrahepatic downregulation of EGP (i.e., by a decrease in glycogenolysis). The FFA induced insulin resistance is physiologically important during starvation by preserving carbohydrate for oxidation in the central nervous system and during pregnancy, where the well recognized accelerated starvation pattern provides carbohydrate for the growing fetus. In obesity, however, there is no need to spare carbohydrate and the FFA induced insulin resistance may result in type 2 diabetes and other cardiovascular risk factors.

9450987

Effect of TGF-beta 1 on PDGF receptors expression in human scar fibroblasts.

This study examined the effect of exogenous TGF -beta1 on platelet derived growth factor alpha and beta (PDGF-alpha, beta) receptor expression in human dermal fibroblasts derived from both normal cutaneous tissues (normal skin [NSk]) and (normal scar [NSc]) and abnormal scar (keloid). TGF-beta and PDGF are present in the early phases of wound healing and are implicated in tissue fibrosis. In this study, replicate samples of NSk, NSc and keloid fibroblasts were grown to subconfluency in DMEM/10% FBS followed by replacement of media with DMEM/0.1%FBS for 24 hrs. One group of cells (NSk, NSc and keloid) were exposed to 10 ng/mL of exogenous TGF-beta1 for 24 hours, while the other group was used as control with no exposure to exogenous TGF-beta1. RadioImmunoBinding assays, Western and Northern blot analysis were performed to examine both PDGF-alpha and PDGF-beta receptor expression at the transcriptional and post-transcriptional levels. cDNA receptor probes were synthesized using polymerase chain reaction (PCR) with selected primer sets derived from published sequences. Beta-actin probe was used as a control to confirm that the same quantity of RNA was used for each experimental condition. TGF-beta1 was found to upregulate the expression of PDGF-alpha receptor for keloid fibroblasts but not for NSk or NSc fibroblasts. No effect was observed for TGF-beta 1 on PDGF-beta receptor expression for any of the cell lines examined.

9450986

Regulation of bile acid synthesis.

Bile acids are important physiological agents required for disposal of cholesterol and absorption of vitamins and fats. Bile acids are synthesized from cholesterol in the liver. Enterohepatic circulation of bile acids is very efficient and plays an important physiological role in lipid absorption and secretion, and regulation of bile acid biosynthesis and cholesterol homeostasis. Conversion of cholesterol to bile acids requires 15 different enzymatic steps. Four cytochrome P450 enzymes play important roles in bile acid biosynthesis. The classic bile acid biosynthesis pathway starts with modification of the sterol ring and followed by side chain cleavage reactions to synthesize cholic acid (CA) and chenodeoxycholic acid (CDCA), the primary bile acids in most species. The first and rate-limiting enzyme in this pathway is cholesterol 7alpha -hydroxylase, a microsomal cytochrome P450, CYP7A. Another microsomal cytochrome P450 sterol 12alpha-hydroxylase (CYP12) is required for the synthesis of cholic acid. Mitochondrial cytochrome P450 sterol 27-hydroxylase (CYP27) catalyzes sterol side chain oxidation to convert C27 sterol to C24 bile acids. An alternative bile acid biosynthesis pathway (acidic) has been known for sometime but only recently has attracted much attention. In this pathway, side chain oxidation precedes modification of the sterol ring. Mitochondrial sterol 27-hydroxylase (CYP27) catalyzes the first reaction and followed by 7alpha-hydroxylation catalyzed by a microsomal oxysterol 7alpha-hydroxylase (CYP7B). Recent advances in purification and cloning of these major enzymes in the pathways have led to better understanding the molecular basis of regulation of bile acid synthesis and physiological role of the alternative pathways.

9450988

Targeted inhibition of hepatitis B virus gene expression: a gene therapy approach.

In this study, we employ antisense RNA technology to block Hepatitis B Virus (HBV) gene expression in cell culture by gene transfer as an approach to block immune recognition and pathogenic sequelae. Retroviral vectors encoding antisense and sense copies of the HBV surface antigen gene (HBsAg) were constructed, respectively. To assay the inhibition of HBV gene expression by antisense RNA, the antisense retroviral construct was co-transfected with HBV expression vector (pTHBV) in hepatoma cell line, HepG2 cells. Expression of surface antigen was assessed by a standard HBsAg assay. The results indicated that HBsAg expression was reduced (40-50%) in antisense co-transfected cells pared to the control vector co-transfected cells. Furthermore, HepG2 was transduced with antisense retroviral vector and transfected with pTHBV. HBsAg expression was reduced 75% in the antisense retrovirus transduced HepG2 cells pared to control vector transduced cells. The retroviral vectors developed in this study can be used to identify the target antigen of cytotoxic T lymphocytes, which contribute to the immune mediated damage in chronic HBV patients. The retroviral mediated antisense gene bined with liver (or hepatocyte) transplant could also provide a molecular targeting approach for treating chronic hepatitis patients.

9450989

Distinct functions of calmodulin are required for the uptake step of receptor-mediated endocytosis in yeast: the type I myosin Myo5p is one of the calmodulin targets.

The uptake step of receptor-mediated endocytosis in yeast is dependent on the calcium binding protein calmodulin (Cmd1p). In order to understand the role that Cmd1p plays, a search was carried out for possible targets among the genes required for the internalization process. Co-immunoprecipitation, two-hybrid and overlay assays demonstrated that Cmd1p interacts with Myo5p, a type I unconventional myosin. Analysis of the endocytic phenotype and the Cmd1p-Myo5p interaction in thermosensitive cmd1 mutants indicated that the Cmd1p-Myo5p interaction is required for endocytosis in vivo. However, the Cmd1p-Myo5p interaction requirement was partially e by deleting the calmodulin binding sites (IQ motifs) from Myo5p, suggesting that these motifs inhibit Myo5p function. Additionally, genetic and biochemical evidence obtained with a collection of cmd1 mutant alleles strongly suggests that Cmd1p plays an additional role in the internalization step of receptor-mediated endocytosis in yeast.

9450990

Ca2+-independent insulin exocytosis induced by alpha-latrotoxin requires latrophilin, a G protein-coupled receptor.

alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.

9450991

Essential role of tubulin-folding cofactor D in microtubule assembly and its association with microtubules in fission yeast.

The main ponents of microtubules are alpha- and beta-tubulins. A group of proteins called cofactors are crucial in the formation of petent tubulin molecules in vitro. Whilst an in vitro role is emerging for these cofactors, their biological functions in vivo remain to be established. In order to understand the fundamental mechanisms that determine cell polarity, we have screened for fission yeast mutants with altered polarity. Here we show that alp1+ encodes a homologue of cofactor D and executes a function essential for cell viability. A temperature-sensitive alp1 mutant shows a variety of defects including abnormal mitoses, loss of microtubule structures, displacement of the nucleus, altered growth polarity and asymmetrical cell division. Overexpression of Alp1 is lethal in wild-type cells, resulting in altered cell shape, but is rescued by co-overexpression of beta-tubulin. Alp1 co-localizes with microtubules, both interphase arrays and mitotic spindles. Furthermore, Alp1 binds to and co-sediments with taxol (paclitaxel)-stabilized porcine microtubules. Our results suggest that, in addition to a function in the folding of beta-tubulin, cofactor D may play a vital role in microtubule-dependent processes as a microtubule-associated protein.

9450992

AIM-1: a mammalian midbody-associated protein required for cytokinesis.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

9450993

Peroxisomal beta-oxidation of polyunsaturated fatty acids in Saccharomyces cerevisiae: isocitrate dehydrogenase provides NADPH for reduction of double bonds at even positions.

The beta-oxidation of saturated fatty acids in Saccharomyces cerevisiae is confined exclusively to the partment of the cell. Processing of mono- and polyunsaturated fatty acids with the double bond at an even position requires, in addition to the basic beta-oxidation machinery, the contribution of the NADPH-dependent enzyme 2,4-dienoyl-CoA reductase. Here we show by biochemical cell fractionation studies that this enzyme is a typical constituent of peroxisomes. As a consequence, the beta-oxidation of mono- and polyunsaturated fatty acids with double bonds at even positions requires stoichiometric amounts of intraperoxisomal NADPH. We suggest that NADP-dependent isocitrate dehydrogenase isoenzymes function in an NADP redox shuttle across the peroxisomal membrane to keep intraperoxisomal NADP reduced. This is based on the finding of a third NADP-dependent isocitrate dehydrogenase isoenzyme, Idp3p, next to the already known mitochondrial and cytosolic isoenzymes, which turned out to be present in the peroxisomal matrix. Our proposal is strongly supported by the observation that peroxisomal Idp3p is essential for growth on the unsaturated fatty acids arachidonic, linoleic and petroselinic acid, which require 2, 4-dienoyl-CoA reductase activity. On the other hand, growth on oleate which does not require 2,4-dienoyl-CoA reductase, and NADPH pletely normal in Deltaidp3 cells.

9450994

Mode of interaction of the single a subunit with the multimeric c subunits during the translocation of the coupling ions by F1F0 ATPases.

