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Biomedical subjects

Y Hong

Publications and source records attributed to Y Hong.

At least 163 records · Page 9Linked to original sources
PubMed1997

Expression of the spinach betaine aldehyde dehydrogenase (BADH) gene in transgenic tobacco plants.

Plasmid pLS9 contains a 1.5-kb of spinach cDNA including its complete open reading frame. The 1.5-kb BADH cDNA was cut from pLS9 using restriction enzyme and was inserted into the expression cassette of plasmid pYH between the CaMV 35S promoter and polyA signal sequence. The 35S-BADH cDNA-polyA fragment of pYH was cloned into a polylinker cloning site of the binary vector pBin19. The resulting plasmid pBinBADH-S was transferred to Agrobacterium tumefacies LBA4404. The tobacco plants were transformed with strain LBA4404 containing pBinBADH-S, and more than ninety kanamycin-resistant transformants were selected. Polymerase chain reaction (PCR) detection showed that more than 60% of the transformed tobacco plants contained the foreign BADH gene. The Western blot analysis, BADH enzymatic assay, specific stain for BADH activity, and the test for salt tolerance showed that BADH gene was normally expressed in the transgenic tobacco plants. The BADH enzymes also presented in chloroplasts and cytosol of the transgenic plants. The transgenic tobacco plants having strong expression of BADH gene had strong ability to tolerate high salt stress.

Aldehyde Dehydrogenase↗
PubMed1997

[Comparison of cardiopulmonary endurance and muscular fitness in teenagers between Hong Kong and inland cities].

A study on the data of the physique investigated in teenagers was carried out between Hong Kong and inland cities to compare their cardiopulmonary endurance and muscular fitness. Results revealed that cardiopulmonary endurance in school teenagers of both sex at different ages in inland cities was better than that in Hong Kong. Muscular strength and endurance of sports performance of teenagers, except for standing long jump, in Hong Kong were weaker than that in inland cities. It suggests that attention should be paid to the involvement of teenagers in physical education with the increase of people's living standard.

Adolescent↗
PubMed1996

RNA polymerase activity catalyzed by a potyvirus-encoded RNA-dependent RNA polymerase.

We have expressed the putative RNA-dependent RNA polymerase encoded by the potyvirus tobacco vein mottling virus (TVMV) in Escherichia coli as a glutathione S-transferase fusion protein. As prepared, the fusion protein possessed the poly(U) polymerase activity that is a hallmark of other picornavirus-encoded polymerases. In addition, this protein was able to utilize full-length TVMV RNA as a template for RNA synthesis. A fusion protein containing a mutation in the highly conserved GDD motif of the polymerase (GDD-->ADD) possessed 7% of the activity of the wild type. Our results confirm that the presumed polymerase encoded by TVMV is in fact an RNA-dependent RNA polymerase and that the GDD motif so widely seen in viral polymerases has an important function in the TVMV protein.

DNA-Directed RNA Polymerases↗
PubMed1996

A glass fiber/diethylaminoethyl double filter binding assay that measures apoptotic internucleosomal DNA fragmentation.

A filter binding assay that measures internucleosomal DNA fragmentation associated with apoptosis is described. The assay is based on a novel principle that consists of using simultaneously two kinds of glass fiber filters to harvest [3H]thymidine-prelabeled cells following their incubation with inducers of apoptosis. One filter, which is neutral, traps intact chromatin and high-molecular-weight DNA. The other filter, which is positively charged with DEAE active groups, traps low-molecular-weight DNA fragments. DNA fragmentation is quantified by measuring the radioactivity retained by each of the filters. The assay was evaluated with the histiocytic lymphoma cell line U937 and the topoisomerase inhibitors camptothecin, etoposide, and doxorubicin. These agents caused a dose-dependent decrease of radioactivity in the neutral filter and a parallel increase of radioactivity in the DEAE filter. Irradiation-induced single strand breaks and topoisomerase-mediated primary DNA damage were not detected by this method. Consistent with the detection of internucleosomal DNA fragmentation, the effects measured by this assay were prevented by the endonuclease inhibitor zinc acetate and by the metabolic inhibitor sodium azide. Results obtained using this assay were validated by observation of DNA ladders on agarose gels and by morphologic examination of apoptotic features. Evaluation of the assay in a mock screen demonstrated that the introduction of the DEAE filter increases the assay sensitivity and eliminates false positives. Thus, this assay may be used in high-throughput screening approaches to discover novel modulators of apoptosis.

