Biomedical subjects
Publications and source records attributed to J Park.
Ketones were asymmetrically transformed to chiral acetates by one-pot processes using a lipase and an achiral ruthenium complex under 1 atm of hydrogen gas in ethyl acetate. Molecular hydrogen was also effective for the transformation of enol acetates to chiral acetates without additional acyl donors with the same catalyst system.
The expression of pro-apoptotic molecules p53 and Bax in the spinal cord of rats with experimental autoimmune encephalomyelitis (EAE) was examined. Apoptosis was confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. TUNEL (+) apoptotic cells were mainly either ED1 (+) macrophages or T-cells in the parenchyma of EAE. Western blot analysis showed that both p53 and Bax expression significantly (P<0. 01) increased in the spinal cords of EAE rats at the peak stage, and thereafter declined. An immunohistochemical study showed that inflammatory cells (notably T cells) in the parenchyma express p53 and Bax, while brain cells, including neurons and glia, were devoid of nuclear staining for these molecules. The nuclear expression of p53 largely matches apoptotic cells in the parenchyma of EAE. These findings suggest that the pro-apoptotic molecules p53 and Bax may play an important role in eliminating T cells in the parenchyma in EAE.
An enzyme-metal combo reaction has been developed for the dynamic kinetic resolution of allylic alcohols in which racemic substrates are transformed by a lipase and a ruthenium complex in the presence of an acyl donor to allylic acetates of high optical purity in over 80% yield.
We have developed a simple, sensitive, specific and reproducible stereoselective high-performance liquid chromatography technique for analytical separation of cisapride enantiomers and measurement of cisapride enantiomers in human plasma. A chiral analytical column (ChiralCel OJ) was used with a mobile phase consisting of ethanol-hexane-diethylamine (35:64.5:0.5, v/v/v). This assay method was linear over a range of concentrations (5-125 ng/ml) of each enantiomer. The limit of quantification was 5 ng/ml in human plasma for both cisapride enantiomers, while the limit of detection was 1 ng/ml. Intra- and inter-day C.V.s did not exceed 15% for all concentrations except at 12.5 ng/ml for EII (+)-cisapride, which was approximately 20 and 19%, respectively. The clinical utility of the method was demonstrated in a pharmacokinetic study of normal volunteers who received a 20 mg single oral dose of racemic cisapride. The preliminary pharmacokinetic data obtained using the method we describe here provide evidence for the first time that cisapride exhibits stereoselective disposition.
Mercury content and distribution as well as its effects on growth and oxidative stress were investigated in 30-day-old tomato seedlings (Lycopersicon esculentum Mill.). The content of Hg increased with external Hg concentrations, and was considerably higher in roots than in shoots. Among the leaves, the mature leaves accumulated more. Excess Hg suppressed biomass production of both roots and shoots and reduced chlorophyll content in leaves. Further, substantial increases of H(2)O(2) content, malondialdehyde formation, and antioxidant enzyme activities such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POX) were observed in Hg-stressed plants in comparison with controls. The results suggest that the phytotoxic effects of Hg in tomato seedlings may be achieved by an enhanced production of active oxygen species (AOS) and subsequent lipid peroxidation.
The increase in neural activity in cat visual cortex associated with eye movements has been thought to reflect a replica of the motor command signal. We examined the timing of the saccade-related increase in neural activity in cat areas 17 and 18 in relation to saccade onset and offset. The increase in activity was temporally coupled to saccade offset rather than onset both for visually guided saccades and for spontaneous saccades in the dark. Overall, it occurred 63 ms after saccade offset, and the peak was higher and sharper for data aligned at saccade offset than for onset. These results are inconsistent with the idea that saccade-related activity in cat visual cortex reflects a copy of the motor command signal.
To understand the roles of p38 mitogen-activated protein kinase (p38 MAPK) isoforms in adult mouse brain, in vivo activities and detailed expression patterns of two p38 isoforms, p38alpha and p38beta, were examined by using biochemical and immunohistochemical analyses. The result indicated that the activity of both p38alpha and p38b MAPKs in normal adult mouse brain was remarkably high, and the nuclear pool of the p38 isoforms was primarily responsible for most of the constitutive p38 MAPK activity in brain. Both p38alpha and p38beta were highly expressed in brain areas including cerebral cortex, hippocampus, cerebellum, and few nuclei of the brainstem. At the subcellular level, p38alpha was distributed in dendrites and in cytoplasmic and nuclear regions of cell body of neurons, which is in contrast to p38beta, since p38beta was preferentially expressed in nucleus of neurons. These results suggest that the p38 pathway may play an important role, not only in inflammation and neuronal cell death as previously suggested, but also in normal physiology of adult mouse brain.
