Search PubMed⌕ Search

Biomedical subjects

P Walter

Publications and source records attributed to P Walter.

At least 289 records · Page 16Linked to original sources
PubMed1987

Evidence for an extended 7SL RNA structure in the signal recognition particle.

The signal recognition particle (SRP) functions in conjunction with the SRP receptor to target nascent ectoplasmic proteins to the protein translocation machinery of the endoplasmic reticulum membrane. SRP is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of 7SL RNA 300 nucleotides long. SRP has previously been visualized by a variety of electron microscopic techniques as a rod-shaped particle 24 nm long and 6 nm wide. We report here microanalysis by electron spectroscopic imaging which localizes the RNA molecule in SRP to primarily the two ends of the particle. These results suggest that the single 7SL RNA molecule spans the length of the particle. Micrographs from a scanning transmission electron microscope permit visualization of unstained SRP with low electron exposure, as well as the direct measurement of the mol. wt of the particle. These micrographs confirm our earlier suggestion that SRP is divided into three structural domains and allow discrimination of the two ends of the structure. The results of both techniques have been combined in a model for the structure of SRP in which we propose the basic orientation of the 7SL RNA. The structure proposed is consistent with the secondary structure predicted for the RNA and with biochemical data.

Microscopy, Electron↗
PubMed1987

Influence of postdiabetic onset time and immunosuppressive treatment on islet grafts in the spontaneous diabetic BB/W rat.

1000 islets from one donor, isolated with a modified neutral red method, were intraportally transplanted from BB/Wistar or Lewis (RT1) donors to short- and long-term diabetic BB/Wistar recipients. Recipients received immunosuppression with cyclosporine i.m.--either postoperative 4 X 25 mg/kg body weight or pre- and postoperative 3 X 10 mg and 4 X 25 mg/kg body weight. Graft survival in short-term diabetic recipients was significantly shorter than in long-term diabetics, whether they were immunosuppressed or not. Especially in recent onset recipients, the additional preoperative immunosuppression with cyclosporine provided longer graft survival than postoperative cyclosporine alone. Histological examinations of islet grafts showed eosinophilic cells in all transplanted islets, in both functioning and clinically rejected grafts of allogenic and "pseudoisogenic" transplanted recipients. a coincidence of eosinophilic cells with the reenactment of the original autoimmune disease in islet grafts seems possible.

Animals↗
PubMed1986

Formation of hexose 6-phosphates from lactate + pyruvate + glutamate by a cell-free system from rat liver.

A cell-free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors and gluconeogenic intermediates at near-physiological concentrations was shown to form hexose 6-phosphates linearly from lactate + pyruvate + glutamate at a rate of 0.82 +/- 0.05 mumol/min per g of liver (mean +/- S.E.M., n = 8, 37 degrees C). The indicated rates were measured between 20 min and 60 min incubation time, when the system was near steady state. Experiments with either [1-14C]lactate or [U-14C]glutamate revealed that the incorporation of radioactive label into hexose 6-phosphates was proportional to the utilization of lactate + pyruvate and of glutamate during incubation and that both served as gluconeogenic substrates at a ratio of about 2:1. When the [ATP]/[ADP] ratio was lowered from 60 to 19 by addition of ATPase, the rate of hexose 6-phosphate formation fell to one-third. This decrease in gluconeogenic flux was mainly due to a decreased flow through the phosphoglycerate kinase step. Hexose 6-phosphate formation could also be decreased by increasing the ratio [NADH]/[NAD+], either by addition of ethanol or by increasing the initial concentration of lactate + pyruvate at a fixed ratio of 10:1. The observed inhibition was linked to a limitation in the availability of oxaloacetate for the phosphoenolpyruvate carboxykinase reaction and to an increased formation of sn-glycerol 3-phosphate. Finally, the rates of hexose 6-phosphate formation in incubations with cytosols from fed rats were only 50% of those observed with cytosols from animals starved for 48 h. One of the limiting steps was found to be the flow through the phosphoenolpyruvate carboxykinase step.

Adenosine Diphosphate↗
PubMed1986

In vitro protein translocation across the yeast endoplasmic reticulum: ATP-dependent posttranslational translocation of the prepro-alpha-factor.

The in vitro synthesized precursor of the alpha-factor pheromone, prepro-alpha-factor, of Saccharomyces cerevisiae was translocated across yeast microsomal membranes in either a homologous or a wheat germ cell free system. Translocated prepro-alpha-factor was glycosylated, sedimented with yeast microsomal vesicles, and was protected from digestion by added protease, but was soluble after alkaline sodium carbonate treatment. Thus prepro-alpha-factor was properly sequestered within yeast microsomal vesicles, but was not integrated into the lipid bilayer. In marked contrast to protein translocation across mammalian microsomal membranes, translocation of prepro-alpha-factor across yeast microsomal membranes could occur posttranslationally. This reaction required protein components in the yeast microsomal fraction that could be inactivated by alkylation or proteolysis, was ATP-dependent, and was insensitive to the presence of a variety of uncouplers and ionophores.