We have recently isolated a mutant (aK220R, aV264E, aI278N) of the Na+-translocating Escherichia coli/Propionigenium modestum ATPase hybrid with a Na+-inhibited growth phenotype on succinate. ATP hydrolysis by the reconstituted mutant ATPase was inhibited by external (N side) NaCl but not by internal (P side) NaCl. In contrast, LiCl activated the ATPase from the N side and inhibited it from the P side. A similar pattern of activation and inhibition was observed with NaCl and the ATPase from the parent strain PEF42. We conclude from these results that the binding sites for the coupling ions on the c subunits are freely accessible from the N side. Upon occupation of these sites, the ATPase es more active, provided that the ions can be further translocated to the P side through a channel of the a subunit. If by mutation of the a subunit this channel es impermeable for Na+, N side Na+ ions specifically inhibit the ATPase activity. These conclusions were corroborated by the observation that proton transport into proteoliposomes containing the mutant ATPase was abolished by N side but not by P side Na+ ions. In contrast, LiCl affected proton translocation from either side, similar to the sidedness effect of Na+ ions on H+ transport by the parent hybrid ATPase. If the ATPase carrying the mutated a subunit was incubated with 22NaCl and ATP, 1 mol 22Na+/mol enzyme was occluded. With the parent hybrid ATPase, 22Na+ occlusion was not observed. The occluded 22Na+ could be removed from its tight binding site by 20 mM LiCl, while incubation with 20 mM NaCl was without effect. Li+ but not Na+ is therefore apparently able to pass through the mutated a subunit and make the entrapped Na+ ions accessible again to the aqueous environment. These results suggest an ion translocation mechanism through F0 that in the ATP hydrolysis mode involves binding of the coupling ions from the cytoplasm to the multiple c subunits, ATP-driven rotation to bring a Na+, Li+, or H+-loaded c subunit into a contact site with the a subunit and release of the coupling ions through the a subunit channel to the periplasmic surface of the membrane.

9450995

Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function.

Preprotein translocase catalyzes membrane protein integration as well plete translocation. Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer. We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence. Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA. Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states. Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics. In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling. Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself. Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane.

9450996

A dominant interfering mutant of FADD/MORT1 enhances deletion of autoreactive thymocytes and inhibits proliferation of mature T lymphocytes.

Members of the tumour necrosis factor receptor family that contain a death domain have pleiotropic activities. They induce apoptosis via interaction with intracellular FADD/MORT1 and trigger cell growth or differentiation via TRADD and TRAF molecules. The impact of FADD/MORT1-transduced signals on T lymphocyte development was investigated in transgenic mice expressing a dominant negative mutant protein, FADD-DN. Unexpectedly, FADD-DN enhanced negative selection of self-reactive thymic lymphocytes and inhibited T cell activation by increasing apoptosis. Thus signalling through FADD/MORT1 does not lead exclusively to cell death, but under certain circumstances can promote cell survival and proliferation.

9450997

A strain-independent postnatal neurodegeneration in mice lacking the EGF receptor.

Mice lacking the epidermal growth factor receptor (EGFR) exhibit strain-dependent phenotypes ranging from placental to postnatal skin, lung and brain defects. After birth, all mutant mice develop a progressive neurodegeneration in the frontal cortex, olfactory bulb and thalamus, characterized by massive apoptosis and upregulation of c-fos. These defects occur in a strain-independent manner, since neither rescue of the placental phenotype by aggregation of diploid 129/Sv EGFR mutant and tetraploid wild-type embryos, nor promotion of lung maturation by transplacental dexamethasone administration alters the course of neurodegeneration. VEGF is not induced during the degenerative process, excluding hypoxia and ischemia as causes of cell death. A migratory disorder is detected in the hippocampus with nests of ectopic neurons, which are also apoptotic. Cerebral cortices from EGFR mutants contain lower numbers of GFAP positive astrocytes, which display reduced proliferation in vitro. Since EGFR is expressed in the affected cell-types, these results define a specific function for EGFR in the proliferation and/or differentiation of astrocytes and in the survival of postmitotic neurons.

9450999

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase.

p85/p110 phosphoinositide 3-kinase (PI3K) is a posed of a p85-regulatory and a p110-catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65-PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85alpha-protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

9451000

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are ponents of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.

9450998

Regulation of Raf-1 kinase by TNF via its second messenger ceramide and cross-talk with mitogenic signalling.

Raf-1 kinase is a central regulator of mitogenic signal pathways, whereas its general role in signal transduction of tumour necrosis factor (TNF) is less well defined. We have investigated mechanisms of Raf-1 regulation by TNF and its messenger ceramide in cell-free assays, insect and mammalian cell lines. In vitro, ceramide specifically bound to the purified catalytic domain and enhanced association with activated Ras proteins, but did not affect the kinase activity of Raf-1. Cell-permeable ceramides induced a marked increase of plexes in cells co-expressing Raf-1 and activated Ras. Likewise, a fast elevation of the endogeneous ceramide level, induced by TNF treatment of human Kym-1 a cells, was followed by stimulation of Ras-Raf-1 association without significant Raf-1 kinase activation. Failure of TNF or ceramide to induce Raf-1 kinase was observed in several TNF-responsive cell lines. Both TNF and exogeneous C6-ceramide interfered with the mitogenic activation of Raf-1 and ERK by epidermal growth factor and down-regulated v-Src-induced Raf-1 kinase activity. TNF also induced the translocation of Raf-1 from the cytosolic to the particulate fraction, indicating that this negative regulatory cross-talk occurs at the cell membrane. Interference with mitogenic signals at the level of Raf-1 could be an important initial step in TNF's cytostatic action.

9451001

Different positioning of the ligand-binding domain helix 12 and the F domain of the estrogen receptor accounts for functional differences between agonists and antagonists.

The estrogen receptor is capable of binding a diverse set of ligands that are broadly categorized as agonists or antagonists, depending on their abilities to induce or interfere with transcriptional responsiveness. We show, using a fusion protein assay for ligand-binding which does not rely on transcriptional responsiveness, that agonists and antagonists differently position the C-terminus of the ligand-binding domain (helix 12) and the F domain. Upon antagonist binding, the F domain interferes with the fusion protein activity. Mutational disruption of helix 12 alters the position of the F domain, imposing interference after agonist or antagonist binding. Genetically selected inversion mutations where only agonists, but not antagonists, induce interference are similarly reliant on helix 12 and F domain positioning. Our results demonstrate that agonists and antagonists differently position helix 12 and implicate the F domain in mechanisms of antagonist action.

9451002

Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation.

The switch from transcriptionally activating MYC-MAX to transcriptionally repressing MAD1-MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1-MAX dimers during differentiation, we disrupted the Mad1 gene by homologous bination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals revealed that cell cycle exit of granulocytic precursors was inhibited following the colony-forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster-forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes were essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes was correlated with pensatory decrease in granulocytic cluster-forming cell survival under apoptosis-inducing conditions. In addition, recovery of the peripheral partment following bone marrow ablation was significantly enhanced in Mad1 knockout mice. Two Mad1-related genes, Mxi1 and Mad3, were found to be expressed ectopically in adult spleen, indicating that functional redundancy and cross-regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation.

9451003

CRP interacts with promoter-bound sigma54 RNA polymerase and blocks transcriptional activation of the dctA promoter.

The cAMP receptor protein (CRP) is an activator of sigma70-dependent transcription. Analysis of the sigma54-dependent dctA promoter reveals a novel negative regulatory function for CRP. CRP can bind to two distant sites of the dctA promoter, sites which overlap the upstream activator sequences for the DctD activator. CRP interacts with Esigma54 bound at the dctA promoter via DNA loop formation. When the CRP-binding sites are deleted, CRP still interacts in a cAMP-dependent manner with the stable Esigma54 plex via protein-protein contacts. CRP is able to repress activation of the dctA promoter, even in the absence of specific CRP-binding sites. CRP affects both the final level and the kinetics of activation. The establishment of the repression and its release by the NtrC activator proceed via slow processes. The kinetics suggest that CRP favours a new form of plex which interconverts slowly with the classical closed intermediate. Only the latter is capable of interacting with an activator to form an open plex. Thus, Esigma54 promoters are responsive to CRP, a protein unrelated to sigma54 activators, and the repression exerted is the direct result of an interaction between Esigma54 and the plex.

9451004

Sequence and structural elements of methylation guide snoRNAs essential for site-specific ribose methylation of pre-rRNA.

Site-specific 2'-O-ribose methylation of eukaryotic rRNAs is guided by small nucleolar RNAs (snoRNAs). The methylation guide snoRNAs carry long plementaries to rRNAs. These antisense elements are located either in the 5' half or in the 3' end region of the snoRNA, and are followed by the conserved D' or D box motifs, respectively. An uninterrupted helix formed between the rRNA and the antisense element of the snoRNA, in conjunction with the adjacent D' or D box, constitute the recognition signal for the putative methyltransferase. Here, we have identified an additional essential box mon to methylation guide snoRNAs, termed the C' box. We show that the C' box functions in concert with the D' box and plays a crucial role in the methyltransfer reaction directed by the upstream antisense element and the D' box. We also show that an internal fragment of U24 methylation guide snoRNA, passing the upstream antisense element and the D' and C' box motifs, can support the site-specific methylation of rRNA. This strongly suggests that the C box of methylation guide snoRNAs plays an essential role in the methyltransfer reaction guided by the 3'-terminal antisense element and the D box of the snoRNA.

9451005

Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the ribosome.

Peptidyl-tRNA dissociation from ribosomes is an energetically costly but apparently inevitable process that panies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the ribosome in a manner related to its proposed function in removing deacylated tRNA during ribosome recycling.

9451006

HMG box proteins bind to four-way DNA junctions in their open conformation.

