Transport of 3-hydroxybutyrate by cultured rat brain astrocytes

It is well established that 3-hydroxybutyrate can serve as an energy source for the brain. Since substrate utilization may be regulated in part by transport across the cellular membrane, we investigated the uptake of 3-hydroxybutyrate by primary cultures of rat brain astrocytes. Measurement of the net uptake indicated a saturable system and a Lineweaver-Burke type plot was consistent with a single carrier-mediated mechanism with a Km of 6.03 mM and a Vmax of 32.7 nmol/30 seconds/mg protein. The rate of uptake at pH 6.2 was more than ten times the rate at pH 8.2, with the rate at pH 7.4 being intermediate between these values, suggesting the possibility of cotransport with H⁺ or exchange with OH^– (antiport). Mersalyl had only a slight effect on the transport of 3-hydroxybutyrate, suggesting that sulfhydryl groups are not involved in the transport of this monocarboxylic acid. Phenylpyruvate and -ketoisocaproate also attenuated the transport, but lactate had only a marginal effect. These results suggest that the utilization of 3-hydroxybutyrate as an energy source by astrocytes is regulated in part by carrier-mediated transport and that the uptake system is different from the lactate transport system.These data were presented in part at the FASEB Meeting, April, 1990     

Lactate oxidation byTreponema pallidum

Whole cells ofTreponema pallidum consumed O₂ with lactate in a glucose-depleted medium.d(–) Lactate caused marked stimulation of O₂ uptake at a rate similar to that with glucose, whereasl(+) lactate resulted in no increase over the reduced rate observed upon glucose depletion. Lactate oxidation was specific for -hydroxy straight-chain acids of 3,4, and 5 carbons. O₂ uptake during lactate oxidation proceeded independently of pyruvate oxidation and required NAD. The product of lactate oxidation was pyruvate.d(–) Lactate-stimulate O₂ uptake was sensitive to chlorpromazine and resistant to amytal and cyanide. Glucose did not inhibit the oxidation of lactate as shown by the additive effect of both substrates on O₂ uptake. Oxidation of glucose, but not lactate, provided energy necesary for motilibty or maintenance of virulence. A mixture of lactate isomers was formed from glucose with thel(+) isomer concentration remaining constant and thed(–) isomer concentration varying inversely with dissolved O₂ concentration. The function of lactate as an oxidizable substrate is apparently quite distinct from that of glucose.     

Histochemical detection of α-d-glucosidases and their molecular forms with 5-Br-4-Cl-3-indoxyl-α-d-glucoside

Summary 5-Br-4-Cl-3-Indoxyl--d-gluco(pyrano)side was found to be the most suitable synthetic substrate for the demonstration of -d-glucosidases in situ. Using an azoindoxyl procedure with hexazotized pararosaniline or new fichsine at pH 5 in freeze-dried celloidine-mounted cryostat sections acid -d-glucosidase (EC 3.2.1.20) was shown for the first time in lysosomes of many cells of fetal and adult rat, mouse, guinea-pig, marmoset and human organs. At pH 6.5, in chloroform-acetone pretreated cryostat sections plasma membrane -d-glucosidases were shown in the brush border of enterocytes of the small and large intestine, in the brush border of proximal renal tubule cells and in the stereocilia of the epididymal duct. In an indigogenic procedure with ferricyanide/ferrocyanide as redox catalysator plasma membrane -d-glucosidases were depicted as well as with the azo-indoxyl method; the demonstration of the acid -d-glucosidase was inferior to that achieved with the azo-indoxyl procedure. Using tetrazolium salts as capture reagent intracellular localization was unsatisfactory. In enterocytes, a localization in the Golgi apparatus was shown by the azo-indoxyl procedure only. Analytical isoelectric focusing revealed organ-dependent differences of plasma membrane and lysosomal -d-glucosidases. Compared with the already existing methods the azo-indoxyl and indigogenic procedures are by far the most suitable techniques.Supported by the German Research Foundation (Sfb 174) and the BMFT (Project CMT 35)     

Immunochemical determination and immunocytological localization of brain-specific protein α-albumin (GFA) in isolated astrocytes

Astrocyte- and neuron-enriched fractions were isolated by preparative centrifugation from rat brain and human brain, obtained at autopsy. The brain-specific protein -albumin (GFA) was studied in these fractions by quantitative immunoradiochemical and by immunocytological methods. It is concluded that morphologically intact astrocytes can be isolated not only from rat, but also from human brain. In the rat, -albumin (GFA) is only found in the astrocyte-enriched fractions; this shows that determination of -albumin (GFA) is useful for the evaluation of cell separation methods for the brain. From the astrocyte-enriched fractions, four times more -albumin (GFA) can be extracted when detergent (0.1% SDS) is used, than when an aqueous extract is made. About ±0.15×10^–6 particles were extracted in 1 ml of either PBS or SDS/PBS. This suggests that -albumin (GFA) exists in at least two forms, one hydrosoluble, and one detergent soluble, possibly structurally bound, form.     

