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1. Fibroblasts cultured from the skin of patients with Sandhoff disease accumulate excessive amounts of sulfated glycosaminoglycans, probably chondroitin sulfate and dermatan sulfate. In addition, Sandhoff fibroblasts show a lengthened turnover time for these compounds. Only little such abnormality in sulfated glycosaminoglycan metabolism has been found in Tay-Sachs fibroblasts. 2. The impaired catabolism of glycosaminoglycans in Sandhoff fibroblasts can be normalized, i.e. corrected, by addition of β-N-acetylhexosaminidase isozyme A from normal human urine to the culture medium. β-N-Acetylhexosaminidase isozyme B from urine is ineffective because it is not taken up by the cells. However, if homogenates of normal or Tay-Sachs fibroblasts are used as a source of the enzymes, metabolic correction is obtained in both instances. When β-N-acetylhexosaminidase isozymes A and B of normal fibroblasts are separated by isoelectric focusing and tested individually, isozyme A exhibits about five times the corrective activity of isozyme B, when compared on the basis of their activity toward the synthetic substrate, 3. Although β-N-acetylhexosaminidase isozyme B participates in the degradation of sulfated glycosaminoglycans in cultured cells, only fibroblast homogenates containing isozyme A release N-acetylglucosamine from [14C]glucosamine-labelled hyaluronic acid oligosaccharides in vitro. [ABSTRACT FROM AUTHOR]

Techniques for preparation of enzyme extracts from Chlorella pyrenoidosa chick are described which yield a stable system which converts adenosine 5'-phosphosulfate to sulfide and/or cysteine in the absence of added active thiols. Since these thiols (such as dithiothreitol) have been shown to yield sulfite, among other products, from the adenosine-5'-phosphosulfate sulfotransferase reaction, we have studied the adenosine 5'-phosphusulfate reducing system in the presence and absence of dithiothreitol. In all cases dithiothreitol increases the activity of the reducing system forming cysteine from adenosine 5'-phosphosulfate. Mutant Sat-2, which cannot grow on sulfate, is shown to lack thiosulfonate reductase activity but to retain sulfite reductase activity. Extracts from this mutant will form sulfide and cysteine only in the presence of dithiothreitol, while wild-type extracts will form these products in presence or absence of dithiothreitol, the activity with dithiothreitol being higher. Mutants Sat-1,3-6 which are blocked in the adenosine-5'-phosphosulfate sulfotransferase step are blocked in the formation of sulfide and cysteine from adenosine 5'-phosphosulfate. Binding studies with adenosine 5'-phosphosulfate indicate that the sulfonyl group is transferred to a carrier to form carrier-S-SO-3 and the available evidence suggests that this carrier is a prosthetic group of the thiosulfonate reductase. On the basis of this evidence, two pathways in the cell-free extracts are thought to be operating. One, in the absence of active thiols, is a pathway consisting of bound intermediates in which the sulfonyl group of adenosine 5'-phosphosulfate is transferred via adenosine-5'-phosphosulfate sulfotransferase to carrier on the thiosulfonate reductase where it is reduced to the thiol level by ferredoxin; the thiol is then transferred via O-acetyl-L-serine sulfhydrylase to form cysteine. The other, or free intermediate pathway operates in the presence of dithiothreitol which releases free sulfite from the adenosine-5'-phosphosulfate sulfotransferase reaction. This sulfite is then reduced via sulfite reductase to sulfide which forms cysteine via O-acetyl-L-serine sulfhydrylase. Since mutant Sat-2, which cannot grow on sulfate nor reduce it in vivo, lacks thiosulfonate reductase activity but yields sulfite reductase activity in extracts, we conclude that the bound intermediate pathway is the preferred pathway of sulfate reduction in vivo; the free intermediate pathway is probably operative only when free sulfite is available either from occasional side reactions in vivo or when sulfite is fed to cells from outside. [ABSTRACT FROM AUTHOR]

