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Biomedical subjects

Y Sun

Publications and source records attributed to Y Sun.

At least 361 records · Page 20Linked to original sources
PubMed1999

SnoN and Ski protooncoproteins are rapidly degraded in response to transforming growth factor beta signaling.

Transforming growth factor beta (TGF-beta) regulates a variety of physiologic processes, including growth inhibition, differentiation, and induction of apoptosis. Some TGF-beta-initiated signals are conveyed through Smad3; TGF-beta binding to its receptors induces phosphorylation of Smad3, which then migrates to the nucleus where it functions as a transcription factor. We describe here the association of Smad3 with the nuclear protooncogene protein SnoN. Overexpression of SnoN represses transcriptional activation by Smad3. Activation of TGF-beta signaling leads to rapid degradation of SnoN and, to a lesser extent, of the related Ski protein, and this degradation is likely mediated by cellular proteasomes. These results demonstrate the existence of a cascade of the TGF-beta signaling pathway, which, upon TGF-beta stimulation, leads to the destruction of protooncoproteins that antagonize the activation of the TGF-beta signaling.

Cell Line↗
PubMed1999

The transcription factors Sp1, Sp3, and AP-2 are required for constitutive matrix metalloproteinase-2 gene expression in astroglioma cells.

Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that contribute to pathological conditions associated with angiogenesis and tumor invasion. MMP-2 is highly expressed in human astroglioma cells, and contributes to the invasiveness of these cells. The human MMP-2 promoter contains potential cis-acting regulatory elements including cAMP response element-binding protein, AP-1, AP-2, PEA3, C/EBP, and Sp1. Deletion and site-directed mutagenesis analysis of the MMP-2 promoter demonstrates that the Sp1 site at -91 to -84 base pairs and the AP-2 site at -61 to -53 base pairs are critical for constitutive activity of this gene in invasive astroglioma cell lines. Electrophoretic gel shift analysis demonstrates binding of specific DNA-protein complexes to the Sp1 and AP-2 sites: Sp1 and Sp3 bind to the Sp1 site, while the AP-2 transcription factor binds the AP-2 element. Co-transfection expression experiments in Drosophilia SL2 cells lacking endogenous Sp factors demonstrate that Sp1 and Sp3 function as activators of the MMP-2 promoter and synergize for enhanced MMP-2 activation. Overexpression of AP-2 in AP-2-deficient HepG2 cells enhances MMP-2 promoter activation. These findings document the functional importance of Sp1, Sp3, and AP-2 in regulating constitutive expression of MMP-2. Delineation of MMP-2 regulation may have implications for development of new therapeutic strategies to arrest glioma invasion.

Animals↗
PubMed1999

CCR5 HIV-1 coreceptor activity. Role of cooperativity between residues in N-terminal extracellular and intracellular domains.

Human (H-) CCR5 is the primary coreceptor for ENV-mediated fusion by R5 strains of human immunodeficiency virus type 1, whereas mouse (M-) CCR5 lacks this function. An array of 23 H/M-CCR5 hybrids containing increasing amounts of H-CCR5 extending from the N terminus generated by random chimeragenesis had a biphasic pattern of coreceptor activity with JRFL and 89.6, revealing active regions in the N-terminal extracellular domain (N-ED) and at the junction of cytoplasmic loop 3. The M-CCR5 mutant in which divergent residues were replaced with the corresponding H-CCR5 N-ED sequence (NyYTsE) gained coreceptor function in fusion but not infection experiments. A M-CCR5 double mutant with substitution of human sequences for divergent residues from the N-ED and cytoplasmic loop 3 had augmented coreceptor activity in fusion assays and gain of function in infection experiments. The SIV-251 ENV utilized H- and M-CCR5 and variants. Flow cytometric analysis of M-CCR5 mutants and bifunctional receptors composed of CD4 domains fused to M-CCR5 mutants excluded the possibility that differences in coreceptor activity resulted from variations in cell surface expression. These results demonstrate that the coreceptor activity of the H-CCR5 N-ED is modulated by intracellular residues, illustrating the complexity of CCR5 requirements for interaction with ENV.

