Effects of vanadyl sulphate on ornithine decarboxylase and progesterone levels in the ovary of rat

The effects of vanadyl sulphate in vitro on the levels of ODC activity and progesterone synthesis in ovaries were studied. The levels of ODC in the ovaries were stimulated with high concentration of vanadyl sulphate and at low concentrations there was no change in the levels of ODC activity. On the contrary progesterone levels were stimulated with low concentrations of vanadyl sulphate and were inhibited at higher concentrations. Vanadyl sulphate showed additional stimulation of ODC activity, when it was added with hCG and caused inhibition of hCG induced progesterone biosynthesis. These results show that the effects of vanadyl sulphate on ODC and progesterone are different.     

Stimulation of MAP kinase and S6 kinase by vanadium and selenium in rat adipocytes

To explore the mechanism underlying the insulin-mimetic actions of vanadium and selenium we examined their effects on the mitogen activated protein/myelin basic protein kinases (MAPK) and ribosomal S6 protein kinases, which are among the best characterized of the kinases that comprise the phosphorylation cascade in insulin signal transduction. We observed a transient activation of MAPK and S6 kinases by insulin in rat adipocytes, while both sodium selenate and vanadyl sulphate produced prolonged activation of the kinases. Vanadyl sulphate stimulated the activity of MAPK and S6 kinase by as much as 6 fold and 15 fold, respectively. Pretreatment of the cells with genistein did not affect the activation of MAPK by insulin, but partially blocked the effects of sodium selenate and vanadyl sulphate. Genistein did not change the activation of S6 kinase by insulin, but blocked the activation in vanadyl sulphate- and sodium selenate-treated-cells, suggesting that a genistein sensitive tyrosine kinase may be involved in the activation by these two compounds. Rapamycin, a specific inhibitor of the p70s6k isoform of S6 kinase, partially reduced the activation of S6 kinase activity by sodium selenate, indicating a role for this kinase in the overall activity of the S6 kinase in sodium selenate-treated cells. A similar trend was noted in vanadyl sulphate-treated cells. Thus, this study supports the involvement of MAPK and S6 kinases in the insulin-mimetic actions of vanadium and selenium.     

Increased accumulation of indole alkaloids by some cell lines of Catharanthus roseus in response to addition of vanadyl sulphate

Vanadyl sulphate (10–500 mg/l), when added to cell suspension cultures of Catharanthus roseus stimulated increased intracellular accumulation of catharanthine and ajmalicine. This response was demonstrated in both flask and fermenter (30 litre) systems. The response varied, and depended upon cell line, concentration of vanadyl sulphate and the stage of the growth phase at which the cells were treated. This process has the potential to increase the yield and reduce the production time for commercially useful secondary plant metabolites.Abbreviations Ajm ajmalicine - Cath catharanthine - CAS ceric ammonium sulphate - VOSO₄ vanadyl sulphate - FW fresh weight - n.d. not detected     

Influence of vanadium on acclimatization of humans to high altitude

 The study was conducted on human volunteers as controls as well as after administration of vanadyl sulphate on induction to high altitude (HA) at 3500 m. The plasma vanadium contents were significantly reduced in the control group on abrupt induction to HA on days 3 and 10, indicating redistribution to other organs/tissues under the stressful situation. In the vanadium salt-treated group, plasma vanadium contents were similar to those obtained at sea-level. Administration of vanadyl sulphate did not act as a diuretic. Moreover the vanadium supplemented group drank more water and also excrete less urine than the control group. Received: 1 November 1995 / Accepted: 9 October 1996     

Vanadium inhibition of serine and cysteine proteases.

A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain.     

