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At least 883 records · Page 49Linked to original sources
PubMed1982

Isolation from thyroid cells or purified plasma membranes with associated actin microfilaments. Proteins bound to actin.

Plasma membranes of thyroid cells were purified from hog thyroid glands following two procedures. Their homogeneity was tested by electron microscopy and by measurements of the activity of membrane-bound enzyme markers. According to the procedure used the membrane fractions obtained present some differences in their morphological features as well as in the repartition of the activities of the membrane-bound enzyme markers. However, whatever the composition of the membrane fraction examined (membrane vesicles, single membrane sheets with junctional complexes), decoration with heavy meromyosin clearly shows the presence of actin filaments attached to these fragments. Analysis of proteins by polyacrylamide gel electrophoresis indicates the presence of about twelve major components with actin. Treatment of membranes with Triton X-100 results in an insoluble core which contains all the actin and most of the major proteins. The selective extraction of these components by buffers differing in their ionic strength, pH, or the presence or absence of ATP X Mg has been used to characterize some of the proteins associated to actin; among them are filamin, myosin, alpha-actinin, tropomyosin.

Actins↗
PubMed1983

The identity of conformational states of reconstituted and native histone octamers.

Histone octamers were reconstituted from the following preparations: (a) natural histone H3-H4 tetramer and histone H2A-H2B dimer, either selectively extracted from chromatin with solutions chloride or prepared by dissociation of the natural octamer; (b) acid-denatured core histones, either an unfractionated mixture or individually purified proteins. Complexes assembled from these histones elute from exclusion chromatography columns with octamer size as verified by cross-linking with dimethylsuberimidate. The reconstituted octamers all crystallize in the same form of helical tubes as the natural octamer.

Animals↗
PubMed1987

Identification of the C-terminally alpha-amidated amino acid in peptides by high-performance liquid chromatography.

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.

Amides↗
PubMed1993

High-yield production of bacteriorhodopsin via expression of a synthetic gene in Escherichia coli.

A gene (bos) coding for bacterioopsin (BO), the apoprotein of bacteriorhodopsin was assembled from chemically synthesized oligonucleotides by a new method of repeated rounds of insertion mutagenesis. The gene sequence was designed for convenient manipulation in future protein engineering experiments. In-frame fusion of bos to the lacZ454 gene allowed high-yield production in Escherichia coli of a beta-Gal454/BO fusion protein, deposited as intracellular inclusion bodies. These were enriched by virtue of their insolubility in 0.5% Triton X-100 and cleaved in aqueous suspension with IgA protease at a specific site provided at the beta-Gal454/BO boundary. Pure BO could be obtained from the mixture of water-insoluble cleavage products by selective extraction into organic solvent. The yield was in the range 30-50 mg pure protein/l culture medium, depending on individual preparation. This material could be used for reconstitution of fully functional bacteriorhodopsin. Taken together, the procedure constitutes a practical basis for the production of genetically engineered bacteriorhodopsins.

Amino Acid Sequence↗
PubMed1994

Cartilage oligomeric matrix protein and thrombospondin 1. Purification from articular cartilage, electron microscopic structure, and chondrocyte binding.

Cartilage oligomeric matrix protein (COMP) and thrombospondin 1 (TSP1) were purified in a native form from normal bovine articular cartilage. The key step in the purification scheme was selective extraction with EDTA-containing buffer. Final separation of these two molecules was achieved by heparin affinity chromatography. Particles viewed by electron microscopy after rotary shadowing and negative staining revealed structures similar to their prototype molecules; from the Swarm rat chondrosarcoma for COMP, or from platelets for TSP1. Attachment of primary bovine chondrocytes to purified matrix proteins was investigated. Cells attached to COMP but not to the structurally related TSP1 indicating separate functions for these proteins in cartilage.

Amino Acid Sequence↗
PubMed1990

Fat transfer for aging skin: technique for rhytids.

Dermatologic surgeons are familiar with many surgical and nonsurgical techniques for the reduction of facial rhytids. Injectable collagen, silicone, and fibril are commonly employed as injectable filler materials. Properly done, fat transfer is another valuable tool for use in facial restoration. Fat transfer is an excellent technique for intervention in the aging process, and is easily managed as an office procedure. We will review the near-century-old history of fat grafting and outline the structural dynamics in the process of facial aging. Our experience from the last 50 fat transfer procedures performed will be reviewed with specific attention to patient selection, extraction and injection technique, complications, and overall results.

