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At least 1,711 records · Page 95Linked to original sources
PubMed1994

Transport of bovine serum albumin across rat jejunum: role of the enteric nervous system.

To assess the mechanisms for movement of antigenically intact macromolecules across small intestinal mucosa, transport kinetics of bovine serum albumin (BSA) uptake and the effect of neural and metabolic inhibition were examined in stripped short-circuited rat jejunum. The mucosa was exposed to BSA, and, after a 50-min equilibration, mucosal-to-serosal movement of immunologically intact BSA was determined by enzyme-linked immunosorbent assay and total BSA by radiolabeled 125I-BSA. Intact BSA uptake demonstrated saturable kinetics. Immunologically intact BSA crossed the intestinal mucosa as 4.5% of total 125I-BSA flux. Colchicine and 4 degrees C significantly reduced uptake of immunologically intact BSA. NaF significantly reduced uptake of immunologically intact BSA and 125I-BSA. Treatment with tetrodotoxin significantly reduced intact BSA uptake, but did not significantly alter total BSA uptake. The muscarinic cholinoceptor antagonist atropine also significantly inhibited transport of intact BSA, whereas the nicotinic cholinoceptor antagonist hexamethonium had no effect. These findings indicate that transport of intact macromolecules across small intestinal mucosa is a saturable energy-dependent process that utilizes the microtubular network and is regulated by the enteric nervous system primarily through cholinergic nerves acting on muscarinic receptors.

Animals↗
PubMed1987

Paroxysmal nocturnal hemoglobinuria type III. Lack of an erythrocyte membrane protein restricting the lysis by C5b-9.

The complement-mediated lysis is inefficient when complement and target cells are homologous with regard to the species. In erythrocytes from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH), the species restriction is lost: PNH-erythrocytes (PNH-E) are susceptible to lysis by human complement. In human erythrocytes (huE) the species restriction is ascribed to an integral membrane protein, designated C8-binding protein (C8bp). In the present study, we tested membranes of PNH-E type III for the presence of C8bp. A protein with C8-binding capacity could not be detected. C8bp, which was isolated from the membrane of huE, inhibited the lysis of PNH-E by C5b-9 as well as the C9 polymerization. Thus, addition of C8bp restored the species restriction in PNH-E. In conclusion, we propose that lack of C8bp might represent the defect in PNH-E type III membranes, which is responsible for their enhanced lytic susceptibility towards lysis by the late complement components.

Complement C8↗
PubMed1993

Neurodevelopmental abnormalities and lactic acidosis in a girl with a 20-bp deletion in the X-linked pyruvate dehydrogenase E1 alpha subunit gene.

We describe a girl with developmental abnormalities of the CNS and a lactic acidosis whose cultured fibroblasts showed a profound deficiency of pyruvate dehydrogenase complex (PDHC) activity (patient = 0.14 nmol/mg protein per minute, controls = 0.7 to 1.1 nmol/mg protein per minute). Immunocytochemistry demonstrated the fibroblast culture to be mosaic, with 14% of cells expressing the PDHC E1 alpha subunit protein in normal amounts and the remaining 86% having no detectable immunoreactive activity. Direct sequencing of cDNA for the X-linked PDHC E1 alpha subunit established that the patient was heterozygous for a 20-bp deletion beginning in the codon for Ser300 of the derived amino acid sequence. The pattern of methylation at the DXS255 locus suggested predominant expression of the X chromosome carrying the mutant allele in the fibroblast culture. There was a good correlation between the residual PDHC activity, the proportion of cells with immunoreactive E1 alpha protein, and the X chromosome inactivation ratio, demonstrating the importance of X-inactivation for expression of this X-linked neurometabolic disease in females.

Acidosis, Lactic↗
PubMed1975

Fatal lung scan in a case of pulmonary hypertension due to obliterative pulmonary vascular disease.

A young woman with pulmonary hypertension due to sclerodermatous pulmonary vascular disease died immediately following the injection of macroaggregated albumin labeled with 99-TC for a perfusion lung scan. Review of the literature suggests that patients with chronic pulmonary hypertension due to an obliterative pulmonary vascular disease are at risk of developing a serious reaction to perfusion lung scanning. Important factors which predispose to an adverse reaction to lung scanning are the size and dose of macroaggregated albumin particles in relation to the total cross-sectional area of the pulmonary vascular bed and a critical arteriolar lumen size.

