The heparin-protein complex of ox liver capsule. Isolation and chemical characterization.
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ABSTRACT:
SUBMITTER: Serafini-Fracassini A
PROVIDER: S-EPMC1187689 | biostudies-other | 1969 Apr
REPOSITORIES: biostudies-other
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Project description:(1) Polysaccharides were isolated from bovine liver capsule by extraction with 2m-potassium chloride followed by precipitation from 0.8m-potassium chloride with cetylpyridinium chloride. Chondroitin sulphate was eliminated by digestion with hyaluronidase. The yield of heparin was approx. 40% of that obtained after extraction of the papain-digested tissue. (2) The macromolecular properties of the hyaluronidase-digested polysaccharide were studied by gel chromatography on Sephadex G-200 of the intact, as well as of the alkali-degraded, material. The results suggested the presence of single heparin chains in addition to a dermatan sulphate proteoglycan. (3) A purified heparin preparation was analysed for amino acids and neutral sugars. Xylose, galactose and serine were found in amounts corresponding to 0.1, 0.2, and 0.4 residue/polysaccharide chain (mol.wt. 7400), respectively. It is suggested that the isolated material had been degraded by a polysaccharidase with endo-enzyme properties.
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Project description:Cryptococcus neoformans is a pathogenic fungus which most commonly affects the central nervous system and causes fatal meningoencephalitis primarily in patients with AIDS. This fungus produces a thick extracellular polysaccharide capsule which is well recognized as a virulence factor. Here, we describe the isolation and characterization of a novel gene, CAP10, which is required for capsule formation. Complementation of the acapsular cap10 mutant produced an encapsulated strain and the deletion of CAP10 from a wild strain resulted in an acapsular phenotype. The molecular mass of the hemagglutinin epitope-tagged Cap10p is about 73 kDa, which is similar to the size predicted from sequence analysis. When CAP10 was fused with a hybrid green fluorescent protein construct, the fluorescence signals appeared as patches in the cytoplasm. Using a reporter gene construct, we found that CAP10 was expressed at high levels in late-stationary-phase cells. In addition, we found that the expression levels of CAP10 are modulated by the transcriptional factor STE12alpha. Deletion of STE12alpha downregulated the expression levels of CAP10 while overexpression of STE12alpha upregulated the expression levels of CAP10. Animal model studies indicate that deletion of the CAP10 gene results in the loss of virulence, and complementation of the acapsular phenotype of cap10 restores virulence. Thus, CAP10 is required for capsule formation and virulence.
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Project description:The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component αT-GTP and β1γ1 subunit complex. Structural information for the Rho*-GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a GαT/Gαi1 chimera (αT*) and β1γ1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to GαT* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β2-adrenergic receptor-GS complex, including a flexible αT* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex.
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Project description:Glycosaminoglycans were extracted from bovine liver capsule with 4 M-guanidinium chloride, resulting in solubilization of approx. 90% of the total uronic acid-containing polysaccharide of the tissue. The extracted polysaccharide was purified and fractionated by anion-exchange chromatography on DEAE-cellulose, density-gradient ultracentrifugation in CsCl and finally gel chromatography on Sepharose 4B. By using these procedures, the two major polysaccharide components, dermatan sulphate and heparin, which constituted 55 and 30% respectively of the total glycosaminoglycan content of the tissue, were separated from each other. Analysis of the macromolecular properties of the two polysaccharides showed that heparin existed exclusively as single polysaccharide chains, whereas dermatan sulphate occurred largely as a proteoglycan (protein content, 74% dry wt.). The purified heparin preparation was subjected to sedimentation-equilibrium ultracentrifugation, indicating a molecular weight of 8800. Analysis for neutral sugars (by g.l.c.) showed 0.1 residue of xylose and 0.2 residue of galactose/polysaccharide chain; serine amounted to 0.3 residue/polysaccharide chain. Reduction of the heparin with NaB3H4 resulted in incorporation of 3H, approximately corresponding to one reducible group/polysaccharide chain. The 3H-labelled sugar residue was liberated by a combination of acid hydrolysis and deaminative cleavage of the polysaccharide with HNO2; it was subsequently identified as an aldonic acid by paper electrophoresis. Most of the heparin chains thus contained a uronic acid residue in reducing position. It is suggested that heparin isolated from bovine liver capsule is a degradation product released from larger molecules by an endo-glycuronidase.
