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PubMed1986

Isolation of ntrA-like mutants of Azotobacter vinelandii.

A number of chlorate-resistant mutants of Azotobacter vinelandii affected in a general control of nitrogen metabolism were isolated. These mutants could not utilize dinitrogen, nitrate, or nitrite as a nitrogen source. The reason for this inability is that they were simultaneously deficient in nitrogenase and nitrate and nitrite reductase activities. They were complemented by a cosmid carrying a DNA fragment of A. vinelandii able to complement ntrA mutants of Escherichia coli, so they seemed to be ntrA-like mutants.

Azotobacter↗

PubMed1998

Physiological and genetic analyses leading to identification of a biochemical role for the moeA (molybdate metabolism) gene product in Escherichia coli.

A unique class of chlorate-resistant mutants of Escherichia coli which produced formate hydrogenlyase and nitrate reductase activities only when grown in medium with limiting amounts of sulfur compounds was isolated. These mutants failed to produce the two molybdoenzyme activities when cultured in rich medium or glucose-minimal medium. The mutations in these mutants were localized in the moeA gene. Mutant strains with polar mutations in moeA which are also moeB did not produce active molybdoenzymes in any of the media tested. moeA mutants with a second mutation in either cysDNCJI or cysH gene lost the ability to produce active molybdoenzyme even when grown in medium limiting in sulfur compounds. The CysDNCJIH proteins along with CysG catalyze the conversion of sulfate to sulfide. Addition of sulfide to the growth medium of moeA cys double mutants suppressed the MoeA- phenotype. These results suggest that in the absence of MoeA protein, the sulfide produced by the sulfate activation/reduction pathway combines with molybdate in the production of activated molybdenum. Since hydrogen sulfide is known to interact with molybdate in the production of thiomolybdate, it is possible that the MoeA-catalyzed activated molybdenum is a form of thiomolybdenum species which is used in the synthesis of molybdenum cofactor from Mo-free molybdopterin.

Bacterial Proteins↗

PubMed2004

Hot spot for a large deletion in the 18- to 19-centisome region confers a multiple phenotype in Salmonella enterica serovar Typhimurium strain ATCC 14028.

Loss of the Salmonella MsbB enzyme, which catalyzes the incorporation of myristate destined for lipopolysaccharide in the outer membrane, results in a strong phenotype of sensitivity to salt and chelators such as EGTA and greatly diminished endotoxic activity. MsbB- salmonellae mutate extragenically to EGTA-tolerant derivatives at a frequency of 10(-4) per division. One of these derivatives arose from inactivation of somA, which suppresses sensitivity to salt and EGTA. Here we show that a second mode of MsbB- suppression is a RecA-dependent deletion between two IS200 insertion elements present in Salmonella enterica serovar Typhimurium strain ATCC 14028 but not in two other wild-type strains, LT2 and SL1344, which lack one of the IS200 elements. This deletion occurs spontaneously in wild-type and MsbB- strain 14028 salmonellae and accounts for about one-third of all of the spontaneous suppressors of MsbB- in strain 14028. It spans the region corresponding to 17.7 to 19.9 centisomes, which includes somA, on the sequenced map of Salmonella LT2 (136 ORFs in that strain; ATCC 14028 and other strains showed variability in this region). In addition to conferring EGTA resistance correlated with somA, the deletion confers a MacConkey galactose resistance phenotype on MsbB- Salmonella, indicating that at least one additional gene (distinct from somA) within the deletion is responsible for this phenotype. In the wild type, the deletion mutant grows with normal exponential growth rate in Luria broth but is chlorate resistant and does not grow on citrate agar. The deletion strains have lost hydrogen sulfide production, nitrate reductase activity, and gas production from glucose fermentation.

Acyltransferases↗

PubMed2006

Identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity.