A role for glycosaminoglycans in the development of collagen fibrils

Extensive data on the glycosaminoglycan (GAG) composition and the collagen fibril diameter distribution have been collected for a diverse range of connective tissues. It is shown that tissues with the smallest diameter collagen fibrils (mass-average diameter less than 60 nm) have high concentrations of hyaluronic acid and that tissues with the largest diameter collagen fibrils (mass-average diameter approximately 200 nm) have high concentrations of dermatan sulphate. It is suggested that the lateral growth of fibrils beyond a diameter of about 60 nm is inhibited by the presence of an excess of hyaluronic acid but that this inhibitory effect may be removed by an increasing concentration of chondroitin sulphate and/or dermatan sulphate. It is also postulated that high concentrations of chondroitin sulphate will inhibit fibril growth beyond a mass-average diameter of approximately 150 nm. Such an inhibition may in turn be removed by an increasing concentration of dermatan sulphate such that it becomes the dominant GAG present in the tissue.     

Specific association of iduronic acid-rich dermatan sulphate with the extracellular matrix of human skin fibroblasts cultured on collagen gels.

Human skin fibroblasts cultured on collagen gels produced two dermatan sulphate species, one, enriched in iduronic acid residues, that bound specifically to the collagenous fibres of the gel, the other, enriched in glucuronic acid, that accumulated in the culture medium. Collagen-binding and collagen-non-binding dermatan sulphates were also produced by cells grown on plastic surfaces, but in these cultures each constituent was released into the growth medium. Net synthesis of dermatan sulphate was 3-fold higher in cells maintained on collagen gels. In contrast, heparan sulphate synthesis was not influenced by the nature of the culture surface. The concentration of heparan sulphate in surface-membrane extracts was similar for cells grown on plastic and on collagen gels, but cells cultured on collagen showed a notable increase in the content of surface-membrane dermatan sulphate. The patterns of synthesis and distribution of sulphated glycosaminoglycans observed in skin fibroblasts maintained on collagen gels may reflect differentiated cellular functions.     

The effect of acid mucopolysaccharides and acid mucopolysaccharide–proteins on fibril formation from collagen solutions

1. The effects of acid mucopolysaccharides and acid mucopolysaccharide-proteins on the size and rate of formation of fibril aggregates from collagen solutions in pH7.6 buffers were studied by turbidimetric and light-scattering methods. 2. Serum albumin, orosomucoid, methylated cellulose, chondroitin sulphate A and chondroitin sulphate C of molecular weight less than 20000, and hyaluronate of molecular weight less than 40000 did not influence rates of fibril formation. Chondroitin sulphate A, chondroitin sulphate C and hyaluronate of high molecular weight retarded the rate of fibril formation. This effect of high-molecular-weight chondroitin sulphate C decreased with increasing ionic strength. Heparin, though of low molecular weight (13000), was highly effective, as was also heparitin sulphate. The chondroitin sulphate-proteins of very high molecular weight were highly effective, despite the fact that for some preparations the component chondroitin sulphate chains had molecular weights much less than 20000. 3. Agents that had delayed fibril formation were also effective in producing an increase in degree of aggregation of fibrillar collagen, as indicated by dissymmetry changes observed in light-scattering experiments at low collagen concentrations. Methylated cellulose and heparin at 2.5mug./ml. were unusual in decreasing aggregation, but heparin at 0.25mug./ml. increased aggregation. Electron microscopy of gels showed fibrils and fibril aggregates with ;normal' collagen spacing and dimensions consistent with the light-scattering results. 4. The rates of electrical transport of agents and of solvent (electro-osmosis) through collagen gels indicated a contribution of molecular entanglement that increased with increase in molecular size of the agents. Electrostatic binding of heparin to collagen was noted. Binding to collagen during fibril formation was also found for heparitin sulphate and a chondroitin sulphate with extra sulphate groups. 5. Electrostatic binding of acid mucopolysaccharide-proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development. Excluded-volume (molecular-entanglement) effects may also be important. These factors operate simultaneously and interact mutually so that precise assessment of their relative importance is difficult.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels.