The Rb protein is the product of the retinoblastoma susceptibility gene and loss of Rb function is detected in many types of human cancers. Rb plays important roles in the regulation of cell proliferation, differentiation, senescence, and apoptotic cell death. Here we show that Rb can physically interact with c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK), thereby inhibiting intracellular signals mediated by JNK/SAPK. Both in vitro binding and in vitro kinase studies suggest that a carboxyl-terminal domain of Rb containing amino acids 768-928 might be crucial for inhibiting JNK/SAPK. In comparison, Rb did not affect enzymatic activity of either extracellular signal-regulated kinase 1 or p38. Ectopically expressed Rb also abrogated the apoptotic cell death induced by ultraviolet radiation or the activation of MEKK1, an upstream kinase that can stimulate the JNK/SAPK cascade. JNK/SAPK inhibition highlights a novel function of Rb, which may provide a new mechanism by which Rb regulates cell death. JNK/SAPK is a major protein kinase that can be stimulated in response to a variety of cellular stresses. Our results, therefore, suggest that Rb, by inhibiting JNK/SAPK, may act as a negative regulator in stress-activated intracellular signaling cascades.
Two mouse genes, Mit1/Lb9 and Copg2, linked to Peg1/Mest on mouse chromosome 6, were identified to be imprinted maternally and paternally, respectively. Mit1/Lb9 encoding untranslated transcripts resides within the intron 20 of Copg2. The gene is maternally imprinted in adult mouse brain, partially imprinted in other tissues. Copg240 kb genomic region, being expressed ubiquitously in mouse tissues with a partial imprinting pattern in embryos, neonates, and adult brain in contrast to maternally imprinted human COPG2. In addition, we identified an antisense transcript of Copg2, Copg2AS, which overlaps 3'-UTRs of Copg2 and Peg1/Mest. The Copg2AS transcript is maternally imprinted in embryos, neonates, and adult tissues.
[reaction: see text] Enol acetates or ketones asymmetrically transformed to chiral acetates in high yields with high optical purities through multistep reactions catalyzed by a lipase and a ruthenium complex. 2,6-Dimethylheptan-4-ol was chosen as a suitable hydrogen donor, and 4-chlorophenyl acetate was used as an acyl donor for the conversion of ketones.
The regulatory mechanisms of the biosynthesis of in vivo poly-beta-hydroxybutyrate [PHB] and poly(3-hydroxybutyrate-3-hydroxyvalerate) [P(3HB-3HV)] of Alcaligenes eutrophus were investigated by using various transformants with enzyme activities that were modified through the transformation of cloned phbCAB genes. The biosynthesis rates of PHB and P(3HB-3HV) were controlled by beta-ketothiolase and acetoacetyl-CoA reductase, and especially by beta-ketothiolase condensing acetyl-CoA or propionyl-CoA. The contents of PHB and P(3HB-3HV) were controlled by PHB synthase, polymerizing 3-hydroxybutyrate to PHB or 3-hydroxybutyrate and 3-hydroxyvalerate to P(3HB-3HV). The molar fraction of 3-hydroxyvalerate in P(3HB-3HV) was also closely connected with PHB synthase. This may be due to the accelerated polymerization between 3-HB from glycolysis pathway and 3-HV converted from propionate supplied as precursor. Enforced beta-ketothiolase and acetoacetyl-CoA reductase to PHB synthase tended to enlarge the size of the PHB and P(3HB-3HV) granules, however, higher activity ratio of PHB synthase to beta-ketothiolase and acetoacetyl-CoA reductase than parent strain tended to induce the number of granules.