Antineoplastic Agents↗
PubMed1996

Resistance to geminivirus infection by virus-induced expression of dianthin in transgenic plants.

Ribosome-inactivating proteins (RIPs) are naturally occurring plant toxins that exhibit antiviral activity against a diverse range of plant and animal viruses. Here, the action of dianthin, a potent RIP isolated from Dianthus caryophyllus, has been exploited to engineer resistance to a plant DNA virus, African cassava mosaic virus (ACMV), in transgenic Nicotiana benthamiana. To achieve this, dianthin has been expressed from the ACMV virion-sense promoter that is transactivated by the product of viral gene AC2. This avoids the need for constitutive expression of the RIP, facilitating the regeneration of phenotypically normal plants, and ensures transgene expression is localized to virus-infected cells. When challenged with ACMV, transgenic plants produce atypical necrotic lesions on inoculated leaves, indicative of dianthin expression, viral DNA accumulation is significantly reduced in these tissues, and plants exhibit attenuated systemic symptoms from which they recover. This phenotype holds for isolates of ACMV but not for other geminiviruses, suggesting that AC2 homologues from the latter are unable to efficiently transactivate the ACMV promoter.

Antiviral Agents↗
PubMed1996

Contribution of peripheral alpha 1A-adrenoceptors to pain induced by formalin or by alpha-methyl-5-hydroxytryptamine plus noradrenaline.

We examined the peripheral adrenergic mechanisms involved in pain induced by alpha-methyl-5-hydroxytryptamine (alpha-methyl-5-HT) plus (+/-)-noradrenaline or prostaglandin E2 and by intraplantar formalin. Agents were injected s.c. into the plantar surface of rats' paws, and the paw lifting and licking response scored. Pain produced by alpha-methyl-5-HT (10 micrograms) plus noradrenaline (10 micrograms) was blocked by pretreatment with the alpha-adrenoceptor antagonists, phentolamine (10 micrograms) and prazocin HCl (alpha 1; 40 micrograms), but not by timolol (beta; 10 micrograms) or idazoxan (alpha 2; 40 micrograms). Phenylepherine, but not clonidine, substituted for noradrenaline to induce pain when combined with alpha-methyl-5-HT. The alpha 1A-adrenoceptor antagonist, WB-4101 (2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane HCl), but not the alpha 1B- adrenoceptor antagonist, chloroethylclonidine, also blocked the pain response produced by alpha-methyl-5-HT plus noradrenaline. Neither of these agents altered pain produced by alpha-methyl-5-HT plus prostaglandin E2 (0.1 microgram). Formalin-induced pain (1%, 50 microliters) was biphasic, and timolol increased the first phase response. The second phase was attenuated by 40% by phentolamine (10 micrograms) injected 10 min before formalin or at the beginning of the second phase; 30 micrograms did not produce a larger effect. Prazosin and WP-4101, but not idazoxan or chloroethylclonidine, also attenuated the second phase. Thus, activation of alpha 1A-adrenoceptors can contribute to pain, but pain induced by alpha-methyl-5-HT plus prostaglandin E2 is independent of adrenergic function, indicating that adrenergic function is not necessary for induction of pain by inflammatory mediators. alpha 1A-Adrenoceptor blockade attenuates pain when administered after development of pain, implying that peripheral adrenergic mechanisms contribute to ongoing maintenance of pain.

Adrenergic alpha-1 Receptor Agonists↗
PubMed1996

Inhibition of protein kinase C suppresses megakaryocytic differentiation and stimulates erythroid differentiation in HEL cells.