Endogenous respiration reflects the energy load imposed by transport of nonmetabolizable substrates and by induced de novo protein synthesis in Rhodotorula glutinis

Uptake of the nonmetabolizable sugars 6-deoxy-d-glucose, l-rhamnose and l-xylose, which are taken up by a common carrier, stimulated significantly cell respiration in Rhodotorula glutinis. The extra oxygen consumption for uptake (0.5–0.7 equivalents O₂/mol transported sugar) was proportional to the uptake rate and was independent of the K _tvalue of the transport system. Sugars that become metabolized after induction, d-arabinose and methyl--d-glucoside, caused a higher stimulation, 1.4 and 3.6 equivalents O₂/mol respectively, which was reduced to 0.6 equivalents O₂/mol when de novo protein synthesis was blocked by cycloheximide. The stimulation of respiration thus includes a fraction related purely to the energy demand for uptake and another one related to the induced de novo protein synthesis. The net uptake-induced respiration boost was similar with all sugars under study irrespective of their transport systems. The estimated energy demand was equivalent to about 2 ATP/sugar molecule. For comparison, the amino acid analogue -aminoisobutyric acid (AIB) was also investigated; the overall energy demand for its uptake corresponded to the equivalent of about 4 ATP/molecule.Abbreviation AIB -aminoisobutyric acid     

Modulation of cholinergic synaptic functions by sialylcholesterol

The effects of sialylcholesterol, a synthetic ganglioside analogue, on cholinergic synaptic functions were investigated using synaptosomes prepared from C57BL/6 mouse brain cortices. Addition of -sialylcholesterol stimulated high K (50mm)-evoked acetylcholine (ACh) release from synaptosomes at concentrations ranging from 1 to 5 µm. The -anomer of the sialyl compound also increased the neurotransmitter release at 5 µm, but the effect was much smaller than that of the -anomer. Beta-sialylcholesterol appeared to increase high-affinity choline uptake and ACh synthesis, resulting in an increment in the release of ACh. On the other hand, -sialylcholesterol did not change the synthetic rate of ACh, and instead it increased the depolarization-induced influx of calcium ions into synaptosomes, while the -anomer did not affect the divalent cation influx. The enhanced calcium influx is thought to increase ACh release from synaptosomes treated with -sialylcholesterol. These results imply that the two anomers of sialylcholesterol may modulate the synaptic membrane machinery differently, that is, the -anomer may activate voltage-dependent calcium channels and the -anomer may facilitate high-affinity choline uptake.In order to evaluate the ameliorating effect of sialylcholesterol, -sialylcholesterol was applied to the synaptosomes from aged mice (34 months old), which have been shown to have a decreased ACh release (Tanakaet al., 1995,J Neurosci Res, in press [1]). The reduced neurotransmitter release recovered to the levels of younger animals, suggesting that sialylcholesterol might have a potential therapeutic use for restoring synaptic function that occurs in aged brains.     

Synthesis of methyl α-isomalto-oligosaccharides specifically deoxygenated at position 2 of the terminal glycopyranosyl unit

Methyl -isomaltoside and methyl -isomaltotrioside specifically deoxygenated at position C-2 of the terminal glucopyranosyl unit were synthesized by trimethylsilyltriflate-mediated condensation of 3,4,6-tri-O-benzoyl-1-O-tert-butyl(dimethyl)silyl-2-deoxy--d-arabino-hexopyranose with suitably blocked derivatives of methyl -d-glucopyranoside and methyl -isomaltoside, respectively.To whom correspondence should be addressed.     

