Search PubMed⌕ Search

Biomedical subjects

P Walter

Publications and source records attributed to P Walter.

At least 199 records · Page 11Linked to original sources
PubMed1993

Discrete nascent chain lengths are required for the insertion of presecretory proteins into microsomal membranes.

Ribosomes synthesizing nascent secretory proteins are targeted to the membrane by the signal recognition particle (SRP), a small ribonucleoprotein that binds to the signal peptide as it emerges from the ribosome. SRP arrests further elongation, causing ribosomes to stack behind the arrested ribosome. Upon interaction of SRP with its receptor on the ER membrane, the translation arrest is released and the ribosome becomes bound to the ER membrane. We have examined the distribution of unattached and membrane-bound ribosomes during the translation of mRNAs encoding two secretory proteins, bovine preprolactin and rat preproinsulin I. We find that the enhancement of ribosome stacking that occurs when SRP arrests translation of these proteins is relaxed in the presence of microsomal membranes. We also demonstrate that two previously described populations of membrane-associated ribosomes, distinguished by their sensitivity to high salt or EDTA extraction, correspond to ribosomes that have synthesized differing lengths of the nascent polypeptide. This analysis has revealed that nascent chain insertion into the membrane begins at distinct points for different presecretory proteins.

Animals↗
PubMed1993

Interleukin-8 production in patients undergoing cardiopulmonary bypass. The influence of pretreatment with methylprednisolone.

Pulmonary dysfunction caused by pulmonary neutrophil sequestration is a frequent postoperative complication in patients undergoing cardiopulmonary bypass (CPB) surgery. It is yet unclear whether treatment with corticosteroids in vivo in these patients can prevent complement-mediated neutrophil activation and sequestration in the lungs. Therefore, we conducted a prospective study in order to investigate whether methylprednisolone (MP) pretreatment (30 mg/kg) could influence the appearance of IL-8 (a recently discovered cytokine with potent neutrophil-chemotactic activity) in the peripheral circulation. We also studied the effects of MP pretreatment on the inflammatory parameters in the bronchoalveolar lavage (BAL) fluid 4 h postoperatively. Although peripheral neutropenia and the rise in IL-8 serum levels was less pronounced in MP-treated than in non-steroid-treated patients, there was no significant difference in albumin, total protein, concentrations of IL-8 and C3a, and the number of neutrophils in the BAL fluid between the two groups. However, when cultured in vitro, alveolar macrophages from patients treated with MP released significantly lower IL-8, both in basal conditions and after stimulation with lipopolysaccharide. Our results show that MP does not prevent (IL-8-mediated) pulmonary neutrophil infiltration after CPB, although it might affect certain aspects of the microvascular lung injury.

Aged↗
PubMed1992

The anticodon triplet is not sufficient to confer methionine acceptance to a transfer RNA.

Previous work suggested that the presence of the anticodon CAU alone was enough to confer methionine acceptance to a tRNA. Conversions of Escherichia coli nonmethionine tRNAs to a methionine-accepting species were obtained by substitutions reconstructing the whole methionine anticodon loop together with preservation (or introduction) of the acceptor stem base A73. We show here that the CAU triplet alone is unable to confer methionine acceptance when transplanted into a yeast aspartic tRNA. Both non-anticodon bases of the anticodon loop of yeast tRNA(Met) and A73 are required in addition to CAU for methionine acceptance. The importance of these non-anticodon bases in other CAU-containing tRNA frameworks was also established. These specific non-anticodon base interactions make a substantial thermodynamic contribution to the methionine acceptance of a transfer RNA.

Anticodon↗
PubMed1992

Fluorescence-detected assembly of the signal recognition particle: binding of the two SRP protein heterodimers to SRP RNA is noncooperative.

