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Biomedical subjects

J Han

Publications and source records attributed to J Han.

At least 307 records · Page 17Linked to original sources
PubMed1996

Effects of volatile anesthetic isoflurane on ATP-sensitive K+ channels in rabbit ventricular myocytes.

It has been suggested that volatile anesthetic, isoflurane mediates cardioprotective effects via activation of the ATP-sensitive K+ (KATP) channels. However, no direct evidence has been provided to define whether isoflurane activates cardiac KATP channels using patch-clamp technique. We examined the effects of isoflurane on the KATP channels in rabbit ventricular myocytes by use of patch-clamp technique. Contrary to the results of the in vivo experiments, isoflurane inhibited the channel activity without a change in the single-channel conductance. Isoflurane decreased the channel activity by a decrease in burst duration and an increase in the inter-burst duration. On the other hand, isoflurane diminished the ATP sensitivity of KATP channels, indicating an increased probability of KATP channel opening for a given concentration of ATP after isoflurane anesthesia. The result supports, at least in part, the hypothesis that isoflurane mediates cardioprotective effects via KATP channel activation.

Anesthetics, Inhalation↗
PubMed1996

Effects of internal cleavages and mutations in the C-terminal region of NIa protease of turnip mosaic potyvirus on the catalytic activity.

The nuclear inclusion protein a (NIa) of turnip mosaic potyvirus is a protease processing the viral polyprotein into functional proteins. It has been shown that the NIa C-terminal 27-kDa protease cleaves itself between Ser-223 and Gly-224 to generate a 25-kDa protein lacking the C-terminal 20 amino acids. We have found a second internal cleavage near the C-terminus resulting in the degradation of the 25-kDa protein into a 24-kDa protein. Substitution of the active site Asp-81 or Cys-151 with Asn or Ser, respectively, prevented the second cleavage, suggesting that the internal cleavage is also due to the proteolytic activity of the NIa protease. This second internal cleavage was found to occur between Thr-207 and Ser-208, eliminating the C-terminal 36 amino acids from the 27-kDa protease. The proteolytic activity of the 24-kDa protein was not detected at all when it was measured using a nonapeptide containing the cleavage site between 6K1 and Cl as a substrate, suggesting that the C-terminal region between residues 208 and 223 contains essential amino acids for the processing of 6K1-Cl polyprotein. The deletion analyses of the C-terminal region revealed that at least 217 amino acids from the N-terminus are required for the catalytic activity of the NIa protease. The point mutation of Trp-212 to Ser, Gly-213 to Ser, or Ile-217 to Asp drastically abolished the catalytic activity, demonstrating that Trp-212, Gly-213, and Ile-217 are important for the processing of 6K1-Cl polyprotein.

Amino Acid Sequence↗
PubMed1996

A major agonist-regulated capping activity in Dictyostelium is due to the capping protein, cap32/34.

Stimulation of starved Dictyostelium amoebae with the chemoattractant cAMP produces a rapid increase in actin nucleation activity at 5 seconds which is cotemporal with an increase in actin assembly and a decrease in Ca(2+)-insensitive capping activity [1]. Further characterization of this capping activity, called aginactin, led to the isolation of an Hsc70 [2]. Here, we demonstrate that purified aginactin contains both Hsc70 and the heterodimeric barbed-end capping protein, cap32/34. Immunoprecipitation of cap32/34 from purified aginactin removes all capping activity while immunoprecipitation of Hsc70 does not, indicating that the capping activity of aginactin is an intrinsic property of cap32/34. Gel filtration and immunoprecipitation assays fail to demonstrate the existence of a stable, high affinity complex between Hsc70 and cap32/34 in either lysate supernatants or aginactin pools but indicate the presence of a transient, ATP-sensitive interaction in cell lysates. Reconstitution experiments with purified Hsc70 and cap32/34 demonstrate that Hsc70 neither stimulates nor inhibits the capping activity of native cap32/34. Furthermore, we measured a Kd of approx. 0.8 nM for the binding of cap32/34 to barbed ends of actin filaments in the absence or presence of Hsc70, in agreement with Kd values measured for purified capping protein from other sources. We conclude, therefore, that cap32/34 is responsible for the capping activity called aginactin and that Hsc70 is not a regulatory cofactor for cap32/34 in Dictyostelium but may function as a chaperone during assembly of the cap32/34 heterodimer.

