Is dehydroepiandrosterone sulfate an anxiolytic agent?

Effects of dehydroepiandrosterone sulfate (DHEAS, 30 mg/kg, i.p., 4 and 28 hours after the injection) were studied in CBA/Lac male mice different in the level of anxiety resulting from repeated social victories (winners) or social defeats (losers) in 10 daily agonistic confrontations. The losers demonstrated high level of anxiety estimated by the "partition" test. The DHEAS and saline injections had different effects on winners, losers, and intact mice. DHEAS prevented the development of anxiety in losers 28 hours after the injection. In these experimental conditions DHEAS exerted no effect on winners. It was concluded that the DHEAS effect depends on the psychoemotional state of an animal. The anxiolytic effect of the exogenous DHEAS may be also characteristic of the endogenous hormone secreted by the adrenal glands and in the central nervous system.     

Heparan sulfate: anchor for viral intruders?

Heparan sulfates (HS) are ubiquitous, polyanionic carbohydrate chains linked to core proteins in cell membranes and extracellular matrices of all eukaryotes. Due to the complex nature of the HS-biosynthesis, a wealth of different structures are produced. These seem to have a well defined distribution in different tissues and cells throughout development. Binding of endogenous proteins with different functional properties such as growth factors, adhesion molecules or enzymes, is one of the functions of HS. Besides interaction with endogenous factors, glycosaminoglycans (GAG) and especially HS have also been demonstrated to function as receptors for a number of different pathogens. What roles may HS play in the pathogenesis and tropism of different intruders like parasites or viruses? What implications does binding of viruses to HS have for the development of drugs or the application of viral vectors for gene targeting? In this review an attempt is made to collect our present knowledge on viral usage of HS and the implications that follow.     

Quantitative analysis of glycosaminoglycans,chondroitin/dermatan sulfate,hyaluronic acid,heparan sulfate,and keratan sulfate by liquid chromatography–electrospray ionization–tandem mass spectrometry

We developed a method using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive “GAGomic” analysis of biological tissues.     

Are thiosulfate and trithionate intermediates in dissimilatory sulfate reduction?

The fate of 35-S during anaerobic metabolism of [35-S]sulfate, [35-S]thiosulfate, and [35-S]sulfate plus unlabeled thiosulfate by washed cell suspensions of Desulfovibrio spp, and of [35-S]thiosulfate by growing D. desulfuricans was examined. The results appear to be inconsistent with the hypothesis that thiosulfate is an intermediate in sulfate reduction. Since thiosulfate was produced from trithionate, the latter is also unlikely to be an intermediate in the reduction pathway. Extracts of D. desulfuricans catalysed exchange between sulfite and the sulfonate group of thiosulfate.     

Cholesterol sulfate in human physiology: what's it all about?

Cholesterol sulfate is quantitatively the most important known sterol sulfate in human plasma, where it is present in a concentration that overlaps that of the other abundant circulating steroid sulfate, dehydroepiandrosterone (DHEA) sulfate. Although these sulfolipids have similar production and metabolic clearance rates, they arise from distinct sources and are metabolized by different pathways. While the function of DHEA sulfate remains an enigma, cholesterol sulfate has emerged as an important regulatory molecule. Cholesterol sulfate is a component of cell membranes where it has a stabilizing role, e.g., protecting erythrocytes from osmotic lysis and regulating sperm capacitation. It is present in platelet membranes where it supports platelet adhesion. Cholesterol sulfate can regulate the activity of serine proteases, e.g., those involved in blood clotting, fibrinolysis, and epidermal cell adhesion. As a result of its ability to regulate the activity of selective protein kinase C isoforms and modulate the specificity of phosphatidylinositol 3-kinase, cholesterol sulfate is involved in signal transduction. Cholesterol sulfate functions in keratinocyte differentiation, inducing genes that encode for key components involved in development of the barrier.The accumulating evidence demonstrating a regulatory function for cholesterol sulfate appears solid; the challenge now is to work out the molecular mechanisms whereby this interesting molecule carries out its various roles.     

