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A 5-year retrospective study (1992-1996) to look at the situation of human tuberculosis was conducted in Querétaro, México. Also, a 6-month study to determine the frequency of gross lesions in dairy cattle at slaughter, and a short experiment to evaluate the effect of sodium borate in the survival of M. bovis in lesions were carried out. The number of cases were 114 in 1992, 211 in 1995, and 174 in 1996. Possible risk factors were: overcrowding, under-nutrition, previous cases of TB in the family, concurrent Diabetes mellitus, poor personal hygiene, smoking, and alcohol abuse. Eighty percent of the cases were pulmonary. The number of cases increase with age, from 5% in patients 10-year old or younger to 42% in patients 50-year old or older. Seventy-two percent were cured, and only 6% die. Persistent coughing was by far the most observed clinical symptom. From 112 acid-fast negative samples, 8.9% were positive by culture. From 1,201 carcasses revised at slaughter, 17% presented TB-gross lesions. Ninety-six percent were localized lesions involving frequently a single organ, mostly retropharyngeal, mediastinal, mesenteric and mandibular lymph. From 102 lesions, 95% were TB-compatible, and 79% were positive to isolation of M. bovis. Most affected animals were female > 2 years old. It was observed that keeping lesions in a 6% sodium borate solution does not affect the diagnosis of M. bovis by culture after 150 days.

Full activation of human liver arginase (EC 3.5.3.1), by incubation with 5 mM Mn2+ for 10 min at 60 degrees C, resulted in increased Vmax and a higher sensitivity of the enzyme to borate inhibition, with no change in the K(m) for arginine. Borate behaved as an S-hyperbolic I-hyperbolic non-competitive inhibitor and had no effect on the interaction of the enzyme with the competitive inhibitors L-ornithine (Ki = 2 +/- 0.5 mM), L-lysine (Ki = 2.5 +/- 0.4 mM), and guanidinium chloride (Ki = 100 +/- 10 mM). The pH dependence of the inhibition was consistent with tetrahedral B(OH)4- being the inhibitor, rather than trigonal B(OH)3. We suggest that arginase activity is associated with a tightly bound Mn2+ whose catalytic action may be stimulated by addition of a more loosely bound Mn2+, to generate a fully activated enzyme form. The Mn2+ dependence and partial character of borate inhibition are explained by assuming that borate binds in close proximity to the loosely bound Mn2+ and interferes with its stimulatory action. Although borate protects against inactivation of the enzyme by diethyl pyrocarbonate (DEPC), the DEPC-sensitive residue is not considered as a ligand for borate binding, since chemically modified species, which retain about 10% of enzymatic activity, were also sensitive to the inhibitor.

S-Nitrosylation of protein thiol groups by nitric oxide (NO) is a widely recognized protein modification. In this study we show that nitrosonium tetrafluoroborate (BF4NO), a NO+ donor, modified the thiol groups of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by S-nitrosylation and caused enzyme inhibition. The resultant protein-S-nitrosothiol was found to be unstable and to decompose spontaneously, thereby restoring enzyme activity. In contrast, the NO-releasing compound S-nitrosoglutathione (GSNO) promoted S-glutathionylation of a thiol group of GAPDH both in vitro and under cellular conditions. The GSH-mixed protein disulfide formed led to a permanent enzyme inhibition, but upon dithiothreitol addition a functional active GAPDH was recovered. This S-glutathionylation is specific for GSNO because GSH itself was unable to produce protein-mixed disulfides. During cellular nitrosative stress, the production of intracellular GSNO might channel signaling responses to form protein-mixed disulfide that can regulate intracellular function.

The objective of the present study was to evaluate two different types of root canal sealers: AH Plus (an epoxy resin-based sealer) and Fill Canal (a zinc oxide-eugenol based sealer). A total of 34 root canals with vital pulp from dogs' premolars were used. After instrumentation, the root canals were filled with gutta-percha and AH Plus or gutta-percha and Fill Canal sealers using a classical technique of lateral condensation. After histological processing, the sections were stained with hematoxylineosin or Mallory's trichrome stain. Inflammatory cells or areas of necrosis were not associated with AH Plus. Hard tissue formation apically to the material was observed in 14 specimens. The Fill Canal sealer presented an inflammatory response of moderate intensity in the periapical region, mainly adjacent to the material.

High resolution 1H nuclear magnetic resonance (NMR) spectroscopy was employed to conduct a multicomponent investigation of the oxidation of salivary biomolecules by peroxoborate present in a tooth-whitening dentifrice formulation. The results acquired demonstrated that peroxoborate gave rise to the oxidative decarboxylation of the hydrogen peroxide scavenger pyruvate, a reaction generating acetate and CO2 as products. Experiments performed on chemical model systems confirmed the oxidative consumption of pyruvate by dentifrice-derived peroxoborate, and also revealed that the salivary electron donors cysteine and methionine (precursors to volatile sulphur compounds), were oxidised to cystine and methionine sulphoxide respectively. The biochemical and periodontal significance of these results is discussed.

The object of the study was to compare two commercial root canal sealers: Ketac-Endo (a glass ionomer cement) and Fill Canal (a zinc oxide-eugenol cement). A total of 34 root canals from dog premolars with vital pulps were used. After instrumentation, the root canals were sealed with Ketac-Endo and Fill Canal cements using gutta-percha and a lateral condensation technique. After 270 days the animals were sacrificed with an anesthetic overdose and the maxillae and mandibles were removed and fixed in formalin for 48 h. After routine histological processing the sections were stained with hematoxylin-eosin and Mallory trichrome stains. Microscopic analysis revealed that Ketac-Endo cement presented better results than Fill Canal cement.

Leukotriene C4 is an arachidonic acid metabolite and an important mediator of inflammation and anaphylaxis that is known to induce production of prostacyclin in endothelial cells. The goal of this study was to examine the signal transduction mechanisms activated by leukotriene C4 stimulation. Formation of inositol phosphates was measured to determine the activation of phospholipase C and pertussis toxin was used to explore the role of G-proteins. Additionally, we evaluated the role of protein kinase C in these events, especially whether there was an interaction between pertussis toxin mediated effects and the activity of protein kinase C. Leukotriene C4 induced a dose- and time-dependent formation of inositol phosphates and prostacyclin. The response to leukotriene C4 was greater than the response to leukotriene D4 even after treatment with L-serine borate complex, suggesting the presence of a specific leukotriene C4 receptor. Exposure to pertussis toxin potentiated, time-dependently, the leukotriene C4 induced formation of inositol phosphates and prostacyclin through a mechanism which was altered by manipulation of protein kinase C activity. The exact mechanism is not clear but our results are consistent with a postulated dual mechanism of phospholipase C control, in which leukotriene C4 induced stimulation is attenuated by a pertussis toxin sensitive G-protein.

