Biomedical subjects

H D Kim

Publications and source records attributed to H D Kim.

At least 19 recordsLinked to original sources

PubMed2001

Homodimers of mutant tryptophan synthase alpha-subunits in Escherichia coli.

Tryptophan synthase alpha-subunit from Escherichia coli functionally exists as a heterotetramer of alpha(2)beta(2) with beta-subunit. While wild-type and mutant (F139W, T24M/F139W, and T24L/F139W) alpha-subunits were expressed as a monomer from recombinant plasmids in Escherichia coli, T24A/F139W, T24S/F139W, and T24K/F139W mutant alpha-subunits were abnormally expressed as soluble homodimers in addition to monomers. Monomers of dimer-forming mutant alpha-subunits retain high affinity to beta-subunit, high activity in stimulating catalytic activities of beta-subunit, and nearly intact content of secondary structure, indicating that the global structures of these monomers are identical to that of F139W alpha-subunit. However, fluorescence spectra of Trp139 and ANS binding indicate that significant perturbations occur in the mutant proteins. Interestingly, these defective properties of monomers caused by residue replacement were partially repaired by the dimer formation. As a result, it is suggested that dimers may be formed by domain or loop swapping, and that residue 24 may play important role in maintaining on-pathway of alpha-subunit folding.

Anilino Naphthalenesulfonates↗

PubMed2001

Tripartite structure of Saccharomyces cerevisiae Dna2 helicase/endonuclease.

In order to gain insights into the structural basis of the multifunctional Dna2 enzyme involved in Okazaki fragment processing, we performed biochemical, biophysical and genetic studies to dissect the domain structure of Dna2. Proteolytic digestion of Dna2 using subtilisin produced a 127 kDa polypeptide that lacked the 45 kDa N-terminal region of Dna2. Further digestion generated two subtilisin-resistant core fragments of approximately equal size, 58 and 60 kDa. Surprisingly, digestion resulted in a significant (3- to 8-fold) increase in both ATPase and endonuclease activities compared to the intact enzyme. However, cells with a mutant DNA2 allele lacking the corresponding N-terminal region were severely impaired in growth, being unable to grow at 37 degrees C, indicating that the N-terminal region contains a domain critical for a cellular function(s) of Dna2. Analyses of the hydrodynamic properties of and in vivo complex formation by wild-type and/or mutant Dna2 lacking the N-terminal 45 kDa domain revealed that Dna2 is active as the monomer and thus the defect in the mutant Dna2 protein is not due to its inability to multimerize. In addition, we found that the N-terminal 45 kDa domain interacts physically with a central region located between the two catalytic domains. Our results suggest that the N-terminal 45 kDa domain of Dna2 plays a critical role in regulation of the enzymatic activities of Dna2 by serving as a site for intra- and intermolecular interactions essential for optimal function of Dna2 in Okazaki fragment processing. The possible mode of regulation of Dna2 is discussed based upon our recent finding that replication protein A interacts functionally and physically with Dna2 during Okazaki fragment processing.

Adenosine Triphosphatases↗

PubMed2001

Activation of heat shock factor 1 by pyrrolidine dithiocarbamate is mediated by its activities as pro-oxidant and thiol modulator.

Pyrrolidine dithiocarbamate (PDTC) is known to inhibit NF-kappa B, which plays a critical role(s) as an immediate early mediator of immune and inflammatory responses. Here we show that PDTC induces heat shock factor 1 (HSF1) activation and heat shock protein expression, while other antioxidants such as butylated hydroxytoluene (BHT), n-propylgallate (PG), ascorbic acid (AA), and N-acetyl-L-cysteine (NAC) do not. Since PDTC exerts other functions than antioxidant, e.g., a pro-oxidant, metal chelator, and thiol group modulator, we examined which of these activities is responsible for the PDTC-induced HSF1 activation. PDTC-induced HSF1 activation was not prevented by metal chelators, EDTAs, indicating that the metal chelating effect of PDTC is not linked to the HSF1 activation. PDTC increased intracellular GSSG level. In addition, PDTC-induced activation of HSF1 was significantly inhibited by NAC and a thiol-reducing agent dithiothreitol (DTT), while it was partially prevented by other antioxidants, AA, BHT, and PG. These results suggest that the activation of HSF1 by PDTC may be due to its activities as pro-oxidant and thiol group modulator rather than anti-oxidant.