It has been shown previously that the fusion glycoprotein of human respiratory syncytial virus (RSV-F) interacts with cellular heparan sulfate. Synthetic overlapping peptides derived from the F-protein sequence of RSV subtype A (strain A2) were tested for their ability to bind heparin using heparin-agarose affinity chromatography (HAAC). This evaluation identified 15 peptides representing eight linear heparin-binding domains (HBDs) located within F1 and F2 and spanning the protease cleavage activation site. All peptides bound to Vero and A549 cells, and binding was inhibited by soluble heparins and diminished by either enzymatic treatment to remove cell surface glycosaminoglycans or by treatment with sodium chlorate to decrease cellular sulfation. RSV-F HBD peptides were less likely to bind to glycosaminoglycan-deficient CHO-745 cells than parental CHO-K1 cells that express these molecules. Three RSV-F HBD peptides (F16, F26, and F55) inhibited virus infectivity; two of these peptides (F16 and F55) inhibited binding of virus to Vero cells, while the third (F26) did not. These studies provided evidence that two of the linear HBDs mapped by peptides F16 and F55 may mediate one of the first steps in the attachment of virus to cells while the third, F26, inhibited infectivity at a postattachment step, suggesting that interactions with cell surface glycosaminoglycans may play a role in infectivity of some RSV strains.

Amino Acid Sequence↗

PubMed2000

Glycosaminoglycan sulfation requirements for respiratory syncytial virus infection.

Glycosaminoglycans (GAGs) on the surface of cultured cells are important in the first step of efficient respiratory syncytial virus (RSV) infection. We evaluated the importance of sulfation, the major biosynthetic modification of GAGs, using an improved recombinant green fluorescent protein-expressing RSV (rgRSV) to assay infection. Pretreatment of HEp-2 cells with 50 mM sodium chlorate, a selective inhibitor of sulfation, for 48 h prior to inoculation reduced the efficiency of rgRSV infection to 40%. Infection of a CHO mutant cell line deficient in N-sulfation was three times less efficient than infection of the parental CHO cell line, indicating that N-sulfation is important. In contrast, infection of a cell line deficient in 2-O-sulfation was as efficient as infection of the parental cell line, indicating that 2-O-sulfation is not required for RSV infection. Incubating RSV with the purified soluble heparin, the prototype GAG, before inoculation had previously been shown to neutralize its infectivity. Here we tested chemically modified heparin chains that lack their N-, C6-O-, or C2-O-sulfate groups. Only heparin chains lacking the N-sulfate group lost the ability to neutralize infection, confirming that N-sulfation, but not C6-O- or C2-O-sulfation, is important for RSV infection. Analysis of heparin fragments identified the 10-saccharide chain as the minimum size that can neutralize RSV infectivity. Taken together, these results show that, while sulfate modification is important for the ability of GAGs to mediate RSV infection, only certain sulfate groups are required. This specificity indicates that the role of cell surface GAGs in RSV infection is not based on a simple charge interaction between the virus and sulfate groups but instead involves a specific GAG structural configuration that includes N-sulfate and a minimum of 10 saccharide subunits. These elements, in addition to iduronic acid demonstrated previously (L. K. Hallak, P. L. Collins, W. Knudson, and M. E. Peeples, Virology 271:264-275, 2000), partially define cell surface molecules important for RSV infection of cultured cells.

Animals↗

PubMed2002

A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland.

Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.

Acids↗

PubMed2000

Expression of N-deacetylase/sulfotransferase and 3-O-sulfotransferase in rat alveolar type II cells.

Basal laminae beneath alveolar type I cells are suggested to contain highly sulfated heparan sulfate-containing proteoglycans (PGs), and cultured type II cells accumulate highly sulfated matrices. To characterize the regulation of PG synthesis during the transition from type II cells to type I cells, we examined mRNA expression of N-deacetylase/sulfotransferase (NST) and 3-O-sulfotransferase (3-OST), two enzymes specific for heparan sulfate synthesis. We found that both freshly isolated and cultured type II cells expressed NST and 3-OST as shown by in situ hybridization. Expression of surfactant-associated protein A, B, and C mRNAs, determined by semiquantitative PCR, decreased during culture. Expression of type I cell marker T1alpha mRNA increased except in cells cultured on an Engelbrecht-Holm-Swarm gel. Expression of NST was dependent on cell density and matrix and was intense in conditions where cells spread fully, whereas 3-OST expression was unchanged in the conditions examined. The PG sulfation inhibitor sodium chlorate significantly inhibited cultured type II cell spreading, and this inhibition was reversed by sodium sulfate. These results suggest that highly sulfated PGs modified by NST are necessary for the spreading of cells during transdifferentiation of type II cells to mature type I cells.

