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X Bi

Publications and source records attributed to X Bi.

53 records · Page 3Linked to original sources
PubMed1997

Mechanism of substrate recognition by the ribotoxin, alpha-sarcin.

The substrate recognition and catalytic mechanisms of alpha-sarcin were explored with kinetic method by using synthetic 25-mer RNA mimicking the alpha-sarcin/ricin loop in 23S rRNA of E. coli ribosomes. The oligomer containing deoxy-G at the site of alpha-sarcin (G14) was a potent competitive inhibitor. The RNA having deoxy-G8 however, increases the Kcat value by about five times but without significant alteration on Km. Surprisingly, the deletion of G8 makes the oligomer become a strong noncompetitive inhibitor of the enzyme. These results suggested that there are at least two sites in the RNA substrate which are recognized by alpha-sarcin, one is the G8 bulge or at around its neighbor and the other is the GAGA in the sarcin/ricin loop of the rRNA.

Base Sequence↗
PubMed1996

Increased expression of cyclin D1 in the adult rat brain following kainic acid treatment.

Recent evidence has implicated aberrant cell cycle regulation as a possible mechanism of apoptosis in non-dividing cells. We previously demonstrated increased expression of the p53 tumor suppressor gene, a prominent cell cycle regulator, in apoptotic neurons. Here we investigated the potential involvement of cyclin D1, a G1 phase cell cycle protein under p53 regulation, in kainic acid-mediated neuronal degeneration. Adult male Sprague-Dawley rats were treated systemically with kainic acid and sacrificed between 1 h and 5 days following seizure onset. Cyclin D1 expression was studied by Western blot analysis and immunohistochemistry using a rabbit polyclonal anti-cyclin D1 antibody. In untreated control rats low levels of cyclin D1 expression were detected in multiple brain regions. Between 8 and 16 h after the onset of kainic acid-induced seizures, increased cyclin D1 immunoreactivity was observed in vulnerable hippocampal pyramidal cells. Five days after seizure onset increased cyclin D1 expression was evident in reactive astrocytes. These results support a role for cyclin D1 in certain neuronal death pathways, and suggest that cyclin D1 has multiple and cell type-specific functions in the central nervous system.

Animals↗
PubMed1996

Two synaptotagmin genes, Syt1 and Syt4, are differentially regulated in adult brain and during postnatal development following kainic acid-induced seizures.

The synaptotagmins together with other vesicle proteins are thought to be essential for the docking and/or fusion of synaptic vesicles with the plasma membrane that occurs following depolarization and calcium influx in presynatic terminals. Syt4, the fourth identified member of the synaptotagmin family, is inducible in PC12 cells by depolarization and secretagogues, and in limbic regions of the adult rat brain by kainic acid-induced seizures. In the present study, we examined the time course of the seizure-induced changes in the expression of Syt4 and Syt1, both in adult animals and during the postnatal period. Syt4 was transiently induced in several structures of the adult rat brain following seizure activity with peak inductions between 4 and 8 h and overal return to control values by 30 h. No induction was observed following seizure activity in 7-day-old animals. The brain regions most sensitive to increased induction were, in decreasing order of sensitivity, hippocampal pyramidal cells dentate granule cells and piriform cortex pyramidal cells. The brain areas showing the greatest Syt4 stimulation in adults were also the areas in which Syt4 was induced by seizures earlier in development. In contrast, Syt1 mRNA was depressed in adult brains following seizure activity, particularly in the dentate granule cells. Our results suggest that the differential regulation of different synaptotagmin genes following excessive neuronal activity might participate in rapid adaptation of subsequent transmitter release.

Animals↗
PubMed1996

Regional distribution and time-course of calpain activation following kainate-induced seizure activity in adult rat brain.

