Microphotometric determination of enzymes in brain sections. II. GABA transaminase

Summary The tetrazolium salt procedure of van Gelder (1965) for the demonstration of GABA transaminase (GABAT; the most important GABA degrading enzyme) was adapted for microphotometric measurements of GABAT activities in brain sections using the hippocampus of rats as selected brain region. The final incubation medium consisted of 50 mM GABA, 5 mM α-ketoglutarate, 7 mM NAD, 10 mM sodium azide, 6 mM nitroblue tetrazolium chloride, 20 mM malonate and 15% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 8.0. There was a linear relationship between GABAT activity and section thickness up to 14 μm and between GABAT activity and reaction time at least up to 20 min (kinetic and end-point measurements). Phenazine methosulfate as an exogenous electron carrier and pyridoxal-5-phosphate as coenzyme of GABAT did not enhance the demonstrable GABAT activities, whereas sodium azide as a blocker of the respiratory chain resulted in an increase of demonstrable enzyme activities. A coreaction of succinate dehydrogenase was excluded by the use of malonate (competitive inhibitor). Using the incubation medium described GABAT activities were demonstrated via the endogenous enzymes succinic semialdehyde dehydrogenase and NADH tetrazolium reductase which were shown to be not rate limiting and seems to be similarly localized as GABAT. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

Microphotometric determination of enzymes in brain sections. III. Glutamate dehydrogenase

An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM L-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities.     

Microphotometric determination of enzymes in brain sections. IV. Isocitrate dehydrogenases

A polyvinyl alcohol-(PVA) containing incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of NAD- and NADP-linked isocitrate dehydrogenases (ICDHs) in cryostat sections of the rat hippocampus. The following incubation medium is recommended for the quantification of NAD- and NADP- (differences in brackets) ICDHs: 100 mM DL-isocitrate, 10 mM sodium azide, 5 mM (4 mM) nitroblue tetrazolium (NBT), 7 mM NAD (4 mM NADP), 10 mM magnesium chloride, 0.25 mM phenazine methosulfate (PMS), with or without 5 mM ADP (without ADP), 23% PVA in 0.05 M Hepes buffer; the final pH was 7.5. With these incubation media a linear response of the reactions lasted at least 20 min. In kinetic and end-point measurements the same level of activities was demonstrable. The use of NaN3 (as a blocker of the respiratory chain) and PMS (as artificial electron carrier) was indispensible for the transfer of all reduction equivalents in the dehydrogenase reactions to the tetrazolium salt NBT. Furthermore, the activation by magnesium ions and the need of PVA to avoid diffusion artefacts of the loosely bound ICDHs were clearly shown. It is concluded that the quantification of ICDHs in situ could be a valuable tool for neurochemical investigations because ICDHs play a role not only in the substrate flux through the tricarboxylic acid cycle but also in providing alpha-ketoglutarate for the formation of glutamate which is an important amino acid in the brain.     

Microphotometric determination of enzymes in brain sections

Summary An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM l-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN₃), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. — The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN₃ as a blocker of the respiratory chain was indispensible, because without NaN₃ most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities. Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-2)     

Microphotometric determination of enzymes in brain sections. V. Glycerophosphate dehydrogenases

An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM L-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.5 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent Km value at 25 degrees C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Microphotometric determination of enzymes in brain sections. I. Hexokinase

A histochemical procedure was established for the microphotometric determination of hexokinase (HK) in sections of the rat hippocampus, which served as an exemplary brain region. For this quantitative procedure, slides were coated with glucose 6-phosphate dehydrogenase (G6PDH) as an auxiliary enzyme and sections were mounted onto this enzyme film. The sections were then incubated with the following adapted incubation medium: 5 mM D-glucose, 1.5 mM NADP, 7.5 mM ATP, 4 mM nitroblue tetrazolium chloride, 10 mM NaN3, 10 mM MgCl2, 0.25 mM phenazine methosulfate, 1 U/ml G6PDH, 22% polyvinyl alcohol in 0.05 M Hepes buffer; the final pH was 7.5. A linear response of the reaction was observed in the initial 10 min of reaction (kinetic and end-point measurements). The relationship between HK activity and section thickness was linear up to 5 microns. The need for such thin sections is discussed in relation to the limited penetration of the auxiliary enzyme into the section. It is concluded that the quantitative demonstration of HK in brain sections could be a valuable tool for studying the local metabolic entrance of glucose in the glycolytic pathway.     

