Biomedical subjects

E Kim

Publications and source records attributed to E Kim.

At least 55 records · Page 3Linked to original sources

PubMed2001

Trifluoroacetic acid activates ATP-sensitive K(+) channels in rabbit ventricular myocytes.

Recent in vivo experimental evidence suggests that isoflurane-induced cardioprotection may involve K(ATP) channel activation during myocardial ischemia. The actual effect of isoflurane on cardioprotective ion conductance, however, such as that mediated by the opening of K(ATP) channels, has been the subject of some controversy in the past. The investigation reported here used a patch-clamp technique to test the hypothesis that a metabolite of isoflurane, trifluoroacetic acid (TFA), contributes to isoflurane-induced cardioprotection via K(ATP) channel activation. TFA enhanced channel activity in a concentration-dependent fashion, exhibiting half-maximal activation at 0.03 mM. TFA increased the number of openings of the channel, but did not affect the single channel conductance of K(ATP) channels. Analysis of open and closed time distributions showed that TFA increased the burst duration and decreased the interburst interval without eliciting changes of less than 5 ms in open and closed time distributions. TFA diminished the ATP sensitivity of K(ATP) channels in a concentration-response relationship for ATP. These results imply that TFA could mediate isoflurane-induced cardioprotection via K(ATP) channel activation during myocardial ischemia and reperfusion.

Adenosine Triphosphate↗

PubMed2001

Regulation of dendritic spine morphology by SPAR, a PSD-95-associated RapGAP.

The PSD-95/SAP90 family of scaffold proteins organizes the postsynaptic density (PSD) and regulates NMDA receptor signaling at excitatory synapses. We report that SPAR, a Rap-specific GTPase-activating protein (RapGAP), interacts with the guanylate kinase-like domain of PSD-95 and forms a complex with PSD-95 and NMDA receptors in brain. In heterologous cells, SPAR reorganizes the actin cytoskeleton and recruits PSD-95 to F-actin. In hippocampal neurons, SPAR localizes to dendritic spines and causes enlargement of spine heads, many of which adopt an irregular appearance with putative multiple synapses. Dominant negative SPAR constructs cause narrowing and elongation of spines. The effects of SPAR on spine morphology depend on the RapGAP and actin-interacting domains, implicating Rap signaling in the regulation of postsynaptic structure.

Actins↗

PubMed2001

ARF binds the C-terminal region of the Escherichia coli heat-labile toxin (LTA1) and competes for the binding of LTA2.

Cholera toxin (CT) and the heat-labile enterotoxin (LT) from Escherichia coli are highly related in terms of structure and biochemical activities and are the causative agents of cholera and traveler's diarrhea, respectively. The pathophysiological action of these toxins requires their activity as ADP-ribosyltransferases, transferring the ADP-ribose moiety from NAD onto the stimulatory, regulatory component of adenylyl cyclase, Gs. This reaction is highly dependent on the protein cofactor, termed ADP-ribosylation factor (ARF), that is itself a 20 kDa regulatory GTPase. In this study, we define sites of interaction between LTA and human ARF3. The residues identified as important to ARF binding include several of those previously shown to bind to the A2 subunit of the toxin and those important to the organization of two flexible loops, previously implicated as regulators of substrate entry. A model for how ARF acts to enhance the catalytic activity is proposed. A critical portion of the overlap between ARF and LTA(2) in binding LTA(1) includes a short region of sequence homology between LTA(2) and the switch II region of ARF. LTA(2) also interacted with ARF effectors in two-hybrid assays, and thus, we discuss the possibility that the LTA(2) subunit may function in cells as a partial ARF mimetic to compete for the binding of ARF to LTA(1) or regulate aspects of the toxin's transport from the cell surface to the ER.

ADP-Ribosylation Factors↗

PubMed2001

Modulation of ATP-sensitive potassium channels by cGMP-dependent protein kinase in rabbit ventricular myocytes.

This investigation used a patch clamp technique to test the hypothesis that protein kinase G (PKG) contributes to the phosphorylation and activation of ATP-sensitive K(+) (K(ATP)) channels in rabbit ventricular myocytes. Nitric oxide donors and PKG activators facilitated pinacidil-induced K(ATP) channel activities in a concentration-dependent manner, and a selective PKG inhibitor abrogated these effects. In contrast, neither a selective protein kinase A (PKA) activator nor inhibitor had any effect on K(ATP) channels at concentrations up to 100 and 10 microm, respectively. Exogenous PKG, in the presence of both cGMP and ATP, increased channel activity, while the catalytic subunit of PKA had no effect. PKG activity was prevented by heat inactivation, replacing ATP with adenosine 5'-O-(thiotriphosphate) (a nonhydrolyzable analog of ATP), removing Mg(2+) from the internal solution, applying a PKG inhibitor, or by adding exogenous protein phosphatase 2A. The effects of cGMP analogs and PKG were observed under conditions in which PKA was repressed by a selective PKA inhibitor. The results suggest that K(ATP) channels are regulated by a PKG-signaling pathway that acts via PKG-dependent phosphorylation. This mechanism may, at least in part, contribute to a signaling pathway that induces ischemic preconditioning in rabbit ventricular myocytes.