Rat liver arginase contains a dimanganese(II,II) center per subunit that is required for catalytic hydrolysis of l-arginine to form urea and l-ornithine. A recent crystallographic study has shown that the Mn2 center consists of two coordinatively inequivalent manganese(II) ions, MnA and MnB, bridged by a water (hydroxide) molecule and two aspartate residues [Kanyo et al. (1996) Nature 383, 554-557]. A conserved residue, His141, is located near the proposed substrate binding region at 4.2 A from the bridging solvent molecule. The present EPR studies reveal that there is no essential alteration of the Mn2 site upon mutation of His141 to an Asn residue, which lacks a potential acid/base residue, while the catalytic activity of the mutant enzyme is 10 times lower vs wild-type enzyme. The binding affinity of l-lysine, l-arginine (substrate), and Nomega-OH-l-arginine (type 2 binders) increases inversely with the pKa of the side chain. Binding of l-lysine is more than 10 times weaker, and the substrate Michaelis constant (Km) is >6-fold greater (weaker binding) in the His141Asn mutant than in wild-type arginase. L-Lysine and Nomega-OH-L-arginine, type 2 binders, induce extensive loss of the EPR intensity, suggesting direct coordination to the Mn2 center. From these data and the pH dependence of type 2 binders, we conclude that His141 functions as the base for deprotonation of the side-chain amino group of L-lysine and the substrate guanidinium group, -NH-C(NH2)2+ and that the unprotonated side chain of these amino acids is responsible for binding to the active site. A different class of inhibitors (type 1), including L-isoleucine, L-ornithine, and L-citrulline, suppresses enzymatic activity, producing only minor change in the zero-field splitting of the Mn2 EPR signal and no change in the EPR intensity, suggestive of minimal conformational transformation. We propose that type 1 alpha-amino acid inhibitors do not bind directly to either Mn ion, but interact with the recognition site on arginase for the alpha-aminocarboxylate groups of the substrate. A new mechanism for the arginase-catalyzed hydrolysis of L-arginine is proposed which has general relevance to all binuclear hydrolases: (1) Deprotonation of substrate l-arginine(H+) by His141 permits entry of the neutral guanidinium group into the buried Mn2 region. Binding of the substrate imino group (>C=NH), most likely to MnB, is coupled to breaking of the MnB-(mu-H2O) bond, forming a terminal aquo ligand on MnA. (2) Proton transfer from the terminal MnA-aqua ligand to the substrate Ndelta-guanidino atom forms the nucleophilic hydroxide on MnA and the cationic NdeltaH2+-guanidino leaving group. Protonation of the substrate -NdeltaH2+-group is likely assisted by hydrogen bonding to the juxtaposed anionic carboxylate group of Glu277. (3) Attack of the MnA-bound hydroxide at the electrophilic guanidino C-atom forms a tetrahedral intermediate. (4) Formation of products is initiated by cleavage of the Cepsilon-NdeltaH2+ bond, yielding urea and L-ornithine(H+).

Single-rooted premolar teeth, stained with blood utilizing the technique devised by Freccia & Peters (1981), were subjected to traditional and non-peroxide bleaching agents. Colour changes were recorded over a period of 7 days using a Speedmaster R75-CP Reflection Densitometer. The most efficient removal of staining occurred after the application of 30% hydrogen peroxide, with sodium perborate being 75% as effective. All bleaching agents realized their optimum efficacy within the first 3 days. A combination of three enzymes (amylase, lipase and trypsin) with disodium edetate was not as effective as the routine bleaching agents; however, the combination did have a modifying effect on the blood stains. It is suggested that other non-peroxide agents should be investigated to determine their efficacy in removing staining from experimentally induced blood-stained teeth.

The purpose of this study was to investigate the in vitro effect of sodium perborate, which is used as a bleaching agent in the treatment of discolored pulpless teeth, on substrate adherence capacity of macrophages. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. As a test of macrophage adhesion, the adherence capacity of macrophages to a plastic surface was determined. Assays were conducted in Eppendorf tubes for 15 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2. The adherence index was calculated. Results showed that sodium perborate decreased in a dose-dependent manner and decreased significantly (p < 0.05) the adherence index of rat peritoneal macrophages. Sodium perborate was less potent than sodium hypochlorite and eugenol in inhibiting macrophage adhesion. The inhibitory effect of sodium perborate on macrophage adhesion further supports the concept that this agent is not implicated in external cervical root resorption associated with intracoronal bleaching.

The caspases are a family of at least 10 human cysteine proteases that participate in cytokine maturation and in apoptotic signal transduction and execution mechanisms. Peptidic inhibitors of these enzymes are capable of blocking cytokine maturation and apoptosis, demonstrating their crucial roles in these processes. We have recently discovered that nitric oxide (NO), produced either extracellularly by NO donors or intracellularly by the inducible nitric oxide synthase, prevented apoptosis in hepatocytes. Caspase-3-like activity was found to be inhibited under these conditions. To investigate further the interaction between NO and caspases, we utilized purified human recombinant caspases and examined the effect of NO on enzymatic activities of different caspases. We report here that of the seven caspases studied, all were reversibly inhibited by NO. Dithiothreitol was able to reverse the NO inhibition, indicating direct S-nitrosylation of caspase catalytic cysteine residue by NO. Our results support the concept that NO is an endogenous regulator of caspase activity.

Kidney function changes after single-dose administration of borocaptate sodium were studied in rats and in patients with brain tumors. Changes of glomerular filtration rate (GFR) measured as 14C-inulin clearance and urine flow rate (UFR) after a slow intravenous injection of BSH (25 and 50 mg/kg b.w., respectively) were investigated in rats under pentobarbital anesthesia. The effect of BSH has been compared with that of its disulfide (BSSB) which is spontaneously generated by oxidation of BSH during storage. It was found that BSH decreases GFR in relation to dose and, in the same way, causes a temporary increase of UFR. On the other hand, BSSB (50 mg/kg) induced a large reversible decrease of GFR as well as a decrease of urine excretion. Measurements of GFR (inulin clearance), renal plasma flow (PAH clearance) and urine excretion were taken in a group of patients with brain tumors in which boron disposition after an infusion of BSH (25 mg/kg b.w. over 1 h) had been studied. An increase in urine production was the dominant effect (up to 200% of the initial value), with the alterations of GFR and RPF being of minor significance except in one patient with a GFR reduction up to almost 50% the original value. Kidney function changes after BSH or BSSB administration are supposedly related to the high retention of BSH in kidney.

