Biomedical subjects

C H Kim

Publications and source records attributed to C H Kim.

At least 109 records · Page 6Linked to original sources

PubMed2000

Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons.

HuC encodes an RNA binding protein homologous to Drosophila elav that serves as an excellent early marker for differentiating neurons. We have characterized the promoter of the zebrafish HuC gene by examining the ability of 5'-upstream fragments to drive expression of green fluorescent protein (GFP) in live embryos. We determined that 2.8 kb of the 5'-flanking sequence is sufficient to restrict GFP gene expression to neurons. The core promoter spans 251 base pairs and contains a CCAAT box and one SP1 sequence but no TATA box is present near the transcription start site. A putative MyT1 binding site and at least 17 E-box sequences are necessary to maintain the neuronal specificity of HuC expression. Interestingly, sequential removal of the putative MyT1 binding site and 14 distal E boxes does not appear to abolish neuronal expression; rather, it leads to a progressive expansion of GFP expression into muscle cells. Further removal of the three proximal E boxes eliminates neuronal and muscle specificity of GFP expression and leads to ubiquitous expression of GFP in the whole body. Identification of key components of the HuC promoter has led to the establishment of a stable zebrafish transgenic line (HuC-GFP) in which GFP is expressed specifically in neurons. We crossed mind bomb (mib) fish with this line to visualize their neurogenic phenotype in live mib(-/-) mutant embryos. This cross illustrates how HuC-GFP fish could be used in the future to identify and analyze zebrafish mutants with an aberrant pattern of early neurons.

Animals↗

PubMed2000

Repressor activity of Headless/Tcf3 is essential for vertebrate head formation.

The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wnt antagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wnt signalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. Here we describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. We provide genetic evidence that a component of the Wnt signalling pathway is essential in vertebrate head formation and patterning.

Animals↗

PubMed2000

Co-injection of restriction enzyme with foreign DNA into the pronucleus for elevating production efficiencies of transgenic animals.

The microinjection method for production of transgenic farm animals requires specialized techniques and results in intolerably low production efficiencies. We investigated whether or not co-injection of foreign DNA constructs with restriction endonuclease into the pronucleus of mouse zygotes would improve the integration frequencies of foreign DNA into the host genome. Two kinds of DNA constructs that have no EcoRI site in their sequences were used for co-microinjection. With reference to the results of experiments in which EcoRI alone was injected at various amounts varying from 10(-9) to 10(-5) U/nucleus, the amount of 5x10(-8) U/nucleus that showed survival rate of 60.6% was used for the co-injection with DNA. Successful transgenesis of co-injected embryos was identified by DpnI-Bal31 digestion method for single embryos and by PCR method for pups born, respectively. The overall efficiency for the integration of foreign DNA in single embryos and live-born pups obtained by the co-injection procedures were 17.9% compared with 9.1% obtained by the injection of DNA alone. The results suggest that co-injection of foreign genes with restriction enzyme may elevate the integration rate of foreign genes into host genomes.

Animals↗

PubMed2000

Azotobacter vinelandii nitrogenases with substitutions in the FeMo-cofactor environment of the MoFe protein: effects of acetylene or ethylene on interactions with H+, HCN, and CN-.