Amino Acid Sequence↗
PubMed1999

[Influence of IL-4 on the expression of TNF-alpha and IL-1 alpha mRNA by PBMCs from patients with subfulminant viral hepatitis before and after treatment].

OBJECTIVE: The aim of this study was to compare the influence of IL-4 on the expression of TNF-alpha and IL-1 alpha mRNA by PBMCs from patients with subfulminant viral hepatitis(SFH) before and after IL-4 treatment. METHODS: The expression levels of TNF-alpha and IL-1 alpha mRNA by PBMCs were assessed by semiquantitative RT-PCR. RESULTS: IL-4 suppressed the expression of both TNF-alpha and IL-1 alpha mRNA by PBMCs from SFH patients before and after treatment dose-dependently. However, their dose-response curves showed significant differences. The inhibitory effect could be observed at a concentration of 100 U/ml and was near to maximum at 1,000 U/ml in acute phase, whereas the same suppressive action of IL-4 was reached at 100 U/ml in recovery phase. When PBMCs were cultured in the presence of IL-4 at a concentration of 100 U/ml, IL-4 down-regulated the production of both TNF-alpha and IL-1 alpha mRNA by acute phase PBMCs only to 18.18-21.98% of control cells(in absence of IL-4), while the inhibitory rates were all near to 50% in recovery phases. Moreover, it was also found that the suppressive effect of IL-4 on the expression of PBMCs TNF-alpha and IL-1 alpha mRNA in the patients with endotoxinemia and HBeAg was significantly decreased in acute phase. CONCLUSIONS: The inhibitory effect of IL-4 on the expression of PBMCs TNF-alpha and IL-1 alpha mRNA is markedly decreased in acute phase as compared with that in recovery phase and this declined response may be related to endotoxinemia and viremia.

Adult↗
PubMed1999

Preimplantation genetic diagnosis for spinal muscular atrophy type I.

OBJECTIVE: Couples with children who have spinal muscular atrophy type I (SMA) face a 25% risk of having affected offspring with spontaneous conception. Preimplantation genetic testing (PGT) is possible for the deletions in the survival motor neuron (SMN) gene that have been identified in 98% of SMA type I cases. PGT would provide new reproductive options for families at risk for SMA. METHODS: Three couples with previously affected children confirmed by DNA testing each underwent in vitro fertilization (IVF) and PGT of the resulting embryos. One or two blastomeres were biopsied from each embryo and analyzed for deletions in exons 7 and 8 of the SMN gene. RESULTS: Nine embryos were predicted to be unaffected, three to be affected, and one embryo could not be interpreted. One of three patients receiving transfer of unaffected embryos became pregnant with twins. CONCLUSIONS: Preimplantation genetic testing provides a means for couples at risk for spinal muscular atrophy type I to reduce their chance of initiating an affected pregnancy.

Adult↗
PubMed1999

Proteolytic fragments of anti-HIV and anti-tumor proteins MAP30 and GAP31 are biologically active.

We analyzed the structural and functional organization of anti-HIV and anti-tumor proteins MAP30 and GAP31 by limited proteolysis with endopeptidases Lys-C and Glu-C (V8). MAP30 and GAP31 are resistant to proteolytic digestion under conditions of as much as 5% (w/w) proteases. In the presence of 10% (w/w) protease, the central regions of the proteins are still resistant to proteolysis, whereas the N- and C-termini are accessible. Peptide fragments were purified by FPLC on Superdex 75 columns, characterized by gel electrophoresis, identified by amino acid sequencing, and analyzed for anti-HIV, anti-tumor, and other biochemical activities. We report here that limited proteolysis yields biologically active fragments of both MAP30 and GAP31. These fragments are active against HIV-1 and tumor cells with EC(50)s in the sub-nanomolar ranges, 0.2-0.4 nM. At the dose levels used in the assays, little cytotoxicity to normal cells was observed. In addition, these fragments remain fully active in HIV-integrase inhibition and HIV-LTR topological inactivation, but not ribosome inactivation. These results demonstrate that the antiviral and anti-tumor activities of MAP30 and GAP31 are independent of ribosome inactivation activity. In addition, we demonstrate that portions of the N- and C-termini are not essential for antiviral and anti-tumor activities, but do appear to be required for ribosome inactivation. These results may provide novel strategies for rational design and targeted development of mimetic antiviral and anti-tumor therapeutics.