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3-6 months old) and aged male rats (24-27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05 M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1, the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio ento-rhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.     

Methods of microphotometric assay of succinate dehydrogenase and cytochrome c oxidase activities for use on human skeletal muscle

Microphotometric assay media for the measurement of succinate dehydrogenase (SDH) and cytochrome oxidase activities in sections of human skeletal muscle have been developed. The optimal constitution of these media was determined experimentally. Factors investigated include the effects of substrate concentration, pH, use of different electron acceptors and electron donors, influence of intermediate electron carriers and tissue-stabilizing agents, effects of inhibitors, the extent of endogenous and non-specific reactions and the linearity of the reactions during the time course of the assays. Optimal assay media (SDH) contained 130 mM succinate, 1.5 mM Nitro Blue tetrazolium, 0.2 mM phenazine methosulphate and 1.0 mM sodium azide in 0.1 m phosphate buffer, pH7.0. Cytochrome oxidase was optimally assayed in media containing 4 mM diaminobenzidine and 100 microns cytochrome c. Reactions in individual muscle fibers were found to be linear for incubation times up to 10 min in SDH assays and for more than 15 min in cytochrome oxidase determinations. Some potential uses of these microphotometric assays in the investigation of human metabolic muscle disorders are discussed.     

Independent Effects of γ-Aminobutyric Acid Transaminase (GABAT) on Metabolic and Sleep Homeostasis

Breakdown of the major sleep-promoting neurotransmitter, γ-aminobutyric acid (GABA), in the GABA shunt generates catabolites that may enter the tricarboxylic acid cycle, but it is unknown whether catabolic by-products of the GABA shunt actually support metabolic homeostasis. In Drosophila, the loss of the specific enzyme that degrades GABA, GABA transaminase (GABAT), increases sleep, and we show here that it also affects metabolism such that flies lacking GABAT fail to survive on carbohydrate media. Expression of GABAT in neurons or glia rescues this phenotype, indicating a general metabolic function for this enzyme in the brain. As GABA degradation produces two catabolic products, glutamate and succinic semialdehyde, we sought to determine which was responsible for the metabolic phenotype. Through genetic and pharmacological experiments, we determined that glutamate, rather than succinic semialdehyde, accounts for the metabolic phenotype of gabat mutants. This is supported by biochemical measurements of catabolites in wild-type and mutant animals. Using in vitro labeling assays, we found that inhibition of GABAT affects energetic pathways. Interestingly, we also observed that gaba mutants display a general disruption in bioenergetics as measured by altered levels of tricarboxylic acid cycle intermediates, NAD⁺/NADH, and ATP levels. Finally, we report that the effects of GABAT on sleep do not depend upon glutamate, indicating that GABAT regulates metabolic and sleep homeostasis through independent mechanisms. These data indicate a role of the GABA shunt in the development of metabolic risk and suggest that neurological disorders caused by altered glutamate or GABA may be associated with metabolic disruption.     

In situ measurements of enzyme activities in the brain

Summary The present review focusses on enzymes involved in the metabolism of amino acid neurotransmitters and the microphotometric determinations of their activities in various layers of the rat hippocampus. The enzymes are NAD-linked isocitrate dehydrogenase (NAD-ICDH), glutamate dehydrogenase (GDH), and GABA transaminase (GABAT), all of which are localized in mitochondria. GDH seems to be restricted to astrocytes, whereas NAD-ICDH and GABAT are localized in neurons as well as in astrocytes. NAD-ICDH is an important enzyme of the tricarboxylic acid cycle and may deliver -ketoglutarate for the formation of glutamate and GABA, which serve as neurotransmitters in the hippocampus. GDH catalyses the interconversion of -ketoglutarate and glutamate, whereas GABAT is the important GABA-degrading enzyme and requires -ketoglutarate for its activity. While differing in their cellular distribution and activity levels, NAD-ICDH, GDH and GABAT are significantly correlated in their hippocampal distribution. Furthermore, developmental and pharmacohistochemical studies suggest that the distribution and activity of astrocytic GDH is correlated with amino-acidergic neurotransmission in the hippocampus. The data reported give further evidence for a metabolic relationship between neurons and astrocytes in the turnover and metabolism of glutamate and GABA.     

