Brief report: impaired processing of prohormones associated with abnormalities of glucose homeostasis and adrenal function.
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Adult↗
Biomedical subjects
C Carr
Publications and source records attributed to C Carr.
Notes from the school CNEP development and research.
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Humans↗
Second-trimester maternal serum screening using alpha-fetoprotein, human chorionic gonadotrophin, and unconjugated oestriol: experience of a regional programme.
Over a 2-year period from January 1991 to December 1992, second-trimester maternal serum screening for Down's syndrome using alpha-fetoprotein (alpha FP), human chorionic gonadotrophin (hCG), and unconjugated oestriol (uE3) was made available to five health districts in East Anglia, with a total population of 1.2 million. Amniocentesis was offered when the risk of Down's syndrome at term was 1:200 or greater. 25,359 singleton pregnancies were screened, representing an uptake of 77 per cent. The recall rate for the 24 per cent of women who had not had a dating scan prior to the test was 9.4 per cent compared with 3.9 per cent for those who had been scanned (P < 0.0005). Seventy-five per cent (36/48) of Down's syndrome pregnancies were detected for a false-positive rate of 4.0 per cent. Twenty-five out of 36 of detected Down's syndrome pregnancies were dated by scan prior to sampling, and in the 11 remaining cases, the dates were confirmed by scan after a high-risk result was obtained. The exclusion of uE3 from the screening protocol would have reduced the detection rate to 52 per cent (25/48) for the same false-positive rate. Eighty-five per cent of women identified at high risk accepted the offer of an amniocentesis. Other fetal abnormalities detected were trisomy 18 (3), trisomy 13 (2), 45,X (6), 69,XXX (5), other chromosome abnormalities (9), open neural tube defects (26), hydrocephalus (7), abdominal wall defects (4), and steroid sulphatase deficiency (6).
Adult↗
Recombinant E. coli-derived tissue factor pathway inhibitor reduces coagulopathic and lethal effects in the baboon gram-negative model of septic shock.
Excessive coagulation is a typical response to the vascular injury occurring in gram negative sepsis. This study evaluated the pharmacological effects of the use of a recombinant Escherichia coli derived form of tissue factor pathway inhibitor (ala-TFPI) in a baboon model of septic shock. Several doses of ala-TFPI were administered either 30 or 120 min after the initiation of a lethal intravenous infusion of E. coli into baboons. Treatment at 30 min with either 2.7 or 7.4 mg/kg of ala-TFPI resulted in the same survival rates and attenuation of both the coagulation response and cellular injury, as measured by clinical chemistry. When administration of ala-TFPI was delayed for 120 min, a dose of ala-TFPI protein continued to provide a benefit to survival. Ala-TFPI reduced the drop in mean systemic arterial pressure compared to control baboons in addition to partially attenuating the coagulopathic response. Baboons given ala-TFPI also maintained lower levels of plasma interleukin-6 (IL-6) and thrombin-antithrombin. These results suggest that the site of action of the protein may involve the later stage components of the coagulation and inflammatory pathways.
Animals↗
The Candida albicans myristoyl-CoA:protein N-myristoyltransferase gene. Isolation and expression in Saccharomyces cerevisiae and Escherichia coli.
Myristoyl-CoA:protein N-myristoyltransferase (NMT) has recently been identified as a target for antiviral and antifungal therapy. Candida albicans is a dimorphic, asexual yeast that is a major cause of systemic fungal infections in immunosuppressed humans. Metabolic labeling studies indicate that C. albicans synthesizes one principal 20-kDa N-myristoyl-protein. The single copy C. albicans NMT gene (ca-NMT1) was isolated and encodes a 451-amino acid protein that has 55% identity with Saccharomyces cerevisiae NMT. C. albicans NMT1 is able to complement the lethal phenotype of S. cerevisiae nmt1 null mutants by directing efficient acylation of the approximately 12 endogenous N-myristoylproteins produced by S. cerevisiae. C. albicans NMT was produced in Escherichia coli, a prokaryote with no endogenous NMT activity. In vitro studies of purified E. coli-derived S. cerevisiae and C. albicans NMTs revealed species-specific differences in the kinetic properties of synthetic octapeptide substrates derived from known N-myristoylproteins. Together these data indicate that C. albicans and S. cerevisiae NMTs have similar yet distinct substrate specificities which may be of therapeutic significance.
Acyltransferases↗
Agonist-dependent, cholera toxin-catalyzed ADP-ribosylation of pertussis toxin-sensitive G-proteins following transfection of the human alpha 2-C10 adrenergic receptor into rat 1 fibroblasts. Evidence for the direct interaction of a single receptor with two pertussis toxin-sensitive G-proteins, Gi2 and Gi3.
