Biomedical subjects

H Park

Publications and source records attributed to H Park.

At least 163 records · Page 9Linked to original sources

PubMed1999

Differential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB mRNA sequence.

Previously we demonstrated that the yield of Cry3A (70 kDa) can be increased as much as 10-fold when cry3A including its upstream STAB-SD mRNA stabilizing sequence is expressed in Bacillus thuringiensis under the control of cyt1A promoters. To determine whether the cyt1A promoters/STAB-SD combination (cyt1AP/STAB) has broader applicability, we used it to synthesize two other Cry endotoxins in the 70-kDa mass range, Cry2A and Cry11A. Combination of cyt1AP/STAB with orfs 2 and 3 of the cry2A operon yielded about 4. 4-fold the amount of Cry2A obtained with the wild-type cry2A operon. The yield of Cry11A obtained with a construct that contained the cyt1AP/STAB, cry11A and the 20-kDa protein gene was 1.3-fold the amount obtained with a construct similar to the wild-type operon. These results demonstrate that the cyt1AP/STAB combination can enhance synthesis of different Cry proteins significantly, but that the level of enhancement varies with the specific protein synthesized.

Animals↗

PubMed1999

Long-term analysis of differentiation in human myoblasts repopulated with mitochondria harboring mtDNA mutations.

Short-term analysis of myogenesis in respiration-deficient myoblasts demonstrated that respiratory chain dysfunction impairs muscle differentiation. To investigate long-term consequences of a deficiency in oxidative phosphorylation on myogenesis, we quantitated myoblast fusion and expression of sarcomeric myosin in respiration-deficient myogenic cybrids. We produced viable myoblasts harboring exclusively mtDNA with large-scale deletions by treating wild-type myoblasts with rhodamine 6G and fusing them with cytoplasts homoplasmic for two different mutated mtDNAs. Recovery of growth in transmitochondrial myoblasts demonstrated that respiratory chain function is not required for recovery of rhodamine 6G-treated cells. Both transmitochondrial respiration-deficient cultures exhibited impaired myoblast fusion. Expression of sarcomeric myosin was also delayed in deficient myoblasts. However, 4 weeks after induction of differentiation, one cell line was able to quantitatively recover its capacity to form postmitotic muscle cells. This indicates that while oxidative phosphorylation is an important source of ATP for muscle development, myoblast differentiation can be supported entirely by glycolysis.

Biomarkers↗

PubMed1999

Synthesis and in vitro cytotoxicity of hexacyclic camptothecin analogues.

A series of C(7)-N-alkylaminoethyl-C(10), C(11)-methylenedioxy- and ethylenedioxy-camptothecin (3a-g, 4a-b) were prepared. Their syntheses and in vitro cytotoxicity were reported. Among 15 derivatives, 3a and 3b showed more potent cytotoxicity than Camptothecin, especially in CAOV-3 cell line.

Antineoplastic Agents↗

PubMed1999

Cloning of a cysteine proteinase gene from Acanthamoeba culbertsoni.

Free living amoeba, including pathogenic Acanthamoeba culbertsoni, are widely distributed in soil and fresh water. It has been found that cysteine proteinases are more active in pathogenic strains of amoeba whereas serine proteinases are found in both pathogenic and nonpathogenic strains. Cysteine proteinases thus play important roles in the pathogenesis of several parasitic infections and have been proposed as targets for the structure-based strategy of drug design. As the first step toward applying this strategy to design inhibitors as antiparasitic agents for A. culbertsoni, we isolated and sequenced the full length clone of a cysteine proteinase gene from A. culbertsoni by performing reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotide primers derived from conserved cysteine proteinase sequences. The 5' and the 3' regions of the cysteine proteinase gene were amplified using the PCR protocol for the rapid amplification of cDNA ends (RACE). It has an open reading frame of 1359 bp. The deduced amino acid sequence has the sequence homology with the cysteine proteinase genes of Paragonimus westermani metacercaria, Schistosoma mansoni, human cathepsin L and Fasciola hepatica, each by 45.3%, 45.9%, 57.9% and 50.8% respectively. Sequence analysis and alignment showed significant similarity to other eukaryotic cysteine proteinases, including the conservation of the cysteine, histidine, and asparagine residues that form the catalytic triad. A 1.5 kbp mRNA was detected on Northern blot analysis using full-length cysteine proteinase cDNA as a probe. The A. culbertsoni cysteine proteinase gene (AcCP2) was found to contain Ex3Rx3Wx2N at the proregion and also a proline/threonine-rich C-terminal extension. Therefore, it has cathepsin L-like characteristics. Phylogenetic analysis based on the amino acid sequences of cysteine proteinase indicated that AcCP2 was closely related with papaya, while it was remotely related with those of Schistosoma.