The bisindolylmaleimide, GF109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide ), a highly selective inhibitor of protein kinase C (PKC), was used to test the role of this enzyme in phorbol ester-induced megakaryocytic differentiation of HEL cells. Treatment of these cells with 10 nmol/L phorbol 12-myristate 13-acetate (PMA) for 3 days caused a complete inhibition of proliferation and a threefold increase in the surface expression of glycoprotein (GP) IIIa, a marker of megakaryocytic differentiation that forms part of the fibrinogen receptor complex, GPIIb/IIIa. A similar effect was observed with phorbol 12,13-dibutyrate, but not with the biologically inactive derivative PMA-4-O-methyl ether. The PMA-induced increase in GPIIIa expression was completely inhibited by GF109203X in a dose-dependent manner (IC50 = 0.5 mumol/L), with a maximal effect at 2.5 to 5.0 mumol/L. GF109203X also blocked the inhibitory effect of PMA on cell growth and inhibited PMA-stimulated phosphorylation of the 47-kD PKC substrate, pleckstrin. Incubation of HEL cells with 25 mumol/L hemin for 3 days caused a fourfold to fivefold increase in expression of the erythroid differentiation marker, glycophorin A. In contrast to the inhibitory effect of GF109203X on GPIIIa expression, hemin induction of glycophorin A was enhanced by this compound. Furthermore, GF109203X alone caused a dose-dependent increase in glycophorin A expression, and induced hemoglobinization. Consistent with these changes, Northern blot analysis revealed that GF109203X treatment reduced the steady-state level of GPIIb mRNA and increased those for glycophorin A and gamma-globin. These results suggest that PKC may act as a developmental switch controlling erythroid/megakaryocytic differentiation.

Aphidicolin↗
PubMed1996

Crosslinked poly(1-vinyl-2-pyrrolidinone) as a vitreous substitute.

A hydrogel with a high water content was produced and tested as a possible vitreous substitute. The polymer (poly[1-vinyl-2-pyrrolidinone]) (PVP) was synthesized by free radical bulk polymerization of 1-vinyl-2-pyrrolidinone (VP) in the presence of 0.25% divinyl glycol (DVG) as a crosslinking agent. The fully hydrated polymer, containing about 98% water, was clear, transparent, autoclavable, and easily injected through a small-gauge needle with minimum fragmentation, and without changes in its optical properties. Dynamic mechanical analysis of the hydrogel indicated a covalently crosslinked elastic network both before and after injection. The resilience of hydrogel decreased after being subjected to shear stress during the injection process. A cytotoxicity bioassay of the hydrogel in vitro, using cultured mouse (Balb/ c-3T3) fibroblasts, showed cytostatic but not cytocidal effects. The hydrogel was injected into the vitreous cavity of rabbits and followed up to 4 weeks. The gel was clinically well tolerated, however opacities in the vitreous body were observed following the insertion of the gel. Histopathological examination revealed no adverse reactions to the retina, but the presence of loose polymer particles indicated the possibility of the biodegradation of the polymer. These results suggest the potential use of crosslinked PVP hydrogels as vitreous substitutes, provided that their biodegradation is not significant, a matter that should be further investigated.

3T3 Cells↗
PubMed1996

Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana.

Calcium-dependent protein kinases (CDPKs) belong to a unique family of enzymes containing a single polypeptide chain with a kinase domain at the amino terminus and a putative calcium-binding EF hands structure at the carboxyl terminus. From Arabidopsis thaliana, we have cloned three distinct cDNA sequences encoding CDPKs, which were designated as atcdpk6, atcdpk9 and atcdpk19. The full-length cDNA sequences for atcdpk6, atcdpk9 and atcdpk19 encode proteins with a molecular weight of 59343, 55376 and 59947, respectively. Recombinant atCDPK6 and atCDPK9 proteins were fully active as kinases whose activities were induced by Ca2+. Biochemical studies suggested the presence of an autoinhibitory domain in the junction between the kinase domain and the EF hands structure. Serial deletion of the four EF hands of atCDPK6 demonstrated that the integrity of the four EF hands was crucial to the Ca2+ response. All the three atcdpk genes were ubiquitiously expressed in the plant as demonstrated by RNA gel blot experiments. Comparison of the genomic sequences suggested that the three cdpk genes have evolved differently. Using antibodies against atCDPK6 and atCDPK9 for immunohistochemical experiments, CDPKs were found to be expressed in specific cell types in a temporally and developmentally regulated manner.

Amino Acid Sequence↗
PubMed1996

Activation of 5-HT2A receptors potentiates pain produced by inflammatory mediators.