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Vanadium distribution in roots and leaves of Phaseolus vulgaris: morphological and ultrastructural effects

In different plant species, vanadium has been considered either as beneficial or as a toxic element, or even as secondary metabolism elicitor, but the mechanisms involved are still not completely understood. In this study, the responses of Phaseolus vulgaris L. cv. Contender roots and leaves to different vanadyl sulfate concentrations were studied. The plants grown hydroponically with V had thicker roots, a less developed main root, and a smaller number of secondary roots than the control plants. The V content in roots and leaves was correlated with V supply concentration but the V content in leaf was always much lower than in the root, which leads us to conclusion that V accumulates in the roots and only small quantities are transferred to the leaves. However, thylakoid disorganisation was observed in the chloroplasts of plants grown with vanadyl sulphate.     

Quantitative measurements of the proton-motive force and its relation to steady state lactose accumulation in Escherichia coli.

1. Developing tail tendons from rats (19-day foetal to 126 days post partum) were examined by electron microscopy after staining for proteoglycan with a cationic copper phthalocyanin dye. Cuprolinic Blue, in a "critical electrolyte concentration" method. Hydroxyproline was measured on papain digests of tendons, from which glycosaminoglycuronans were isolated, characterized and quantified. 2. Mean collagen fibril diameters increased more than 10-fold with age according to a sigmoid curve, the rapid growth phase 2 being during 30-90 days after conception. Fibril periodicities were considerably smaller (50-55 nm) in phases 1 and 2 than in phase 3 (greater than 62 nm). 3. Dermatan sulphate is the main glycosaminoglycuronan in mature tendon. Chondroitin sulphate and hyaluronate preponderate in foetal tissue. 4. Proteoglycan was seen around but not inside collagen fibrils. Proteoglycan and collagen were quantified from electron micrographs. Their ratios behaved similarly to uronic acid/hydroxyproline and hyaluronate/hydroxyproline ratios, which decreased rapidly around birth, and then levelled off to a low plateau coincident with the onset of rapid growth in collagen fibril diameter. 5. Dermatan sulphate/hydroxyproline ratios suggest that the proteoglycan orthogonal array around the fibril is largely dermatan sulphate. In the foetus hyaluronate and chondroitin sulphate exceed that expected to be bound to collagen. 6. An inhibiting action of chondroitin sulphate-rich proteoglycan on fibril diameter growth is suggested. 7. The distributions of hyaluronate, chondroitin sulphate and dermatan sulphate are discussed in the light of secondary structures suggested to be present in hyaluronate and chondroitin sulphate, but not in dermatan sulphate.     

Age-Related Changes in Collagen and Glycosaminoglycans of Rat Gingiva1

Age-related changes in collagen and glycosaminoglycans (GAGs) of rat gingiva were studied biochemically and histologically. The components of isolated GAGs were identified as dermatan sulphate, hyaluronic acid, and heparan sulphate. In histological studies, hyaluronic acid was present in all regions of the gingiva, whereas dermatan sulphate and heparan sulphate were present only in gingival connective tissue. However, there was no significant difference in the relationship between aging, and the content or distribution of GAGs. On the other hand, histological findings showed that collagen fibers were markedly increased in number and the vascular composition was decreased with increasing age. In biochemical studies, the content of collagen, especially of insoluble collagen, was greatly increased with age, whereas collagen biosynthesis and collagenolytic activity were markedly decreased. In addition, lysyl oxidase activity was also significantly decreased with age. The results indicate that the rate of collagen turnover decreases and collagen fibers increase in stability in rat gingiva with increasing age. These observed age-related changes may affect the ability of gingiva to respond to local irritants.     

Chelation therapy and vanadium: effect on reproductive organs in rats

Present investigation was planned to evaluate the therapeutic effectiveness of chelating agents against vanadium intoxication on blood and reproductive organs of rats. Male and female albino rats were injected vanadyl sulphate (7.5 mg/kg, po, for 21 days, 5 days in a week). Chelating agents tiron (T) alone and in combination with lipoic acid (LA), vitamin E (vit E) and selenium (Se) were given for 2 days/week. With the administration of vanadyl sulphate to rats fructose level in seminal vesicles was significantly (P< or =0.05) declined. The activities of alkaline phosphatase and adenosine triphosphatase were also decreased, whereas glycogen content and acid phosphatase activity increased in testis, seminal vesicles, ovaries and uterus after toxicant exposure. Significant changes in serum transaminases, serum alkaline phosphatase and lactate dehydrogenase were recouped by chelation therapy. Lipid peroxidation, reduced glutathione level and triglycerides levels altered significantly after exposure to vanadium in rats. The ultrastructural damage in spermatogenic stages in treated animals showed recovery pattern after therapy. Co-treatment with antioxidants restored these activities. The most effective combination was tiron + selenium followed by tiron + vitamin E, and tiron + lipoic acid.     

