Enzymes In Mouse Brain Research Articles

Protective effect of Cyperus esculentus (tiger nut) extract against scopolamine-induced memory loss and oxidative stress in mouse brain.

Objectives The juice extract of Cyperus esculentus commonly known as tiger nuts (TINUT) is widely used for its numerous health promoting effects including alleviation of symptoms associated with neurological disorders. Herein, we investigated the influence of the aqueous extract of C.esculentus on cognitive disorder and the underlying changes in acetylcholinesterase (AChE) activity and oxidative stress biomarkers in mice exposed to scopolamine. Methods C.esculentus (50-200mg/kg) or saline (10mL/kg) was given alone or with scopolamine 30min after, to male Swiss mice (6/group) daily for seven days. We evaluated the cognitive performance using Y-maze and object recognition on day seven post-treatment. Brains of the animals were afterwards processed for spectrophotometric determination of AChE activity and contents of oxidative stress biomarkers (malondialdehyde [MDA], glutathione [GSH], catalase, superoxide dismutase and nitrite). Results The extract improves cognitive function and also upturned scopolamine amnesia in mice. The extract markedly reduced brain AChE, MDA, and nitrite contents in mice injected with scopolamine (p<0.05). It also attenuated scopolamine-induced deregulated GSH contents and antioxidant enzymes in mouse brain. Conclusions The results of this study suggest that regular consumption of TINUT might offer beneficial effects in memory-related disorders.

Effects of Long-Term Supplementation with Aluminum or Selenium on the Activities of Antioxidant Enzymes in Mouse Brain and Liver

The aim of this study was to investigate the effects of aluminum (Al) or selenium (Se) on the “primary” antioxidant defense system enzymes (superoxide dismutase, catalase, and glutathione reductase) in cells of mouse brain and liver after long-term (8-week) exposure to drinking water supplemented with AlCl3 (50 mg or 100 mg Al/L in drinking water) or Na2SeO3 (0.2 mg or 0.4 mg Se/L in drinking water). Results have shown that a high dose of Se increased the activities of superoxide dismutase and catalase in mouse brain and liver. Exposure to a low dose of Se resulted in an increase in catalase activity in mouse brain, but did not show any statistically significant changes in superoxide dismutase activity in both organs. Meanwhile, the administration of both doses of Al caused no changes in activities of these enzymes in mouse brain and liver. The greatest sensitivity to the effect of Al or Se was exhibited by glutathione reductase. Exposure to both doses of Al or Se resulted in statistically significant increase in glutathione reductase activity in both brain and liver. It was concluded that 8-week exposure to Se caused a statistically significant increase in superoxide dismutase, catalase and glutathione reductase activities in mouse brain and/or liver, however, these changes were dependent on the used dose. The exposure to both Al doses caused a statistically significant increase only in glutathione reductase activity of both organs.

Dynamic Change of Hydrogen Sulfide After Traumatic Brain Injury and its Effect in Mice

Hydrogen sulfide (H2S) is a lipid-soluble, endogenously produced gaseous messenger molecule collectively known as gasotransmitter. Over the last several decades, gasotransmitters have emerged as potent cytoprotective mediators in various models of tissue and cellular injury. In this study, we performed a weight-drop traumatic brain injury (TBI) model in adult mice and investigated changes of H2S and its possible role in the pathogenesis after TBI. Expression of Cystathionine-β-synthase (CBS) mRNA as H2S-producing enzymes in mouse brain was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). From the results of RT-PCR, it was found that the expression of CBS was down-regulated in mouse brain cortex and hippocampus after brain injury. Western blot analysis revealed that CBS was present in normal mouse brain cortex and the hippocampus. It gradually decreased, reached its lowest level and then increased. Hydrogen sulfide in the cortex and hippocampus exhibited dynamic changes after brain injury, in parallel with CBS mRNA and protein expression. Moreover, pretreatment with the H2S donor (NaHS) could protect the neuron against the injury induced by TBI. Noticeably, the H2S donor NaHS could reduce TBI-induced injury assessed with lesion volume. These data suggested that H2S may have a therapeutic potential against neuron damage.