Acanthamoeba↗

PubMed1999

A new sulfated beta-galactan from clams with anti-HIV activity.

A new polysaccharide composed of galactan sulfate with a beta-(1-->3)-glycosidic linkage has been isolated from the marine clam species Meretrix petechialis. The polysaccharide was homogeneous in its composition containing D-galactose. The glycosidic linkage was examined by 2D DQF-COSY and 2D NOESY spectroscopy. The coupling constant of anomeric proton was 7.8 Hz, suggesting a beta-galacto configuration. The downfield shift of H-2 of galactose residue demonstrated the presence of 2-O-sulfonate group. TQF-COSY confirmed that the C-6 position was substituted with a sulfonate group. The anti-HIV activity of the polysaccharides has been evaluated by the inhibition of syncytia formation. The fusion index and percentage fusion inhibition of sulfated galactan were 0.34 and 56% at 200 micrograms/mL.

Animals↗

PubMed1999

Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (alphaIIbbeta3) and other proteins from hamster-mouse interspecies hybridomas.

To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from beta3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.

Animals↗

PubMed1999

Aromatic hydrocarbon nuclear translocator as a common component for the hypoxia- and dioxin-induced gene expression.

Aromatic hydrocarbon nuclear translocator (Arnt) is an ubiquitously expressed protein that contains basic helix-loop-helix (bHLH) and Per-AhR-Arnt-Sim (PAS) motifs. Other bHLH-PAS proteins, hypoxia-inducible factor-1alpha (HIF-1alpha) and aromatic hydrocarbon receptor (AhR) mediate hypoxia- and dioxin-signal pathway, respectively. Arnt has been identified as a heterodimerization partner for AhR. AhR/Arnt heterodimer binds the regulatory region of xenobiotic-induced genes and activates their transcription. Here, in vivo results provide evidence that Arnt is involved in not only xenobiotic- but also hypoxia-induced transcriptional activation. In hypoxic condition, Arnt dimerizes with HIF-1alpha to make HIF-1alpha/Arnt heterodimer which is able to bind hypoxia-responsive DNA elements. The HIF-1alpha/Arnt heterodimer functions as a transactivator for hypoxia-inducible genes. Given that the expression of Arnt is limited, HIF-1alpha may compete with AhR for recruiting Arnt as a heteromeric partner. Consistent with this idea, the results indicate that the hypoxic activation of HIF-1alpha reduces dioxin-induced AhR's function on the dioxin-responsive reporter gene and the endogenous gene.

Animals↗

PubMed1999

Cysteine carboxyl O-methylation of human placental 23 kDa protein.

C-Terminal carboxyl methylation of a human placental 23 kDa protein catalyzed by membrane-associated methyltransferase has been investigated. The 23 kDa protein substrate methylated was partially purified by DEAE-Sephacel, hydroxyapatite and Sephadex G-100 gel filtration chromatographies. The substrate protein was eluted on Sephadex G-100 gel filtration chromatography as a protein of about 29 kDa. In the absence of Mg2+, the methylation was stimulated by guanine nucleotides (GTP, GDP and GTPgammaS), but in the presence of Mg2+, only GTPgammaS stimulated the methylation which was similar to the effect on the G25K/rhoGDI complex. AFC, an inhibitor of C-terminal carboxyl methylation, inhibited the methylation of human placental 23 kDa protein. These results suggests that the substrate is a small G protein different from the G25K and is methylated on C-terminal isoprenylated cysteine residue. This was also confirmed by vapor phase analysis. The methylated substrate protein was redistributed to membrane after in vitro methylation, suggesting that the methylation of this protein is important for the redistribution of the 23 kDa small G protein for its putative role in intracellular signaling.

Cysteine↗

PubMed1999

Oral vaccination of animals with antigens encapsulated in alginate microspheres.

Most infectious diseases begin at a mucosal surface. Prevention of infection must therefore consider ways to enhance local immunity to prevent the attachment and invasion of microbes. Despite this understanding, most vaccines depend on parenterally administered vaccines that induce a circulating immune response that often does not cross to mucosal sites. Administration of vaccines to mucosal sites induces local immunity. To be effective requires that antigen be administered often. This is not always practical depending on the site where protection is needed, nor comfortable to the patient. Not all mucosal sites have inductive lymphoid tissue present as well. Oral administration is easy to do, is well accepted by humans and animals and targets the largest inductive lymphoid tissue in the body in the intestine. Oral administration of antigen requires protection of antigen from the enzymes and pH of the stomach. Polymeric delivery systems are under investigation to deliver vaccines to the intestine while protecting them from adverse conditions that could adversely affect the antigens. They also can enhance delivery of antigen specifically to the inductive lymphoid tissue. Sodium alginate is a readily available, inexpensive polymer that can be used to encapsulate a wide variety of antigens under mild conditions. Orally administered alginate microspheres containing antigen have successfully induced immunity in mice to enteric (rotavirus) pathogens and in the respiratory tract in cattle with a model antigen (ovalbumin). This delivery system offers a safe, effective means of orally vaccinating large numbers of animals (and perhaps humans) to a variety of infectious agents.

