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Biomedical subjects

Y Hong

Publications and source records attributed to Y Hong.

At least 127 records · Page 7Linked to original sources
PubMed1998

Evolutionary relationships among putative RNA-dependent RNA polymerases encoded by a mitochondrial virus-like RNA in the Dutch elm disease fungus, Ophiostoma novo-ulmi, by other viruses and virus-like RNAs and by the Arabidopsis mitochondrial genome.

The nucleotide sequence (2617 nucleotides) of virus-like double-stranded (ds) RNA 3a in a diseased isolate, Log1/3-8d2 (Ld), of the ascomycete fungus Ophiostoma novo-ulmi has been determined. One strand of the dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 718 amino acids, and the complementary strand contains two smaller ORFs with the potential to encode proteins of 178 and 182 amino acids, respectively. The large ORF contains 12 UGA codons which code for tryptophan in ascomycete mitochondria and has a codon bias typical of mitochondrial genes, consistent with the localization of Ld dsRNAs within the mitochondria. The amino acid sequence contains motifs characteristic of RNA-dependent RNA polymerases (RdRps). This putative RdRp was shown to be related to putative RdRps of mitochondrial dsRNAs of another ascomycete and a basidiomycete fungus and also to a putative RdRp encoded by the mitochondrial genome of Arabidopsis thaliana. In multiple sequence alignments, the fungal mitochondrial dsRNA-encoded RdRp-like proteins formed a cluster, ancestrally related to the RdRps of the yeast 20S and 23S RNA replicons and of the positive-stranded RNA bacteriophages of the Leviviridae family, but distinct from RdRps of other families and genera of fungal RNA viruses and related plant and animal RNA viruses. Northern blot analysis with RNA 3a strand-specific probes indicated that nucleic acid extracts of Ld contain more single-stranded (positive-stranded) RNA than dsRNA, consistent with an evolutionary relationship between RNA 3a and positive-stranded RNA phages.

Amino Acid Sequence↗
PubMed1998

Identification of an alpha helical motif sufficient for association with papillomavirus E6.

We recently identified a cellular protein named E6BP or ERC-55 that binds cancer-related papillomavirus E6 proteins (Chen, J. J., Reid, C. E., Band, V., and Androphy, E. J. (1995) Science 269, 529-531). By construction of a series of deletion mutants, the region of E6BP that is necessary and sufficient for complex formation with human papillomavirus type 16 E6 has been mapped to a 25-amino acid domain. The corresponding peptide was synthesized and found by nuclear magnetic resonance spectroscopy to bind calcium and fold into a classical helix-loop-helix EF-hand conformation. Additional deletion mutagenesis showed that 13 amino acids that form the second alpha helix mediated E6 association. Alanine replacement mutagenesis indicated that amino acids of this helix were most important for E6 binding. Alignment of this alpha helical E6 binding peptide with the 18-amino acid E6 binding region of E6AP (Huibregtse, J. M., Scheffner, M., and Howley, P. M. (1993) Mol. Cell. Biol. 13, 4918-4927) and the first LD repeat of another E6-binding protein, paxillin (Tong, X., and Howley, P. M. (1997) J. Biol. Chem. 272, 33373-33376), revealed substantial similarities among these E6 binding domains. The extent of homology and the mutational data define the peptide as an E6 binding motif.

Amino Acid Sequence↗
PubMed1998

Production of medakafish chimeras from a stable embryonic stem cell line.

Embryonic stem (ES) cell lines provide a unique tool for introducing targeted or random genetic alterations through gene replacement, insertional mutagenesis, and gene addition because they offer the possibility for in vitro selection for the desired, but extremely rare, recombinant genotypes. So far only mouse blastocyst embryos are known to have the competence to give rise to such ES cell lines. We recently have established a stable cell line (Mes1) from blastulae of the medakafish (Oryzias latipes) that shows all characteristics of mouse ES cells in vitro. Here, we demonstrate that Mes1 cells also have the competence for chimera formation; 90% of host blastulae transplanted with Mes1 cells developed into chimeric fry. This high frequency was not compromised by cryostorage or DNA transfection of the donor cells. The Mes1 cells contributed to numerous organs derived from all three germ layers and differentiated into various types of functional cells, most readily observable in pigmented chimeras. These features suggest the possibility that Mes1 cells may be a fish equivalent of mouse ES cells and that medaka can be used as another system for the application of the ES cell technology.

Animals↗
PubMed1998

Biodegradation in vitro and retention in the rabbit eye of crosslinked poly(1-vinyl-2-pyrrolidinone) hydrogel as a vitreous substitute.

