Biomedical subjects

Y W Yang

Publications and source records attributed to Y W Yang.

At least 37 records · Page 2Linked to original sources

PubMed2000

Glucose-responsive gene delivery in pancreatic Islet cells via recombinant adeno-associated viral vectors.

PURPOSE: Recent progress in genetic engineering presents the possibility of providing physiologically regulated glucose metabolism in individuals with diabetes. The objective of this study is to explore the feasibility of obtaining glucose dependent gene expression in the pancreatic beta-cell lines via recombinant adeno-associated virus type 2 (rAAV) mediated gene transfer. METHODS: Two transcription cassettes containing the luciferase gene under the control of the rat insulin I gene promoter and the enhanced green fluorescent protein (EGFP) open reading frame under the control of the immediate early gene promoter of human cytomegalovirus (CMV) were placed in series between the inverted terminal repeats (ITRs) of AAV. The rAAV vectors produced were used to transduce pancreatic beta-cell line grown in the absence or presence of various concentrations of glucose. Luciferase activity assays were performed at 72 hr post-transduction. RESULTS: Glucose-responsive reporter gene expression was obtained in both calcium phosphate transfected HIT-T15 and betaHC-9 cells, demonstrating regulated luciferase gene expression under control of the insulin gene promoter. At MOI of 100, rAAV-transduced betaHC-9 cells exhibited glucose-dependent luciferase activities, which were approximately 4.3 fold higher than those transfected by the calcium phosphate coprecipitation method at 20 mM glucose. CONCLUSIONS: Delivery of the insulin gene promoter via rAAV was shown in this study to result in glucose-dependent control of the reporter gene expression. The results suggest that rAAV is an efficient viral vector for gene transfer into the pancreatic islet cells.

Animals↗

PubMed2000

Gene delivery via polyomavirus major capsid protein VP(1), isolated from recombinant Escherichia coli.

In this study we attempted to investigate the feasibility of transferring exogenous DNA into the recipient host via polyomavirus major capsid protein VP(1) pseudocapsids generated from recombinant Escherichia coli, designated PyVP(1,E), both in vitro and in vivo. NIH-3T3 and FR3T3 cells were transfected with pCMV beta and pPyMT-1 plasmid DNA, respectively. In vitro DNA transfection was carried out via Pseudofect- and PyVP(1,E)-mediated methods, or by co-precipitation with calcium phosphate. Expression of beta-galactosidase and PyMT reporter genes was examined by Western-blot analysis. Parallel experiments were performed in vivo by direct injection of pCMV beta and pPyMT-1 plasmid DNA, complexed with PyVP(1,E), into the livers of Wistar rats, followed by Western-blot analysis and histochemical staining. The results obtained from in vitro transfection experiments showed that expression of the reporter genes can be detected in the recipient cells at 48 h post-transfection. PyVP(1,E) was shown to exhibit similarly efficient in vitro DNA transfection properties to Pseudofect, which was obtained from recombinant baculovirus.

3T3 Cells↗

PubMed1999

Molecular phylogenetic studies of Brassica, rorippa, arabidopsis and allied genera based on the internal transcribed spacer region of 18S-25S rDNA.

The phylogenetic relationships of nine genera in four tribes of the family Brassicaceae were estimated from the sequences of the internal transcribed spacer region (ITS) of the 18S-25S nuclear ribosomal DNA. The entire ITS region of 16 accessions belonging to 10 species of seven genera was sequenced. Eight published sequences of Brassicaceae were also used. A total of 27 sequences were included in this study; four of them were found to be pseudogenes. Both the neighbor-joining and the parsimony trees suggest that the nine genera can be divided into three groups: (1) Arabidopsis, Cardaminopsis, Capsella, and Lepidium; (2) Rorippa and Cardamine; and (3) Brassica, Sinapis, and Raphanus. In contradiction to the proposal that Cardaminopsis and Arabidopsis be put into an expanded tribe Arabideae, our data show that these two genera are more closely related to Capsella and Lepidium (tribe Lepidieae) than to Rorippa and Cardamine (tribe Arabideae). Further, our data show that within the tribe Brassiceae, Raphanus is more closely related to B. nigra than to the B. oleracea/B. rapa clade. This result is in agreement with the nuclear data obtained in several studies, but is in conflict with the RFLP data of mitochondrial and chloroplast DNA. As pointed out by previous authors, it is possible that Raphanus is a hybrid between the B. nigra and B. oleracea/B. rapa lineages with the latter as the maternal parent.

