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PubMed1997

Extracellular matrix heparan sulfate modulates endothelial cell susceptibility to Staphylococcus aureus.

The ability of extracellular matrix heparan sulfate to alter the susceptibility of human endothelial cells to S. aureus was investigated. Endothelial cells grown on extracellular matrix synthesized by S. aureus-infected endothelial cells were more susceptible to subsequent staphylococcal infection than endothelial cells grown on the extracellular matrix synthesized by untreated endothelial cells. Endothelial cells were more susceptible to S. aureus infection when 1) grown on heparitinase-treated extracellular matrix that removed heparan sulfate chains, 2) grown on extracellular matrix produced by chlorate-treated endothelial cells that reduced sulfation in the matrix heparan sulfate proteoglycans, 3) grown on heparan sulfate purified from extracellular matrix elaborated by infected endothelial cells, and 4) endothelial cells were chlorate-treated and therefore expressed desulfated cellular heparan sulfate proteoglycans. Extracellular matrix produced by S. aureus-infected endothelial cells contained heparan sulfate proteoglycans with reduced sulfation. The altered extracellular matrix with reduced sulfated heparan sulfate proteoglycans signalled the uninfected endothelial cells to produce under sulfated cellular heparan sulfate proteoglycans that increased S. aureus adherence to the endothelial cells.

Bacterial Adhesion↗

PubMed1998

Glycosaminoglycans bind granulocyte-macrophage colony-stimulating factor and modulate its mitogenic activity and signaling in human osteoblastic cells.

We recently demonstrated that granulocyte-macrophage colony-stimulating factor (GM-CSF) is an autocrine growth factor for human osteoblastic (hOB) cells. Since GM-CSF is a member of the heparin-binding factor family, we examined the interactions between GM-CSF and glycosaminoglycans (GAGs) present in the osteoblast microenvironment. Using a bioassay in which the mitogenic activity of recombinant human (rh) GM-CSF was measured after incubation in the presence of an hOB cell layer or extracellular matrix (ECM) produced by these cells, we showed that rhGM-CSF binds to GAG components present in the ECM and that the bound rhGM-CSF retains its ability to stimulate hOB cell proliferation. Heparan sulfate compounds on the hOB cell surface were also found to sequester GM-CSF. Moreover, treatment with sodium chlorate, an inhibitor of GAG sulfation, suppressed the mitogenic activity of rhGM-CSF on hOB cells. This inhibitory effect was rescued by a low dose of heparin. Heparin was also found to promote the effect of rhGM-CSF on hOB cell proliferation, allowing nonmitogenic high doses of rhGM-CSF to stimulate hOB cell growth. Western blot analysis showed that undersulfation of cellular GAGs by chlorate inhibited the increased tyrosine phosphorylation of proteins involved in GM-CSF signaling in cloned immortalized hOB cells. The data demonstrate that GM-CSF binds to proteoglycans on the hOB cell surface and in ECM produced by these cells and that the bound GM-CSF is biologically active. Furthermore, this study shows that cellular proteoglycans play an essential role in GM-CSF signaling and biological activity in hOBs.

Cell Division↗

PubMed1994

Amphiregulin-dependent proliferation of cultured human keratinocytes: autocrine growth, the effects of exogenous recombinant cytokine, and apparent requirement for heparin-like glycosaminoglycans.