Acetylcysteine↗

PubMed2001

Extracellular signal-regulated kinase/90-KDA ribosomal S6 kinase/nuclear factor-kappa B pathway mediates phorbol 12-myristate 13-acetate-induced megakaryocytic differentiation of K562 cells.

Two signaling pathways, the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK)-dependent pathway and the nuclear factor-kappaB (NF-kappaB)-dependent pathway, have been known to mediate megakaryocytic differentiation of K562 cells induced by phorbol 12-myristate 13-acetate (PMA). In this study, we examined whether 90-kDa ribosomal S6 kinase (RSK), known as a substrate of ERK/MAPK and a signal-inducible IkappaBalpha kinase, would link two pathways during the differentiation. RSK1 was activated in a time- and dose-dependent manner during the PMA-induced differentiation. Overexpression of wild-type or dominant inhibitory mutant (D205N) of RSK1 enhanced or suppressed PMA-stimulated NF-kappaB activation and megakaryocytic differentiation as shown by morphology, nonspecific esterase activity, and expression of the CD41 megakaryocytic marker, respectively. In addition, overexpression of the dominant inhibitory mutant (S32A/S36A) of IkappaBalpha inhibited PMA-stimulated and RSK1-enhanced megakaryocytic differentiation, indicating that NF-kappaB mediates a signal for megakaryocytic differentiation downstream of RSK1. PMA-stimulated activation of ERK/MAPK, RSK1, and NF-kappaB and the PMA-induced megakaryocytic differentiation were prevented by pretreatment with PD98059, a specific inhibitor of the mitogen-activated ERK kinase (MEK). Therefore, these results demonstrate that the sequential ERK/RSK1/NF-kappaB pathway mediates PMA-stimulated megakaryocytic differentiation of K562 cells.

Amino Acid Substitution↗

PubMed2001

Modulations of soleus H-reflex excitability during gait initiation: central versus peripheral influences.

Soleus and tibialis anterior electromyogram (EMG) and soleus H-reflexes were recorded from the stance limb of an individual who suffered a traumatic peroneal nerve injury and of four nonimpaired individuals during gait initiation. The control subjects also initiated walking after swaying forward (sway-gait initiation), which eliminated the initial tibialis anterior activation. During the initial period of gait initiation, H-reflexes were depressed to 43% of standing values during normal-gait initiation and 86% during sway-gait initiation in the nonimpaired subjects. H-reflexes of the nerve-injured subject were depressed to 37%, even though no tibialis anterior EMG was observed. The findings support the view that reciprocal inhibition of the soleus during a task, which normally involves tibialis anterior activation, is due to a centrally mediated process.

Adult↗

PubMed2001

Characterization of antidepressant-like effects of p-synephrine stereoisomers.

We previously reported that p-synephrine has antidepressant-like activity in the murine models of forced swimming and tail suspension. In the present study, we characterized antidepressant-like effects of p-synephrine stereoisomers in both in vivo and in vitro systems. In the tail suspension test, S-(+)-p-synephrine (3 mg/kg, p.o.) reduced the duration of immobility, while R-(-)-p-synephrine (0.3-3 mg/kg, p.o.) had no effect. S-(+)-p-synephrine (0.3, 1 and 3 mg/kg, p.o.) and R-(-)-p-synephrine (1 mg/ kg and 3 mg/kg, p.o.) significantly reversed the reserpine (2.5 mg/kg, i.p.)-induced hypothermia. S-(+)-p-synephrine was more effective than R-(-)-p-synephrine in inhibition of both [3H]noradrenaline uptake in rat cerebral cortical slices (maximal inhibition 85.7 +/- 7.8% vs. 59.8 +/- 4.3%; EC50 5.8 +/- 0.7 microM vs. 13.5 +/- 1.2 microM) and [3H]nisoxetine binding (Ki 4.5 +/- 0.5 microM vs. 8.2 +/- 0.7 microM). In contrast, R-(-)-p-synephrine was more effective than S-(+)-p-synephrine in stimulation of [3H]noradrenaline release from rat cerebral cortical slices (maximal stimulation 23.9 +/- 1.8% vs. 20.1 +/- 1.7%; EC50 8.2 +/- 0.6 microM vs. EC50 12.3 +/- 0.9 microM). The stimulatory effect of R-(-)-p-synephrine on [3H]noradrenaline release was inhibited by nisoxetine (100 nM), but tetrodotoxin (1 microM) and elimination of extracellular calcium had no effect. It is suggested that S-(+)-p-synephrine has more effective antidepressant-like activity than R-(-)-p-synephrine.