Design,synthesis and in vitro bactericidal/fungicidal screening of some vanadyl(IV)complexes with mono- and di-substituted ONS donor triazoles

Abstract

A new series of anti-bacterial and anti-fungal mono- and di-substituted triazoles (L¹)–(L⁶) have been synthesized and characterized on the basis of their physical, spectral and analytical data. The ligands (L¹)–(L⁶) on reaction with vanadyl(IV) sulphate led to the formation of vanadyl(IV) metal complexes (1)–(4). The structure of the complexes has been established on the basis of their physical, spectral and elemental analyses data. The synthesized ligands and their vanadyl(IV) complexes have been screened in vitro for anti-bacterial activity against six bacterial species such as, Escherichia coli (ATCC 25922), Shigella flexneri (ATCC 12022), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (ATCC 14028), Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6051) and for in vitro anti-fungal activity against six fungal strains, Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata. The screening results showed the vanadyl complexes to be more bactericidal/fungicidal against one or more bacterial/fungal species. The synthesized compounds were also subjected to brine shrimp bioassay for scrutinizing their cytotoxicity.     

Effects of heparin and dextran sulphate on the production of collagen and protein in diabetic and non-diabetic human skin fibroblast cultures

Addition of heparin and dextran sulphate to human skin fibroblasts in cell cultures caused an increase in [3H]-proline incorporation into collagen and total protein in the culture medium by cells derived from nondiabetics. Cells from type 2 diabetic subjects were significantly less affected by dextran sulphate addition, suggesting altered regulatory mechanisms for collagen production in these cells. Addition of chondroitin sulphate caused a dose-dependent increase in labelled collagen, indicating a possible role for this glycosaminoglycan as modulator of collagen deposition.     

The stabilization of in vivo assembled collagen fibrils by proteoglycans/glycosaminoglycans

The effects of proteoglycans/glycosaminoglycans on the thermal stability of in vivo assembled collagen fibrils have been examined. The shrinkage temperature of tendon collagen was found to be linearly dependent on the concentration of chondroitin sulphate in the surrounding fluid. Enzymic pretreatment of articular cartilage, to reduce its glycosaminoglycan content, resulted in decreased stability of the collagen present. The stability of the collagen in hyaluronidase-treated cartilage was found to be higher when measured in a solution of chondroitin sulphate (30 g/dl) than in buffer alone. The results of this study demonstrate that the proteoglycans stabilize collagen fibrils in tissues such as articular cartilage.     

Comparison of the electron spin echo envelope modulation (ESEEM) for human lactoferrin and transferrin complexes of copper(II) and vanadyl ion

Copper(II) and vanadyl ions were bound to human milk lactoferrin or serum transferrin with carbonate or oxalate as the synergistic anion. Electron spin echo envelope modulation (ESEEM) due to nitrogen of a coordinated histidine imidazole was observed for both the copper and vanadyl complexes. For both metals, the modulation frequencies in the Fourier transforms of the data were similar for the two proteins and were weakly dependent on anion. When data in D2O/glycerol-d3 were compared with data in H2O/glycerol, the deep deuterium modulation indicated multiple exchangeable protons in the vicinity of the metals with at most one proton within about 2.9 A of the metal. The distribution of exchangeable protons around the metals as probed by ESEEM was the same, within experimental uncertainty, for the copper or vanadyl complexes with either carbonate or oxalate as the anion. When 13C-labeled oxalate was used as the synergistic anion, 13C-ESEEM was observed for both the copper and vanadyl complexes of lactoferrin and transferrin. The deeper 13C modulation for copper and vanadyl transferrin [13C]oxalate than for vanadyl transferrin [13C]carbonate suggests that both ends of the oxalate are bound to the metal in the transferrin and lactoferrin complexes.     