Amphiregulin, a member of the epidermal growth factor family with heparin binding affinity, functions as a natural regulator of keratinocyte growth. Autocrine signaling by amphiregulin and the effects of exogenous recombinant cytokine were studied in serum-free cultures of human neonatal keratinocytes. A metabolic inhibitor of proteoglycan sulfation was used to assess the role of cellular heparin-like glycosaminoglycans in amphiregulin-dependent growth. Keratinocytes plated at > 10(3) cells/cm2 grew in an autocrine manner in the absence of exogenous epidermal growth factor or amphiregulin. Incubation of keratinocytes with an amphiregulin-blocking antibody indicated that approximately 70% of autocrine growth is mediated by endogenous amphiregulin. Proliferation potential in the presence of recombinant human amphiregulin was dose dependent and saturable and above approximately 1 ng/ml was comparable to that achieved with similar concentrations of epidermal growth factor. Sodium chlorate, which blocks glycosaminoglycan sulfation, reversibly inhibited epidermal growth factor-dependent proliferation by 42%, exogenous amphiregulin-dependent proliferation by 75%, and autocrine growth by 95%; concurrent incubation with 1-100 micrograms/ml heparin partially reversed this inhibition. Exogenous heparin in the absence of chlorate, however, nearly completely inhibited growth under autocrine conditions and in the presence of recombinant amphiregulin. Structure-function studies indicate that the polymerization level, high sulfate group density, and possibly iduronic acid content of heparin-like moieties correlate with their inhibitory activity. Collectively, these observations indicate that amphiregulin is the major autocrine factor for keratinocytes and demonstrate that exogenous amphiregulin is an effective growth promoting factor with molar potency similar to that of epidermal growth factor. Autocrine and paracrine signaling by amphiregulin may require cellular heparin-like glycosaminoglycans, presumably as matrix or membrane proteoglycans, whereas soluble glycosaminoglycans inhibit signaling, possibly by competing for cytokine binding.

Amphiregulin↗

PubMed1994

Modifications in the synthesis of membrane-associated chondroitin sulfate proteoglycans in hemopoietic progenitor cells are accompanied by alterations in their adhesive properties.

In vitro studies in our laboratory have indicated that murine hemopoietic progenitor cell (HPC) lines, irrespective of their differentiation stage, synthesize and accumulate in the cell membrane a unique species of chondroitin sulfate proteoglycan (CS-PG). It has been postulated that CS-PG participates in HPC adhesion to pericellular stromal fibronectin by interacting with its heparin-promoting binding region. To further support this contention, we first attempted to modify CS-PG synthesis in HPC by the use of chlorate and p-nitrophenyl beta-D-xyloside, which inhibit sulfation and glycosaminoglycan (GAG) addition in proteoglycans, respectively. We then studied the effect that these modifications may have in the adhesive capacity of HPC to interact with fibronectin and its cell- and heparin-promoting binding chymotryptic fragments. Treatment with chlorate which resulted in a decreased sulfation of membrane-associated 35 S-labeled CS-PG, as judged by ion exchange chromatography, did not affect HPC adhesion to fibronectin or its fragments. However, beta-xyloside treatment which reduces the abundance of membrane-associated CS-PG, as evidenced by molecular sieve chromatography, produced a major and specific decrease in HPC adhesion to the heparin-promoting binding fragment of fibronectin. These results indicate that CS-PG are involved in HPC interaction with fibronectin, in a mode that seems to be dependent on the differentiation stage of HPC.

Animals↗

PubMed1995

Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate.

Proteoglycans have been implicated in the clustering of acetylcholine receptors (AChRs) on cultured myotubes and at the neuromuscular junction. We report that the presence of chondroitin sulfate is associated with the ability of cultured myotubes to form spontaneous clusters of AChRs. Three experimental manipulations of wild type C2 cells in culture were found to affect both glycosaminoglycans (GAGs) and AChR clustering in concert. Chlorate was found to have dose-dependent negative effects both on GAG sulfation and on the frequency of AChR clusters. When extracellular calcium was raised from 1.8 to 6.8 mM in cultures of wild-type C2 myotubes, increases were observed both in the level of cell layer-associated chondroitin sulfate and in the frequency of AChR clusters. Culture of wild-type C2 myotubes in the presence of chondroitinase ABC eliminated cell layer-associated chondroitin sulfate while leaving heparan sulfate intact and simultaneously prevented the formation of AChR clusters. Treatment with either chlorate or chondroitinase inhibited AChR clustering only if begun prior to the spontaneous formation of clusters. We propose that chondroitin sulfate plays an essential role in the initiation of AChR clustering and in the early events of synapse formation on muscle.