Wild-type and three altered Azotobacter vinelandii nitrogenase MoFe proteins, with substitutions either at alpha-195(His) (replaced by alpha-195(Asn) or alpha-195(Gln)) or at alpha-191(Gln) (replaced by alpha-191(Lys)), were used to probe the interactions of HCN and CN(-), both of which are present in NaCN solutions at pH 7.4, with nitrogenase. The first goal was to determine how added C(2)H(2) enhances the rate of CH(4) production from HCN reduction by wild-type nitrogenase. In the absence of C(2)H(2), wild-type Mo-nitrogenase showed a declining total electron flux, which is an overall measure of all products formed, as the NaCN concentration was increased from 1 to 5 mM, whereas the rates of both CH(4) and NH(3) production increased with increasing NaCN concentration. The NH(3) production rate exceeded the CH(4) production rate up to 5 mM NaCN, at which point they became equal. The "excess NH(3)" likely arises from the two-electron reduction of HCN to CH(2)=NH, some of which is released and hydrolyzed to HCHO plus NH(3). With added C(2)H(2), the rate of CH(4) production increased but only until it equaled that of NH(3) production, which remained unchanged. In addition, total electron flux was decreased even more at each NaCN concentration by C(2)H(2). The increased CH(4) production did not arise from the added C(2)H(2). The lowered total electron flux with C(2)H(2) present would decrease the affinity of the enzyme for HCN, making it a poorer competitor for the binding site. Thus, less CH(2)=NH would be displaced, more CH(2)=NH would undergo the full six-electron reduction, and the rate of CH(4) production would be enhanced. A second goal was to gain mechanistic insight into the roles of the amino acid residues in the alpha-subunit of the MoFe protein at positions alpha-191 and alpha-195 in substrate reduction. At 5 mM NaCN and in the presence of excess wild-type Fe protein, the specific activity for CH(4) production by the alpha-195(Asn), alpha-195(Gln), and alpha-191(Lys) MoFe proteins was 59%, 159%, and 6%, respectively, of that of wild type. For the alpha-195(Asn) MoFe protein, total electron flux decreased with increasing NaCN concentration like wild type. However, the rates of both CH(4) and NH(3) production were maximal at 1 mM NaCN, and they remained unequal even at 5 mM NaCN. With the alpha-195(Gln) MoFe protein, the rates of production of both CH(4) and NH(3) were equal at all NaCN concentrations, and total electron flux was hardly affected by changing the NaCN concentration. With the alpha-191(Lys) MoFe protein, the rates of both CH(4) and NH(3) production were very low, but the rate of NH(3) production was higher, and both rates slowly increased with increasing NaCN concentration. A hypothesis, which is based on the varying apparent affinities of the altered MoFe proteins for HCN and CN(-), is advanced to explain the higher rate of NH(3) production versus the rate of CH(4) production and the effect of increasing NaCN concentration on electron flux to products. A new method for CH(3)NH(2) quantification showed that all four MoFe proteins produced CH(3)NH(2). Added CO significantly inhibited both CH(4) and NH(3) production from HCN with all MoFe proteins except for the alpha-191(Lys) MoFe protein, which still manifested its very low rate of NH(3) production but without CH(4) production. All of the MoFe proteins responded differently to the addition of C(2)H(2) to reactions containing NaCN. With the alpha-195(Asn) MoFe protein, added C(2)H(2) decreased the rates of both CH(4) and NH(3) production, but the rate of NH(3) production decreased much less. C(2)H(2) also exacerbated the inhibition of electron flux. With the alpha-195(Gln) MoFe protein, added C(2)H(2) decreased the rates of both CH(4) and NH(3) production substantially and about equally. C(2)H(2) also eliminated the slight decrease in total electron flux that was caused by NaCN. Added C(2)H(2) hardly affected the alpha-191(Lys) MoFe protein. (ABSTRACT TRUNCA

Acetylene↗

PubMed2000

A retroviral RNA kissing complex containing only two G.C base pairs.

The dimerization of viral RNA through noncovalent interactions at their 5' ends is a key step in the life cycle of retroviruses. In Moloney murine leukemia virus, three stem-loops are important in this process. One is a self-complementary tetraloop (H1), but the other two stem-loops (H2, H3) contain highly conserved GACG tetraloops that are not self-complementary sequences. Using two-dimensional NMR, we determined the structure of the H3 stem-loop. Surprisingly, it forms a stable, homodimeric kissing complex through only two intermolecular G small middle dotC base pairs. Cross-strand interactions of the adenines adjacent to the intermolecular G small middle dotC base pairs, plus unusual strong electrostatic interactions around the base pairs, contribute to the unexpected stability. This structure shows how even stem-loops without self-complementary sequences can facilitate the intermolecular recognition between two identical RNAs, and thus initiate dimerization and encapsidation of retroviral RNAs.

Adenine↗

PubMed2000

Effect of Jindangwon on streptozotocin-induced diabetes.