Adenosine Triphosphate↗

PubMed2001

Protein tyrosine phosphatase PTP1 negatively regulates Dictyostelium STATa and is required for proper cell-type proportioning.

The protein tyrosine phosphatase PTP1, which mediates reversible phosphorylation on tyrosine, has been shown to play an important regulatory role during Dictyostelium development. Mutants lacking PTP1 develop more rapidly than normal, while strains that overexpress PTP1 display aberrant morphology. However, the signalling pathways involved have not been characterised. In reexamining these strains, we have found that there is an inverse correlation between levels of PTP1 activity, the extent of tyrosine phosphorylation on Dictyostelium STATa after treatment with cAMP, and the proportion of the slug population exhibiting STATa nuclear enrichment in vivo. This suggests that PTP1 acts to attenuate the tyrosine phosphorylation of STATa and downstream STATa-mediated pathways. Consistent with this, we show that when PTP1 is overexpressed, there is increased expression of a prestalk cell marker at the slug posterior, a phenocopy of STATa null slugs. In ptp1 null strains, STATa tyrosine phosphorylation and nuclear enrichment in the slug anterior is increased. There is also a change in the prestalk to prespore cell ratio. Synergy experiments suggest that this is due to a cell-autonomous defect in forming the subset of prespore cells that are located in the anterior prespore region.

Animals↗

PubMed2001

Environmental modulation of autoimmune arthritis involves the spontaneous microbial induction of T cell responses to regulatory determinants within heat shock protein 65.

Both genetic and environmental factors are believed to be involved in the induction of autoimmune diseases. Adjuvant arthritis (AA) is inducible in susceptible rat strains by injection of Mycobacterium tuberculosis, and arthritic rats raise T cell responses to the 65-kDa mycobacterial heat-shock protein (Bhsp65). We observed that Fischer 344 (F344) rats raised in a barrier facility (BF-F344) are susceptible to AA, whereas F344 rats maintained in a conventional facility (CV-F344) show significantly reduced incidence and severity of AA, despite responding well to the arthritogenic determinant within Bhsp65. The acquisition of protection from AA can be circumvented if rats are maintained on neomycin/acidified water. Strikingly, naive unimmunized CV-F344 rats but not BF-F344 rats raised T cell responses to Bhsp65 C-terminal determinants (BCTD) (we have previously shown that BCTD are involved in regulation of acute AA in the Lewis rat); however, T cells of naive CV-F344 and BF-F344 gave a comparable level of proliferative response to a mitogen, but no response at all to an irrelevant Ag. Furthermore, adoptive transfer into naive BF-F344 rats of splenic cells of naive CV-F344 rats (restimulated with BCTD in vitro) before induction of AA resulted in a considerably reduced severity of AA. These results suggest that spontaneous (inadvertent) priming of BCTD-reactive T cells, owing to determinant mimicry between Bhsp65 and its homologues in microbial agents in the conventional environment, is involved in modulating the severity of AA in CV-F344 rats. These results have important implications in broadening understanding of the host-microbe interaction in human autoimmune diseases.

Adoptive Transfer↗

PubMed2001

Syntheses and evaluation of pyrido[2,3-dlpyrimidine-2,4-diones as PDE 4 inhibitors.

The syntheses and in vitro evaluation of a new series of pyrido[2,3-d]pyrimidine-2,4-diones bearing substituents at C-3 and/or C-4 positions on the pyridine ring are described. Some of these compounds, especially 51 and 6f, were found to be potent phosphodiesterase 4 (PDE 4) inhibitors exhibiting improved ratio of PDE 4 inhibitory activity:rolipram binding assay (RBA).