Is N-sulfation just a gateway modification during heparan sulfate biosynthesis?

Several biologically important growth factor-heparan sulfate (HS) interactions are regulated by HS sulfation patterns. However, the biogenesis of these combinatorial sulfation patterns is largely unknown. N-Deacetylase/N-sulfotrasferase (NDST) converts N-acetyl-d-glucosamine residues to N-sulfo-d-glucosamine residues. This enzyme is suggested to be a gateway enzyme because N-sulfation dictates the final HS sulfation pattern. It is known that O-sulfation blocks C5-epimerase, which acts immediately after NDST action. However, it is still unknown whether O-sulfation inhibits NDST action in a similar manner. In this article we radically change conventional assumptions regarding HS biosynthesis by providing in vitro evidence that N-sulfation is not necessarily just a gateway modification during HS biosynthesis.     

Neuroprotective–neurotrophic effect of endogenous dehydroepiandrosterone sulfate during intense stress exposure

Recent reports demonstrate neurotrophic properties of dehydroepiandrosterone sulfate (DHEAS) in men at rest, as well as profound neurotrophic responses to stress in both men and women. Little is known of neuroprotective–neurotrophic effects of DHEAS during stress exposure, either in men or women. This translational study was designed to examine neuroprotective–neurotrophic effects of DHEAS throughout intense stress exposure in healthy men and women. The study took place within a stressful 12-day military survival course. Utilizing a longitudinal cross-sectional repeated measures design, One hundred sixteen healthy active-duty military personnel (80% male) were studied before, during, and 24 h after the course. The dependent variable was the neurotrophin salivary nerve growth factor (sNGF). In terms of total hormone output, the effect of DHEAS on sNGF was mediated by testosterone. Unlike testosterone or cortisol, DHEAS reliably predicted sNGF at each time point, and change in DHEAS predicted change in sNGF across time points. Baseline DHEAS predicted total sNGF output across the stress trajectory. Consistent with preclinical as well as cross-sectional human research, this study demonstrates neuroprotective–neurotrophic effects of DHEAS in healthy men and women exposed to intense stress. Results are evaluated in relation to established criteria for causation.     

MDA-MB-231 breast cancer cell viability,motility and matrix adhesion are regulated by a complex interplay of heparan sulfate,chondroitin−/dermatan sulfate and hyaluronan biosynthesis

Proteoglycans and glycosaminoglycans modulate numerous cellular processes relevant to tumour progression, including cell proliferation, cell-matrix interactions, cell motility and invasive growth. Among the glycosaminoglycans with a well-documented role in tumour progression are heparan sulphate, chondroitin/dermatan sulphate and hyaluronic acid/hyaluronan. While the mode of biosynthesis differs for sulphated glycosaminoglycans, which are synthesised in the ER and Golgi compartments, and hyaluronan, which is synthesized at the plasma membrane, these polysaccharides partially compete for common substrates. In this study, we employed a siRNA knockdown approach for heparan sulphate (EXT1) and heparan/chondroitin/dermatan sulphate-biosynthetic enzymes (β4GalT7) in the aggressive human breast cancer cell line MDA-MB-231 to study the impact on cell behaviour and hyaluronan biosynthesis. Knockdown of β4GalT7 expression resulted in a decrease in cell viability, motility and adhesion to fibronectin, while these parameters were unchanged in EXT1-silenced cells. Importantly, these changes were associated with a decreased expression of syndecan-1, decreased signalling response to HGF and an increase in the synthesis of hyaluronan, due to an upregulation of the hyaluronan synthases HAS2 and HAS3. Interestingly, EXT1-depleted cells showed a downregulation of the UDP-sugar transporter SLC35D1, whereas SLC35D2 was downregulated in β4GalT7-depleted cells, indicating an intricate regulatory network that connects all glycosaminoglycans synthesis. The results of our in vitro study suggest that a modulation of breast cancer cell behaviour via interference with heparan sulphate biosynthesis may result in a compensatory upregulation of hyaluronan biosynthesis. These findings have important implications for the development of glycosaminoglycan-targeted therapeutic approaches for malignant diseases.     