Amidohydrolases↗

PubMed2004

Chondroitin sulfate in palatal wound healing.

Chondroitin sulfate is up-regulated in granulation tissue during wound healing. To investigate the role of chondroitin sulfate in the wound-healing process after surgical repair of cleft palate, we isolated and cultured rabbit palatal fibroblasts. Treatment with chondroitin-6-sulfate resulted in a dose-dependent increase in cell adhesion and cell proliferation, whereas the reverse effects were seen after chondroitinase degradation of chondroitin sulfate. The biological actions of chondroitin sulfate appeared to be dependent on the presence and position of sulfate groups. Inhibition of glycosaminoglycan sulfation by chlorate treatment led to reduced cell adhesion and cell proliferation and a slower rate of wound closure in vitro. Furthermore, exposure to chondroitin-4-sulfate resulted in a dose-dependent reduction in cell adhesion. Together, these results show that chondroitin sulfate is involved in palatal wound healing.

Analysis of Variance↗

PubMed2003

Glycosaminoglycans in human retinoblastoma cells: heparan sulfate, a modulator of the pigment epithelium-derived factor-receptor interactions.

BACKGROUND: Pigment epithelium-derived factor (PEDF) has binding affinity for cell-surface receptors in retinoblastoma cells and for glycosaminoglycans. We investigated the effects of glycosaminoglycans on PEDF-receptor interactions. RESULTS: 125I-PEDF formed complexes with protease-resistant components of medium conditioned by human retinoblastoma Y-79 cells. Using specific glycosaminoglycan degrading enzymes in spectrophotometric assays and PEDF-affinity chromatography, we detected heparin and heparan sulfate-like glycosaminoglycans in the Y-79 conditioned media, which had binding affinity for PEDF. The Y-79 conditioned media significantly enhanced the binding of 125I-PEDF to Y-79 cell-surface receptors. However, enzymatic and chemical depletion of sulfated glycosaminoglycans from the Y-79 cell cultures by heparitinase and chlorate treatments decreased the degree of 125I-PEDF binding to cell-surface receptors. CONCLUSIONS: These data indicate that retinoblastoma cells secrete heparin/heparan sulfate with binding affinity for PEDF, which may be important in efficient cell-surface receptor binding.

Chlorates↗

PubMed1994

Insulin-like growth factor-binding protein-3 (IGFBP-3) concentration in rat Sertoli cell-conditioned medium is regulated by a pathway involving association of IGFBP-3 with cell surface proteoglycans.

Rat Sertoli cells in culture produce insulin-like growth factor-I (IGF-I), and IGF-binding protein-3 (IGFBP-3) is the predominant IGFBP. Previous studies suggested that the concentration of IGFBP-3 in Sertoli cell-conditioned medium is regulated by a clearance pathway involving association of IGFBP-3 with the Sertoli cell surface. The purpose of these studies was to characterize further the nature of the interaction of IGFBP-3 with the cell surface and to examine how this interaction might be regulating IGFBP-3 concentrations in conditioned medium. Recombinant nonglycosylated human IGFBP-3 was added to cultured Sertoli cells derived from 15-day-old rats, and the change in concentration over time in the medium was measured by [125I]IGF-I ligand blot analysis and Western blot analysis using an antiserum specific for human IGFBP-3. Chloroquine, an inhibitor of lysosomal enzymes involved in internalization of proteins and reduction of temperature, inhibited IGFBP-3 reduction in Sertoli cell medium. Soluble heparin, at a half-maximal concentration of approximately 5 microgram/ml, inhibited the decrease in IGFBP-3 in the medium. In addition, soluble heparin decreased the amount of IGFBP-3 detectable on the cell surface, as determined by performing ligand blots on plasma membrane preparations. Finally, pretreatment of Sertoli cells with sodium chlorate, an inhibitor of cell surface proteoglycan sulfation, also retarded the decrease in recombinant IGFBP-3. Taken together, these data suggest that an important mechanism influencing the concentration of IGFBP-3 3 in Sertoli cell-conditioned medium is a pathway involving IGFBP-3 interaction with the cell surface proteoglycans.