Arabidopsis↗

PubMed1999

Rates of nucleotide substitution in angiosperm mitochondrial DNA sequences and dates of divergence between Brassica and other angiosperm lineages.

We obtained 16 nucleotide sequences ( approximately 1400 bp each) of the first intron of the mitochondrial (mt) gene for NADH subunit 4 (nad4) from 10 species of Brassicaceae. Using these new sequences and five published sequences from GenBank, we constructed a phylogenetic tree of the Brassicaceae species under study and showed that the rate of nucleotide substitution in the first intron of nad4 is very low, about 0.16-0.23 x 10(-9) substitution per site per year, which is about half of the silent rate in exons of nad4. The ratios of substitution rates in this intron, ITS, and IGS are approximately 1:23:73, where ITS is the nuclear intergenic spacer between 18S and 25S rRNA genes and IGS is the intergenic spacer of 5S rRNA genes. A segment (335 bp) in the first intron of nad4 in Brassicaceae species that is absent in wheat was considered as a nonfunctional sequence and used to estimate the neutral rate (the rate of mutation) in mtDNA to be 0.5-0.7 x 10(-9) substitution per site per year, which is about three times higher than the substitution rate in the rest of the first intron of nad4. We estimated that the dates of divergence are 170-235 million years (Myr) for the monocot-dicot split, 112-156 Myr for the Brassicaceae-Lettuce split, 14.5-20.4 Myr for the Brassica-Arabidopsis split, and 14.5-20.4 Myr for the Arabidopsis-Arabideae split.

Base Sequence↗

PubMed1999

Conformational changes of polyomavirus major capsid protein VP1 in sodium dodecyl sulfate solution.

Conformations of polyomavirus (Py) major capsid protein VP1 were analyzed by circular dichroism (CD) and fluorescence spectroscopy in the presence of sodium dodecyl sulfate (SDS). Binding of PyVP1 to SDS induced marked conformational changes of PyVP1, which were reflected on the CD and fluorescence spectra. Abrupt changes in both optical properties occurred within the narrow ranges of SDS concentrations with the transition midpoints closely related to SDS micelle formation. Analysis of circular dichroism spectra showed that the contents of alpha-helices, beta-sheets, beta-turns and random coils in PyVP1 varied upon addition of SDS, demonstrating the exquisite sensitivity of the conformations of the protein to the environment. The interactions of PyVP1 with SDS were shown to be dependent on the ionic strength of the protein solution, suggesting that both hydrophobic and electrostatic forces contribute to the PyVP1-SDS complex formation. The SDS-induced conformational changes of PyVP1 appeared to be a two-stage process.

Capsid↗

PubMed1998

Stability of polyomavirus major capsid protein VP1 under denaturants guanidine hydrochloride and urea.

The guanidine hydrochloride (GdnHCl)- and urea-induced equilibrium denaturation of recombinant polyomavirus (Py) major capsid protein VP1 was studied by circular dichroism and fluorescence spectroscopy. Both secondary and tertiary structures of PyVP1 were shown to be disrupted in the presence of denaturants. Although the far-UV circular dichroism (CD) spectra of PyVP1 in the denaturants exhibit similar two-phase transition as those obtained from the fluorescence measurements, the unfolding of PyVP1 in GdnHCl was shown to be more complex than a similar two-state mechanism. The presence of unfolding intermediates is manifested by the noncoincidence of transitions when detected by different probes. The unfolding intermediate appeared to be stabilized by 1 M NaCl. Addition of Ca2+ and 2-mercaptoethanol does not show significant effect on the conformational stability of PyVP1. Unfolding of PyVP1 in GdnHCl was shown to be an irreversible process.