Previous results from our laboratory indicate that serotonin (5-HT) potentiates pain produced by other inflammatory mediators. To characterize the receptor subtype(s) mediating this synergistic effect of 5-HT, selective 5-HT agonists were injected, alone or with noradrenaline (NA) or prostaglandin E2 (PGE2), into the plantar surface of the paws of rats. The behavioural response (favouring, elevation and licking the paw) was recorded using the rating scale developed to quantify formalin-induced pain. The 5-HT1A and 5-HT3 agonists, 8-OH-DPAT and 2-methyl-5-HT, respectively, produced only transient responses by themselves and did not interact with PGE2 or NA. The 5-HT2 agonists, alpha-methyl-5-HT and DOI, also produced transient responses alone, but induced lifting and licking of the injected paw lasting more than 30 min when combined with PGE2 or NA. The lifting and licking response produced by 5-HT plus PGE2 was not altered by intraplantar pretreatment with the 5-HT1A and 5-HT3 antagonists, BMY 7378 and tropisetron, but was attenuated by the 5-HT2A/2C antagonist ketanserin. The pain response produced by alpha-methyl-5-HT plus PGE2 was blocked by pretreatment with the 5-HT2A/2C antagonists ketanserin and ritanserin, and the 5-HT2A antagonist spiperone (MPE50 values 1.4, 7.7 and 0.06 nmol, respectively). The second phase of the response to intraplantar formalin was also attenuated by ketanserin, ritanserin and spiperone (MPE50 values 11.3, 21.8 and 0.23 nmol, respectively). These data imply that 5-HT2A antagonists may be effective peripherally acting analgesics or analgesic adjuncts in pain associated with 5-HT release from platelets, such acute injury and, perhaps, some chronic pain states.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1996

Pluripotency and differentiation of embryonic stem cell lines from the medakafish (Oryzias latipes).

Small aquarium fish, like the medaka and zebrafish, offer an excellent opportunity to combine embryological, genetic and molecular analyses of vertebrate development. Pluripotent embryonic stem (ES) cells have enormous potential to study the totipotency and differentiation of cells and provide s bridge linking in vitro manipulations of the genome. In this report we describe the establishment, pluripotency and differentiation of medaka ES-like cell lines (MES). The MES cells exhibit stable growth over 18 months of culture with 100 passages using defined culture conditions in the absence of feeder layer cells. They have a normal karyotype and form colonies of densely packed, alkaline phosphatase-positive cells resembling undifferentiated mouse ES cells. In suspension culture they form embryoid bodies, and under appropriate conditions, differentiate into a variety of cell types.

Alkaline Phosphatase↗
PubMed1996

Identification of plant cytoskeletal, cell cycle-related and polarity-related proteins using Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has been used to identify Arabidopsis thaliana proteins that may play a role in cell shape maintenance or cell cycle regulation. An Arabidopsis thaliana cDNA library was constructed in pREP5N vector under the control of the inducible nmt1 promoter and transformed into S. pombe. Expression of the A. thaliana sequences was induced and clones showing severe morphological changes were identified and analysed. Comparison of the sequences of the inserts with the sequence data bases revealed that several cDNAs encode proteins known to play a role in function of the cytoskeleton, the cell cycle and establishment of cell polarity. These include alpha-1, alpha-2, alpha-6 and beta-6 tubulins, myosin heavy chain-like protein, ubiquitin conjugating enzymes UBC9 (E2), 26S protease subunits, Ranbinding protein, myb protein, PRL1 gene product and rho protein. Approximately 30% of the clones encode novel sequences. The results suggest that S. pombe phenotypic screening can be used to identify plant proteins involved in cell shape maintenance and regulation during cell cycle and development.

Actin Depolymerizing Factors↗
PubMed1996

Immunoglobulin G subclass responses of children during infection with Onchocerca volvulus.

To characterize the patterns of immunoglobulin G (IgG) subclass and IgE reactivity during the early stages of onchocerciasis, sera were collected from 224 children (age groups, 2 to 5, 6 to 10, and 11 to 15 years) residing in a region of Sierra Leone where Onchocerca volvulus is endemic, and these samples were tested by enzyme-linked immunosorbent assay for their reactivity to adult antigens (OvAg) and against four recombinant proteins (OV11, OV27, OV29, and OV16). Over 88% of the samples contained detectable levels of anti-OvAg IgG. In samples from microfilaria (MF)-positive children, IgG4 responses were significantly elevated and constituted on average 39, 35 and 28% of the total IgG responses for the age groups of 2 to 5, 6 to 10, and 11 to 15 years, respectively. For MF-negative individuals, the mean contributions of IgG4 to the total IgG response were 11% (2 to 5 years), 27% (6 to 10 years), and 56% (11 to 15 years). OvAg-specific IgE was detectable in the sera from both MF-negative and MF-positive individuals. To increase the specificity of the response, recombinant antigens OV11, OV27, and OV29 were tested individually or as a cocktail. Nearly 50% of the MF-negative children and 85% of the MF-positive children had detectable levels of IgG against at least one of the recombinant antigens. Only a small portion of the IgG against the recombinant peptides was IgG4. The prevalence of IgG against OV16 in samples from MF-negative children was 51%, and that for MF-positive children was 75%. The general profile of the humoral immune responses mounted by both MF-positive and a large percentage of the MF-negative children during the initial phases of infection with O. volvulus is similar to the profile reported for adults harboring chronic O. volvulus infections. These results suggest that very quickly after infection, the interactions between parasite and host result in an immunological environment that may contribute to the maintenance of a long-term, chronic infection.