Animals↗

PubMed1994

Heparan sulfate proteoglycans as transducers of FGF-2 signalling.

The fibroblast growth factor-2 (FGF-2) low-affinity binding sites, heparan sulfate proteoglycans (HSPGs), function as modulators of FGF-2 activity. It is noteworthy that HSPG binding protects FGF-2 from denaturation and proteolytic degradation, provides a matrix-bound or cell-surface reservoir of this factor for the cells and is required for the activation of FGF high-affinity receptors. In our study we investigated the biological meaning of FGF-2 internalization mediated through its low-affinity binding sites, HSPGs. Using as model system L6 myoblasts lacking endogenous FGF receptors (FGFRs), we demonstrated that these cells internalize FGF-2 efficiently through an HSPG-mediated pathway. FGF-2 internalization occurring through HSPGs was paralleled by an increase in the activity of urokinase plasminogen activator (u-PA). The u-PA-inducing activity of FGF-2 was strictly correlated to its internalization, as chlorate treatment, which causes a strong inhibition of FGF-2 internalization, abrogated the u-PA-inducing activity of FGF-2. In addition, expression of functional FGF high-affinity receptors (FGFR-1) did not enhance u-PA in L6 myoblasts upon FGF-2 stimulation. According to our results we propose that FGF-2 internalization mediated through HSPGs may transduce FGF-2 signalling such as u-PA-activity stimulation. Thus, HSPGs may act as direct transducers of FGF signalling and indeed, different FGF-signalling pathways must exist.

3T3 Cells↗

PubMed1999

Differential regulation of hepatocyte growth factor/scatter factor by cell surface proteoglycans and free glycosaminoglycan chains.

Hepatocyte growth factor interacts with both heparan and dermatan sulphates, in addition to its specific signalling receptor, Met. However, the extent of glycosaminoglycan involvement in its biological activity remains uncertain. We have investigated the effects of exogenous glycosaminoglycan addition upon hepatocyte growth factor-stimulated motility of Madin-Darby canine kidney cells. Exogenous heparan/dermatan sulphate chains behave similarly as either potentiators or inhibitors of cell motility (depending upon the assay). Specific heparan sulphate oligosaccharides, of octasaccharide or larger, elicit similar effects, though with reduced potency. Additionally we have investigated the motility of cells made completely deficient in functional proteoglycans by metabolic inhibition of glycosaminoglycan sulphation, using chlorate. Such cells are completely unresponsive to hepatocyte growth factor, both in terms of downstream phosphorylation of mitogen-activated protein kinase and actual cell motility, though they do remain responsive to phorbol ester. Interestingly, although cell responsiveness to hepatocyte growth factor is not restored by exogenous heparan/dermatan sulphate chains, it is by an immobilised heparan sulphate proteoglycan substratum. These findings suggest that hepatocyte growth factor activity is not only critically dependent upon the presence of glycosaminoglycan, but specifically requires an intact proteoglycan structure located in close apposition to cell surface Met.

Absorption↗

PubMed2002

Syndecan-4 mediates antithrombin-induced chemotaxis of human peripheral blood lymphocytes and monocytes.

