Effects of organic sulfur compounds on extraction and determination of inorganic sulfate

The common methods for determining inorganic soil sulfate may be affected by the extraction of sulfate from organic sulfur compounds such as ester sulfates. In order to test this, various synthetic organic sulfur compounds (ranging from ester sulfates to sulfonates and C-bonded sulfur) were extracted with deionized water or with two common sulfate extractors (0.5 M NaHCO₃ and 0.02 M NaH₂PO₄). Similar amounts of dissolved sulfate were detected in all extracts of the aromatic ester sulfate hydroxyquinoline sulfate. Sulfate was not released from aliphatic ester sulfates or C-bonded sulfur. Ion chromatography was compared to a turbidimetric method for the determination of sulfate. The latter method, based on BaSO₄-precipitation, appeared to be unsuitable for determining sulfate in organically influenced solutions. Barium precipitated sulfate as well as ester sulfates. Furthermore, the photometry of BaSO₄ was influenced by specific absorption of dissolved organic compounds, leading to a misinterpretation of the sulfate concentration in the solution.     

Incorporation of 35SO 4 2− into sediments of three New York lakes

Intact sediment cores were obtained from three New York lakes in May, July, and October 1981. Radioactive S (as ³⁵SO ₄ ²⁻ ) was added to the overlying water and cores were incubated without atmospheric exchange for one week near lake bottom temperatures. Headspace flux of 0₂ as an index of sediment respiration rates varied among lakes and seasonally within lakes. Acidic South Lake had the lowest respiration rate at all seasons and also the smallest net incorporation of the ³⁵SO ₄ ²⁻ . Summer net isotope transformation into ester sulfate and non-HI reducible S (pyrite and C-bonded S) constituents was 88.6%, 89.4%, and 59.7% of total sediment isotope for Oneida, Deer, and South, respectively. Seasonal variation of net isotope incorporation was observed in each lake as were differences in ³⁵SO ₄ ²⁻ partitioning into major S pools. Of the S constituents analyzed, HCl digestible S (volatile sulfides) was the smallest pool, while ester sulfate and non-HI reducible S together accounted for greater than 50% of S isotope transformation in all lakes. In addition, ester sulfate is the major product of dissolved SO ₄ ²⁻ transformation and its formation results in less alkalinity generation than the formation of non-HI reducible S constituents. Thus ester sulfate transformation processes must be considered in calculating alkalinity generation by lake sediments. Financial support provided by Office of Water Research Technology (Project No. 13-096-NY). Financial support provided by Office of Water Research Technology (Project No. 13-096-NY).     

Stimulation of heparan sulfate proteoglycan synthesis and secretion during G1 phase induced by growth factors and PMA

Fetal calf serum (FCS) and PMA (phorbol 12-myristate-13-acetate) specifically stimulate the synthesis of heparan sulfate proteoglycan in endothelial cells. Staurosporine and n-butanol, kinase inhibitors, abolish the PMA effect. Forskolin and 8-bromo adenosine 3′:5′-cyclic monophosphate, activators of, respectively, adenylate cyclase and protein kinase A cannot reproduce the PMA effect. The kinetics of cell entry into S phase of the endothelial cells was determined by DNA synthesis ([³H]-thymidine and Br-dU incorporation), and flow cytometry. The mitogenic effect of fetal calf serum is abolished by PMA. Also, PMA pre-treatment inhibits the enhanced synthesis of heparan sulfate proteoglycan after a second PMA exposure. Remarkably, the stimulation of heparan sulfate proteoglycan synthesis by fetal calf serum and PMA seems to be mainly restricted to G₁ phase. Therefore fetal calf serum and PMA cause an enhanced synthesis of heparan sulfate proteoglycan, and PMA causes a cell cycle block at G₁ phase. J. Cell. Biochem. 70:563–572, 1998. © 1998 Wiley-Liss, Inc.     

Purification of human and avian influenza viruses using cellulose sulfate ester (Cellufine Sulfate) in the process of vaccine production

Affinity chromatography using sulfated, spherical cellulose beads (Cellufine Sulfate) was assessed for purification of influenza A and influenza B viruses. Recovery rates of viruses eluted from the beads were high for all tested virus strains. This method was also useful for removing chicken egg-derived impurities from allantoic fluids containing influenza viruses; the hemagglutination activity per amount of protein in the eluted sample was significantly higher than that in the applied sample. These results suggest that use of Cellufine Sulfate is a practical method for primary purification of influenza viruses in the process of influenza vaccine production.     