Effect of fluoxetine and pergolide on expression of nucleoside transporters and nucleic‐related enzymes in mouse brain

Nucleoside transporter (NT) and nucleic-related enzyme (NRE) play key roles in the physiology of nucleosides and the pharmacology of its analogs in mammals. In this study, we examined the effect of fluoxetine, a selective serotonin reuptake inhibitor, and pergolide, a dopamine D receptor agonist, on the expression of NTs and NREs in mouse brain. It was confirmed by the detection of corresponding mRNAs that three equilibrative nucleoside transporter (ENT1-3) isoforms, concentrative nucleoside transporter 2 (CNT2), CNT3, adenosine kinase (AK), and apyrase, but not CNT1, were expressed in brain tissue. Based on an assessment by mRNA determination, the cerebral expression of CNT2 was found to be increased by administration of fluoxetine and pergolide to mice. Furthermore, pergolide increased the expression of ENT2. However, fluoxetine and pergolide had no significant effect on the expression of mRNA for other NTs, AK, and apyrase. Therefore, we concluded that the expression of several NT isoforms, but not NREs, in mouse brain was affected by treatment with fluoxetine and pergolide.

Nrf2 transcription factor gene regulates basal transcription of mitochondrial superoxide dismutase enzyme in mouse brain

Evidence suggests that the Nrf2 transcription factor participates in the regulation of expression of genes that contain functional antioxidant responsive elements (AREs) in their promoter regions. Previous studies have shown that induction of glutathione-S- transferases (GST) and NADPH quinone reductase 1 (NQO1) by t-butylated hydroxy anisole (BHA) is impaired in the livers of Nrf2(-/-) mice. Basal expression of certain antioxidant enzymes is also lower in the livers of Nrf2 (-/-) mice. Results indicate that Nrf2 contributes to basal expression but not inducible expression of mitochondrial superoxide dismutase. SOD2 level was affected in the Nrf2(-/-) and about 2-fold lower than the Nrf2(+/+) mouse control. The dietary additives caused a small induction of SOD2 in the Nrf2(-/-) mouse brain, ethoxyquin and kahwoel palmitate each induced SOD2 marginally, while oltipraz and indole-3-carbinol caused 1.5 fold induction in the Nrf2(-/-) mouse brain. In contrast, there was no obvious effect on SOD2 in the Nrf2(+/+) mouse brain by any of the chemicals used .

Effects of cyclosporin A, FK506 and rapamycin on calcineurin phosphatase activity in mouse brain

Calcineurin is a Ca2+/calmodulin-dependent protein phosphatase expressed at high levels in brain. The immunosuppressive drugs cyclosporin A and FK506, but not rapamycin are specific inhibitors of calcineurin, the inhibitory effects of which have been elucidated in the immune system. Here by using these compounds as inhibitors, we assayed the enzyme in mouse brain after injection of 12.5 nmol cyclosporin A, FK506, or rapamycin into the left lateral ventricle of mouse brain. Data from calcineurin activity assay suggest that infusion of cyclosporin A or FK506, rather than rapamycin inhibited calcineurin activity in brain and in a substrate noncompetitive manner, which is revealed by the in vitro enzyme kinetic analysis. Cyclosporin A or FK506 injected into brains also affected the inhibitory effects of cyclosporin A or FK506 added to brain extracts on calcineurin activity. The results may be ascribed to the decreased free immunophilin in brain after infusion of corresponding immunosuppressant, or the fact that two immunophilin-immunosuppressant complexes have not completely identical interaction sites on calcineurin.

An assay method for glycogen debranching enzyme using new fluorogenic substrates and its application to detection of the enzyme in mouse brain.

An assay method for glycogen debranching enzyme involving fluorogenic dextrins as substrates was developed. Two dextrins were prepared from 6-O-alpha-D-glucosyl-alpha-cyclodextrin and glucose by taking advantage of the action of Bacillus macerans cyclodextrin glucanotransferase, and converted by pyridylamination to fluorogenic derivatives. Structural analysis of the fluorogenic dextrins by FAB-MS, partial acid hydrolysis, and glucoamylase digestion revealed that they were Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1- 4Glcalpha1-4Glc-PA (FD6) and Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1- 4Glcalpha1-4Glcalpha1-4G lc-PA (FD7). Using the glycogen debranching enzyme from rabbit muscle, FD6 and FD7 were, respectively, hydrolyzed to PA-maltopentaose and PA-maltohexaose, in addition to glucose, showing that these two fluorogenic dextrins are suitable substrates for assaying the glycogen debranching enzyme. An assay method involving the separation and quantification by HPLC of the characteristic fluorogenic products was successfully applied to determination of the distribution of the enzyme activity in mouse cerebrum.