Cells, Cultured↗

PubMed2004

The role of Ynt1 in nitrate and nitrite transport in the yeast Hansenula polymorpha.

Ynt1 is the only high-affinity nitrate uptake system in Hansenula polymorpha. Nitrate uptake was directly correlated with the Ynt1 levels and shown to be independent of nitrate reductase (NR) activity levels. Ynt1 failed to transport chlorate and, as a result, strains lacking YNT1 were sensitive to chlorate, as is the wild-type. Nitrite uptake in a wild-type strain was partially inhibited by nitrate to levels shown by a YNT1-disrupted strain in which, in turn, nitrite transport was not inhibited by nitrate. It is concluded that nitrite uptake takes place by two different transport systems: Ynt1 and a nitrite-specific transporter(s). The nitrite-specific transport system was induced by nitrate; consistently, no induction was observed in strains lacking the transcription factor YNA1, which is involved in nitrate and nitrite induction of the nitrate assimilatory structural genes. Ynt1 presents its optimal rate for nitrite uptake at pH 6, while pH 4 was optimal for the specific nitrite uptake system(s). At pH 5.5, the contribution of Ynt1 to high-affinity nitrate and nitrite uptake was around 95% and 60%, respectively. The apparent Km of Ynt1 for nitrate and nitrite is in the microM range, as is the specific nitrite uptake system for nitrite. The analysis of the effect of the reduced nitrogen sources on nitrate assimilation revealed that glutamine inactivates nitrate and nitrite transport, dependent on Ynt1, but not the nitrite-specific system.

Anion Transport Proteins↗

PubMed1997

Role of fibronectin and sulfated proteoglycans in endothelial cell migration on a cultured smooth muscle layer.

Endothelial cell (EC) migration is essential for the healing of denudation injuries to the vessel wall. This event occurs in vivo in a pericellular environment and should be regulated in part by juxtacrine and paracrine interactions with underlying smooth muscle cells (SMCs). To investigate the EC migration behavior over SMCs under direct cell-cell contact conditions, we have utilized human umbilical vein endothelial cells (HUVECs) grown to confluence on microcarrier beads and radiolabeled with chromium-51. The EC carrying beads were spread over a confluent human aortic SMC monolayer and cocultured for 48 hr, allowing the EC to migrate from beads onto the underlying SMC monolayer in the presence of sodium chlorate, cycloheximide (CHM), or anti-fibronectin monoclonal antibody (anti-FN mAb). The level of EC migration was quantified by counting the amount of radioactivity on the SMC layer after the removal of the beads. The presence of a SMC layer enhanced EC migration more than threefold (P < 0.05). Furthermore, EC migration was inhibited from 30 to 80% (P < 0.05) by sodium chlorate, CHM, and anti-FN mAb in a dose-dependent fashion. This model has shown that smooth muscle cells augment endothelial motility. Both fibronectin and sulfated proteoglycans released by ECs and SMCs likely play an important role in the regulation of EC migration behavior over SMCs. The method described of using radiolabeled ECs on microcarrier beads should prove to be a useful tool in the study of cell migration in a heterotypic cellular environment.

Antibodies↗

PubMed1994

Heterologous transformation of Zalerion arboricola.