Actins↗
PubMed1996

The primary structure of p38 gamma: a new member of p38 group of MAP kinases.

We have identified a third member of the p38 group of MAP kinase termed p38 gamma. The cDNA for this MAP kinase encodes an 367 amino acid polypeptide that is slightly greater than 60% identical to p38 and p38 beta. Expression of its mRNA is primarily in muscle with no detectable expression in Northern blots using RNA from other tissues. In contrast to p38 and p38 beta, p38 gamma, fails to phosphorylate ATF-2 or MAPKAP kinase 2 but does like other MAP kinases phosphorylate MBP. In vivo kinase assays using protein extracts from skeletal muscle reveal that a 7-kDa protein is phosphorylated by p38 gamma but not by other members of this group of kinases. We suggest p38 gamma may have unique functions when compared with other members of the p38 group due to its restricted tissue expression and apparent substrate preferences.

Amino Acid Sequence↗
PubMed1996

Activation of p38 in stimulated human neutrophils: phosphorylation of the oxidase component p47phox by p38 and ERK but not by JNK.

Incubation of human neutrophils with FMLP, a chemotactic peptide, or PMA, a stimulator of protein kinase C, resulted in the activation of p38, a proline-directed kinase. Previous studies had shown that extracellular signal-regulated kinase (ERK), another proline-directed kinase, was activated with similar kinetics in neutrophils stimulated with FMLP and PMA (1, 2). Because one possible target for these proline-directed kinases is p47phox, a component of the respiratory burst oxidase, we examined the phosphorylation of this protein by p38 and ERK, as well as JNK, another proline-directed kinase present in neutrophils. We found that both p38 and ERK phosphorylated p47phox at the same site and at similar rates, but that p47phox was not a substrate for JNK. These data show that p38, like ERK, can be activated in neutrophils exposed to an appropriate stimulus, and that some but not all proline-directed kinases are able to participate in the phosphorylation of a protein essential for normal neutrophil function.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Localization of human elav-like neuronal protein 1 (Hel-N1) on chromosome 9p21 by chromosome microdissection polymerase chain reaction and fluorescence in situ hybridization.

Hel-N1 is a member of the highly conserved elav family of neuronal genes. It shares considerable sequence homology with HuD, another human member, and both genes are expressed in brain. HuD was recently mapped to chromosome 1p34. Here, we have utilized chromosome microdissection polymerase chain reaction and fluorescence in situ hybridization to map Hel-N1 to chromosome 9p21. The different chromosomal locations of these homologous genes underscore their distinct identities.

Chromosome Mapping↗
PubMed1996

Characterization of the structure and function of a new mitogen-activated protein kinase (p38beta).

Mitogen-activated protein (MAP) kinase cascades represent one of the major signal systems used by eukaryotic cells to transduce extracellular signals into cellular responses. Four MAP kinase subgroups have been identified in humans: ERK, JNK (SAPK), ERK5 (BMK), and p38. Here we characterize a new MAP kinase, p38beta. p38beta is a 372-amino acid protein most closely related to p38. It contains a TGY dual phosphorylation site, which is required for its kinase activity. Like p38, p38beta is activated by proinflammatory cytokines and environmental stress. A comparison of events associated with the activation of p38beta and p38 revealed differences, most notably in the preferred activation of p38beta by MAP kinase kinase 6 (MKK6), whereas p38 was activated nearly equally by MKK3, MKK4, and MKK6. Moreover, in vitro and in vivo experiments showed a strong substrate preference by p38beta for activating transcription factor 2 (ATF2). Enhancement of ATF2-dependent gene expression by p38beta was approximately20-fold greater than that of p38 and other MAP kinases tested. The data reported here suggest that while closely related, p38beta and p38 may be regulated by differing mechanisms and may exert their actions on separate downstream targets.

Activating Transcription Factor 2↗
PubMed1996

HLA-DR4-IE chimeric class II transgenic, murine class II-deficient mice are susceptible to experimental allergic encephalomyelitis.