Protein-RNA and protein-protein interactions involved in the assembly of the signal recognition particle (SRP) were examined using fluorescence spectroscopy. Fluorescein was covalently attached to the 3'-terminal ribose of SRP RNA following periodate oxidation, and the resulting SRP RNA-Fl was reconstituted into a fluorescent SRP species that was functional in promoting translocation of secretory proteins across the membrane of the endoplasmic reticulum. Each of the two protein heterodimers purified from SRP elicited a substantial change in fluorescein emission upon association with the modified RNA. The binding of SRP9/14 to singly-labeled SRP RNA-Fl increased fluorescein emission intensity by 41% at pH 7.5 and decreased its anisotropy from 0.18 to 0.16. The binding of SRP68/72 increased the fluorescein anisotropy from 0.18 to 0.23 but did not alter the emission intensity of SRP RNA-Fl. These fluorescence changes did not result from a direct interaction between the dye and protein because the fluorescein remained accessible to both iodide ions and fluorescein-specific antibodies in the complexes. The spectral changes were elicited by specific SRP RNA-protein interactions, since (i) the SRP9/14- and SRP68/72-dependent changes were unique, (ii) an excess of unlabeled SRP RNA, but not of tRNA, blocked the fluorescence changes, and (iii) no emission changes were observed when SRP RNA-Fl was titrated with other RNA-binding proteins. Each heterodimer bound tightly to the RNA, since the Kd values determined spectroscopically and at equilibrium for the SRP9/14 and the SRP68/72 complexes with SRP RNA-Fl were less than 0.1 and 7 +/- 3 nM, respectively. The binding affinity of SRP68/72 for SRP RNA-Fl was unaffected by the presence of SRP9/14, and hence the binding of the heterodimers to SRP RNA is noncooperative in the absence of SRP54 and SRP19. The SRP protein heterodimers therefore associate randomly and independently with SRP RNA to form domains in the particle that are distinct both structurally and functionally. Any cooperativity in SRP assembly would have to be mediated by SRP54 and/or SRP19.

Animals↗
PubMed1992

Binding of the yeast tRNA(Met) anticodon by the cognate methionyl-tRNA synthetase involves at least two independent peptide regions.

As for Escherichia coli methionine tRNAs, the anticodon triplet of yeast tRNA(Met) plays an important role in the recognition by the yeast methionyl-tRNA synthetase (MetRS), indicating that this determinant for methionine identity is conserved in yeast. Efficient aminoacylation of the E. coli tRNA(Met) transcript by the heterologous yeast methionine enzyme also suggests conservation of the protein determinants that interact with the CAU anticodon sequence. We have analysed by site-directed mutagenesis the peptide region 655 to 663 of the yeast MetRS that is equivalent to the anticodon binding region of the E. coli methionine enzyme. Only one change, converting Leu658 into Ala significantly reduced tRNA aminoacylation. Semi-conservative substitutions of L658 allow a correlation to be drawn between side-chain volume of the hydrophobic residue at this site and activity. The analysis of the L658A mutant shows that Km is mainly affected. This suggests that the peptide region 655 to 663 contributes partially to the binding of the anticodon, since separate mutational analysis of the anticodon bases shows that kcat is the most critical parameter in the recognition of tRNA(Met) by the yeast synthetase. We have analysed the role of peptide region (583-GNLVNR-588) that is spatially close to the region 655 to 663. Replacements of residues N584 and R588 reduces significantly the kcat of aminoacylation. The peptide region 583-GNLVNR-588 is highly conserved in all MetRS so far sequenced. We therefore propose that the hydrogen donor/acceptor amino acid residues within this region are the most critical protein determinants for the positive selection of the methionine tRNAs.

Amino Acid Sequence↗
PubMed1992

New kinetic parameters for rat liver arginase measured at near-physiological steady-state concentrations of arginine and Mn2+.