Does PAPS generation determine the overall sulfate conjugation in human platelets?

The formation of 3'-phospho-adenosine-5'phosphosulfate (PAPS) depends on essentially two enzymic reactions catalysed by ATP-sulfurylase and APS-kinase (adenosine 5'-phosphosulfate-kinase). In this paper, PAPS generation by human platelets was determined by the transfer of 35sulfate from PAP35S formed in vitro to N-acetyldopamine (NADA), using the phenolsulfotransferase extracted from rat liver. A pre-requisite of this quantitative procedure was the prior inhibition of the sulfate-activating system in the latter enzyme preparation. This was accomplished by the addition of 10 mM EDTA and 14 mM pyrophosphate. The PAPS-generating system of human platelets exhibited two pH peaks with higher activity at pH 8 than pH 6. Optimal concentrations of ATP and Mg++ at 7 mM were required for the two reactions. PAPS generation so measured showed a highly significant correlation with the overall sulfate conjugation of NADA: a correlation coefficient of 0.96 was established from data obtained from 60 platelet preparations of normal subjects.     

Chloroplast sulfate transport in green algae – genes,proteins and effects

This review summarizes evidence at the molecular genetic, protein and regulatory levels concerning the existence and function of a putative ABC-type chloroplast envelope-localized sulfate transporter in the model unicellular green alga Chlamydomonas reinhardtii. From the four nuclear genes encoding this sulfate permease holocomplex, two are coding for chloroplast envelope-targeted transmembrane proteins (SulP and SulP2), a chloroplast stroma-targeted ATP-binding protein (Sabc) and a substrate (sulfate)-binding protein (Sbp) that is localized on the cytosolic side of the chloroplast envelope. The sulfate permease holocomplex is postulated to consist of a SulP–SulP2 chloroplast envelope transmembrane heterodimer, flanked by the Sabc and the Sbp proteins on the stroma side and the cytosolic side of the inner envelope, respectively. The mature SulP and SulP2 proteins contain seven transmembrane domains and one or two large hydrophilic loops, which are oriented toward the cytosol. The corresponding prokaryotic-origin genes (SulP and SulP2) probably migrated from the chloroplast to the nuclear genome during the evolution of Chlamydomonas reinhardtii. These genes, or any of its homologues, have not been retained in vascular plants, e.g. Arabidopsis thaliana, although they are encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the Photosystem II D1 reaction center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in Chlamydomonas reinhardtii is discussed along with its impact on the repair of Photosystem II from a frequently occurring photo-oxidative damage and H₂-evolution related metabolism in this green alga.     

Microenvironment of tryptophan residues in β-lactoglobulin derivative polypeptide-sodium dodecyl sulfate complexes

The changes of microenvironment of tryptophan residues in β-lactoglobulin A and its cyanogen bromide (CNBr) fragments with the binding of sodium dodecyl sulfate (SDS) were studied with measurements of the rates of N-bromosuccinimide (NBS) modification reactions by stopped-flow photometry. Two tryptophan residues of carboxyamidomethylated (RCM) β-lactoglobulin A in the states of their complexes with SDS were clearly distinguishable by their differences in NBS modification rates. We confirmed by experiments with CNBr fragments containing tryptophan residue. The modification rates of Trp 19 in RCM β-lactoglobulin A-SDS complexes were about 10-fold smaller than those expected for tryptophan residues exposed entirely to the aqueous solvent. The Trp 61 was hardly changed. The change of rate constants for Trp 19 was virtually consistent with those observed when N-acetyl-l-tryptophan ethylester was dissolved in SDS micelles. For various species of polypeptide-SDS complexes, all tryptophan residues were reactive to NBS and also, for some of them, the differences in NBS modification rates were observed between tryptophan residues on a common polypeptide chain. These results suggest micellar and heterogeneous bindings of SDS to polypeptides.     