The HMG box is an 80 amino acid domain found in a variety of eukaryotic chromosomal proteins and transcription factors. Binding to DNA is associated with recognition of structural distortion or manipulation of DNA structure. All the HMG box domains bind to four-way DNA junctions, which must therefore present some feature that mon to the binding targets of this wide variety of proteins. Since the four-way junction can itself adopt a variety of structures depending upon conditions, it is important to determine in which form it exists plexes with HMG boxes. We find that a single HMG box domain is bound exclusively to the open square form of the junction and that conditions that stabilize the stacked X structure significantly lower affinity for the HMG box. We suggest that the HMG domain binds to one arm of the junction in the minor groove at the point of strand exchange and we present a model for the structure of plex.

9451007

A helix-turn-helix structure unit in human centromere protein B (CENP-B).

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1. We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3.

9451008

Pankinetoplast DNA structure in a primitive bodonid flagellate, Cryptobia helicis.

The mitochondrial DNA (mtDNA) of a primitive kinetoplastid flagellate Cryptobia helicis posed of 4.2 kb minicircles and 43 kb maxicircles. 85% and 6% of the minicircles are in the form of supercoiled (SC) and relaxed (OC) monomers, respectively. The remaining minicircles (9%) constitute catenated posed of both the SC and OC molecules. Minicircles contain bent helix and sequences homologous to the minicircle conserved sequence blocks. Maxicircles encode typical mitochondrial genes and are not catenated. The mtDNA, which we describe with the term 'pankinetoplast DNA', is spread throughout the mitochondrial lumen, where it is associated with multiple electron-lucent loci. There are approximately 8400 minicircles per pankinetoplast-mitochondrion, with the pan-kDNA representing approximately 36% of the total cellular DNA. Based on the similarity of the C.helicis minicircles to plasmids, we present a theory on the formation of the kDNA network.

9451009

Phosphoglycosylation: a new structural class of glycosylation?

There are a number of different glycoproteins that have been identified relatively recently which contain oligosaccharides linked to serine or threonine in a peptide backbone via phosphodiesters. It is possible that these glycoproteins may form an alternative structural class of glycosylation. This modification has been referred to as phosphoglycosylation (Mehta et al., 1996; J. Biol. Chem., 271, 10897-10903), and has been reported in slime molds and several unicellular parasites. In this review, examples of phosphoglycosylation from different biological sources are discussed. Those which are well characterized have been found to be highly variable with respect to the glycan moiety, while sharing mon features. An experimental approach detailing how to determine whether a protein is phosphoglycosylated is also presented.

9451010

Studies on the binding site of the galactose-specific agglutinin PA-IL from Pseudomonas aeruginosa.

The binding properties of Pseudomonas aeruginosa agglutinin-I (PA-IL) with glycoproteins (gps) and polysaccharides were studied by both the biotin/avidin-mediated microtiter plate lectin-binding assay and the inhibition of agglutinin-glycan interaction with sugar ligands. Among 36 glycans tested for binding, PA-IL reacted best with two glycoproteins containing Galalpha1-->4Gal determinants and a human blood group ABO precursor equivalent gp, but this lectin reacted weakly or not at all with A and H active gps or sialylated gps. Among the mammalian disaccharides tested by the inhibition assay, the human blood group Pkactive Galalpha1-->4Gal, was the best. It was 7.4-fold less active than melibiose (Galalpha1-->6Glc). PA-IL has a preference for the alpha-anomer in decreasing order as follows: Galalpha1-->6 >Galalpha1-->4 >Galalpha1-->3. Of the monosaccharides studied, the phenylbeta derivatives of Gal were much better inhibitors than the methylbeta derivative, while only an insignificant difference was found between the Galalpha anomer of methyl- and p -NO2-phenyl derivatives. From these results, it can be concluded that bining size of the agglutinin is as large as a disaccharide of the alpha-anomer of Gal at nonreducing end and plementary to Galalpha1-->6Glc. As for bining site of PA-IL toward the beta-anomer, the size is assumed to be less than that of Gal; carbon-6 in the pyranose form is essential, and hydrophobic interaction is important for binding.

9451011

Identification and analysis of a class 2 alpha-mannosidase from Aspergillus nidulans.

A Class 2 alpha-mannosidase gene was cloned and sequenced from the filamentous fungus Aspergillus nidulans. A portion of the gene was amplified using degenerate oligonucleotide primers which were designed based on similarity between the Saccharomyces cerevisiae vacuolar and rat ER/cytosolic Class 2 protein sequences. The PCR amplification product was used to isolate the full length gene, and DNA sequencing revealed a 3383 bp coding region containing three introns. The predicted 1049 amino acid reading frame contained six potential N-glycosylation sites and encoded a protein of 118 kDa. The protein sequence did not appear to encode a typical fungal signal sequence or membrane spanning domain. Although the cellular location of the A.nidulans mannosidase was not determined, experimental evidence suggested that it was located within a subcellular organelle. The Matchbox sequence similarity matrix indicated that the A.nidulans protein sequence was more highly similar to the rat ER/cytosolic (Rij = 0.33) and S.cerevisiae vacuolar alpha-mannosidases (Rij = 0.43) than the rat and yeast sequences were to each other (Rij = 0.29). These three enzymes were found to be distantly related to other Class 2 sequences, pose a third subgroup of Class 2 alpha-mannosidases, as shown by ClustalW sequence alignment.

9451012

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection.

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 microm recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.

9451013

Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3.

A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin-3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta-strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, -2, and -3. Minimized docked models were generated for each mutant plex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of puted protein-carbohydrate interaction energies for each lectin-oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.

9451014

Purification of galectin-3 from ovine placenta: developmentally regulated expression and immunological relevance.

Galectins, beta-galactoside-binding lectins, are extensively distributed in the animal kingdom and share some basic molecular properties. Galectin-3, a member of this family, is generally associated with differentiation, morphogenesis, and metastasis. In this study, galectin-3 was isolated from ovine placental cotyledons round the middle of the gestation period by lactose extraction followed by affinity chromatography on lactosyl-agarose, and separated from galectin-1 by size exclusion chromatography on a Superose 12 column. Under native conditions this lectin behaved as a monomer with an apparent molecular weight of approximately 29,000 and an isoelectric point of 9.0. The partial amino acid sequence of the peptides obtained by tryptic digestion of this protein followed by HPLC separation showed striking homology with other members of the galectin-3 subfamily. Furthermore, ovine placental galectin-3 exhibited specific mitogenic activity toward rat spleen mononuclear cells. Besides, this protein strongly reacted with a rabbit antiserum raised against a chicken galectin. Results obtained by Western blot analysis showed that its expression was greatly decreased in term placenta with respect to the middle of the gestation period, suggesting a regulated expression throughout development.

9451015

The oligomerization of a family of four genetically clustered human gastrointestinal mucins.

Mucins are synthesized and secreted by many epithelia. They plex glycoproteins that offer cytoprotection. In their functional configuration, mucins form oligomers by a biosynthetic process that is poorly understood. A family of four human gastrointestinal mucin genes (MUC2, MUC5AC, MUC5B, and MUC6) is clustered to chromosome 11p15.5. To study oligomerization of these related mucins, we performed metabolic labeling experiments with [35S]amino acids in LS174T cells, and isolated mucin precursors by specific immunoprecipitations that were analyzed on SDS-PAGE. Each of the precursors of MUC2, MUC5AC, MUC5B, and MUC6 formed a single species of disulfide-linked homo-oligomer within 1 h after pulse labeling. Based on apparent molecular masses, these oligomeric precursors were most likely dimers. Inhibition of vesicular RER-to-Golgi transport, with brefeldin A and CCCP, did not affect the dimerization of MUC2 precursors, localizing dimerization to the RER. O-Glycosylation of MUC2 followed dimerization. Inhibition of N-glycosylation by tunicamycin retarded, but did not inhibit, dimerization, indicating that N-glycans play a role in efficient dimerization of MUC2 precursors. Based on sequence homology, the ability of MUC2, MUC5AC, MUC5B and MUC6 to dimerize most likely resides in their C-terminal domains. Thus, the RER-localized dimerization of secretory mucins likely proceeds by similar mechanisms, which is an essential step in the formation of the human gastrointestinal mucus-gels.

9451016

Cloning and functional expression of the human GlcNAc-1-P transferase, the enzyme for the committed step of the dolichol cycle, by heterologous complementation in Saccharomyces cerevisiae.

The gene for the human dolichol cycle GlcNAc-1-P transferase (ALG7/GPT) was cloned by screening a human lung fibroblast cDNA library. The library was constructed in a Saccharomyces cerevisiae expression vector, and the positive clone was identified plementation of the conditional lethal S.cerevisiae strain YPH-A7-GAL. This strain was constructed by replacing the endogenous promoter of the GPT-gene by the stringently regulated GAL1-promoter. This construct allows to specifically suppress the endogenous enzyme activity. The insert of the positive clone displayed an open reading frame of 1200 nucleotides, coding for a putative protein of 400 amino acids with a calculated molecular weight of 44.7 kDa. The deduced protein sequence shows a homology of over 90% pared with other mammalian GPT sequences, thus resembling the close phylogenetic relationship between mammalian species. This homology however decreases to 40-50% pared to more distantly related organisms such as S.cerevisiae , Schizosaccharomyces pombe , or Leishmania amazonensis . Biochemical characterization of the binant protein showed that it is functionally expressed in the S.cerevisiae strain YPH-A7-GAL. GlcNAc- and GlcNAc2-PP-Dolichol biosynthesis could be shown with isolated S.cerevisiae membranes from cells harboring the binant plasmid and grown on glucose thus suppressing transcription of the endogenous gene. Synthesis could be stimulated by dolicholphosphate and was inhibited by tunicamycin. These results show that we have cloned the human GlcNAc-1-P transferase by plementation in S. cerevisiae, a strategy that may be useful for the cloning and characterization of glycosyltransferases from a variety of organisms.

