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At least 253 records · Page 14Linked to original sources
PubMed1994

Different expression of beta-N-acetylhexosaminidase in mouse tissues.

The expression of genes encoding for the alpha and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase was investigated in different mouse tissues. It was found, using fluorogenic substrates, that the amounts of alpha and beta subunits were not the same in different tissues: alpha-subunit was more abundant in the brain, beta-subunit in epididymis and brain. The different isoenzyme patterns and specific activities in mouse tissues are due to the differences in the amount of hexosaminidase subunits. The mRNA, evaluated by Northern blotting analyses, revealed a greater expression of alpha-subunit in the testis and of beta-subunit in the brain and epididymis. The results indicate, therefore, that gene expression and the amount of subunits are in good relationship for beta-subunit, whereas there is no correlation for alpha-subunit.

Animals↗
PubMed1994

GM2 activator protein expression in mouse tissues.

The GM2 activator protein is an essential cofactor of hexosaminidase A in the degradation of GM2 ganglioside and it is responsible for the variant AB of GM2 gangliosidosis in man. In this study GM2 activator protein and its mRNA were determined in different mouse tissues. It was found that this protein is expressed mostly in the spleen and testis followed by brain and kidney which represent the main source in man. It is also interesting that in mouse testis there is a higher expression of the alpha subunit of hexosaminidase, thus suggesting a relationship between alpha subunit and GM2 activator protein. Furthermore the results indicate that the expression of GM2 activator protein is regulated, at least in part, at the transcriptional level.

Animals↗
PubMed1996

Nitric oxide relaxes rabbit corpus cavernosum smooth muscle via a potassium-conductive pathway.

We tested the hypothesis that acetylcholine-induced relaxation in cavernosal tissue is the result of nitric oxide production that alters K+ conductance. In the organ bath, acetylcholine- and sodium nitroprusside-induced relaxation of corpus cavernosum were significantly attenuated by tetraethylammonium. Basal [K+]i in cavernosal smooth muscle cells was 102 +/- 11 mM using a K+-sensitive fluorescent dye. Acetylcholine produced a decrease in [K+]i to 74 +/- 10 (n > 4, P < 0.05). Tetraethylammonium pretreatment blunted the acetylcholine- and sodium nitroprusside-induced decrease in [K+]i by 82%, (n > 5, P < 0.001), respectively. L-NMMA blunted the acetylcholine- and sodium nitroprusside-induced fall in [K+]i by 93% and 83% (n > 4, P < 0.001), respectively. These data suggest that acetylcholine-mediated cavernosal smooth muscle cells relaxation occurs through nitric oxide release with activation of K+ conductance.

Acetylcholine↗
PubMed1997

Enhancement of catalytic activity by gene truncation: activation of L-aspartase from Escherichia coli.

Aspartase from Escherichia coli is activated by proteolysis at the carboxy-terminal. A systematic study has been undertaken with the goal of identifying the amino acids in this region that influence the catalytic activity of aspartase. Stop codons have been introduced at various positions to prematurely truncate the aspA gene that encodes for aspartase by sequentially eliminating each of the polar and charged amino acids in this region. The affinity of the enzyme for its substrate aspartic acid decreases systematically as each functionally significant amino acid is eliminated. However, enhanced catalytic activity (up to 2.5 times the kcat for native aspartase) is observed for those truncation mutants that end in a positively charged carboxy-terminal amino acid. The precise position of the proteolytic activation of aspartase has been defined, and this covalent activation has been shown to be independent of the allosteric activation of aspartase that is also observed.

Allosteric Regulation↗
PubMed1998

Detection of benzodiazepine receptor ligands in small libraries of flavone derivatives synthesized by solution phase combinatorial chemistry.

Solution phase combinatorial synthesis of flavone derivatives and evaluation of their affinity for the central benzodiazepine receptors is described. The libraries preparation is simple and provides a convenient method for rapid compound generation and screening. Thirty one new compounds were obtained of which the most promising, as high affinity benzodiazepine receptor ligands, were 6-bromo-3'-fluoroflavone; 6,3'-dichloroflavone; 6-bromo-3'-chloroflavone and 6-chloro-3'-bromoflavone.

Animals↗
PubMed1999

6-Methyl-3'-bromoflavone, a high-affinity ligand for the benzodiazepine binding site of the GABA(A) receptor with some antagonistic properties.

6-Methyl-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the GABA(A) receptor (BDZ-bs) with Ki values between 10 and 50 nM in different brain regions. The GABA ratio of 1.03 for [(3)H]flunitrazepam binding to cerebral cortex, 0.76 for cerebellum, 0.7 for hippocampus, 0.7 for striatum, and 0.8 for spinal cord indicated an antagonistic or weak inverse agonistic profile of 6-methyl-3'-bromoflavone on BDZ-bs. Unlike classical benzodiazepines, it had no anticonvulsant, anxiolytic, myorelaxant, sedative, amnestic or motor incoordination effects. However, it antagonized the muscle relaxant, the sedative effect, and the changes in locomotor activity induced by diazepam. Taken together, these findings suggest that 6-methyl-3'-bromoflavone has an antagonistic profile on the BDZ-bs.

