Biomedical subjects

Y Hong

Publications and source records attributed to Y Hong.

At least 19 recordsLinked to original sources

PubMed1999

Regulation of protein phosphatase 2A activity by heat shock transcription factor 2.

Heat shock transcription factor (HSF) mediates the stress-induced expression of heat shock protein genes (hsp). However, HSF is required for normal cell function even in the absence of stress and is important for cell cycle progression, but the mechanism that mediates these effects of HSF is unknown. Here, it is shown that a member of the HSF family, HSF2, interacts with the PR65 (A) subunit of protein phosphatase 2A (PP2A). HSF2 binding to PR65 blocks its interaction with the catalytic subunit, due to competition between HSF2 and catalytic subunit for the same binding site in PR65. In addition, overexpression of HSF2 stimulates PP2A activity in cells, indicating the relevance of HSF2 as a regulator of PP2A in vivo. These results identify HSF2 as a dual function protein, capable of regulating both hsp expression and PP2A activity. This could function as a mechanism by which hsp expression is integrated with the control of cell division or other PP2A-regulated pathways.

Animals↗

PubMed1999

Expression of a functional N-methyl-D-aspartate-type glutamate receptor by bone marrow megakaryocytes.

Better understanding of hemostasis will be possible by the identification of new lineage-specific stimuli that regulate platelet formation. We describe a novel functional megakaryocyte receptor that belongs to a family of ionotropic glutamate receptors of the N-methyl-D-aspartate (NMDA) subtype responsible for synaptic neurotransmission in the central nervous system (CNS). Northern blotting and reverse-transcriptase polymerase chain reaction (RT-PCR) studies identified expression of NMDAR1 and NMDAR2D type subunit mRNA in rat marrow, human megakaryocytes, and MEG-01 clonal megakaryoblastic cells. Immunohistochemistry and in vivo autoradiographic binding of the NMDA receptor-specific antagonist MK-801 confirmed that megakaryocytes expressed open channel-forming NMDA receptors in vivo. Western blots indicated that megakaryocyte NMDAR1 was either unglycosylated or only glycosylated to low levels, and of identical size to CNS-type NMDAR1 after deglycosylation with endoglycosidase F/peptide-N-glycosidase F. In functional studies, we demonstrated that NMDA receptor activity was necessary for phorbol myristate acetate (PMA)-induced differentiation of megakaryoblastic cells; NMDA receptor blockade by specific antagonists significantly inhibited PMA-mediated increases in cell size, CD41 expression, and adhesion of MEG-01 cells. These results provide evidence for a novel pathway by which megakaryocytopoiesis and platelet production may be regulated.

Animals↗

PubMed1999

Rac homologues and compartmentalized phosphatidylinositol 4, 5-bisphosphate act in a common pathway to regulate polar pollen tube growth.

Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-delta1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process.

Amino Acid Sequence↗

PubMed1999

Sequences downstream of the RNA initiation site of the HTLV type I long terminal repeat are sufficient for trans-activation by human cytomegalovirus immediate-early proteins.

Human T cell leukemia virus type I infection is associated with a low incidence of morbidity in the form of adult T cell leukemia and neurologic disease, suggesting that there are other factors determining the pathogenic outcome of infection. We found that HCMV could infect various human cell lines known to be susceptible to HTLV-I infection, including T cell lines already harboring HTLV-I, and that HCMV infection could highly activate gene expression from the HTLV-I LTR. In addition, the coexpression of IE1 and IE2 genes of HCMV increased transcription from the HTLV-I LTR. The deletion analysis indicated that the entire U3 region is not required, but that the 216-bp region from +101 to +316 is sufficient for activation of the LTR by IE1 and IE2. These results suggest that HCMV IE proteins may affect the level of HTLV-I gene expression in coinfected individuals by interacting with HTLV-I LTR sequences.

Antigens, Viral↗

PubMed1999

Tumorigenicity of mouse thymoma is suppressed by soluble type II transforming growth factor beta receptor therapy.