Adipose Tissue↗
PubMed1996

Molecular and immunological characterization of the major outer membrane proteins of Brucella.

The major outer membrane proteins (OMPs) of Brucella spp. were initially identified in the early 1980s by selective extraction techniques and classified according to their apparent molecular mass as 36-38 kDa OMPs or group 2 porin proteins and 31-34 kDa and 25-27 kDa OMPs which belong to the group 3 proteins. Variation in apparent molecular mass is essentially due to association with peptidoglycan subunits of different sizes. Two genes, omp2a and omp2b, which are closely linked in the Brucella genome, and which share a great degree of homology (> 85%), encode the 36 kDa porin proteins, now named Omp2a and Omp2b proteins respectively. Two genes code for the group 3 OMPs and are named omp25 and omp31. The predicted amino acid sequences of omp25 and omp31 share 34% identity. Furthermore, all Brucella major OMPs share amino acid sequence homology with the major OMPs RopA or RopB of Rhizobium leguminosarum, which supports the close genetic relationship of brucellae with members of the alpha-2 subdivision of the class Proteobacteria. Another characteristic common to the major OMPs of R. leguminosarum and Brucella is that they are tightly, probably covalently, associated with the peptidoglycan. The major OMP genes display diversity among Brucella species, biovars and strains allowing their differentiation, and the polymorphic markers identified have brought new insights into the evolutionary development of the genus Brucella, antigenic variability of brucellae, and future prospects in the field of vaccine development.

Antigenic Variation↗
PubMed1980

Radioimmunoassay for doxepin and desmethyldoxepin.

A simple and sensitive radioimmunoassay (RIA) for the determination of doxepin and desmethyldoxepin in plasma or serum has been developed using a previously reported antiserum to the tricyclic anti-depressants. Before assay, doxepin is separated from desmethyldoxepin with selective extraction at different pH values enabling each to be measured specifically. 3H-imipramine is used as tracer. By using the extraction procedure doxepin and desmethyldoxepin concentrations can be measured down to 9 nmol/1 from a 0.1 ml sample. If necessary, sensitivity can be doubled by taking a 0.2 ml sample to extraction. Recoveries of doxepin and desmethyldoxepin were quantitative when the drugs were added at different concentrations to normal, pooled human plasma and the inter- and intra-assay coefficients of variation did not exceed 9%. The concentrations obtained from patient samples by the present RIA correlated well with those by high-pressure liquid chromatography. The RIA was also shown to be useful in pharmacokinetic single dose studies with doxepin.

Antibody Specificity↗
PubMed1976

Methods for study of fluphenazine kinetics in man.

Fluphenazine and its principal metabolites, fluphenazine sulphoxide, and 7-hydroxyfluphenazine were identified and quantified in human plasma, urine and faeces following intramuscular and oral administration of 14C-fluphenazine dihydrochloride. The presence of a conjugate fraction was also noted. Unmetabolized fluphenazine was selectively extracted into n-heptane. The metabolites were separated by solvent extraction into toluene. Conjugates were hydrolysed back to fluphenazine, fluphenazne sulphoxide and 7-hydroxyfluphenazine. Fluphenazine and fluphenazine conjugates were also measured in the urine of patients receiving long term non-radioactive fluphenazine decanoate therapy. The urinary excretion rate of the conjugate fraction was systematically related to the plasma concentration, regardless of urine flow rate or pH, providing a convenient method for the assessment of fluphenazine kinetics by urinary excretion studies not involving administration of labelled drug.

Administration, Oral↗
PubMed1977

The identification and analysis of mexiletine and its metabolic products in man.

Sensitive and specific gas-liquid chromatographic methods were developed for the analysis of mexiletine and its metabolites in urine of man. The identity of the g.l.c. peaks was established by mass-spectrometry. The hydroxylamine (Va) was qualitatively identified and determined quantitatively after conversion to the more stable oxime (Vb). Selective extraction procedures, t.l.c. and derivatization with hexamethyldisilazane (HMDS) and trifluoroacetic anhydride (TFA) were used in the qualitative identification of the major metabolites (VI-IX), particularly in distinguishing the basic products VI and VII from their corresponding alcoholic products VIII and IX. The limit of detection of the g.l.c. method was 6 to 12 ng ml-1 for compounds I-IV, and 40 to 50 ng ml-1 for compounds Vb-IX.