A Pi Form of Glutathione‐S‐Transferase Is a Myelin‐and Oligodendrocyte‐Associated Enzyme in Mouse Brain

The pi form of glutathione-S-transferase (GST), previously found to be oligodendrocyte associated, has also been found in myelin. In the brains of adult mice, immunocytochemical staining for a pi form of GST was observed in myelinated fibers, as well as oligodendrocytes. In contrast, and as previously found in rats, positive immunostaining for mu forms occurred in astrocytes and not in oligodendrocytes or myelinated fibers. Double immunofluorescence staining strengthened the conclusion that pi was in oligodendrocytes and myelin in mouse brains. The results of enzyme assays performed on brain homogenates and purified myelin indicated that GST specific activities in mouse brain myelin were almost as high (0.8-fold) as those in mouse brain homogenates. Low, but reproducible, GST activities were also observed in myelin purified from rat brains, in which pi had been demonstrated in oligodendrocytes and mu in astrocytes. The pi form was also stained by the immunoblot technique in myelin purified from mouse brain. It was concluded that pi is a myelin associated, as well as oligodendrocyte associated, enzyme in mouse brain, and possibly also in rat brain. This is the first report of localization of GSTs in mouse brain and of GST in myelin.

Slow tight-binding inhibition of prolyl endopeptidase by benzyloxycarbonyl-prolyl-prolinal

Prolyl endopeptidase is a serine proteinase that specifically cleaves peptides on the carboxy side of proline residues. Wilk & Orlowski [(1983) J. Neurochem. 41, 69-75] have shown that benzyloxycarbonyl-prolyl-prolinal (Z-prolyl-prolinal) is a potent inhibitor of prolyl endopeptidase. We show that Z-prolyl-prolinal is a slow-binding inhibitor of mouse brain prolyl endopeptidase with Ki 0.35 +/- 0.05 nM. Kinetic analysis indicates that the mechanism is a simple, but slow, reversible equilibrium between free and bound enzyme (E + I in equilibrium EI) with rate constants for association (kon) and dissociation (koff) of 1.6 X 10(5) M-1.s-1 and approx. 4 X 10(-5) s-1 respectively. Slow-binding inhibition is dependent on the presence of the aldehyde group since the alcohol (Z-prolyl-prolinol) is a rapid and 50,000-fold poorer inhibitor (Ki 19 microM). Prolyl endopeptidase from human brain is also inhibited by Z-prolyl-prolinal with kinetics similar to those of the mouse brain enzyme.

Isolation of a cDNA likely to encode a novel Ca 2+-dependent/calmodulin-stimulated protein phosphatase

Complementary DNA encoding a novel protein phosphatase catalytic subunit has been isolated from a rabbit brain library. The deduced protein sequence is more similar to the major Ca 2+-dependent/calmodulin-stimulated protein phosphatase (2B) in brain (55% identity) than to protein phosphatases 1 and 2A (38–39% identity). A putative calmodulin-binding domain is present C-terminal to the catalytic domain, which closely resembles that of the mouse brain enzyme. These findings represent the first indication that at least two distinct Ca 2+-dependent/calmodulin-stimulated protein phosphatases are present in mammalian brain.

Effect of acrylamide and related compounds on glycolytic enzymes in mouse brain in vitro.

The effects of acrylamide and eight analogues on mouse brain glycolytic enzymes were studied in vitro. Most of the neurotoxic and non-neurotoxic analogues showed inhibitory effects on enolases. Their inhibitory constants, I50, for both mouse brain total enolases and purified bovine neuron specific enolase were similar for each analogue, being lowest for N,N'-methylene-bis-acrylamide and highest for methacrylamide. A neurotoxic compound, N-isopropylacrylamide, and a non-neurotoxic compound, N,N'-methylene-bis-acrylamide, showed non-competitive inhibition towards mouse brain total enolase. After preincubation of anologues in vitro for 90 min with hepatic S-9 fraction prepared from normal mice, their inhibitory potency towards brain total enolase was also seen. When the S-9 fraction was prepared from mice pretreated with phenobarbital, the inhibitory potency was not different from that of the control. After preincubation with S-9 fraction prepared from either diethyl maleate-, or piperonyl butoxide-pretreated mice the inhibitory potency was significantly greater than with non-pretreated control mice. Many of the test analogues also showed inhibition of glyceraldehyde 3-phosphate dehydrogenase activity, the highest inhibitory potency being seen with acrylamide, but no compounds inhibited phosphofructokinase.