A novel type of arylsulfotransferase

Arylsulfotransferase catalyzes the transfer of a sulfate group from 3-phosphoadenosine-5-phosphosulfate (PAPS) to a phenolic acceptor substrate. We discovered a novel type of sulfotransferase from an anaerobic bacterium of human intestine, Eubacterium A-44. In the bacterial enzyme PAPS did not serve as a donor and all alcohols did not as acceptors. The new arylsulfotransferase was purified 185-fold from a crude extract of sonicated bacteria to homogeneity. The enzyme (MW 315 kd) was composed of four identical subunits (MW 80 kd) whose N-terminal amino acid was arginine, and its optimal pH and pI were 8–9 and 3.9, respectively. The enzyme catalyzed stoichiometric transfer of a sulfate group from a phenol sulfate ester to other phenols, with strict specificity. With tyramine as an acceptor, p-acetylphenyl sulfate was the best donor, followed by 4-methylumbelliferyl sulfate and p-nitrophenyl sulfate. With p-nitrophenyl sulfate as a donor, naphthol was the best acceptor, followed by estradiol, phenol, tyrosine methylester, tyramine, and epinephrine in decreasing order. Only the 4-position of catecholamines was specifically sulfated. Naturally occurring phenolic compounds, such as flavone, chalcone, and xanthone, were sulfated as well. Tyrosine-containing peptides were enzymatically sulfated: enkephalin, LH-RH, vasopressin, angiotensins, proctorin, CCK-8, and phyllocaerulein were sulfated with high yields. The novel sulfotransferase is expected to be applicable to enzymatic O-sulfation of tyrosine-containing hormones. The ³⁵S-labeled sulfate group from (³⁵S)p-nitrophenyl sulfate was incorporated into a tyrosyl residue at the active site of the enzyme (2 mole ³⁵S/mole of enzyme). The enzyme was inactivated by diethylpyrocarbamate and TLCK, chemical modifying agents for a histidyl residue. The reaction mechanism of arylsulfotransferase was proposed as follows: a donor substrate combines a histidyl residue with concomitant release of a phenolic compound. The sulfate group of the histidyl residue transfers to a tyrosyl residue, and then to an acceptor with the binding of another donor substrate to the histidyl residue.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Cycling of inorganic and organic sulfur in peat from Big Run Bog,West Virginia

Total S concentration in the top 35 cm of Big Run Bog peat averaged 9.7 mol·g — wet mass^–1 (123 mol·g dry mass^–1). Of that total, an average of 80.8% was carbon bonded S, 10.4% was ester sulfate S, 4.5% was FeS₂­S, 2.7% was FeS­S, 1.2% was elemental S, and 0.4% was SO₄ ^2–­S. In peat collected in March 1986, injected with³⁵S­SO₄ ^2– and incubated at 4 °C, mean rates of dissimilatory sulfate reduction (formation of H₂S + S⁰ + FeS + FeS₂), carbon bonded S formation, and ester sulfate S formation averaged 3.22, 0.53, and 0.36 nmol·g wet mass^–1·h^–1, respectively. Measured rates of sulfide oxidation were comparable to rates of sulfate reduction. Although dissolved SO₄ ^2– concentrations in Big Run Bog interstitial water (< 200 µM) are low enough to theoretically limit sulfate reducing bacteria, rates of sulfate reduction integrated throughout the top 30–35 cm of peat of 9 and 34 mmol·m^–2·d^–1 (at 4 °C are greater than or comparable to rates in coastal marine sediments. We suggest that sulfate reduction was supported by a rapid turnover of the dissolved SO₄ ^2– pool (average turnover time of 1.1 days). Although over 90% of the total S in Big Run Bog peat was organic S, cycling of S was dominated by fluxes through the inorganic S pools.     