9451017

Conserved structural features in eukaryotic and prokaryotic fucosyltransferases.

Fucosyltransferases are the enzymes transferring fucose from GDP-Fuc to Gal in an alpha1,2-linkage and to GlcNAc in alpha1,3-, alpha1,4-, or alpha1,6-linkages. Since all fucosyltransferases utilize the same nucleotide sugar, their specificity will probably reside in the recognition of the acceptor and in the type of linkage formed. A search of nucleotide and protein databases yielded more than 30 sequences of fucosyltransferases originating from mammals, chicken, nematode, and bacteria. On the basis of protein sequence similarities, these enzymes can be classified into four distinct families: (1) the alpha-2-fucosyltransferases, (2) the alpha-3-fucosyltransferases, (3) the mammalian alpha-6-fucosyltransferases, and (4) the bacterial alpha-6-fucosyltransferases. Nevertheless, using the sensitive hydrophobic cluster analysis (HCA) method, conserved structural features as well as a consensus peptide motif have been clearly identified in the catalytic domains of all alpha-2 and alpha-6-fucosyltranferases, from prokaryotic and eukaryotic origin, that allowed the grouping of these enzymes into one superfamily. In addition, a few amino acids were found strictly conserved in this family, and two of these residues have been reported to be essential for enzyme activity for a human alpha-2-fucosyltransferase. The alpha-3-fucosyltransferases constitute a distinct family as they lack the consensus peptide, but some regions display similarities with the alpha-2 and alpha-6-fucosyltranferases. All these observations strongly suggest that the fucosyltransferases share mon structural and catalytic features.

9451018

Localization of LewisX, sialyl-LewisX and alpha-galactosyl epitopes on glycosphingolipids in lens tissues.

Mammalian lens contains several neutral and acidic glycosphingolipids, the core structures of which are ganglio-, neolacto-, globo-, and isoglobo-series sugar chains. Old World monkey lens shows positions similar to those of human cataractous lens, in particular the presence of Lewisxand sialyl-Lewisxepitopes and the absence of alpha-galactosyl epitope. Dog and pig lenses contain globotriaosylceramide and the sialyl-Lewisxcontaining neolactotetraosylceramide, respectively, which were found in primate lens, together with the alpha-galactosyl epitope containing neolactotetraosylceramide. Thin-layer chromatography immunostaining revealed the enrichment of some neolacto-series glycosphingolipids in the cortical and nuclear fibers, but not in lens epithelia, of dog, pig, and Japanese monkey lenses. Immunohistochemical studies confirmed the expression of Lewisx, sialyl-Lewisx, and alpha-galactosyl epitopes in the inner cortical and nuclear fibers, in association with the differentiation and maturation of lens epithelial cells to lens fibers. Glycobiological approaches thus suggested that some neolacto-series glycosphingolipids are involved in lens fiber development, in which the physiological roles of the alpha-galactosyl epitope are evolutionarily replaced by the Lewisxand sialyl-Lewisxepitopes in Old World monkeys and humans.

9451019

Glucosylated N-acetyllactosamine O-antigen chain in the lipopolysaccharide from Helicobacter pylori strain UA861.

The O-antigen chain from the lipopolysaccharide of Helicobacter pylori strain UA861 was determined to posed of an elongated type 2 N -acetyllactosamine backbone, -[-->3)-beta-D-Gal-(1-->4)-beta-D-GlcNAc-(1-]n-->, with approximately half of the GlcNAc units carrying a terminal alpha-d-Glc residue at the O -6 position. The O-chain of H.pylori UA861 was terminated by a N -acetyllactosamine [beta-D-Gal-(1-->4)-beta-D-GlcNAc] (LacNAc) epitope and did not express terminal Lewis X or Lewis Y blood-group determinants as previously found in other H.pylori strains. The absence of terminal Lewis X and Lewis Y blood-group epitopes and the replacement of Fuc by Glc as a side chain in the O-chain of H.pylori UA861 represents yet another type of lipopolysaccharide structure from H.pylori species. These structural differences in H.pylori lipopolysaccharide molecules carry implications with regard to possible different pathogenic events between strains and respective hosts.

9451020

Mycobacterial lipoarabinomannan: an extraordinary lipoheteroglycan with profound physiological effects.

Detailed structural and functional studies over the last decade have led to current recognition of the mycobacterial lipoarabinomannan (LAM) as a phosphatidylinositol anchored lipoglycan with diverse biological activities. Fatty acylation has been demonstrated to be essential for LAM to maintain its functional integrity although the focus has largely been on the arabinan motifs and the terminal capping function. It has recently been shown that the mannose caps may be involved not only in attenuating host immune response, but also in mediating the binding of mycobacteria to and subsequent entry into macrophages. This may further be linked to an intracellular trafficking pathway through which LAM is thought to be presented by CD1 to subsets of T-cells. The implication of LAM as major plex (MHC)-independent T-cell epitope and the ensuing immune response is an area of intensive studies. Another recent focus of research is the biosynthesis of arabinan which has been shown to be inhibitable by the anti-tuberculosis drug, ethambutol. The phenomenon of truncated LAM as synthesized by ethambutol resistant strains provides an invaluable handle for dissecting the array of arabinosyltransferases involved, as well as generating much needed structural variants for further structural and functional studies. It is hoped that with more systematic investigations based on clinical isolates and human cell lines, the true significance of LAM in the immunopathogenesis of tuberculosis and leprosy can eventually be explained.

9451022

Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4.

We have employed a direct radiolabel binding assay to investigate the interaction between3H-heparin and binant envelope glycoproteins, rgp120s, derived from several different isolates of HIV-1. Comparable dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN and SF-2. Under identical experimental conditions the binding of3H-heparin to a binant soluble form of the cellular receptor for gp120, CD4, is negligible. The binding of3H-heparin to rgp120 peted for by excess unlabeled heparin and certain other, but not all, glycosaminoglycan and chemically modified heparins. Of a range of such polysaccharides tested, ability pete with3H-heparin for binding was strictly correlated with inhibition of HIV-1 replication in vitro. Those possessing potent anti-HIV-1 activity were petitors, whereas those having no or little anti-HIV-1 activity were petitors. Scatchard analysis indicates that the K d of the interaction between heparin and rgp120 is 10 nM. Binding studies conducted in increasing salt concentrations confirm that the interaction is ionic in nature. Synthetic 33-35 amino acid peptides based on the sequence of the V3 loop of gp120 also bind to heparin with high affinity. V3 loop peptides that are cyclized due to terminal cysteine residues show more selective binding than their uncyclized counterparts. Overall, these data demonstrate further that heparin exerts its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1, rather than its cellular receptor, CD4. This study confirms that the V3 loop of gp120 is the site at which heparin exerts its anti-HIV-1 activity. Moreover, it reveals that high affinity binding to heparin is shared by all four rgp120s examined, despite amino acid substitutions within the V3 loop.

9451021

The role of site-specific N-glycosylation in secretion of soluble forms of rabies virus glycoprotein.

Rabies virus glycoprotein is important in the biology and pathogenesis of neurotropic rabies virus infection. This transmembrane glycoprotein is the only viral protein on the surface of virus particles, is the viral attachment protein that facilitates virus uptake by the infected cell, and is the target of the host humoral immune response to infection. The extracellular domain of this glycoprotein has N-glycosylation sequons at Asn37, Asn247, and Asn319. Appropriate glycosylation of these sequons is important in the expression of the glycoprotein. Soluble forms of rabies virus glycoprotein were constructed by insertion of a stop codon just external to the transmembrane domain. Using site-directed mutagenesis and expression in transfected eukaryotic cells, it was possible pare the effects of site-specific glycosylation on the cell-surface expression and secretion of transmembrane and soluble forms, respectively, of the same glycoprotein. These studies yielded the surprising finding that although any of the three sequons permitted cell surface expression of full-length rabies virus glycoprotein, only the N-glycan at Asn319 permitted secretion of soluble rabies virus glycoprotein. Despite its biological and medical importance, it has not yet been possible to determine the crystal structure of the full-length transmembrane form of rabies virus glycoprotein which contains heterogeneous oligosaccharides. The current studies demonstrate that a soluble form of rabies virus glycoprotein containing only one sequon at Asn319 is efficiently secreted in the presence of the N-glycan processing inhibitor 1-deoxymannojirimycin. Thus, it is possible to purify a conformationally relevant form of rabies virus glycoprotein that contains only one N-glycan with a substantial reduction in its microheterogeneity. This form of the glycoprotein may be particularly useful for future studies aimed at elucidating the three-dimensional structure of this important glycoprotein.

9451023

Lex glycosphingolipids-mediated cell aggregation.

Glycoconjugates bearing oligosaccharide Lex, Galbeta1-->4(Fucalpha1-->3)GlcNAcbeta1-->3R, are found on the surface of several cell types. Although recent studies have indicated that Lexon both glycosphingolipids (GSL) and polylactosaminoglycans can mediate under certain experimental conditions Lex-Lexinteractions, cell-cell interactions based exclusively on LexGSLs have not been demonstrated. In this study we show that preincubation of nonaggregating rat basophilic leukemia (RBL) cells with purified LexGSLs resulted in incorporation of the GSLs into plasma membrane, as determined by immunostaining, and formation of aggregates in the presence of Ca2+; no aggregates were formed after preincubation of the cells with globoside or sphingomyelin. Lex-mediated aggregation was inhibited by removal of Ca2+or by addition of lactofucopentaose III but not by lactose or lacto-N-fucopentaose II. In a mixture of Lex-positive and Lex-negative RBL cells most of the aggregates posed exclusively of Lex-positive cells. bined data suggest that interactions between LexGSL on opposite cell surfaces are strong enough to allow formation of stable cell-cell contacts.