A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenosine Diphosphate Ribose↗
Disulfide assignments in recombinant mouse and human interleukin 4.
The disulfide pairings of mouse and human interleukin 4 (IL-4) proteins have been determined. The purified proteins, synthesized by recombinant DNA technology, are fully active as judged by their ability to stimulate an appropriate biological response in a variety of functional assays. Peptide maps were produced by digesting the proteins with pepsin and separating the resulting fragments by reverse-phase HPLC using linear acetonitrile-TFA gradients. Cystine-containing peptides were identified by determining which reverse-phase peaks showed an altered elution pattern after reduction. These peptides were purified further and defined by composition and sequence analysis. Three sets of disulfide-linked peptides were consistently identified for each protein. For mouse IL-4, the first and fifth, second and fourth, and third and sixth cysteines are joined. The disulfide bonds in human IL-4 are between the first and sixth, second and fourth, and third and fifth cysteines. A large double-loop region within the central three-fifths of each protein is stabilized by these bonds. Sequence analysis of the peptides containing the third and fifth cysteines of human IL-4 also demonstrated that only one of the potential N-glycosylation sites is used by C127 mammary tumor cells. Complete alkylation of mouse IL-4 under mild conditions completely destroyed its biological activity in a hematopoietic precursor cell proliferation assay.
Amino Acid Sequence↗
Chronic exposure of rat glioma C6 cells to cholera toxin induces loss of the alpha-subunit of the stimulatory guanine nucleotide-binding protein (Gs).
Rat glioma C6 BU1 cells were treated in tissue culture with cholera toxin. Incubation of membranes derived from these cells with fresh cholera toxin and [32P]NAD+ failed to promote incorporation of radioactivity into polypeptides corresponding to forms of Gs alpha. This is generally assumed to reflect prior ADP ribosylation of these polypeptides in vivo using endogenous NAD+ as substrate. However, immunological studies with anti-peptide antisera which identify all forms of Gs alpha demonstrated that concentrations of this polypeptide were now substantially reduced in the membranes. This effect was specific for Gs alpha as neither the alpha-subunits of the pertussis toxin-sensitive G-proteins Gi2 and Gi3, nor the beta subunit common to the various G-proteins were lost in parallel. Pertussis toxin-catalysed ADP ribosylation did not cause the downregulation of Gs alpha nor of the alpha-subunits of Gi2 or Gi3 although it did cause ADP ribosylation of the entire complement of both Gi2 and Gi3 in the membranes. Despite the reduction in levels of immunoreactive Gs alpha from the membranes of cholera toxin-treated cells, no alterations in levels of mRNA corresponding to this G-protein were noted.
Adenosine Diphosphate Ribose↗
Evolution of the glucose dehydrogenase gene in Drosophila.
The glucose dehydrogenase genes (Gld) of Drosophila melanogaster, of D. pseudoobscura, and of D. virilis have been isolated and compared with each other in order to identify conserved and divergent aspects of their structure and expression. The exon/intron structure of Gld is conserved. The Gld mRNAs are similar, with a range of 2.6-2.8 kb among the three species. All three species exhibit peaks of Gld expression during every major developmental stage, although considerable variation in the precise timing of these peaks exists between species. Interspecific gene transfer experiments demonstrate that the regulation and function of the D. pseudoobscura Gld is similar enough to the homologous gene in D. melanogaster to substitute for its essential role in the eclosion process. Comparison of the putative promoter sequences has identified both shared and divergent sequence elements which are likely responsible, respectively, for the conserved and divergent patterns of expression observed. The entire coding sequences of the pseudoobscura and melanogaster Gld genes are presented and shown to encode a 612-amino-acid pre-protein. The inferred amino acid sequences are 92% conserved between the two species. In general the intronic regions of Gld are unusually well conserved.
Amino Acid Sequence↗
Parathyrin assay. An analytical evaluation of two commercial Immuno Radiometric Assay kits.
We have performed a comparative evaluation of two Immuno Radiometric Assay (IRMA) kits for parathyrin against an existing Radioimmuno Assay (RIA) technique for the measurement of intact parathyrin. The analytical evaluation which was performed in line with the ECCLS recommended kit evaluation protocol showed a marginal improvement in precision with the new assays. There was a substantial improvement in the theoretical limit of detection utilising the IRMA kits although it may prove difficult to realize this improvement in practice with individual samples because of differing protein matrices. The evaluation also demonstrated the degree of parallelism and range of linearity of both kits as well as inaccuracies when compared with the International Reference Preparation for parathyrin (IRP 79/500) as the accepted standard. The demonstration of lack of agreement between measured and kit assigned results for standards when cross-over studies between kits were performed may highlight a possible contributing factor to inaccuracy. Alternatively there may be a difference of antisera avidity within the kits for intact parathyrin. Whilst minor differences in sample stability were demonstrated between the kits, the sample stability was much improved compared to that for intact parathyrin measurement by RIA. The correlation studies showed a degree of correlation consistent with other comparisons similarly performed.