Chemoenzymatic preparation of dermatan sulfate oligosaccharides as arylsulfatase B and α-L-iduronidase substrates

Dermatan sulfate was partially depolymerized with chondroitin ABC lyase to obtain an oligosaccharide mixture from which an unsaturated disulfated tetrasaccharide was purified and characterized using nuclear magnetic resonance spectroscopy and electrospray ionization mass spectrometry. Chemical removal of the unsaturated uronate residue with mercuric acetate, followed by de-4-O-sulfation with arylsulfatase B (N-acetylgalactosamine 4-sulfatase) and N- acetylhexo-saminidase catalyzed removal of the 2-acetamido-2-deoxy-D-galactospyranosyl residue at the non-reducing end afforded a monosulfated disaccharide of the structure -L-idopyranosyluronic acid (13)-,-D-2-acetamido-2-deoxy-4-O-sulfo galactopyranose. This monosulfated disaccharide serves as a substrate for mammalian -L-iduronidase as demonstrated using fluorophore assisted carbohydrate electrophoresis.     

Sequence analysis ofp-hydroxyphenyl-O-β-d-xyloside initiated and radio-iodinated dermatan sulfate from skin fibroblasts

To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated withp-hydroxyphenyl--d-xylopyranoside and [³H]galactose, and free [³H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After¹²⁵I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on theN-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-Å, Havsmark B, Silverberg I (1990)Biochem J 269:381–8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.Abbreviations GAG glycosaminoglycans - CS chondroitin sulfate - DS dermatan sulfate - Ser serine - Xyl d-xylose - Gal d-galactose - GlcA d-glucuronic acid - IdoA l-iduronic acid - GalNAc N-acetyl-d-galactosamine - GlcNAc N-acetyl-d-glucosamine - HexA 4-deoxy-l-threo-hex-4-enopyranosyluronic acid - HO-Phe p-hydroxyphenyl group - HO-Phe-Xyl p-hydroxyphenyl-O--d-xylopyranoside - O₂N-Phe-Xyl p-nitrophenyl--d-xylopyranoside - OSO₃ ester sulfate - PAGE polyacrylamide gel electrophoresis - HPLC high performance liquid chromatography - FPLC fast performance liquid chromatography - LC standard liquid chromatography     

A study of the effect of sodium dodecyl sulfate on bovine β-casein self-association

The effect of low concentrations of sodium dodecyl sulfate on the self-association of β-casein in solution has been reinvestigated at neutral pH by using instrinsic fluorescence measurements, analytical ultracentrifugation, gel filtration chromatography, and the fluorescent properties of the probe, anilinonaphthalene sulfonate. Sodium dodecyl sulfate was found to interact with the protein so that the normal equilibrium between monomers and micellelike polymers was displaced toward polymer formation. At higher concentrations of sodium dodecyl sulfate, the β-casein polymers became smaller while the monomer-polymer equilibrium remained displaced toward polymer formation. It seems likely that there is a limited number of sites on the β-casein molecule that bind sodium dodecyl sulfate strongly. As a consequence of this binding, the balance of electrostatic and hydrophobic forces is altered to increase the degree of self-association at low concentrations of sodium dodecyl sulfate, despite the increase in net negative charge per protein monomer.     

Isolation of sulfate‐reducing bacteria from deep sediment layers of the pacific ocean