To elucidate the relatively short retention of crosslinked poly(1-vinyl-2-pyrrolidinone) hydrogels in the eye when used as potential vitreous substitutes, a 14C-labeled hydrogel was produced and subjected to both in vitro biodegradation assays and in vivo experiments. The polymer was synthesized by the free-radical copolymerization of 99% 1-vinyl-2-pyrrolidinone with 1% 14C-methyl methacrylate in the presence of ethylene glycol dimethacrylate (0.1%) as crosslinking agent. The in vitro protocol for assessing the biodegradation included the incubation of hydrogel with hydrolases (trypsin or collagenase), followed by examination of changes in its physical characteristics and by monitoring its residual radioactivity, as well as by detection of possible degradation products. Within the maximum duration of experiments (4 weeks), none of the procedures indicated biodegradation of polymer. The hydrogel was also injected into the vitreous humor of rabbits and followed up to 4 weeks. Residual radioactivity measurements of the vitreous contents indicated that 50% of the polymer was removed by the end of this period. Histopathologic examination revealed cell infiltrates of the mononuclear phagocyte system in both vitreous and retinal tissue. A possible phagocyte-mediated mechanism for the dissipation of hydrogel is discussed.

Animals↗
PubMed1998

Novel structures of two virus-like RNA elements from a diseased isolate of the Dutch elm disease fungus, Ophiostoma novo-ulmi.

The nucleotide sequences of 2 of the 10 mitochondrial double-stranded (ds) RNA segments in a diseased isolate, Log 1/3-8d2 (Ld), of Ophiostoma novo-ulmi, RNA-7 (1057 nucleotides) and RNA-10 (317-330 nucleotides), have been determined. Both RNAs are A-U-rich, but in Southern and Northern blots, no hybridization with mitochondrial DNA or RNA could be detected. Only very short open reading frames were found in both RNAs. As most of its sequence is unrelated to any of the other Ld dsRNAs, RNA-7 may be regarded as a satellite RNA. Northern blotting detected a full-length single-stranded (ss) form of RNA-7 in nucleic acid extracts from Ld. The 5'- and 3'-terminal 39 nucleotides of ssRNA-7 are imperfect inverted complementary repeats of each other, which could cause ssRNA-7 to form a panhandle structure. In addition, the 5'-terminal nucleotides 1-28 and 3'-terminal nucleotides 1032-1057 of ssRNA-7 each contained inverted complementary sequences, allowing the possibility for each terminus to form separate stem-loop structures. The combination of these two structural features has not been found previously in any dsRNA or ssRNA virus. RNA-10 was shown to have an unusual structure, consisting of a mosaic of sequences derived from regions of the 5'- and 3'-termini, or just the 5'-terminus, of RNA-7, RNA-10 has a high degree of inverted complementarity, with the potential to be folded into a very stable hairpin structure. A model for the formation of RNA-10 is presented, involving replicase-driven strand switching between (-)-strand and (+)-strand templates during RNA synthesis, followed by utilization of the nascent strand as a primer and template to form a snap-back RNA.

Ascomycota↗
PubMed1998

ATP-dependent interaction of human mismatch repair proteins and dual role of PCNA in mismatch repair.

DNA mismatch repair ensures genomic stability by correcting biosynthetic errors and by blocking homologous recombination. MutS-like and MutL-like proteins play important roles in these processes. In Escherichia coli and yeast these two types of proteins form a repair initiation complex that binds to mismatched DNA. However, whether human MutS and MutL homologs interact to form a complex has not been elucidated. Using immunoprecipitation and Western blot analysis we show here that human MSH2, MLH1, PMS2 and proliferating cell nuclear antigen (PCNA) can be co-immunoprecipitated, suggesting formation of a repair initiation complex among these proteins. Formation of the initiation complex is dependent on ATP hydrolysis and at least functional MSH2 and MLH1 proteins, because the complex could not be detected in tumor cells that produce truncated MLH1 or MSH2 protein. We also demonstrate that PCNA is required in human mismatch repair not only at the step of repair initiation, but also at the step of repair DNA re-synthesis.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Thrombopoietin stimulates VEGF release from c-Mpl-expressing cell lines and haematopoietic progenitors.