9451024

New conformational constraints in isotopically (13C) enriched oligosaccharides.

Multidimensional heteronuclear NMR studies have been applied to the resonance assignment and conformational analysis of 13C-enriched Neu5Acalpha2-3Galbeta1-4Glc. It is demonstrated that three-dimensional ROESY-HSQC experiments provide through-space distance restraints which cannot be observed with conventional homonuclear 1H techniques due to resonance overlap. In particular, connectivities demonstrating the existence of the "anti" conformation about the Galbeta1-4Glc glycosidic linkage are unambiguously observed. It is shown that 13C isotopic enrichment of the trisaccharide at a level >95% enables straightforward measurement of trans-glycosidic 1H-13C and 13C-13C coupling constants and a Karplus-type relation is derived for the latter. In total 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theoretical puted from a 5 ns molecular dynamics simulation of the glycan.

9451025

Genetic tailoring of N-linked oligosaccharides: the role of glucose residues in glycoprotein processing of Saccharomyces cerevisiae in vivo.

In higher eukaryotes a quality control system monitoring the folding state of glycoproteins is located in the ER and posed of the proteins calnexin, calreticulin, glucosidase II, and UDP-glucose: glycoprotein glucosyltransferase. It is believed that the innermost glucose residue of the N- linked oligosaccharide of a glycoprotein serves as a tag in this control system and therefore performs an important function in the protein folding pathway. To address this function, we constructed Saccharomyces cerevisiae strains which contain nonglucosylated (G0), monoglucosylated (G1), or diglucosylated (G2) glycoproteins in the ER and used these strains to study the role of glucose residues in the ER processing of glycoproteins. These alterations of the oligosaccharide structure did not result in a growth phenotype, but the induction of the unfolded protein response upon treatment with DTT was much higher in G0 and G2 strains pared to wild-type and G1 strains. Our results provide in vivo evidence that the G1 oligosaccharide is an active oligosaccharide structure in the ER glycoprotein processing pathway of S.cerevisiae. Furthermore, by analyzing N- linked oligosaccharides of the constructed strains we can directly show that no general glycoprotein glucosyltransferase exists in S. cerevisiae.

9451026

Abnormal synthesis of mannose 1-phosphate derived carbohydrates in carbohydrate-deficient glycoprotein syndrome type I fibroblasts with phosphomannomutase deficiency.

In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] mannose into mannose phosphates, GDP-mannose, GDP-fucose, dolichol-P-mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol-P-mannose, and nascent glycoproteins. The incorporation of radioactivity into mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (</=20%), but their size was severely reduced, mostly containing five or fewer residues. As a consequence, truncated oligosaccharides were transferred to newly synthesized glycoproteins. The metabolic changes can be explained by a deficiency of phosphomannomutase activity, which was reduced to </=10% of control.

9451027

Concanavalin A distorts the beta-GlcNAc-(1-->2)-Man linkage of beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man upon binding.

Carbohydrate recognition by proteins is a key event in many biological processes. Concanavalin A is known to specifically recognize the pentasaccharide core (beta-GlcNAc-(1-->2)-alpha- Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man-(1-->6)]-Man) of N-linked oligosaccharides with a Ka of 1.41 x 10(6 )M-1. We have determined the structure of concanavalin A bound to beta-GlcNAc-(1-->2)-alpha-Man-(1-->3)-[beta-GlcNAc-(1-->2)-alpha-Man- (1-->6)]-Man to 2.7A. In six of eight subunits there is clear density for all five sugar residues and a well ordered binding site. The pentasaccharide adopts the same conformation in all eight subunits. The binding site is a continuous extended cleft on the surface of the protein. Van der Waals interactions and hydrogen bonds anchor the carbohydrate to the protein. Both GlcNAc residues contact the protein. The GlcNAc on the 1-->6 arm of the pentasaccharide makes particularly extensive contacts and including two hydrogen bonds. The binding site of the 1-->3 arm GlcNAc is much less extensive. Oligosaccharide recognition by Con A occurs through specific protein carbohydrate interactions and does not require recruitment of adventitious water molecules. The beta-GlcNAc-(1-->2)-Man glycosidic linkage PSI torsion angle on the 1-->6 arm is rotated by over 50 degrees from that observed in solution. This rotation is coupled to disruption of interactions at the monosaccharide site. We suggest destabilization of the monosaccharide site and the conformational strain reduces the free energy liberated by additional interactions at the 1-->6 arm GlcNAc site.

9451028

Changes in ganglioside composition of photoreceptors during postnatal maturation of the rat retina.

To examine at which stage the unusual position observed in adult retinal photoreceptor cells was established, and to see whether ganglioside changes could be correlated to distinct maturational events, quantitative and qualitative variations in gangliosides within pure sheets of photoreceptors during postnatal differentiation and aging of retina were studied. Retinas were separated into ponent layers, (particularly photoreceptor layers uncontaminated by other neuronal types) by exploiting a technique of mechanical separation by vibratome. We extracted lipids from the cell membranes and analyzed the position by high performance thin layer chromatography. The data show that from the earliest recordable postnatal age (6 days) until late in life (18 months), photoreceptors contain low quantities of lipid-bound N-acetyl neuraminic acid and a simplified ganglioside pared to inner retinal neurons. Specific ganglioside changes occur within photoreceptor cells during postnatal maturation and aging, with downregulation of a-pathway GM1 and overlapping upregulation of b-pathway GD1b taking place during the period corresponding to outer segment formation, correlating with the onset of retinal function.

9451029

Calf thymus high mobility group proteins are nonenzymatically glycated but not significantly glycosylated.

Over the past decade, there have been many reports suggesting the presence plex carbohydrates on nuclear and cytoplasmic proteins in mammalian cells. Some of the most often cited of these reports deal with the glycosylation of the high mobility group (HMG) proteins. These are relatively abundant chromosomal proteins that are known to be associated with nucleosomes and actively transcribed regions of chromatin. The original report describing HMG protein glycosylation presented several lines of evidence suggesting that these proteins are glycosylated, including positional analysis and periodic-acid Schiff staining. We have attempted to repeat these observations with more highly purified protein than was utilized in the original study. Using positional analysis performed by high pH anion exchange chromatography coupled to pulsed-amperometric detection, we saw no evidence for significant glycosylation of these proteins. In addition, we found no evidence for the presence of O-GlcNAc, a well known form of nuclear glycosylation. The HMG proteins did react with periodate, suggesting the presence of a modification containing cis-diols on the protein. Several tryptic peptides isolated from HMG 14 and 17 which retained the periodate reactivity had mon lysine residues, suggesting a potential modification of the straightepsilon-amino groups of lysines such as nonenzymatic glycation. Western blot analysis of the HMG proteins using anti-advanced glycation endproducts (AGE) antibodies confirmed the presence of glycation products on the HMG proteins.

9451030

Induction of sialic acid 9-O-acetylation by diverse gene products: implications for the expression cloning of sialic acid O-acetyltransferases.

Sialic acids can be modified by O-acetyl esters at the 7- and/or 9-position, altering recognition by antibodies, lectins and viruses. 9(7)-O-acetylation is mediated by a sialic acid-specific O-acetyltransferase, which has proven difficult to purify. Two groups have recently isolated cDNAs possibly encoding this enzyme, by expression cloning of human melanoma libraries in COS cells expressing the substrate ganglioside GD3. Pursuing a similar approach, we have isolated additional clones that can induce 9-O-acetylation. One clone present in a melanoma library encodes a fusion protein between a bacterial tetracycline resistance gene repressor and a sequence reported to be part of the P3 plasmid. Expression of the open reading frame is necessary for inducing 9-O-acetylation, indicating that this is not a reaction to the introduction of bacterial DNA. Another clone from a rat liver cDNA library induced 9-O-acetylation on COS cells expressing alpha2-6-linked sialic acids, and encodes an open reading frame identical to the Vitamin D binding protein. However, truncation at the 5' end eliminates the amino-terminal hydrophobic signal sequence, predicting cytosolic hyperexpression of a truncated protein. Thus, diverse types of cDNAs can indirectly induce sialic acid 9-O-acetylation in the COS cell system, raising the possibility that the real enzyme may posed of multiple subunits which would not be amenable to expression cloning. Importantly, the cDNAs we isolated are highly specific in their ability to induce 9-O-acetylation either on alpha2-6-linked sialic acids of glycoproteins (truncated vitamin D binding protein) or on the alpha2-8-linked sialic acids of gangliosides (Tetrfusion protein). These data confirm our prior suggestion that a family of O-acetyltransferases with distinctive substrate specificities exists in mammalian systems.

9451031

Differential binding of lectins IL-2 and CSL to candida albicans and cancer cells.