Bacterial populations exist at great depths in marine sediments, but little is known about the type and characteristics of organisms in this unique bacterial environment. Cascadia Margin sediments from the Pacific Ocean have deep bacterial activity and bacterial populations, which are stimulated around a gas hydrate zone (215–225 m below sea floor [mbsf]). Bacterial sulfate reduction is the dominant anaerobic process within these sediments, and the depth distribution of sulfate‐reducing activity corresponds with distributions of viable sulfate‐reducing bacteria (SRB). Anaerobically stored sediments from this site were used to isolate sulfate‐reducing bacteria using a temperature‐gradient system, elevated pressure and temperatures, different media, and a range of growth substrates. A variety of enrichments on lactate were obtained from 0.5 and 222 mbsf, with surprisingly more rapid growth from the deeper sediments. The temperature range of enrichments producing strong growth from 222 mbsf was markedly wider than those from the near surface sediment (15–45°C and 9–19°C, respectively). This presumably reflects a temperature increase in deeper sediments. Only a few of these enrichments were successfully isolated due to very slow or no growth on subculture, despite the use of a wide range of different media and growth conditions. Psychrophilic and mesophilic sulfate‐reducing isolates were obtained from 0.5 m depth. As the minimum growth temperature of the mesophile (probably a Desulfotomaculum sp.) was above the in situ temperature of 3°C, it must have been present in the sediment as spores. A larger number of isolates (23) was obtained from 222 mbsf, and these barophilic SRB were closely related (based on 16S rRNA gene analysis), but not identical to, Desulfovibrio profundus, recently isolated from deep sediments from the Japan Sea. Bacteria related to D. profundus may be widespread in deep marine sediments.     

Hyaluronidase 1 and β-hexosaminidase have redundant functions in hyaluronan and chondroitin sulfate degradation

Hyaluronan (HA), a member of the glycosaminoglycan (GAG) family, is a critical component of the extracellular matrix. A model for HA degradation that invokes the activity of both hyaluronidases and exoglycosidases has been advanced. However, no in vivo studies have been done to determine the extent to which these enzymes contribute to HA breakdown. Herein, we used mouse models to investigate the contributions of the endoglycosidase HYAL1 and the exoglycosidase β-hexosaminidase to the lysosomal degradation of HA. We employed histochemistry and fluorophore-assisted carbohydrate electrophoresis to determine the degree of HA accumulation in mice deficient in one or both enzyme activities. Global HA accumulation was present in mice deficient in both enzymes, with the highest levels found in the lymph node and liver. Chondroitin, a GAG similar in structure to HA, also broadly accumulated in mice deficient in both enzymes. Accumulation of chondroitin sulfate derivatives was detected in mice deficient in both enzymes, as well as in β-hexosaminidase-deficient mice, indicating that both enzymes play a significant role in chondroitin sulfate breakdown. Extensive accumulation of HA and chondroitin when both enzymes are lacking was not observed in mice deficient in only one of these enzymes, suggesting that HYAL1 and β-hexosaminidase are functionally redundant in HA and chondroitin breakdown. Furthermore, accumulation of sulfated chondroitin in tissues provides in vivo evidence that both HYAL1 and β-hexosaminidase cleave chondroitin sulfate, but it is a preferred substrate for β-hexosaminidase. These studies provide in vivo evidence to support and extend existing knowledge of GAG breakdown.     

The role of sulfate‐reducing bacteria in the deposition of sedimentary uranium ores

The formation of many important sediment‐hosted uranium ore deposits is thought to have resulted from the reduction of relatively soluble uranyl ion—U(VI)—to insoluble uranium (IV) oxides and silicates by aqueous sulfide species. This study focused on the influence that the sulfate‐reducing bacteria Desulfovibrio desulfuricans (ATCC 7757) has on this process. Preliminary studies showed that bacterial growth was not inhibited by concentrations of uranyl ion up to 100 mg U per liter. More detailed studies showed that sulfate‐reducing bacteria have an influence on uranyl ion removal beyond the simple production of the aqueous sulfide reductant. Comparative studies of bacterial cultures containing high densities of the sulfate reducers with bacterial cell‐free but otherwise identical media showed that the bacteria themselves enhance uranium removal from solution. At pH 8.0, no reaction was observed in H₂S‐bearing cell‐free media, whereas at the same H₂S concentration, the uranyl ion decreased markedly in the presence of the bacteria. At pH 7.0, some uranium removal occurred in the absence of bacteria, but it was much more rapid in their presence. We postulate that these effects are due to the ability of bacterial cell walls to adsorb uranium. Adsorption to surfaces is known from independent studies to enhance uranium reduction, and evidently this two‐step adsorption‐reduction mechanism is occurring in our experiments. We conclude that sulfate‐reducing and other bacteria may play a significant role in the geochemical cycling of uranium.