Capsid↗

PubMed1998

Circular dichroism and fluorescence studies of polyomavirus major capsid protein VP1.

The conformational changes of polymavirus (Py) major capsid protein VP1 in solution by the solution pH, addition of calcium, and ionic strength were examined by circular dichroism (CD) and fluorescence spectroscopy. Comparison of the predicted secondary structures of PyVP1 and simian virus (SV) 40 by the methods of Chou-Fasman, Garnier et al., and Yang method are presented. Hydropathicity, surface probability, and chain flexibility of PyVP1 were computer-analyzed by the methods of Kyte and Doolittle, Emini et al., and Karplus and Schulz, respectively. The CD measurements indicate that the secondary structure of PyVP1 is little dependent on its concentration, Ca2+ concentration, and ionic strength, but is strongly pH dependent. Fluorescence studies showed that emission spectra of PyVP1 are also pH-dependent. At extreme acidic and alkaline pH, the fluorescence intensity of PyVP1 is decreased and the emission maximum is red-shifted. The fluorescence of PyVP1 is quenched by the presence of CsCl, KI, and acrylamide. The analyses of the modified Stern-Volmer plots indicate that five of seven tryptophan residues in PyVP1 are located on the surface of the protein, among which two are accessible to Cs+ and the other three are accessible to I- . The two others are buried more deeply in the interior of the protein molecule.

Acrylamide↗

PubMed1998

Topography of low skin resistance points (LSRP) in rats.

Based on the electrical properties of the skin, a method employing the unijunction transistor (UJT) relaxation oscillator for detecting low skin resistance points (LSRP) was developed in this study. By means of this instrumentation, the topography of the LSRP in Wistar rats was developed. All the LSRP in the rats were found to be bilaterally and symmetrically distributed except those points located on the dorsal midline (i.e., governor vessel, GV) and the ventral midline (i.e., conception vessel, CV). The resistances of the LSRP on these two major vessels, including 14 CV points and 17 GV points of six rats were experimentally determined to be in the ranges of 179.4 +/- 41.2 K omega and 152.5 +/- 32.2 K omega, respectively. The resistances of the GV points were found in general to be lower than those of the CV points. Most non-LSRP, on the other hand, exhibited resistances of greater than 420 K omega. It is noted that the resistances of most LSRP increased yet still retained a separate identity within thirty minutes after the death of the animals, but the low resistance properties of some LSRP gradually disappeared thereafter and could not be detected by the relaxation oscillator.

Acupuncture Points↗

PubMed1997

Calcium phosphate as a gene carrier: electron microscopy.

Transfection of DNA into cultured cells by the calcium phosphate (CaPi)-mediated method provides a means of examining gene functions. Several factors in preparing the CaPi-DNA co-precipitates were speculated to be correlated with the transfection efficiency. To achieve a better understanding of the steps of CaPi formation and the effect of other components in the medium, this study was undertaken to examine the morphological changes of CaPi in the transfection medium. Transmission electron microscopy (TEM) was employed to investigate the reaction products of CaPi precipitates prepared in the presence or absence of plasmid DNA, Dulbecco's modified Eagle medium and calf serum. The effect of pH of the HEPES-buffered saline (HBS) in which CaPi was formed was also examined. Here we report the observations we obtained from TEM. The results showed that CaPi precipitates underwent several steps of conversion in the presence of other components in the cell culture medium. The pH of the HBS in which CaPi precipitates were formed also exerts a profound effect on the morphology of the CaPi solid phase, indicating that the associated compositional and structural changes occurred during the maturation of CaPi in the DNA transfection medium.

Calcium Phosphates↗

PubMed1997

A preliminary analysis of antineoplastic activity of parvovirus MVMp NS-1 proteins.