Heparin localization and fine structure regulate Burkitt's lymphoma growth

Burkitt's lymphoma (BL) is a B-cell malignancy associated with the Epstein-Barr virus (EBV). Mounting evidence has implicated heparan sulfate proteoglycans and heparan sulfate-like glycosaminoglycans (HSGAGs) in the initiation, severity, and progression of the malignancy. The importance of HSGAGs in regulating BL cell growth was therefore examined. Extracellular exogenous heparin inhibited cell growth >30%, while heparin internalized with poly(beta-amino ester)s promoted proliferation up to 58%. The growth-modulating effects of heparin and internalized heparin were dependent on cell surface HSGAGs, PI3K, and Erk/Mek. Treatment of cells with protamine sulfate or with heparinases potently inhibited proliferation, with the greatest effects induced by heparinase I. Cell surface HSGAGs therefore play an important role in regulating BL proliferation and may offer a potential target for therapeutic intervention.     

Hydroxycinnamic acid esters from Polygala chamaebuxus

Three new glycosides have been isolated from the aerial parts of Polygala chamaebuxus by preparative liquid chromatography on an axially-compressed silica column. The compounds were identified as 1,3-diesters of β-D-fructofuranosyl-α-D-glucopyranoside with sinapoyl, feruloyl and acetyl ester moieties on the furanose ring. The structures were elucidated by a combination of chemical and spectroscopic methods (D/CI-MS, ¹H and ¹³C NMR).     

Pregnenolone sulfate, a naturally occurring excitotoxin involved in delayed retinal cell death

The present study was designed to investigate the neurosteroid pregnenolone sulfate (PS), known for its ability to modulate NMDA receptors and interfere with acute excitotoxicity, in delayed retinal cell death. Three hours after exposure of the isolated and intact retina to a 30-min PS pulse, DNA fragmentation as assessed by genomic DNA gel electrophoresis and a modified in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method appeared concurrently with an increase in superoxide dismutase (SOD) activity and thiobarbituric acid-reactive substances (TBARS) levels. At 7 h, the increased amount of DNA laddering was accompanied by a higher number of TUNEL-positive cells in the inner nuclear and ganglion cell layers. Necrotic signs were characterized by DNA smear migration, lactate dehydrogenase (LDH) release, and damage mainly in the inner nuclear layer. PS-induced delayed cell death was markedly reduced by the NMDA receptor antagonists 4-(3-phosphonopropyl)-2-piperazinecarboxylic acid and 3alpha-hydroxy-5beta-pregnan-20-one sulfate but completely blocked after concomitant addition of the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Steroids with antioxidant properties (progesterone, dehydroepiandrosterone and its sulfate ester, and 17beta-estradiol) differently prevented PS-induced delayed cell death. Cycloheximide treatment protected against DNA fragmentation and LDH release but failed to prevent the rise in SOD activity and TBARS level. We conclude that a brief PS pulse causes delayed cell death in a slowly evolving apoptotic fashion characterized by a cycloheximide-sensitive death program downstream of reactive oxygen species generation and lipid peroxidation, turning into secondary necrosis in a retinal cell subset.     

Isolation and structure of a chlorosis-inducing toxin of Pseudomonas phaseolicola

A toxin, named phaseolotoxin, which causes leaf-chlorosis in bean leaves has been isolated from liquid cultures of Pseudomonas phaseolicola, and purified. Its structure is (N^δ-phosphosulphamyl)ornithylalanylhomoarginine.     

EJ-ras oncogene transfection of endothelial cells upregulates the expression of syndecan-4 and downregulates heparan sulfate sulfotransferases and epimerase