Animals↗
PubMed1999

Phosphatidylcholine-dependent phospholipase C in rat liver chromatin.

Phosphatidylcholine-dependent phospholipase C is an enzyme which hydrolyses phosphatidylcholine giving origin to diacylglicerol and phosphorylcholine. Diacylglicerol has many effect and activates also protein kinase C. Since the presence of protein kinase C in the hepatocyte nuclei and the existence of a phospholipidic fraction in the chromatin have been demonstrated, we investigated if phosphatidylcholine-dependent phospholipase C could be present in the nuclei. The results obtained have shown the presence of this enzyme in the chromatin fraction which differs with respect to that of nuclear membrane in pH and Km. The activity has been also evaluated during liver regeneration. In the chromatin an increase of activity has been shown 12 h and 30 h after hepatectomy, i.e. at the beginning of hepatocyte S-phase. No similar behaviour has been observed in the nuclear membrane. It has been suggested that diacylglicerol, produced by the hydrolysis of chromatin phosphatidylcholine, may have a role in initiating DNA synthesis through the prolonged activation of the nuclear form of protein kinase C.

Animals↗
PubMed2000

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone: new additions to the 6,3'-disubstituted flavone family of high-affinity ligands of the brain benzodiazepine binding site with agonistic properties.

6,3'-dibromoflavone and 6-nitro-3'-bromoflavone inhibited [(3)H]flunitrazepam binding to the benzodiazepine binding site of the gamma amino butyric acid receptor complex with K(i) values between 17 and 36 nM in different brain regions. Their gamma amino butyric acid ratio for [(3)H]flunitrazepam binding to cerebral cortex membranes indicated partial agonistic properties. Both compounds had similar pharmacological effects: they produced anxiolytic-like effects at low doses but did not alter locomotor activity or muscle tonicity; sedation was caused only at doses higher than 30 mg/kg in mice. These synthetic flavone derivatives join an existing family of 6,3'-disubstituted flavone compounds with high affinity for the benzodiazepine binding site and partial agonistic profiles.

Animals↗
PubMed2002

Transcriptional regulation of the human tumor suppressor p14(ARF) by E2F1, E2F2, E2F3, and Sp1-like factors.

The human ARF/INK4a locus encodes two cell cycle inhibitors, p16(INK4a) and p14(ARF), by using separate promoters. A variety of mitogenic stimuli upregulate ARF but a direct modulation at the transcriptional level has been reported only for E2F-1. We show here that the ARF promoter is strongly responsive also to E2F2 and E2F3, thus providing a strong support to their suggested role in the induction of apoptosis. Through the usage of both deletion mutants and/or site-directed mutants, we surprisingly found that none of the four putative E2F consensus sites is strictly necessary for the upregulation of ARF expression, as a minimal deletion mutant, lacking all the putative E2F binding sites, is still transactivated by E2F. Moreover, our data suggest that the ARF promoter is regulated by E2F through both direct binding to the promoter sequences and indirectly, probably by being tethered to the ARF promoter by Sp1-like factors.

3T3 Cells↗
PubMed2002

Synthesis and evaluation of alternative substrates for arginase.

Two novel carboxyl-containing arginase substrates, 4-guanidino-3-nitrobenzoic acid and 4-guanidino-2-nitrophenylacetic acid, have been synthesized and found to give enhanced catalysis and dramatically lower K(m) values relative to 1-nitro-3-guanidinobenzene, a substrate designed for use in a chromophoric arginase assay. To more efficiently mimic the natural substrate, a series of sulfur analogs of L-arginine were synthesized and kinetically characterized. The parent compound, L-thioarginine, with the bridging guanidinium nitrogen of L-arginine replaced with sulfur, functions as efficiently as the natural substrate. The desamino analog shows extremely low turnover, while the k(cat) of the descarboxy analog is only 75-fold lower than that of arginine. These results suggest that the bridging nitrogen of L-arginine is not important for either substrate binding or catalysis, while the alpha-carboxyl group facilitates substrate binding, and the alpha-amino group is necessary for efficient catalysis. Isothiourea homologs previously reported to be nitric oxide synthase inhibitors have been found to undergo a rapid non-enzymatic rearrangement to a species that is probably the true inhibitor.

Animals↗
PubMed1996

Phospholipids and nuclear RNA.

It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of phospholipids in DNA synthesis and RNA transcription.

Animals↗
PubMed1997

Choline base exchange activity in rat hepatocyte nuclei and nuclear membranes.