The inhibitory effects of the traditional herbal medicine Jindangwon (JDW) on streptozotocin (ST)-induced diabetic mellitus were studied using the ST-treated diabetic model. Glucokinase activity of pancreatic islets was severely impaired by ST treatment. However, when ST-treated islets were treated with 1 mg/ml of JDW, the enzyme activities of glucokinase and hexokinase were protected, glucose-6-phosphatase was not. When the effects of JDW on ST-induced ATP/ADP ratio of islets were assayed, JDW was effective in restoring of ATP/ADP ratio. In addition, ST decreased the enzyme activities of PDH, while JDW had a protective effect on the enzyme. ST-induced cGMP accumulation was significantly inhibited by JDW treatment. Furthermore, ST-induced nitrite formation was significantly inhibited by JDW treatment. JDW also showed the suppressed nitrite production in ST-treated pancreatic islet cells. When the islets (200/condition) were treated with ST (5 mM for 30 min), and then JDW was added to the ST-treated cells, 1.0 mg/ml of JDW showed the activated and recovered aconitase activity in pancreatic islet cells. When the effect of ST on the gene expression of pancreatic GLUT2 and glucokinase were examined, the level of GLUT2 and glucokinase mRNA in pancreatic islets was significantly decreased. However, JDW protected and improved the expression of protein and genes, indicating that JDW is effective on ST-induced inhibition of gene expression of GLUT2, glucokinase and proinsulin in islets. These results suggested that JDW is effective in this model to treat ST-induced diabetes.

Adenosine Diphosphate↗

PubMed2000

Role of lipid peroxidation and poly(ADP-ribose) polymerase activation in oxidant-induced membrane transport dysfunction in opossum kidney cells.

This study was undertaken to examine the role of lipid peroxidation and poly(ADP-ribose) polymerase (PARP) activation in H(2)O(2)-induced inhibition of Na(+)-dependent phosphate (Na(+)-Pi) uptake in opossum kidney (OK) cells. H(2)O(2) inhibited Na(+)-Pi uptake in a dose-dependent manner. H(2)O(2)-induced inhibition of Na(+)-Pi uptake was prevented by dithiothreitol and glutathione. A potent antioxidant, DPPD, had no effect on H(2)O(2) inhibition of Na(+)-Pi uptake, despite completely inhibiting lipid peroxidation induced by H(2)O(2). However, in primary cultured rabbit proximal tubular cells, the effect of H(2)O(2) on Na(+)-Pi uptake was significantly prevented by DPPD, suggesting a species difference in the role of lipid peroxidation in the inhibition of Na(+)-Pi uptake occurring with H(2)O(2). t-Butylhydroperoxide (tBHP) caused the inhibition of Na(+)-Pi uptake that was prevented by DPPD in OK cells and rabbit proximal tubular cells. The PARP inhibitor 3-aminobenzamide completely protected the inhibition of Na(+)-Pi uptake induced by H(2)O(2) but not by tBHP. H(2)O(2)-induced ATP depletion was prevented by 3-aminobenzamide but not by DPPD. tBHP-induced ATP depletion was prevented by DPPD, whereas it was not altered by 3-aminobenzamide. Effects of H(2)O(2) and tBHP on Na(+)-Pi uptake and ATP depletion were prevented by an iron chelator, deferoxamine, suggesting that the oxidants inhibit Na(+)-Pi uptake through an iron-dependent mechanism. The extent of DNA damage by tBHP was similar to that by H(2)O(2). These results indicate that the effect of H(2)O(2) on membrane transport function in OK cells is associated with PARP activation but not lipid peroxidation, whereas the effect of tBHP is associated with lipid peroxidation.

Animals↗

PubMed2000

Molecular cloning of a gene encoding the thermoactive levansucrase from Rrahnella aquatilis and its growth phase-dependent expression in Eescherichia coli.