Antithrombin inhibits chemokine-induced migration of neutrophils by activating heparan sulfate proteoglycan-dependent signaling. Whether antithrombin affects migration of other types of leukocytes is not known. We investigated the effects of antithrombin on spontaneous and chemokine-triggered migration of lymphocytes and monocytes from human peripheral blood in modified Boyden chamber micropore filter assays. Lymphocyte and monocyte populations from human peripheral blood were purified using magnetic antibody cell sorting. The signaling mechanisms required for antithrombin-dependent migration were studied using signaling enzyme blockers. Expression of heparan sulfate proteoglycan core protein was studied by RT-PCR and flow cytometry. The antithrombins used were Kybernin P from human plasma and a monoclonal-antibody-purified preparation from this plasma. Pretreatment of lymphocytes and monocytes with antithrombin inhibited chemotaxis toward optimal concentrations of interleukin-8 or Rantes (regulated upon activation normal T-cell expressed and activated) at concentrations of antithrombin as low as 10 nU/ml. In the absence of the chemokines, direct exposure of cells to gradients of antithrombin stimulated migration. Effects of antithrombin were abolished by pretreating cells with heparinase-1, chondroitinase, sodium chlorate and anti-syndecan-4 antibodies. Expression of syndecan-4 mRNA and protein in monocytes and lymphocytes was demonstrated in RT-PCR and anti-syndecan-4 immunoreactivity assays, respectively. In the presence of pentasaccharide, antithrombin lost its effect on cells. Data indicate that antithrombin directly inhibits chemokine-stimulated migration of monocytes and lymphocytes via the effects of its heparin-binding site on cell surface syndecan-4 by activation of protein kinase C and Rho signaling.

Antibodies, Monoclonal↗

PubMed2005

Development of a modified positive selection medium that allows to isolate Aspergillus oryzae strains cured of the integrated niaD-based plasmid.

The nitrate reductase gene (niaD) is the most frequently utilized as a selectable marker for homologous integration at the niaD locus of Aspergillus oryzae. In this study we developed a method for curing of the niaD-based plasmid integrated on the A. oryzae genome. Positive selection using a modified chlorate medium containing leucine as a nitrogen source enabled efficient isolation of the strains deficient in nitrate assimilation from the niaD(+) transformant. PCR analysis of the strains confirmed that the homologously integrated plasmid carrying the h2b-egfp fusion gene was cured by intrachromosomal recombination which was accompanied by the loss of the EGFP-fluorescence.

Aspergillus nidulans↗

PubMed1999

Proteoglycan involvement in polyamine uptake.

We have evaluated the possible role of proteoglycans in the uptake of spermine by human lung fibroblasts. Exogenous glycosaminoglycans behaved as competitive inhibitors of spermine uptake, the most efficient being heparan sulphate (Ki=0.16+/-0.04 microM). Treatment of fibroblasts with either heparan sulphate lyase, p-nitrophenyl-O-beta-D-xylopyranoside or chlorate reduced spermine uptake considerably, whereas chondroitin sulphate lyase had a limited effect. Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine resulted in an increase of cell-associated heparan sulphate proteoglycans exhibiting higher affinity for spermine. The data indicate a specific role for heparan sulphate proteoglycans in the uptake of spermine by fibroblasts. Spermine uptake by pgsD-677, a mutant Chinese hamster ovary cell defective in heparan sulphate biosynthesis, was only moderately reduced (20%) compared with wild-type cells. Treatment of mutant cells with the above-mentioned xyloside resulted in a greater reduction of endogenous proteoglycan production as well as a higher inhibition of spermine uptake than in wild-type cells. Moreover, treatment with chondroitin sulphate lyase resulted in a selective inhibition of uptake in mutant cells, indicating a role for chondroitin/dermatan sulphate proteoglycans in the uptake of spermine by these cells. Fibroblasts, made growth-dependent on exogenous spermine by alpha-difluoromethylornithine treatment, were growth-inhibited by heparan sulphate or beta-D-xyloside, which might have future therapeutical implications.

Animals↗

PubMed2000

Cellular degradation of free and inhibitor-bound tissue-type plasminogen activator--requirement for a co-receptor?