Glycinergic neurotransmission is mediated via inhibitory glycine receptors (GlyRs) which are heterogeneous during development. Electrophysiological studies performed on recombinant GlyRs have identified different pharmacological properties and attributed them to differences in their subunit composition. Here, we report on age-related changes in the response properties of native GlyRs in the mammalian brain. Whole-cell patch-clamp recordings were obtained from neurons of the medial nucleus of the trapezoid body (MNTB), a major relay station in the mammalian auditory brainstem. Experiments were performed in acute medullary slices of rats between postnatal day (P) 1 and P15, a period during which synapse maturation occurs. Glycine-induced currents were present throughout the period under investigation and displayed age-related modifications in their amplitude, kinetic characteristics, and sensitivity to drugs. Current amplitudes and GlyR desensitization behavior increased with age. The alpha 1 subunit-specific GlyR antagonist cyanotriphenylborate (CTB) was barely effective in reducing glycine-induced currents during the first few postnatal days, yet a significant increase of the inhibitory effect occurred after the first postnatal week. This finding indicates that alpha 1 subunit-containing GlyRs become expressed only postnatally in the MNTB. Picrotoxin, which most effectively blocks recombinant alpha 2-homooligomers, reduced glycine-induced currents in neonatal MNTB neurons, suggesting that alpha 2-homooligomers may form native GlyR isoforms. Our results show that the physiology and pharmacology of GlyRs in the auditory brainstem underlie age-related changes which are most probably produced through a replacement of "neonatal" alpha 2 subunits with "adult" alpha 1 subunits.

Concentrations of the synthetic androgen R1881 that correspond to physiologically relevant concentrations of 5alpha-dihydrotestosterone are capable of altering the activity of gamma-glutamyl transpeptidase (GGT) in human prostate carcinoma cells. GGT activity of the androgen-responsive prostate cancer cell line LNCaP increases >50% above that of the control after a 72-h exposure to 1 nM R1881. This elevation in GGT activity occurs as early as 48 h after treatment and is maintained for at least 96 h. Loss of glutathione (GSH) from media and accumulation of intracellular GSH of cells pretreated with 1 nM R1881 occur at a higher rate than in control cells, suggesting that a greater rate of GSH salvage is associated with the increased GGT activity. Immunohistochemical staining detects an increase in GGT-positive staining in cells treated with 1 nM R1881 for 72 h. Steady-state mRNA levels for GGT are elevated above those of the control 24-72 h after treatment. R1881 has no effect on the GGT activity of the androgen-independent prostate cell line DU145. Growth of LNCaP but not DU145 cells is inhibited by 1 nM R1881 compared to that of the control. Inhibitors of GGT activity, acivicin and serine-borate, are capable of dampening or blocking the effect of R1881 on growth. Growth of LNCaP cells treated with 1 nM R1881 plus 100 mM glycylglycine, a stimulator of GGT activity, is inhibited to a greater extent than the growth of LNCaP cells treated with R1881 alone. These data demonstrate that androgens can elevate GGT activity and increase GGT mRNA and protein levels in human prostate carcinoma cells. In addition, compounds able to alter GGT activity are capable of altering androgen-related growth effects.

Two derivatives of carborates, sodium 5,6-dichloro-7,8-dicarbanonaborate (CB-Cl) and sodium 5-mercapto-7,8-dicarbanonaborate (CB-SH) were found to inhibit endogenous as well as glucose-induced respiration of the yeast Saccharomyces cerevisiae. Both substances slightly increased endogenous acid production, were neutral toward H(+)-ATPase-associated acidification but pronouncedly inhibited the K(+)-stimulated acidification. The same effects were observed also with an ATPase-deficient mutant of the yeast. The ATP-hydrolyzing activity of yeast plasma membranes in vitro was severely reduced. The membrane potential was substantially increased toward more negative values. The H(+)-symporting uptake of glutamic acid was considerably decreased, that of adenine was diminished much less. The effects of the dicarbanonaborates are obviously pleiotropic but their inhibition of ATP hydrolysis and of uptake of H(+)-symported substances, on the one hand, and absolute lack of effect on ATPase-catalyzed acidification, on the other, pose an unresolved problem.

The large subunit core of ribulose-bisphosphate carboxylase from Synechococcus PCC 6301 expressed in Escherichia coli in the absence of its small subunits retains a trace of carboxylase activity (about 1% of the kcat of the holoenzyme) (Andrews, T. J (1988) J. Biol. Chem. 263, 12213-12219). During steady-state catalysis at substrate saturation, this residual activity diverted approximately 10% of the reaction flux to 1-deoxy-D-glycero-2,3-pentodiulose-5-phosphate as a result of beta elimination of inorganic phosphate from the first reaction intermediate, the 2,3-enediol form of ribulose bisphosphate. This indicates that the active site's ability to stabilize and/or retain this intermediate is compromised by the absence of small subunits. Epimerization and isomerization of the substrate resulting from misprotonation of the enediol intermediate were not significantly exacerbated by lack of small subunits. The residual carboxylating activity partitioned product between pyruvate and 3-phosphoglycerate in a ratio similar to that of the holoenzyme, indicating that stablization of the penultimate three-carbon aci-acid intermediate is not perturbed by lack of small subunits. The underlying instability of the five-carbon enediol intermediate was revealed, even with the holoenzyme, under conditions designed to lead to exhaustion of substrate CO2 (and O2). When carboxylation (and oxygenation) stalled upon exhaustion of gaseous substrate, both spinach and Synechococcus holoenzymes continued slowly to beta eliminate inorganic phosphate from and to misprotonate the enediol intermediate. With carboxylation and oxygenation blocked, the products of these side reactions of the enediol intermediate accumulated to readily detectable levels, illustrating the difficulties attendant upon ribulose-P2 carboxylase's use of this reactive species as a catalytic intermediate.