Vascular endothelial growth factor (VEGF) production was analysed in megakaryocytic cell lines and CD34+ haematopoietic progenitors following treatment with thrombopoietin (TPO). In CMK cells TPO caused a time- and dose-dependent increase in the levels of VEGF released into the medium. A similar effect was observed in UT-7/mpl cells transfected with the TPO receptor c-Mpl, but not in parental UT-7 cells. In CD34+ haematopoietic progenitor cell cultures TPO stimulated VEGF mRNA expression and VEGF protein release. Production of VEGF in CD34+ cultures increased with TPO-induced megakaryocytic differentiation, but not with erythroid or myelomonocytic differentiation induced respectively by erythropoietin and granulocyte-macrophage colony-stimulating factor. These results demonstrate that TPO stimulates VEGF release in c-Mpl-expressing cells and suggest that this process is an integral feature of the megakaryocytic differentiation programme.

Animals↗
PubMed1998

Protein kinase C mediates the mitogenic action of thrombopoietin in c-Mpl-expressing UT-7 cells.

Protein kinase C (PKC) has been implicated in signal transduction events elicited by several hematopoietic growth factors. Thrombopoietin (TPO) is the major regulator of megakaryocytic lineage development, and its receptor, c-Mpl, transduces signals for the proliferation and differentiation of hematopoietic progenitors. In this study we have examined the effect of TPO on the subcellular distribution of PKC (a measure of enzyme activation) in a growth factor-dependent pluripotent hematopoietic cell line that was engineered to express the c-Mpl receptor (UT-7/mpl). In addition, we have assessed the significance of this activation for the induction of both mitogenesis and differentiation. Using a PKC translocation assay, TPO was found to stimulate a time- and dose-dependent increase in the total content of PKC activity present in the membrane fraction of UT-7/mpl cells (maximum increase = 2.3-fold above basal level after 15 minutes with 40 ng/mL TPO, EC50 = 7 ng/mL). Accordingly, a decrease of PKC content in the cytosolic fraction was observed. Immunoblot analysis using PKC isotype-specific antibodies showed that TPO treatment led to a marked increase of the Ca2+/diacylglycerol-sensitive PKC isoforms alpha and beta found in the membrane fraction. In contrast, the subcellular distribution of these isoforms did not change after treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF). Exposure of UT-7/mpl cells to the selective PKC inhibitor GF109203X completely inhibited the PKC activity associated to the membrane fraction after TPO treatment, and blocked the mitogenic effect of TPO. In contrast, GF109203X had no effect on the TPO-induced expression of GpIIb, a megakaryocytic differentiation antigen. Downregulation of PKC isoforms alpha and beta to less than 25% of their initial level by treatment with phorbol 12,13-dibutyrate also abolished the TPO-induced mitogenic response, but had no significant effect when this response was induced by GM-CSF. Taken together, these findings suggest that (1) TPO stimulates the activation of PKC, (2) PKC activation mediates the mitogenic action of TPO, and (3) PKC activation is not required for TPO-induced expression of megakaryocytic surface markers.

Biological Transport↗
PubMed1998

Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase.

Spinach (Spinacea oleracea L.) nitrate reductase (NR) is inactivated by phosphorylation on serine-543, followed by binding of the phosphorylated enzyme to 14-3-3 proteins. We purified one of several chromatographically distinct NRserine-543 kinases from spinach leaf extracts, and established by Edman sequencing of 80 amino acid residues that it is a calcium-dependent (calmodulin-domain) protein kinase (CDPK), with peptide sequences very similar to Arabidopsis CDPK6 (accession no. U20623; also known as CPK3). The spinach CDPK was recognized by antibodies raised against Arabidopsis CDPK. Nitrate reductase was phosphorylated at serine-543 by bacterially expressed His-tagged CDPK6, and the phosphorylated NR was inhibited by 14-3-3 proteins. However, the bacterially expressed CDPK6 had a specific activity approx. 200-fold lower than that of the purified spinach enzyme. The physiological control of NR by CDPK is discussed, and the regulatory properties of the purified CDPK are considered with reference to current models for reversible intramolecular binding of the calmodulin-like domain to the autoinhibitory junction of CDPKs.

Amino Acid Sequence↗
PubMed1998

Efficiency of cell culture derivation from blastula embryos and of chimera formation in the medaka (Oryzias latipes) depends on donor genotype and passage number.