The EC rabbit endothelial cell line was transfected with the EJ-ras oncogene (EJ-ras EC). EJ-ras EC cells display over expression of the Ras oncogene, morphological changes and deregulation of the cell cycle, becoming more densely populated and serum-independent. In addition, EJ-ras-transfectant cells show higher levels of the syndecan-4 mRNA. In addition to the increase in the core protein, a parallel increase in the glycosylation of the syndecan-4 protein, a proteoglycan that bears heparan sulfate chains, also occurs. This increase is observed both for the heparan sulfate proteoglycan synthesized by the cells and for that secreted to the culture medium. This enhancement in heparan sulfate synthesis was observed through metabolic labeling of the cells, immunoprecipitation of syndecan-4 and heparitinases treatment. Furthermore, the EJ-ras-transfectant cells do not exhibit decreased synthesis of heparan sulfate during the G(1)-S phase transition, as observed for the parental cell line. Also, heparan sulfate synthesis is not stimulated by PMA as displayed by parental endothelial cells. Significant structural changes of heparan sulfate, such as decreased O-sulfation, were observed in the EJ-ras-transfected cells. Decreases in the mRNA levels of some enzymes (glucuronosyl C-5 epimerase, iduronosyl-2-O-sulfotransferase, glucosaminyl-6-O-sulfotransferase-1 and N-deacetylase/N-sulfotransferase-1), involved in the biosynthetic pathway of heparan sulfate, were also observed. The results suggest that overexpression of the EJ-ras oncogene alters the cell cycle, through signal transduction cascades, upregulates the expression of syndecan-4, and downregulates enzymes involved in the heparan sulfate biosynthesis related to chain modification, leading to the structural changes of the heparan sulfate syndecan-4 proteoglycan in endothelial cells.     

Histone phosphorylation in phorbol ester stimulated and β-adrenergically stimulated mouse epidermis in vivo and characterization of an epidermal protein phosphorylation system

Under certain physiological conditions a change i n the phosphorylation of histones in mouse epidermis in vivo was observed. Thus a single local application of the tumor-promoting mitogen 12-O-tetradecanoylphorbol-13-acetate caused a long-lasting increase of histone H1 phosphorylation which paralleled stimulated cell proliferation. Injection of the antimitotic β-adrenergic agonist isoproterenol led to a temporatory decrease in the rate of phosphorylation of H1, H2A and H2b immediately after cyclic AMP accumulation. A complete protein phosphorylation system could be demonstrated in mouse epidermis homogenates. The following enzyme activities were partially purified and characterized: a cyclic AMP-dependnet histone kinase; a ‘casein kinase’ and an ‘unsopecific’ protein kinase; a histone-specific protein phosphatase; and two ‘unspecific’ phosphoprotein phosphatases. In addition, a stimulatory effect of cyclic GPM on histone phosphorylation was observed. The enzymes were found to be predominantly localized in the 105 000 × g supernatant, but a small proportion of protein kinase and phosphatase activity could be regularly demonstrated in cell nuclei.     

Simultaneous quantitative determination of eight triterpenoid monoesters from flowers of 10 varieties of Calendula officinalis L. and characterisation of a new triterpenoid monoester

Dichloromethane extracts of dried flowers of Calendula officinalis contain eight known bioactive triterpendiol monoesters, namely, faradiol-3-O-palmitate, faradiol-3-O-myristate, faradiol-3-O-laurate, arnidiol-3-O-palmitate, arnidiol-3-O-myristate, arnidiol-3-O-laurate, calenduladiol-3-O-palmitate and calenduladiol-3-O-myristate. These pentacyclic terpenoids have been quantified simultaneously using reversed-phase HPLC with isocratic elution and internal standardisation. Of the 10 varieties of C. officinalis investigated, Calypso Orange Florensis produced the highest amounts of the bioactive monoesters, followed by Fiesta Gitana Gelb and May Orange Florensis. The lipophilic extract from the flowers of Calypso Orange Florensis variety also contained low levels of the newly characterised calenduladiol-3-O-laurate.     

Novel indolizine derivatives with unprecedented inhibitory activity on human farnesyltransferase

The rational structural modification of new substituted indolizin-3-yl(phenyl)methanones 1a–i, 2a–i and 3a–i has greatly improved human farnesyltransferase inhibition. The para-bromophenyl analog 2f bearing an ester unit on the indolizine ring demonstrates the highest inhibition potential, with IC₅₀ value of 1.3 ± 0.2 μM. The amidic series 1a–i proves to be the most promising for future modulations, particularly at the triple bond level.     

