Thermal activity of the isolated brain fragment

The paper deals with thermovisional studies of thermal activity of hippocampus transplants placed in the eye anterior chamber of the rat. The method permits investigation into the dynamics of temperature field in the nervous tissue directly. In the transplants with synchronous spontaneous activity of large neuron groups there were found zones of high thermoactivity with mean amplitude 0.1 degree C, as well as thermopassive zones with temperature variations not above 0.03 degree C. Under certain conditions transfer of heat-up regions along the transplant surface, synchronous thermoactivation of single zones and chaotic regime of thermoactivation were observed. Causes of the observed effects are analysed. The method applied opens a new approach to the studies of space and time organization of the nervous tissue functioning in the course of its living activity.     

Effect of calcium glucarate on beta-glucuronidase activity and glucarate content of certain vegetables and fruits

Glucarate is normally present in tissues and body fluids and is in equilibrium with D-glucaro-1,4-lactone, a natural inhibitor of beta-glucuronidase activity. Dietary calcium glucarate, a sustained-release from of glucarate, elevates the blood level of D-glucaro-1,4-lactone which suppresses blood and tissue beta-glucuronidase activity. A single dose of CaG (4.5 mmole/kg body weight) inhibited beta-glucuronidase activity in serum and liver, lung, and intestinal microsomes by 57, 44, 37, and 39%, respectively. A chronic administration of calcium glucarate (4% in diet) also decreased beta-glucuronidase activity in intestinal and liver microsomes. Maximal inhibition of beta-glucuronidase activity in serum was observed from 12 noon to 2:00 PM. In contrast, maximum inhibition of beta-glucuronidase activity in intestinal and liver microsomes occurred during mornings, although a secondary depression in intestinal microsomes also occurred around 4 PM. A 4% calcium glucarate supplemented diet also inhibited beta-glucuronidase activity by 70% and 54%, of the bacterial flora obtained from proximal (small intestine) and distal (colon) segments of intestine, respectively. Due to the potential effect of dietary glucarate on net glucuronidation and on other metabolic pathways, glucaric acid levels in various foods were determined. The glucaric acid content varied from a low of 1.12-1.73 mg/100 g for broccoli and potatoes to a high of 4.53 mg/100 g for oranges.     

Metabolic activity and enzyme induction in rat fecal microflora maintained in continuous culture

The enzyme activity of the rat hindgut microflora maintained in an anaerobic two-stage continuous culture was compared with that of rat cecal contents. A qualitative comparison (API ZYM) showed a high degree of similarity between the two populations. Quantitative determinations showed that azoreductase, beta-glucosidase, nitrate reductase, and nitroreductase activities were comparable, and that beta-glucuronidase activity was very low in the culture. beta-Glucuronidase, beta-glucosidase, and nitrate reductase activities were induced within the culture by their respective substrates. Bile acids influenced microbial activity in vitro, with cholic acid inducing beta-glucosidase, azoreductase, and beta-glucuronidase activities and decreasing nitrate reductase activity. Chenodeoxycholic acid increased beta-glucosidase and beta-glucuronidase activities and decreased azoreductase, nitrate reductase, and nitroreductase activities in vitro. These studies demonstrate that the rat hindgut microflora may be successfully cultured in vitro and suggest control mechanisms that regulate the metabolic activity of these organisms in vivo.     