To investigate the development of HLA-DR-associated autoimmune diseases, we generated transgenic (Tg) mice with HLA-DRA-IE alpha and HLA-DRB1*0401-IE beta chimeric genes. The transgene-encoded proteins consisted of antigen-binding domains from HLA-DRA and HLA-DRB1*0401 molecules and the remaining domains from the IE(d)-alpha and IE(d)-beta chains. The chimeric molecules showed the same antigen-binding specificity as HLA-DRB1*0401 molecules, and were functional in presenting antigens to T cells. The Tg mice were backcrossed to MHC class II-deficient (IA beta-, IE alpha-) mice to eliminate any effect of endogenous MHC class II genes on the development of autoimmune diseases. As expected, IA alpha beta or IE alpha beta molecules were not expressed in Tg mice. Moreover, cell-surface expression of endogenous IE beta associated with HLA-DRA-IE alpha was not detectable in several Tg mouse lines by flow cytometric analysis. The HLA-DRA-IE alpha/HLA-DRB1*0401-IE beta molecules rescued the development of CD4+ T cells in MHC class II-deficient mice, but T cells expressing V beta 5, V beta 11, and V beta 12 were specifically deleted. Tg mice were immunized with peptides, myelin basic protein (MBP) 87-106 and proteolipid protein (PLP) 175-192, that are considered to be immunodominant epitopes in HLA-DR4 individuals. PLP175-192 provoked a strong proliferative response of lymph node T cells from Tg mice, and caused inflammatory lesions in white matter of the CNS and symptoms of experimental allergic encephalomyelitis (EAE). Immunization with MBP87-106 elicited a very weak proliferative T cell response and caused mild EAE. Non-Tg mice immunized with either PLP175-192 or MBP87-106 did not develop EAE. These results demonstrated that a human MHC class II binding site alone can confer susceptibility to an experimentally induced murine autoimmune disease.

Amino Acid Sequence↗
PubMed1996

Involvement of the switch 2 domain of Ras in its interaction with guanine nucleotide exchange factors.

While Ras proteins are activated by stimulated GDP release, which enables acquisition of the active GTP-bound state, little is known about how guanine nucleotide exchange factors (GEFs) interact with Ras to promote this exchange reaction. Here we report that mutations within the switch 2 domain of Ras (residues 62-69) inhibit activation of Ras by the mammalian GEFs, Sos1, and GRF/CDC25Mm. While mutations in the 62-69 region blocked upstream activation of Ras, they did not disrupt Ras effector functions, including transcriptional activation and transformation of NIH 3T3 cells. Biochemical analysis indicated that the loss of GEF responsiveness of a Ras(69N) mutant was due to a loss of GEF binding, with no change in intrinsic nucleotide exchange activity. Furthermore, structural analysis of Ras(69N) using NMR spectroscopy indicated that mutation of residue 69 had a very localized effect on Ras structure that was limited to alpha-helix 2 of the switch 2 domain. Together, these results suggest that the switch 2 domain of Ras forms a direct interaction with GEFs.

3T3 Cells↗
PubMed1996

C-Fos proteins are not involved in the activation of preproenkephalin gene expression in rat brain by peripheral electric stimulation (electroacupuncture).

The present work was designed to study the role of the oncogene product c-Fos in activating the transcription of preproenkephalin (PPE) gene following a kind of peripheral electric stimulation known as electroacupuncture (EA) stimulation. The temporal patterns of rat brain c-fos and PPE mRNA expression were evaluated using the method of Northern blotting, showing that c-fos mRNA expression, which peaked at 2 h after the termination of EA, was always ahead of the PPE mRNA expression which began at 4 h and peaked at 48 h after EA. The methods of immunocytochemistry (ICC) and in situ hybridization (ISH) techniques were combined to identify the co-existence of c-Fos protein and PPE mRNA at the cellular level. The results showed that only a small percentage of PPE mRNA-containing neurons depicts Fos-like immunoreactive nuclei. These findings suggest that c-Fos protein may not be involved in the activation of brain PPE gene transcription induced by peripheral electric stimulation.

Animals↗
PubMed1996

Sulfhydryl redox modulates ATP-sensitive K+ channels in rabbit ventricular myocytes.

The properties of sulfhydryl redox modulation of the ATP-sensitive K+ K(ATP) channel have been examined in rabbit ventricular myocytes, using the patch-clamp technique. The sulfhydryl oxidizing agent 5.5'-dithio bis-(2-nitro-benzoic acid) (DTNB) induced an inhibition of the channel activity without change in the single channel conductance. DTNB had no effect on the inhibitory action by ATP. Analysis of the open and closed time distributions showed that DTNB decreased the life time of bursts and increased the interburst interval without changes in open and closed time distributions shorter than 5 ms. N-ethylmaleimide (NEM), a substance that reacts with sulfhydryl groups of cysteine residues in proteins, induced an irreversible closure of the channel. The results suggested that changes in the sulfhydryl redox also modulate K(ATP) channel activity of the K(ATP) channel in rabbit ventricular myocytes.