Hypoxia-Induced Lipid Peroxidation in Rat Brain and Protective Effect of Carnitine and Phosphocreatine

The exposure to hypobaric hypoxia increased lipid peroxidation (as indicated by thiobarbituric acid-reactive substances [TBARS] in rat brain. Plasma lactate/pyruvate ratio was used as a marker of hypoxia. We compared the protective effect of -tocopherol with the effect of l-carnitine or phosphocreatine. Rats pretreated with -tocopherol, l-carnitine, or phosphocreatine had lower TBARS levels after the exposure to hypobaric hypoxia. However, lactate/pyruvate ratio was improved only in rats pretreated with l-carnitine or phosphocreatine. We conclude from our data that, contrary to -tocopherol, protective effects of l-carnitine and phosphocreatine administrations are due to their regulation of metabolic reactions during hypobaric hypoxia rather than to their scavenger activity.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

Glucose inhibition of galactose-induced synthesis of β-galactosidase in Streptomyces violaceus

Various carbon compounds inhibited galactose induced synthesis of a -galactosidase activity in Streptomyces violaceus. Glucose and 2-deoxyglucose, but not methyl--d-glucose, caused inhibition of galactose uptake activity. In addition, glucose, or one of its metabolites, inhibited the synthesis of the glactose uptake system. Therefore it is concluded that the main inhibitory activity of glucose on galactose induced enzyme synthesis is exerted through inducer exclusion. Other carbon sources, such as d-ribose, d-gluconate, cellobiose or dl--glycerophosphate, did not inhibit uptake of the inducer galactose and may exert their effect through catabolite repression, inactivation or direct enzyme inhibition.     

Transport of maltose by Pseudomonas fluorescens W

The system for uptake of maltose in Pseudomonas fluorescens W was inducible. Using a mutant strain unable to hydrolyze maltose, it was shown that maltose was taken up unaltered against a concentration gradient. Uptake of ¹⁴C maltose was only significantly inhibited by nonradioactive maltose or maltotriose. These were the only sugars that could displace accumulated radioactive maltose in the strain unable to hydrolyze maltose. Uptake exhibited saturation kinetics and was inhibited by energy poisons, indicating that this system was one of active transport. Sulfhydryl-binding reagents reversibly inhibited maltose uptake. No transport ability was lost when cells were subjected to osmotic shock. Using the protein-binding dye 7-diazonium-1,3-naphthalene disulfonate a protein or proteins located in or external to the cell membrane was implicated in maltose transport. The hydrolysis of p-nitrophenyl--D-glucoside (PNPG) was used as an indirect measure of transport ability since penetration of PNPG, not its hydrolysis, was the rate-limiting step.Abbreviations PNPG paranitrophenyl--D-glucoside - NDS 7-diazonium-1,3-naphthalene disulfonic acid - PMB p-hydroxymercuribenzoate - MBS p-chloromercuriphenylsulfonic acid - PCMB p-chloromercuribenzoate - CCCP carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - HOQNO 2-heptyl-4-hydroxyquinoline-N-oxide     

Cation effects on the conformations of muscle and non-muscle α-actinins

We examined the effects of changing KCl concentration on the secondary structures of -actinins using circular dichroism (CD), 1,1-bis(4-anilino) naphthalene-5,5-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mm KH₂PO₄, pH 7.5, increasing KCl from 0 to 120 mm led to decreases in -helix conformation for brain, platelet and heart -actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mm KCl, 0.65 mm calcium produced small changes in the CD spectra of both brain and platelet -actinin, but had no effect on heart -actinin. bisANS fluorescence of all three -actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mm KCl increasing concentrations of Ca²⁺ or Mg²⁺ did not have significant effects on the bisANS fluorescence of any -actinin. Digestion of brain, platelet and heart -actinins with -chymotrypsin showed an increase of proteolytic susceptibility in 120 mm KCl. These experiments also showed that increasing the concentration of Ca²⁺ or Mg²⁺ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mm KCl. The results suggest that -actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different -actinins.     

Induction,purification and characterisation of arabinases produced by Aspergillus niger