Further characterization of tumor-promoter-mediated activation of protein kinase C

Tumor promoting phorbol esters are able to activate Ca 2+-sensitive, phospholipid-dependent protein kinase (protein kinase C) in a reconstituted system. Indol alkaloid teleocidin, a tumor promoter, has been found to be as potent as tumor promoters from the series of phorbol esters and mezerein in activating the mouse brain enzyme. Chemically unrelated tumor promoters such as tetrachlorodibenzo-p-dioxin, anthralin and phenobarbital are devoid of effect. Diacylglycerol 1,2 diolein strongly activated the enzyme whereas 1,3 diolein like 1,2 distearin were poor activators and 1,3 distearin was inactive. Although tumor-promoter-or diacylglycerol-mediated activation of protein kinase C was observed in the presence of 0.5mM EGTA, the reaction requires traces of Ca 2+. Tumor promoters did not prevent inhibitory action of antipsychotic phenothiazines and local anesthetics but appear to increase IC 50 of these drugs.

A coupled assay for histidine decarboxylase: in vivo turnover of this enzyme in mouse brain.

A sensitive coupled assay for histidine decarboxylase has been developed. This method involved conversion of [3H]histidine into [3H]histamine by the enzyme sample, with methylation of this product in situ, catalysed by the enzyme histamine N-methyltransferase, to yield [3H]N-tele-methylhistamine. The radioactive product was separated from the substrate by (i) extraction into chloroform, (ii) ion-exchange chromatography and (iii) liquid cation-exchange extraction. The "no tissue" assay blank comprised 0.0007% of the substrate radioactivity. Sample material with a histidine decarboxylase activity of as little as 0.14 fmol/min/ml (measured at 1 microM histidine) gave double the blank value. More than 50 assays could be performed in one day. This assay was used to determine the in vivo changes in mouse brain histidine decarboxylase activity following irreversible inhibition with (+) alpha-fluoromethylhistidine (alpha-FMH). From the time course of recovery of enzyme activity the half-life of histidine decarboxylase in vivo was calculated to be 53 h.

Fatty acid activation in the brains of neurologically mutant mice

1. 1. Palmitoyl-CoA synthetase activity was assayed in subfractions of control and Quaking, Jimpy, Shiverer and Trembler mouse brain. 2. 2. Mouse brain palmitoyl-CoA synthetase activity is not altered during myelination. 3. 3. Mouse brain enzyme activity (homogenate 1.5 ± 0.3 nmol palmitoyl carnitine/min/per mg protein crude mitochondria 0.6 ± 0.1 nmol/min/per mg protein and microsomes 1.9 ± 0.3 nmol/min/per mg protein) does not differ markedly from rat and rabbit brain activity. 4. 4. The lesions of the above mutants which affect myelination and lipid synthesis do not include the enzyme palmitoyl-CoA synthetase.

Genetic control of mitochondrial malic enzyme in mouse brain.

A mitochondrial malic enzyme (EC 1.1.1.40; L-malate: NADP/sup +/ oxidoreductase (oxalacetate-decarboxylating) has been extensively purified from mouse heart and brain. The molecular weight of the enzyme was determined in the analytical ultracentrifuge to be 250,000 to 253,000. Rabbit antiserum has been prepared against the purified heart enzyme. The antiserum is specific for the mitochondrial form of malic enzyme and cross-reacts completely with the enzyme from brain. An examination of the specific activity of the enzyme in heart and brain mitochondrial pellets of 10 inbred strains showed that all had essentially identical heart activities, whereas the brain activities fell into two distinct classes. F/sub 1/ hybrid animals between a high activity strain (C3H) and a low activity strain (C57BL/10) had an exactly intermediate level of activity. Using the antiserum, it has been shown that the tissue-specific difference in the specific activity of the enzyme in brains of C3H and C57BL/10 mice is due to the presence of more enzyme molecules in one of the strains than in the other. Genetic analysis has established that the level of expression of mitochondrial malic enzyme in brain is determined by a single locus, designated Mdr, which exists in two allelic forms. Mdr alleles havemore » no effect on the expression of the enzyme in heart.« less

Rat and mouse brain histamine N-methyltransferase: modulation by methylated indoleamines.

Abstract—A purification procedure for rat and mouse brain histamine N‐methyltransferase (HMT, EC 2.1.1.8) is described which achieves the preparation of 87‐fold purified rat brain and 166‐fold purified mouse brain enzyme. The purified HMT (MW 29,000) is inhibited by a number of physiologically and pharmacologically active amines, among them several methylated indoleamines, at concentrations above 5 ± 10‐6M. At concentrations below 1 ± 10‐7M, most of the methylated indoleamines stimulate HMT, provided histamine is maintained at, or close to, its optimal concentration as an HMT substrate, namely 1 ± 10‐5M. A study of the nature of the inhibitory process revealed a non‐competitive inhibition of HMT by dopamine as against a competitive inhibition of the enzyme by most methylated indoleamines. Increasing the concentration of histamine beyond the optimal value, i.e. to inhibitory levels, resulted in less stimulation. The findings support the notion that MSO elicits the formation in selected brain cells of supranormal amounts of several methylated indoleamines which are able to stimulate HMT (and possibly other methyltransferases, see Salaset al., 1977), thereby causing the depletion of the cerebral levels of S‐adenosyl‐L‐methionine, reported previously (Schatz &amp; Sellinger, 1975b).