We used an enhanced luminescence technique to study the response of rat tissues, such as liver, heart, muscle and blood, to oxidative stress and to determine their antioxidant capacity. As previously found for liver homogenate, the intensity of light emission (E) of tissue homogenates and blood samples, stressed with sodium perborate, is dependent on concentration, and the dose-response curves can be described by the equation E = a.C/exp(b.C). The b value depends on the antioxidant defence capability of the tissues. In fact, it increases when homogenates are supplemented with an antioxidant, and is correlated with tissue antioxidant capacity, evaluated by two previously set up methods both using the same luminescence technique. Our results indicate that the order of antioxidant capacity of the tissues is liver > blood > heart > muscle. The a value depends on the systems catalysing the production of radical species. In fact, it is related to the tissue level of hemoproteins, which are known to act as catalysts in radical production from hydroperoxides. The equation proposed to describe the dose-response relation is simple to handle and permits an immediate connection with the two characteristics of the systems analysed which determine their response to the pro-oxidant treatment. However, the equation which best describes the above relation for all the tissues is the following: E = alpha. C/exp(beta.C delta). The parameter delta assumes values smaller than 1, which seem to depend on relative amounts of tissue hemoproteins and antioxidants. The extension of the analysis to mitochondria shows that they respond to oxidative stress in a way analogous to the tissues, and that the adherence of the dose-response curve to the course predicted from the equation E = a.C/exp(b.C) is again dependent on hemoprotein content.

We incubated human paranasal sinus mucosa in tissue culture with each of leukotrienes C4, D4, and E4 (LTC4, LTD4, and LTE4). Ciliary beat frequency (CBF) was measured photoelectrically, and the concentrations of leukotrienes in the incubation medium were determined over time. LTC4 significantly decreased CBF with 2-h, 4-h, and 8-h exposures to 10(-6) M, 10(-8) M, and 10(-10) M, respectively. Moreover, LTC4 dose dependently reduced CBF to 81.4% of the initial value after 6-h exposure to 10(-6) M, to 82.5% after 8-h exposure to 10(-8) M, and to 89.7% after 12-h exposure to 10(-10) M. LTD4 also exhibited progressive ciliary inhibition, while LTE4 had a minimal effect on CBF. In the medium, LTC4 was changed to LTD4 and further to LTE4. gamma-Glutamyl transpeptidase (gamma-GTP), an enzyme that converts LTC4 to LTD4, was detected in the mucosa. Serine-borate complex, an inhibitor of gamma-GTP, blocked the inhibitory effect of LTC4 on CBF. These findings suggest that LTC4 may induce ciliary inhibition indirectly by conversion to LTD4.

Dicarbanonaborates inhibit the mitochondrial cytochrome c oxidase activity. In contrast to mitochondrial ATPase or glycerol phosphate dehydrogenase, inhibition of cytochrome c oxidase was not competitive and the residual, drug-insensitive activity was higher. These results indicate that dicarbanonaborates inhibit various mitochondrial membrane-bound enzymes through different mechanisms.

A method is described for analysis of the tetracycline antibiotic minocycline using capillary zone electrophoresis. Potential impurities are 4-epiminocycline, 6-deoxy-6-demethyltetracycline, 7-didemethylminocycline, 7-monodemethylminocycline and 9-minocycline. Method development was undertaken with a mixture consisting of minocycline and its related substances mentioned above. Using a fused silica capillary, the type of buffer and its pH and concentration were investigated. In all cases 1 mM EDTA was added to prevent metal ion complexation. Instrumental parameters such as capillary temperature and applied voltage were optimised. The effects of the sample solvent and of organic modifiers in the buffer were also investigated. The following method is proposed: capillary: fused silica, l = 38 cm, L = 44 cm, 50 microns i.d.; buffer: 25 mM sodium tetraborate, 1 mM EDTA at pH 11.75; voltage, 13 kV; temperature, 15 degrees C; UV detection performed at 254 nm. Relative standard deviations, linearity, LOD and LOQ are reported and compared with those of liquid chromatography.

The polyether-macrolide antibiotic, boromycin, was isolated as a potent anti-human immunodeficiency virus (HIV) antibiotic from a fermentation broth of Streptomyces sp. A-3376. Boromycin was found to strongly inhibit the replication of the clinically isolated HIV-1 strain as well as the cultured strain in in vitro laboratory experiments. The mechanism for the anti-HIV activity of boromycin is suggested to involve blocking the later stage of HIV infection, and probably the maturity step for replication of the HIV molecule.

We screened 125 Pseudomonas strains from our culture collection for the production of hydantoinase activity using DL-phenylhydantoin as a substrate. Pseudomonas desmolyticum NCIM 2112 was found to be the best hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) producer. The enzymatic reactions were carried out using 18-20-h grown cells in nutrient broth and 5-phenylhydantoin as the substrate. Optimization studies for the biotransformation reaction were performed to increase product yield. The optimum pH and temperature for D(-)N-carbamoylphenylglycine production were 9.5 and 30 degrees C, respectively. Biotransformation under these alkaline conditions allowed the complete conversion of 27.0 g l-1 of DL-phenylhydantoin to 26.5 g l-1 of N-carbamoylphenylglycine within 24 h, with a molar yield of 90%. The hydantoinase involved in this biotransformation process was strictly D-stereospecific, because the product isolated was pure D(-)N-carbamoylphenylglycine. This pure product was further chemically converted to D(-)phenylglycine using nitrous acid with an 80% chemical yield. Thus, the overall conversion efficiency of DL-5-phenylhydantoin to D(-)phenylglycine was found to be 65-68%.

Glycine, strychnine and certain chloride channel blockers were reported to protect cells against lethal cell injury. These effects have been attributed to interactions with membrane proteins related to CNS glycine gated chloride channel receptors. We have investigated the pharmacology of these actions. Madin-Darby canine kidney (MDCK) epithelial cells were depleted of adenosine triphosphate (ATP) by incubation in glucose free medium containing a mitochondrial uncoupler. Medium Ca2+ was adjusted to 100 nM in the presence of an ionophore such that intracellular Ca2+ did not increase, and Ca(2+)-related injury mechanisms were inhibited. This permitted more sensitive quantitation of protection against cell injury attributable to glycine or other agents whose actions might be related to those of the amino acid. Two classes of compounds showed cytoprotective activity in this system: (1) ligands at chloride channel receptors, such as glycine, strychnine and avermectin B1a; (2) chloride channel blockers, including cyanotriphenylboron and niflumic acid, both of which are known to bind to channel domains of CNS glycine receptors. Morphological and functional studies showed that the compounds preserved plasma membrane integrity, but permitted cell swelling. Substitution of medium chloride by gluconate, or chloride salts by sucrose, did not substantially modify lethal damage or its prevention by glycine or other drugs. The compounds did not modify ATP declines. At least for some compounds, cytoprotection appeared to be specific to structural features on the molecules. These observations are consistent with the hypothesis that a plasma membrane protein related to glycine-gated chloride channel receptors plays a significant role in cell injury, but indicate that the mechanisms of injury and protection by compounds active in this system are not related to chloride fluxes.