Systemic injection of kainic acid (KA) in adult rat elicits a pattern of neuronal pathology which exhibits several features of human temporal lobe epilepsy. KA-induced seizure activity is accompanied by the activation of the calcium-dependent protease calpain in limbic structures. In the present study, we evaluated the spatio-temporal activation of calpain after the onset of seizure activity by immunohistochemistry using an antibody for the spectrin breakdown product (sbdp) generated by calpain-mediated spectrin proteolysis. In addition, we compared the changes in sbdp immunoreactivity with those in immunoreactivity to subunits of the Glu/AMPA receptors (GluR1 and GluR2/3). One hour after seizure onset, sbdp accumulation was observed in selected interneurons in stratum oriens and in the hilus of the dentate gyrus. By 4 h, sbdp immunoreactivity was prominent in dendritic fields of the hippocampus as well as in neurons in thalamus and piriform cortex. By 8 h, sbdp immunoreactivity had disappeared from interneurons but was localized in pyramidal cell bodies in hippocampus. Intense labeling of cell bodies and dendritic fields persisted until 5 days following KA treatment. Changes in GluR subunit immunoreactivity were mirror images of those seen for sbdp. In general, increased sbdp immunoreactivity in dendritic fields was associated with decreased GluR1 immunoreactivity. However, increased sbdp immunoreactivity in neuronal perikarya was also associated with increased GluR immunoreactivity. These results indicate that calpain activation following seizure onset exhibits a specific spatio-temporal pattern, with activation in restricted interneurons preceding widespread activation in pyramidal neurons. Calpain activation also precedes neuronal pathology and could thus represent an initial trigger for neuronal pathology. Finally, the results suggest that calpain activation produces rapid alterations in GluR subunit properties which could be involved in the hyperexcitability observed following seizure activity.

Animals↗
PubMed1996

A replicational model for DNA recombination between direct repeats.

DNA rearrangement (recombination) mediated by direct repeats is a major cause of genome instability. In Escherichia coli, direct repeats in close proximity can mediate efficient recA-independent intramolecular recombination, which produces multiple products. Using plasmid substrates, three basic forms of products have been revealed: the monomeric deletion product and two dimeric products. The frequency of recombination has been shown to be affected by structural factors such as the length of the repeat and the distance between the repeats. We show here that these factors also affect the relative abundance of each form of the product. Recombination between very short tandem repeats yields exclusively the monomeric product. Lengthening the repeats increases the abundance of the dimeric products. Increasing the distance separating the repeats sharply reduces the formation of the monomeric product. These results can be explained by a model for DNA rearrangement (recombination) involving DNA replication. We propose that misalignment of the repeats at the replication fork creates a recombinogenic intermediate that can be differentially processed to form the three basic products. The proposed sister-strand recombination mediated by direct repeats might be a general mechanism for deletion and/or amplification of repeated sequence in both prokaryotic and eukaryotic genomes.

DNA Replication↗
PubMed1996

DNA rearrangement mediated by inverted repeats.

Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement. We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli. We show that inverted repeats can mediate an efficient and recA-independent recombination event. Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrangement. Moreover, this recombination is dramatically reduced by increasing the distance separating the repeats. These results can be readily explained by a model involving reciprocal switching of the leading and lagging strands of DNA replication within the inverted repeats, which leads to the formation of a Holliday junction. Reciprocal strand switching during DNA replication might be a common mechanism for genome rearrangement associated with inverted duplication.

Antiporters↗
PubMed1996

The C-terminal domain of glutamate receptor subunit 1 is a target for calpain-mediated proteolysis.

The AMPA receptors are glutamate-gated ion channels mediating synaptic transmission at the majority of excitatory synapses in the mammalian CNS. They are composed of four subunits (GluR1-4) which exist in two alternatively spliced variants (flip and flop) and are generally considered to form pentameric receptors. The transmembrane structure of the receptors remains a matter of controversy as some data suggest a transmembrane topology consisting of five, four, or three membrane spanning regions. Some receptor properties have been shown to be regulated by phosphorylation processes as well as by the phospholipid environment. More recently, we have shown that calcium treatment of thin (10 microns) frozen-thawed brain sections resulted in profound modifications of the immunochemical properties of the AMPA receptors. More specifically, immunolabelling of the AMPA receptors with antibodies directed against the C-terminal domain of GluR1 and GluR2/3 was markedly decreased in dendritic fields following such treatment at 35 degrees C. This effect was temperature-dependent and completely blocked by inhibitors of the calcium-dependent proteases calpains, and we suggested that calpains are involved in the regulation of AMPA receptor properties. The results of the present study demonstrate that calpain activation produces a partial proteolysis in the C-terminal domain of the receptors and generates a new receptor species with an apparent molecular weight of 103,000 mol. wt. Sequence analysis of the GluR1 C-terminal domain suggests a couple of cleavage sites for calpains. These results are of particular interest considering the body of evidence implicating calpains and changes in excitatory amino acid receptors in mechanisms of synaptic plasticity as well as in neurodegenerative processes.