A cytosolic cell-free system from rat liver containing the last three enzymes of the urea cycle, a number of cofactors and the substrates aspartate and citrulline was shown to synthesize urea at near-physiological rates ranging between 0.40 and 1.25 mumol/min per g of liver. This system was used to determine the kinetic parameters for arginase. With saturating amounts of Mn2+ (30 microM), arginine remained at a steady-state concentration of 5-35 microM depending on the aspartate and citrulline supply. Vmax. at micromolar arginine concentrations was between 1.10 and 1.25 mumol/min per g of liver, the K0.5 (arginine) between 6.0 and 6.5 microM and positive co-operativity was observed (Hill coefficient 2). Omission of Mn2+ caused a significant accumulation of arginine during the incubation, suggesting a regulatory effect of arginase. Under these conditions, Vmax. was 1.10-1.65 mumol/min per g of liver and the Km (arginine) increased up to 14.4-21.1 microM. The apparent Ka for Mn2+ in the presence of physiological concentrations of ATP, Mg2+ and arginine was calculated to be maximally 8 microM. Initial-velocity experiments with millimolar arginine concentrations as the direct substrate gave the following results, which are in good agreement with literature data. In the absence of Mn2+, Vmax. was 71.3 mumol/min per g of liver and the Km (arginine) 1.58 mM. With 30 microM-Mn2+, Vmax. was 69.4 mumol/min per g of liver and the Km (arginine) decreased to 0.94 mM. On the basis of our results, we propose the presence of high-affinity and low-affinity sites for arginine on rat liver arginase and postulate that alterations in arginase activity arising from changes in the concentration of arginine and of the cofactor Mn2+ may contribute to the regulation of ureagenesis in vivo.

Adenosine Triphosphate↗
PubMed1992

Evidence for peptide transport across microsomal membranes.

Antigenic peptides bound to class I molecules of the major histocompatibility complex (MHC) are recognized by T-cell receptors during development of an antiviral immune response. T cells respond to peptides derived from cytoplasmic viral proteins as well as viral membrane proteins, indicating that a pathway exists for the transport of proteins or peptides from the cytosol into the compartment(s) where the MHC class I molecules assemble. To investigate this pathway, we have developed an in vitro assay for the transport of peptides into microsomal vesicles. This assay provides evidence for the transport of chemically synthesized peptides (13-21 amino acids) containing N-linked glycosylation acceptor sequences, which serve as glycosylation substrates. Their transport results in depletion of the pool of available dolichol high-mannose oligosaccharides in the lumen of the microsomal vesicles. We have observed transport of peptides derived from antigenic human immunodeficiency virus gag and influenza B nucleoprotein sequences, but transport of a third randomly selected peptide was not detected, suggesting specificity of the transport process. We were not able to demonstrate ATP dependence of this peptide transport process by using apyrase and an ATPase inhibitor. This result was unexpected in light of the recent identification of MHC-linked genes with homology to ATP-binding cassette transporters, which have been proposed to mediate peptide transport.

Amino Acid Sequence↗
PubMed1992

SEC65 gene product is a subunit of the yeast signal recognition particle required for its integrity.

Protein targeting to the endoplasmic reticulum (ER) in mammalian cells is catalysed by the signal recognition particle (SRP), which consists of six protein subunits and an RNA subunit. Saccharomyces cerevisiae SRP is a 16S particle, of which only two subunits have been identified: a protein subunit, SRP54p, which is homologous to the mammalian SRP54 subunit, and an RNA subunit, scR1 (ref. 3). The sec65-1 mutant yeast cells are temperature-sensitive for growth and defective in the translocation of several secreted and membrane-bound proteins. The DNA sequence of the SEC65 gene suggests that its product is related to mammalian SRP19 subunit and may have a similar function. Here we show that SEC65p is a subunit of the S. cerevisiae SRP and that it is required for the stable association of another subunit, SRP54p, with SRP. Overexpression of SRP54p suppresses both growth and protein translocation defects in sec65-1 mutant cells.

Biological Transport, Active↗
PubMed1992

In vivo modulation of total and mitochondrial glutathione in rat liver. Depletion by phorone and rescue by N-acetylcysteine.