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Project description:The synthesis of bona fide organometallic CeIV complexes is a formidable challenge given the typically oxidizing properties of the CeIV cation and reducing tendencies of carbanions. Herein, we report a pair of compounds comprising a CeIV - Caryl bond [Li(THF)4][CeIV(κ2-ortho-oxa)(MBP)2] (3-THF) and [Li(DME)3][CeIV(κ2-ortho-oxa)(MBP)2] (3-DME), ortho-oxa = dihydro-dimethyl-2-[4-(trifluoromethyl)phenyl]-oxazolide, MBP2- = 2,2'-methylenebis(6-tert-butyl-4-methylphenolate), which exhibit CeIV - Caryl bond lengths of 2.571(7) - 2.5806(19) Å and strongly-deshielded, CeIV - Cipso 13C{1H} NMR resonances at 255.6 ppm. Computational analyses reveal the Ce contribution to the CeIV - Caryl bond of 3-THF is ~12%, indicating appreciable metal-ligand covalency. Computations also reproduce the characteristic 13C{1H} resonance, and show a strong influence from spin-orbit coupling (SOC) effects on the chemical shift. The results demonstrate that SOC-driven deshielding is present for CeIV - Cipso 13C{1H} resonances and not just for diamagnetic actinide compounds.
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Project description:Staphylococcus epidermidis releases factors that activate the HIV-1 long terminal repeat, induce cytokine release, and activate nuclear factor B in cells of macrophage lineage. The active material had a mass of 34,500 daltons, was inactivated by proteases and partitioned into the phenol layer on hot aqueous phenol extraction, and thus was termed phenol-soluble modulin (PSM). High performance liquid chromatography (HPLC) of crude PSM yielded two peaks of activity designated PSM peak 1 and peak 2. MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) mass spectroscopy indicated the presence of two components in peak 1, which were designated PSM and PSM. Peak 2 contained a single component, designated PSM. Separation of PSM and PSM in peak 1 could be achieved by a second HPLC procedure. The structure of each component was determined by amino acid sequence analysis and identification and sequencing of their genes. PSM, PSM, and PSM were 22-, 44-, and 25-amino acid, respectively, strongly hydrophobic polypeptides. PSM was identified as Staphylococcus epidermidis delta toxin, whereas PSM and PSM exhibited more distant homology to previously described staphylococcal toxins. They appeared to exist as a complex or aggregate with activity greater than the component parts. The properties of the S. epidermidis PSMs suggest that they may contribute to the systemic manifestations of Gram-positive sepsis.
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Project description:Mutations in amyloid precursor protein (APP) are associated with familial Alzheimer's disease. Recent development suggests that homo- and heterodimerization of APP and APP-like proteins (APLPs), which are enhanced by heparan sulfate binding, may play a role in signal transduction and cell adhesion. Despite efforts to model heparin binding based on known apo crystal structures, the mechanism of heparin-induced APP/APLP dimerization has not been established experimentally. Here we report the crystal structure of a complex between heparin and the E2 domain of APLP1, which harbors the conserved high affinity heparin binding site of the full-length molecule. Within the asymmetric E2:heparin complex, the polysaccharide is snugly bound inside a narrow groove between the two helical subdomains of one protein protomer. The nonreducing end of the sugar is positioned near the protein's 2-fold axis, making contacts with basic residues from the second protomer. The inability of the E2 dimer to accommodate two heparin molecules near its symmetry axis explains the observed 21 binding stoichiometry, which is confirmed by isothermal titration calorimetric experiment carried out in solution. We also show that, at high concentrations, heparin can destabilize E2 dimer, probably by forcing into the unoccupied binding site observed in the 21 complex. The binding model suggested by the crystal structure may facilitate the design of heparin mimetics that are capable of modulating APP dimerization in cells.