Assay and subcellular localization of the arylsulphatases in rat brain

—The properties and subcellular localization of type I (nitrophenyl) and type II (nitrocatechol) arylsulphatases were investigated in brain tissue of the rat, and optimal assay conditions were established. Sulphate, phosphate and sulphite ions inhibited the nitrocatechol sulphatases; nitrophenyl sulphatase was inhibited only by sulphite. The presence of latent enzyme activity was demonstrated for the nitrocatechol sulphatases, beta-glucuronidase, and beta-glycerophosphatase in rat and mouse brain homogenates. These hydrolases were highly sensitive to mechanical and osmotic damage; and Triton X-100 was very effective in releasing their latent (bound) activities, a finding suggestive of a lysosomal localization. Activity of nitrophenyl sulphatase was unaffected by osmotic changes or Triton X-100, characteristics suggesting a membranous association for this enzyme. Total activity of nitrophenyl sulphatase was approximately twice as great in canine gray matter as in canine white matter; the converse obtained for beta-glucuronidase activity. Values for total enzymic activity of the nitrocatechol sulphatases in canine white and gray matter were similar. Fractionation of homogenates from rat brain by differential centrifugations and separation of crude mitochondrial fractions by sucrose density gradient centrifugations revealed the following: (1) most of the nitrocatechol sulphatase activity (93 per cent) and all of the nitrophenyl sulphatase activity were sedimentable; (2) crude mitochondrial fractions exhibited the highest relative specific activity (RSA = 1·38) for the nitrocatechol sulphatases, whereas microsomal fractions displayed the highest RSA for nitrophenyl sulphatase (1·89); (3) the lightest fraction (A + B) and the densest fraction (E) from the sucrose density gradient contained most of the activity for both the type I and type II arylsulphatases, whereas the RSA of cytochrome oxidase was greatest in the intermediate density regions (fractions C and D); (4) the highest RSA for beta-glucuronidase and beta-glycerophosphatase occurred in gradient fraction C; (5) appreciable activity of beta-glycerophosphatase was found in a nerve ending fraction (M₃). It is suggested that the hydrolases in heterogeneous tissue like brain might be associated with lysosomal particles of differing enzyme compositions and varying populations, and that the data on distribution lend credence to the concept of bimodal and possible trimodal particle affinity for the hydrolases of brain tissues.     

Metabolic activity and enzyme induction in rat fecal microflora maintained in continuous culture.

The enzyme activity of the rat hindgut microflora maintained in an anaerobic two-stage continuous culture was compared with that of rat cecal contents. A qualitative comparison (API ZYM) showed a high degree of similarity between the two populations. Quantitative determinations showed that azoreductase, beta-glucosidase, nitrate reductase, and nitroreductase activities were comparable, and that beta-glucuronidase activity was very low in the culture. beta-Glucuronidase, beta-glucosidase, and nitrate reductase activities were induced within the culture by their respective substrates. Bile acids influenced microbial activity in vitro, with cholic acid inducing beta-glucosidase, azoreductase, and beta-glucuronidase activities and decreasing nitrate reductase activity. Chenodeoxycholic acid increased beta-glucosidase and beta-glucuronidase activities and decreased azoreductase, nitrate reductase, and nitroreductase activities in vitro. These studies demonstrate that the rat hindgut microflora may be successfully cultured in vitro and suggest control mechanisms that regulate the metabolic activity of these organisms in vivo.     

Systemic and central nervous system correction of lysosomal storage in mucopolysaccharidosis type VII mice

Mucopolysaccharidosis (MPS) type VII patients lack functional beta-glucuronidase, leading to systemic and central nervous system dysfunction. In this study we tested whether recombinant adenovirus that encodes beta-glucuronidase (Adbetagluc), delivered intravenously and into the brain parenchyma of MPS type VII mice, could provide long-term transgene expression and correction of lysosomal distension. We also tested whether systemic treatment with the immunosuppressive anti-CD40 ligand antibody, MR-1, affected transgene expression. We found substantial plasma beta-glucuronidase activity for over 9 weeks after gene transfer in the MR-1- treated group, with subsequent decline in activity corresponding to a delayed anti-beta-glucuronidase antibody response. At 16 weeks, near wild-type amounts of beta-glucuronidase activity and striking reduction of lysosomal pathology were detected in livers from mice that had received either MR-1 cotreatment or control antibody. In the lung and kidney, beta-glucuronidase activity was markedly higher for the MR-1-treated group. beta-Glucuronidase activity in the brain persisted independently of MR-1 treatment. Activity was intense in the injected hemisphere and was also evident in the noninjected cortex and striatum, with dramatic improvements in storage deposits in areas of both hemispheres. These results indicate that prolonged enzyme expression from transgenes delivered to deficient liver and brain can mediate pervasive correction and illustrate the potential for gene therapy of MPS and other lysosomal storage diseases.     