Human gastric cancer MKN-45 cells were transfected with pULB 3238, a plasmid carrying MVMp NS-1 gene with its original P4 promoter replaced by the glucocorticoid inducible promoter MMTV-LTR. After the integration and expression of NS-1 gene, some of the transfectants died, while others remained alive, but the growth features of survived cells were changed. For further study on the antineoplastic function of parvoviral NS-1 protein in vivo, transgenic mice carrying NS-1 genes were established by conventional method. Among 4 founders, one of them was found to be able to transmit the transgene to around 50% of their offsprings. RT-PCR was performed to indicate the expression of NS-1 gene in transgenic mice and its mRNA appeared in a variety of tissues. The expression of integrated NS-1 gene may correlate with the decreased incidence of tumor induced in vivo by chemical carcinogens.

Animals↗

PubMed1997

Studies of DEAE-dextran-mediated gene transfer.

DEAE-dextran-mediated gene transfer was studied for the introduction of pSV2neo DNA into Fisher-rat 3T3 (FR3T3) cells. Zeta (zeta) potentials of the DEAE-dextran-DNA complexes and FR3T3 cells were found to be dependent on the concentration of DEAE-dextran in the medium. The maximum transfection efficiency occurred at a DEAE-dextran/DNA ratio of 50:1 or thereabouts. The interaction between DNA and cells is determined by the adsorption process. The results obtained, along with the correlation between the kinetic adsorption behaviour of 3H-labelled DNA and the transfection efficiency, indicated that adsorption of DEAE-dextran-DNA complexes to the negatively charged cell surfaces, due to electrostatic and dispersion attraction, plays the decisive role in determining the DNA transfection efficiencies.

3T3 Cells↗

PubMed1996

Effect of polyionic compounds on the adsorption of polyoma virus.

The inhibitory effect of polyionic compounds, including dextran sulfates and poly-L-lysines of various molecular weight, on the cytopathogenicity of polyoma virus was studied. Poly-L-lysines were found to be more potent inhibitors of polyoma virus than dextran sulfates in UC1B cells. The 50% effective concentrations (EC50) were found to be inversely related to the molecular weight of the polymers. The higher the molecular weight of poly-L-lysine, the more effective was the polymer in inhibiting virus-induced cytopathogenicity. Incubation of the cells with poly-L-lysines was found to increase the zeta potential of the cells, whereas no significant change of the electrokinetic behavior was observed for dextran sulfate-treated cells, indicating that poly-L-lysines were adsorbed to the cells and prevented virus adsorption through steric hindrance effect. Studies using 3H-labeled virions confirmed that poly-L-lysines inhibit virus adsorption to the host cells. The results obtained from this study indicated that poly-L-lysines bind preferentially to UC1B cells through electrostatic interactions, whereas dextran sulfates are not effective inhibitors of polyoma virus.

Animals↗

PubMed1996

Heparin inhibition of insulin-like growth factor-binding protein-3 binding to human fibroblasts and rat glioma cells: role of heparan sulfate proteoglycans.

The mitogenic action of insulin-like growth factors (IGFs) on target cells is determined by interaction with signaling IGF-I receptors and modulated by interactions with IGF-binding proteins (IGFBPs). IGFBP-3, an abundant IGFBP that binds IGF-I and IGF-II with high affinity, can form soluble inhibitory complexes with the IGFs that prevent them from binding to IGF-I receptors. Alternatively, IGFBP-3 can bind to the cell surface and possibly potentiate IGF action or act independently of the IGFs. Previous studies showed that heparin inhibited IGFBP-3 binding to the cell surface and increased its accumulation in the medium, suggesting that it might act as a competitive inhibitor of IGFBP-3 binding to structurally similar heparan sulfate proteoglycans on the cell surface. We evaluated this hypothesis by binding 125I-labeled recombinant glycosylated human IGFBP-3 to human fetal skin fibroblasts (GM-10) and to C6 rat glioma cells at 12 C. Heparin inhibited [125I]IGFBP-3 binding more effectively than chondroitin sulfate and dextran sulfate. Complete digestion of cell surface heparan sulfate and chondroitin sulfate glycosaminoglycans using heparitinase and chondroitinase ABC, however, did not significantly decrease IGFBP-3 binding. Quantitative removal was demonstrated by analysis of parallel cultures of cells whose glycosaminoglycans had been biosynthetically labeled using Na2 35SO4. These results suggested that IGFBP-3 did not bind to heparan sulfate glycosaminoglycans on the cell surface, and that the inhibition of IGFBP-3 binding by heparin most likely resulted from its direct interaction with the heparin-binding domains of IGFBP-3. When [125I]IGFBP-3 was incubated with GM-10 fibroblasts or C6 glioma cells at 37 C for 4 h, only 10% of the bound ligand remained associated with the cell surface; approximately 90% of the cell-associated radio-activity was internalized and could be recovered in lysates of acid-washed cells. Incubation with IGF-I or heparin decreased the total cell-associated radioactivity, but did not affect internalization. These results suggest that direct interaction of heparin or IGF-I with IGFBP-3 inhibits its ability to bind to the surface of GM-10 fibroblasts and C6 glioma cells.