Embryonic stem (ES) cells from early vertebrate embryos only rarely retain their full developmental potential under in vitro culture conditions, but undergo differentiation and lose their ability for chimeric embryogenesis. This is reflected by the fact that the ES cell technology to date could only be fully developed in mice. In the fish Oryzias latipes, the medaka, one ES-like cell line, MES1, has been established which gives rise to a high frequency of somatic chimeras but a low degree of chimerism. Here we have tested the effect of donor genotype and cultivation time on the efficiency of cell culture derivation and on chimera formation. The HB12A, HB32C and HNI strains of medaka most efficiently and reproducibly give rise to blastula-derived cell cultures that produce pigmented chimeras in albino hosts. Seven chimeras grew to male or female adults with normal fertility, although none of them showed obvious donor germline contribution. During prolonged in vitro propagation the frequency of chimeras and the degree of chimerism dropped to a value retained in the long-term cultured MES1 cells. Obviously, genetic factors in host/donor compatibility and physiological changes during prolonged in vitro culture may compromise, but do not abolish, the developmental potential of medaka ES-like cells. Thus, elucidation of conditions that will expand the developmental potential of medaka blastula cell cultures should lead to a further improvement towards establishment of the ES cell technology in medaka.

Animals↗
PubMed1998

Hypoxia and endothelin-1 induce VEGF production in human vascular smooth muscle cells.

Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is a secreted mitogen for vascular endothelial cells, and it promotes vascular permeability and neovascularization in vivo. We investigated the mechanisms by which low oxygen tension modulates the expression of VEGF in human aortic vascular smooth muscle cells (h-SMC) in vitro. Moreover, we measured VEGF levels in the cultured medium with or without endothelin-1 (ET-1) using a newly developed, highly sensitive, enzyme-linked immunosorbent assay. Hypoxia resulted in a substantial induction of VEGF transcripts at 3 and 24 hr. VEGF levels were significantly higher when h-SMC were cultured in medium containing ET-1 than when cultured in medium without ET-1. In conclusion, hypoxia and ET-1 constitute potent stimuli for VEGF production in h-SMC.

Cell Hypoxia↗
PubMed1998

Use of a neural network to predict stone growth after shock wave lithotripsy.

OBJECTIVES: To determine whether a neural network is superior to standard computational methods in predicting stone regrowth after shock wave lithotripsy (SWL) and to determine whether the presence of residual fragments, as an independent variable, increases risk. METHODS: We reviewed the records of 98 patients with renal or ureteral calculi treated by primary SWL at a single institution and followed up for at least 1 year; residual stone fragment growth or new stone occurrence was determined from abdominal radiographs. A neural network was programmed and trained to predict an increased stone volume over time utilizing input variables, including previous stone events, metabolic abnormality, directed medical therapy, infection, caliectasis, and residual fragments after SWL. Patient data were partitioned into a training set of 65 examples and a test set of 33. The neural network did not encounter the test set until training was complete. RESULTS: The average follow-up period was 3.5 years (range 1 to 10). Of 98 patients, 47 had residual stone fragments 3 months after SWL; of these 47, 8 had increased stone volume at last follow-up visit. Of 51 patients stone free after SWL, 4 had stone recurrence. Coexisting risk factors were incorporated into a neural computational model to determine which of the risk factors was individually predictive of stone growth. The classification accuracy of the neural model in the test set was 91%, with a sensitivity of 91%, a specificity of 92%, and a receiver operating characteristic curve area of 0.964, results significantly better than those yielded by linear and quadratic discriminant function analysis. CONCLUSIONS: A computational tool was developed to predict accurately the risk of future stone activity in patients treated by SWL. Use of the neural network demonstrates that none of the risk factors for stone growth, including the presence of residual fragments, is individually predictive of continuing stone formation.

Follow-Up Studies↗
PubMed1998

In vitro interactions between a potyvirus-encoded, genome-linked protein and RNA-dependent RNA polymerase.

Recent studies have shown that potyvirus VPg/ proteinases and RNA-dependent RNA polymerases are capable of protein-protein interactions in yeast cells. We have extended these studies in vitro. We found that tobacco vein mottling virus (TVMV) VPg is retained on glutathione-Sepharose matrices if co-incubated with a glutathione S-transferase (GST)-NIb fusion protein, but not with GST, which is suggestive of a direct physical interaction between these two proteins. However, a mutation in the VPg (Y1860S) that eliminates virus infectivity and the interaction in yeast cells had little effect on the in vitro interaction. We also found that the TVMV VPg and NIa proteins are capable of stimulating the polymerase activity of the NIb protein. Since this stimulatory activity is retained when the proteinase domain of the NIa is removed, we conclude that the VPg is the moiety responsible for the stimulation of polymerase activity. As with the interaction revealed by co-purification, the Y1860S mutation had little or no effect on the stimulation of polymerase activity. Moreover, the VPg was able to stimulate a mutant NIb with an altered 'GDD' motif. Our studies thus provide two lines of evidence indicative of in vitro interactions between the TVMV VPg and NIb proteins.

