Biomedical subjects
Publications and source records attributed to M J Kim.
[reaction: see text] Prochiral ketoximes were asymmetrically transformed to optically active amines in the acetylated forms by coupled lipase/palladium catalysis in the presence of an acyl donor under 1 atm of hydrogen.
The relation between the quaternary structure and the substrate specificity of Thermus maltogenic amylase (ThMA) has been investigated. Sedimentation diffusion equilibrium ultracentrifugation and gel filtration analyses, in combination with the crystal structure determined recently, have demonstrated that ThMA existed in a monomer/dimer equilibrium. The truncation of ThMA by removing the N-terminal domain, which is composed of 124 amino acid residues, resulted in the complete monomerization of the enzyme (ThMADelta124) accompanied by a drastic decrease in the activity for beta-cyclodextrin (beta-CD) and a relatively smaller reduction of the activity for starch. Despite the overall low activity of ThMADelta124, the activity was higher toward starch than beta-CD, and the ratio of the specific activities toward these substrates was approximately 100 fold higher than that of wild-type ThMA. Furthermore, the addition of KCl to wild-type ThMA shifted the monomer/dimer equilibrium toward the monomer. In the presence of 1.0 M KCl, the relative activity of ThMA toward beta-CD decreased to 74%, while that for soluble starch increased to 194% compared to the activities in the absence of KCl. Thus, the ThMA monomer and dimer are both inferred to be enzymatically active but with a somewhat different substrate preference. Kinetic parameters of the wild-type and truncated enzymes also are in accordance with the changes in their specific activities. We thus provide evidence in support of a model, which shows that the relative multisubstrate specificity of ThMA is influenced by the monomer/dimer equilibrium of the enzyme.
How the 5'-terminus of the template affects RNA synthesis by viral RNA replicases is poorly understood. Using short DNA, RNA and RNA-DNA chimeric templates that can direct synthesis of replicase products, we found that DNA templates tend to direct the synthesis of RNA products that are shorter by 1 nt in comparison to RNA templates. Template-length RNA synthesis was also affected by the concentration of nucleoside triphosphates, the identity of the bases at specific positions close to the 5'-terminus and the C2'-hydroxyl of a ribose at the third nucleotide from the 5'-terminal nucleotide. Similar requirements are observed with two bromoviral replicases, but not with a recombinant RNA-dependent RNA polymerase. These results begin to define the interactions needed for the viral replicase to complete synthesis of viral RNA.
In the present study, we used immunohistochemistry to investigate the effects of transient global ischemia on pituitary adenylate cyclase activating polypeptide (PACAP) immunoreactivity in gerbil brain regions. PACAP immunoreactivity had significantly decreased in the pyramidal cells of CA1 subfield susceptible to ischemic insult at 1-4 days after transient global ischemia. On the contrary, PACAP immunoreactivity had not changed in the pyramidal cell bodies of more resistant CA3 subfield after ischemic injury. In the Purkinje cell layer, PACAP immunoreactivity had significantly decreased 1 day following transient ischemia, and had increased 2 days after ischemia. The first demonstration of the postischemic localizations of PACAP should allow us to gain a more fundamental rationale for developing methods of treating ischemic brain damage with neuroprotective peptides such as PACAP.
The present study examined the spatial localizations of voltage-gated K(+) (Kv) channels in the rat brain following transient focal ischemia, using immunohistochemistry. Increased expression of Kv1.2 was obvious in the cerebral cortex, dentate gyrus, amygdala, and hypothalamic areas at 3 days following ischemic insults. There was a significant increase in Kv1.2 immunoreactivity in several cortical regions, including cingulate cortex, infralimbic cortex, dorsal peduncular cortex and piriform cortex. On the contrary, Kv1.2 immunoreactivity had not significantly increased in the hippocampal CA1-3 regions although moderate Kv1.2 immunoreactivity was found in the cell bodies and processes of some neurons. Potentially the first demonstration of spatial changes in Kv1.2 channel expression could provide important molecular basis for altered neuronal excitability after ischemic brain injury.
5'-upstream region of the phospholipase C-beta2 gene, 810 bp, was cloned and characterized. S1 nuclease mapping and primer extension analyses revealed that a single transcriptional start site locates at 284 nucleotides upstream from the beginning of translation. The 5-upstream region lacks both TATA motif and typical initiator sequence, but retains GC-rich segment. Two putative regulatory regions, a negative region (-636/-588) and a positive region (-98/ -13) were identified in the upstream region of PLC-beta2 gene. We suggest that the transcription of PLC-beta2 may be regulated by binding of regulatory proteins to the negative and/or positive regulatory regions located in the upstream of the gene.
