Biomedical subjects

Ki-Hyeong Rhee

Publications and source records attributed to Ki-Hyeong Rhee.

8 recordsLinked to original sources

PubMed2004

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306.

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.

5-Aminolevulinate Synthetase↗

PubMed2004

Cyclic dipeptides exhibit synergistic, broad spectrum antimicrobial effects and have anti-mutagenic properties.

Cyclic dipeptides are known to have antiviral, antibiotic and antitumour properties. The aim of this study was to determine the combined effects of cyclo(L-leucyl-L-prolyl) and cyclo(L-phenylalanyl-L-prolyl) on the growth of vancomycin-resistant enterococci (VRE) and pathogenic yeasts, as well as determining their anti-mutagenic effects. This drug combination was especially effective against five VRE strains: Enterococcus faecium (K-99-38), E. faecalis (K-99-17), E. faecalis (K-99-258), E. faecium (K-01-312) and E. faecalis (K-01-511) with MIC values of 0.25-1 mg/l. It was also effective against Escherichia coli, Staphylococcus aureus, Micrococcus luteus, Candida albicans and Cryptococcus neoformans with MIC values of 0.25-0.5 mg/l. In addition, the cyclic dipeptides exerted anti-mutagenic activity against Salmonella typhimurium TA98 and TA100 strains in a Salmonella mutation assay. The number of mutant colonies of S. typhimurium strains TA98 and TA100 induced by exposure to AF-2 (0.2 microg/plate) decreased in a concentration-dependent manner in the presence of the two cyclic dipeptides (correlation 0.72 and 0.78, respectively). Here, for the first time, we report synergistic effects of the cyclic dipeptides [cyclo(L-leu-L-pro) and cyclo(L-phe-L-pro)] in inhibiting the growth of pathogenic microorganisms, as well as their anti-mutagenic effects in Salmonella strains.

Anti-Bacterial Agents↗

PubMed2003

Differential distribution of GABA(B1) and GABA(B2) receptor mRNAs in the rat brain.

Gamma-aminobutyric acid receptors (GABA(B)Rs) are G-protein coupled receptors mediating the slow-onset and prolonged synaptic actions of GABA in the central nervous system (CNS). There are two subtypes, GABA(B1)R and GABA(B2)R, that are though to form heterodimers, with the GABA(B1)R subunit essential for ligand binding and the GABA(B2)R subunit for cell surface localization. We have analyzed the distribution of GABA(B1)R and GABA(B2)R transcripts by in situ hybridization. The two transcripts were generally expressed in parallel, and the highest levels were detected in the piriform cortex, hippocampus, medial habenula, and olfactory bulb. There was moderate expression in several thalamic nuclei and the cortex area. In contrast to the distribution of GABA(B2)R mRNA, GABA(B1)R gave weak signals throughout the thalamus, piriform cortex, and field CA2 of the hippocampus, but strong signals in the septum, superior colliculus, retrosplenial cortex area, and field CA1 of the hippocampus. Thus GABA(B2)R and GABA(B1)R mRNA expression overlapped considerably, consistent with the suggestion that the two subtypes assemble to form a heterodimer. There are nevertheless differences in their expression levels that suggest at the existence of unidentified receptor subtypes.

Animals↗

PubMed2003

Inhibition of complement activation by recombinant Sh-CRIT-ed1 analogues.

Sh-CRIT-ed1 is a potent anti-complement peptide that inhibits the classical complement-activation pathway by interfering with the formation of the C3-convertase complex, C4b2a. C2 is an essential serum glycoprotein that provides the catalytic subunit of the C3 and C5 convertases of the classical pathways of complement activation. Because only in its C4-bound state is C2a capable of cleaving its physiological protein substrates C3 and C5, the interaction of Sh-CRIT-ed1 with C2 plays a decisive role of inhibition in the classical complement-activation process. However, the role of individual Sh-CRIT-ed1 amino acid residues in C2 binding is not fully understood. We constructed nine recombinant Sh-CRIT-ed1 (rSh1) analogues, substituted at conserved residues, and evaluated their anti-complement and C2-binding activities. Results from glutathione S-transferase (GST) pull-down and haemolytic assays suggested that residues 10K, 17E, 19K and 26Y are critical for the interaction of rSh1 with C2. We then constructed an improved anti-complement peptide by duplicating Sh-CRIT-ed1 C-terminal motifs (17H-26Y). This linear homodimer (rH17d) was more potent than rSh1 with respect to binding to C2 and anti-complement activity (the 50% inhibitory concentration value was approximately equal 1.2 micro m versus approximately equal 6.02 micro m for rSh1). Furthermore, rH17d showed higher anti-complement activity in vivo, providing additional evidence that this duplication is a more effective inhibitor of complement activation than rSh1. Taken together, these results identify four key residues in rSh1 and strongly suggest that rH17d is a potent inhibitor of complement activation that may have therapeutic applications.

Amino Acid Sequence↗

PubMed2002

Caffeine-induced neuronal death in neonatal rat brain and cortical cell cultures.

We examined the potential neurotoxicity of caffeine and. Intraperitoneal administration of caffeine (50 mg/kg, 3 times a day) produced neuronal death in various brain areas of neonatal rats 24 h later. Caffeine at doses > 300 microM was also neurotoxic in murine cortical cell cultures. Caffeine-induced neuronal death was accompanied by cell body shrinkage and attenuated by anti-apoptotic drugs including cycloheximide, high potassium, and growth factors. Two necrotic pathways, excitotoxicity and oxidative stress, did not mediate caffeine neurotoxicity. The pro-apoptotic protease caspase-3 was activated to mediate neuronal death following exposure to caffeine. The present findings suggest that caffeine may cause caspase-3-dependent neuronal cell apoptosis in neonatal rat as well as.