A heterologous DNA-mediated transformation system was developed for the pneumocandin-producing fungus Z. arboricola that was based on either conferral of hygromycin B resistance or complementation of a nitrate reductase mutant. Hygromycin-resistant transformants were selected with plasmid pCSN43 which contains the E. coli hygromycin B phosphotransferase gene under the control of Aspergillus nidulans trpC transcription signals. Transformation frequencies were about four transformants per microgram of circular DNA and could be improved four- to six-fold by linearizing the transforming DNA. The transformants differed from one another with respect to the copy number of the integrated plasmid and the site of integration. Adding an autonomously-replicating sequence (AMA1) from A. nidulans to pCSN43 enhanced transformation three-fold and produced, in addition, numerous abortive transformants. However, it is unlikely that the AMA1 sequence promoted plasmid replication in Z. arboricola. Nitrate reductase mutants of Z. arboricola were isolated by positive selection on chlorate-containing medium, and one mutant was subsequently transformed with pSTA700 which contains the nitrate reductase gene (niaD) from Cephalosporium acremonium. Introduction of the niaD gene restored sensitivity to chlorate in the mutant; therefore, using the niaD gene as a selectable marker provides a system for both positive and negative selection. To our knowledge, this is the first report describing transformation of a member of the genus Zalerion.

Acremonium↗

PubMed1999

Alterations of matrix- and cell-associated proteoglycans inhibit osteogenesis and growth response to fibroblast growth factor-2 in cultured rat mandibular condyle and calvaria.

Matrix and cell surface proteoglycans (PGs) may play important roles in the control of cellular actions of heparan-binding growth factors such as fibroblast growth factor (FGF) during chondrogenesis and osteogenesis. In this study, we used 4-methylumbelliferyl-beta-d-xyloside, an inhibitor of PG synthesis, and sodium chlorate, a competitive inhibitor of glycoconjugate sulfation, to determine the functional consequences of alterations of PG metabolism on osteogenesis and on FGF actions in neonatal rat condyle and calvaria in vitro. Biochemical analysis showed that beta-d-xyloside (1 mM) or chlorate (15 mM) treatment for 1-8 days inhibited cellular PG synthesis by 60-80% in condyle and calvaria, as evaluated by [35S]sulfate incorporation. Histochemistry and immunohistochemistry showed that the inhibition of PG synthesis by beta-d-xyloside resulted in reduced incorporation of chondroitin sulfate into cartilage and bone matrix. This was associated with a 75% reduction in cell growth in condyle, determined by DNA synthesis, and in collagenous matrix synthesis in condyle and calvaria, evaluated by tritiated proline incorporation and type I collagen immunohistochemistry. Morphological and quantitative autoradiographic analyses also showed that inhibition of PG synthesis by beta-d-xyloside blocked bone matrix formation by perichondral progenitor cells in condyles and by osteoblasts in calvaria. In addition, alteration of PG metabolism blocked the mitogenic response to rhFGF-2 in calvaria. The data show that functional proteoglycans are essential for osteogenesis and for the growth response to FGF-2 during osteogenic differentiation in vitro.

Animals↗

PubMed1992

Inhibition of synthesis of rat parotid secretory proteoglycan in a gland slice system.

The chondroitin sulphate contained within the secretory granules of the rat parotid gland and its saliva was shown to be in the form of a proteoglycan by using inhibitors of proteoglycan synthesis in a gland slice system. Gland slices were incubated in either p-nitrophenyl-beta-D-xyloside or chlorate in the presence of both [3H]-leucine and [35S]-sulphate. The slices were next homogenized and either the 250 g supernatant fraction (for initial experiments) or secretory granule-containing fractions were isolated. Protein and proteoglycans of these fractions were precipitated in 10% trichloracetic acid (TCA), and glycosaminoglycans in cetylpyridinium chloride. [3H]-leucine and [35S]-sulphate were quantitated in each type of precipitate by scintillation counting. The results showed that 1 mM xyloside had no effect on protein or glycosaminoglycan synthesis but blocked incorporation of radiosulphate into TCA-precipitable material. Sixteen mM chlorate almost totally inhibited incorporation of radiosulphate into glycosaminoglycan and TCA-precipitable material. These findings demonstrate that the rat parotid secretory chondroitin sulphate is indeed a proteoglycan because its synthesis is blocked by the protein-core analogue acceptor, p-nitrophenyl-beta-D-xyloside. This system offers opportunities for exploring the functional role of chondroitin sulphate proteoglycan in this salivary gland.