Dose-Response Relationship, Drug↗
Myristic acid is the NH2-terminal blocking group of the 43-kDa protein of Torpedo nicotinic post-synaptic membranes.
The NH2-terminal blocking group of the 43-kDa peripheral membrane protein (43-kDa protein) of Torpedo post-synaptic membranes has been identified as myristic acid. To identify that blocking group pure 43-kDa protein was digested with trypsin and the blocked tryptic peptide was isolated by reverse phase HPLC. That peptide coeluted with and had the same amino acid composition as a synthetic peptide, myristoyl-Gly-Gln-Asp-Gln-Thr-Lys, the structure of the amino terminus predicted from the protein sequence deduced from a cDNA clone. The presence of myristate was confirmed by the precise molecular mass of the peptide, 886.5266, determined by fast atom bombardment mass spectroscopy.
Amino Acids↗
Recombinant human erythropoietin treatment may improve quality of life and cognitive function in chronic hemodialysis patients.
Medical, psychological, and social adaptation (quality of life) as well as cognitive function were studied in 15 chronic stable hemodialysis patients before the onset of treatment with recombinant human erythropoietin (r-HuEPO), 1 month after stabilization of normal hematocrit levels, and 10 to 15 months after treatment onset. After r-HuEPO treatment, subjects had significantly higher hematocrits, markedly improved energy levels, and marginally improved global health. r-HuEPO treatment was also associated with progressively decreased levels of subject mood disturbance and dialysis-related stresses. Subjects had no increased participation in paid employment and only minimally increased participation in social and leisure activities at posttreatment data points. There was no significant improvement in cognitive function after treatment. r-HuEPO treatment appears to be associated with higher energy levels, significant psychological benefits, and minimal improvements in social adaptation. The effects on cognitive function merit further study.
Adaptation, Psychological↗
A novel 87,000-Mr protein associated with acetylcholine receptors in Torpedo electric organ and vertebrate skeletal muscle.
To identify proteins associated with nicotinic postsynaptic membranes, mAbs have been prepared to proteins extracted by alkaline pH or lithium diiodosalicylate from acetylcholine receptor-rich (AChR) membranes of Torpedo electric organ. Antibodies were obtained that recognized two novel proteins of 87,000 Mr and a 210,000:220,000 doublet as well as previously described proteins of 43,000 Mr, 58,000 (51,000 in our gel system), 270,000, and 37,000 (calelectrin). The 87-kD protein copurified with acetylcholine receptors and with 43- and 51-kD proteins during equilibrium centrifugation on continuous sucrose gradients, whereas a large fraction of the 210/220-kD protein was separated from AChRs. The 87-kD protein remained associated with receptors and 43-kD protein during velocity sedimentation through shallow sucrose gradients, a procedure that separated a significant amount of 51-kD protein from AChRs. The 87- and 270-kD proteins were cleaved by Ca++-activated proteases present in crude preparations and also in highly purified postsynaptic membranes. With the exception of anti-37-kD antibodies, some of the monoclonals raised against Torpedo proteins also recognized determinants in frozen sections of chick and/or rat skeletal muscle fibers and in permeabilized chick myotubes grown in vitro. Anti-87-kD sites were concentrated at chick and rat endplates, but the antibodies also recognized determinants present at lower site density in the extrasynaptic membrane. Anti-210:220-kD labeled chick endplates, but studies of neuron-myotube cocultures showed that this antigen was located on neurites rather than the postsynaptic membrane. As reported in other species, 43-kD determinants were restricted to chick endplates and anti-51-kD and anti-270-kD labeled extrasynaptic as well as synaptic membranes. None of the cross reacting antibodies recognized determinants on intact (unpermeabilized) myotubes, so the antigens must be located on the cytoplasmic aspect of the surface membrane. The role that each intracellular determinant plays in AChR immobilization at developing and mature endplates remains to be investigated.
Animals↗
The implications for everyday life of incident self-reported visual decline among people over age 65 living in the community.
We examined the consequences of incident self-reported vision loss among a cohort of community-living people aged 66 years and older. Those with visual decline were older than those reporting constant excellent or good vision, but not different in any other demographic characteristic, the use of formal support services, the use of health services, or ADL (activities of daily living) functioning. Controlling for age and sex, vision loss was associated with unmet instrumental ADL needs such as housekeeping, grocery shopping, and food preparation, and with physical and emotional disabilities. We discuss the implications of these disabilities in terms of the challenges to the aging and blindness service delivery systems.