Animals↗

PubMed1995

Induction of erythroid differentiation by 5-fluorouracil in K562 leukemia cells.

K562 cell line, established from a patient in the blast crisis of chronic myeloid leukemia, expresses high levels of c-myc and bcr/abl gene products. Exposure of K562 cells to 5-fluorouracil (5-FU) resulted in a marked benzidine-positive erythroid differentiation with concomitant reduction in cell proliferation. No change in c-myc mRNA or protein levels occurred during 96 h of drug treatment. In contrast, a biphasic change of p210bcr/abl and the abl-associated kinase activities was observed upon treatment with 5-FU. The change in p210bcr/abl expression may be mediated at the translational level, since the steady-state level and the enzymatic activity of p210bcr/abl are reduced, whereas bcr/abl mRNA levels are unaltered. The results are consistent with the existence of pleiotropic differentiation pathways in K562 cells.

Cell Differentiation↗

PubMed1994

Insulin-like growth factor-I and insulin stimulate the synthesis of IGF-binding protein-2 in a human embryonic kidney cell line.

Insulin and insulin-like growth factor (IGF)-I are thought to be major metabolic regulators of IGF-binding protein-2 (IGFBP-2). We have examined the regulation of IGFBP-2 expression by IGF-I and insulin in 293 cells, a cell line derived from human embryonic kidney. The predominant 34 kDa IGFBP in media conditioned by unstimulated 293 cells was identified as IGFBP-2 by immunoprecipitation. IGFBP-2 levels were increased 6 to 7-fold following incubation with IGF-I, IGF-II or insulin for 48 h. A corresponding increase in IGFBP-2 mRNA was not observed, suggesting that regulation occurred at the translational or post-translational level. The stimulation of IGFBP-2 by IGF-I and insulin was reversibly abolished by incubation with protein synthesis inhibitors such as cycloheximide. Biosynthetic labeling of quiescent 293 cells using [35S]cysteine indicated that incubation with insulin or IGF-I for 24 h increased the synthesis of total cell proteins (predominantly intracellular) and IGFBP-2 (predominantly secreted) to a similar extent (2- to 4-fold). These results suggest that the increase in IGFBP-2 secreted by 293 cells after incubation with IGF-I or insulin largely results from a general stimulation of protein synthesis.

Carrier Proteins↗

PubMed1993

Developmental regulation of insulin-like growth factor binding protein-2 in chick embryo serum and vitreous humor.