Animals↗

PubMed2004

Under-sulfation by PAPS synthetase inhibition modulates the expression of ECM molecules during chondrogenesis.

Sulfation of proteoglycans is an important post-translational modification in chondrocytes. We previously found that 3'-phosphoadenosine 5'-phosphosulfate (PAPS) synthetase-2 levels increased more than 10-fold during mesenchymal cell chondrogenesis. Given that PAPS is the sole sulfur donor, and is produced only by PAPS synthetase in all cells, increased expression of PAPS synthetase-2 should be a prerequisite for increased sulfation activity of chondrocytes. We found that sodium chlorate, a specific inhibitor of PAPS synthetase, inhibited proteoglycan sulfation during chondrogenesis. In contrast, sodium chlorate unexpectedly induced early expression of type II collagen and increased the number of cartilage nodules during chondrogenesis. Inhibition of sulfation also accelerated the down-regulation of N-cadherin and fibronectin during chondrogenesis. These findings suggest that sulfation has an important regulatory role in coordinating the timely expression of extracellular matrix molecules during chondrogenesis, and that under-sulfation may cause the breakdown of this coordination, leading to premature chondrogenesis.

Amino Acid Sequence↗

PubMed2006

Chondroitin sulfate A chains enhance platelet derived growth factor-mediated signalling in fibrosarcoma cells.

Platelet derived growth factor is involved in the autocrine growth stimulation of malignant cells, the stimulation of angiogenesis and the recruitment and regulation of tumor fibroblasts. PDGF has been shown to physically interact with glycosaminoglycans which are abundant in the fibrosarcoma cell microenvironment. Aim of the present study was to examine the effects of glycosaminoglycans on the mitogenic function of platelet derived growth factor in two human fibrosarcoma cell lines (B6FS, HT1080). For this purpose exogenously added glycosaminoglycans, regulators of endogenous glycosaminoglycan synthesis (sodium chlorate as selective inhibitor and beta-D-xyloside as a stimulator) and specific glycosidases to cleave cell-associated glycosaminoglycans, were utilized. Platelet derived growth factor demonstrated a growth stimulating effect on B6FS, whereas no effect was evident on HT1080 fibrosarcoma cells. Beta-D-xyloside had no effect on the basal level or the platelet derived growth factor-induced cell proliferation, whereas sodium chlorate severely reduced the basal level of proliferation in both cell lines. Significant co-stimulatory effects of chondroitin sulfate A in combination with platelet derived growth factor BB on the growth of HT1080 and B6FS cells were found. The co-stimulatory effect of chondroitin sulfate A was not due to transcriptional up regulation of platelet derived growth factor receptors genes, but rather to more efficient signalling of tyrosine kinase receptors. In conclusion, this study shows that chondroitin sulfate A can enhance the mitogenic activity of platelet-derived growth factor in fibrosarcoma cells utilizing a pathway which involves tyrosine kinases. This result introduces a new modulating role for chondroitin sulfate in signalling pathways critical for cancer growth.

Becaplermin↗

PubMed1998

Role of heparan sulphate proteoglycans in the regulation of human lactoferrin binding and activity in the MDA-MB-231 breast cancer cell line.