Changes in the rate of production of superoxide anion in tails of Rana catesbeiana tadpoles during spontaneous and triiodothyronine-induced metamorphosis

1. The time course of changes in the rate of production of superoxide anion (O2) was compared with that of beta-glucuronidase in tails of Rana catesbeiana tadpoles during spontaneous and triiodothyronine (T3)-induced metamorphosis. 2. Superoxide production increases in tadpole tail tissue undergoing regression in vivo during spontaneous metamorphosis and in response to T3 treatment. 3. The specific activity of beta-glucuronidase rises three-fold relative to control levels in tadpoles treated with T3 in vivo. 4. The time of onset of the rise in beta-glucuronidase activity precedes the onset of tissue regression and the onset of the increase in superoxide production precedes the rise in beta-glucuronidase activity.     

GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.

We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.     

Clinical use of the embryonic brain tissue grafts in epilepsy

Brain and spinal nerve tissue grafts of 5- to 10-week human embryos obtained during legal abortions were transplanted in the last 2.5 years to 15 patients with epilepsy and other paroxysmal disorders. Different forms of epilepsy were found in these patients. The latter were examined to determine their somatic, neurological, and psychological status. This made it possible to divide the patients into groups. Embryonic nerve tissue transplantation (ENTT) was found to be among efficient ways of treatment of patients with different forms of epilepsy. The positive results differed by degree and attested to the marked recovery activity of the impaired central nervous system (CNS). In patients with epilepsy resistant to medical treatment, embryonic nerve tissue implantation was found to cause a stable remission lasting from eight months to 2.5 years in 12 patients and to significantly decrease the frequency of seizures in three patients. Improvement of cognitive functions was observed in all cases. The changes in EEG were always positive, sometimes lagging behind the clinical effect, though more often progressing parallel to it. The possible biological mechanisms causing the above positive effect are discussed.     

Effect of vanadate on (Na+-K+)-stimulated ATPase activity in the brain of rats of different ages

Experiments were carried out on infant rats aged five days and on adult rats (of both sexes) to investigate vanadate inhibition of (Na+-K+)ATPase activity in various parts of the brain. Vanadate was administered in 10(-5), 10(-7), 10(-8), 10(-9) and 10(-10) mol/l concentration. The enzyme activity and the effect of vanadate were studied in the tissue of the cerebral cortex, subcortical formations and the medulla oblongata. It was demonstrated that an inhibitory effect of vanadate on ouabain-sensitive ATPase could be determined in the brain of very young rats, i.e. in the immature nervous tissue. It was further demonstrated that the inhibitory effect of vanadate (in low concentrations) was significantly more potent in the nervous tissue of adult rats than in the CNS tissue of 5-day-old animals. Lastly, attention is drawn to certain differences in the sensitivity of ouabain-sensitive ATPase to the action of vanadate indifferent parts of the CNS in both the given age groups.     

beta-Glucuronidase in the bull seminal plasma and reproductive organs

The biochemical distribution of beta-glucuronidase activity was studied in different reproductive organs, seminal plasma and spermatozoa of the bull. The highest specific activity was found in the epididymis, where the activity seemed to be mostly in nonsecretory and only partly in secretory form. A molecular weight of 340 X 10(3) to 360 X 10(3) was recorded for beta-glucuronidase in the bull seminal plasma and different reproductive organs with gel filtration on Sepharose 6B. In chromatofocusing four activity areas (CF-1 to CF-4) were usually obtained for beta-glucuronidase in the bull seminal plasma. The major peak CF-2 (also in the different reproductive organs) had a pI value of 5.6-5.3 and the two minor activity areas CF-1 and CF-3 had pI values of 6.0-5.8 and 5.2-4.5, respectively. Peak CF-4 eluted with a NaCl gradient after the Polybuffer elution and possibly represents an enzyme form incompletely detached from negatively charged cellular material. Isoelectric focusing on polyacrylamide gel confirmed the heterogeneity of beta-glucuronidase, since several activity bands were detected in the secretion of the different parts of the epididymis. beta-Glucuronidase activities CF-1, CF-2 and CF-3 had similar pH activity profiles (pH optimum around pH 3.0-4.0) and response to thermal inactivation at 50 degrees C. The multiple beta-glucuronidase activities of the bull seminal plasma are proposed to derive mainly from the secretion of the cauda epididymidis.     