The induction of arabinases in Aspergillus niger N400 was studied on different simple and complex carbon sources. Sugar beet pulp was found to be an inducer of three arabinan degrading enzymes (-l-arabinofuranosidase A, -l-arabinofuranosidase B and endoarabinase). These enzymes were purified from A. niger culture fluid after growth of the fungus in medium employing sugar beet pulp as the carbon source and were characterised both physico-chemically (Mw 83 000, 67 000, 43 000 Da and, pI 3.3, 3.5 and 3.0 for -l-arabinofuranosidases A and B and endo-arabinase, respectively) and kinetically (K _m on p-nitrophenyl--l-arabinofuranoside 0.68 and 0.52 mM for -l-arabinofuranosidases A and B, resp.; K _m on sugar beet arabinan 0.24 and 3.7 g/l for -l-arabinofuranosidase B and endoarabinase, resp.). The amino acid compositions of the three enzymes were determined also. The enzymic properties were compared with those of arabinases purified from a commerical A. niger enzyme preparation. Differences were found though the kinetic data suggest considerable similarity between the enzymes from the different sources. Antibodies raised in mice against the three enzymes were found to be highly specific and no crossreactivity with other proteins present in culture filtrates was observed. A mixture of these antibodies has been used to analyze specific induction of these individual enzymes on simple and complex substrates by Western blotting.Abbreviation PNA p-nitrophenyl--l-arabinofuranoside     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The -isopropylmalate synthase (EC 4.1.3.12) from Alcaligenes eutrophus H 16 was inhibited by l-leucine and -ketoisocaproate. The extent of inhibition was influenced by substrate- and inhibitor concentrations as well as by the pH. Intermediary plateaus, which always appeared in the inhibition curves, suggested cooperative effects. The maximal Hill coefficient was found to be two. At low concentrations of leucine the inhibition mechanism was of the competitive type with respect to substrate acetyl coenzyme A and of the noncompetitive type with respect to substrate -ketoisovalerate. The inhibition was specifically relieved by the addition of valine or isoleucine. The anomalous effect of temperature on enzyme activity was diminished by leucine. The Arrhenius energy of the reaction increased from about 11 kcal/mole in the absence of leucine to about 18 kcal/mole in the presence of leucine. The further addition of valine reversed this effect. The physiological relevance of the -ketoisocaproate-mediated inhibition is discussed.Abbreviations IPM -isopropylmalate - KIC -ketoisocaproate - KIV -ketoisovalerate - DTNB 5:5 Dithiobis-(2-nitrobenzoe acid)     

Ochrobactrum anthropi NCIMB 40321: a new biocatalyst with broad-spectrum l-specific amidase activity

Of 125 microorganisms that were able to use -hydroxy acid amides as sole nitrogen source, Ochrobactrum anthropi NCIMB 40321 was selected for its ability to hydrolyse racemic amides l-selectively. The substrate specificity of whole O. anthropi cells is remarkably wide and ranges from -H--amino-, -alkyl--amino, N-hydroxy--amino acid amides to -hydroxy-acid amides. After 50% conversion, both the l-acids formed and the remaining d-amides were present in >99% enantiomeric excess, and ammonia accumulated in stoichiometric amounts. Using mandelic acid amide as a model substrate, the hydrolysis was optimized. Optimal rates were observed at pH 8.5 at 50°C. At higher temperatures the initial rate was even higher; however, fairly rapid inactivation occurred.     

Studies on the mode of action of the antibacterial agent diethyltin dichloride onEscherichia coli

The mode of action of diethyltin dichloride as a representative of the antibacterial dialkyl-substituted tin compounds was studied forEscherichia coli. The oxygen uptake by resting cells ofEicoli with glucose, lactate, pyruvate or glutamate as substrates is strongly inhibited by diethyltin dichloride at a concentration of 0.08mm which is still below the minimum inhibitory concentration for growth on a rich medium. Inhibition of oxygen uptake with glucose or lactate as substrates is accompanied by an increase in the amount of pyruvic acid accumulating. A slight rise in the concentration of -ketoglutaric acid results from the inhibition of succinate and malate oxidation by diethyltin dichloride, while inhibition of -keto acid production is obtained with glutamate as a substrate. Anaerobically, with glucose as a substrate, ethanol formation as well as the production of carbon dioxide and hydrogen are inhibited by diethyltin dichloride. In many respects the effects of diethyltin dichloride on metabolism resemble those of arsenite.The inhibition of pyruvate metabolism probably results in a lack of acetyl-CoA, and this is suggested to be a possible cause of growth inhibition.     

Biosynthesis of 5-(p-aminophenyl)-1,2,3,4-tetrahydroxypentane by methanogenic bacteria

In an attempt to establish the nature of the ammonium-assimilation products which mediate the inhibition by ammonium of nitrate uptake in cyanobacteria, the effect of different amino acids on nitrate utilization by intact Anacystis nidulans cells has been assayed. To exclude an indirect inhibition of nitrate uptake through the ammonium which the amino acids might release, the cells were pretreated with l-methionine-d,l-sulfoximine (MSX), a potent inactivator of glutamine synthetase. Under these conditions, several l-amino acids, but not the corresponding d-isomers, affected nitrate utilization to a variable extent, causing inhibitions ranging between 20 and 80% when added at 20 mM concentration.For most of the inhibitory amino acids, including l-isoleucine, l-leucine and l-valine, a correlation was found between their ability to act as amino group donors to -ketoglutarate, in reactions catalyzed by A. nidulans cell-free extracts, and their inhibitory effect on nitrate utilization. l-Glutamine, l-asparagine and glycine, being effective inhibitors of nitrate utilization, were poor substrates for the transaminating activity to -ketoglutarate, however. The possible role of the latter amino acids as mediators in the ammonium-promoted inhibition of nitrate uptake is discussed.Abbreviations MSX l-methionine-d,l-sulfoximine - MTA-5 mixed alkyltrimethylammonium bromide - Mops morpholinopropane sulfonic acid     