Many types of tumor cells overexpress transforming growth factor beta (TGF-beta), which is believed to promote tumor progression. We hypothesized that overexpression of the extracellular region of the type II TGF-beta receptor (soluble TbetaRII) would compete for or block TGF-beta binding to TbetaRs on immune cells, preventing TGF-beta-mediated immunosuppression and consequently resulting in the eradication of tumor cells. We tested this in the mouse thymoma cell line EL4, which has been reported to suppress cellular immunity by secreting a large amount of TGF-beta. Transduction of EL4 with recombinant retrovirus encoding soluble TbetaRII resulted in the secretion of heterogeneously glycosylated, 25 to 35 kDa truncated TbetaRII. Inoculation of 1 x 10(4) to 5 x 10(4) soluble TbetaRII-modified EL4 cells (EL4/Ts, EL4 cells transduced with recombinant retrovirus encoding soluble TbetaRII and neomycin resistance gene) s.c. to mice showed reduced tumorigenicity, as indicated by lower overall tumor incidence (7%, 1 of 14; P < 0.001) compared with unmodified EL4 (100%, 9 of 9) or vector-modified EL4 cells (EL4/neo, EL4 cells transduced with recombinant retrovirus encoding neomycin resistance gene; 100%, 4 of 4). Administration of mitomycin C-treated EL4/Ts cells (1 x 10(6)) after EL4 inoculation (1 x 10(4)) reduced tumor incidence from 100% (5 of 5 in mice inoculated with mitomycin C-treated EL4/neo) to 40% (4 of 10, P < 0.05), indicating that supply of soluble TbetaRII could actually block TGF-beta-mediated tumorigenesis. In vitro tumor cytotoxicity assays revealed 3-5-fold higher cytotoxic activity with lymphocytes from EL4/Ts-bearing mice compared with those from EL4- or EL4/neo-bearing mice, indicating that the observed tumor rejection was mediated by restoration of the tumor-specific cellular immunity. These data suggest that expression of soluble TbetaRII is an effective strategy for treating highly progressive tumors secreting TGF-beta.

Animals↗

PubMed1999

The bovine papillomavirus type 1 E6 oncoprotein sensitizes cells to tumor necrosis factor alpha-induced apoptosis.

Expression of viral proteins may result in susceptibility of cells to the cytotoxic effect of Tumor Necrosis Factor Alpha (TNF). While murine C127 cells containing the bovine papillomavirus type 1 (BPV-1) genome were reported to exhibit increased TNF sensitivity, the gene(s) responsible was not identified. The BPV-1 E6 oncoprotein induces tumorigenic transformation of murine C127 cells and stimulates transcription when targeted to a promoter. BPV-1 E6 was introduced into C127 cells (PBE6) by retroviral infection and stable clones were isolated. These cells showed increased apoptosis in response to TNF, as measured by several criteria. TNF-induced apoptosis in PBE6 cells was accompanied by increased release of arachidonic acid, indicating that phospholipase A2 was activated. We also provide evidence that BPV-1 E6 mediated-sensitization of cells to TNF-induced apoptosis can occur in the absence of p53.

Animals↗

PubMed1999

Genetic effects for plasma factor VII levels independent of and in common with triglycerides.

BACKGROUND: Coagulation factor VII has been demonstrated as a potential risk factor for cardiovascular disease. Both genes and non-genetic factors are related to plasma levels of factor VII. However, the extent to which genetic effects influence variability in plasma factor VII levels is unknown. Further, increased levels of plasma factor VII are associated with serum triglycerides, yet the reason for this association is not fully understood. METHODS AND RESULTS: Quantitative genetic analyses were applied to evaluate the relative importance of genetic and different environmental influences on plasma factor VII levels and to test the significance of genetic and environmental factors in common to factor VII and triglycerides in 215 pairs of middle-aged and elderly twins, of whom 104 were reared apart and 120 were women. Genetic influences were found to account for 57% of the individual differences in plasma factor VII levels, whereas shared-rearing and residual-familial environmental factors were not significant. Furthermore, a significant genetic correlation of 0.38 was found between factor VII and triglycerides, but the environmental correlation between these two measures was not significant. Genetic factors in common to factor VII and triglycerides explain about 7% of the total variance for factor VII. CONCLUSION: The present study suggests that there are substantial genetic influences on plasma factor VII levels. Furthermore, genetic effects explain the phenotypic association between factor VII and triglycerides.

Aged↗

PubMed1998

Cholinergic cell expression in the developing rat medial septal nucleus in vitro is differentially controlled by GABAA and GABAB receptors.

The early appearance and relative abundance of GABAergic neurons in basal forebrain cholinergic nuclei like the medial septum suggest that the maturation of the later developing cholinergic neurons in these nuclei may be controlled by GABA. To examine this possibility, the effects of both exogenous GABA and specific GABA receptor agonists, as well as that of endogenous GABA on the phenotypic expression and survival of the cholinergic neurons in primary cultures from the fetal rat medial septum, were studied. Treatment of these cultures for six days with GABA significantly decreased the enzymatic activity of choline acetyltransferase (EC 2.3.1.6) (ChAT) in a dose-dependent manner. This response to exogenous GABA was blocked by bicuculline, mimicked by muscimol and slightly potentiated by saclofen. Consistent with this latter observation, the GABAB receptor agonist, baclofen, dose-dependently increased septal ChAT activity. However, while the effect of baclofen on cholinergic expression was lost in the absence of glia, the suppressive effects of GABA or muscimol were more marked. Acetylcholinesterase (EC 3.1.1.7) (AChE) expression in mixed neuronal-glial cultures, was, like ChAT activity, increased or decreased in intensity with the inclusion of baclofen or muscimol, respectively. Although the number of AChE positive neurons in muscimol-treated cultures was significantly lower than that in controls, no changes in neither neuronal nor general cell viability were noted. Finally, as GABAA or GABAB receptor antagonists bicuculline and picrotoxin or saclofen, when applied alone to mixed cultures, increased or decreased ChAT activity, respectively, it appears that endogenous GABA, tonically released in the developing septum, may, via specific receptor types, differentially control the biochemical maturation of the cholinergic neurons.