Effect of hydrocortisone and dopamine on activity of beta-glucuronidase in lung parenchyma during experimental septic shock]

An effect of hydrocortisone and dopamine on beta-glucuronidase activity in the lung tissue in experimental septic shock was analysed. The study involved 30 mongrel dogs. Septic shock was induced with i.v. E. coli endotoxin. Total and free activity of beta-glucuronidase was determined with Talalay et al. technique in pulmonary parenchyma homogenate, mitochondrial-lysosomal fraction and supernatant. It was found, that hydrocortisone and dopamine given together increase the stability of the pulmonary lysosomes indicating a value of such management of the septic shock in man.     

Characterization of the defective beta-glucuronidase activity in canine mucopolysaccharidosis type VII

Canine mucopolysaccharidosis type VII results from deficient activity of lysosomal beta-glucuronidase. Residual enzymatic activity (0.2-1.7% of normal) was detected in tissue homogenates from affected dogs. In contrast, serum and urine from affected animals had up to 15% residual activity. To further characterize the nature of the defective enzyme, hepatic beta-glucuronidase was partially purified from normal and MPS VII dogs for determination of their physical and kinetic properties. About 65% of the total beta-glucuronidase in normal canine liver required detergent for solubilization (i.e., membrane-associated), whereas only 22% of the residual activity in canine MPS VII liver was membrane-associated. Compared to the normal hepatic enzyme, the Km towards 4-methylumbelliferyl-beta-glucuronide was markedly increased in MPS VII dogs (i.e., 0.48 versus greater than 2.5 mmol/l). In contrast, the thermo-, cryo-, and pH stability properties, as well as the pH optimum (approximately 4.6), were essentially unaffected. In addition, the canine MPS VII hepatic residual activity was unresponsive to sulfhydryl reducing reagents and divalent cations, despite the fact that incubation of normal canine beta-glucuronidase with dithiothreitol and magnesium and/or calcium enhanced the activity more than 15-fold.     

Aldehyde dehydrogenase activity in the barrier brain structures

The histochemical method was used to study the aldehyde dehydrogenase (EC 1.2.1.3.; ALDH) activity in capillaries and glial structures of different regions in the rat central nervous system (CNS). The occurrence of three metabolic barriers for aldehydes on systemic level in the CNS has been shown. They are: the barrier between blood and the nervous tissue (represented by capillary endothelium and surrounding astrocytes ALDH), that between blood and cerebrospinal fluid (ALDH in ependymocytes of vascular plexus), and that between cerebrospinal fluid and nervous tissue (ALDH of ependymocytes covering brain cavities). On the single microregions level a similar barrier is between interstitial fluid and neurons (ALDH of satellite oligodendrocytes).     

Oxygen toxicity in the nervous tissue: Comparison of the antioxidant defense of rat brain and sciatic nerve

Nervous tissue, central and peripheral, is, as any other, subject to variations in oxygen tension, and to the attack of different xenobiotics; these situations may promote the generation of activated oxygen species of free radical character. Results are presented showing that the content of total glutathione (GSH) in brain is 10-fold that found in the sciatic nerve of the rat (2620 vs. 261 nmol/g wet weight, respectively). The existence of a relatively high superoxide dismutase activity in peripheral nervous tissue, when compared with brain or liver, in combination with the DT-diaphorase activity detected in the sciatic nerve might represent an effective defense mechanism against quinone toxicity, as is also discussed. Nervous tissue, both central and peripheral lack Se-independent GSH peroxidase activity. Finally, the activities of other glutathione-related enzymes studied in the sciatic nerve are very low, when compared with the central nervous tissue, thus suggesting a higher susceptibility of peripheral tissue to oxidative stress damage, since GSH concentration and/or any GSH-related enzymatic activities, e.g. GSH peroxidase or glutathione disulfide reductase, might become limiting.     