1. The inhibition of the 5-serine O-sulphate-degrading activity of pig liver has been investigated. Compounds that acted as competitive inhibitors all possessed the basic stereochemistry associated with the L-serine O-sulphate molecule. 2. In general the most efficient competitive inhibitors were those possessing an interproton distance of 0.75 nm, with two electronegative ends or alternatively one electronegative end and the negative end of a dipole. 3. O-Phospho-L-serins is an atypical inhibitor since it obeys non-competitive inhibitor kinetics even though from theoretical considerations it should act as a competitive inhibitor. 4. 3-Chloro-L-alanine methyl ester is a substrate for the enzyme. 5. 4-Iodoacetamidosalicy]Je acid acts as an irreversible inhibitor of the system. 6. The probable topography of the active site and the disposition of the substrate at the active site is discussed. [ABSTRACT FROM AUTHOR]

By incubation with washed mitochondria from guinea pig liver [7α-³H]dehydroepiandrosterone sulfate can be converted into a ³H-labelled lipophile sulfoconjugate. Concerning the overall reaction a Km-value of approximately 6 μM was derived from the substrate concentration curve. Optimal yields of lipophile steroid sulfoconjugates were obtained at a pH of 8.0. Of the various coenzymes tested CTP appeared to be essential for the formation of lipophile sulfoeonjugates. Whereas magnesium ions stimulated the conversion of [7α-³H]dehydroepiandrosterone sulfate, the biosynthesis of lipophile steroid sulfoconjugates was inhibited by sodium fluoride or p-chloromercuribenzoate. [ABSTRACT FROM AUTHOR]

Presents a study on free and sulphate sulphur contents and Potential of Hydrogen (pH) values in soil profiles from barren flats and mangrove swamps of Keneba in the lower Gambia river basin. Concentrations of soluble sulphate and free sulphur content in layers of soils under vegetation and barren flats; Distribution in depth and range in pH of fresh and air dried mangrove soils; Variations in the levels and pH of water table of a barren flat during the dry season.

1. Lipoamide dehydrogenase binds sodium dodecylsulphate in its dimeric state at 25 °C in two phases. The first phase, 0-0.3 g/g protein, is bound purely to hydrophobic sites; the second binding phase, 0.3-1.4 g/g protein is less well characterized and is influenced by the ionic strength and the dissociation of a protonated group with pK 6.6. The effects can be reversed by cooling to 0 °C. 2. The spectral and kinetic parameters are only affected during the second binding phase, maximal effects are obtained, with the exception of fluorescence parameters, at 1.4 g/g protein. The changes m the absorption spectrum indicate that different parts of the flavin absorption spectrum are influenced by unequal dodecylsulphate concentrations. Fluorescence polarisation shows that FAD becomes less tightly bound. Upon prolonged incubation it dissociates. 3. Binding of dodecylsulphate has a large influence on the hydrodynamic parameters. Viscosity measurements indicate the existence of asymmetrical molecules; the sedimentation constant declines from 5.8 S to 2.6 S after an increase to 8.9 S. The results show that a dimeric dodecylsulphate · enzyme complex can be reactivated by removal of dodecylsulphate at 0 °C, the monomeric dodecylsulphate · protein complex remains reactive. [ABSTRACT FROM AUTHOR]

The steroids in meconium from normal male and female newborn infants have been studied by gas chromatography-mass spectrometry. Practically all steroids were excreted as mono- and disulphates. The approximate concentrations of steroid mono- and disulphates were 430 and 470 μg per g of meconium. Twenty unsaturated and fourteen saturated steroids were completely identified. Most of the unsaturated steroids had a 3β-hydroxy-Δ5 structure. In addition, seven saturated steroids were tentatively identified. No significant qualitative differences were found in the steroid composition of meconium from male and female newborns. The results are discussed in relation to present knowledge of steroid metabolism in the foeto-placental unit. [ABSTRACT FROM AUTHOR]