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2001

Limited accumulation of damaged proteins in l-isoaspartyl (D-aspartyl) O-methyltransferase-deficient mice.

l-Isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1) can initiate the conversion of damaged aspartyl and asparaginyl residues to normal l-aspartyl residues. Mice lacking this enzyme (Pcmt1-/- mice) have elevated levels of damaged residues and die at a mean age of 42 days from massive tonic-clonic seizures. To extend the lives of the knockout mice so that the long term effects of damaged residue accumulation could be investigated, we produced transgenic mice with a mouse Pcmt1 cDNA under the control of a neuron-specific promoter. Pcmt1 transgenic mice that were homozygous for the endogenous Pcmt1 knockout mutation ("transgenic Pcmt1-/- mice") had brain PCMT1 activity levels that were 6.5-13% those of wild-type mice but had little or no activity in other tissues. The transgenic Pcmt1-/- mice lived, on average, 5-fold longer than nontransgenic Pcmt1-/- mice and accumulated only half as many damaged aspartyl residues in their brain proteins. The concentration of damaged residues in heart, testis, and brain proteins in transgenic Pcmt1-/- mice initially increased with age but unexpectedly reached a plateau by 100 days of age. Urine from Pcmt1-/- mice contained increased amounts of peptides with damaged aspartyl residues, apparently enough to account for proteins that were not repaired intracellularly. In the absence of PCMT1, proteolysis may limit the intracellular accumulation of damaged proteins but less efficiently than in wild-type mice having PCMT1-mediated repair.

Animals↗

PubMed2001

Tropomyosin-troponin regulation of actin does not involve subdomain 2 motions.

Dynamic properties of F-actin structure prompted suggestions (Squire, J. M., and Morris, E. P. (1998) FASEB J. 12, 761-771) that actin subdomain 2 movements play a role in thin-filament regulation. Using fluorescently labeled yeast actin mutants Q41C, Q41C/C374S, and D51C/C374S and azidonitrophenyl putrescine (ANP) Gln(41)-labeled alpha-actin, we monitored regulation-linked changes in subdomain 2. These actins had fully regulated acto-S1 ATPase activities, and emission spectra of regulated Q41C(AEDANS)/C374S and D51C(AEDANS)/C374S filaments did not reveal any calcium-dependent changes. Fluorescence energy transfer in these F-actins mostly occurred from Trp(340) and Trp(356) to 5-(2((acetyl)amino)ethyl)amino-naphthalene-1-sulfonate (AEDANS)-labeled Cys(41) or Cys(51) of adjacent same strand protomers. Our results show that fluorescence energy transfer between these residues is similar in the mostly blocked (-Ca(2+)) and closed (+Ca(2+)) states. Ca(2+) also had no effect on the excimer band in the pyrene-labeled Q41C-regulated actin, indicating virtually no change in the overlap of pyrenes on Cys(41) and Cys(374). ANP quenching of rhodamine phalloidin fluorescence showed that neither Ca(2+) nor S1 binding to regulated alpha-actin affects the phalloidin-probe distance. Taken together, our results indicate that transitions between the blocked, closed, and open regulatory states involve no significant subdomain 2 movements, and, since the cross-linked alpha-actin remains fully regulated, that subdomain 2 motions are not essential for actin regulation.

Actins↗

PubMed2001

Effects of nicotine on APP secretion and Abeta- or CT(105)-induced toxicity.

Several lines of evidence indicated that overexpression or aberrant processing of amyloid precursor protein (APP) is causally related to Alzheimer's disease (AD). Amyloid precursor protein is principally cleaved within the amyloid beta protein domain to release a large soluble ectodomain (APPs), known to have a wide range of trophic functions. The central hypothesis guiding this review is that nicotine may play an important role in APP secretion and protection against toxicity induced by APP metabolic fragments (beta-amyloid [Abeta], carboxyl terminal [CT]). Findings from our experiments have shown that nicotine enhances the release of APPs, which has neurotrophic and neuroprotective activities in concentration-dependent (>50 micromol/L) and time-dependent (>2 hours) manners. In addition, pretreatment of nicotine (>10 micromol/L for 24 hours) partially prevented Abeta or CT(105)-induced cytotoxicity in primary cultured neuron cells, and the effects of nicotine-induced protection were inhibited by the pretreatment with a nicotine alpha-bungarotoxin. Nicotine (>10 micromol/L for 24 hours) partially inhibited CT(105)-induced cytotoxicity when PC12 cells was transfected with CT(105). From these results, we proposed that nicotine or nicotinic receptor agonist treatment might improve the cognitive functions not only by supplementation of cholinergic neurotransmission, but also by protecting Abeta- or CT(105)-induced neurotoxicity probably through the increased release of APPs and the activation of nicotinic receptors.

Alzheimer Disease↗

PubMed2001

Tetragonal crystalline carbon nitrides: theoretical predictions.