Increase of enzyme activities following the in vitro peroxidation of normal human red blood cells

Red blood cells (RBCs) from 15 normal human blood samples were incubated with different concentrations of hydrogen peroxide in sodium azide, and the effects of the peroxidation on several glycolytic and nucleotidic enzyme activities were investigated. The release of malonyl dialdehyde (MDA) and methemoglobin formation were used as indicators of RBC peroxidation. The increase of H2O2, final concentration from 0.1 to 5 mmol/l, resulted in a progressive rise of almost all glycolytic enzyme activities, especially those of aldolase (200% of normal at 1 mmol/l), phosphoglycerate kinase (140%), phosphoglycerate mutase (136%), pyruvate kinase (130%) and glutathione peroxidase (130%), and in a decrease of glucose-6-phosphate dehydrogenase (68%) and pyrimidine-5-nucleotidase (23%). The addition of beta-mercaptoethanol to the incubation medium abolished only the effect of 1 mmol/l H2O2 on glucose-6-phosphate dehydrogenase.     

Studies on the GABAergic system in astrocytoma and neuroblastoma cells in culture

The GABAergic system was investigated in C-6 astrocytoma cells and C-1300 neuroblastoma cells in culture and compared to that in mouse brain. The activities of glutamate decarboxylase, GABA-transaminase, succinic semialdehyde dehydrogenase and glutamate dehydrogenase were measured. In the cultured cells, only glutamate dehydrogenase activity was equal or greater than that of mouse cerebral cortex. Glutamate decarboxylase in both cell lines was 2%, while GABA-transaminase and succinic semialdehyde dehydrogenase activities were less than 20% of those found in brain. In spite of the disparate enzyme activities, GABA, glutamate, and -ketoglutarate concentrations were similar in the cell lines and cerebral cortex. The anticonvulsant drugs sodium valproate and aminooxyacetic acid increased cortical GABA concentrations but either had no effect or decreased GABA in the cells in a complete medium. The convulsant isoniazid decreased GABA in mouse brain but had no effect in either cell line. In the absence of pyridoxal in the medium, some drug effects could be induced in the cultured cells. It is concluded that the differing responses of the GABAergic system in the mouse brain and cell lines may be attributed in part to the fact that the cells do not represent an integrated system and are of tumor origin.     

Microphotometric determination of enzymes in brain sections

Summary An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM l-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.05 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent K _m value at 25°C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Studies on the optimalisation and standardisation of the light microscopical succinate dehydrogenase histochemistry

The present investigation was undertaken in order to establish an optimal tissue pretreatment and an optimal incubation medium for the histochemical demonstration of succinate dehydrogenase (E.C. 1.3.99.1). The investigations were performed on steroid producing (testicle, adrenal gland) and steroid dependent (Fallopian tube) tissues. We studied the influences fo formalin fixation, acetone, magnesium ions, cyanides, electron carries (phenazine methosulfate, menadione coenzyme Q10), osmolarity, substrate concentration and inhibitors (oxalacetate, oxalate, malonate, 4-chloromercuribenzoic acid). The following procedure yields blameless morphological integrity and enzyme localization as well as optimal SDH-activity: Freezing of tissue cubes (diameter less than 5 mm) in propane cooled with liquid nitrogen or in melting freon. Incubation of 5 micrometer cryostat sections in narrow jars in the following medium (38.5 ml):--10 ml of 0.2 M sodium phosphate buffer pH 7.6 (52 mM).--18 mg tetranitro-BT in 0.5 ml dimethylformamide and aqua bidest. ad 10 ml (0.5 mM).--2.6 mg KCN in 16 ml aqua bidest. (1 mM).--540 mg succinate (disodium salt, hexahydrate) in 2 ml aqua bidest. (52 mM).--3 mg PMS (phenazine methosulfate) in 0.5 ml aqua bidest. (0.25 mM). The incubation medium has an osmolarity of 440 mosm. The incubation is carried out for 10 min at 37 degree C in darkness. To avoid non specific formazan deposits in lipid containing tissues a preincubation of the cryostat sections in 100% acetone at--22 degree C or--40 degree C for 7--10 min and an incubation time of 20--30 min is recommended. Control incubations adduced proof at the specificity of the SDH demonstration. Parallel incubation without PMS in order to determine indirectly the content of endogenous CoQ10 is further recommended.     