Adrenergic Uptake Inhibitors↗

PubMed2001

Investigation of juxtasellar and cerebellopontine angle meningiomas and neurogenic tumours: two-phase helical CT.

We performed two-phase helical CT in 31 patients with juxtasellar region and cerebellopontine angle tumours to evaluate its usefulness in differentiating meningiomas from neurogenic tumours. After the intravenous injection of 90 ml contrast medium at 3 ml/s, axial helical images were obtained with delays of 30 and 120 s. After the delayed axial images, we acquired coronal images. Changes in attenuation were assessed visually and quantitatively (by comparing the attenuation in Hounsfield units). There were 17 meningiomas and 14 neurogenic tumours, all pathologically proven. Two-phase helical CT showed a decrease in attenuation in 15 (88%) meningiomas and an increase in 14 (100%) neurogenic tumours from early to delayed axial images. Coronal images showed a decrease in attenuation in all 17 meningiomas and an increase in 13 (93%) of the neurogenic tumours. The mean HU and their ratios were significantly different between meningiomas and neurogenic tumours.

Adult↗

PubMed2001

The mechanism of action of coculture on embryo development in the mouse model: direct embryo-to-cell contact and the removal of deleterious components.

OBJECTIVE: To elucidate the mechanism for the mode of action of coculture by the use of a coculture system for mouse one-cell embryos with human oviductal epithelial cells. DESIGN: Prospective, controlled in vitro experimental study. SETTING: Academic research laboratory. ANIMAL(S): Female ICR strain mice aged between 6 and 8 weeks. INTERVENTION(S): Flushed one-cell embryos were cultured in human tubal fluid medium alone (control), in coculture system with human oviductal cells, in five kinds of conditioned media, and in a contactless coculture system using a cell-culture insert. MAIN OUTCOME MEASURE(S): The percentage of the embryos developed to hatching blastocyst stage and the level of superoxide anion in the supernatant from each culture condition. RESULT(S): The rates of embryo development to the hatching blastocyst stage were significantly higher in the coculture group (43%) than in the control group (none) (P <.05). The embryo development rate in the control group was similar to that of the embryos in the five kinds of conditioned media. The effects of coculture on embryo development disappeared in the contactless coculture group. The level of superoxide anion was significantly reduced in the coculture group compared to the control group. CONCLUSION(S): The present coculture system overcomes the two-cell block in vitro and improves the embryo development. The beneficial effect may be a result of direct cell-to-cell contact between the embryo and helper cells and the removal of deleterious components from medium, rather than a result of embryotrophic factors.

Animals↗

PubMed2001

Anti-inflammatory effect of the aqueous extract from Lonicera japonica flower is related to inhibition of NF-kappaB activation through reducing I-kappaBalpha degradation in rat liver.

We investigated the anti-inflammatory effects of aqueous extract from Lonicera japonica flower (AELJ), a traditional skin rash drug, in lipopolysaccharide (LPS)-induced rat liver sepsis. Immunoblot analysis showed that the level of nuclear factor (NF)-kappaBp65 was rapidly up-regulated and inhibitory (I)-kappaBalpha was down-regulated by LPS challenge. However, AELJ inhibited the increase of NF-kappaBp65 and degradation of I-kappaBalpha in the liver of LPS-challenged rats. Immunohistochemical analysis of rat hepatocytes showed that LPS-induced inflammatory responses, involving degradation of I-kappaBalpha and induction of NF-kappaBp65, tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS), are partially inhibited by pretreatment with AELJ. These results suggest that AELJ may act as a therapeutic agent for inflammatory disease through a selective regulation of NF-kappaB activation.