New tetragonal phases of crystalline carbon nitride (CN) and their atomic structures have been identified using a self-consistent-charge density-functional tight-binding method. A tetragonal rocksalt structure provides theoretical support to recent experimental evidence for a stoichiometric CN phase with tetragonal symmetry. A body-centered tetragonal CN phase with 1:1 stoichiometry is predicted to be highly stable and of interesting atomic structure with complicated C-C and N-N dimerizations along the c axis. The cubic-to-tetragonal transitions are carefully examined to understand the underlying mechanism.

Journal Article↗

PubMed2001

Sharpin, a novel postsynaptic density protein that directly interacts with the shank family of proteins.

The Shank family of proteins (also termed CortBP, ProSAP, or Synamon) is highly enriched in the postsynaptic density (PSD) of excitatory synapses in brain. Shank contains multiple domains for protein-protein interactions, including ankyrin repeats, SH3 domain, PDZ domain, SAM domain, and an extensive proline-rich region. We have identified a novel protein, termed Sharpin, that directly interacts with the ankyrin repeats of Shank. Sharpin is enriched in the PSD and forms a complex with Shank in heterologous cells and brain. Immunostaining reveals the presence of Sharpin at excitatory synapses and its colocalization with Shank. While the C-terminal half of Sharpin interacts with Shank, the N-terminal half of Sharpin mediates homomultimerization. Considering the fact that the ankyrin repeats and the SH3 domain of Shank can be truncated by alternative splicing, these results define Sharpin as a novel PSD protein that may regulate the complexity of the Shank-based protein network in an alternative splicing-dependent manner.

Adaptor Proteins, Signal Transducing↗

PubMed2001

A method for assessing the regional vibratory pattern of vocal folds by analysing the video recording of stroboscopy.

Stroboscopy and kymography have been used to examine the motional abnormality of vocal folds and to visualise their regional vibratory pattern. In a previous study (Laryngoscope, 1999), we introduced the conceptual idea of videostrobokymography, in which we applied the concept of kymography on the pre-recorded video images using stroboscopy, and showed its possible clinical application to various disorders in vocal folds. However, a more detailed description about the software and the mathematical formulation used in this system is needed for the reproduction of similar systems. The composition of hardwares, user-interface and detail procedures including mathematical equations in videostrobokymography software is presented in this study. As an initial clinical trial, videostrobokymography was applied to the preoperative and postoperative videostroboscopic images of 15 patients with Reinke's edema. On preoperative examination, videostrobokymograms showed irregular pattern of mucosal wave and, in some patients, a relatively constant glottic gap during phonation. After the operation, the voice quality of all patients was improved in acoustic and aerodynamic assessments, and videostrobokymography showed clearly improved mucosal waves (change in open quotient: mean +/- SD= 0.11 +/- 0.05).

Female↗

PubMed2001

The truncated cytoplasmic tail of HLA-G serves a quality-control function in post-ER compartments.

In contrast to the current model of MHC class I trafficking, which predicts that once a MHC class I molecule leaves the ER, it moves to the cell surface by bulk flow, we show that HLA-G that is loaded with suboptimal peptides is retrieved from post-ER compartments to the ER. Loading of HLA-G with high-affinity peptides abrogates this retrieval due to the lack of binding affinity to coatomer. Moreover, the loss of the endocytosis motif in the truncated cytoplasmic tail results in the prolonged half-life of HLA-G on the cell surface. Our findings reveal that surface expression of HLA-G can be further regulated in post-ER compartments and that the truncated cytoplasmic tail plays a critical role in such quality-control mechanisms.

Amino Acid Motifs↗

PubMed2001

Mre11 protein complex prevents double-strand break accumulation during chromosomal DNA replication.

Mre11 complex promotes repair of DNA double-strand breaks (DSBs). Xenopus Mre11 (X-Mre11) has been cloned, and its role in DNA replication and DNA damage checkpoint studied in cell-free extracts. DSBs stimulate the phosphorylation and 3'-5' exonuclease activity of X-Mre11 complex. This induced phosphorylation is ATM independent. Phosphorylated X-Mre11 is found associated with replicating nuclei. X-Mre11 complex is required to yield normal DNA replication products. Genomic DNA replicated in extracts immunodepleted of X-Mre11 complex accumulates DSBs as demonstrated by TUNEL assay and reactivity to phosphorylated histone H2AX antibodies. In contrast, the ATM-dependent DNA damage checkpoint that blocks DNA replication initiation is X-Mre11 independent. These results strongly suggest that the function of X-Mre11 complex is to repair DSBs that arise during normal DNA replication, thus unraveling a critical link between recombination-dependent repair and DNA replication.

Amino Acid Sequence↗

PubMed2001

Efficient gene transfer of VSV-G pseudotyped retroviral vector to human brain tumor.