Protective effect of inhibitors of succinate dehydrogenase on wheat seedlings during osmotic stress

The effect of malonate and sedaxane, a compound with the fungicidal effect which act as succinate dehydrogenase inhibitors, on the resistance of etiolated wheat seedlings (Triticum aestivum L.) to osmotic stress caused by 12% PEG 6000 solution, was studied. The presowing treatment of seeds with 0.3 mM sedaxane solution significantly reduced the inhibitory effect of osmotic stress on seedling growth. The protective effect of 10 mM malonate was significant when it was added to the incubation medium of the roots; the effect of preseeding treatment with malonate was less significant. Unlike malonate, malate had no positive effect on seedling growth under osmotic stress. The activity of succinate dehydrogenase and the hydrogen peroxide content decreased in seedlings after the treatment of roots with malonate and sedaxane. Pretreatment with sedaxane and the addition of malonate to the incubation medium of roots prevented the accumulation of a lipid peroxidation product, malondialdehyde, which is caused by osmotic stress, and increased peroxidase activity. It was concluded that the stress-protective effect of sedaxane and malonate on wheat seedlings might be due to the inhibition of succinate dehydrogenase-dependent formation of reactive oxygen species and the prevention of oxidative cell damage.     

Quantitative dehydrogenase histochemistry with exogenous electron carriers (PMS,MPMS, MB)

Summary The relative efficiencies of phenazine methosulfate (PMS), 1-methoxy-phenazine methosulfate (MPMS) and Meldola Blue (MB) as electron carriers were determined biochemically (non-enzymic NADH-tetrazolium salt-test) and by quantitative histochemistry (heart and kidney slices; succinate dehydrogenase, SDH; lactate dehydrogenase, LDH). MPMS developed the highest electron transfer velocity in biochemical assays. The reaction was independent of the pH value between 7.0–8.5. PMS and MB always showed a lower transfer ability in biochemical tests which was higher with iodonitrotetrazolium chloride (INT) than with nitro blue tetrazolium chloride (NBT). A distinct pH dependence was demonstrable with MB in this respect, preferentially using INT as tetrazolium salt.Quantitative histochemical results with electron carriers are often at variance with biochemical ones. MPMS leads to somewhat higher demonstrable activities only in the determination of the NAD-dependent LDH, whereas MB results in somewhat higher LDH activity than PMS (reaction medium with agarose). MB and PMS yielded almost equally high activities in the demonstration of the flavoprotein-dependent SDH using a reaction medium with agarose. With an aqueous reaction medium, PMS resulted in higer SDH activities than MB. MPMS always had the lowest efficiency in electron transfer ability using an aqueous or agarose containing reaction medium (SDH). With PVA in the reaction medium (SDH determination) PMS was clearly superior to MPMS. MB showed only a small transfer activity under these conditions because PVA seems to bind MB almost completely. It is concluded that in histochemistry an appropriate electron carrier and electron carrier concentration must be determined for different incubation conditions, tissues, tissue preparations and dehydrogenases studied. General statements about the efficiency or inefficiency of an electron carrier as a result of only one incubation condition does not seem to be justified.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Formate dehydrogenase. Subunit and mechanism of inhibition by cyanide and azide.