Animals↗

PubMed2001

Lateralizing ability of single-voxel proton mr spectroscopy in hippocampal sclerosis: comparison with mr imaging and positron emission tomography.

BACKGROUND AND PURPOSE: Proton MR spectroscopy (MRS) is still in the early stages in the evaluation of epilepsy, and comparisons with MR imaging and positron emission tomography (PET) in the same patients have rarely been documented. The purpose of this study was to evaluate the lateralizing ability of single-voxel MRS in comparison with MR imaging and PET in patients with hippocampal sclerosis. METHODS: Thirty-three patients with intractable temporal lobe epilepsy whose MR imaging diagnosis was unilateral hippocampal sclerosis and who underwent anterior temporal lobectomy and had good postsurgical outcome over 1-year follow-up were included in the study. MR spectra were obtained from the hippocampus bilaterally, using the point-resolved spectroscopy sequence. Metabolite ratios of NAA/Cho and NAA/Cr were calculated from the relative peak height measurements. An NAA/Cho ratio of 0.8 or less and an NAA/Cr ratio of 1.0 or less were regarded as abnormal. The MRS results were compared retrospectively with those of MR imaging and PET as to the ability to lateralize the epileptogenic focus. RESULTS: The sensitivity of MRS and PET (concordance with MR imaging) was 85% each in the lateralization of the ipsilateral lesion side. Bilateral abnormalities were seen in 30% of the patients. False-lateralization rates for MRS and PET were 3% and 6%, respectively. The concordance rate of MRS and PET was 73%, when comparing the results of the ipsilateral lesion side. CONCLUSION: MRS may be used as an adjunct tool in the evaluation of hippocampal sclerosis, like PET, although its sensitivity has to be improved and the clinical significance of bilateral abnormality is still to be determined.

Adolescent↗

PubMed2000

Fluorescence quenching: A tool for single-molecule protein-folding study.

By using titin as a model system, we have demonstrated that fluorescence quenching can be used to study protein folding at the single molecule level. The unfolded titin molecules with multiple dye molecules attached are able to fold to the native state. In the native folded state, the fluorescence from dye molecules is quenched due to the close proximity between the dye molecules. Unfolding of the titin leads to a dramatic increase in the fluorescence intensity. Such a change makes the folded and unfolded states of a single titin molecule clearly distinguishable and allows us to measure the folding dynamics of individual titin molecules in real time. We have also shown that fluorescence quenching can signal folding and unfolding of a small protein with only one immunoglobulin domain.

Animals↗

PubMed2000

Implication of a small GTPase Rac1 in the activation of c-Jun N-terminal kinase and heat shock factor in response to heat shock.

Heat shock induces c-Jun N-terminal kinase (JNK) activation as well as heat shock protein (HSP) expression through activation of the heat shock factor (HSF), but its signal pathway is not clearly understood. Since a small GTPase Rac1 has been suggested to participate in the cellular response to stresses, we examined whether Rac1 is involved in the heat shock response. Here we show that moderate heat shock (39-41 degrees C) induces membrane translocation of Rac1 and membrane ruffling in a Rac1-dependent manner. In addition, Rac1N17, a dominant negative mutant of Rac1, significantly inhibited JNK activation by heat shock. Since Rac1V12 was able to activate JNK, it is suggested that heat shock may activate JNK via Rac1. Similar inhibition by Rac1N17 of HSF activation in response to heat shock was observed. However, inhibitory effects of Rac1N17 on heat shock-induced JNK and HSF activation were reduced as the heat shock temperature increased. Rac1N17 also inhibited HSF activation by l-azetidine-2-carboxylic acid, a proline analog, and heavy metals (CdCl)), suggesting that Rac1 may be linked to HSF activation by denaturation of polypeptides in response to various proteotoxic stresses. However, Rac1N17 did not prevent phosphorylation of HSF1 in response to these proteotoxic stresses. Interestingly, a constitutively active mutant Rac1V12 did not activate the HSF. Therefore, Rac1 activation may be necessary, but not sufficient, for heat shock-inducible HSF activation and HSP expression, or otherwise a signal pathway(s) involving Rac1 may be indirectly involved in the HSF activation. In sum, we suggest that Rac1 may play a critical role(s) in several aspects of the heat shock response.