Neutral steroid glucuronides and mono- and disulphates in faeces and urine of normal adult males were analysed before and during phthalylsulphathiazole treatment ir order to clarify whether faecal and urinary excretion of C19 and C21 steroids is modified by reduction of the intestinal microflora. For the same purpose, the unconjugated neutral steroids in faeces were analysed, too. During the sulphonamide treatment, the profile of faecal neutral steroid sulphates became very similar to that in bile. The amount of sulphate-conjugated neutral steroids, especially disulphates, was increased and the pattern of the faecal steroids indicated a decrease in the oxidoreducing and epimerising activities of the intestinal contents. No neutral steroid glucuronides were detected in faeces either before or during the sulphonamide treatment. The urinary excretion of neutral steroid conjugates was very little affected by the administration of the drug. However, most of the 16-hydroxylated C19 steroid monosulphates were excreted in urine in larger amounts during the treatment than before it.The resets obtained indicate that in normal adult subjects changes in the composition of the intestinal microflora greatly modify faecal steroid excretion, but seem to be of very little importance for the urinary output of a large number of neutral steroid conjugates. [ABSTRACT FROM AUTHOR]

Copolymers with different contents of adenine and N1-methyl adenine residues have been studied, polyadenylic acid being methylated by sulfate. Thermal stability, circular dichroism and light scattering measurements are in favor of a two-strand structure at neutral pH. Thermal stability of these products is much less sensitive to ionic strength than that of most polynucleotides, and in some cases independent of ionic strength. These copolymers interact or not with poly(U), depending on the percentage of N1-methyl adenine residues. Copolymers with high contents of methyladenine interact with poly(A) at neutral pH. These copolymers do not bind DNA-dependent RNA polymerase. Some results at acid pH are also reported. [ABSTRACT FROM AUTHOR]

The steroids in urine from a pregnant woman have been studied by gas chromatography-mass spectrometry. The major part, 64%, of the steroids identified occurred in the glucuronide fraction, whereas the free, monosulphate and disulphate fractions contained 24%, 5% and 7%, respectively. The compounds had the following general structures: 3-hydroxy-17β-pregnan-20-one, 3α-hydroxy-5β, 17α-pregnan-20-one, 3α-hydroxy-5α-androstan-17-one, preganane-3, 20-diol, 3α, 21-dihydroxy-5β-pregnan-20-one, 3α, 16α-dihydroxy-5β-pregnan-20-one, 3α, 15β-dihydroxy-pregnan-20-one, 5-pregnane-3β, 20α-diol, 5α-pregnane-3, 20α, 21-triol, 5-androstene-3β, 16, 17-triol, oestriol, 15-hydroxyoestriol and pregnane-3, 16, 20, 21-tetrol. In the free and glucuronide fractions the C21O2 steroids mainly has a5β configuration whereas about 90% of the mono- and di-sulphurylated C21O2 steroids had a 5α structure. The results are discussed in relation to what is known about faecal excretion of steroids during pregnancy. [ABSTRACT FROM AUTHOR]

Gas-liquid chromatography, radio-gas chromatography and gas chromatography-mass spectrometry were used to identify steroid metabolites in the monosulphate and disulphate fractions of faeces from germfree female rats and in the monosulphate fraction of faeces from conventional female rats after intraperitoneal administration of [4-14C]pregnenolone and [4-14C]-corticosterone. The germfree rats excreted the following steroids: monosulphate fraction: 3β, 17α-dihydroxy-5α-pregnan-20-one, 5α-pregnane-3α, 16α, 20α-triol, 2α, 3β, 16α-trihydroxy-5α-pregnan-20-one and 3α, 15β, 16α-trihydroxy-5α-pregnan-20-one; disulphate fraction: 3α- and 3β, 11β, 21-trihydroxy-5α-pregnan-20-one. The following steroids were identified in faeces from conventional rats: oestriol, 3α, 15α(and 15β)-dihydroxy-5α, 17α (and 17β)-pregnan-20-one-3α 19-dihydroxy-5α, 17α-pregnan-20-one, 5α-pregnane-3α, 16α, 20-triol, 3β, 11α-dihydroxy-5α-pregnan-20-one, 2α, 3α-dihydroxy-5α, 17α-pregnan-20-one (tentatively identified), and 3α, 17α-dihydroxy-5α-pregnan-20-one. The different steroid patterns in faeces from germfree and conventional rats are explained by the metabolic reactions carried out by the intestinal flora. Radio-gas chromatography showed that 3α- and 3β, 11β, 21-trihydroxy-5α-pregnan-20-one and 3β, 11α-dihydroxy-5α-pregnan-20-one were metabolites of corticosterone. The formation of 3β, 11α-dihydroxy-5α-pregnan-20-one and of the 17α-pregnane derivatives by intestinal microorganisms is discussed. [ABSTRACT FROM AUTHOR]

