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Biomedical subjects

G Walter

Publications and source records attributed to G Walter.

At least 19 recordsLinked to original sources
PubMed1992

Expression of the A subunit of protein phosphatase 2A and characterization of its interactions with the catalytic and regulatory subunits.

The alpha form of the A subunit of human protein phosphatase 2A was expressed in insect cells following infection with a recombinant baculovirus. A alpha was expressed as a soluble protein that comprised approximately 10% of total cellular protein. The expressed A alpha subunit was purified by chromatography on amino-hexyl-Sepharose and Mono Q with a yield of 2 mg/500-ml culture. The recombinant protein had the same apparent molecular mass as the bovine cardiac protein and was devoid of myosin light chain phosphatase activity. Biological activity of expressed A was assessed by assays of complex formation with the catalytic (C) and B subunits, purified from bovine cardiac tissue, and by inhibition of phosphatase activity. Purified A alpha had a high apparent affinity for C (IC50 = 0.10 nM) and bound with a stoichiometry of 1 mol of A/mol of C. Interaction of A alpha with the catalytic subunit caused a maximal inhibition of myosin light chain and phosphorylase phosphatase activities of 50 and 79%, respectively. The AC complex prepared by reconstitution of recombinant A alpha with C had the same electrophoretic mobility in nondenaturing polyacrylamide gels and the same elution volume when chromatographed on a size exclusion column as the native AC complex purified from cardiac muscle. Similar chromatographic profiles were also observed for the heterotrimer reconstituted from recombinant A alpha, purified B and C, and the native bovine cardiac heterotrimeric holoenzyme. Cross-linking of the native enzyme and the reconstituted heterotrimer generated the same pattern of high molecular weight species. Immunological analyses of these complexes demonstrated that distinct cross-linked forms composed of ABC, AC, AB, and BC were obtained. These results suggest that each of the three subunits of protein phosphatase 2A forms direct contacts with both of the others.

Animals↗
PubMed1992

The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops.

We have used the polymerase chain reaction and VH family-based primers to clone and sequence 74 human germline VH segments from a single individual and built a directory to include all known germline sequences. The directory contains 122 VH segments with different nucleotide sequences, 83 of which have open reading frames. The directory indicates that the structural diversity of the germline repertoire for antigen binding is fixed by about 50 groups of VH segments: each group encodes identical hypervariable loops. The directory should help in mapping the VH locus, in estimating somatic mutation and VH segment usage and in designing and constructing synthetic antibody libraries.

Amino Acid Sequence↗
PubMed1992

Structural repertoire of the human VH segments.

The VH gene segments produce the part of the VH domains of antibodies that contains the first two hypervariable regions. The sequences of 83 human VH segments with open reading frames, from several individuals, are currently known. It has been shown that these sequences are likely to form a high proportion of the total human repertoire and that an individual's gene repertoire produces about 50 VH segments with different protein sequences. In this paper we present a structural analysis of the amino acid sequences produced by the 83 segments. Particular residue patterns in the sequences of V domains imply particular main-chain conformations, canonical structures, for the hypervariable regions. We show that, in almost all cases, the residue patterns in the VH segments imply that the first hypervariable regions have one of three different canonical structures and that the second hypervariable regions have one of five different canonical structures. The different observed combinations of the canonical structures in the first and second regions means that almost all sequences have one of seven main-chain folds. We describe, in outline, structures of the antigen binding site loops produced by nearly all the VH segments. The exact specificity of the loops is produced by (1) sequence differences in their surface residues, particularly at sites near the centre of the combining site, and (2) sequence differences in the hypervariable and framework regions that modulate the relative positions of the loops.

Amino Acid Sequence↗
PubMed1992

Hadamard spectroscopic imaging technique as applied to study human calf muscles.

In vivo results obtained by the B1 insensitive Hadamard spectroscopic imaging multivolume technique used with a surface coil are shown. The functional behavior of different human calf muscles during exercise was determined and the Pi/PCr ratio in each calf muscle, during steady-state conditions, was measured as a function of work level. Different levels of metabolic and physical activity were observed at the three calf muscles.