Chromatography, Gas↗
PubMed1964

HISTONE STAINING WITH AMMONIACAL SILVER.

Under controlled conditions, ammoniacal silver (A-S) stains the bands of Dipteran salivary gland chromosomes in a precise and selective manner. Such staining is dependent upon the histone content of the band, as shown by the effects of selective extraction and blocking of the histones. The effects of qcid extraction and HONO solution on the A-S staining of different bands suggest that specific genes may have particular histones associated with them.

Ammonium Compounds↗
PubMed2001

Antigenic characterization of the fish pathogen Flavobacterium psychrophilum.

Flavobacteria are a poorly understood and speciated group of commensal bacteria and opportunistic pathogens. The psychrotroph Flavobacterium psychrophilum is the etiological agent of rainbow trout fry syndrome and bacterial cold water disease, septicemic diseases that heavily impact salmonids. Consequently, two verified but geographically diverse isolates were characterized phenotypically and biochemically. A facile typing system was devised which readily discriminated between closely related species and was verified against a pool of recent prospective isolates. F. psychrophilum was found to be enveloped in a loosely attached, strongly antigenic outer layer comprised of a predominant, highly immunogenic, low-molecular-mass carbohydrate antigen as well as several protein antigens. Surface-exposed antigens were visualized by a combination of immunoflourescence microscopy, immunogold transmission, and thin-section electron microscopy and were discriminated by Western blotting using rabbit antisera, by selective extraction with EDTA-polymyxin B agarose beads, and by extrinsic labeling of amines with sulfo-N-hydoxysuccinimide-biotin and glycosyl groups with biotin hydrazide. The predominant approximately 16 kDa antigen was identified as low-molecular-mass lipopolysaccharide (LPS), whereas high-molecular-mass LPS containing O antigen was not as prevalent on whole cells but was abundant in culture supernatants. Rainbow trout convalescent antisera recognized both molecular mass classes of LPS as well as a predominant approximately 20-kDa protein. This study represents the first description at the molecular level of the surface characteristics and potential vaccine targets of confirmed F. psychrophilum strains.

Animals↗
PubMed1990

Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins.

The pathogenic spirochete Borrelia burgdorferi contains a set of integral membrane proteins which were selectively extracted into the detergent phase upon solubilization of intact B. burgdorferi with the nonionic detergent Triton X-114. Virtually all of these hydrophobic proteins were recognized by antibodies in pooled sera from patients with chronic Lyme arthritis, demonstrating that proteins partitioning into the detergent phase of Triton X-114 encompass the major B. burgdorferi immunogens. Furthermore, most of these immunogenic proteins, including the previously characterized OspA and OspB membrane antigens, could be biosynthetically labeled when B. burgdorferi was incubated in vitro with [3H]palmitate. The OspA and OspB antigens were radioimmunoprecipitated from [3H]palmitate-labeled detergent-phase proteins with monoclonal antibodies, and [3H]palmitate was recovered unaltered from these proteins after sequential alkaline and acid hydrolyses. The combined results provide formal confirmation that the major B. burgdorferi immunogens extracted by Triton X-114 are lipoproteins. The demonstration that B. burgdorferi integral membrane antigens are lipoproteins may explain the basis of their immunogenicity and may help to improve our understanding of the surface topology of B. burgdorferi.

Antigens, Bacterial↗
PubMed1994

Purification, pore-forming ability, and antigenic relatedness of the major outer membrane protein of Shigella dysenteriae type 1.