Adenosine Triphosphate↗
PubMed1986

The chicken oestrogen receptor sequence: homology with v-erbA and the human oestrogen and glucocorticoid receptors.

A chicken oviduct cDNA clone containing the complete open reading frame of the oestrogen receptor (ER) has been isolated and sequenced. The mol. wt of the predicted 589-amino acid protein is approximately 66 kd which is very close to that of the human ER. Comparison of the human and chicken amino acid sequences shows that 80% of their amino acids are identical. There are three highly conserved regions; the second and third of which probably represent the DNA- and hormone-binding domains of the receptor. The putative DNA-binding domain is characterised by its high cysteine and basic amino acid content, and the hormone-binding domain by its overall hydrophobicity. These two domains of homology are also present in the human glucocorticoid receptor (GR) and the product of the avian erythroblastosis virus (AEV) gene, v-erbA, indicating that c-erbA, the cellular counterpart of v-erbA, belongs to a multigene family of transcriptional regulatory proteins which bind steroid-related ligands. The first highly conserved ER region is not present in the truncated v-erbA gene, but shares some homology with the N-terminal end of the GR. The function of the v-erbA gene product is discussed in relation to its homology with the ER and GR sequences.

Amino Acid Sequence↗
PubMed1986

Cloning of the human oestrogen receptor cDNA.

Poly A+ RNA isolated from the human breast cancer cell line MCF-7 was fractionated by sucrose gradient centrifugation and those fractions enriched in oestrogen receptor (ER) mRNA were used to prepare randomly primed cDNA libraries in the lambda gt11 vectors. Clones corresponding to the ER were isolated from both libraries after screening with either ER monoclonal antibodies (lambda gt11) or synthetic oligonucleotide probes designed from two peptide sequences of purified ER (lambda gt10). Five cDNA clones were isolated by antibody screening and five after screening with synthetic oligonucleotides. The two largest ER cDNA clones, lambda OR3 (1.3 kbase) and lambda OR8 (2.1 kbase), isolated using antibodies and oligonucleotides, respectively, were able to enrich selectively for ER mRNA by hybrid-selection. Furthermore, lambda OR8 contains DNA sequences which cross-hybridize with each of the other ER cDNA clones. These results demonstrate that the clones isolated correspond to the ER mRNA sequence. Using lambda OR8 as a hybridization probe revealed a single poly A+ RNA band of approx. 6.2 kbase in the ER containing human breast cancer cell lines MCF-7 and T47D. In contrast, no hybridization was seen in the human ER-cell line HeLa. The same probe hybridizes to a chicken gene which is expressed in oviduct tissue as a 7.5 kbase poly A+ RNA.

Animals↗
PubMed1986

Photocrosslinking of the signal sequence of nascent preprolactin to the 54-kilodalton polypeptide of the signal recognition particle.

Photoreactive moieties were incorporated into nascent polypeptides in a wheat germ protein-synthesizing system by using a plasmid-derived preprolactin mRNA and a Lys-tRNA analog, N epsilon-(5-azido-2-nitrobenzoyl)-Lys-tRNA (epsilon ANB-Lys-tRNA). The presence of the abnormally large amino acid side chains in the nascent chains did not impair function: complete preprolactin chains were synthesized in the absence of the signal recognition particle (SRP), elongation was arrested in the presence of SRP, and SRP-dependent translocation across the membrane of the endoplasmic reticulum and signal peptidase cleavage were observed in the presence of salt-extracted microsomes. Photolysis of elongation-arrested ribosomes resulted in several light- and epsilon ANB-Lys-tRNA-dependent crosslinks. By using antibodies specific for each of the proteins, one covalent complex was shown to be a photocrosslink between the preprolactin nascent chain and the 54-kDa protein subunit of SRP. This demonstrates that the N-terminal end of a secretory protein is located adjacent to the SRP in elongation-arrested ribosomes and strongly suggests that the signal sequence is recognized by and binds to the 54-kDa subunit of SRP. The other photocrosslinks involve as-yet-unidentified proteins in the large ribosomal subunit, indicating that this method of incorporating probes provides a powerful approach to examining the environment and interactions of the nascent chain during translation and translocation across the membrane of the endoplasmic reticulum. The Lys-tRNA analog also successfully photoaffinity-labeled the Escherichia coli elongation factor Tu (EF-Tu) in the epsilon ANB-Lys-tRNA.EF-Tu.GTP ternary complex.

Affinity Labels↗
PubMed1986

The signal recognition particle receptor is a complex that contains two distinct polypeptide chains.

Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (alpha-subunit, SR alpha), a 30-kD beta-subunit (SR beta). When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are approximately 30 kD in size and are immunologically distinct from each other, as well as from SR alpha and SRP proteins. One of the 30-kD proteins (SR beta) forms a tight complex with SR alpha in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR alpha or SR beta. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SR beta is an integral component of SRP receptor. The presence of SR beta was previously masked by proteolytic breakdown products of SR alpha observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR alpha. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR alpha and SR beta. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.