Humans↗
PubMed1992

Murine monoclonal antibodies directed against the transmembrane protein gp41 of human immunodeficiency virus type 1 enhance its infectivity.

Monoclonal antibodies (MAbs) were raised against the transmembrane protein (TM) gp41 of human immunodeficiency virus type 1 (HIV-1, strain HTLV-IIIB). The reactivity of three TM-specific MAbs was investigated in several tests, ELISA, immunostaining of Western blots, immunofluorescence and an alkaline phosphatase-anti-alkaline phosphatase assay. Epitope mapping was done by using overlapping gp41 peptides produced as Escherichia coli fusion proteins and synthetic peptides. In an in vitro assay, all three MAbs showed enhancing effects on HIV-1 infection after single or repeated treatment with the purified MAbs at concentrations of 6 to 25 micrograms/ml. The enhancing domain is located between amino acids 724 and 752 of the env protein sequence. Homologous peptides based on this sequence were used for analysis of sera from 100 individuals at different stages of HIV infection to evaluate the relevance of antibodies against this region to the prognosis of disease. No antibodies reactive with this region were found in ELISA, indicating that this domain is not immunogenic in humans.

Amino Acid Sequence↗
PubMed1992

The psychiatrist in American cartoons, 1941-1990.

The portrayal of psychiatrists in cartoons from the United States was examined. A total of 404 cartoons from 1941 to 1990 were obtained and arranged chronologically. The appearance, behaviour, treatment methods and efficacy of the cartoon psychiatrist were assessed. It was found that there has been little change in these parameters over the past 50 years. This finding is partly explained by the constraints of the cartoon medium and by the workings of humour and stereotyping. Psychiatric cartoons from other countries and those drawn by psychiatrists themselves do not depart from the American pattern. The differences between the representation of psychiatrists in the cartoon and in other media, such as the film, are highlighted. It is postulated that the cartoon serves to stabilize the more mutable portrayals of psychiatrists in other media. Further studies are suggested.

Cartoons as Topic↗
PubMed1992

Identification of binding sites on the regulatory A subunit of protein phosphatase 2A for the catalytic C subunit and for tumor antigens of simian virus 40 and polyomavirus.

Protein phosphatase 2A is composed of three subunits: the catalytic subunit C and two regulatory subunits, A and B. The A subunit consists of 15 nonidentical repeats and has a rodlike shape. It is associated with the B and C subunits as well as with the simian virus 40 small T, polyomavirus small T, and polyomavirus medium T tumor antigens. We determined the binding sites on subunit A for subunit C and tumor antigens by site-directed mutagenesis of A. Twenty-four N- and C-terminal truncations and internal deletions of A were assayed by coimmunoprecipitation for their ability to bind C and tumor antigens. It was found that C binds to repeats 11 to 15 at the C terminus of A, whereas T antigens bind to overlapping but distinct regions of the N terminus. Simian virus 40 small T binds to repeats 3 to 6, and polyomavirus small T and medium T bind to repeats 2 to 8. The data suggest cooperativity between C and T antigens in binding to A. This is most apparent for medium T antigen, which can only bind to those A subunit molecules that provide the entire binding region for the C subunit. We infer from our results that B also binds to N-terminal repeats. A model of the small T/medium T/B-A-C complexes is presented.

Amino Acid Sequence↗
PubMed1992

Contribution of skeletal muscle atrophy to exercise intolerance and altered muscle metabolism in heart failure.