The mechanism for inactivation of the KIP family cyclin-dependent kinase inhibitor (CDKI) genes, the p21, p27, and p57 genes, in gastric cancer cells was tested by treating the cells with either the DNA demethylation agent, 5-aza-2'-deoxycytidine or the histone deacetylase inhibitor, n-butyric acid or trichostatin A. RNA expression of the gene was determined by reverse transcription PCR. The p21 gene was activated only by histone deacetylase inhibitor. The p57 gene was activated by histone deacetylase inhibitors in all of the gastric cancer cell lines and by 5-aza-2'-deoxycytidine in five of eight gastric cell lines. However, the p27 gene was not inactivated in gastric cancer cell lines. The methylation status of the promoter of the p21 and p57 genes was also tested by digestion with the methylation-sensitive restriction enzymes and a subsequent PCR. The promoter of the p21 gene has no methylation. The promoter of the p57 gene is, however, methylated in five of eight gastric cancer cell lines as expected from the result of the treatment with 5-aza-2'-deoxycytidine. Formation of the inactive chromatin through histone deacetylation seems to be the general mechanism for inactivation of both the p21 and the p57 genes in gastric cancer cells. Hypermethylation of promoter region seems to be an alternative pathway for inactivation of the p57 gene.
BACKGROUND: Tinnitus is a prevalent condition for which patients may seek treatment with acupuncture since no conventional treatment has been shown to be effective. OBJECTIVE: To summarize and critically review all randomized controlled trials on the efficacy of acupuncture as a treatment for tinnitus. DATA SOURCES: Four independent computerized literature searches (MEDLINE, Cochrane Controlled Trials Register, Embase, and CISCOM) were conducted in December 1998 using the key words acupuncture and tinnitus. STUDY SELECTION: All randomized controlled trials that compared any form of acupuncture with any control intervention in the treatment of tinnitus were included. DATA EXTRACTION: Data were extracted by 2 authors independently. The methodological quality of the included randomized controlled trials was assessed using the Jadad score. DATA SYNTHESIS: Six randomized controlled trials were included in the review, 4 of which used crossover design. Four studies used manual acupuncture and 2 used electroacupuncture. Five of 6 studies used inconsistent acupoints. Three studies scored 3 points or more on the Jadad scale. MAIN OUTCOME MEASURES: Outcome measurements were visual analog scale scores for loudness, annoyance, and awareness of tinnitus; subjective severity scale scores for tinnitus; or Nottingham Health Profile scores. RESULTS: Two unblinded studies showed a positive result, whereas 4 blinded studies showed no significant effect of acupuncture. CONCLUSION: Acupuncture has not been demonstrated to be efficacious as a treatment for tinnitus on the evidence of rigorous randomized controlled trials.
We previously reported that two nuclear factor 1-like elements mediated the transcription of the rat p53 gene. A 40-kDa protein was shown to bind to these elements, which was different from common NF1 family proteins. In this study, the biochemical properties of the 40-kDa binding protein were investigated. The metal ion dependency of the protein was examined with various chelators; the protein was proved to require Mg(2+) for maximum DNA-binding activity. The binding protein was highly resistant to ionic strength and denaturant. The protein-DNA complex was reduced at high NaCl concentration, but residual DNA-binding activity remained. Even 2 M urea did not completely eliminate the formation of protein-DNA complex. DNA-binding activity of the protein was also stable at high temperature. Treatment of the protein-DNA complex with increasing concentrations of proteinase K or trypsin demonstrated the existence of a protease-resistant DNA-bound core. These biochemical properties provide new insight into the 40-kDa NF1-like nuclear factor.