In this paper, 1-O-hexadecyl and 1-O-octadecyl-2-acetyl-sn-glycero-3- phosphorylcholine (C16-AGEPC and C18-AGEPC) in the platelet activating factor of human blood were analyzed by micellar electrokinetic capillary chromatography (MECC) with indirect ultraviolet absorption detection at 254 nm. The optimum running buffer for the separation contained 50 mmol/l sodium dodecy sulphate, 20 mmol/l potassium hydrogen phthalate, 10 mmol/l borax and 3 mol/l urea (pH 6.8). The separation was completed within 10 min. The detection limits of C16-AGEPC and C18-AGEPC were the same, i.e. 60 ng/ml (k = 3). The analytical precision (n = 6) was 2.8-3.0% and 1.4-1.7% for the determination (peak height mode) and for the measurement of the migration times, respectively. The application of this method to the clinical samples was demonstrated.

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10(-6)M to 10(-9)M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10(-8)M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a gamma-glutamyl transpeptidase (gamma-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by gamma-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.

The mutagenic activity of more than 120 antimicrobial agents and protective components was investigated. Only Kathon showed a consistent increase in revertant counts in the Ames test on Salmonella typhimurium. The hereditary bleaching test on Euglena gracilis used for detecting extranuclear mutations, showed positive results for Kathon, triethanolamine and diamine silver tetraborate.

Putrescine uptake in type II pneumocytes is a carrier-mediated active process. Our hypothesis was that oligoamines might be taken up into the cell at least in part by gamma-glutamyltransferase (gamma-GT). This was investigated in rat type II pneumocytes 24 hr after their isolation. Preexposure to 125 microM L-buthionine-[SR]-sulfoximine (BSO) or 100 microM diethylmaleate (DEM), both of which affect intracellular glutathione (GSH) only, were found to decrease GSH by 85% (p < 0.05) and 62%, respectively (p < 0.05), without change in [3H]-putrescine uptake. Preexposure to 20 microM N-ethylmaleimide (NEM), which affects intra- and extracellular GSH, decreased intracellular GSH by 79% (p = 0.015) and putrescine uptake by 39% (p = 0.03). Selective extracellular GSH depletion by 10 microM copper-o-phenanthroline complex (CuP) led to a decrease in putrescine uptake of 41% (p = 0.001), while intracellular GSH remained unchanged. Specific inhibition of gamma-GT by 5-20 mM serine-borate or 5 mM acivicin gave similar degrees of putrescine uptake inhibition (39.5% and 40.5%). The kinetic properties of the putrescine uptake system in the presence of acivicin and serine-borate indicated that the Vmax decreased by 25%, while Km remained unchanged. In experiments with pure gamma-GT, the oligoamines putrescine, spermidine and spermine, and cystamine proved to be acceptor substrates for gamma-GT, all having similar efficiencies (Vmax/Km); methylglyoxal-bis-(guanyl-hydrazone) and paraquat were not accepted. As extracellular GSH is required for gamma-GT, and because its extracellular depletion inhibits putrescine uptake as much as specific inhibition of gamma-GT, we suggest that 30-40% of the putrescine uptake in type II pneumocytes occurs by gamma-GT and that, therefore, at least two systems are involved in the uptake of putrescine.

The inhibitory effect of mercapto- and chlorodicarbanonaborates on mitochondrial, FAD-linked, L-glycerol-3-phosphate dehydrogenase activity was tested. The mercaptodicarbanonaborate was found to be a more potent inhibitor than the chloro derivative. The Ki of mercapto- and chlorodicarbanonaborates were 0.8 M and 480 M, respectively. Whereas the mercaptodicarbanonaborate inhibited the enzyme activity competitively, the inhibitory effect of chloro derivative was noncompetitive. The inhibitory effect of mercaptodicarbanonaborate was completely prevented by addition of bovine serum albumin.

An enhanced luminescence technique was used to monitor the response of liver homogenates stressed with sodium perborate. Rat liver homogenates were subjected to oxidative stress with sodium perborate, and the light signals, generated by a suitable system, containing luminol and compounds producing enhancement of light emission such as sodium benzoate and indophenol, were detected by a luminometer. The intensity of light emission (E) was found dependent on homogenate concentration (C). When C increased, E at first increased as well and, then, decreased rapidly. The graphic expression of this phenomenon resulted as a curve that can be described by the equation: E = a.C/exp(b.C). It is proposed that the a value represents the capacity of the tissue to catalyze the production of .OH radical species. The b value might be related to the capacity of the tissue to scavenge such radicals, since it increases when homogenates are supplemented with antioxidants and decreases when homogenates are treated with prooxidant. The results obtained by supplementing homogenates with iron containing substances, or using model systems, suggest that cell substances catalyzing the luminescent reaction, such as the hemoproteins, are "scavengers" as well as radical producers. The concentration-emission curve obtained with suitable model system is described by the equation: E = a.C/exp(b.Ck). It is suggested that, using the k value, information can be obtained on the relative capacity of hemoproteins and antioxidant systems to interact with .OH radicals.

We studied the expression of glycine receptor (GlyR) subunits and gephyrin in the adult rat retina. Reverse transcribed RNA was amplified by polymerase chain reaction (RT-PCR) with primers designed to recognize GlyR alpha 1, alpha 2, alpha 3, beta subunits, and gephyrin. Using RNA isolated from the whole retina, signals for all four GlyR subunits and gephyrin could be observed. In rod bipolar cells, in contrast, we detected a subset of GlyR subunits, alpha 1 and beta, and no gephyrin. Patch-clamp recording employing two subtype-specific blockers of the GlyR, picrotoxinin and cyanotriphenylborate (CTB), indicated that the GlyR in rod bipolar cells is a heteromeric protein composed of the alpha 1 and beta subunit. Moreover, the absence of detectable amounts of gephyrin mRNA suggests that the anchor protein is not required for the function of GlyRs in rod bipolar cells.

The influence of thyroid hormone administration on liver glutathione (GSH) extraction in the isolated perfused liver was studied in fed rats for a period of 1-7 days following a single dose of 0.1 mg 3,5,3'-triiodothyronine (T3)/kg. T3 treatment led to an early and transient calorigenic response, as well as an enhancement in liver GSH removal, reaching a maximal effect at 2 days after hormone administration, which was normalized in the 3- to 7-day period studied. Addition of the gamma-glutamyltransferase (gamma-GT) inhibitor DL-serineborate (4 mM) to the perfusate abolished the increase in the hepatic removal of GSH elicited by T3, and enhanced the sinusoidal concentration of GSH, studied at 2 days after hormone administration. These data support the role of hepatic basolateral gamma-GT ectoactivity in the depletion of portally added and liver-derived GSH as an adaptive response to recover GSH levels after reduction by T3-induced oxidative stress.