Glycosaminoglycans show a specific periodic interaction with type I collagen fibrils

Current wisdom on intermolecular interactions in the extracellular matrix assumes that small proteoglycans bind collagen fibrils on highly specific sites via their protein core, while their carbohydrate chains interact with each other in the interfibrillar space. The present study used high-resolution scanning electron microscopy to analyse the interaction of two small leucine-rich proteoglycans and several glycosaminoglycan chains with type I collagen fibrils obtained in vitro in a controlled, cell-free environment. Our results show that most ligands directly influence the collagen fibril size and shape, and their aggregation into thicker bundles. All chondroitin sulphate/dermatan sulphate glycosaminoglycans we tested, except chondroitin 4-sulphate, bound to the fibril surface in a highly specific way and, even in the absence of any protein core, formed regular, periodic interfibrillar links resembling those of the intact proteoglycan. Only intact decorin, however, was able to organize collagen fibrils into fibres compact enough to mimic in vitro the superfibrillar organization of natural tissues. Our data indicate that multiple interaction patterns may exist in vivo, may explain why decorin- or biglycan-knockout organisms show milder effects than can be expected, and may lead to the development of better, simpler engineered biomaterials.     

Interactions between bovine cornea proteoglycans and collagen.

Two types of proteoglycan subunits were obtained from bovine cornea, the first mainly composed of proteochondroitin sulphate and the second of proteokeratan sulphate. These two fractions can be obtained from the tissue as an aggregate, and are able to recombine each other after separation, to re-form the original structure. In order to investigate collagen-proteoglycan interactions, type-I collagen was isolated from bovine cornea by pepsin digestion followed by 3.5% (w/v) NaCl precipitation, and was then linked to CNBr-activated Sepharose 4B. Two identical columns were prepared, the first filled with collagen coupled to Sepharose 4B, the second with free Sepharose 4B. The two proteoglycan subunits and the aggregate were chromatographed on the two gels under the same conditions; the elution profiles showed that both the aggregate and the proteochondroitin sulphate subunit are retarded by the collagen coupled to Sepharose. No interaction, however, occurred when proteokeratan sulphate subunit was run through the columns. Chondroitinase digestion of the proteoglycan samples confirmed that chondroitin sulphate chains are mainly responsible for the interaction with collagen; their removal, in fact, completely abolishes any differences between the chromatographic behaviour on the collagen-Sepharose and the control columns.     

Electron tomography reveals multiple self-association of chondroitin sulphate/dermatan sulphate proteoglycans in Chst5-null mouse corneas

The spatial distribution of collagen fibrils in the corneal stroma is essential for corneal transparency and is primarily regulated by extrafibrillar proteoglycans, which are multi-functional polymers that interact with hybrid type I/V collagen fibrils. In order to understand more about proteoglycan organisation and collagen associations in the cornea, three-dimensional electron microscopy reconstructions of collagen-proteoglycan interactions in the anterior, mid and posterior stroma from a Chst5 knockout mouse, which lacks a keratan sulphate sulphotransferase, were obtained. Both longitudinal and transverse section show sinuous, oversized proteoglycans with near-periodic, orthogonal off-shoots. In many cases, these proteoglycans traverse over 400nm of interfibrillar space interconnecting over 10 collagen fibrils. The reconstructions suggest that multiple chondroitin sulphate/dermatan sulphate proteoglycans have aggregated laterally and, possibly, end-to-end, with orthogonal extensions protruding from the main electron-dense stained filament. We suggest possible mechanisms as to how sulphation differences may lead to this increase in aggregation of proteoglycans in the Chst5-null mouse corneal stroma and how this relates to proteoglycan packing in healthy corneas.