A levansucrase gene (lsrA) from Rahnella aquatilis ATCC33071 was isolated from a genomic library and the nucleotide sequence of the lsrA structural gene was determined. lsrA is composed of 1248 bp and encodes 415 amino acid residues with a calculated molecular mass of 45.9 kDa. Although the amino acid sequence of lsrA gene showed good conservation with the sequences of reported levansucrases and of the conserved regions thought to be implicated in the enzyme activity, comparison of the deduced amino acid sequences certified the dissimilarity of the proteins from Gram-negative and Gram-positive bacteria. The lsrA gene was expressed from its own promoter in Escherichia coli in an active form. The lsrA expression in E. coli-pRL1CPR was affected by the growth phase of cells: it was repressed in the early phase of growth, but was significantly stimulated during the entrance of cells into the late phase of growth. The growth-phase-dependent fashion of lsrA expression was altered in a constitutive-like fashion by the deletion of an upstream region of lsrA (pNd137), suggesting that the growth-phase dependent expression of lsrA was mediated by the deleted upstream region.

Amino Acid Sequence↗

PubMed2000

The CC chemokine CK beta-11/MIP-3 beta/ELC/Exodus 3 mediates tumor rejection of murine breast cancer cells through NK cells.

CK beta-11 chemoattracts T cells, B cells, dendritic cells, macrophage progenitors, and NK cells and facilitates dendritic cell and T cell interactions in secondary lymphoid tissues. We hypothesized that expression of CK beta-11 in tumor cells may generate antitumor immunity through these interactions. After transduction with the retroviral vector L(CK beta 11)SN, the murine breast cancer cell line C3L5 (C3L5-CK beta 11) showed expression of retroviral mRNA by Northern analysis and production of functional CK beta-11 by chemotaxis of human NK cells to C3L5-CK beta 11 supernatant. Only 10% of mice injected with C3L5-CK beta 11 developed tumors, compared with 100% of mice injected with a transduced control C3L5 line (C3L5-G1N). Importantly, the in vitro growth characteristics of the CK beta-11-transduced cell line were unaffected, suggesting the difference in growth in vivo was a result of chemokine production. Vaccination with C3L5-CK beta 11 partially protected animals from parental C3L5 challenge. Immunodepletion with anti-asialo-GM1 or anti-CD4 during C3L5-CK beta 11 vaccination significantly reduced CK beta-11 antitumor activity compared with control and anti-CD8-treated groups. Splenocytes from NK-depleted animals transferred the acquired immunity generated with C3L5-CK beta 11 vaccination, while splenocytes from the CD4-depleted animals did not. These results indicate, for the first time, that expression of CK beta-11 in a breast cancer cell line mediates rejection of the transduced tumor through a mechanism involving NK and CD4+ cells. Furthermore, CK beta-11-transduced tumor cells generate long-term antitumor immunity that requires CD4+ cells. These studies demonstrate the potential role of CK beta-11 as an adjuvant in stimulating antitumor responses.

Animals↗

PubMed2000

Biphasic effects of dithiocarbamates on the activity of nuclear factor-kappaB.

Dithiocarbamates are well-known antioxidants and nuclear factor-kappaB (NF-kappaB) inhibitors. Recently, they have been characterized as zinc ionophores. Concentration-dependent biphasic effects of dithiocarbamates on NF-kappaB activity have been widely reported. We studied the mechanism of this phenomenon in relation to Zn(2+) influx. Two dithiocarbamates, pyrrolidine dithiocarbamate and diethyldithiocarbamate, showed concentration-dependent biphasic effects in inhibiting NF-kappaB activation in cerebral endothelial cells. These unique effects of dithiocarbamates on NF-kappaB were tightly linked to their ability to elevate intracellular Zn(2+)500 microM), dithiocarbamates started to lose their ability to promote Zn(2+) influx and to inhibit NF-kappaB activation. These results might provide insight into the appropriate use of dithiocarbamates in various disorders.

Animals↗

PubMed2000

Azotobacter vinelandii nitrogenases containing altered MoFe proteins with substitutions in the FeMo-cofactor environment: effects on the catalyzed reduction of acetylene and ethylene.