We investigated the role of epithelium in smooth muscle contraction induced by leukotriene C4 (LTC4) in isolated human trachea. The contractile response to LTC4 was potentiated by a gamma-glutamyl transpeptidase (gamma-GTP) inhibitor L-serine borate and by removal of the epithelium. Both L-serine borate (4.5 x 10(-2) M) and removal of the epithelium shifted the concentration-response curves to LTC4 to lower concentrations by 1.2 and 1.0 log units, respectively. Incubation of cultured human tracheal epithelial cells with either LTC4 or LTD4 resulted in the formation of LTD4 or LTE4. The isolated epithelial cells and cultured epithelial cells contained gamma-GTP activity. Histochemical study indicated that gamma-GTP-like activity was present in the epithelial, submucosal, and smooth muscle layers. These findings suggest that the epithelium modulates the contractile response to LTC4 in human trachea by LT-degrading enzymes.

Interphase fluorescence in situ hybridization (FISH) has been shown to be a means to map DNA sequences relative to each other in the 100 kb to 1-2 Mb genomic-separation range. At distances below 0.1 Mb, probe sites are infrequently resolved in interphase chromatin. In the 0.1- to 1-Mb range, interphase chromatin can be modeled as a freely flexible chain. The mean square interphase distance between two probes is proportional to the genomic separation between the probes on the linear DNA molecule. Above 1-2 Mb, the relationship between interphase distance and genomic separation changes abruptly and appears to level off. We have used alkaline-borate treatment to expand the capability of interphase FISH mapping. We show here that alkaline-borate treatment increases nuclear diameter, the interphase distance between probes on homologous chromosomes, and the distance between probes on the same chromosome. We also show that the mean square distance between hybridization sites in borate-treated nuclei is proportional to genomic separation up to 4 Mb. Thus, alkaline-borate treatment enhances the capability of interphase FISH mapping by increasing the absolute distance between probes and extending the range of the simple relationship between interphase distance and genomic separation.

New antibiotics were isolated from the culture broth of the myxobacterium, Sorangium cellulosum, strain So ce 678. The antibiotics were active against Gram-positive bacteria and mammalian cells. They were named tartrolon A and B. Tartrolon B contains a boron atom. The boron binding region of tartrolon is identical with that of boromycin and aplasmomycin.

Bovine liver dihydrodiol dehydrogenase (DD2) was inactivated following pseudo-first order manner by the treatment of 5 mM 2,3-butanedione (BD) as functions of incubation-time and concentration. Anionic substrates or analog which have a carboxyl group, D-glucuronate, p-carboxybenzaldehyde and D-glycerate, protected DD2 efficiently. But, the other substrates or coenzymes and their analogs did not show any protection on the inactivation, i.e., D,L-glyceraldehyde, D-erythrose, NADP+, NAD+, 2',5'-ADP, 2'-AMP. Results of kinetic analyses suggest that the inactivated enzyme lost its binding ability to anionic substrates. The inactivated enzyme was reactivated very effectively by removing excess BD by gelfiltration for 30 min.

Background: Since the effect of exogenous glutathione (GSH) on overall hepatic GSH homeostasis is not known, the present study investigated the changes in the hepatic, biliary, and plasmatic GSH levels during GSH administration in intact rats., Methods: An exteriorized biliary-duodenal fistula was established, and GSH (1 mmol/kg over 2 h) or saline was administered intraperitoneally to rats with or without pretreatment with 5 mmol/kg L-serine borate, an inhibitor of gamma-glutamyltransferase (GGT)., Results: Three hours after GSH administration, biliary GSH efflux and bile flow rose from 104.7 +/- 5.6 to 290.6 +/- 8.6 micrograms/ml bile and from 20.2 +/- 1.3 to 30.2 +/- 2.1 microliters/min, respectively; GSH-treated rats also showed increased liver (35%) and posthepatic vein plasma (68%) GSH concentrations compared with controls. By contrast, in rats pretreated with the GGT inhibitor GSH administration appeared to be devoid of any effect, except for a modest biliary GSH increase., Conclusions: This study indicates that significant changes occur in the hepatic GSH homeostasis after intraperitoneal GSH administration. The activity of hepatic GGT, most likely through degradation of circulating GSH, followed by an increase in cysteine availability, seems to account, at least partially, for the reported effects.

The Na,K-ATPase is a multifunctional system anchored in the membrane of eukaryotic cells; it is responsible for the establishment and regulation of the Na/K balance of cell and organism by a stoichiometric mechanism linking Na extrusion to K uptake and ATP hydrolysis. The receptor for cardioactive steroids such as digoxin and ouabain is located at the extracellular surface of the system. Conversely, palytoxin, the most potent animal toxin, exerts its toxic effect by creating nonspecific leaks in the cell membrane leading to K-efflux and influx of Na and Ca ions. Ouabain prevents the pore-forming action of palytoxin in cells and therefore Na,K-ATPase is suspected to be the common receptor of ouabain and palytoxin. We have developed an artificial membrane system to determine structure-function relationships and ligand interactions of purified Na,K-ATPase: two-sided, bi-directional ATP-filled liposomes. In this system, ATP-driven 86Rb accumulation, arrest of 86Rb-uptake by ouabain, and palytoxin-induced 86Rb-leak were measured successively in the same preparation. Ouabain prevented the leak when the enzyme was ouabain-sensitive (rabbit kidney) but not when it was ouabain-resistant (rat kidney). On the basis of these data in conjunction with conformational analyses, allosteric conformational competition for the ouabain-palytoxin antagonism is proposed.

The inhibitory glycine receptor is a ligand-gated ion-channel protein existing in different homo- and heterooligomeric isoforms. Here we show that the chloride channel of the recombinant alpha 1-subunit homooligomeric glycine receptor is efficiently blocked by cyanotriphenylborate (CTB) with a concentration effecting 50% inhibition (IC50) of 1.3 microM in the presence of 50 microM glycine. The antagonistic effect of CTB is noncompetitive, use dependent, and more pronounced at positive membrane potentials, suggesting open-channel block. In contrast to alpha 1-subunit receptors, alpha 2-subunit homooligomers are resistant to CTB (IC50 >> 20 microM). By exchanging the channel-lining transmembrane segment M2 of the alpha 1 polypeptide by that of the alpha 2 polypeptide, we could transfer this resistance to alpha 1 channels, indicating that a single glycine residue at position 254 of the alpha 1 subunit is critical for CTB sensitivity. The blocker did not affect the cation-selective channel of the nicotinic acetylcholine receptor. Thus, CTB may prove useful as a tool to probe the subunit structure of native glycine receptors in mammalian neurons.