The demonstration that interleukin 2 (IL-2) is a lectin specific for oligomannosides allows to understand a new function for this cytokine: as a bifunctional molecule when bound to its receptor ss, IL-2 associates the latter which the plex, interacting with oligosaccharides of CD3 through its carbohydrate-recognition domain (Zanetta et al. , 1996, Biochem. J., 318, 49-53). This induces the tyrosine phosphorylation of the IL-2R beta by ++p56(lck) , the first step of the IL-2-dependent signaling. Since this specific association is disrupted in vitro by oligomannosides with five and six mannose residues, we made the hypothesis that pathogenic cells or microorganisms could bind IL-2, consequently disturbing the IL-2-dependent response. This study shows that the pathogenic yeast Candida albicans (in contrast with nonpathogenic yeasts) binds high amounts of IL-2 as did cancer cells. In contrast with cancer cells, yeasts do not bind the Man6GlcNAc2-specific lectin CSL, an endogenous "amplifier of activation signals" (Zanetta et al. , 1995, Biochem. J., 311, 629-636).

9451032

Biotinyl-l-3-(2-naphthyl)-alanine hydrazide derivatives of N-glycans: versatile solid-phase probes for carbohydrate-recognition studies.

Biotinyl-oligosaccharides are a relatively new generation of saccharide probes that enable immobilization of desired oligosaccharides on streptavidin matrices for studies of carbohydrate-protein interactions. Here we describe the facile preparation of biotinyl-l-3-(2-naphthyl)-alanine hydrazide (BNAH) derivatives of oligosaccharides, containing a strong UV absorbing and fluorescent group, in which the ring of the reducing-end monosaccharide is nonreduced. We evaluate reactivities of immobilized BNAH- N -glycans with plant lectins that recognize aspects of the oligosaccharide core or outer-arms. We make parisons with 2-amino-6-amidobiotinyl-pyridine (BAP) derivatives obtained by reductive amination, and 6-(biotinyl)-aminocaproyl-hydrazide (BACH) derivatives which have a longer spacer-arm. N -Glycan-BNAH and-BAP derivatives have, parable reactivities with lectins which recognize N -glycan outer-arms or the trimannosyl core, but only BNAH and BACH derivatives are bound by lectins which recognize the non-reduced core. Moreover, with Pisum sativum agglutinin (PSA) which additionally requires the fucosyl- N- glycan-asparaginyl core for high affinity binding, the immobilized BNAH derivative (which is an alanine hydrazide beta-glycoside) can substitute for the natural beta-glycosylasparaginyl core, whereas the BACH derivative (aminocaproyl-hydrazide-beta-glycoside) is less effective. BNAH is a derivatization reagent of choice, therefore, for solid phase carbohydrate-binding experiments with immobilized N -glycans.

9451033

Molecular and biological characterization of rabbit mannan-binding protein.

Mannan-binding protein (MBP) is a member of the collectin family of protein. There are two types of MBP, MBP-A and MBP-C, which were found in rodent (rats and mice), rhesus monkey, and cynomolgus monkey, while chimpanzee and human have only one MBP. It was considered that the loss of one MBP gene occurred during hominoid evolution. In this article two rabbit MBP, a liver and serum MBP, were characterized biologically and genetically. Analyses by SDS-PAGE under reduced condition and their amino acid sequences of both MBPs showed that they have a same molecular weight of 32 kDa and their amino acid sequences were identical. A serum MBP has a higher ability to plement than does a liver MBP; however, a liver MBP inhibits hemagglutination by influenza virus as strongly as a serum MBP does. cDNA clones encoding the rabbit MBP were isolated from a rabbit cDNA liver library using whole cDNA of mouse MBP-C as a probe. The cDNA carried an insert of 744 bp coding for a protein of 247 acid residues with a signal peptide of 22 residues. The deduced amino acid sequence of the cDNA was identical to that of amino acid sequences of the 32 kDa proteins determined here. Northern blot analysis showed that mRNA transcripts of about 0. 9 and 3.0 kb were expressed only in the liver. The analysis of the phylogenetic tree of rabbit and bovine MBPs and other collectins indicates that the loss of MBP gene occurred not only during hominoid evolution but also at some points after the separation of birds and mammals.

9451034

Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization.

Sialylation is a biosynthetic process occurring in the partments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to binant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the binant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.

9451035

Trafficking and localization studies of recombinant alpha1, 3-fucosyltransferase VI stably expressed in CHO cells.

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3-fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble binant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.

9451036

Human endometrial MUC1 carries keratan sulfate: characteristic glycoforms in the luminal epithelium at receptivity.

MUC1 is a high molecular mass, highly glycosylated epithelial apical glycoprotein that has been shown to exhibit both adhesive and anti-adhesive properties. Its expression in human glandular endometrial epithelium is transcriptionally regulated with the highest levels in the mid secretory phase, the "receptive" period during which implantation occurs. We demonstrate that endometrial MUC1 carries highly sulfated lactosaminoglycan chains recognized by monoclonal antibody (Mab) 5D4, and the sialokeratan sulfate epitope recognized by Mab D9B1. These glycans are hormonally regulated in endometrium, and show increased abundance in the secretory phase, but detailed evaluation of their distribution shows important differences. The 5D4 epitope is abundant at the luminal epithelial surface until the implantation phase, when it disappears, first from patches of cells, then altogether. D9B1 binding sites are retained in the luminal epithelium at receptivity. These data show that endometrial MUC1 carries sulfated lactosaminoglycans. They identify the luminal partment as a site of unique MUC1 glycosylation and independent regulation. Glycosylation and the negative charge associated with sialo- and sulfoglycans may be important in the regulation of embryo attachment.

9451037

Synthesis of neoglycoconjugates containing deaminated neuraminic acid (KDN) using rat liver alpha2,6-sialyltransferase.

2-Keto-3-deoxy-D- glycero -D- galacto -nononic acid (KDN) was introduced into asialotransferrin and N -acetyllactosamine (LacNAc) from CMP-KDN by using rat liver Galbeta1-->4GlcNAc alpha2, 6-sialyltransferase to form KDN-transferrin and KDN-LacNAc. These structures contain terminal KDNalpha2-->6Gal-residues, a glycotope that has not yet been described in natural glycoconjugates. KDN was transferred to all four Gal residues in asialotransferrin by this enzyme. The incorporation efficiency of KDN from CMP-KDN into asialotransferrin was about half that of Neu5Ac from CMP-Neu5Ac, based on the V max/ K m values for these donor substrates, 0.0527 min-1and 0.119 min-1, respectively. The KDNalpha2-->6Gal linkage was resistant to exosialidase treatment, in contrast to the sensitivity of the Neu5Acalpha2-->6Gal linkage. Interestingly, Sambucus sieboldiana agglutinin (SSA) was shown to prefer KDN-transferrin to the corresponding Neu5Ac-transferrin, as estimated by slot-blot analysis. The use of an alpha2,6-sialyltransferase to synthesize neoglycoproteins containing KDN has not been previously reported. Their facile synthesis using CMP-KDN and sialyltransferases with different specificities offers new possibilities to study the function of neo-KDN-glycoconjugates, and to explore their use in glycotechnology.

9451039

Efficiency of a high-titer retroviral vector for gene transfer into skeletal myoblasts.

Genetic transformation of skeletal myoblasts for myocardial repair is dependent on an efficient gene transfer system that integrates the genes of interest into the genome of the target cell and its progeny. The aim of this investigation was to evaluate the use of a new retrovirally based gene transfer system for this purpose.

9451038

Direct utilization of mannose for mammalian glycoprotein biosynthesis.

Direct utilization of mannose for glycoprotein biosynthesis has not been studied because cellular mannose is assumed to be derived entirely from glucose. However, animal sera contain sufficient mannose to force uptake through glucose-tolerant, mannose-specific transporters. Under physiological conditions this transport system provides 75% of the mannose for protein glycosylation in human hepatoma cells despite a 50- to 100-fold higher concentration of glucose. This suggests that direct use of mannose is more important than conversion from glucose. Consistent with this finding the liver is low in phosphomannose isomerase activity (fructose-6-P<->mannose-6-P), the key enzyme for supplying glucose-derived mannose to the N-glycosylation pathway. [2-3H] Mannose is rapidly absorbed from the intestine of anesthetized rats and cleared from the blood with a t1/2of 30 min. After a 30 min lag, label is incorporated into plasma glycoproteins, and into glycoproteins of all organs during the first hour. Most (87%) of the initial incorporation occurs in the liver, but this decreases as radiolabeled plasma glycoproteins increase. Radiolabel in glycoproteins also increases 2- to 6-fold in other organs between 1-8 h, especially in lung, skeletal muscle, and heart. These organs may take up hepatic-derived radiolabeled plasma glycoproteins. Significantly, the brain, which is not exposed to plasma glycoproteins, shows essentially no increase in radiolabel. These results suggest that mammals use mannose transporters to deliver mannose from blood to the liver and other organs for glycoprotein biosynthesis. Additionally, contrary to expectations, most of the mannose for glycoprotein biosynthesis in cultured hepatoma cells is derived from mannose, not glucose. Extracellular mannose may also make a significant contribution to glycoprotein biosynthesis in the intact organism.

9451040

Effect of volume reduction on lung transplant timing and selection for chronic obstructive pulmonary disease.

End-stage chronic obstructive pulmonary disease has traditionally been treated with lung transplantation. For 2 years, our lung transplantation program has placed patients with appropriate criteria for lung transplantation and volume reduction into a prospective management algorithm. These patients are offered the lung volume reduction option as a "bridge" to "extend" the eventual time to transplantation. We examine the results of this pilot program.

9451042

Aerosol cyclosporine prevents acute allograft rejection in experimental lung transplantation.

The incidence of acute rejection and the morbidity of systemic cyclosporine (INN: cyclosporine) after lung transplantation is significant. Experimental evidence suggests that the allograft locally modulates the immune mechanisms of acute rejection. The purpose of this study was to determine whether aerosolized cyclosporine would prevent acute cellular rejection, achieve effective graft concentrations with low systemic drug delivery, and locally affect production of the inflammatory cytokines involved in acute rejection.