Radio-gas chromatography and gas chromatography-mass spectrometry were used to study unconjugated metabolites of administered [4-14C] corticosterone and [4-14C]pregnenolone in faeces from germfree and conventional female rats. 3α-Hydroxy-5α, 17α-pregnan-20-one, 3α-hydroxy-5α, 17β-pregnan-20-one, 3β-hydroxy-5α, 17α-pregnan-20-one, 3β, 15β-dihydroxy-5α-pregnan-20-one, 3β, 15α-dihydroxy-5α-pregnan-20-one and 3α, 16α-dihydroxy-5α-pregnan-20-one were characterized as metabolites of pregnenolone whereas 3α, 20β-dihydroxy-5α-pregnan-11-one and 3β, 20β-dihydroxy-5α-pregnan-11-one were identified as metabolited of corticosterone in the conventional rats. Two other 3, 15-dihydroxypregnan-20-one isomers, tentatively identified as the 3α, 5α, 15α (and 15β) epimers, were also found. In addition 3β-hydroxy-5-androsten-17-one, 5α-androstane-3β, 17β-diol, 5α-androstane-3&Alpha, 7α, 17β-triol, 3α (and 3β), 19-dihydroxy-5α-pregnan-20-one and 5α-pregnane-3α (and 3β), 20α-diol were recovered in the free steroid fraction in faeces from conventional female rats. The amounts of these steroids were too small to permit a study of the degree of labelling. Although some radioactivity was present in the free steroid fraction from the germfree animals no steroid could be identified in this fraction. The findings are discussed with particular reference to enzyme activities of the intestinal flora. [ABSTRACT FROM AUTHOR]

The steroid glucuronide, and mono- and disulphate fractions were obtained from human bile by chromatography on Sephadex LH-20. The conjugates in the glucuronide fraction were hydrolysed with β-glucuronidase and the mono- and disulphate fractions were solvolysed. The steroids liberated were fractionated on silicic acid and by thin layer chromatography and analysed by gas-liquid chromatography and gas chromatography-mass spectrometry. The following C21O3 and C21O4 steroids were identified: 5α-pregnane-3α,17α,2oα-triol, 5β-pregnane-3α,17α,20αtriol, 5-pregnene-3α,17α,20α-triol, 5-pregnene-3β,17α,20α-triol, 5α-pregnane-3α,16α,20α-triol, 3α,21-dihydroxy-5β-pregnan-20-one, 3α,11β,21-trihydroxy-5α-pregnan-20-one, 3α,11β,21-trihydroxy-5β-pregnan-20-one and 3α,21-dihydroxy-5β-pregnane-11,20-dione. Concentrations of these steroids were measured in fistula or gall-bladder bile samples from five subjects. [ABSTRACT FROM AUTHOR]