Altered MoFe proteins of Azotobacter vinelandii Mo-nitrogenase, with amino acid substitutions in the FeMo-cofactor environment, were used to probe interactions among C(2)H(2), C(2)H(4), CO, and H(2). The altered MoFe proteins used were the alpha-195(Asn) or alpha-195(Gln) MoFe proteins, which have either asparagine or glutamine substituting for alpha-histidine-195, and the alpha-191(Lys) MoFe protein, which has lysine substituting for alpha-glutamine-191. On the basis of K(m) determinations, C(2)H(2) was a particularly poor substrate for the nitrogenase containing the alpha-191(Lys) MoFe protein. Using C(2)D(2), a correlation was shown between the stereospecificity of proton addition to give the products, cis- and trans-C(2)D(2)H(2), and the propensity of nitrogenase to produce ethane. The most extensive loss of stereospecificity occurred with nitrogenases containing either the alpha-195(Asn) or the alpha-191(Lys) MoFe proteins, which also exhibited the highest rate of ethane production from C(2)H(2). These data are consistent with the presence of a common ethylenic intermediate on the enzyme, which is responsible for both ethane production and loss of proton-addition stereochemistry. C(2)H(4) was not a substrate of the nitrogenase with the alpha-191(Lys) MoFe protein and was a poor substrate of the nitrogenases incorporating either the wild-type or the alpha-195(Gln) MoFe protein, both of which had a low V(max) and high K(m) (120 kPa). Ethylene was a somewhat better substrate for the nitrogenase with the alpha-195(Asn) MoFe protein, which exhibited a K(m) of 48 kPa and a specific activity for C(2)H(6) formation from C(2)H(4) 10-fold higher than the others. Neither the wild-type nitrogenase nor the nitrogenase containing the alpha-195(Asn) MoFe protein produced cis-C(2)D(2)H(2) when turned over under trans-C(2)D(2)H(2). These results suggest that the C(2)H(4)-reduction site is affected by substitution at residue alpha-195, although whether the effect is related to the substrate-reduction site directly or is mediated through disturbance of the delivery of electrons/protons is unclear. Ethylene inhibited total electron flux, without uncoupling MgATP hydrolysis from electron transfer, to a similar extent for all four A. vinelandii nitrogenases. This observation indicates that this C(2)H(4) flux-inhibition site is remote from the C(2)H(4)-reduction site. Added CO eliminated C(2)H(4) reduction but did not fully relieve its electron-flux inhibition with all four A. vinelandii nitrogenases, supporting the suggestion that electron-flux inhibition by C(2)H(4) is not directly connected to C(2)H(4) reduction. Thus, C(2)H(4) has two binding sites, and the presence of CO affects only the site at which it binds as a substrate. When C(2)H(2) was added, it also eliminated C(2)H(6) production from C(2)H(4) and also did not relieve electron-flux inhibition fully. Thus, C(2)H(2) and C(2)H(4) are likely reduced at the same site on the MoFe protein. Two schemes are presented to integrate the results of the interactions of C(2)H(2) and C(2)H(4) with the MoFe proteins.

Acetylene↗

PubMed2000

Mx mRNA expression and RFLP analysis of rainbow trout Oncorhynchus mykiss genetic crosses selected for susceptibility or resistance to IHNV.

Three interferon-inducible Mx genes have been identified in rainbow trout Oncorhynchus mykiss and their roles in virus resistance have yet to be determined. In mice, expression of the Mx1 protein is associated with resistance to influenza virus. We report a study to determine whether there was a correlation between the expression of Mx in rainbow trout and resistance to a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV). A comparison of Mx mRNA expression was made between different families of cultured rainbow trout selected for resistance or for susceptibility to IHNV. A trout-specific Mx cDNA gene probe was used to determine whether there was a correlation between Mx mRNA expression and resistance to the lethal effects of IHNV infection. Approximately 99% of trout injected with a highly virulent strain of the fish rhabdovirus, IHNV, were able to express full length Mx mRNA at 48 h post infection. This is markedly different from the expression of truncated, non-functional Mx mRNA found in most laboratory strains of mice, and the ability of only 25% of wild mice to express functional Mx protein. A restriction fragment length polymorphism (RFLP) assay was developed to compare the Mx locus between individual fish and between rainbow trout genetic crosses bred for IHNV resistance or susceptibility. The assay was able to discriminate 7 distinct RFLP patterns in the rainbow trout crosses. One cross was identified that showed a correlation between homozygosity at the Mx locus and greater susceptibility to IHN-caused mortality.