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In the present study, we used immunohistochemistry and Western blot analysis to determine region-specific changes in the distribution of voltage-gated calcium channels (VGCCs) in aged rat cerebellum. Age-dependent changes in the staining intensity of the alpha(1C) and alpha(1D) subunits were prominent in the Purkinje cells, whereas there was no change in the expression of the alpha(1A) and alpha(1B) subunits. In the aged rat, in particular, immunoreactivity for the alpha(1C) subunits were increased in the dendrites as well as in the cell bodies of Purkinje cells. On the other hand, decreases in immunoreactivity for alpha(1A) and alpha(1D) subunits were found in the molecular or granular layers. However, only alpha(1D) subunit immunoreactivity was decreased in the aged cerebellum membrane by Western blot analysis that, while not addressing regional specificity, further confirmed an age-related decrease in alpha(1D) subunit. These age-related changes in alpha(1D) subunit expression might reflect a gradual loss of regulation for L-type Ca(2+) channels in the senescent period. The first demonstration of age-related alterations in VGCC expression may provide useful data for future investigations on aging and neurodegenerative diseases.
The glycerol-3-phosphate (G3P) transporter, GlpT, from Escherichia coli mediates G3P and inorganic phosphate exchange across the bacterial inner membrane. It possesses 12 transmembrane alpha-helices and is a member of the Major Facilitator Superfamily. Here we report overexpression, purification, and characterization of GlpT. Extensive optimization applied to the DNA construct and cell culture has led to a protocol yielding approximately 1.8 mg of the transporter protein per liter of E. coli culture. After purification, this protein binds substrates in detergent solution, as measured by tryptophan fluorescence quenching, and its dissociation constants for G3P, glycerol-2-phosphate, and inorganic phosphate at neutral pH are 3.64, 0.34, and 9.18 microM, respectively. It also shows transport activity upon reconstitution into proteoliposomes. The phosphate efflux rate of the transporter in the presence of G3P is measured to be 29 micromol min(-1) mg(-1) at pH 7.0 and 37 degrees C, corresponding to 24 mol of phosphate s(-1) (mol of protein)(-1). In addition, the glycerol-3-phosphate transporter is monomeric and stable over a wide pH range and in the presence of a variety of detergents. This preparation of GlpT provides ideal material for biochemical, biophysical, and structural studies of the glycerol-3-phosphate transporter.
In the present study, we have investigated the spatial and temporal distribution of voltage-gated calcium channels in the gerbil model of global cerebral ischemia using immunohistochemistry. Distinct localizations of P-type (alpha(1A)), N-type (alpha(1B)), and L-type (alpha(1C) and alpha(1D)) Ca(2+) channels were observed in the hippocampus at days 1-5 after ischemic injury. However, increased expression of N-type Ca(2+) channels was detectable in brain regions vulnerable to ischemia only at days 2 and 3 after ischemic injury. The pyramidal cell bodies of CA1-3 areas and the granule cell bodies of the dentate gyrus were intensely stained at days 2 and 3 following ischemic injury. Transient changes in N-type Ca(2+) channel expression were also observed in the affected cerebral cortex and striatum at days 2 and 3 after ischemic injury. Although the present study has not addressed the multiple mechanisms contributing to the intracellular free Ca(2+) concentration ([Ca(2+)](i)) increase in the ischemic brain, the first demonstration of the transient increase in N-type Ca(2+) channels may prove useful for future investigations.
Quinolone-resistant Escherichia coli (QREC) strains are being isolated with increasing frequency. From 1993 to 1998, 40 cases of QREC bacteremia were observed in a teaching hospital; 25 episodes (63.5%) were community-acquired. The incidence of QREC bacteremia increased steadily, from 6.7% to 24.6% during 5 years, and correlated with the significantly increased use of fluoroquinolones (P = .003, r = 0.98). When the 40 QREC bacteremic patients were compared with 80 patients with bacteremia due to quinolone-susceptible E. coli, prior fluoroquinolone use was the only independent risk factor for QREC bacteremia (P = .001). A high APACHE II score was the only independent risk factor for death. The rate of multidrug resistance of QREC was much higher (60%) than that of quinolone-susceptible isolates (13.8%). Pulsed-field gel electrophoresis patterns of these isolates were diverse. Therefore, the isolates revealed little evidence of clonal spread and may have emerged in direct response to the selective pressure exerted by prior fluoroquinolone use.
[reaction: see text] Lipase-catalyzed transesterifications in ionic liquids proceeded with markedly enhanced enantioselectivity. It was observed that lipases were up to 25 times more enantioselective in ionic liquids than in conventional organic solvents.