Acetylcholinesterase↗

PubMed1998

Kinetic analysis of the interaction of actin-depolymerizing factor (ADF)/cofilin with G- and F-actins. Comparison of plant and human ADFs and effect of phosphorylation.

The thermodynamics and kinetics of actin interaction with Arabidopsis thaliana actin-depolymerizing factor (ADF)1, human ADF, and S6D mutant ADF1 protein mimicking phosphorylated (inactive) ADF are examined comparatively. ADFs interact with ADP.G-actin in rapid equilibrium (k+ = 155 microM-1.s-1 and k- = 16 s-1 at 4 degreesC under physiological ionic conditions). The kinetics of interaction of plant and human ADFs with F-actin are slower and exhibit kinetic cooperativity, consistent with a scheme in which the initial binding of ADF to two adjacent subunits of the filament nucleates a structural change that propagates along the filament, allowing faster binding of ADF in a "zipper" mode. ADF binds in a non-cooperative faster process to gelsolin-capped filaments or to subtilisin-cleaved F-actin, which are structurally different from standard filaments (Orlova, A., Prochniewicz, E., and Egelman, E. H. (1995) J. Mol. Biol. 245, 598-607). In contrast, the binding of phalloidin to F-actin cooperatively inhibits its interaction with ADF. The ADF-facilitated nucleation of ADP.actin self-assembly indicates that ADF stabilizes lateral interactions in the filament. Plant and human ADFs cause only partial depolymerization of F-actin at pH 8, consistent with identical functions in enhancing F-actin dynamics. Phosphorylation does not affect ADF activity per se, but decreases its affinity for actin by 20-fold.

Actin Depolymerizing Factors↗

PubMed1998

GFP expression in Drosophila tissues: time requirements for formation of a fluorescent product.

We analyzed the acquisition of fluorescence of a GFP fusion protein expressed from gene constructs designed to regulate either the time of translation of the protein or the time of gene transcription. Both gene constructs expressed a fusion protein with wild-type GFP appended to the N-terminus of the Bicoid (Bcd) transcription factor. One gene construct, P[gfp-bcd] expressed an mRNA that was initially translated at the time of egg deposition. GFP-Bcd fluorescence could first be detected in anterior nuclei in the embryo at syncytial blastoderm stage, within 1.5-2.5 h after translation commenced. The second gene construct, P[hs-gfp-bcd], utilized the hsp70 promoter to regulate the induction of gene expression. In larval salivary glands, GFP-Bcd fluorescence could first be detected in nuclei after a 40-min recovery period following a 10-min heat shock induction of gene expression. These estimates of the time required for the acquisition of a fluorescent GFP product in Drosophila tissues are less than suggested by previously published reports.

Animals↗

PubMed1998

In vivo chiro-inositol metabolism in the rat: a defect in chiro-inositol synthesis from myo-inositol and an increased incorporation of chiro-[3H]inositol into phospholipid in the Goto-Kakizaki (G.K) rat.

We report our comparative studies of myo- and chiro-[3H]inositol distribution in control nondiabetic Wistar and spontaneous nonobese insulin-resistant Type II diabetic G.K. rat tissues following 78-h labeling period. From various tissue extracts, free inositols, inositol phosphates, and inositol phospholipids were isolated and analyzed. Our findings demonstrate (1) a significant difference in the metabolism of myo- and chiro-inositol in normal Wistar and G.K. rats, (2) a severe defect in conversion of myo-[3H]inositol to chiro-[3H]inositol at the inositol phospholipid level of insulin-sensitive tissues of the G.K. rat, (3) an increased incorporation of myo-[3H]inositol into purified inositol phospholipids of the G.K. rat consistent with a decreased conversion to chiro-[3H]inositol, (4) the presumed presence of a pathway which incorporates chiro-[3H]inositol into inositol phospholipids, and which is overactive in the G.K. rat compared to the Wistar rat, and (5) no or minimal conversion of chiro-[3H]inositol to myo-[3H]inositol.

Animals↗

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↗