Blotting, Western↗
PubMed1996

Risk factors for initiation of drug use among young males in southwest China.

To identify risk factors for drug use among young males in southwest China a cross-sectional survey and a non-concurrent cohort study were conducted. Demographic, behavioral and drug-use information was collected from young males aged 18-29 years. The non-concurrent cohort included the period 1 January 1991 to 1 August 1994. A total of 1548 subjects were interviewed. The cumulative incidence increased between 1991 and 1993. Multivariate analysis identified the following significant risk factors for drug use: being divorced/widowed/separated, having been encouraged by friends/others to try drugs, smoking cigarettes, belonging to the Jingpo ethnic group, and having a family member who used drugs in 1991. More than 6 years of education was a protective factor for drug use. Drug use in the community was associated with having been encouraged by friends or others to try drugs (71%), to smoking cigarettes (50%), and to belonging to the Jingpo group (24%). The results are consistent with community based prevention approaches which should be studied carefully because of the unique cultural and epidemiological situation in China.

Adolescent↗
PubMed1996

Risk factors for intravenous drug use and sharing equipment among young male drug users in Longchuan County, south-west China.

OBJECTIVES: To identify the risk factors for intravenous drug use and sharing of equipment in Longchuan County in south-west China. METHODS: Demographic information and sexual and drug-use-related behavior between 1 January 1991 and 1 August 1994 were collected retrospectively from a cohort of young male drug users aged 18-29 years in 82 villages. RESULTS: A total of 433 drug users were identified. The cumulative incidence of intravenous drug use was 40.0% during the 3.7-year study period. The annual incidence increased from 10% in 1991 to over 30% in 1994. Risk factors for intravenous drug use among drug users, according to the multivariate model, included having had premarital/extramarital sex [odds ratio (OR), 1.5; 95% confidence interval (CI), 1.01-2.3], having a family member who used drugs in 1991 (OR, 1.8; 95% CI, 1.1-2.9), and currently not married (OR, 1.6; 95% CI, 0.98-2.7). Being Buddhist protected against intravenous drug use (OR, 0.4; 95% CI, 0.2-0.9). The population attributable fraction was 30% for not being currently married, 17% for having had premarital/extramarital sex and 14% for having a family member who used drugs. The risk factor for sharing of equipment was being of Jingpo ethnicity (OR, 5.8; 95% CI, 2.5-13.8). The average incidence of sharing equipment was 19.6% per year. The population attributable fraction for sharing equipment was 58.5% for being Jingpo. CONCLUSIONS: The incidence of intravenous drug use and sharing equipment is increasing. Therefore, it is urgent that vigorous, effective intervention programs be initiated in southern Yunnan. Unmarried, sexually promiscuous Jingpo drug users with a family history of drug use should be especially targeted. Given the problems of transport and communication in this remote area of China, intervention programs which use existing social, governmental and community networks should be implemented.

Adolescent↗
PubMed1995

Cloning the P. falciparum gene encoding PfEMP1, a malarial variant antigen and adherence receptor on the surface of parasitized human erythrocytes.

Plasmodium falciparum-infected human erythrocytes evade host immunity by expression of a cell-surface variant antigen and receptors for adherence to endothelial cells. These properties have been ascribed to P. falciparum erythrocyte membrane protein 1 (PfEMP1), an antigenically diverse malarial protein of 200-350 kDa on the surface of parasitized erythrocytes (PEs). We describe the cloning of two related PfEMP1 genes from the Malayan Camp (MC) parasite strain. Antibodies generated against recombinant protein fragments of the genes were specific for MC strain PfEMP1 protein. These antibodies reacted only with the surface of MC strain PEs and blocked adherence of these cells to CD36 but without effect on adherence to thrombospondin. Multiple forms of the PfEMP1 gene are apparent in MC parasites. The molecular basis for antigenic variation in malaria and adherence of infected erythrocytes to host cells can now be pursued.

Amino Acid Sequence↗
PubMed1995

Specific stimulation of recA-independent plasmid recombination by a DNA sequence at a distance.