Animals↗

PubMed2000

Direct binding of hepatitis B virus X protein and retinoid X receptor contributes to phosphoenolpyruvate carboxykinase gene transactivation.

The X gene product of the human hepatitis B virus (HBx), a major factor responsible for hepatitis and hepatocellular carcinoma, modulates transactivation by a variety of transcription factors. Herein, expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene was found to be regulated transcriptionally by HBx through two distinct promoter regions. The cAMP response element (CRE)-1 site within the proximal promoter region mediated the HBx-induced transactivation of the PEPCK gene through C/EBP alpha and ATF-2. A retinoid X receptor (RXR) response element within the distal promoter region also contributed to the HBx-induced transactivation. Consistent with these results, HBx directly interacted with RXR, and the interaction interfaces were localized to the transactivation domain of HBx and the ligand binding domain of RXR.

Activating Transcription Factor 2↗

PubMed2000

Uroguanylin treatment suppresses polyp formation in the Apc(Min/+) mouse and induces apoptosis in human colon adenocarcinoma cells via cyclic GMP.

The enteric peptides, guanylin and uroguanylin, are local regulators of intestinal secretion by activation of receptor-guanylate cyclase (R-GC) signaling molecules that produce cyclic GMP (cGMP) and stimulate the cystic fibrosis transmembrane conductance regulator-dependent secretion of Cl- and HCO3-. Our experiments demonstrate that mRNA transcripts for guanylin and uroguanylin are markedly reduced in colon polyps and adenocarcinomas. In contrast, a specific uroguanylin-R-GC, R-GCC, is expressed in polyps and adenocarcinomas at levels comparable with normal colon mucosa. Activation of R-GCC by uroguanylin in vitro inhibits the proliferation of T84 colon cells and elicits profound apoptosis in human colon cancer cells, T84. Therefore, down-regulation of gene expression and loss of the peptides may interfere with renewal and/or removal of the epithelial cells resulting in the formation of polyps, which can progress to malignant cancers of the colon and rectum. Oral replacement therapy with human uroguanylin was used to evaluate its effects on the formation of intestinal polyps in the Min/+ mouse model for colorectal cancer. Uroguanylin significantly reduces the number of polyps found in the intestine of Min/+ mice by approximately 50% of control. Our findings suggest that uroguanylin and guanylin regulate the turnover of epithelial cells within the intestinal mucosa via activation of a cGMP signaling mechanism that elicits apoptosis of target enterocytes. The intestinal R-GC signaling molecules for guanylin regulatory peptides are promising targets for prevention and/or therapeutic treatment of intestinal polyps and cancers by oral administration of human uroguanylin.

Adenocarcinoma↗

PubMed2000

Identification of p53 gene mutations in breast cancers and their effects on transcriptional activation function.

Somatic mutations in the p53 tumor suppressor gene are the most common genetic alterations found in human malignancies. In the present study, we studied 36 primary human breast carcinomas, using a polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and sequencing analysis of exons 2 through 9 for the presence of p53 gene mutations. Six of 36 (17%) breast cancers contained mutations within the core domain of the p53 protein responsible for sequence-specific DNA binding (codons 102-292); all 5 missense mutations clustered between codons 240 and 291 (codons 240, 243, 250, 285, and 291), whereas one nonsense mutation occurred at codon 199. By using recombinant PCR in vitro mutagenesis, we introduced point mutations at codons 199 from Gly to stop (gly199stop), 240 from Ser to Ile (ser240Ile), 250 from Pro to Ala (pro250ala), 285 from Glu to Lys (glu285lys), and 291 from Lys to Asn (lys291asn), and all the p53 sequences were subcloned into the CMVneoBam vector under the control of the cytomegalovirus (CMV) promoter. To test whether the mutants p53 were functionally wild-type (wt) or mutant, we transfected them to p53-null Saos-2 cells with a reporter plasmid containing a p53-responsive element, and performed chloramphenicol acetyltransferase (CAT) assay. Transient CAT assay for transcriptional activation revealed that one group, including gly199stop, ser240ile, glu285lys, and lys291asn, abolished the transcriptional activity, whereas the other group, including pro250ala, retained stronger transcriptional transactivation activity than that of wt p53.