Enzymatic synthesis of terpenyl esters by transesterification with fatty acid vinyl esters as acyl donors by Trichosporon fermentans lipase

Enzymatic synthesis of terpenyl esters by esterification or transesterification with fatty acid vinyl esters as acyl donors by celite-adsorbed lipase of Trichosporon fermentans was investigated. In direct esterification of geraniol, the lipase showed high reactivity toward fatty acids with carbon chains longer than C-8, but little reactivity toward fatty acids with shorter chains. With fatty acid vinyl esters as acyl donors, the lipase catalysed the synthesis of geranyl and citronellyl esters with carbon chains shorter than C-6 in with yields of >90% molar conversion. Time course, effects of added water, temperature and substrate concentration were studied for the synthesis of geranyl acetate. Molar conversion yield reached 97.5% after 5 h incubation at 30–40°C with the addition of 3% water. In this reaction, no inhibition by substrates such as geraniol and vinyl acetate was observed.     

Glycosaminoglycan structures required for strong binding to midkine,a heparin-binding growth factor

Midkine (MK), a heparin-binding growth factor, binds strongly to oversulfated structures in chondroitin sulfates (CSs) and heparan sulfate. To elucidate the carbohydrate structure actually involved in the strong binding, dissected brains from 13-day mouse embryos were incubated with [14C]-glucosamine. The labeled glycosaminoglycans were fractionated by MK-agarose affinity chromatography to a weakly binding fraction, which was eluted by 0.5 M NaCl, and a strongly binding fraction, which was eluted by higher NaCl concentrations. Among the unsaturated disaccharides released from the strongly binding fraction by chondroitinase ABC, DeltaDi-diSE with 4,6-disulfated N-acetylgalactosamine accounted for 32.3%, whereas its content was lower in the weakly binding fraction. Artificial CS-E structure was formed using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid or recombinant human enzyme. Analysis of the products and their interaction with MK revealed that E units without 3-O-sulfation of glucuronic acid are sufficient for strong binding, provided that they are present as a dense cluster. Among the sulfated disaccharides released by heparitinase digestion, the trisulfated one, DeltaDiHS-triS, was the most abundant in the strongly binding fraction and was lower in the weakly binding fraction. Together with results of previous studies, we concluded that the multivalent trisulfated heparin-like unit is another structure involved in strong binding to MK.     

Quantitative colorimetric and gas chromatoographic determination of arecaidine propargyl ester

Arecaidine propargyl ester (APE) is a potent muscarinic agonist often used in pharmacological studies. To date, no sensitive quantitative analytical method for APE has been published. In this study, two methods for the quantitative determination of APE are compared: a colorimetric assay, based on the formation of the corresponding ferric(III)-hydroxamic acid complex, and a direct gas chromatographic method, using arecoline as the internal standard. The latter method was found to be more precise. The utility of the gas chromatographic assay was further demonstrated in a stability study of the drug in the biological fluid aqueous humor of rabbits.     

Solid angle as steric parameter of acyclic saturated groups

With the early aim of quantifying steric consequences of chirality, efforts to define a nonempirical steric parameter of chemical groups are reported. Steric hindrance of a reacting center by any acyclic saturated R group has been characterized by a geometric “axial steric parameter”: the solid angle of R. When the group is a “symmetric top substituent” (i.e., when all the terminal atoms are equivalent), the solid angle matches the solid angle of a cone envelope of R. The definition of this cone is compared with Tolman's definition of a ligand cone in organometallic complexes. The chemical significance of this parameter is shown by an excellent correlation with the Dubois' experimental steric parameter E′_s. Modeling steric repulsion by the cone of R, and correcting solid angles for conformational effects, only 3 empirical coefficients are needed to calculate 33 values of E′_s with less than 10% error. The cone model is suggested to be relevant within the limits of random and independent free rotations about all the bonds in the C–R group. A separation between “axial cone steric hindrance” and other steric effects is proposed. The basic model and the corrections proposed allow the conformational features of esters to be discussed.     

Presence of Conjugated Catecholamines in Rat Brain: A New Method of Analysis of Catecholamine Sulfates

Abstract: A new method of measuring catecholamine (CA) sulfate permitted us to detect its presence in rat brain for the first time. The procedure consisted of separating the CA sulfate from the free CA by alumina adsorption followed by passage through Dowex, and measuring the CA sulfate by a radioenzymatic assay in the presence of a sulfatase. This method permitted demonstration of the presence of dopamine sulfate, and occasionally, of norepinephrine and epinephrine sulfate in the hypothalamus, striatum, and hippocampus of rat brain.