Amino Acid Sequence↗
PubMed1998

Clinical results of implantation of the Chirila keratoprosthesis in rabbits.

AIMS/BACKGROUND: An ideal keratoprosthesis (KPro) would closely resemble a donor corneal button in terms of its surgical handling, optics, and capacity to heal with host tissue in order to avoid many of the complications associated with the KPros which are currently in clinical use. This study was carried out to assess the long term clinical outcomes on implantation of the core and skirt poly(2-hydroxyethyl methacrylate) KPro in animals. METHODS: 20 KPros were made and implanted as full thickness corneal replacements into rabbits and followed for up to 21 months to date. RESULTS: 80% of the prostheses have been retained, with a low incidence of complications such as cataract, glaucoma, and retroprosthetic membrane formation which are frequently associated with KPro surgery. CONCLUSIONS: KPros of this type may offer promise in the treatment of patients for whom penetrating keratoplasty with donor material carries a poor prognosis. Refinement of the KPro and further animal trials, including implantation into abnormal corneas, are however mandatory before human implantation could be planned.

Animals↗
PubMed1998

Familial clustering of insulin and abdominal visceral fat: the HERITAGE Family Study.

Abdominal visceral fat (AVF) is an obesity-related phenotype thought to be associated with insulin resistance, diabetes mellitus, and atherosclerosis. Significant genetic influences on both AVF and insulin levels have been reported. However, information is lacking as to whether common genetic influences on AVF and insulin levels exist. AVF was assessed by computed tomography scan, and fasting insulin was measured by RIA in 512 members of 98 sedentary Caucasian families participating in the HERITAGE Family Study. Baseline data, collected before exercise training, were used in the present investigation. A bivariate familial correlation model was applied to evaluate whether there are familial influences that are common to insulin and AVF before and after adjustment for total fat mass (FM), and to assess the overall heritability of insulin and AVF. The maximal heritability for AVF, before and after adjustment for total FM, was 42% and 50%, respectively; and for insulin, it was 21%. Interestingly, 29% of the familial influences on insulin were also common to AVF, whereas 14% of the familial influences on AVF were shared by insulin. Furthermore, after AVF was adjusted for total FM, these common familial influences were increased to 48% and 20%. Genes and/or familial nongenetic factors with pleiotropic effects seem to influence both AVF and plasma insulin levels to a certain degree. Genes involved in the regulation of lipid storage and mobilization in the abdominal fat depot are potential candidates for these genetic pleiotropic effects.

Adipose Tissue↗
PubMed1998

Developmental regulation of a cyclin-dependent kinase inhibitor controls postembryonic cell cycle progression in Caenorhabditis elegans.

C. elegans cki-1 encodes a member of the CIP/KIP family of cyclin-dependent kinase inhibitors, and functions to link postembryonic developmental programs to cell cycle progression. The expression pattern of cki-1::GFP suggests that cki-1 is developmentally regulated in blast cells coincident with G1, and in differentiating cells. Ectopic expression of CKI-1 can prematurely arrest cells in G1, while reducing cki-1 activity by RNA-mediated interference (RNAi) causes extra larval cell divisions, suggesting a role for cki-1 in the developmental control of G1/S. cki-1 activity is required for the suspension of cell cycling that occurs in dauer larvae and starved L1 larvae in response to environmental signals. In vulva precursor cells (VPCs), a pathway of heterochronic genes acts via cki-1 to maintain VPCs in G1 during the L2 stage.

Animals↗
PubMed1998

[Isolation and identification of Triterpenoid compounds in the fruits of Chaenomeles lagenaria (Loisel.) Koidz].

OBJECTIVE: The fruit of Chaenomeles lagenaria is a commonly used drug in Chinese traditional medicine. It is especially effective on rheumatism. The current work is to find out the chemical basis of its pharmacological activity. METHOD: All compounds were extracted with organic solvents and separated by silica gel column or thin layer chromatography, and then purified by recrystalization. The structures were elucidated on the basis of chemical reactions and spectral data analysis. RESULT: In addition to the compounds reported in our previous work, three triterpenoid compounds were separated and identified as 3-O-acetyl ursolic acid, 3-O-acetyl pomolic acid and betulinic acid respectively. CONCLUSION: It is obvious that triterpenoid compounds are dominant among the chemical constituents of C. lagenaria. But it is still to be clarified whether they are the chemical basis of the drug's pharmacological activity.

Drugs, Chinese Herbal↗