9451041

Total respiratory support from swine lungs in primate recipients.

The use of nonhuman lung donors, such as swine, has the potential to provide an unlimited supply of organs. However, hyperacute rejection has prevented pulmonary xenotransplantation.

9451043

Ex vivo liposome-mediated gene transfer to lung isografts.

Gene therapy is a promising strategy to modify ischemia-reperfusion injury and rejection after transplantation. We evaluated variables that may affect ex vivo gene transfer to rat lung isografts.

9451044

Postoperative portable chest radiographs: optimum use in thoracic surgery.

Daily portable chest radiographs are routinely ordered in many institutions after thoracic surgery. Our purpose was to assess the efficacy and cost of this practice and to determine the optimum use of postoperative x-ray studies.

9451045

Massive hiatus hernia: evaluation and surgical management.

Paraesophageal hernias represent advanced degrees of sliding hiatus hernia with intrathoracic displacement of the intraesophageal junction. Gastroesophageal reflux disease occurs in most cases, resulting in acquired short esophagus, which should influence the type of repair selected.

9451046

Aggressive surgical management in localized pulmonary mycotic and nonmycotic infections for neutropenic patients with acute leukemia: report of eighteen cases.

To prevent hemoptysis and relapse during subsequent chemotherapy-induced neutropenia in patients with localized forms of invasive pulmonary aspergillosis, we adopted an aggressive surgical approach.

9451047

Thirty-day operative mortality for thoracotomy in lung cancer.

The 30-day operative mortality for thoracotomy in lung cancer is described herein.

9451049

Left ventricular dysfunction after open repair of simple congenital heart defects in infants and children: quantitation with the use of a conductance catheter immediately after bypass.

Quantification of myocardial injury after the simplest pediatric operations by load-independent indices of left ventricular function, using conductance and Mikro-Tip pressure catheters (Millar Instruments, Inc., Houston, Tex.) inserted through the left ventricular apex.

9451050

Reconstructive surgery in congenital mitral valve insufficiency (Carpentier's techniques): long-term results.

Previous publications have stressed the benefits of mitral valve repair over mitral valve replacement in children. However, munications have reported the long-term results and none with a follow-up of more than 10 years. This article reports our results in a series of 145 patients operated on for congenital mitral valve insufficiency by means of the same technique (Carpentier's technique) in a single center.

9451051

Measurement of cerebral blood flow during cardiopulmonary bypass with near-infrared spectroscopy.

A novel noninvasive method for repeatedly measuring cerebral blood flow during cardiopulmonary bypass by near-infrared spectroscopy is described. The reproducibility of the method is investigated and parison is made with an established technique.

9451052

Resting coronary flow and coronary flow reserve in human infants after repair or palliation of congenital heart defects as measured by positron emission tomography.

Coronary physiology in infants with congenital heart disease remains unclear. Our objective was to better understand coronary physiology in infants with congenital heart disease.

9451053

Evidence for rejection of homograft cardiac valves in infants.

Concern about the durability of small homograft cardiac valves has been expressed by surgeons, and evidence has been found that homograft valves evoke a recipient immune response. We reviewed our experience with homograft valves for evidence of rejection.

9451054

Pathology of explanted cryopreserved allograft heart valves: comparison with aortic valves from orthotopic heart transplants.

We sought to determine the morphology, mechanisms of deterioration, cellular viability, extracellular matrix integrity, and the role of immune responses in the dysfunction of cryopreserved aortic and pulmonic valve allografts.

9451056

Mortality and cerebral outcome in patients who underwent aortic arch operations using deep hypothermic circulatory arrest with retrograde cerebral perfusion: no relation of early death, stroke, and delirium to the duration of circulatory arrest.

Our goal was to investigate factors for mortality and cerebral e in patients with aneurysm of the aortic arch.

9451057

Effect of the Cox maze procedure on the secretion of atrial natriuretic peptide.

The Cox maze procedure has been confirmed to be effective in curing atrial fibrillation. Some authors have reported severe fluid retention after the Cox maze procedure and have suggested decreased secretion of atrial natriuretic peptide as a possible mechanism. This study was designed (1) to examine the serial changes in atrial natriuretic peptide after the Cox maze procedure pared with changes occurring after coronary artery bypass grafting and (2) to elucidate any differences in atrial natriuretic peptide levels between patients with transient recurrence of atrial fibrillation after the Cox maze procedure and those without recurrence of atrial fibrillation.

9451058

The "H" graft: an alternative approach for performing minimally invasive direct coronary artery bypass.

Minimally invasive direct coronary artery bypass permits arterial revascularization without cardiopulmonary bypass, potentially decreasing associated morbidity. The procedure is, however, technically challenging and associated with significant postoperative pain resulting from retraction through the small incision. METHODS AND PATIENT SELECTION: From December 1996 to April 1997, eight patients underwent grafting of the left anterior descending coronary artery by use of a short segment of right inferior epigastric artery attached proximally to the side of an in situ left internal thoracic artery. We have termed this procedure the "H" graft MIDCAB.

9451060

Proximal aortic perfusion for complex arch and descending aortic disease.

Cannulation of the femoral artery is used routinely for hypothermic circulatory arrest operations on the aortic arch. A two-stage approach is advocated bined arch and descending aortic disease. These methods are associated with important neurologic injury through embolism or malperfusion. We therefore changed to a central cannulation technique through extended left thoracotomy.

9451059

Saphenous vein graft protection: effects of c-myc antisense.

Saphenous vein grafting is associated with extensive medial remodeling, characterized by cellular proliferation, loss of smooth muscle cells, and an inflammatory response. In this study, we examined whether unfavorable responses to vein grafting could be modified by the intraoperative application of c-myc antisense oligomers.

9451062

Cardiopulmonary effects of 7.2% saline solution compared with gelatin infusion in the early postoperative period after coronary artery bypass grafting.

We report a clinical study on the use of 7.2%, 2400 mOsm/L, hypertonic saline pared with gelatin in early postoperative period after coronary artery bypass surgery.

9451061

Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart.

Vascular endothelial growth factor (VEGF), a potent angiogenic mediator, can be delivered to targeted tissues by means of a replication-deficient adenovirus (Ad) vector. We hypothesized that direct administration of Ad vector expressing the plementary deoxyribonucleic acid (AdGVVEGF121.10) into regions of ischemic myocardium would enhance collateral vessel formation and improve regional perfusion and function.

9451063

Downstream defects in beta-adrenergic signaling and relation to myocyte contractility after cardioplegic arrest.

Transient left ventricular dysfunction can occur after hypothermic, hyperkalemic cardioplegic arrest and is associated with decreased beta-adrenergic receptor responsiveness. Occupancy of the beta-adrenergic receptor activates adenylate cyclase, which phosphorylates the L-type Ca2+ channel-enhancing myocyte contractility. The goal of this study was to identify potential mechanisms that contribute to the defects in the beta-adrenergic receptor signaling cascade after cardioplegic arrest.

9451064

Relative induction of heat shock protein in coronary endothelial cells and cardiomyocytes: implications for myocardial protection.

Induction of the 70 kd heat shock protein in the heart is known to exert a protective effect against postischemic mechanical and endothelial dysfunction. However, the exact site of induction and the mechanisms involved remain unknown. The aim of this study was to investigate the relative capacity of endothelial and myocardial cells to express the 70 kd heat shock protein in response to heat stress, as well as their significance.

9451065

Preconditioning human cardiomyocytes and endothelial cells.

The effects of simulated "ischemia" and "reperfusion" were evaluated in cell cultures of human ventricular cardiomyocytes and human saphenous vein endothelial cells.

9451066

Operations on the thoracic aorta and hypothermic circulatory arrest: is aprotinin safe?

The safety of aprotinin, especially when used with profound hypothermic circulatory arrest, is still a matter of intense debate despite its presumed salutary effects on blood loss. Many investigators have reported toxic renal effects of high-dose aprotinin in such patients, but no prospective, randomized study has been conducted. To assess the potential detrimental effect of aprotinin on renal function and its putative reduction of blood loss, 50 patients undergoing thoracic aortic operations with the use of profound hypothermic circulatory arrest were randomly assigned to receive either low-dose aprotinin (1 x 10(6) kallikrein activation units) or placebo.

9451067

Optimal flow rates for integrated cardioplegia.

Antegrade cardioplegic delivery may be impaired by coronary occlusions, whereas retrograde delivery of cardioplegic solution may be inhomogeneous, leading to an accumulation of lactate and hydrogen ions, the products of anaerobic metabolism. Integrated cardioplegia using continuous retrograde cardioplegia and antegrade infusions pleted vein grafts washes out metabolites accumulated in regions inadequately perfused by retrograde cardioplegia alone. To determine the flow rates required to achieve the greatest washout, pared a high flow rate (200 ml/min) to a low flow rate (100 ml/min).

9451129

The role of exercise in diabetes management.

Monitor blood glucose level before, during and for up to 24 hours after exercise. Ensure refined carbohydrate snack is taken prior to exercise. Reduce insulin dosage if possible. Inject insulin away from any exercising muscle. Remember that glycogen stores are replenished in two phases: immediately after the exercise and two to three hours later. These are the key risk times for hypoglycaemia. If blood glucose control is poor (14 mmol/litre or higher) prior to exercise, the 'stress' effect of the exercise may cause further increases in the blood sugar level unless control is achieved.

9451141

Mould spores: the unusual suspects in hay fever.