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Project description:Freshly isolated intact ox neurofilaments have been incubated with copper(II)-o-phenanthroline complex to induce thiol cross-linking between the two largest (apparent Mr 205 000 and 158 000) polypeptide components. Subsequent tryptic digestion shows that the thiol bonds formed between these polypeptides are distributed exclusively among 'rod-domain' fragments that remain associated with intact sedimentable filaments. These observations suggest that the polypeptide chains of the two largest neurofilament components are closely arranged within the backbone but are separate from one another in more peripheral regions. Soluble protofilaments derived from neurofilament disassembly at low ionic strength and high pH have also been cross-linked via thiol bonds in order to determine the polypeptide arrangement within these structures. All three neurofilament polypeptides cross-link more readily when in the form of protofilaments than when in the form of fully assembled filaments, and the pattern of cross-linked complexes formed is different. Analysis of one of these complexes shows that at least some of the protofilaments are composed of oligomers containing both the 72 000- and the 158 000-Mr neurofilament polypeptides arranged in close proximity.
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Project description:Two Klebsiella bacteriophages K5-2 and K5-4, which are able to infect and grow on either capsular types K30/K69 and K5 or K8 and K5 of Klebsiella strains, were isolated and characterized. Each phage contained two open reading frames (ORFs), which encoded two putative capsule depolymerases, respectively. The first ORF encoded tail fiber proteins, which have K30/K69 depolymerase and K8 depolymerase activities. The second ORF encoded hypothetical proteins, which are almost identical in amino acid sequences, and have K5 depolymerase activity. Alcian blue staining of enzyme-treated capsular polysaccharides (CPS) showed that purified depolymerases can cleave purified Klebsiella CPS in vitro and liberate monosaccharaides. Capsule K5 deletion mutants were not lysed by either phage, suggesting that the capsule was essential for phage infection. Bacterial killing was observed when incubated Klebsiella strains with phages but not with purified depolymerases. Treatment with the K5-4 phage significantly increased the survival of mice infected with a K. pneumoniae K5 strain. In conclusion, two dual host-specific Klebsiella phages and their tailspikes exhibit capsule depolymerase activity were characterized. Each phage and phage-encoded depolymerase has specificity for capsular type K30/K69, K8 or K5, and could be used for the typing and treatment of K. pneumoniae infection.
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Project description:Bamboo shavings, the outer or intermediate layer of bamboo stems, are the bulk of by-products produced in bamboo processing. In this study we investigated the isolation, chemical characterization, and immunostimulatory activity in vitro of the hemicelluloses from bamboo shavings. Shavings were first pretreated by steam explosion. The optimal pretreatment was found to be steam explosion at 2.2 MPa for 1 min. Following this pretreatment, the yield of hemicelluloses reached (2.05±0.22)% (based on the dry dewaxed raw materials), which was 5.7-fold higher than that of untreated samples. Bamboo-shavings hemicellulose (BSH) was then prepared by hot water extraction and ethanol precipitation from the steam-exploded shavings. Purification of BSH by anion-exchange chromatography of diethylaminoethanol (DEAE)-sepharose Fast Flow resulted in a neutral fraction (BSH-1, purity of 95.3%, yield of 1.06%) and an acidic fraction (BSH-2, purity of 92.5%, yield of 0.79%). The weight-average molecular weights (Mw) of BSH-1 and BSH-2 were 12 800 and 11 300 g/mol, respectively. Chemical and structural analyses by Fourier transform infrared spectroscopy (FT-IR), 1D (1H and 13C) and 2D (heteronuclear single quantum correlation (HSQC)) nuclear magnetic resonance (NMR) spectra revealed that BSH-1 was O-acetylated-arabinoxylan and BSH-2 was O-acetylated-(4-O-methylglucurono)-arabinoxylan. BSH-1 had a higher content of acetyl groups than BSH-2. For the immunomodulatory activity in vitro, BSH and BSH-2 significantly stimulated mouse splenocyte proliferation while BSH-1 had no effect; BSH, BSH-1, and BSH-2 markedly enhanced the phagocytosis activity and nitric oxide production of the murine macrophage RAW264.7 in a dose-dependent manner. Our results suggest that the water-extractable hemicelluloses from steam-exploded bamboo shavings are naturally acetylated and have immunostimulatory activity.
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