We previously demonstrated that lactoferrin increases breast cell sensitivity to natural killer cell cytotoxicity whereas haematopoietic cells are unaffected by lactoferrin. It has been described that lactoferrin binds to various glycosaminoglycans. Compared to haematopoietic cells, breast cancer cells and particularly the breast cell line MDA-MB-231, possess a high level of proteoglycans. Scatchard analysis of 125I-lactoferrin binding to MDA-MB-231 cells revealed the presence of two classes of binding sites: a low affinity site with a Kd of about 700 nM and 3.9 x 10(6) sites and a higher affinity class with a Kd of 45 nM and 2.9 x 10(5) sites per cell. To investigate the potential regulation of lactoferrin activity by proteoglycans expressed on the MDA-MB-231 cells, we treated these cells with glycosaminoglycan-degrading enzymes or sodium chlorate, a metabolic inhibitor of proteoglycan sulphation. We showed that chondroitinase treatment has no effect, while heparinase or chlorate treatment significantly reduces both the binding of lactoferrin to cell surface sulphated molecules such as heparan sulphate proteoglycans (HSPG) and the affinity of lactoferrin for the higher affinity binding sites. The modulation of the lactoferrin binding was correlated with a decrease in lactoferrin activities on both MDA-MB-231 cell sensitisation to lysis and proliferation. Taken together, these results suggest that the presence of adequately sulphated molecules, in particular HSPG, is important for lactoferrin interaction and activity on the breast cancer cells MDA-MB-231.

Breast Neoplasms↗

PubMed2002

Undersulfation of proteoglycans and proteins alter C6 glioma cells proliferation, adhesion and extracellular matrix organization.

Proteoglycans are considered to be important molecule in cell-microenvironment interactions. They are overexpressed in neoplastic cells modifying their growth and migration in hosts. In this work we verified that undersulfation of proteoglycans and other sulfated molecules, induced by sodium chlorate treatment, inhibited C6 glioma cells proliferation in a dose-dependent way. This effect was restored by the addition of exogenous heparin. We could not detect significant cell mortality in our culture condition. The treatment also impaired in a dose-dependent manner, C6 cell adhesion to extracellular matrix (ECM) proteins (collagen IV, laminin and fibronectin). In addition, sodium chlorate treatment altered C6 glioma cell morphology, from the fibroblast-like to a more rounded one. This effect was accompanied by increased synthesis of fibronectin and alterations in its extracellular network organization. However, we could not observe modifications on laminin organization and synthesis. The results suggest an important connection between sulfation degree with important tumor functions, such as proliferation and adhesion. We suggest that proteoglycans may modulate the glioma microenvironment network during tumor cell progression and invasion.

Animals↗

PubMed2000

Role of tyrosine sulfation and serine phosphorylation in the processing of procholecystokinin to amidated cholecystokinin and its secretion in transfected AtT-20 cells.

Mammalian procholecystokinin (pro-CCK) is known to have three sulfated tyrosine residues, one of which is present in the CCK 8 moiety and two additional residues present in the carboxyl-terminal extension. In the present study, inhibition of tyrosine sulfation by sodium chlorate decreased the secretion of processed CCK 8 in CCK-expressing endocrine cells in culture. It was then demonstrated that when each of these tyrosines individually, as well as all three together, was mutated to phenylalanine and expressed in endocrine cells, CCK was still processed and secreted. However, the amount of CCK secreted varied with the type of mutation. Substitution of Phe to Tyr in CCK 8 reduced the quantity of secreted CCK 8 by 50%, and when all the sulfated Tyr were mutated to Phe the quantity of secreted CCK was reduced by about 70%, similar to what is observed with chlorate treatment. Changing of the putative phosphorylation site serine to alanine does not affect the processing. Serine phosphorylation at this site may play a functional role in regulatory events. Our results demonstrate that tyrosine sulfation alters the amount of secretion but is not an absolute requirement for the processing and secretion of CCK in this cell line. Tyrosine sulfation of CCK may still be important for its solubility, stabilization, and/or functional interaction.