Amino Acid Sequence↗
PubMed1999

Macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) matrix as an anion-exchange resin for protein adsorption.

A novel macroporous poly(glycidyl methacrylate-triallyl isocyanurate-divinylbenzene) matrix was prepared by a radical suspension copolymerization. The matrix contained epoxy groups, so diethylaminohydroxypropyl groups were coupled to the matrix, leading to an anion-exchange resin. We studied the components, surface and pore structures of the anion-exchange resin by Fourier transform infared spectroscopy and scanning electron microscopy (SEM). SEM observations showed that the resin abounded in macropores as large as 3 to 8 microns both in the surface and the interior. The back-pressure of the column packed with the resin was modest even at a high flow-rate (60.2 cm/min). Then, bovine serum albumin (BSA) was used as a model protein to examine the adsorption properties of the anion-exchange resin. The results showed that under optimum conditions the resin had a capacity as high as 22.8 mg BSA/g wet resin, or 68.7 mg/g dry resin. The adsorbed protein could be desorbed by increasing the liquid phase ionic strength. Most importantly, the matrix had little nonspecific adsorption for BSA before introducing the ion-exchange groups.

Adsorption↗
PubMed1999

Differential effects of cAMP in neurons and astrocytes. Role of B-raf.

Mitogen-activated protein kinase (MAPK) activation provides cell type-specific signals important for cellular differentiation, proliferation, and survival. Cyclic AMP (cAMP) has divergent effects on MAPK activity depending on whether signaling is through Ras/Raf-1 or Rap1/B-raf. We found that central nervous system-derived neurons, but not astrocytes, express B-raf. In neurons, cAMP activated MAPK in a Rap1/B-raf-dependent manner, while in astrocytes, cAMP decreased MAPK activity. Inhibition of MAPK in neurons decreased neuronal growth factor-mediated survival, and activation of MAPK by cAMP analogues rescued neurons from death. Furthermore, constitutive expression of B-raf in astrocytoma cells increased MAPK activation, as seen in neurons, and enhanced proliferation. These data provide the first experimental evidence that B-raf is the molecular switch which dominantly permits differential cAMP-dependent regulation of MAPK in neurons versus astrocytes, with important implications for both survival and proliferation.

Animals↗
PubMed1999

Biodistribution of free and N-(2-hydroxypropyl)methacrylamide copolymer-bound mesochlorin e(6) and adriamycin in nude mice bearing human ovarian carcinoma OVCAR-3 xenografts.

The purpose of this study was to examine the biodistribution of the photosensitizing drug, mesochlorin e(6) monoethylenediamine (Mce(6)), and the antineoplastic agent, adriamycin (ADR), as well as their N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates in female nu/nu athymic mice bearing human ovarian carcinoma OVCAR-3 xenografts. The levels of Mce(6) and HPMA copolymer-bound Mce(6) in tissues were assayed spectrophotometrically, while the levels of ADR and HPMA copolymer-bound ADR were determined using high-performance liquid chromatography. It appeared that the circulation lifetimes of HPMA copolymer-bound Mce(6) and ADR were three times more than those of the drugs in the free form. The concentrations of the HPMA copolymer-conjugated drugs in tumor reached maximum levels 18 h post injection. Intravenous injection routinely gave higher tissue levels of the drugs than intraperitoneal administration at time intervals less than 24 h. The biodistribution of the HPMA copolymer-bound drugs in tumor-bearing mice was significantly different from that of the free drugs, which is important in optimizing the treatment protocols. In particular, the HPMA copolymer-conjugated drugs accumulated at significantly higher levels in tumor tissues. This effect is attributed to the increased vascular permeability and reduced lymphatic drainage characteristic of tumor tissues [enhanced permeability and retention (EPR) effect].