BACKGROUND: The purpose of this study was to investigate the prevalence of skeletal muscle atrophy and its relation to exercise intolerance and abnormal muscle metabolism in patients with heart failure (HF). METHODS AND RESULTS: Peak VO2, percent ideal body weight (% IBW), 24-hour urine creatinine (Cr), and anthropometrics were measured in 62 ambulatory patients with HF. 31P magnetic resonance spectroscopy (MRS) and imaging (MRI) of the calf were performed in 15 patients with HF and 10 control subjects. Inorganic phosphorus (Pi), phosphocreatine (PCr), and intracellular pH were measured at rest and during exercise. Calf muscle volume was determined from the sum of the integrated area of muscle in 1-cm-thick contiguous axial images from the patella to the calcaneus. A reduced skeletal muscle mass was noted in 68% of patients, as evidenced by a decrease in Cr-to-height ratio of less than 7.4 mg/cm and/or upper arm circumference of less than 5% of normal. Calf muscle volume (MRI) was also reduced in the patients with HF (controls, 675 +/- 84 cm3/m2; HF, 567 +/- 112 cm3/m2; p less than 0.05). Fat stores were largely preserved with triceps skinfold of less than 5% of normal and/or IBW of less than 80% in only 8% of patients. Modest linear correlations were observed between peak VO2 and both calf muscle volume per meter squared (r = 0.48) and midarm muscle area (r = 0.36) (both p less than 0.05). 31P metabolic abnormalities during exercise were observed in the patients with HF, which is consistent with intrinsic oxidative abnormalities. The metabolic changes were weakly correlated with muscle volume (r = -0.42, p less than 0.05). CONCLUSIONS: These findings indicate that patients with chronic HF frequently develop significant skeletal muscle atrophy and metabolic abnormalities. Atrophy contributes modestly to both the reduced exercise capacity and altered muscle metabolism.

Exercise↗
PubMed1992

Studies on human porin. VIII. Expression of "Porin 31HL" channels in the plasmalemma of the acute-lymphoblastic-leukemia cell line KM3 as revealed by light- and electron-microscopy.

First electron microscopy data on the expression of the VDAC "Porin 31HL" in the plasmalemma of a eucaryotic cell are presented. In a light and electron microscopic study we demonstrate the expression of the porin channel in the outer cell membrane of the pre-B lymphocyte type acute-lymphoblastic-leukemia cell line KM3. Monoclonal mouse anti-"Porin 31HL" antibodies were applied in indirect immunofluorescence or immunogold labelling experiments. The results confirm our early topological data on the expression of porin channels in the cytoplasmic membrane of different human cell types as revealed on the light microscopy level. Interestingly, only a pre-embedding immunoreaction approach was successful for gold particle labelling of the plasmalemma. This again is in agreement with our recent data on the accessibility of the acetylated N-terminal part of "Porin 31HL" molecules on the outer cell surface.

Antibodies, Monoclonal↗
PubMed1991

Metabolic heterogeneity in human calf muscle during maximal exercise.

Human skeletal muscle is composed of various muscle fiber types. We hypothesized that differences in metabolism between fiber types could be detected noninvasively with 31P nuclear magnetic resonance spectroscopy during maximal exercise. This assumes that during maximal exercise all fiber types are recruited and all vary in the amount of acidosis. The calf muscles of seven subjects were studied. Two different coils were applied: an 11-cm-diameter surface coil and a five-segment meander coil. The meander coil was used to localize the 31P signal to either the medial or the lateral gastrocnemius. Maximal exercise, consisting of rapid plantar flexions, resulted in an 83.7% +/- 7.8% decrease of the phosphocreatine pool and an 8-fold increase of the inorganic phosphate (Pi) pool. At rest the Pi pool was observed as a single resonance (pH 7.0). Toward the end of the first minute of exercise, three subjects showed three distinct Pi peaks. During the second minute of exercise the pH values stabilized at 7.12 +/- 0.12, 6.63 +/- 0.15, and 6.27 +/- 0.23. The same pattern was seen when the signal was collected from the medial or lateral gastrocnemius. In four subjects only two distinct Pi peaks were observed. The Pi peaks had differing relative areas in different subjects, but they were reproducible in each individual. This method allowed us to study the appearance and disappearance of the different Pi peaks, together with the changes in pH. Because multiple Pi peaks were seen in single muscles they most likely identify different muscle fiber types.