Recombination between directly repeated DNA sequences can occur via both recA-dependent and recA-independent mechanisms. They are differentially affected by the length of the repeat and the distance separating the repeats, respectively. Interestingly, a 623 base-pairs long DNA sequence of the plasmid pBR322 was found to stimulate specifically recA-independent recombination between tandem repeats. Analysis of this stimulating sequence has revealed the following features. (1). It is cis-acting. (2). No specific region of it appears to be essential for the effect. Moreover, a neutral sequence of comparable size was able to substitute for the sequence in influencing recombination. (3). The sequence affects recombination between tandem repeats within the tetA but not the bla gene of pBR322. (4). The sequence exerts its effect in a position-dependent manner. (5). It changes not only the frequency but also the products of recombination. Our results provide an example that recA-independent recombination can be influenced by DNA sequences at a distance.

Antiporters↗
PubMed1995

Neuronal localization of metallothioneins in rat and human spinal cord.

The immunocytochemical localization of the metallothioneins (MT), a family of low molecular weight, heavy metal binding proteins, was investigated in rat and human spinal cord using two distinct antibodies. Polyclonal antibodies were raised to the peptide (SCKCKECKCTS), an epitope common to all human MT isoforms. The monoclonal antibody, E9 which recognizes a different epitope of the MT protein, was also used. Similar results were obtained with either antibody. Both antibodies recognized authentic MT from rabbit liver and horse kidney on Western blots. Immunoreactivity was localized primarily in the cytoplasm of motoneurons in the anterior horn of cervical and lumbar cord. However nuclear staining was present in some motoneurons in both rat and human cord. Intense nuclear, as well as cytoplasmic staining was seen in astrocytes. Capillary endothelia, ependymal cells, arachnoid and pia were also positive for MT.

Amino Acid Sequence↗
PubMed1994

Calpain-mediated regulation of AMPA receptors in adult rat brain.

Changes in AMPA receptors have been proposed to underlie changes in synaptic efficacy in hippocampus and other brain structures. Activation of calpain has also been discussed as a potential mechanism to produce lasting modifications of synaptic structure and function. We report here that preincubation of thin (10 microns) frozen rat brain sections with calcium at physiological temperature changes the immunological properties of AMPA receptors, an effect totally blocked by calpain inhibitors. Immunocytochemistry indicates that in situ calpain activation produces a decreased immunoreactivity for GluR1, and to a lesser extent for GluR2/3, in the neuropil throughout the brain and an increased immunoreactivity in cell bodies, particularly in hippocampus. Western blots of calcium-treated sections suggest that the decreased immunoreactivity for GluR subunits is due to partial proteolysis. These results strongly suggest the involvement of calpain in the regulation of glutamatergic synapses.

Animals↗
PubMed1994

Hypernegative supercoiling of the DNA template during transcription elongation in vitro.

Supercoiled plasmid DNAs with negative superhelicity several times higher than normal have been isolated from Escherichia coli topA mutants. The formation of these hypernegatively supercoiled plasmid DNAs is apparently induced by transcription. We show that hypernegatively supercoiled plasmid DNAs isolated from topA mutants contain R-loop(s). To study the mechanism of formation of hypernegatively supercoiled plasmid DNA, we have been able to reproduce hypernegatively supercoiled DNA in vitro using purified RNA polymerase and DNA gyrase. The formation of hypernegatively supercoiled plasmid DNA template in vitro is shown to require transcription elongation and is tightly linked to R-loop formation. We propose that one of the roles of topoisomerase I is to suppress R-loop formation during transcription elongation.

DNA Topoisomerases, Type I↗
PubMed1994

recA-independent and recA-dependent intramolecular plasmid recombination. Differential homology requirement and distance effect.

We show that recombination between directly repeated sequences in plasmids occurs via both recA-independent and recA-dependent mechanisms in Escherichia coli. They are differentially affected by two factors, the distance separating the homologies and the size of the homology. Recombination between tandem duplications up to 300 base-pairs shows virtually no recA dependence. Increasing the size of the duplications beyond 300 base-pairs gradually increases the recA dependence. Furthermore, insertion of a sizable DNA sequence in between the duplications, substantially increases the recA dependence. We conclude that increasing the distance separating the homologous regions preferentially inhibits the recA-independent recombination. On the other hand, shortening of the homology preferentially inhibits recA-dependent recombination. Consequently, recombination between short tandem duplications is totally recA-independent.

Base Sequence↗