Animals↗
PubMed1986

Prolonged reversal of diabetes in the rat by transplantation of allogeneic islets from a single donor and cyclosporine treatment.

Islets were isolated using neutral red as a dye to exclude lymphatic and acinar impurities in the transplant preparation. In a one donor-one recipient model, intraportally transplanted isogeneic islets survived indefinitely. Mean graft survival could be prolonged from 5 to 90 days, using a 3-dose rejection prophylaxis with CsA in allografts of (DA [RT1a]----Lew (RT 1l). Histological examination revealed intact islets in the grafted normoglycemic animals. Immunohistochemical staining of these islets showed B cells with insulin-rich cytoplasma. Blood glucose levels and the results of i.v. glucose tolerance tests 50 days after transplantation are presented. The different outcomes of our experiments are discussed.

Animals↗
PubMed1986

The influence of (+/-)-15-deoxyspergualin on experimental transplantation and its immunopharmacological mode of action.

15-Deoxyspergualin, a novel structure which was described to have anti-tumor activity, was tested on its influence on the survival time of transplanted organs. Prolongation of graft survival was demonstrated in rat skin (LEW----Fischer), islet cells heart and kidney transplantation (DA----LEW) as well as in allogeneic tumor transplantation in mice (C57Bl/6----Balb/c). Especially in the kidney transplantation a long-lasting transplant survival time was observed, indicating induction of tolerance. Examination of its immunopharmacological properties showed that the mononuclear phagocytes and not the lymphocytes are primarily affected by the substance, thus expressing a new mode of action for a substance being effective in transplantation immunology.

Animals↗
PubMed1986

"Dermodress", a new temporary skin substitute: pilot study on donor sites.

"Dermodress" is a new temporary biological dressing (heterograft) composed of a pure neonatal bovine collagen I. The authors present their first trial with this skin substitute. Ten consecutive patients who required skin grafts were included in the pilot test group. The "Dermodress" was tested on their donor sites and compared with "Furacin" gauze. It was found that "Dermodress" provided a covering that adhered excellently to debrided surfaces, without disturbing the healing processes underneath, for up to three weeks after application. The results point to the potential value of the "Dermodress" for open wounds.

Adolescent↗
PubMed1985

Cytoplasmic methionyl-tRNA synthetase from Bakers' yeast. A monomer with a post-translationally modified N terminus.

Methionyl-tRNA synthetase has been purified from a yeast strain carrying the MES1 structural gene on a high copy number plasmid (pFL1). The purified enzyme is a monomer of Mr = 85,000 in contrast to its counterpart from Escherichia coli which is a dimer made up of identical subunits (Mr = 76,000; Dardel, F., Fayat, G., and Blanquet, S. (1984) J. Bacteriol. 160, 1115-1122). The yeast enzyme was not amenable to Edman's degradation indicating a blocked NH2 terminus. Its primary structure as derived from the DNA sequence (Walter, P., Gangloff, J., Bonnet, J., Boulanger, Y., Ebel, J.P., and Fasiolo, F. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2437-2441) has been confirmed using the fast atom bombardment-mass spectrometric method. This method was applied to tryptic digests of the carboxymethylated enzyme and the corresponding data provided extensive coverage of the translated DNA sequence, thus confirming its correctness. The ambiguity concerning which of the three NH2-terminally located methionine codons is the initiation codon was easily resolved from peptides identified in this region. It was possible to show that the first methionine had been removed and that the new NH2 terminus, serine, had been acetylated. A comparison between the yeast and E. coli sequences shows that the former has an N-terminal extension of about 200 residues as compared to the latter. It also lacks the C-terminal domain which is responsible for the dimerization of the E. coli methionyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1985

[Successful treatment of superior mesenteric artery thrombosis with local high-dose urokinase therapy].

A 56 year old man presented with increasing abdominal pain. He suffered from arterial occlusive disease with occlusion of the right A. iliaca communis. Angiography revealed partial thrombotic occlusion of the superior mesenteric artery. Urokinase (UK) at a dose of 150 IU/kg X minutes and heparin (1,000 U/h) was infused through the 7F angiographic catheter for 180 minutes. After 70 min of treatment, angiography showed improvement, and after 120 min the thrombus was nearly completely lysed. A stenosis of approximately 50% was still present after 180 min. Two hours after treatment the patient was pain free without analgesics. Laboratory studies showed systemic fibrinogenolysis, but fibrinogen was still within the upper normal range. Only slight systemic fibrinolytic activity (less than 5 IU UK/ml) could be determined. However, alpha 2-antiplasmin was depleted. The catheter was drawn 15 h after thrombolysis without bleeding. While under concurrent heparin and phenprocoumon therapy, the patient developed an infected gluteal hematoma as a result of i.m. injections prior to this treatment. A repeat angiography approximately one month after thrombolysis revealed further improvement and patency. The patient is well and free of abdominal angina and under oral anticoagulant therapy.

Drug Therapy, Combination↗