Adenine Nucleotides↗
PubMed1991

Increased expression of pp60c-src protein-tyrosine kinase during peripheral nerve regeneration.

Since little is known about the intracellular changes that take place in response to Schwann cell-neuron interactions that occur during neurite outgrowth and myelination, we investigated the expression of a protein-tyrosine kinase, pp60c-src, during peripheral nerve regeneration through a silicone tube. Segments of regenerated nerve, extracted at various times following nerve-transection, showed an induction of in vitro c-src kinase activity as measured by autophosphorylation of immunoprecipitated pp60c-src. This activity occurred at 7 days following nerve transection coincident with the onset of neurite outgrowth in vivo. This kinase activity, which peaked out between 21 and 35 days and decreased thereafter, appeared to be associated with axonal growth and myelination, but not mitogenesis in the tube. Analysis of c-src proteins levels by Western blot showed a similar expression profile as that of the kinase activity. Qualitatively, the expression of an immunoreactive c-src band, migrating slightly slower than pp60, was detected in extracts of regenerating nerve segments as well as in the corresponding L4 and L5 dorsal root ganglia. This protein may be the CNS neuronal-specific form (pp60+) of the c-src protein. In situ hybridization revealed that Schwann cells and sensory and motor neurons associated with the regenerated sciatic nerve were positive for c-src mRNA during regeneration possibly accounting for the increased src protein expression during regeneration. Since the increased expression of pp60c-src in regenerated nerve segments coincides with both axonal sprouting and myelination, our findings suggest that the c-src protein may play a role in Schwann cell-neuron interactions which facilitate the occurrence of these events during regeneration. In addition, although pp60+ is generally not detectable in the mature PNS, our findings show that this protein may be induced during conditions of PNS differentiation which promote neurite outgrowth.

Animals↗
PubMed1991

Immunoglobulin VH and VK genes of the BALB/c anti-foot-and-mouth disease virus (O1) VP1 response: cloning, characterization and transgenic mice.

Hybridomas producing monoclonal antibodies of different isotypes were isolated from BALB/c antibody responses to the capsid protein VP1 of the foot-and-mouth disease virus (FMDV) strain O1. According to antigen binding measured by ELISA a weak-binding (81D10, IgM) and a strong-binding antibody (113C12, IgG2a) were selected. As RNA sequencing of productive immunoglobulin VH and VK genes turned out, both chains of the weak-binding antibody (81D10) are encoded by germline (i.e. not mutated) genes whereas the gene encoding the strong-binding antibody (113C12) k chain is mutated at several sites. Therefore, rearranged VH and VK genes of 81D10 were cloned, expressed in immunoglobulin non-producing plasmacytoma cells, and mice transgenic for the 81D10 k gene were produced. These mice provide a first step in the development of a transgenic mouse model for genetical investigations in the affinity maturation of anti-viral immunoglobulin variable genes.

Animals↗
PubMed1991

Protein phosphatases and DNA tumor viruses: transformation through the back door?

Cellular transformation by many oncogenic viruses is mediated by alterations in signal transduction pathways that control normal growth and proliferation. Common targets for many transforming viruses are pathways regulated by protein phosphorylation. The biochemical control of proteins in these pathways is a dynamic process that is regulated by the relative activities of protein kinases and phosphatases. Although there are numerous examples of viral oncogenes that encode protein kinases (Hunter, 1991), until recently there has been no evidence linking altered phosphatase activity to transformation. In this review we describe a novel mechanism, utilized by small DNA tumor viruses, in which viral oncogenes bind to and regulate a cellular protein serine/threonine phosphatase. The currently available evidence indicates that alteration of phosphatase activity and subsequent changes in phosphorylation levels is an important step in transformation by these viruses.

Antigens, Neoplasm↗
PubMed1991

Control of protein phosphatase 2A by simian virus 40 small-t antigen.