Adult↗
PubMed2002

Dissecting functional interactions in coagulation protein complexes by use of NMR spectroscopy.

The blood coagulation cascade can be considered as a system of well-orchestrated protein activation reactions involving and leading to the formation of large macromolecular assemblies. NMR investigations performed during the last six years have focused on the structural, motional and binding properties of some protein domains and interfaces critical for the formation of these protein complexes, outlining sophisticated intermolecular adaptations. The studied protein domains are either single molecules or covalently-linked heterodimers of the epidermal growth factor (EGF) homology domains, calcium-binding EGF domains and gamma-carboxyglutamic(Gla)-containing domains responsible for calcium-dependent binding to cell membranes. The characterized binding interfaces have included those between thrombin and fibrinogen, between thrombin and thrombomodulin, between factor VIIIa and the cell membrane, between tissue factor and factor VIIa, and most recently between factor Va and prothrombin. The obtained results indicate that the regulation of blood coagulation by protein and low molecular weight cofactors may involve a significant degree of protein folding transitions with changes in molecular and conformational motions coupled to enzymatic activities. This new level of complexity of the molecular processes controlling coagulation may lead to novel strategies for the development of more effective therapeutic anticoagulants.

Binding Sites↗
PubMed2000

Formation of protease-resistant prion protein in cell-free systems.

In transmissible spongiform encephalopathies (TSE) or prion diseases, the endogenous protease-sensitive prion protein (PrP-sen) of the host is converted to an abnormal pathogenic form that has a characteristic partial protease resistance (PrP-res). Studies with cell-free reactions indicate that the PrP-res itself can directly induce this conversion of PrP-sen. This PrP-res induced conversion reaction is highly specific in ways that might account at the molecular level for TSE species barriers, polymorphism barriers, and strains. Not only has this reaction been observed using mostly purified PrP-sen and PrP-res reactants, but also in TSE-infected brain slices. The conversion mechanism appears to involve both the binding of PrP-sen to polymeric PrP-res and a conformational change that results in incorporation into the PrP-res polymer.

Animals↗
PubMed1992

Glycophorin A facilitates the expression of human band 3-mediated anion transport in Xenopus oocytes.

The effects of human red cell glycophorin A (GPA) on the expression of the human erythrocyte anion transporter (band 3, AE1) has been examined in Xenopus oocytes. The coexpression of GPA with band 3 increased stilbene disulfonate-sensitive chloride transport into the oocytes. The effect of GPA was particularly noticeable at low band 3 concentrations and less marked at high band 3 cRNA concentrations. The enhancement of chloride transport was specific to GPA and was not observed when either glycophorin B or glycophorin C was coexpressed with band 3. Immunoprecipitations of whole oocyte homogenates showed the amount of band 3 synthesized was not affected by GPA at subsaturating cRNA concentrations. More band 3 was detected at the oocyte surface by immunoprecipitation when GPA was also expressed. Chymotrypsin treatment of intact oocytes was also used to assess surface band 3 and greater cleavage of band 3 by chymotrypsin was observed when GPA was present. Band 3 synthesis and assembly into canine pancreatic microsomes in the reticulocyte cell-free translation system was not altered by cotranslation of GPA. We suggest that GPA facilitates the translocation of band 3 to the plasma membrane at some point during band 3 biosynthesis in Xenopus oocytes. However, GPA is not essential for the expression of band 3 in red cells, since GPA-deficient individuals have apparently normal levels of band 3. Other GPA-independent mechanisms must also allow translocation of band 3 to the surface membrane in erythroid cells and oocytes. GPA may affect the rate of accumulation of band 3 at the cell surface, rather than the final level in the plasma membrane.

Animals↗
PubMed2004

Thermodynamic cooperativity and kinetic proofreading in DNA damage recognition and repair.