Ethanol intake has been shown to cause a rapid increase of the concentration of androst-5-ene-3β,17β-diol 3-sulphate and a simultaneous decrease of the concentration of dehydroepiandrosterone sulphate in plasma. The ratio between the 17β-hydroxysteroid and the 17-ketosteroid increased with the ethanol concentration up to 30-50 mg ethanol/100 ml blood when a 6 to 10-fold increase was obtained. The ratios between the 3-sulphates of 5α-androstane-3α,17β-diol and androsterone and between the monosulphates of 5α-androstane-3β,17β-diol and 3β-hydroxy-5α-androstan-17-one also increased during ethanol metabolism. Deuterium was rapidly incorporated into the D-ring of the three C19 steroid diols, reaching about 20 atom per cent excess 10 to 20 min after oral administration of 0.1 g [1-²H2]ethanol per kg body weight. The concentrations of steroids with a sulphate group at C-17 did not change and deuterium was not incorporated into these steroids or into the 17-ketosteroid sulphates. The ratio between the 3-sulphates of 5-pregnene-3β,20α-diol and pregnenolone increased only slightly and incorporation of deuterium into the side chain of the C21 diol reached a maximum of about 4 atom per cent excess about 1 hour after the ethanol administration. The results indicate that the ratio between 3-sulphates of 17β-hydroxysteroids and the corresponding 17-ketosteroids in plasma is determined by the NADH/NAD+ ratio in the liver cell cytoplasm. They also indicate that the 3-sulphate of 5-pregnene-3β,20α-diol is not formed from pregnenolone sulphate at the same site or with the use of the same coenzyme molecules as the 17β-hydroxysteroids. [ABSTRACT FROM AUTHOR]

The sulphate of 3β-hydroxypregn-5-en-20-one is now reported to be a substrate for crystalline 20β-hydroxysteroid:NAD oxidoreductase prepared from Streptomyces hydrogenans. Utilization of this steroid sulphate was not independent of activity towards pregn-4-ene-3,20-dione or 3β-acetoxypregn-5-en-20-one, but the total rate of reaction for equimolar mixtures of 3β-hydroxypregn-5-en-20-one sulphate and either pregn-4-ene-3,20-dione or 3β-acetoxypregn-5- en-20-one was less than that calculated for the simplest type of mutual competitive inhibition, particularly at low substrate concentrations. The Michaelis constant for NADH was similar (about 5 μM) with each of these compounds as substrate. 3β-Hydroxypregn-5-en-20-one, and its 17α-hydroxy derivative behaved anomalously, and their behaviour is discussed. 3β,17α,21-Trihydroxypregn-5-en-20-one was a good substrate and had kinetic constants similar to those of 17α,21-dihydroxypregn-4-ene-3,20-dione. [ABSTRACT FROM AUTHOR]

When ferric myoglobin reacts with alkyl sulphates, its absorption spectrum changes into one characteristic of a haemichrome or parahaematin. The reaction equilibrium was studied over a range of protein concentrations using dodecyl sulphate and other alkyl sulphates with different chain lengths. The results obtained from spectrophotometric measurements and equilibrium dialysis experiments are not in agreement with those of other authors who. from an anal sis of spectral changes only, supported the hypothesis of an all-or-none type of reaction whereby 18 detergent. anions were bound to one molecule of ferric myoglobin. From the results we conclude that the first stage of the association of dodecyl sulphate to ferric myoglobin at pH 8.0 involves the binding of one molecule of detergent without alteration of the myoglobin absorption characteristics. In the second phase a further 3 or 4 molecules of dodecyl sulphate become associated. This causes the ferric myoglobin to be converted into its ferric myochrome, which exhibits the characteristic haemichrome absorption spectrum. The binding of additional dodecyl sulphate molecules to the myochrome then proceeds in numerous discrete steps, so that a great number of complexes occur simultaneously in the reaction medium. The experimental evidence indicates that the ferric myoglobin retains its native configuration in these complexes, provided the number of bound dodecyl sulphate anions does not exceed 50. The association compounds can be described as low-spin coordination complexes, in which a second imidazole originating from histidine E7, occupies the sixth coordination site of the iron atom. [ABSTRACT FROM AUTHOR]