The [formula omitted] inhibition of gaba-transaminase by gabaculine

The naturally occurring amino acid gabaculine ((−)-5-amino-1,3 cyclohexadiene carboxylic acid) is a potent irreversible inhibitor of mouse brain γ-aminobutyric acid (GABA)-α-ketoglutaric acid transaminase. When administered I.P. gabaculine, irreversibly inhibits the mouse brain enzyme in a time dependent fashion. Concomitant with this inhibition is a rise in endogenous brain GABA levels. Administration of gabaculine at a concentration of 100 mg/kg mouse leads to the complete inhibition of the enzyme after 4 hrs. Brain levels of GABA continually rise after the administration of the drug. After 20 hrs they are 15–20 times higher than levels in the untreated animals.

Analysis of the forms of acetylcholinesterase from adult mouse brain.

The solubilization of 80% of the acetylcholinesterase activity of mouse brain was performed by repeated 2h incubations of homogenates at 37 degrees C in an aqueous medium. Analysis of the soluble extract by gel filtration on Sephadex G-200 showed that up to 80% of the enzyme activity was eluted in a peak which was estimated to consist of molecules of about 74000mol.wt. This peak was called the monomer form of the enzyme. After 3 days at 4 degrees C, the soluble extract was re-analysed and was eluted from the column in four peaks of about 74000, 155000, 360000 and 720000 mol.wt. Since the total activity of the enzyme in these peaks was the same as that in the predominantly monomer elution profile of fresh enzyme, we concluded that the monomer had aggregated, possibly into dimers, tetramers and octomers. Extracts of the enzyme were analysed by polyacrylamide-gel electrophoresis and the resulting multiple bands of enzyme activity on gels were shown to separate according to their molecular sizes, that is by molecular sieving. All these forms had similar susceptibilities to the inhibitors eserine, tetra-isopropyl pyrophosphoramide and compound BW 284c51 [1,5-bis-(4-allyldimethylammoniumphenyl)pentan-3-one dibromide]. Thus the forms of the enzyme in mouse brain which can be detected by gel filtration and polyacrylamide-gel electrophoresis may all be related to a single low-molecular-weight form which aggregates during storage. This supports similar suggestions made for the enzyme in other locations.

Some immunochemical properties and species specificity of GABA-α-ketoglutarate transaminase from mouse brain

Antibody against a purified preparation of GABA-transaminase from mouse brain was produced in rabbits after injection of 15, 30 or 600 μg of the antigen. The antibody was used to study the species specificity of this enzyme. Brain GABA-transminase was prepared from the following species: human, calf, mouse, rat, guinea pig, rabbit, quail, pigeon, frog and trout. In double diffusion tests the antibody was found to cross-react with the enzyme from human, calf, mouse, rat, guinea pig, rabbit and frog. An excess of antibody inhibited the enzyme from mouse brain to an extent of 55% and the same result was obtained with the enzyme from rat, guinea pig and rabbit. The enzymes from calf and human were inhibited 30% and 10%, respectively. Microcomplement fixation tests showed that only the enzyme from rat brain may be very similar to the mouse brain enzyme in terms of protein structure. Accordingly, this enzyme seems to be highly species specific.

The effect of ?-amanitin on passive and active avoidance acquisition in mice

α-Amanitin, a specific and potent inhibitor of form II DNA-dependent RNA polymerase, produced greater than 98% inhibition of the enzyme in mouse brain within 2 h of intracerebroventricular (icv.) injection. Mice were given one trial passive avoidance training and retested on the task 4 h later. Mice treated with α-amanitin 2 h before training or immediately after training demonstrated a retention deficit when compared to non-injected or saline injected controls. Active avoidance was trained for 1 h using a Sidman schedule with a drumturning response. Performance during the last 15 min of training was compared to performance in the first 15 min of a retesting session, 4 h after training. α-Amanitin, 2 h prior to training reduced the number of responses, per cent escapes and per cent avoidances in the retesting session. Post-training injection of α-amanitin significantly reduced the number of responses and per cent avoidances. Rotarod and spontaneous motor activity were not affected by α-amanitin. Whole body temperature was slightly and transiently reduced in icv. administration of α-amanitin.