The aim of the present work was to modulate in vivo the level of hepatic mitochondrial glutathione (GSH). Rats were given phorone (diisopropylidene acetone), which in vivo becomes enzymatically conjugated to GSH, and were subsequently treated with N-acetylcysteine (NAC) to rescue GSH. In liver homogenate, a rapid and biphasic (T1/2 less than or equal to 15 min and 1.5 hr) drop of GSH was observed upon phorone administration. NAC treatment led to a restoration (T1/2 about 1 hr) of GSH in the homogenate above control values within 3 hr. The mitochondrial GSH level decreased with T1/2 of about 1.5 hr upon phorone treatment, and was 75% restored by NAC treatment within 3 hr. Hydroperoxide-induced mitochondrial pyridine nucleotide oxidation and Ca2+ release were impeded in GSH-depleted organelles, and NAC treatment restored these processes. The GSH status had no influence on mitochondrial pyridine nucleotide oxidation and Ca2+ released induced by alloxan, which reacts directly and non-enzymatically with pyridine nucleotides. It is concluded that NAC is able to rescue mitochondrial GSH in vivo and restore important mitochondrial functions. The data suggest that NAC may be a useful antidote in oxidative stress-related diseases.

Acetylcysteine↗
PubMed1992

Protein targeting to and translocation across the membrane of the endoplasmic reticulum.

Several approaches are currently being taken to elucidate the mechanisms and the molecular components responsible for protein targeting to and translocation across the membrane of the endoplasmic reticulum. Two experimental systems dominate the field: a biochemical system derived from mammalian exocrine pancreas, and a combined genetic and biochemical system employing the yeast, Saccharomyces cerevisiae. Results obtained in each of these systems have contributed novel, mostly non-overlapping information. Recently, much effort in the field has been dedicated to identifying membrane proteins that comprise the translocon. Membrane proteins involved in translocation have been identified both in the mammalian system, using a combination of crosslinking and reconstitution approaches, and in S. cerevisiae, by selecting for mutants in the translocation pathway. None of the membrane proteins isolated, however, appears to be homologous between the two experimental systems. In the case of the signal recognition particle, the two systems have converged, which has led to a better understanding of how proteins are targeted to the endoplasmic reticulum membrane.

Animals↗
PubMed1992

Mutants in three novel complementation groups inhibit membrane protein insertion into and soluble protein translocation across the endoplasmic reticulum membrane of Saccharomyces cerevisiae.

We have isolated mutants that inhibit membrane protein insertion into the ER membrane of Saccharomyces cerevisiae. The mutants were contained in three complementation groups, which we have named SEC70, SEC71, and SEC72. The mutants also inhibited the translocation of soluble proteins into the lumen of the ER, indicating that they pleiotropically affect protein transport across and insertion into the ER membrane. Surprisingly, the mutants inhibited the translocation and insertion of different proteins to drastically different degrees. We have also shown that mutations in SEC61 and SEC63, which were previously isolated as mutants inhibiting the translocation of soluble proteins, also affect the insertion of membrane proteins into the ER. Taken together our data indicate that the process of protein translocation across the ER membrane involves a much larger number of gene products than previously appreciated. Moreover, different translocation substrates appear to have different requirements for components of the cellular targeting and translocation apparatus.

Biological Transport↗
PubMed1992

Signal recognition particle receptor is important for cell growth and protein secretion in Saccharomyces cerevisiae.

In mammalian cells, the signal recognition particle (SRP) receptor is required for the targeting of nascent secretory proteins to the endoplasmic reticulum (ER) membrane. We have identified the Saccharomyces cerevisiae homologue of the alpha-subunit of the SRP receptor (SR alpha) and characterized its function in vivo. S. cerevisiae SR alpha is a 69-kDa peripheral membrane protein that is 32% identical (54% chemically similar) to its mammalian homologue and, like mammalian SR alpha, is predicted to contain a GTP binding domain. Yeast cells that contain the SR alpha gene (SRP101) under control of the GAL1 promoter show impaired translocation of soluble and membrane proteins across the ER membrane after depletion of SR alpha. The degree of the translocation defect varies for different proteins. The defects are similar to those observed in SRP deficient cells. Disruption of the SRP101 gene results in an approximately sixfold reduction in the growth rate of the cells. Disruption of the gene encoding SRP RNA (SCR1) or both SCR1 and SRP101 resulted in an indistinguishable growth phenotype, indicating that SRP receptor and SRP function in the same pathway. Taken together, these results suggest that the components and the mechanism of the SRP-dependent protein targeting pathway are evolutionarily conserved yet not essential for cell growth. Surprisingly, cells that are grown for a prolonged time in the absence of SRP or SRP receptor no longer show pronounced protein translocation defects. This adaptation is a physiological process and is not due to the accumulation of a suppressor mutation. The degree of this adaptation is strain dependent.