Previous studies have suggested that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) undergoes covalent modification of an active site thiol by a NO.-induced [32P]NAD(+)-dependent mechanism. However, the efficacy of GAPDH modification induced by various NO donors was found to be independent of spontaneous rates of NO. release. To further test the validity of this mechanism, we studied the effects of nitrosonium tertrafluoroborate (BF4NO), a strong NO+ donor. BF4NO potently induces GAPDH labeling by the radioactive nucleotide. In this case, the addition of thiol significantly attenuates enzyme modification by competing for the NO moiety in the formation of RS-NO. Peroxynitrite (ONOO-) also induces GAPDH modification in the presence of thiol, consistent with the notion that this species can transfer NO+ (or NO2+) through the intermediacy of RS-NO. However, the efficiency of this reaction is limited by ONOO- -induced oxidation of protein SH groups at the active site. ONOO- generation appears to account for the modification of GAPDH by SIN-1. Thus, S-nitrosylation of the active site thiol is a prequisite for subsequent post-translational modification with NAD+, and emphasizes the role of NO+ transfer in the initial step of this pathway. Our findings thus provide a uniform mechanism by which nitric oxide and related NO donors initiate non-enzymatic ADP-ribosylation (like) reactions. In biological systems, endogenous RS-NO are likely to support the NO group transfer to thiol-containing proteins.

Objective: The purpose of this study was to investigate the activity of gamma-glutamyl transpeptidase (gamma-GTP, E.C. 2.3.2.2) in rat endometrium (day 5 of pregnancy). Since gamma-GTP is an enzyme involved in the translocation of amino acids from fluids toward tissues, these substrates are necessary for anabolic processes., Methods and Results: The presence of statistically higher activity of gamma-GTP in rat (Sprague-Dawley) implantation sites (1.06 nmol/mg protein/min) than in nondecidualized (0.87 nmol/mg protein/min) tissues was demonstrated. The intrauterine administration of L-serine-borate complex (5 mM) during day 5 of pregnancy arrested 91.6% of rat embryonic development (day 18). This inhibitory effect was not present when borate or L-serine was administered separately. The L-serine-borate complex also inhibited (by 88%) the gamma-GTP in vitro., Conclusion: The inhibition of gamma-GTP by L-serine-borate complex might be considered as a new approach to the arrest of biological processes in differentiation or development.

The activity of guanidino compounds as alternate substrates for rat liver arginase is critically dependent on the length of the amino acid side chain and the substituents about C-alpha. In addition to L-arginine, the enzyme catalyzes the hydrolysis of L-argininamide, L-canavanine, L-homoarginine, L-argininic acid, and agmatine. The kcat values for these substrates are 15- to 5000-fold slower than the kcat for L-arginine. Guanidobutyrate, D-arginine, and NG-methyl-L-arginine are not substrates. Competitive inhibition by the products L-ornithine and urea indicates a rapid-equilibrium random mechanism for the enzyme. Despite the requirement for added divalent cations in the activation of the enzyme, metal chelators such as EDTA and citrate do not inhibit the enzyme. These results suggest that the metal site is not readily accessible to solvent. Multiple inhibition experiments with the noncompetitive inhibitor borate demonstrate that borate and urea bind in a mutually exclusive manner, while L-ornithine and borate can bind simultaneously to the enzyme. Borate inhibition is proposed to arise from chelation of Mn(II) in the binuclear Mn(II) center, thus displacing a metal-bound water molecule that is responsible for nucleophilic attack on the guanidium carbon.

We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human gamma-glutamyltransferase (GGT) activity. We now report the utilization of V79HGGT as a model system for studying human GGT. The papain-solubilized recombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N-terminal sequencing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native human enzymes. The circular dichroism analysis indicated an alpha-helical content of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutathione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell line, depending on, but differentially affected by, alteration of the culture medium. Elevated GGT activity was associated with a 2.5-fold reduced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Possible mechanisms involved are proposed. Our findings pointed out that, among the GSH-related enzymes, GGT could constitute an important factor determining the steady-state content of GSH.

gamma-L-glutaminyl-4-hydroxy-3-iodobenzene (I-GHB), a novel iodinated analog of gamma-L-glutaminyl-4-hydroxybenzene (GHB), demonstrates greater anti-tumor activity in human and in murine melanoma cell lines. These phenolic amides are substrates for gamma-glutamyltranspeptidase (GGTP; E.C. 2.3.2.2), a cell-membrane-associated ecto-enzyme which is elevated in a number of tumor systems. We now present data to show that the growth-inhibitory activity of I-GHB and GHB may be mediated via GGTP-catalyzed reactions. The growth-inhibitory activity of I-GHB and GHB in pigmented B16-BL6 melanoma cells was blocked significantly by rabbit anti-rat GGTP polyclonal antibodies. The combination of L-serine and sodium borate, a specific transition-state inhibitor of GGTP, as well as acivicin, a glutamine antagonist and irreversible GGTP inhibitor, inhibited the killing of BL6 cells by GHB and I-GHB. To further define the role of GGTP expression in the regulation of phenolic amide cytotoxicity, GGTP-negative Chinese hamster ovary cells (CHO-K1) were transfected with a functional rat renal cDNA representing the full-length GGTP transcript. I-GHB and GHB were significantly more cytotoxic in GGTP cDNA transfected Chinese hamster ovary (CHO-K1-GGTP) cells than in non-transfected CHO-K1 cells. The combination of L-serine and sodium borate blocked the cytotoxic activity of these pro-drugs and also inhibited GGTP-catalyzed formation of polymerized products from these phenolic amides in intact BL6 melanoma and CHO-K1-GGTP cells. Furthermore, melanin formation from GHB was not observed in non-transfected CHO-K1 cells lacking GGTP expression. The combined data strongly suggest that GGTP-catalyzed hydrolysis of the anti-tumor pro-drugs I-GHB and GHB to 4-aminophenols mediates the expression of antitumor activity.