Animals↗

PubMed2000

Reduced glutathione oxidation ratio and 8 ohdG accumulation by mild ischemic pretreatment.

A critical role of oxidative stress has been implicated in ischemic brain damage. Mild ischemic pretreatment and/or synthesis of heat shock proteins (HSPs) has been suggested to protect against oxidative brain damage. However, experimental support of this suggestion have proven to be difficult partly because sensitive indices to assess oxidative consequences of ischemic brain damage were few. In this study, we have attempted to establish biochemical assay systems to quantitate oxidative brain damage following ischemia. We produced experimental brain ischemia in the Mongolian gerbil (Meriones unguiculatus) and examined the hippocampus for ischemic brain damage. The results obtained from ischemic gerbil hippocampus demonstrated that oxidative brain damage can be quantitated by determining glutathione oxidation ratio together with the accumulation of the oxidative DNA damage product, 8-hydroxy-2'-deoxyguanosine (8 ohdG). Our results also demonstrated a role for mild ischemic pretreatment and synthesis of HSPs against oxidative brain damage. We showed that mild 2-min ischemic pretreatment reduced the degree of both glutathione oxidation ratio and 8 ohdG accumulation in gerbil hippocampus subsequent to 10 min ischemic challenge. We also showed that the accumulation of HSP70 was closely associated with the reduction of oxidative brain damage. To our knowledge, this is the first report to investigate glutathione redox states and oxidative DNA damage levels to evaluate a protective role of mild ischemic pretreatment and HSP synthesis following brain ischemia. Our data validate the previous suggestions and provide new additional data that argue for the protective role of mild ischemic pretreatment and HSP70 synthesis against oxidative brain damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2000

Structural comparison of zebrafish Elav/Hu and their differential expressions during neurogenesis.

The present communication reports the isolation and characterization of three new zebrafish elav/Hu (Kim, C.-H., Ueshima, E., Muraoka, O., Tanaka, H., Yeo, S.-Y., Huh, T.-L. and Miki, N., Zebrafish elav/HuC homologue as a very early neuronal marker. Neurosci. Lett., 216 (1996) 109-112) homologues, HuA, HuD and HuG. While HuA and HuG showed weak and ubiquitous expressions, HuD, as well as HuC, were specifically expressed in the neuronal cells. The first expression of HuD was detectable of the 10-somite stage, that is, several hours later than HuC. After 24 h of embryonic development, although HuD and HuC expressions overlapped overall, the cells expressing HuD were restricted to subsets of the HuC-positive neuronal cells in the brain and spinal cord. These differentially regulated spatial and temporal expression patterns implied distinct roles for HuC and HuD in neuronal determination and neuronal differentiation, respectively.

Amino Acid Sequence↗

PubMed2000

Novel biphasic effect of pyrrolidine dithiocarbamate on neuronal cell viability is mediated by the differential regulation of intracellular zinc and copper ion levels, NF-kappaB, and MAP kinases.

Nuclear factor kappaB (NF-kappaB) is a transcription factor involved in the expression of a wide range of genes, most of which code for proteins that play a role in immunity and inflammation. Pyrrolidine dithiocarbamate (PDTC) is a well-known inhibitor of NF-kappaB. Although its mechanism of action is conferred by its antioxidant property, other mechanisms by which PDTC can act as a prooxidant, metal chelator, and free thiol group modulator have recently been suggested. Here we report that PDTC caused a dual effect on cell viability in neuronal rat pheochromocytoma (PC12) cells, depending on its concentration. Increase of intracellular zinc and copper ion levels selectively potentiated the cytotoxic PDTC effect in a dose-dependent manner, and thiol reagents, such as glutathione and N-acetylcysteine, as well as divalent metal-chelating reagents, such as EDTA and bathocuproline disulfonic acid, blocked its cell death effect. The differential effect of PDTC on cell viability correlates well with the inhibition of NF-kappaB activities. In addition, PDTC differentially activated microtubule-associated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), but not p38, depending on its dose, and the coaddition of glutathione (GSH), other antioxidants, and metal ions also modulated their activities. Furthermore, stable Bcl-2 expression blocked the PDTC-induced cell death. These results suggest that the thiol groups and free zinc and copper ion levels are important for the novel biphasic PDTC effect on cell viability, which is associated with the differential activation of NF-kappaB and MAP kinases.