Distribution of beta-glucosidase and beta-glucuronidase activity and of beta-glucuronidase gene gus in human colonic bacteria

beta-Glycosidase activities present in the human colonic microbiota act on glycosidic plant secondary compounds and xenobiotics entering the colon, with potential health implications for the human host. Information on beta-glycosidases is currently limited to relatively few species of bacteria from the human colonic ecosystem. We therefore screened 40 different bacterial strains that are representative of dominant bacterial groups from human faeces for beta-glucosidase and beta-glucuronidase activity. More than half of the low G+C% Gram-positive firmicutes harboured beta-glucosidase activity, while beta-glucuronidase activity was only found in some firmicutes within clostridial clusters XIVa and IV. Most of the Bifidobacterium spp. and Bacteroides thetaiotaomicron carried beta-glucosidase activity. A beta-glucuronidase gene belonging to family 2 glycosyl hydrolases was detected in 10 of the 40 isolates based on degenerate PCR. These included all nine isolates that gave positive assays for beta-glucuronidase activity, suggesting that the degenerate PCR could provide a useful assay for the capacity to produce beta-glucuronidase in the gut community. beta-Glucuronidase activity was induced by growth on d-glucuronic acid, or by addition of 4-nitrophenol-glucuronide, in Roseburia hominis A2-183, while beta-glucosidase activity was induced by 4-nitrophenol-glucopyranoside. Inducibility varied between strains.     

The distribution of tyrosyltubulin ligase in brain and other tissues

Tyrosyltubulin ligase is widely distributed. It is present in all brain areas, synaptic endings, all of the nonnervous tissues that were examined and in birds as well as mammals. Brain is the only tissue that contains large amounts of endogenous tyrosylatable tubulin. In nervous tissues, ligase activity is not correlated with regions of high catecholamine synthetic capacity.     

Activity of lactate dehydrogenase in the brain cortex and hippocampus of Mongolian gerbils after global ischemia and reperfusion injuries

Cerebral ischemia results in severe derangements of energy metabolism in the nervous tissue including activation of glycolytic pathway. Activity of cytosolic lactate dehydrogenase (LDH) in the specific brain structures remains unclear. The recent study was aimed at investigation into the LDH activity in the cytoplasm of both hippocampal and cortical neurons in Mongolian gerbils (Meriones unguiculatus) at different durations of reperfusion after global ischemia. Analysis showed that the activity of LDH in pyramidal neurons of various hippocampal areas and neurons of II, III and V cortical layers after 7-minute forebrain ischemia depended on both localization of the neurons and duration ofreperfusion. In general, the changes in postischemic cytosolic LDH activity include significant decrease in the LDH activity 2 days after reperfusion with varying degree of recovery on day 7 of reperfusion.     

Biochemical and structural brain alterations in female mice with cerebral pyruvate dehydrogenase deficiency

Pyruvate dehydrogenase complex (PDC) deficiency is an inborn metabolic disorder associated with a variety of neurologic abnormalities. This report describes the development and initial characterization of a novel murine model system in which PDC deficiency has been introduced specifically into the developing nervous system. The absence of liveborn male and a roughly 50% reduction in female offspring following induction of the X-linked mutation indicate that extensive deficiency of PDC in the nervous system leads to pre-natal lethality. Brain tissue from surviving females at post-natal days 15 and 35 was shown to have approximately 75% of wild-type PDC activity, suggesting that a threshold of enzyme activity exists for post-natal survival. Detailed histological analyses of brain tissue revealed structural defects such as disordered neuronal cytoarchitecture and neuropil fibers in grey matter, and reduced size of bundles and disorganization of fibers in white matter. Many of the histologic abnormalities resemble those found in human female patients who carry mutations in the X-linked ortholog. These findings demonstrate a requirement for PDC activity within the nervous system for survival in utero and suggest that impaired pyruvate metabolism in the developing brain can affect neuronal migration, axonal growth and cell-cell interactions.