High D-glucose does not affect binding of α-tocopherol to human erythrocytes

The role of -tocopherol uptake system in human erythrocyte in the uptake of plasma -tocopherol has been suggested. However no information is available on -tocopherol uptake activity of human erythrocytes in the presence of high levels of D-glucose which is known to lead to pathological alterations in different cells including human erythrocytes. Therefore, in order to examine the effect of D-glucose on the binding of -tocopherol to human erythrocytes, the binding characteristics of -tocopherol to these cells were established first. Binding of [3H]-tocopherol to human erythrocytes was both saturable and specific. Scatchard analysis of -tocopherol binding to these cells showed the presence of two independent classes of binding sites with widely different affinities. The high affinity binding sites had a dissociation constant (Kd1) of 90 nM with a binding capacity (n1) of 900 sites per cell, whereas the low affinity binding sites had a dissociation constant (Kd2) of 5.2 M and a binding capacity (n2) of 105,400 sites per cell. Trypsin treatment abolished all the -tocopherol binding activity. Competition for the binding of -tocopherol to human erythrocytes was effective with other homologues of -tocopherol (-tocopherol, -tocopherol and -tocopherol) and their potency was almost equal to -tocopherol itself. The order of preference was -tocopherol > -tocopherol -tocopherol -tocopherol. Incubation of human erythrocytes with various concentrations of D-glucose did not affect -tocopherol uptake activity. Our data demonstrate the presence of an -tocopherol uptake system in human erythrocytes and that the -tocopherol uptake activity is not modulated by the presence of D-glucose.     

Functional assessment of genetic variants of α1-Antitrypsin

Summary The specific activity of thirteen genetic variants of the protease inhibitor ₁-antitrypsin (₁-AT) has been determined. Elastase inhibitor activity was assayed using protein substrates (elastin and gelatin) and the synthetic substrate N-tert-butoxycarbonyl-l-alanine p-nitrophenyl ester. The synthetic substrate -N-benzoyl-dl-arginine p-nitroanilide HCl was used to assay trypsin inhibitor activity. The specific activity of ₁-AT was expressed as serum inhibition/immunological concentration of ₁-AT. Sera of PI type FM had reduced specific activity with elastase, but not with trypsin. With the possible exception of MP, no other variants showed significant differences in specific activity when compared with normal PI type M.This research was supported by the Medical Research Council of Canada (No. MA 5426)     

Studies of the mechanisms of toxicity of the administration of recombinant tumor necrosis factor α in normal and tumor-bearing mice

Summary Tumor-bearing mice have a greater sensitivity to the acute lethal effects of the administration of high-dose recombinant human tumor necrosis factor (rhTNF-) compared to normal, non-tumor-bearing mice. We studied whether or not the presence of tumor per se was responsible for the enhanced rhTNF- toxicity. Tumor-bearing mice underwent tumor excision or sham operation before the systemic administration of rhTNF at staged times (0.5–24 h) following surgery. There was little survival difference between sham-operated tumor-bearing mice and tumor-bearing mice undergoing tumor excision (at 24 h, treatment with 12 µg rhTNF-, survival:sham-operated tumor bearers = 0/12, excised tumor-bearers = 0/12;P ² <0.01 compared to non-tumor-bearers). Mice without tumors receiving sham operation, had minimal toxicity (10 of 12 mice surviving). The injection of 3 ml Ringer's lactate i.p. before i.v. rhTNF- therapy increased survival in tumor-bearing animals; following pretreatment with Ringer's lactate 30/42 mice survived 12 µg rhTNF- compared to 6/42 surviving a similar rhTNF- dose without hydration (P ² <0.001). Since the production of oxygen free-radical metabolites has been postulated to play a role in the acute toxicity of rhTNF-, bismuth subnitrate was used to induce the enzyme metallothionein to act as a natural scavenger for these metabolites. Daily oral bismuth subnitrate treatments improved survival of mice with MCA-106 or MCA-102 sarcoma and of mice without tumors, with higher rhTNF- doses (12–20 µg), without reducing the therapeutic effect of rhTNF- against the weakly immunogenic MCA-106 sarcoma. These studies suggest methods for reducing the toxicity of rhTNF- administration in clinical trials.