The low density lipoprotein receptor-related protein (LRP) is a multiligand clearance receptor that removes free tissue-type plasminogen activator (t-PA) or complexes of t-PA with plasminogen activator inhibitor type 1 (PAI-1) from the blood circulation or the pericellular space. Co-receptors are essential for LRP-mediated clearance of several ligands (e.g. glycosaminoglycans for thrombin/protease nexin and lipoprotein lipase, and the urokinase receptor for urokinase/PAI-1 complexes). The present study was undertaken to investigate whether LRP-mediated t-PA clearance requires a co-receptor as well. In five cell lines from different organs and species degradation of t-PA and t-PA/PAI-1 was mediated by LRP (or LRP-like receptors). No degradation of t-PA and t-PA/PAI-1 occurred in THP-1 or U-937 human monocyte-like cells, despite the presence of functional LRP. As glycosaminoglycans can bind t-PA and PAI-1 we investigated whether they are involved in t-PA/PAI-1 degradation. Pre-treatment of COS cells or HT1080 cells with chlorate, an inhibitor of glycosaminoglycan sulfation, did not decrease t-PA/PAI-1 degradation. Furthermore, CHO cells genetically deficient in glycosaminoglycans efficiently degraded t-PA/PAI-1. Thus it is unlikely that glycosaminoglycans are co-receptors for degradation of t-PA or t-PA/PAI-1. This study indicates that THP-1 and U-937 cells lack a critical component (co-receptor?) for the LRP-mediated degradation of t-PA.

Animals↗

PubMed1990

Sulfation of von Willebrand factor.

von Willebrand factor (vWF) is a multimeric adhesive glycoprotein essential for normal hemostasis. We have discovered that cultured human umbilical vein endothelial cells incorporate inorganic sulfate into vWF. Following immunoisolation and analysis by polyacrylamide or agarose gel electrophoresis, metabolically labeled vWF was found to have incorporated [35S]-sulfate into all secreted multimer species. The time course of incorporation shows that sulfation occurs late in the biosynthesis of vWF, near the point at which multimerization occurs. Quantitative analysis suggests the presence, on average, of one molecule of sulfate per mature vWF subunit. Virtually all the detectable sulfate is released from the mature vWF subunit by treatment with endoglycosidases that remove asparagine-linked carbohydrates. Sulfated carbohydrate was localized first to the N-terminal half of the mature subunit (amino acids 1 through 1,365) by partial proteolytic digestion with protease V8; and subsequently to a smaller fragment within this region (amino acids 273 through 511) by sequential digestions with protease V8 and trypsin. Thus, the carbohydrate at asparagine 384 and/or 468 appears to be the site of sulfate modification. Sodium chlorate, an inhibitor of adenosine triphosphate-sulfurylase, blocks sulfation of vWF without affecting either the ability of vWF to assemble into high molecular weight multimers or the ability of vWF multimers to enter Weible-Palade bodies. The stability of vWF multimers in the presence of an endothelial cell monolayer also was unaffected by the sulfation state. Additionally, we have found that the cleaved propeptide of vWF is sulfated on asparagine-linked carbohydrate.

Chlorates↗

PubMedDate not supplied

[Refinement of the methods of diffractometric analysis in relation to the preparation surface exposed to X rays].

The aim of the study was to assess the degree of X-ray diffractometric response using preparations having a surface exposed to X-rays other than 2 cm2, which is standard for sample-holders supplied with a goniometer, coupled to a commercially available analytic apparatus. Two samples of commercial talc were examined with dust size below 30 microns (and having different content of "respirable" or "alveolar" dust classes), as regards chlorate, talc and asbestos constituents and quartz. Twelve different experimental conditions of the preparations permitted X-ray exposure of surfaces between 6.25 and 500 mm2. For the low angle positions it was possible to obtain a substantial improvement in the overall diffractometric response by increasing the surface of the preparations; however, in each case, a reduction in the surface produced, in addition to a better definition of the peaks, an increase in the diffracted intensities per unit of surface of the preparation; this is clearly related to the collection spectrum of the radiant energy of the incident beam in space, and leads to the possibility of reducing to an order of magnitude of milligrams the mass of samples that can still be submitted to qualitative and quantitative diffractometric analysis.

Asbestos↗