Animals↗
PubMed1999

Targeting tumor vasculature endothelial cells and tumor cells for immunotherapy of human melanoma in a mouse xenograft model.

An immunotherapy treatment for cancer that targets both the tumor vasculature and tumor cells has shown promising results in a severe combined immunodeficient mouse xenograft model of human melanoma. The treatment involves systemic delivery of an immunoconjugate molecule composed of a tumor-targeting domain conjugated to the Fc effector domain of human IgG1. The effector domain induces a cytolytic immune response against the targeted cells by natural killer cells and complement. Two types of targeting domains were used. One targeting domain is a human single-chain Fv molecule that binds to a chondroitin sulfate proteoglycan expressed on the surface of most human melanoma cells. Another targeting domain is factor VII (fVII), a zymogen that binds with high specificity and affinity to the transmembrane receptor tissue factor (TF) to initiate the blood coagulation cascade. TF is expressed by endothelial cells lining the tumor vasculature but not the normal vasculature, and also by many types of tumor cells including melanoma. Because the binding of a fVII immunoconjugate to TF might cause disseminated intravascular coagulation, the active site of fVII was mutated to inhibit coagulation without affecting the affinity for TF. The immunoconjugates were encoded as secreted molecules in a replication-defective adenovirus vector, which was injected into the tail vein of severe combined immunodeficient mice. The results demonstrate that a mutated fVII immunoconjugate, administered separately or together with a single-chain Fv immunoconjugate that binds to the tumor cells, can inhibit the growth or cause regression of an established human tumor xenograft. This procedure could be effective in treating a broad spectrum of human solid tumors that express TF on vascular endothelial cells and tumor cells.

Adenoviridae↗
PubMed1999

Wild-type but not Alzheimer-mutant amyloid precursor protein confers resistance against p53-mediated apoptosis.

Amyloid precursor proteins (APPs) are expressed in multiple organs and cell types in diverse species. Their conservation across species and high abundance in brain and the association of various APP missense mutations with autosomal dominant forms of familial Alzheimer's disease (FAD) suggest important roles for APP in the central nervous system. However, the basic functions of APP in the central nervous system remain largely unknown. To assess potential effects of APP on neuronal death and survival, we transfected APP-deficient rat neuroblastoma cells (B103) with DNA constructs encoding wild-type or FAD-mutant human APP. Wild-type, but not FAD-mutant, APP effectively protected cells against apoptosis induced by ultraviolet irradiation, staurosporine, or p53. Wild-type APP also strongly inhibited p53 DNA-binding activity and p53-mediated gene transactivation, whereas FAD-mutant APP did not. We conclude that APP protects neuronal cells against apoptosis by controlling p53 activation at the post-translational level. Disruption of this function by mutations or alterations in APP processing could enhance neuronal vulnerability to secondary insults and contribute to neuronal degeneration.

Alzheimer Disease↗
PubMed1999

Penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus: kinetic characterization of its interactions with beta-lactams using electrospray mass spectrometry.