Microfungi are microscopic plants that lack chlorophyll and depend on plant or animal material for nourishment. Moulds will only proliferate in a humid atmosphere. Moulds produce vast numbers of small spores, varying in diameter from 2 to 5 microns, which e airborne. mon moulds in the UK include Cladosporium, Alternaria and Aspergillus, of which several species are pathogenic to humans. The frequency of mould allergy is uncertain, but it appears to be higher in children than in adults. Wet weather favours mould growth, and sunny, windy weather favours spore release, while snow reduces both considerably. In warm, humid climates, fungi are present in large quantities all the year round. In temperate zones, spore counts are highest during the late summer. Indoor exposure largely depends upon the level of humidity. Mould growth can be immense in badly constructed houses and can contribute to sick-house syndrome. Occupational exposure can occur during manufacture of bread, cheese, beer and wine. In recent years, the use of moulds has been extended to include antibiotic, enzyme and steroid manufacture. Inhalation of small quantities of mould spores can evoke an IgE response and asthma. Massive exposure to some moulds growing in the airway can evoke an IgE and IgG response in people with bronchopulmonary aspergillosis. The inhalation of large amounts of mould antigen in organic dust can cause an IgG response and extrinsic allergic alveolitis

9451156

Caring for caregivers.

In home health nursing, the whole family is the client. In a recent issue of Home Care Provider, Knox and Thobaben discussed the need to identify the significant family members who influence health care resources and decisions. Whatever their relationship to the patient, these individuals frequently provide his or her primary care.

9451157

Home health reimbursement and the 1997 Budget Act.

The Balanced Budget Act of 1997, which was passed by Congress on July 28, was signed into law by President Clinton on August 5. The legislation contains a 5-year plan to balance the federal budget and reduces Medicare expenditures by $115 billion during that period. Home health cuts total $16.2 billion; hospice accounts for $200 million in additional reductions. In addition, the agreement contains significant cuts for home medical equipment (HME) and oxygen.

9451158

Assessment of the child: what's different?

As home health nurses, we see a lot of adult patients. Our skills may be pretty sharp when assessing these adults, but when a child is sick in the home, it is important to consider the differences in assessing this child and an adult.

9451159

Practical tips for incontinence.

Incontinence of urine and feces is monly encountered problem, particularly among bedridden elders. Although incontinence is often a transient problem precipitated by the onset of an unrelated medical condition or use of certain medications, incontinent patients are at high risk for developing both perineal dermatitis and pressure ulcers resulting from friction and shear injury.

9451160

Special home visitation program provides long-term benefits.

In recent years, home visitation services have been widely promoted as a means of preventing health and developmental problems in children from vulnerable families. The U.S. Advisory Board of Child Abuse and Neglect, for example, mended that home visitation services be made available to all parents of newborns to prevent child abuse and neglect.

9451161

Controlling the spread of antibiotic-resistant organisms.

Gram-negative bacilli (GNB), enterococci, and staphylococci mon members of the human microflora and are often the cause of plications in the elderly, the severely ill, and the promised patient. In addition, patients with invasive devices (e.g., trachs, feeding tubes, catheters), open wounds, and those whose normal microbial flora have been altered by antimicrobial therapy are also at risk for infection with these microorganisms. Urinary tract infection, bacteremia, and infected wounds are the principal anatomic sites involved.

9451162

Storing parenteral medication at home.

The storage requirements for parenteral products vary with the characteristics of the medication in the product. Temperature, light, moisture, position of the container, type of infusion fluid, and exposure to other chemical substances are the major determinants of the stability of parenteral medication stored in the patient's home.

9451171

Nutrition screening tool development and utilization for home care patients.

Caring for individuals with nutritional problems is a part of nursing practice. Nurses often struggle with how to categorize nutrition-related maladies. Signature Home Care (now a subsidiary of Integrated Health Services) modified an existing tool to assist nurses in their assessment of nutrition status. This article describes the process of adapting the Nutrition Screening Initiative and implementing the screening tool.

9451172

Who are these patients and what services do they receive?

The purpose of this study was to develop baseline information concerning older adults who use psychiatric home healthcare services, identify ponents of interventions provided over the course of treatment, and identify es associated with this type of home care. This review focused on patients 65 years or older with Medicare coverage and a primary or secondary diagnosis of depression. Retrospectively, 107 charts were reviewed, beginning with those of patients most recently discharged. These psychiatric home care patients plex healthcare problems, required family caregivers and multiple home healthcare services, and experienced variable es. These findings support the need for more information regarding specific interventions provided to patients and their family caregivers and what effect these interventions have on patient and caregiver es.

9451188

The complete blood count: physiologic basis and clinical usage.

plete blood count is one of the most frequently ordered laboratory tests in medicine. ponents are the red blood cell count, hemoglobin, hematocrit, red blood cell indices (including the mean cell volume, mean cell hemoglobin, mean cell hemoglobin concentration, and red cell distribution width), reticulocyte count, white blood cell count and differential, and platelet count. To aid in understanding the multiple uses of plete blood count, the article discusses the function, life cycle, and physiology of the various ponents. In addition, the characteristics of the various tests, including their strengths and limitations, are presented. This information can be helpful in difficult diagnoses and in monitoring treatments for hematologic disorders and other medical problems.

9451189

Umbilical cord blood transplants: treatment for selected hematologic and oncologic diseases.

Umbilical cord blood transplantation is a rapidly growing form of treatment for many types of cancer and hematologic disorders. The concepts behind the use of umbilical cord blood transplantation are based on information gained from experience in bone marrow transplantation. Previously discarded as human waste, the blood in the umbilical cord remnant and the placenta has been observed to be rich in hematopoietic stem cells. Techniques for collecting these stem cells from the placenta may vary among the institutions, physicians, and other health care providers, including midwives and nurse practitioners, involved with this procedure. This source of hematopoietic stem cells in transplantation has many advantages, disadvantages, and controversies associated with its use.

9451190

Disseminated intravascular coagulation in pregnancy: an update.

Disseminated intravascular coagulation is an imbalance between the coagulation and fibrinolytic systems. Hematologic changes that occur during pregnancy make this group of patients susceptible to triggers that upset the balance. Understanding normal hemostasis and the pathophysiology of disseminated intravascular coagulation, as well as conditions that are likely to precipitate this condition, may allow for early detection. Closely monitoring the patient's clinical parameters and laboratory tests can help guide supportive care as the triggers are identified and treated.

9451191

Systemic lupus erythematosus in pregnancy.

For the pregnant woman with systemic lupus erythematosus (SLE), there is a potential for profound effects on perinatal e. Because SLE is a multisystem disease, there are numerous effects on and nursing implications for the mother, the fetus, and the newborn that require individual case management. The article discusses SLE in pregnancy as well as appropriate clinical management and therapeutic intervention to assist the perinatal and neonatal nurse in caring for the mother, fetus, and neonate.

9451192

Recombinant human erythropoietin as a treatment for anemia of prematurity.

Anemia of prematurity (AOP) is an exaggerated form of the normal physiologic anemia of infancy. The primary pathophysiologic abnormality implicated in the development of AOP is inadequate production of erythropoietin, whose function is the regulation of red blood cell production. Recent studies into the safety and efficacy of binant human erythropoietin in infants with AOP have demonstrated consistently a rise in hematocrits and reticulocyte counts, fewer blood transfusions, reduced transfused volume of blood per kilogram body weight, and a decrease in bioavailable iron. Current dose mendations are 200 U/kg subcutaneously or intravenously daily or every other day.

9451193

Neonatal disseminated intravascular coagulation.

Bleeding in the neonate can be a life-threatening emergency. Quick action is necessary to determine the cause of bleeding, which determines how the infant will be treated. Disseminated intravascular coagulation (DIC) is uncontrolled, simultaneous bleeding and clotting occurring as a secondary disorder in sick neonates. Knowledge of plex physiologic mechanisms at work to maintain hemostasis contributes to the proper nursing care of infants at risk for DIC and better es.

9451194

Hyperbilirubinemia: new approaches to an old problem.

Hyperbilirubinemia continues to be mon problem of the term and near-term neonate. Each year more than 60% of infants born in the United States develop jaundice, making it difficult at times to differentiate jaundice due to pathologic reasons from jaundice due to physiologic ones. Now, the knowledge that bilirubin may even have a beneficial role as an antioxidant has caused a great deal of renewed interest in the management of jaundice. The American Academy of Pediatrics has published revised guidelines for management of neonatal jaundice that have resulted in much discussion. The article reviews current theories of bilirubin production and clearance, noninvasive and nonintrusive techniques to assess and care for jaundice, and newer concepts of preventive and treatment modalities from a nursing perspective.

9451196

Soft tissue problems associated with rheumatic disease.

Rheumatic disease and associated soft tissue problems pass a large number of syndromes and account for a high percentage of visits to primary care practitioners. This article describes the symptoms, causes, and treatments for five of the problems monly encountered: bursitis, tendinitis, carpal tunnel syndrome, myofascial pain syndrome, and fibromyalgia. Effective management requires a structured history, physical examination, and definitive diagnosis that distinguishes the soft tissue problem from a joint problem and an inflammatory syndrome from a noninflammatory syndrome. The overriding principle is self-management of treatments that focuses on relief of pain, maintenance of function, and avoidance of factors that cause recurrence or exacerbation of the problem. Medications, physical therapies, biomechanical aids, and exercise strategies, along with cognitive-behavioral techniques for the more chronic problems, are all known to decrease symptoms and to assist patients in returning to normal functioning.