Adolescent↗
PubMed1996

The major surface protein complex of Treponema denticola depolarizes and induces ion channels in HeLa cell membranes.

The oral spirochete Treponema denticola is closely associated with periodontal diseases in humans. The 53-kDa major surface protein (Msp) located in the outer membrane of T. denticola serovar a (ATCC 35405) has both pore-forming activity and adhesin activity. We have used standard patch clamp recording methods to study the effects of a partially purified outer membrane complex containing Msp on HeLa cells. The Msp complex was free of the chymotrypsin-like proteinase also found in the outer membrane of T. denticola. Msp bound to several HeLa cell proteins, including a 65-kDa surface protein and a 96-kDa cytoplasmic protein. The Msp complex depolarized and increased the conductance of the HeLa cell membrane in a manner which was not strongly selective for Na+, K+, Ca2+, and Cl- ions. Cell-attached patches of HeLa cell membrane exposed to Msp complex exhibited short-lived channels with a slope conductance of 0.4 nS in physiologically normal saline. These studies show that Msp binds both a putative epithelial cell surface receptor and cytoplasmic proteins and that the Msp complex can form large conductance ion channels in the cytoplasmic membrane of epithelial cells. These properties may contribute to the cytopathic effects of T. denticola on host epithelial cells.

Anions↗
PubMed1996

Functional and physical interactions between the Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1: Effects on EBV transcription and lytic replication.

The Epstein-Barr virus (EBV) proteins BZLF1 and BMRF1 are both essential for lytic EBV replication. BZLF1 is a transcriptional activator which binds directly to the lytic origin of replication (oriLyt) and plays a critical role in the disruption of viral latency. The BMRF1 protein is required for viral polymerase processivity. Here we demonstrate that the BMRF1 gene product functions as a transcriptional activator and has direct (as well as indirect) interactions with the BZLF1 gene product. The BMRF1 gene product activates an essential oriLyt promoter, BHLF1, but does not activate two other early EBV promoters (BMRF1 and BHRF1). Direct interaction between the BMRF1 and BZLF1 gene products requires the first 45 amino acids of BMRF1 and the bZip domain of BZLF1. The effect of the BZLF1-BMRF1 interaction on early EBV transcription is complex and is promoter specific. The oriLyt BHLF1 promoter is activated by either the BZLF1 or BMRF1 gene product alone and is further activated by the combination of the BZLF1 and BMRF1 gene products. Enhanced activation of BHLF1 transcription by the BMRF1-BZLF1 combination does not require direct interaction between these proteins. In contrast, BZLF1-induced activation of the BMRF1 promoter is inhibited in the presence of the BMRF1 gene product. A point mutation in the BZLF1 protein (amino acid 200), which prevents in vitro interaction with the BMRF1 protein but which does not reduce BZLF1 transactivator function, allows the BZLF1 protein to activate the BMRF1 promoter equally well in the presence or absence of the BMRF1 gene product. Therefore, direct interaction between the BZLF1 and BMRF1 proteins may inhibit BZLF1-induced transcription of the BMRF1 promoter. BZLF1 mutated at amino acid 200 is as efficient as wild-type BZLF1 in promoting replication of an oriLyt plasmid. However, this mutation reduces the ability of BZLF1 to induce lytic replication of the endogenous viral genome in D98/HE-R-1 cells. Our results indicate that functional and physical interactions between the BMRF1 and BZLF1 proteins may modulate the efficiency of lytic EBV infection. The BMRF1 gene product clearly has a transcriptional, as well as replicative, role during lytic EBV infection.

Antigens, Viral↗