Amides↗

PubMed1997

Glypican-3 is a binding protein on the HepG2 cell surface for tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a primary regulator of the initiation of blood coagulation. TFPI is internalized and degraded by HepG2 cells through the low-density-lipoprotein receptor-related protein (LRP) but also binds another molecule present on the cell surface at approx. 10-fold the abundance of LRP [Warshawsky, Broze and Schwartz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 6664-6668]. When HepG2 cells are washed with heparin or dextran sulphate, a substance that binds TFPI is removed from the cell surface and can be detected in a slot-blot assay. Preincubation with trypsin destroys the reactivity of the TFPI-binding component in the slot-blot assay, suggesting that it is a protein. In addition, when the sulphation of glycosaminoglycans (GAGs) is prevented by growing the HepG2 cells in the presence of 30 mM sodium chlorate, TFPI binding is unaffected, whereas the binding of bovine lipoprotein lipase, a protein known to associate with cell-surface GAGs, falls to 50% of control levels. Dextran sulphate washes of HepG2 cells grown in sodium chlorate have an equal reactivity in slot-blot experiments to that of non-treated cells, suggesting that GAGs are not totally responsible for the binding activity observed. By using the slot blot to follow binding activity and conventional protein purification techniques, a protein species that migrates at 40 kDa after reduction was identified in the HepG2 cell wash. The binding of this protein to TFPI was confirmed with immobilized TFPI. Amino acid sequence analysis identified this protein species as a proteolytic fragment of glypican-3 (also called OCI-5), a member of the glypican family of glycosylphosphatidylinositol-anchored proteoglycans.

Amino Acid Sequence↗

PubMed2002

Hepatocyte growth factor/scatter factor binds to small heparin-derived oligosaccharides and stimulates the proliferation of human HaCaT keratinocytes.

Hepatocyte growth factor/scatter factor (HGF/SF) acts via a dual receptor system consisting of the MET tyrosine kinase receptor and heparan sulfate or dermatan sulfate proteoglycans. In optical biosensor binding assays, competition by oligosaccharides for binding of HGF/SF to immobilized heparin showed that disaccharides failed to compete, whereas tetrasaccharides inhibited HGF/SF binding (IC(50) 8 microg/ml). The inhibitory potency of the oligosaccharides increased as their length increased by successive disaccharide units, to reach a maximum (IC(50) 1 microg/ml) at degree of polymerization (dp) 10. In binding assays, HGF/SF was found to bind directly to oligosaccharides as small as dp 4, and the binding parameters were similar for oligosaccharides of dp 4-14 (k(a) 2.2-45.3 x 10(6) m(-1) s(-1), k(d) 0.033-0.039 s(-1), and K(d) 9-16 nm). In human keratinocytes, HGF/SF stimulated DNA synthesis, and this was dependent on a sustained phosphorylation of p42/44(MAPK). In chlorate-treated and hence sulfated glycosaminoglycan-deficient HaCaT cells, the stimulation of DNA synthesis by HGF/SF was almost abolished. Heparin-derived oligosaccharides from dp 2 to dp 24 were added together with HGF/SF to chlorate-treated cells to determine the minimum size of oligosaccharides able to restore HGF/SF activity. At restricted concentrations of oligosaccharides (4 ng/ml), HGF/SF required decasaccharides, whereas at higher concentrations (100 ng/ml) even tetrasaccharides were able to partly restore DNA synthesis. The results suggest that HGF/SF binds to a tetrasaccharide and that although this is sufficient to enable the stimulation of DNA synthesis, longer oligosaccharides are more efficient, perhaps by virtue of their ability to bind more easily other molecules.

Binding, Competitive↗

PubMed1977

Aerobic and anaerobic bacterial respiration monitored by electrodes.

A technique is described by which both oxygen and nitrate (or nitrate or chlorate) levels were continuously monitored during bacterial respiration. Paracoccus (Micrococcus) denitrificans and Escherichia coli oxidizing succinate rapidly ceased to reduce nitrate when oxygen was available, and equally rapidly commenced nitrate reduction when all the oxygen had been consumed. By contrast, membrane vesicles isolated from P. denitrificans reduced oxygen and nitrate simultaneously. The respiratory nitrate reductase in intact cells of P. denitrificans appeared to be inacessible to chlorate present in the reaction medium, and it is suggested that the nitrate reductase is orientated on the plasma membrane so that nitrate gains access from the inner (cytosolic) face.

Aerobiosis↗