Soluble, monomeric simian virus 40 (SV40) small-t antigen (small-t) was purified from bacteria and assayed for its ability to form complexes with protein phosphatase 2A (PP2A) and to modify its catalytic activity. Different forms of purified PP2A, composed of combinations of regulatory subunits (A and B) with a common catalytic subunit (C), were used. The forms used included free A and C subunits and AC and ABC complexes. Small-t associated with both the free A subunit and the AC form of PP2A, resulting in a shift in mobility during nondenaturing polyacrylamide gel electrophoresis. Small-t did not interact with the free C subunit or the ABC form. These data demonstrate that the primary interaction is between small-t and the A subunit and that the B subunit of PP2A blocks interaction of small-t with the AC form. The effect of small-t on phosphatase activity was determined by using several exogenous substrates, including myosin light chains phosphorylated by myosin light-chain kinase, myelin basic protein phosphorylated by microtubule-associated protein 2 kinase/ERK1, and histone H1 phosphorylated by protein kinase C. With the exception of histone H1, small-t inhibited the dephosphorylation of these substrates by the AC complex. With histone H1, a small stimulation of dephosphorylation by AC was observed. Small-t had no effect on the activities of free C or the ABC complex. A maximum of 50 to 75% inhibition was obtained, with half-maximal inhibition occurring at 10 to 20 nM small-t. The specific activity of the small-t/AC complex was similar to that of the ABC form of PP2A with myosin light chains or histone H1 as the substrate. These results suggested that small-t and the B subunit have similar qualitative and quantitative effects on PP2A enzyme activity. These data show that SV40 small-antigen binds to purified PP2A in vitro, through interaction with the A subunit, and that this interaction inhibits enzyme activity.

Animals↗
PubMed1991

Dephosphorylation of simian virus 40 large-T antigen and p53 protein by protein phosphatase 2A: inhibition by small-t antigen.

Simian virus 40 (SV40) large-T antigen and the cellular protein p53 were phosphorylated in vivo by growing cells in the presence of 32Pi. The large-T/p53 complex was isolated by immunoprecipitation and used as a substrate for protein phosphatase 2A (PP2A) consisting of the catalytic subunit (C) and the two regulatory subunits, A and B. Three different purified forms of PP2A, including free C, the AC form, and the ABC form, could readily dephosphorylate both proteins. With both large-T and p53, the C subunit was most active, followed by the AC form, which was more active than the ABC form. The activity of all three forms of PP2A toward these proteins was strongly stimulated by manganese ions and to a lesser extent by magnesium ions. The presence of complexed p53 did not affect the dephosphorylation of large-T antigen by PP2A. The dephosphorylation of individual phosphorylation sites of large-T and p53 were determined by two-dimensional peptide mapping. Individual sites within large-T and p53 were dephosphorylated at different rates by all three forms of PP2A. The phosphates at Ser-120 and Ser-123 of large-T, which affect binding to the origin of SV40 DNA, were removed most rapidly. Three of the six major phosphopeptides of p53 were readily dephosphorylated, while the remaining three were relatively resistant to PP2A. Dephosphorylation of most of the sites in large-T and p53 by the AC form was inhibited by SV40 small-t antigen. The inhibition was most apparent for those sites which were preferentially dephosphorylated. Inhibition was specific for the AC form; no effect was observed on the dephosphorylation of either protein by the free C subunit or the ABC form. The inhibitory effect of small-t on dephosphorylation by PP2A could explain its role in transformation.

Antigens, Polyomavirus Transforming↗
PubMed1991

Constant expression and activity of protein phosphatase 2A in synchronized cells.

The levels of the A, B, and C subunits of protein phosphatase 2A in extracts from synchronized embryonic bovine tracheal cells were determined by immunoblotting with subunit-specific antibodies. A constant amount of each subunit was found in resting cells as well as in growing cells from all stages of the cell cycle. The phosphatase activity of protein phosphatase 2A was also constant. A quantitative comparison showed that the A and C subunits were present in similar amounts, whereas the B subunit was present at a significantly lower level. Together, the A, B, and C subunits represented approximately 0.2% of the total cellular protein.

Animals↗