Amino Acid Sequence↗
PubMed1992

Analysis of African Burkitt's and high-grade B cell non-Burkitt's lymphoma for Epstein-Barr virus genomes using in situ hybridization.

We investigated the association of Epstein-Barr virus (EBV) with African cases of Burkitt's lymphoma (BL) and high grade B cell non-Burkitt's lymphoma (non-BL) occurring in areas where BL is endemic. The presence of EBV genomes was analysed in 24 cases using in situ hybridization with a 35S-labelled EBV probe applied to paraffin sections. EBV DNA was detected in each of 10 cases of BL in which technically satisfactory results were obtained, the virus being homogeneously distributed in all identifiable tumour cells. Two other cases of BL could not be evaluated because of technical problems. In contrast, EBV DNA was not detected in any case of high-grade non-BL (10 centroblastic and two immunoblastic lymphomas). These results confirm previous reports of the strong association of EBV with endemic BL, but suggest that the virus is not important in the pathogenesis of other types of African high-grade B cell lymphoma from regions where BL is endemic.

Burkitt Lymphoma↗
PubMed1992

C-terminal sequences can inhibit the insertion of membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae.

We have constructed three gene fusions that encode portions of a membrane protein, arginine permease, fused to a reporter domain, the cytoplasmic enzyme histidinol dehydrogenase (HD), located at the C-terminal end. These fusion proteins contain at least one of the internal signal sequences of arginine permease. When the fusion proteins were expressed in Saccharomyces cerevisiae and inserted into the endoplasmic reticulum (ER), two of the fusion proteins placed HD on the luminal side of the ER membrane, but only when a piece of DNA encoding a spacer protein segment was inserted into the fusion joint. The third fusion protein, with or without the spacer included, placed HD on the cytoplasmic side of the membrane. These results suggest that (i) sequences C-terminal to the internal signal sequence can inhibit membrane insertion and (ii) HD requires a preceding spacer segment to be translocated across the ER membrane.

Alcohol Oxidoreductases↗
PubMed1992

[Risks and benefits of thrombendarterectomy in bilateral internal carotid artery stenosis].

Among the patients who were operated for cerebrovascular insufficiency, one third suffered from bilateral stenoses of the internal carotid artery. In a retrospective analysis of 47 patients who were operated in consecution and of 28 patients who refused operation and were treated conservatively, the outcome was compared to cases with a one-sided stenosis. 31 patients were operated on one side and 16 on both sides. 28 of the surgically treated cases were followed up for an average of 32 months. The morbidity of 3.2% of the bilateral cases was twice as high as that of the unilateral cases. A permanent neurological deficit was observed in 2 patients after reconstruction of the asymptomatic but higher grading stenosis, but in none of the 18 cases after reconstruction of the symptomatic side although the stenosis of the other side was not removed. Patients with bilateral stenoses of the internal carotid artery should therefore be operated first on the symptomatic side. The frequently concomitant coronary artery disease of these patients prevents an improvement of the survival period in the group of bilaterally operated patients. The success in these cases concerns only the quality of life. The risk of a subsequent stroke in the 28 not operated cases with bilateral stenoses was 14.6% and therefore much higher than in the group of operated patients. Increase of the stenoses during the observation period is a prognostically unfavorable sign. Low dose treatment with platelet aggregation inhibitors (less than 500 mg ASS) increased the risk of an apoplectic insult.

Carotid Artery, Internal↗
PubMed1992

[The celiac trunk compression syndrome: a rare disease picture].

We report the case of a 49-year-old woman who complained of strong epigastric pain, emesis, diarrhea, and a weight reduction of 10 kg. The diagnosis of a coeliac axis compression syndrome was made based on a typical angiographic picture. Half a year after the operative resection of the median arcuate ligament and the coeliac plexus, the patient is completely symptom-free and her weight has increased by 4 kg. In our opinion, the most important prerequisite for the indication to operate is the careful exclusion of all other possible diagnoses in combination with all the typical findings.

Angiography↗