Adult↗

PubMed2000

Furosemide stimulates K transport in HCD57 erythroid cells.

We examined the influence of serum and furosemide on K movement and cell volume in HCD57 cells, a murine erythroleukemia cell line, which require erythropoietin (EPO) for survival. We found that maintenance of cell volume depends on the concentration of serum in the culture medium. In isotonic medium containing 20% serum, HCD57 cells maintain their steady-state volume. In contrast, the cells shrink progressively as medium serum content is reduced. In serum-free medium, raising external K to 75 mm prevents cell shrinkage and a further increase in K to 145 mm results in swelling, revealing a role for K permeability in the regulation of cell volume. Of particular interest has been a serendipitous finding with furosemide. Below an external K concentration of 2.1 +/- 0.3 mm in medium containing 2% serum, furosemide inhibits K uptake, probably stemming from its well known inhibitory action on KCl cotransport. However, above that K concentration, furosemide stimulates K uptake in a dose-dependent manner. Moreover, furosemide potentiates cell shrinkage induced by serum withdrawal. These findings suggest that the transport machinery mediating cellular shrinkage, once primed by serum depletion, becomes receptive to a second stimulus.

Animals↗

PubMed2000

Involvement of SPARC in in vitro differentiation of skeletal myoblasts.

SPARC (secreted protein acidic and rich in cysteine) is an extracellular Ca(2+)-binding glycoprotein associated with the morphogenesis and remodeling of various tissues. Here, involvement of SPARC in the myogenesis of skeletal myoblasts was investigated in vitro. First, the differential expression of SPARC mRNA during the myogenesis was initially identified by a differential display reverse transcription (DDRT)-PCR method. The expression of the SPARC gene was significantly up-regulated during the differentiation of C2C12 mouse myoblasts. Second, the treatment with anti-SPARC antibody almost completely prevented the differentiation of myoblasts. Third, the treatment with EGTA, a Ca(2+) chelator that is known to inhibit the fusion of C2C12 myoblasts, reversibly inhibited the up-regulation of SPARC gene expression. On the other hand, the treatment with A23187, a Ca(2+) ionophore, rapidly and dramatically increased the level of SPARC transcript. Taken together, these results suggest that SPARC may play a critical role(s) in the morphological change of myoblasts, and that the expression of SPARC gene may be controlled by Ca(2+)-dependent pathway in myogenesis.

Animals↗

PubMed2000

Identification of a novel human member of the DEAD box protein family.

The cDNA library of human pancreatic islets was screened with sera from patients with insulin-dependent diabetes mellitus (IDDM). From the library screening, we isolated a novel cDNA, RNA helicase-like protein (RHELP), which exhibited strong sequence homology to p68 RNA helicase, a prototypic member of the DEAD (Asp-Glu-Ala-Asp) box protein family. Sequence analysis of the cDNA revealed that RHELP contained DEAD sequence motif and other conserved motifs of the DEAD box protein family, indicating that RHELP is a new member of this family. DEAD box-containing proteins are involved in the RNA processing, ribosome assembly, spermatogenesis, embryogenesis, and cell growth and division. RHELP showed 42% and 44% amino acid sequence identity to human p68 RNA helicase and yeast DBP2 RNA helicase, respectively, among the DEAD box protein family. Northern blot analysis revealed that RHELP is expressed in most tissues including the liver, lung, tonsil, thymus, and muscle in addition to the pancreatic islets. In vivo or in vitro functions of RHELP as a putative RNA helicase and its potential role as a diabetic autoantigen need to be further investigated.

Amino Acid Sequence↗