Animals↗

PubMed2002

A hot pepper cDNA encoding ascorbate peroxidase is induced during the incompatible interaction with virus and bacteria.

Capsicum annuum L. is infected by a number of viruses, including the tobacco mosaic virus (TMV). To study the defense-related genes that are induced by TMV in hot peppers, the pepper plant, which is susceptible to P1.2 but resistant to the P0 pathotype of TMV, was inoculated with TMV-P0. Differential screening isolated the genes that were specifically up- or down-regulated during the hypersensitive response (HR). The CaAPX1 cDNA clone that putatively encodes a polypeptide of cytosolic ascorbate peroxidase was selected as an up-regulated gene. It was isolated for further study. The full-length cDNA for CaAPX1, which is 972 bp long, contained the open-reading frame of 250-amino acid residues. A genomic Southern blot analysis showed that there were only limited copies of the CaAPX1 gene in the hot pepper genome. In hot pepper cv. Bugang, which is resistant to TMV-P0 and susceptible to TMV-P1.2, the CaAPX1 gene transcript was accumulated by TMV-P0, but not by TMV-P1.2 inoculation. CaAPX1 transcripts began to accumulate 24 h post-inoculation of TMV-P0, and increased gradually until 96 h. To investigate whether each transcript is induced by other stimuli, the plants were treated with various chemicals and wounding. A striking induction of the CaAPX1 transcript was observed at 2 h. It subsided 12 h after salicylic acid (SA), ethephon, and methyl jasmonate (MeJA) treatments. The response of the gene upon other pathogen infection was also examined by a bacterial pathogen (Xanthomonas campestris pv. vesicatoria race 3) inoculation. The CaAPX1 gene was induced in a hot pepper (C. annuum cv. ECW 20R) that was resistant to this bacterial pathogen, but not in a susceptible hot pepper (C. annuum cv. ECW). These results suggest the possible role(s) for the CaAPX1 gene in plant defense against viral and bacterial pathogen.

Amino Acid Sequence↗

PubMed2002

Refolding of the catalytic and hinge domains of human MT1-mMP expressed in Escherichia coli and its characterization.

The catalytic and hinge domain (Tyr112-Ile318) of the human membrane type-1 matrix metalloproteinase (MT1-MMP; MMP-14), containing hexa-histidines at the C-terminus (chMT1-MMP), was overexpressed in Escherichia coli. The expressed polypeptide was almost exclusively found in the inclusion body, and then purified by a single Ni2+-NTA agarose column chromatography after solubilization with 6 M urea. During refolding, the 26.9 kDa chMT1-MMP was processed to a 24.3 kDa intermediate form and then to a 22.2 kDa mature form. By Western blot analysis and mass spectrometry combined with N-terminal sequencing, the intermediate form was identified as a mixture of the Tyr112-Thr299 with a translation-initiating methionine and Ile114-Thr299, and that the mature form corresponds to Ile114-Pro290. These results demonstrate that the mature form was generated by successive autoproteolysis of the N- and C-terminal sites between Thr299-Thr300, Ala113-Ile114, and Pro290-Thr291 during refolding. Catalytic activity of the mature chMT1-MMP was demonstrated by a peptide cleavage assay. In addition, it has gelatinolytic activity and is able to activate proMMP-2 to the mature MMP-2. These results indicate that the refolded chMT1-MMP retains characteristics of MT1-MMP.

Base Sequence↗

PubMed2002

Isolation and characterization of Streptomyces sp KH-614 producing anti-VRE (vancomycin-resistant enterococci) antibiotics.

The actinomycete strain KH-614 was antagonistic to vancomycin-resistant enterococci (VRE). Based on the diaminopimelic acid (DAP) type, morphological and physiological characteristics examined by scanning electron microscopy (SEM), KH-614 was confirmed as belonging to the genus Streptomyces. Based on the 16S rDNA nucleotide sequences, Streptomyces sp. KH-614 was found to have a relationship with Streptomyces lydicus. The production of antibiotic from this strain was most favorable when cultured in glucose, polypeptone, yeast extract (PY) medium for 6 days at 27 degrees C. The antibiotic was identified as a cyclo(L-leucyl-L-prolyl) by comparing it with the reported spectral data including MS and NMR. Cyclo(leu-pro) was found to be active against twelve VRE strains, including E. faecium (vanA, vanB), and E. faecalis (vanA, vanB), that had been isolated over a period three years (1998-2000). Cyclo(leu-pro) was especially effective against VRE strains such as E. faecalis (K-99-34), E. faecalis (K-00-184), E. faecalis (K-00-221), and the MIC values were 12.5 microg/ml. Moreover, cyclo(leu-pro) was effective against three leukemic cell lines at concentrations below 100 microg/ml. At 100 mg/ml cyclo(leu-pro), K562, HL60, and U937 leukemic cell lines showed growth inhibition of 95, 91, and 93%, respectively. In a normal cell line, MDBK, cyclo(leu-pro) exerted 24% growth inhibition at a concentration of 100 microg/ml, and showed no inhibitory activity at concentrations below 10 microg/ml. These results indicate that cyclo(leu-pro) is a potential anti-leukemic and anti-VRE agent.

Animals↗