In this study, we evaluated the effects of pervanadate, a tyrosine phosphatase inhibitor, on the regulation and function of heat-shock factor 1 (HSF1) in HeLa cells. We showed that 50-100 microM pervanadate induced the hyperphosphorylation of the latent HSF1, as demonstrated by a retarded mobility of the HSF1 protein in SDS-polyacrylamide gel electrophoresis and as supported by the reversal of this mobility shift upon treatment of the cell extract with acid phosphatase. Pervanadate by itself had no effect on the monomeric stoichiometry and DNA-binding activity of HSF1. Upon heat shock, the pervanadate-induced hyperphosphorylated HSF1 formed DNA-binding trimers and translocated into the nuclear compartment. At high concentration (approximately 500 microM), pervanadate also induced the tyrosine phosphorylation of many cellular proteins and blunted the heat-induced transcription of hsp 70. N-acetyl cysteine inhibited these effects of pervanadate, suggesting a redox-based mechanism for its activity. Analysis of the activation of mitogen-activated protein kinases (MAPKs) using antibodies specific for the phospho-form (activated) of the kinases in Western blot showed that pervanadate activated extracellular signal-regulated kinase (ERK1/2), c-Jun-N-terminal kinase 1/2 (JNK1/2), and p-38 kinase. Pharmacological inhibitors of the ERK1/2 kinase pathway or the p38 kinase had little or no effect on the pervanadate-induced hyperphosphorylation of HSF1. Our results show that hyperphosphorylation of hHSF1 can occur prior to and independent of other events involved in the activation of hHSF1. The possibility that activation of the MAPK signaling cascade, notably JNK, may contribute to the hyperphosphorylation of human HSF1 (hHSF1) is discussed.
Following seizures, heat shock protein (HSP)-70 is induced in various brain regions. Since zinc that can induce HSP-70 in various cell systems is enriched in certain glutamatergic terminals and translocates to postsynaptic neurons with seizures, we examined the possibility that HSP-70 induction in the epileptic brain is mediated by synaptic zinc. Adult rats were injected intraperitoneally with kainate to induce seizures. Seizures were halted 3 h after the kainate administration by the injection of phenytoin. Staining of brain sections with zinc-specific fluorescent dye TFL at 24 h after the kainate injection revealed a one-to-one correlation between dense TFL fluorescence and acidophilic neuronal degeneration in the hippocampus. Subsequent staining with anti-HSP-70 antibody, however, revealed that more numerous neurons than degenerating neurons exhibited HSP-70 immunoreactivity. Most of the HSP-70(+) neurons were not stained with acid fuchsin but exhibited mild zinc fluorescence in the cytoplasm. Intraventricular injection of CaEDTA attenuated neuronal death as well as the HSP-70 induction in a dose-dependent manner. Supporting the specificity of zinc rather than calcium as the inducer of HSP-70 in neurons, exposure to zinc but not to a calcium ionophore or excitotoxins increased expression of HSP-70 mRNA and protein in cultured cortical neurons. The present results suggest that not only selective neuronal death, but also HSP-70 induction in neurons after seizures, is mediated by the translocation of endogenous synaptic zinc.
A type of oxidative DNA damage, 8-hydroxyguanine (8-OH-Gua) formation, and the activity for its subsequent repair, 8-OH-Gua endonuclease activity, were examined in an ischemia-reperfusion model of isolated rat hearts. The level of 8-OH-Gua in myocardial DNA was measured by a high performance liquid chromatography (HPLC) equipped with an electrochemical detector, and the 8-OH-Gua endonuclease activity was analysed by the endonuclease nicking assay using a synthetic double-stranded oligonucleotide containing an 8-OH-Gua residue as a substrate. The Langendorff-perfused rat hearts were subjected to 30 or 60 min of global ischemia, followed by reperfusion with an oxygenated or a nitrogenated Krebs-Henseleit solution. The 8-OH-Gua content in the DNA of the ischemic hearts reperfused with an oxygenated solution was three to four times higher than that of the control hearts. The levels of 8-OH-Gua did not increase either in the ischemic hearts reperfused with a nitrogenated solution or in the ischemic-reperfused hearts treated with SOD, mannitol or allopurinol. When the myocardial extract was incubated with the 8-OH-Gua-containing oligonucleotide substrate, a specific cleavage at the site of an 8-OH-Gua residue was detected. The endonuclease activity responsible for this cleavage increased two-fold in the ischemic-reperfused hearts, compared to the control. This study demonstrates that the formation of 8-OH-Gua in DNA as well as the level of its repair process, 8-OH-Gua endonuclease activity, increase in the ischemic-reperfused rat hearts in response to oxidative stress due to higher levels of oxygen free radicals.
This study describes the synthesis, in vitro evaluation and molecular modeling study of novel compounds for the inhibition of TNF-alpha production. Among these compounds, 2-[3-(cyclopentyloxy)-4-methoxyphenyl]-1-isoindolinone (9) was selected as a lead compound and its pyridine derivative 10 was more potent in activity and safer than rolipram.