The chick embryo is a useful vertebrate model for studying developmental embryogenesis. Insulin-like growth factor I (IGF-I), a potent mitogen, is thought to contribute to the general growth of the embryo as an endocrine factor, and as a paracrine factor to the development of the early embryo and of specific organs such as the eye. Recent data suggest that a family of at least six IGF binding proteins (IGFBPs) complex IGF-I and modulate its biological actions. In the present study, we examine the expression of IGFBPs in chicken serum and vitreous humor at different stages of embryonic development, and compare it with that of IGF-I. As determined by ligand blotting, the predominant IGFBP in chick serum and vitreous humor between embryonic days 4 and 22 (E4-E22) is a 30 kDa IGFBP. This IGFBP was specifically immunoprecipitated by a polyclonal antiserum raised against rat IGFBP-2, the predominant IGFBP in fetal human and rat serum. Although IGFBP-2 is present in both chick fluids at all times examined, serum IGFBP-2 increased progressively between E10-E22, whereas vitreous IGFBP-2 was highest during eye organogenesis (E4-E8). This suggests that vitreous IGFBP-2 is synthesized locally. Like serum IGFBP-2, levels of immunoreactive IGF-I in serum are higher in the second week of embryogenesis than the first. Despite this correlation, changes in IGFBP-2 do not appear to be regulated by IGF-I: (a) serum IGF-I decreases after day 15, whereas IGFBP-2 levels remain stable until hatching; (b) vitreous IGF-I, like serum IGF-I, is higher in the second week of embryogenesis, whereas vitreous IGFBP-2 is highest in the first week; (c) embryos cultured ex ovo express IGFBP-2 at E15-E19, although they lack the normal mid-embryogenesis surge in IGF-I. We conclude that vitreous IGFBP-2 is synthesized locally in the eye, and that the expression of IGFBP-2 in chick embryos is not directly regulated by IGF-I.

Animals↗

PubMed1993

Cyclic adenosine monophosphate stimulates insulin-like growth factor binding protein-4 and its messenger ribonucleic acid in a clonal endothelial cell line.

Endothelial cells regulate the passage of insulin-like growth factors (IGFs) or IGFs complexed to IGF-binding proteins (IGFBPs) from plasma to the extravascular space, and in addition respond to plasma and tissue IGFs. The IGFBPs are thought to determine the availability and localization of IGFs to tissues, and to inhibit or potentiate their biological activity. Because of the potential importance of the IGF system in endothelial cell pathophysiology, and because IGFBPs modulate IGF action, we have characterized the IGFBPs synthesized by a clonal endothelial cell line (BPE-1) established from bovine parathyroid microvessels. The only IGFBPs identified by ligand blotting in media conditioned by BPE-1 cells were N-glycosylated 28 kilodalton and non-N-glycosylated 24 kilodalton IGFBP-4 species. Both forms were immunoprecipitated by antibodies to human IGFBP-4. Northern blot hybridization of BPE-1 RNA identified messenger RNAs for IGFBP-4 and IGFBP-6, but not for IGFBP-1, 2, 3, or 5. Agents that increase cAMP including forskolin, (Bu)2cAMP, isobutyl-methylxanthine, and histamine increased IGFBP-4 and IGFBP-4 messenger RNA in BPE-1 cells 2- to 5-fold and 2- to 3-fold, respectively, similar to results previously reported in human osteosarcoma cells and fibroblasts. Increased IGFBP-4 was detected in BPE-1 media 6 h after forskolin addition and was maximal after 24 h. Maximal stimulation (6- to 9-fold) was observed with 1-30 microM forskolin. IGFBP-4 also was the predominant IGFBP synthesized by two other endothelial cell lines, a clonal cell line established from bovine bone microvessels, and a polyclonal cell line established from calf pulmonary artery. Further study is required to determine the role of endothelial cell IGFBP-4 on endothelium and adjacent cells, and on the transport of IGFs from plasma to specific subendothelial sites.

1-Methyl-3-isobutylxanthine↗

PubMed1992

[Trimethylsilylation of testosterone, epi-testosterone and determination of their ratio in urine by GC-MS].

The trimethylsilylation of testosterone and epitestosterone was discussed in detail in this report. Both derivative conditions under which testosterone and epi-testosterone were prepared into TMS-derivatives in the presence of mercaptoethanol as an antioxidizing agent and method for the analysis of the ratio of testosterone to epi-testosterone in urine, based on GC-MS, had been established. The conditions of detection were: carrier gas was helium, derivatives were separated with SE-54 fused silica capillary column, using temperature program and detected by using multiple ion detection mode in which the ion of m/z 432 was the monitoring ion. The method is rapid, sensitive and specific. For the ratio of testosterone to epi-testosterone (testosterone: 20 ng/microliters), there is a linearity between ratio 1:1 and 10:1 (r = 0.998), the limit of detection for testosterone and epi-testosterone is 1 ng, and the minimum concentration of detection in urine is 8 ng/ml.

Antioxidants↗