Adenosine Triphosphate↗
PubMed1996

The E1B 19K protein blocks apoptosis by interacting with and inhibiting the p53-inducible and death-promoting Bax protein.

The E1B 19K protein is a potent apoptosis inhibitor and the putative adenovirus Bcl-2 homolog. To investigate the mechanism of apoptosis regulation, 19K-interacting cellular proteins were identified using the yeast two-hybrid system, and Bax was one of seven 19-K interacting clones. Residues 50-78 of Bax containing a conserved region designated Bcl-2 homology region 3 (BH3) were sufficient for specific binding to both the E1B 19K and Bcl-2 proteins. The Bax-E1B 19K interaction was detectable in vitro and in lysates from mammalian cells, and Bax expression antagonized E1B 19K protein function. bax mRNA and protein levels were p53-inducible with kinetics identical to that of p21/Waf-1/Cip-1, and E1B 19K and Bcl-2 expression did not affect Bax or p21/Waf-1/Cip-1 accumulation. In cells where p53 was mutant, Bax expression induced apoptosis, suggesting that Bax was sufficient for apoptosis, and acted downstream of p53. p53 may simultaneously activate the transcription of genes required for both growth arrest (p21/Waf-1/Cip-1) and death (bax), and E1B 19K and Bcl-2 may act distally and function through interaction with and antagonism of Bax to prevent apoptosis. With the death pathway disabled, induction of growth arrest by p53 can then be manifested.

Adenovirus E1B Proteins↗
PubMed1996

Characterization of the structure and function of a novel MAP kinase kinase (MKK6).

Mitogen-activated protein (MAP) kinases require dual phosphorylation on threonine and tyrosine residues in order to gain enzymatic activity. This activation is carried out by a family of enzymes known as MAP kinase kinases (MKKs or MEKs). It appears that there are at least four subgroups in this family; MEK1/MEK2 subgroup that activates ERK1/ERK2, MEK5 that activates ERK5/BMK1, MKK3 that activates p38, and MKK4 that activates p38 and Jun kinase. Here we describe the characteristics of a new MKK termed MKK6. The clones we isolated encode two splice isoforms of human MKK6 comprised of 278 and 334 amino acids, respectively, and one murine MKK6 with 237 amino acids. Sequence information derived from cDNA cloning indicated that MKK6 is most closely related to MKK3. The functional data revealed from co-transfection assays suggests that MKK6, like MKK3, selectively phosphorylates p38. Unlike the previously described MKKs (or MEKs), MKK6 exists in a variety of alternatively spliced isoforms with distinct patterns of tissue expression. This suggests novel mechanisms regulating activation and/or function of various forms of MKK6.

Amino Acid Sequence↗
PubMed1996

Blockade of the ATP-sensitive potassium channel by taurine in rabbit ventricular myocytes.

Using patch-clamp techniques, the effects of taurine on properties of ATP-sensitive K+ (KATP) channel of rabbit ventricular myocytes were examined. Intracellular taurine (20 mM) markedly depressed the KATP channel activity. The taurine concentration for half-inhibition (apparent Kd) was 13.5 mM with a Hill coefficient, n, of 1.3. Intracellular taurine caused channel inhibition without affecting channel inhibition by ATP. In control conditions, the ATP concentration for half-inhibition (Ki) and n were 73 microM and 1.2 (n = 6), respectively. In the presence of taurine, Ki and n were 81 microM and 1.3 (n = 6), respectively. Analysis of the open and closed time distributions showed that taurine decreased the life time of bursts and increased the inter-burst interval and/or reduced the number of functional channels. 2,4-Dinitrophenol (DNP) activated KATP channel after a lag period. This lag period was much longer after pretreatment with taurine (6.6 +/- 1.2 min, n = 5) than in the absence of taurine (2.8 +/- 1.5 min, n = 12). When DNP was removed in the bath solution, channel activity showed a gradual reduction with time and this process was facilitated by the presence of external taurine (20 mM). From these results it is suggested that taurine blocks KATP channel activity in dose-dependent manner and the depletion of taurine during myocardial ischemia contribute to the early activation of the KATP channel.

2,4-Dinitrophenol↗