Formate dehydrogenase (EC 1.2.1.2) prepared from peas (Pisum sativum) was a two-subunit enzyme. The enzyme accelerated the formation of an NAD+-cyanide compound having an adsorption band at 330 nm. The enzyme was able to bind one NAD+ molecule per each subunit but only 1 mole of NAD+-cyanide compound was formed per two subunits. The complex of NAD+, cyanide, and the enzyme was very stable and had no catalytic activity. Azide inhibited the formate dehydrogenase reaction in two different ways. By incubation of the enzyme with azide in the presence of NAD+, half of its catalytic activity was lost. The remaining activity was also inhibited by azide but this inhibition was removed competively by formate. Contrary to the case of cyanide the inhibition by azide could be removed by dialysis and no spectral species due to the addition compound of NAD+ and azide could be observed. The data from double recipricol plots of the initial velocity and the formate concentration led to a conclusion that formate dehydrogenase has two sites with about equal catalytic activity. The Km for formate was different for the two catalytic sites (1.67 and 6.25 mM) but the difference was not noticeable in the case of the Km for NAD+.     

GABA(A)-mediated toxicity of hippocampal neurons in vitro

In the present study, we examined whether the elevation of GABA by gamma-vinyl-GABA protects cultured rat fetal hippocampal neurons against toxicity induced by a 20-min incubation with 100 microM L-glutamate. Neither a 24-h pretreatment nor posttreatment with gamma-vinyl-GABA (100 microM) had any neuroprotective effects, as determined by counting microtubule-associated protein-2 positive cells and lactate dehydrogenase assay 24 h after the glutamate treatment. Unexpectedly, gamma-vinyl-GABA alone induced a 20% loss of microtubule-associated protein-2-positive cells in a culture that was grown in medium containing 25 mM KCl. The toxic effect of gamma-vinyl-GABA was mimicked by a 24-h treatment with GABA (100 microM) and the GABA(A) receptor agonist, muscimol (10 microM), but not the GABA(B) receptor agonist, baclofen (10 microM). The GABA(A) receptor antagonist, bicuculline (10 microM), protected against gamma-vinyl-GABA and GABA-evoked toxicity. Neither gamma-vinyl-GABA nor GABA was toxic in culture medium containing 15 mM KCl. These data indicate that, under depolarizing conditions, an increased GABA level is toxic for a subpopulation of developing hippocampal neurons in vitro. The effect is GABA(A) receptor-mediated. These data provide a new view for understanding neurodegenerative processes, and raise a question of the safety of therapies aimed at increasing GABA concentration following brain insults, especially in immature brains.     

Effect of some metabolic inhibitors on citric acid production by Aspergillus niger

Stationary cultures of Aspergillus niger grown on a synthetic medium have been used to study the effect of some metabolic inhibitors on citric acid production. Addition of 0.05 to 1 mM sodium malonate or 0.01 to 0.1 mM potassium ferricyanide, iodoacetate, sodium azide, sodium arsenate or sodium fluoride stimulated citric acid production (3.6 to 45%), but not total titratable acids. Addition of higher concentrations (0.2 to 10 mM) of later inhibitors caused a marked inhibition of fungal growth and citric acid production. The implications of these preliminary findings are discussed.     

Enzyme cytochemistry of unfixed leukocytes and bone marrow cells using polyvinyl alcohol for the diagnosis of leukemia

Cytochemical methods for the demonstration of enzyme activities in blood and bone marrow cells were systematically improved by the addition of an inert polymer, polyvinyl alcohol (PVA), to the incubation medium and by using optimized reaction media. The methods investigated were tetrazolium salt methods for lactate, glucose-6-phosphate, succinate and glutamate dehydrogenase, the indoxyl-tetrazolium salt method for alkaline phosphatase, the diaminobenzidine method for peroxidase, and diazonium salt methods for chloroacetate esterase, beta-glucosaminidase, beta-glucuronidase, acid phosphatase, and dipeptidylpeptidase II and IV. PVA in the media preserved the morphology of cells very well and prevented leakage of large molecules such as enzymes from the cells. Therefore, fixation or long periods of air-drying prior to incubation leading to substantial loss of enzyme activity could be avoided. A brief period of drying (2 min at 37 degrees C) of the cell preparations just before the incubation was sufficient for making the cells permeable. Localization of enzyme activities was very precise and precipitation of the final reaction product was confined to sites which are known to contain the enzyme under study (granules, mitochondria, lysosomes). These advantages advocate the use of PVA in haematological enzyme cytochemistry and especially for diagnosis of leukemia.