In the present study, we have examined the expression of voltage-gated calcium channels in a rat model of transient focal ischemia using immunohistochemistry. Increased expression of class C L-type Ca2+ channels was clearly detected in reactive astrocytes in each region of the hippocampus 7 days after ischemic injury. On the contrary, class D L-type Ca2+ channels were not expressed in reactive astrocytes under these conditions. These patterns were also observed in reactive astrocytes in the affected cerebral cortex and fiber tracts. Our study showed the spatial and temporal localization of class C L-type Ca2+ channels in reactive astrocytes in ischemic rat brain, for the first time. The present studies may provide useful data for future investigations to understand the role of Ca2+ channels in reactive astrocytes following ischemia or glutamate toxicity.
The homozygous anorexia mutant (anx/anx) mice present with premature death during the third or fourth postnatal week: this phenotype is caused by a lethal mutation, anx, on chromosome 2, which has an autosomal recessive mode of inheritance. These animals also present phenotypically with decreased food intake, weight loss, and neurological deficits such as hyperactivity, body tremors, uncoordinated gait, and head weaving. In order to investigate changes in the occurrence of cell proliferation and apoptosis in the dentate gyrus of the hippocampus of anx/anx mice, 5-bromo-2'-deoxyuridine (BrdU) immunohistochemistry and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay were performed in this study. In addition, the volume of the dentate gyrus was estimated via stereological analysis. anx/anx mice showed significantly higher numbers of both BrdU- and TUNEL-positive cells in the dentate gyrus than those of the control mice. Furthermore, the volume of the dentate gyrus of anx/anx mice was significantly reduced compared to that of the control mice.
Copy-choice RNA recombination occurs during viral RNA synthesis when the viral transcription complex switches templates. We demonstrate that RNA-dependent RNA polymerase from bovine viral diarrhea virus and the replicases from three plant-infecting RNA viruses can produce easily detectable recombination products in vitro by switching templates during elongative RNA synthesis. Template sequence and/or structure, and NTP availability affected the frequency of template switch by the transcription complex. Our results provide biochemical support for copy-choice recombination and establish assays for mechanistic analyses of intermolecular RNA recombination in vitro.
The gamma-aminobutyric acid (GABA) transporter from Escherichia coli was homologously overexpressed and purified to homogeneity with a yield of 1.0 mg per liter culture. The purification procedure consists of a cobalt affinity column, proteolytic cleavage of His- and myc-tags, and size-exclusion chromatography. The purified transporter exists as a monomer in FOS-Choline 12 detergent, with a Stokes radius of 45 A for the protein-detergent complex. In detergent solution the protein binds substrates, as indicated by tryptophan fluorescence quenching. Its dissociation constants (K(d)) for GABA, muscimol and nipecotic acid are 13.8, 13.3 and 27.9 microM, respectively. This protein preparation provides ideal starting materials for future biochemical, biophysical and structural studies of the GABA transporter.
We have revealed age-related changes in the expression patterns of Kv1.1 and Kv1.2 in the rat cerebellum for the first time. In the aged rat, immunoreactivity for Kv1.1 was increased in the cell bodies of Purkinje cells, while the staining intensity was significantly decreased in the granule cells. The cell bodies of cerebellar output neurons showed strong Kv1.1 immunoreactivity in the nucleus medialis, interpositus and lateralis of the aged rat. Kv1.2 immunoreactivity was found in some interneurons with their processes in this region of the aged rat. Image analysis demonstrated that immunoreactivities for Kv1.1 and Kv1.2 were increased specifically in the cell bodies of cerebellar output neurons of the aged rat. This study may provide useful data for future investigations on the channels that cause brain diseases and age-related disorders.
Voltage-gated K(+) (Kv) channels are critical for a wide variety of processes, and play an essential role in neurons. In the present study, we have demonstrated a unique pattern of expression of the six Kv1 channel subunits in the rat cerebellum, for the first time. The greatest concentration of Kv1.2 was found in the basket cell axon plexus and terminal regions around the Purkinje cells. Relatively weak immunoreactivity for Kv1.1 was also found in this area. The somatodendritic Purkinje cell areas were intensely stained with anti-Kv1.5 antibodies. In the cerebellar nuclei, the cell bodies of cerebellar output neurons showed strong Kv1.5 and Kv1.6 immunoreactivities in the nucleus medialis, interpositus and lateralis. Interestingly, Kv1.2 immunoreactivity was found in some neurons with their processes. Our immunohistochemical results may support the notion that the formation of heteromultimeric Kv channels possibly represents an important contribution to the generation of Kv channel diversity in the brain, especially in the cerebellum.