Administration, Oral↗

PubMed1999

Identification and characterization of the genes for two topoisomerase I-interacting proteins from Saccharomyces cerevisiae.

The two-hybrid system was used to identify yeast genes encoding proteins that interact with DNA topoisomerase I. Two new genes were found and named TOF1 and TOF2. The Tof1 protein has 1238 amino acids and no obvious homologues in databases. The Tof2 protein has 771 amino acids; it is quite closely related to another yeast protein, the product of an uncharacterized ORF. Both tof1 and tof2 null mutants, and tof1-tof2 double mutants, grow normally and have normal levels of topoisomerase I activity. TOF2 shows various genetic interactions with TOP1 and HPR1. The implications of these interactions for TOF2 function are discussed.

DNA Topoisomerases, Type I↗

PubMed1999

Synthesis of superporous hydrogels: hydrogels with fast swelling and superabsorbent properties.

We have been interested in the synthesis of hydrogels with fast swelling kinetics and superabsorbent properties. To increase the water absorption rate, interconnected pores were introduced to the hydrogels. Since the pore size in the dried hydrogels is in the order of hundreds of micrometers, these hydrogels are called "superporous" hydrogels. Superporous hydrogels were synthesized by crosslinking polymerization of various vinyl monomers in the presence of gas bubbles formed by the chemical reaction of acid and NaHCO3. The polymerization process was optimized to capture the gas bubbles inside the synthesized hydrogels. The use of the NaHCO3/acid system allowed easy control of timing for gelation and foam formation. We found that PF127 was the best foam stabilizer for most of the monomer systems used in our study. Scanning electron microscope (SEM) pictures showed interconnected pores forming capillary channels. The capillary channels, which were critical for fast swelling, were preserved during drying by dehydrating water-swollen hydrogels with ethanol before drying. The ethanol-dehydrated superporous hydrogels reached equilibrium swelling within minutes. The equilibrium swelling time could be reduced to less than a minute with the use of a wetting agent. In our study, water moisture was used as a wetting agent since the amount of moisture content in the dried hydrogels easily could be controlled. Preparation of superporous hydrogels using the right blowing system, foam stabilizer, drying method, and wetting agent makes it possible to reduce the swelling time to less than a minute regardless of the size of the dried gels. The superporous hydrogels can be used where fast swelling and superabsorbent properties are critical.

Absorption↗

PubMed1999

Use of sulfite resistance in Saccharomyces cerevisiae as a dominant selectable marker.

Two S. cerevisiae genes were found to exhibit dominant phenotypes useful for selecting transformants of industrial and laboratory strains of S. cerevisiae. FZF1-4, which confers sulfite resistance, was originally isolated and identified as RSU1-4, but the two genes are shown here to be allelic. Cysteine 57 in wild-type Fzf1p was found to be replaced by tyrosine in Fzf1-4p. Multicopy SSU1, which also confers sulfite resistance, was found to be somewhat less efficient. In both cases, a period of outgrowth in non-selective medium following transformation was found to be necessary. The number of transformants obtained was found to be strain-dependent, and also to depend on the sulfite concentration used during selection. Undesirable background growth of non-transformants was not observed at cell densities as high as 2.5 x 10(7)/plate. In two ura3 laboratory strains where selection for URA3 was applied independently of that for sulfite, the transformation efficiency for sulfite resistance was about 50% that for uracil prototrophy.

Anion Transport Proteins↗

PubMed1999

Experimental rabies virus infection of p75 neurotrophin receptor-deficient mice.

The low-affinity neurotrophin (NT) receptor, p75(NTR), has complex biologic functions. A recent report provided evidence that the p75(NTR) is a rabies virus receptor in cultured BSR cells. We studied the experimental infection of 6-day-old p75(NTR)-deficient mice with the challenge virus standard strain of fixed rabies virus inoculated intracerebrally. The mice developed a fatal encephalitis. There were morphologic changes of apoptotic cell death involving neurons in widespread areas of the brain, which were associated with in situ evidence of oligonucleosomal DNA fragmentation. The findings were very similar to those that we previously reported in wild-type ICR mice of the same age. If the p75(NTR) is an important receptor of rabies virus in animal hosts, then a greater effect on the clinical and pathologic features of rabies virus-infected p75(NTR)-deficient mice would have been expected.

Animals↗

PubMed1999

Neuromuscular control of esophageal peristalsis.