The stability of beta-galactosidase from Aspergillus oryzae in water-miscible organic solvents in different buffers at various pH values ranging from 4.6 to 8.0 was studied. The stability of the enzyme in all six organic solvents studied was dependent on pH and on the type of buffer ions present. At a given pH, destabilization by organic solvents was highest in sodium borate buffer. The destabilization of beta-galactosidase by these solvents could be reversed by addition of sugars or polyhydroxy compounds exclusively in sodium borate, suggesting a role of borate ions in stabilization. A similar effect of addition of mannitol was observed on deactivation of beta-galactosidase by N, N-dimethylformamide (DMF). Exclusively in sodium borate, at pH 8.0, the addition of mannitol (0.02 M) not only prevented the deactivation by DMF (8%, v/v) but increased the enzyme activity to the level at its optimum pH. Since beta-galactosidase from Aspergillus oryzae is a glycoprotein, complexation of the borate ions to the carbohydrate part may result in change in protein conformation, which, without leading to denaturation or inactivation of the enzyme, may facilitate interaction of the organic solvents with the enzyme leading to its denaturation. Such a denaturation is probably prevented by addition of polyhydroxy compounds, which appear to compete favorably with the carbohydrate moiety of the protein in complexing with borate ions. This should result in the enzyme regaining its native conformation.

The effect of different buffer anions on the maximal velocity (Vmax) and Michaelis constant (Km) of the first reaction between horse cytochrome c and beef cytochrome c oxidase was studied. The Michaelis constant for the high affinity phase varies little as the buffer anions borate, phosphate, succinate and citrate were employed, but Vmax varies significantly. At the ionic strength I = 0.1 and pH 7.4 the Vmax increases in the order as following: borate < phosphate < succinate < > citrate. citrate. The differences in maximal activities in the presence of the anions may result from the changes in anion binding side on cytochrome c that affect its interaction with cytochrome c oxidase. The effect of the phosphate, succinate and citrate anions on the electron transfer reaction between cytochrome c and cytochrome c oxidase is important as a control of the respiration process in mitochondria at physiological conditions.

The present study was designed to determine the mechanism(s) underlying the excitatory effects of several sulfidopeptide leukotrienes (LTs) on the muscularis mucosae in three regions of the rabbit colon. Proximal colonic muscularis mucosae was refractory to LTs C4, D4, and E4. In addition, it exhibited no responses to prostaglandin (PG) E2 and only a minimal contractile response to PGF2 alpha. Mid and distal colonic muscularis mucosae each responded to LTs C4, D4, and E4 and PGs E2 and F2 alpha with concentration-dependent contractions. In both regions, responses to LTD4 and LTE4 were abolished by indomethacin (10(-6) M) pretreatment. LTC4-induced responses were reduced approximately 50% by this procedure. The residual contraction to LTC4 was resistant to both tetrodotoxin (10(-6) M) and atropine (10(-6) M). In separate experiments, responses to LTC4 were also reduced by approximately 50% if LTC4 conversion to LTD4 and LTE4 was first prevented by L-serine borate (45 mM) in combination with L-cysteine (10 mM). It is concluded that proximal colonic muscularis mucosae lacks the appropriate functional excitatory LT and PG receptors. On mid and distal colonic muscularis mucosae, the actions of LTD4 and LTE4 and, in part, LTC4 are the result of PG production, whereas LTC4 has an additional direct action, possibly mediated through a selective LTC4 receptor.

Pig gastric mucin, a highly glycosylated glycoprotein, inhibits angiotensin-converting enzyme (ACE) with an IC50 of 2 mM-neutral hexose content. Pig submaxillary mucin at 2.3 mM inhibits by 73%. To determine whether the oligosaccharide moieties of the mucins contribute to this inhibition, oligosaccharides were prepared from each mucin by reductive beta-elimination and their effects on enzyme activity determined. Total oligosaccharides from gastric mucin inhibited enzyme activity with an IC50 of 0.3 mM based on the neutral hexose content of the oligosaccharide solution. Fractions isolated from gastric mucin by chromatography on DEAE-cellulose and Bio-Gel P-2 inhibited ACE with IC50 values ranging from 2 to 16 mM-oligosaccharide. Larger oligosaccharides inhibited with lower IC50 values than did smaller oligosaccharides. Fractions of average molecular mass 1100 and 740 Da prepared from submaxillary mucin inhibited with IC50 values of 40 and 80 mM-oligosaccharide respectively. Monosaccharides commonly present in serum and membrane glycoproteins were also tested for their effect on ACE. Galactose, N-acetylglucosamine, N-acetylgalactosamine and glucosamine were inhibitory. N-Acetylneuraminic acid stimulated the activity of ACE. Fucose, ethylene glycol and sucrose had no effect on the activity of the enzyme. The influences of different buffers, ion concentrations, pH and substrate structure on the effect of carbohydrate on enzyme activity were also evaluated. The extent of inhibition by the monosaccharide galactose was strongly influenced by buffer ion and substrate concentration. The effects of the oligosaccharide moieties and intact mucins were less sensitive to assay conditions.

Inhibitors of gamma-glutamyl transpeptidase (mixture of serine and borate--13 mM, kainic acid--5 mM and 6-diazo-5-oxo-L-norleucine--2 mM) significantly suppressed glutamate uptake into cultured neurones and glial cells. The simultaneous application of any of these inhibitors with ouabain resulted in a further decline in glutamate uptake. It can be speculated that gamma-glutamyl transpeptidase significantly contributes to glutamate transport into nerve cells in the early period of brain development until the Na(+)-K(+)-gradient is fully constituted.

The possibility that boron and molybdenum anions can influence sex steroid metabolism by forming complexes with catechol estrogens has been studied in vitro. The formation of 2-methoxyestrone (2-OHE1 2-Me) from 2-hydroxyestrone (2-OHE1) by catechol-O-methyltransferase (COMT) was followed by measuring the transfer of the radiolabeled methyl group from S-adenosylmethionine. In the presence of both sodium tetraborate and sodium molybdate using a phosphate buffer medium, the formation of 2-OHE1 2-Me decreased as the anion:2-OHE1 molar ratio was increased. However, the reverse effect was observed when using a tris buffer medium and further investigation showed that phosphate and sulphate also enhanced COMT activity in a tris buffer medium. Boric acid affinity medium, used as a substitute for borate salt, also showed a negative relationship with enzyme activity in a phosphate buffer medium, and inhibition of methylation was more marked than with the free anion. Erythrocytes contain appreciable amounts of COMT, which is mostly responsible for the rapid O-methylation of catechol estrogens in blood. The methylation of a simple catechol compound, 1,2-dihydroxybenzene (pyrocatechol) was therefore studied using rat red blood cell lysates. Methylation was inhibited in a concentration-related manner by borate, as found in the studies of 2-OHE1. It is possible that high dietary intakes of boron or molybdenum could regulate the rate of catabolism, or even the metabolic fate of the major estrogens.