Animals↗

PubMed2000

Short-term safety and tolerability of antihypertensive agents in Korean patients: an observational study.

PURPOSE: In order to assess the short-term safety and tolerability of newly prescribed antihypertensive monotherapies in Korean patients, a prospective study was carried out. METHODS: A total of 1181 patients were observed from July 1997 to August 1999. At 2, 4, 8 and 12 weeks after initiation of antihypertensive monotherapy, the patients were evaluated for whether treatment was to be continued and the development of any adverse reactions. RESULTS: During the first 12 weeks, the percentage of subjects continuing with their initial angiotensin converting enzyme inhibitors (ACEI) (40.7%) was substantially lower than that of subjects continuing with angiotensin II receptor antagonists (ARA) (66.2%), alpha-blockers (63.0%), calcium channel blockers (CCB) (61.3%), beta-blockers (55.8%), and diuretics (53.5%), respectively (P < 0.01). The following adverse reactions were detected: cough (7.5%), headache (6.1%), dizziness (3.9%), flushing (3.7%) and impotence (2.4%). Cough, headache, and flushing were more frequent in women, and impotence and loss of libido were more frequent in men (P < 0.05). The incidence of cough associated with ACEI (27.9%) was higher than that associated with the other classes of drugs (P < 0.01). CONCLUSIONS: The tolerability of antihypertensive medication was highest in Korean patients treated with ARAs, followed by CCBs, beta-blockers, diuretics, alpha-blockers, and ACEIs. The incidences of adverse reactions were different by sex, age and drug class.

Adrenergic beta-Antagonists↗

PubMed2000

Refolding and purification of Zymomonas mobilis levansucrase produced as inclusion bodies in fed-batch culture of recombinant Escherichia coli.

Zymomonas mobilis levansucrase was overproduced by the fed-batch culture of recombinant Escherichia coli harboring a novel expression system that is constitutively expressed by the promoter from the Rahnella aquatilis levansucrase gene. Most of the levansucrase was produced as inclusion bodies in the bacterial cytoplasm, accounting for approximately 20% of the total cellular protein. Refolding after complete denaturation by high concentrations of urea or guanidine hydrochloride was not successful, resulting in large amounts of insoluble aggregates. During the development of the refolding method, it was found that direct solubilization of the inclusion bodies with Triton X-100 reactivated the enzyme, with a considerable refolding efficiency. About 65% of inclusion body levansucrase was refolded into active levansucrase in the renaturation buffer containing 4% (v/v) Triton X-100. The in vitro refolded enzyme was purified to 95% purity by single-step DEAE-Sepharose ion exchange chromatography. Triton X-100 was removed by this ion exchange chromatography.

Amino Acids↗

PubMed2000

Eligibility of antigenic-peptide-pre-loaded and fixed adhesive peripheral blood cells for induction of cytotoxic T lymphocytes from cancer patients with elevated serum levels of carcinoembryonic antigen.

The inducibility of cytotoxic T lymphocytes (CTL) that react with carcinoembryonic antigen (CEA) was tested in cancer patients with elevated (more than 5 ng/ml) serum CEA levels when antigen presentation was carried out with paraformaldehyde-fixed adhesive peripheral blood mononuclear cells (PBMC) from the patient that had been pre-loaded with CEA652(9), an HLA-A2402-restricted tumor antigenic peptide derived from CEA. By culturing fresh autologous PBMC on the fixed cell layer in medium containing interleukin-1, -2, -4 and -6. three out of eight patients developed CTL. The CTL from two of these patients killed CEA-protein-producing gastric cancer cells carrying HLA-A2402 and the cells from the remaining patient killed CEA-non-producing stomach cancer cells pre-loaded with CEA652(9). The results suggest that a single antigenic peptide on the fixed adhesive cells will allow the ex vivo induction of peptide-reactive CTL that are easier to handle and allow antigen presentation without tedious preculture of the "professional" antigen-presenting dendritic-cells.

Adult↗