A retroviral vector constructed from the murine leukemia virus (MLV) can only express transgenes in cells undergoing mitosis, indicating its suitability as a delivery vehicle for cancer gene therapy. However, the transduction efficiency (TE) of retroviruses embedding endogenous envelope proteins in human cancer cells was found to be unsatisfactory. Recently, several research groups have demonstrated the feasibility of a retroviral vector pseudotyped with a vesicular stomatitis virus G (VSV-G) protein. In this study, the potential of VSV-G pseudotyped MLV-based retrovirus was examined as a delivery vehicle in a variety of human cancer cells including brain tumor cells in vitro and in vivo. The transduction efficiency of the 293T/G/GP/LacZ retrovirus in cell culture was superior in most cancer cells, particularly in brain tumor cells, compared with that of other retroviruses, such as PA317- or PG13-derived. The relative growth rate and phosphatidylserine expression level on the plasma membrane of target cells mainly influenced the transduction efficiency of VSV-G pseudotyped retrovirus, which suggested that both the relative growth rate and phosphatidylserine expression level were major determinants of TE. Furthermore, 293T/G/GP/LacZ could efficiently transduce human cancer cells regardless of the presence of chemical additives, whereas in other retroviruses, cationic chemical additives such as polybrene or liposomes were essential during virus infection. Finally, an average of 10% gene expression was routinely obtained exclusively in the tumor mass when 293T/G/GP/LacZ concentrated by simple ultracentrifugation was directly administrated to pre-established brain tumors in animal models (U251-N nu/nu mice or C6 Wistar rats). All told, the present study suggests that the VSV-G pseudotyped retrovirus is a suitable vector for brain tumor gene therapy.

Brain Neoplasms↗

PubMed2001

All angiogenesis is not the same: Distinct patterns of response to antiangiogenic therapy in experimental neuroblastoma and Wilms tumor.

BACKGROUND/PURPOSE: Neuroblastoma and Wilms tumor exhibit different patterns of metastasis, invasion, and therapeutic response. Vascular endothelial growth factor (VEGF) is an angiogenic factor expressed in both tumors. The authors hypothesized that because the clinical behavior of these tumors differs, the response to anti-VEGF therapy would be distinct, and tumor vascular architectures would reflect this distinction. METHODS: Xenografts were induced by intrarenal injection of cultured cells in athymic mice. After 1 week, anti-VEGF antibody or vehicle were administered for 5 weeks before sacrifice. Additional animals were maintained for 3 weeks after termination of antibody injections to assess rebound growth of tumors. Fluorescein angiography was performed in selected animals. RESULTS: Neuroblastoma control and treated tumor weights were not significantly different (1.48 g v 0.77 g, P =.34). By comparison, as previously reported, antibody-treated Wilms tumors were growth inhibited. Angiograms of treated (but not control) neuroblastomas displayed novel rounded structures at vessel branches, which the authors term terminal vascular bodies (TVBs). Wilms tumor vessels displayed no such alteration. CONCLUSIONS: Neuroblastoma xenografts are less effectively suppressed by anti-VEGF antibody than Wilms tumors. Neuroblastoma vascular architecture displays a novel alteration during antibody administration, which attenuates when antibody is withdrawn. These studies suggest that angiogenesis is differently regulated in experimental neuroblastoma and Wilms tumor.

Angiogenesis Inhibitors↗

PubMed2001

Highly specific antiangiogenic therapy is effective in suppressing growth of experimental Wilms tumors.

BACKGROUND/PURPOSE: Pathologic angiogenesis in tumors is a potential target for novel therapies. Vascular endothelial growth factor (VEGF) is an angiogenic promoter present in a wide variety of human tumors. VEGF is expressed as 4 isoforms; one of these, VEGF165, predominates in human tumors. The authors hypothesized that antagonism of VEGF165 by a specific aptamer would block tumor growth in an experimental model of Wilms tumor. METHODS: VEGF isoform expression in clinical (n = 2) and experimental tumors were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Tumors were induced in NCR nude mice (n = 32) by intrarenal injection of 10(6) cultured Wilms tumor cells. At 1 week, aptamer (n = 16) or vehicle (n = 16) treatment was started and continued daily for 5 weeks. RESULTS: At 6 weeks tumors weighed 84% less in treated versus control animals (0.69 v 4.41 g; P <.028), without observed adverse effects and similar to suppression previously reported using nonisoform-specific anti-VEGF antibody (94% to 96%). CONCLUSIONS: Anti-VEGF165 aptamer effectively suppressed primary tumor growth in experimental animals with no observed adverse effects. Development of highly specific antiangiogenic therapies may be of particular benefit to pediatric patients.

Angiogenesis Inhibitors↗