The pathological changes in murine runts induced by neonatal thymectomy, the graft-versus-host reaction, neonatal infection with reovirus type 3, treatment with bacterial endotoxin, bacterial vaccine and cortisone acetate are described. It is shown that while each inducer produces its own characteristic features, others are common to all classes of runts. We suggest that these shared attributes result from the action of bacterial endotoxin. This, we believe, plays a more important role in the genesis of all murine runting than autoimmune reactions which act, if at all, during the chronic phase of these syndromes. [ABSTRACT FROM AUTHOR]

Carrageenin prevents the reaction of guinea-pig C′ with sensitized red blood cells, probably by a direct inactivation of the C′1. The anti-complementary effect of carrageenin is antagonized by protamine sulphate. Marked reduction of circulating C′ activity in guinea-pigs resulting from intravenous injection of carrageenin, does not alter their susceptibility to acute or protracted anaphylactic shock. The C′ activities of human, guinea-pig, rabbit, rat and monkey sera are all inhibited in vitro by carrageenin. [ABSTRACT FROM AUTHOR]

Carrageenin prevents the reaction of guinea-pig C′ with sensitized red blood cells, probably by a direct inactivation of the C′1. The anti-complementary effect of carrageenin is antagonized by protamine sulphate. Marked reduction of circulating C′ activity in guinea-pigs resulting from intravenous injection of carrageenin, does not alter their susceptibility to acute or protracted anaphylactic shock. The C′ activities of human, guinea-pig, rabbit, rat and monkey sera are all inhibited in vitro by carrageenin. [ABSTRACT FROM AUTHOR]

The polar movement of IAA has been examined in 5-mm root segments of Brassica oleracea and Helianthus annuus. The movement was studied partly with IAA-1-14C and partly with IAA-5-³H In both plants a slight acropetal flux of 14C and IAA-³H was found through the segments. The recovered radioactivity in the agar receiver blocks and in the receiver end of the segments increased as a function of time. A large portion of the applied IAA was converted on the cut surfaces and in the tissues of the segments. Chromatographic analysis indicated different destruction products when estimated by scintillation counting and by spraying with indole reagent (DMCA). Chromatograms run in isopropanol : ammonia : water, 8 : 1 : 1, yielded three different substances, one spot near the starting line and one near the front, neither of which has been identified. Finally there was a spot with Rf 0.4-0.6, probably representing IAA.

Gradients of tritiated water, 35S-sulphate and 32P-phosphate, were established in isolated segments of willow stems. Sieve tube exudate was collected as honeydew from the high activity end of the segment. After girdling the stem a few centimeters from the site of sieve tube puncture, the specific activity of 35S and 32P in the honeydew rose, whilst the specific activity of tritium remained constant. These findings indicate that prior to girdling, unlabelled sulphates and phosphates were contributing to the honeydew, whilst there had been no detectable contribution by unlabelled water from the low activity end of the segment. The data support the conclusions drawn from previous experiments by Peel et al. (1969), that water is relatively immobile in sieve tubes of willow when compared to solutes. [ABSTRACT FROM AUTHOR]

A method for the cultivation of Zygnema in quantity on agar plates is described. Axenic filaments were chopped with razor blades, and 4 ml of the suspension of short filaments in nutrient medium were transferred with a syringe to 1% agarized nutrient medium plates. Filamentous growth uniformly covered agar plates in 10 days or less. [ABSTRACT FROM AUTHOR]

A time course effect of intravenous papain on the chondromucoprotein complex of rabbit ear cartilage was followed chemically. There was a decrease of chondroitin sulfate. A and non-sulfated glycosaminoglycans (GAH) up to the moment of maximal collapse of the rabbits ears. This was shown by a decrease in the content of hexuronic acids and hexosamines. During the repair of the matrix, there was an increase in non-sulfated GAG, which preceded the synthesis of chondroitin sulfate A. [ABSTRACT FROM AUTHOR]