In humans UV-induced cyclobutane thymine dimers are excised by the joint action of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF.ERCC1. Yet, in vitro assays show that none of these six factors is capable of detectably discriminating thymine dimer-containing DNA from undamaged DNA. We show how two elementary principles in macromolecular recognition, (1). cooperativity and (2). kinetic proofreading, are utilized to confer specificity to the repair system where none exists at the individual repair factor level and enable human cells to excise thymine dimers with a physiologically relevant specificity and at a biologically acceptable rate.

DNA Damage↗
PubMed1992

Monovalent cation selectivity for ATP-dependent association of the glucocorticoid receptor with hsp70 and hsp90.

We have reported previously that incubation of the immunopurified transformed hormone-free glucocorticoid receptor with rabbit reticulocyte lysate reconstitutes the receptor complex with hsp90 and that reconstitution is accompanied by concomitant repression of DNA binding activity and regeneration of the steroid binding conformation (Scherrer, L. C., Dalman, F. C., Massa, E., Meshinchi, S., and Pratt, W. B. (1990) J. Biol. Chem. 265, 21397-21400). In this work we further characterize this system by defining the small M(r) components of reticulocyte lysate required for both structural and functional reconstitution of the receptor-hsp90 complex. Reconstitution is ATP-dependent and there is a direct relationship between the extent of hsp90 binding to the receptor and the number of specific steroid binding sites that are generated. Dialysis of reticulocyte lysate inactivates its reconstituting activity. Addition of an ATP-regenerating system or readdition of small M(r) lysate components (in the form of a Centricon C30 filtrate) has little effect, but the presence of both restores full reconstituting activity to dialyzed lysate, as assayed by steroid binding activity and by the binding of hsp90 and hsp70 to the receptor. The small M(r) activity is heat-stable, and it can be completely replaced by NH+4, K+, and Rb+, with K+ producing a maximal effect at the concentration normally present in undialyzed lysate. Na+ and Li+ have no reconstituting activity. This ion selectivity demonstrates that a monovalent cation binding site is involved in receptor heterocomplex reconstitution. It is intriguing that the protein unfoldase (e.g. clathrin uncoating ATPase) activity of hsp70 is known to have a similar monovalent cation dependence, and that under all conditions where hsp90 becomes bound to the receptor, we find that hsp70 is also bound.

Adenosine Triphosphate↗
PubMed1989

Proteolytic processing of the beta-subunit of the lysosomal enzyme, beta-hexosaminidase, in normal human fibroblasts.

We have characterized the proteolytic processing of the beta-subunit of beta-hexosaminidase by identifying the amino termini of the various forms synthesized in cell-free translation and in cultured human fibroblasts. The procedures used had been developed for similar studies of the alpha-subunit (Little, L. E., Lau, M. M. H., Quon, D. V. K., Fowler, A. V., and Neufeld, E. F. (1988) J. Biol. Chem. 263, 4288-4292). Radioactive amino acids were incorporated biosynthetically into the different forms of the beta-subunit, which were isolated by immunoprecipitation, gel electrophoresis, and electroelution, and analyzed by automated Edman degradation. Translation by reticulocyte lysate in the presence of canine pancreas microsomes gave a product with alanine 43 at the amino terminus. The lysate could initiate translation at methionine 1 or methionine 13, depending on the SP6 mRNA provided. The product of signal peptidase action, the precursor form of the beta-subunit with amino-terminal alanine 43, was found in NH4+-induced secretions of cultured fibroblasts; intracellularly, this form was trimmed of two additional amino acids. The mature form was found to consist of three polypeptides joined by disulfide bonds; the amino termini were found to be valine 48, threonine 122, and lysine 315. Thus, in contrast to the alpha-subunit, the mature form of the beta-subunit of beta-hexosaminidase is derived from the precursor by internal proteolytic nicking rather than by removal of a large amino-terminal peptide segment.

Amino Acid Sequence↗
PubMed1988

Glucose permease of Escherichia coli. Purification of the IIGlc subunit and functional characterization of its oligomeric forms.