The major outer membrane protein (MOMP), the most abundant outer membrane protein, was purified to homogeneity from Shigella dysenteriae type 1. The purification method involved selective extraction of MOMP with sodium dodecyl sulfate in the presence of 0.4 M sodium chloride followed by size exclusion chromatography with Sephacryl S-200 HR. MOMP was found to form hydrophilic diffusion pores by incorporation into artificial liposome vesicles composed of egg yolk phosphatidylcholine and dicetylphosphate, indicating that MOMP of S. dysenteriae type 1 exhibited significant porin activity. However, the liposomes containing heat-denatured MOMP were barely active. The molecular weight of MOMP found by size exclusion chromatography was 130,000, and in sodium dodecyl sulfate-10% polyacrylamide gel it moved as an oligomer of 78,000 molecular weight. Upon boiling, fully dissociated monomers of 38,000 molecular weight were seen for S. dysenteriae type 1. However, among the four Shigella spp., the monomeric MOMP generated upon boiling ranged from 38,000 to 35,000 in molecular weight. Antibody raised in BALB/c mice immunized with MOMP of S. dysenteriae type 1 reacted strongly with purified MOMP of S. dysenteriae type 1 in an enzyme-linked immunosorbent assay (ELISA). The antibody reacted with whole-cell preparations of S. dysenteriae type 1 in an ELISA, suggesting that MOMP possessed surface components. Moreover, MOMP could be visualized on the bacterial surface by immunoelectron microscopy with anti-MOMP antibody. S. dysenteriae type 1 MOMP-specific immunoglobulin eluted from MOMP bound to a nitrocellulose membrane was found to cross-react with MOMP preparations of S. flexneri, S. boydii, and S. sonnei, indicating that MOMPs were antigenically related among Shigella species. The strong immunogenicity, surface exposure, and antigenic relatedness make MOMP of Shigella species an immunologically significant macromolecule for study.

Animals↗
PubMed2002

Plasma membrane cholesterol modulates cellular vacuolation induced by the Helicobacter pylori vacuolating cytotoxin.

The Helicobacter pylori vacuolating cytotoxin (VacA) induces the degenerative vacuolation of mammalian cells both in vitro and in vivo. Here, we demonstrate that plasma membrane cholesterol is essential for vacuolation of mammalian cells by VacA. Vacuole biogenesis in multiple cell lines was completely blocked when cholesterol was extracted selectively from the plasma membrane by using beta-cyclodextrins. Moreover, increasing plasma membrane cholesterol levels strongly potentiated VacA-induced vacuolation. In contrast, inhibiting de novo biosynthesis of cholesterol with lovastatin or compactin had no detectable effect on vacuolation. While depletion of plasma membrane cholesterol has been shown to interfere with both clathrin-mediated endocytosis and caveola-dependent endocytosis, neither of these two internalization pathways was found to be essential for vacuolation of cells by VacA. Depleting plasma membrane cholesterol attenuated the entry of VacA into HeLa cells. In addition, beta-cyclodextrin reagents blocked vacuolation of cells that were either preloaded with VacA or had VacA directly expressed within the cytosol. Collectively, our results suggest that plasma membrane cholesterol is important for both the intoxication mechanism of VacA and subsequent vacuole biogenesis.

Animals↗
PubMed1972

Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium.

A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction.

Bacillus megaterium↗
PubMed1977

Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant.

nov-12, a novobiocin-resistant mutant of Bacillus licheniformis ATCC 9945, grows as long chains of cells, a characteristic of autolytic-deficient (Lyt-) mutants. Isolated walls from nov-12 autolyzed at a rate equal to 5% of that displayed by wild-type walls, thus confirming the Lyt- phenotype. Protein-free nov-12 walls displayed marked resistance to, and also failure to bind, added autolysin solubilized from wild-type walls. Comparison of isolated cell walls revealed a deficiency in teichuronic acid in the mutant. Lesser differences were observed in walls of this strain, including a reduction in galactose, an increase in the proportion of peptidoglycan, and small quantitative differences in peptidoglycan composition though the proportions of protein and teichoic acid were similar in walls of both strains. Autolytic sensitivity was studied in walls in which protein, teichoic acid, and teichuronic acid were removed successively by selective extraction procedures. Autolysis of wild-type walls was unaffected by removal or protein or teichoic acid, but teichuronic acid removal rendered wild-type walls as insensitive to autolysis as mutant walls had been throughout. Therefore, in this mutant, deficiency in teichuronic acid alone leads to the Lyt- phenotype and, hence, activity and binding of autolysin(s) are dependent upon teichuronic acid but not teichoic acid. Also, the potential rate of autolysis of cell walls in this organism was correlated with the proportion of teichuronic acid in the wall. The possible significance of these findings with respect to control of autolysis and cell separation is discussed.

Acetylgalactosamine↗
PubMed1981

Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida.

Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.

Aeromonas↗