Activities of Daily Living↗
Human erythroleukemia cells express functional thromboxane A2/prostaglandin H2 receptors.
The human erythroleukemia (HEL) cell line is a cultured hematopoietic cell line reported to express platelet membrane glycoproteins and alpha-2 adrenergic receptors. The present studies were designed to determine if functional thromboxane A2 (TXA2)/prostaglandin H2 (PGH2) receptors exist in HEL cells. Radioligand binding assays were performed using [125I]PTA-OH, a TXA2/PGH2 receptor antagonist. Scatchard analysis revealed one class of binding sites for 1-PTA-OH with a Kd = 122 +/- 18 nM and maximum binding = 1.7 +/- 0.3 x 10(5) sites/cell. Competition for [125I]PTA-OH binding with the TXA2/PGH2 receptor agonists SQ26655 and U46619 revealed one class of binding sites for SQ26655 with a Kd = 17 nM and two classes of binding sites for for U46619 with a Kd = 45 nM for the high-affinity site and a Kd = 450 nM for the low-affinity site. Competition for [125I]PTA-OH by the steroisomeric TXA2/PGH2 receptor antagonists L657925 and L657926 revealed two classes of binding sites for the more potent L657925 with a Kd = 8 nM for the high-affinity site and a Kd = 400 nM for the low-affinity site whereas L657926 bound to one class of sites with a Kd = 380 nM. Stimulation of the TXA2/PGH2 receptor by SQ26655 and U46619 resulted in concentration-dependent increases in [Ca++], as measured by FURA-2 fluorescence, with EC50 values of 28 +/- 2 and 149 +/- 32 nM, respectively. I-PTA-OH, L657925 and L657926 antagonized this response to U46619 with IC50 values similar in rank potency to that seen in the binding studies.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium↗
A survey of physicians' attitudes and behavior toward PMS.
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Attitude↗
Acetylcholine receptor-associated 43K protein contains covalently bound myristate.
Torpedo electroplaque and vertebrate neuromuscular junctions contain high levels of a nonactin, 43,000-Mr peripheral membrane protein referred to as the 43K protein. 43K protein is associated with the cytoplasmic face of postsynaptic membranes at areas of high acetylcholine receptor density and has been implicated in the establishment and/or maintenance of these receptor clusters. Cloning of cDNAs encoding Torpedo 43K protein revealed that its amino terminus contains a consensus sequence sufficient for the covalent attachment of the rare fatty acid myristate. To examine whether 43K protein is, in fact, myristoylated, mouse muscle BC3H1 cells were metabolically labeled with either [35S]cysteine or [3H]myristate and immunoprecipitated with a monospecific antiserum raised against isolated Torpedo 43K protein. In cells incubated with either precursor, a single labeled species was specifically recovered that comigrated on SDS-PAGE with 43K protein purified from Torpedo electric organ. Approximately 95% of the 3H labeled material released from [3H]myristate-43K protein by acid methanolysis was extractable in organic solvents and eluted from a C18 reverse-phase HPLC column exclusively at the position of the methyl myristate internal standard. Thus, 43K protein contains authentic myristic acid rather than an amino or fatty acid metabolite of [3H]myristate. Myristate appears to be added to 43K protein cotranslationally and cannot be released from it by prolonged incubation in SDS, 2-mercaptoethanol, or hydroxylamine (pH 7.0 or 10.0), characteristics consistent with amino terminal myristoylation. Covalently linked myristate may be responsible for the high affinity of purified 43K protein for lipid bilayers despite the absence of a notably hydrophobic amino acid sequence.
Animals↗
The development of the jamming avoidance response in the weakly electric fish, Eigenmannia.
The jamming avoidance response (JAR) in young weakly electric fish, Eigenmannia, develops at the onset of a functional electrosensory phase-coding system, a neural pathway that is critical for the performance of the JAR. Size (measured in head to tail length) seems to be the best predictor of the onset of the JAR. A distinguishable JAR value (0.15 Hz or greater) develops in fish at a length of 12-15 mm, and its strength continues to increase with maturity until it approaches an adult value (8-20 Hz) at a length of about 45 mm. The JAR is not dependent upon social interactions, as it can be performed correctly upon first stimulation by animals raised in individual aquaria from the egg stage. Preliminary studies suggest that there are anatomical correlates to the development of the JAR behavior. As the JAR strengthens with age, there is a concomitant increase in the number of giant cells and a development of the commissural plexus in lamina 6 of the torus semicircularis. Giant cells play a pivotal role in the phase comparison circuit. Both phase and amplitude information play a role in the proper performance of the JAR, but the discrete nature of the phase comparison circuit allows the correlation between the development of the JAR and an essential part of the phase comparison circuit (lamina 6 of the torus) to be observed in Eigenmannia.
Animals↗