The membrane subunit (IIGlc) of the glucose permease has been purified from overproducing Escherichia coli. About 2 mg of pure protein was obtained from 10 g (wet weight) of cells. IIGlc of E. coli and Salmonella typhimurium are functionally indistinguishable. A small difference was revealed, however, by a monoclonal antibody which neutralizes glucose phosphorylation activity of IIGlc from S. typhimurium, but does not cross-react with IIGlc of E. coli. A dimeric form of purified IIGlc can be detected by chemical cross-linking and by zonal sedimentation at 4 degrees C. Upon mild oxidation a disulfide bond is formed between the subunits of the dimer. Oxidized IIGlc is more stable than the reduced form but is inactive because it cannot be phosphorylated by the cytoplasmic subunit (IIIGlc) of the glucose permease. Cys-421 could be identified as the oxidation-sensitive residue, using a novel assay to detect IIIGlc-dependent phosphorylation of nitrocellulose-bound IIGlc that has been purified by gel electrophoresis. No dimeric form of phosphorylated IIGlc could be detected. Because phosphorylated IIGlc is a catalytic intermediate it is concluded that catalytically active IIGlc is a monomer and that the dimeric form is an artefact observed only with purified resting IIGlc. That IIGlc is active as a monomer is further supported by the observation that monomeric IIGlc catalyzes phosphoryl exchange between glucose and glucose 6-phosphate at equilibrium and that an excess of inactive IIGlc with a serine replacing Cys-421 does not interfere with the activity of wild-type IIGlc as would be expected if interaction between the subunits in a dimer were essential for activity.

Escherichia coli↗
PubMed1977

[Comparative characteristics of cell-free filtrates of Shigella flexneri of different virulence].

It was shown for the first time that the virulent Sh. flexneri strain grown on Luria broth differed from the avirulent one by the yield of readily released surface-located complexes--lipopolysaccharide (determined by rhamnose) and protein into the filtrate. There was no distinct correlation between the strain virulence and the content of rhamnose-determined lipopolysaccharide in the filtrate; growing bacteria in the presence of Ca and Mg ions had no significant influence on the lipopolysaccharide release into the filtrate. Protein release into the cell-free filtrate was thrice that in the virulent shigella strain than in the avirulent one. When bacteria were grown in the presence of Ca ions protein release from the virulent strain increased 1.5-fold and changed but little in the avirulent culture. Cell-free filtrates of the virulent strain produced toxic action on L tissue culture cells; in conjunctival infection of guinea pigs they caused some reduction of the LD50 of the virulent strain and sharply aggravated the course of the infectious process. Heating of the filtrate at 100 degrees C for 15 min decreased their toxic action on L cells. The data obtained indicated that the active biological factor revealed in the virulent strain of Sh. flexneri was protein or its derivative.

Animals↗
PubMed1978

[Comparative study of the effect of different rifamycins on bacterial cell metabolism and on the RNA-polymerase reaction in a cell-free system].

The results of the study on the inhibitory effect of a number of rifamycin derivatives, such as rifamycin B, rifamycin O, rifamycin, rifamycin A, 25-desacetylrifampicin and rifampicin are presented. It was shown that rifampicin had the highest inhibitory effect on the synthesis of RNA in the cells of E. coli and Staph. aureus. It inhibited the above process by 93.0 and 98.8 per cent respectively. The data on the cells of Staph. aureus, as well as the data on comparison of the inhibitory effect of rifampicin derivatives with respect to the RNA-polymerase reaction in acellular systems are presented.

Cell-Free System↗
PubMed1984

[Molecular organization of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria. Chemical modification with bifunctional reagents in the study of oligomeric forms].

The molecular organization of adrenocortical cytochrome P-450scc has been investigated using chemical modification with bifunctional imidates. The oligomeric organization of cytochrome P-450scc in solution has been shown. The application of dimethyl-3,3'-dithiobispropioimidate and subsequent cleavage of the modified products by reducing agents revealed the presence of two types of intramolecular cross-links: "short" at the distance of 3,0 A between the amino groups of lysine residues and "long" ones at a distance of 11,9 A. The analysis of the products, obtained by limited proteolysis of the oligomeric forms of the cross-linked cytochrome P-450, by two-dimensional electrophoresis has shown that the cross-links are formed between the functional domain (fragment F1) and domain responsible for the interaction with the phospholipid membrane (fragment F2). A model for cytochrome P-450scc molecular organization has been suggested on the basis of the obtained results.

Adrenal Cortex↗