Previous investigations have demonstrated the presence of phospholipids as a component of chromatin; however the mechanism of their synthesis, namely if they are synthesized in the nuclei or in the cytoplasm (microsomal fraction), from where they may eventually be transported to the nucleus, has not yet been clarified. The phosphatidylcholine, for example, can be formed, albeit in a limited amount, by an interconversion reaction between bases. The aim of the present research was to ascertain the presence of the enzyme complex responsible for this reaction in hepatocyte nuclei and in isolated nuclear membrane. The incorporation of [14C]-choline in phosphatidylcholine was assayed in microsomes, hepatocyte nuclei, liver nuclei and nuclear membranes of rat liver. The reaction was Ca(2+)-dependent and the specific activity was higher in microsomes but was present, albeit at a low level, also in nuclei and in nuclear membranes. Possible contaminations were excluded by specific microsomal markers and by the reaction time course. In fact, the nuclear reaction reached the maximum level slowly with respect to microsomes. Since the phosphatidylcholine extracted from the nuclei show an enrichment in unsaturated fatty acids of monoenoic fraction, such as oleic acid, the difference in reaction kinetics has been tentatively explained as due to the phosphatidylcholine fatty acid content. The presence of this base exchange enzyme complex may allow a fast change in chromatin phospholipid composition.

Animals↗
PubMed1995

Leishmania amazonensis: multidrug resistance in vinblastine-resistant promastigotes is associated with rhodamine 123 efflux, DNA amplification, and RNA overexpression of a Leishmania mdr1 gene.

A vinblastine-resistant Leishmania amazonensis cell line (RV100) which exhibits cross-resistance to the unrelated drug adriamycin, and thus is considered to be multidrug resistant (MDR), was isolated after stepwise selection with increasing concentrations of vinblastine. This phenotype was partially reverted by the calcium channel antagonist verapamil. Drug transport studies using the hydrophobic fluorescent dye rhodamine 123 demonstrated that the MDR cell line has a reduced dye accumulation due to an increased efflux. Furthermore, DNA and RNA hybridization studies demonstrated that a gene (lamdr1), homologous to ldmdr1 and lemdr1, was overexpressed and amplified within 27 kb extrachromosomal DNA circles (V-circles) in these cells. An independent cell line, RA5000, which was selected for resistance to adriamycin and was not cross-resistant to vinblastine, accumulated normal levels of rhodamine 123 and did not contain amplified DNA or overexpressed RNA of mdr-related sequences.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Crystallization and preliminary X-ray studies of L-aspartase from Escherichia coli.

Single crystals of L-aspartate ammonia-lyase (L-aspartase) from Escherichia coli have been obtained by microdialysis at room temperature using polyethylene glycol 3350 and sodium acetate as co-precipitants. The crystals exhibit the symmetry of space group P2(1)2(1)2 with a = 156.5 A, b = 147.6 A, c = 102.5 A and diffract at least to 2.8 A.

Aspartate Ammonia-Lyase↗
PubMed1999

Structure of aspartate-beta-semialdehyde dehydrogenase from Escherichia coli, a key enzyme in the aspartate family of amino acid biosynthesis.

Aspartate beta-semialdehyde dehydrogenase (ASADH) lies at the first branch point in an essential aspartic biosynthetic pathway found in bacteria, fungi and the higher plants. Mutations in the asd gene encoding for ASADH that produce an inactive enzyme are lethal, which suggests that ASADH may be an effective target for antibacterial, herbicidal and fungicidal agents. We have solved the crystal structure of the Escherichia coli enzyme to 2.5 A resolution using single isomorphous replacement and 3-fold non-crystallographic symmetry. Each monomer has an N-terminal nucleotide-binding domain and a dimerisation domain. The presence of an essential cysteine locates the active site in a cleft between the two domains. The functional dimer has the appearance of a butterfly, with the NADP-binding domains forming the wings and the dimerisation domain forming the body.A histidine residue is identified as a likely acid/base catalyst in the enzymic reaction. Other amino acids implicated in the enzymic activity by mutagenesis are found in the active site region and define the substrate binding pocket.

Amino Acid Sequence↗
PubMed1999

The "anxiety state" and its relation with rat models of memory and habituation.

Rats selected as "anxious", "nonanxious," or normal according to their behavior in an elevated plus maze were submitted to memory tasks and the densities of central benzodiazepine receptors in the amygdala and the hippocampus were studied. Anxious rats exibited better retention scores in the inhibitory avoidance task while nonanxious rats exibited worse retention scores in inhibitory and two-way active avoidance tasks compared to normal rats. No significant differences were detected in the retention scores for habituation to an open field. Nonanxious rats presented a lower benzodiazepine receptor density in the hippocampus but not in the amygdala compared to the other groups. These data suggest that the benzodiazepine receptors are involved in the effect of "anxiety" or emotional states on memory storage processes.

Amygdala↗
PubMed2002

Aversive experiences are associated with a rapid and transient activation of ERKs in the rat hippocampus.

The extracellular signal-regulated kinases (ERKs) family of mitogen-activated protein kinases (MAPKs) has been shown to participate in memory formation. We recently found that a hippocampal ERK/MAPK cascade is required for memory formation of an inhibitory avoidance training in rats. Here we reported that this learning task is accompanied by a rapid increase in the phosphorylation of hippocampal p44 MAPK. A single mild foot shock produced a similar effect and three consecutive foot shocks provoked the activation of both p44 and p42 MAPKs. In contrast, a brief exposure to the training box or the habituation to an open field did not alter hippocampal ERK/MAPK levels. Together, these findings indicate that aversive behavioral experiences induced a rapid and transient activation of ERK/MAPKs in the hippocampus.

Animals↗