The esophagus is a muscular conduit connecting the pharynx and the stomach. Its function is controlled by an intrinsic nervous system and by input from the central nervous system through the vagus nerve. Peristalsis in its striated muscle is directed by sequential vagal excitation arising in the brain stem, whereas peristalsis in its smooth muscle involves complex interactions among the central and peripheral neural systems and the smooth muscle elements of the esophagus. The peripheral neuronal elements responsible for producing esophageal off-response, relaxation of the lower esophageal sphincter, and hyperpolarization of the circular esophageal muscle cells reside in the myenteric plexus of the esophagus. For many years these nerves were considered nonadrenergic and noncholinergic because the inhibitory neurotransmitter released on their activation was unknown. We now know that nitric oxide or a related compound is that inhibitory neurotransmitter. The primary excitatory neurotransmitter controlling esophageal motor function is acetylcholine. Some disorders of esophageal motor function, including diffuse esophageal spasm and achalasia, may result from defects in or an imbalance between these excitatory and inhibitory neuromuscular systems.

Acetylcholine↗

PubMed1999

In vitro system to study role of blood flow on nitric oxide production and cell signaling in endothelial cells.

Through the approaches illustrated in this article we have demonstrated how molecular signaling events can be elucidated in cells responding to physiological forces. With the recent findings that endothelial form of nitric oxide synthase is associated with proteins such as caveolin and the availability of these cDNA constructs, this methodology allows a possible avenue to determine the physiological significance of such associations and the regulation of NO formation in response to shear stress.

Animals↗

PubMed1999

Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ.

Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.

Amino Acid Sequence↗

PubMed1999

Enhanced basophil histamine release and neutrophil chemotactic activity predispose grain dust-induced airway obstruction.

BACKGROUND: The pathogenic mechanism of grain dust (GD)-induced occupational asthma (OA) remains unclear. OBJECTIVE: To understand further the mechanism of GD-induced OA. METHODS: Fifteen employees working in a same GD industry, complaining of work-related respiratory symptoms, were enrolled and were divided into two groups according to the GD-bronchoprovocation test (BPT) result: six positive responders were grouped as group III, nine negative responders as group II and five healthy controls as group I. Serum GD-specific immunoglobulin (Ig)E (sIgE), specific IgG (sIgG) and specific IgG4 (sIgG4) antibodies were detected by enzyme-linked immunosorbent assay. Basophil histamine release was measured by the autofluorometric method, and changes of serum neutrophil chemotactic activity were observed by the Boyden chamber method. RESULTS: For clinical parameters such as degree of airway hyperresponsiveness to methacholine, duration of respiratory symptoms, exposure duration, and prevalences of serum sIgE, sIgG and sIgG4 antibodies, there were no significant differences between group II and III (P > 0.05, respectively). Serum neutrophil chemotactic activity increased significantly at 30 min and decreased at 240 min after the GD-BPT in group III subjects (P < 0.05, respectively), while no significant changes were noted in group II subjects (P > 0.05). Basophil histamine release induced by GD was significantly higher in group III than those of group I or group II (P < 0.05, respectively), while minimal release of anti-IgG4 antibodies was noted in all three groups. CONCLUSIONS: These results suggest that enhanced basophil histamine release and serum neutrophil chemotactic activity might contribute to the development of GD-induced occupational asthma.

Adult↗

PubMed1999

Neutrophil activation following TDI bronchial challenges to the airway secretion from subjects with TDI-induced asthma.

BACKGROUND: The immunopathological mechanism for occupational asthma induced by toluene diisocyanate (TDI) remains to be further clarified. There have been few reports suggesting involvement of neutrophils in inducing bronchoconstriction after TDI inhalation. OBJECTIVES: To further understand the role of neutrophils in the pathogenesis of TDI-induced asthma. MATERIALS AND METHODS: Eight TDI-induced asthmatic subjects were classified as group I, and five exposed workers who had complained of work-related symptoms and worked in the same workplace, but showed negative bronchial challenges were enrolled as controls (group II). Serum neutrophil chemotactic activity during TDI bronchial challenge test was measured by the Boyden chamber method. Induced sputum was collected before and after the TDI bronchial challenge test. The myeloperoxidase (MPO) and interleukin (IL) -8 levels in the sputum were measured using RIA and ELISA. RESULT: Serum neutrophil chemotactic activity significantly increased at 10 min (P = 0.01), then decreased at 60 min (P = 0.02) and remained unchanged for up to 420 min (P = 0.07) in group I subjects, while no significant changes were found in group II subjects (P > 0.05). MPO and IL-8 were abundantly present in the sputum of all the TDI-induced asthmatic subjects and they increased significantly at 420 min after the bronchial challenges (P = 0.02, P = 0.03, respectively), while no significant changes were noted in group II subjects (P > 0.05). CONCLUSION: These findings support the view that activated neutrophils may contribute to bronchoconstriction induced by TDI which may be associated with IL-8 release.

Adult↗