Penicillin-binding protein 2a (PBP2a) is the primary beta-lactam resistance determinant of methicillin-resistant Staphylococcus aureus (MRSA). MecA, the gene coding for PBP2a, was cloned with the membrane-anchoring region at the N-terminus deleted. The truncated protein (PBP2a) was overexpressed in Escherichia coli mostly in the soluble form accounting for approximately 25% of soluble cell protein and was purified to homogeneity. The purified protein was shown to covalently bind beta-lactams in an 1:1 ratio as determined by electrospray mass spectrometry. A novel method based on HPLC-elctrospray mass spectrometry has been developed to quantitatively determine the formation of the covalent adducts or acyl-PBP2a complexes. By using this method, combined with kinetic techniques including quench flow, we have extensively characterized the interactions between PBP2a and three beta-lactams and determined related kinetic parameters for the first time. The apparent first-order rate constants (ka) of PBP2a acylation by benzylpenicillin showed a hyperbolic dependence on the concentration of benzylpenicillin. This is consistent with the mechanism that the binding of the penicillin to PBP2a consists of reversible formation of a Michaelis complex followed by formation of the penicilloyl-PBP2a adduct, and allowed the determination of the individual kinetic parameters for these two steps, the dissociation constant Kd of 13.3 mM and the first-order rate constant k2 of 0.22 s-1. From these values, the second-order rate constant k2/Kd, the value reflecting the overall binding efficiency of a beta-lactam, of 16.5 M-1 s-1 was obtained. The fairly high Kd value indicates that benzylpenicillin fits rather poorly into the protein active site. Similar studies on the interaction between PBP2a and methicillin revealed k2 of 0.0083 s-1 and Kd of 16.9 mM, resulting in an even smaller k2/Kd value of 0.49 M-1 s-1. The rate constants k3 for deacylation of the acyl-PBP2a complexes, the third step in the interactions, were measured to be <1.5 x 10(-)5 s-1. These results indicate that the resistance of PBP2a to penicillin inactivation is mainly due to the extremely low penicillin acylating rate in addition to the low association affinity, but not to a fast rate of deacylation. Acylation of PBP2a by a high-affinity cephalosporin, Compound 1, also followed a saturation curve of ka versus the compound concentration, from which k2 = 0.39 s-1, Kd = 0.22 mM, and k2/Kd = 1750 M-1 s-1 were obtained. The 100-fold increase in the k2/Kd value as compared with that of benzylpenicillin is mostly attributable to the decreased (60-fold) Kd, indicating that the cephalosporin fits much better to the binding pocket of the protein.

Acylation↗
PubMed1999

Expression of the c-myc proto-oncogene is essential for HIV-1 infection in activated T cells.

We previously found that activation of primary CD4+ T cells via both the T cell antigen receptor (TCR) and CD28 is required for HIV-1 DNA to be translocated from the cytoplasm to the nucleus. Here we report that expression of c-Myc protein in CD4+ T cells is induced only after such costimulation. In addition, cyclosporin A not only inhibits nuclear import of HIV-1 DNA but also inhibits expression of c-Myc protein. Because of these correlations, we tested whether c-Myc is necessary for nuclear import of HIV-1 DNA. Specific c-myc antisense, but not sense or non-sense, phosphorothioate oligodeoxynucleotides selectively induced the accumulation of two NH2-terminally truncated c-Myc proteins and abolished HIV-1 genome entry into host nuclei. Consequently, both virus replication and HIV-1-induced apoptotic cell death were inhibited. Synthesis of viral full-length DNA was not affected. Specific c-myc antisense oligonucleotide inhibited HIV-1 infection under conditions that did not affect cell cycle entry or proliferation. Thus, c-Myc appears to regulate HIV-1 DNA nuclear import via a mechanism distinct from those controlling entry into the cell cycle.

CD4-Positive T-Lymphocytes↗
PubMed1999

HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma.

Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae. This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers. Promoter region and one pseudogene of hRad17 have been identified. Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.

Animals↗
PubMed1999

p38 kinase mediates UV-induced phosphorylation of p53 protein at serine 389.

The p53 tumor suppressor protein is a transcription factor that plays a key role in the process of apoptosis and the cell's defense against tumor development. Activation of p53 occurs, at least in part, by phosphorylation of its protein. Very recently it has been reported that UV induced a functional activation of p53 via phosphorylation at serine 389. Here, we report that the UV-induced phosphorylation of p53 at serine 389 is mediated by p38 kinase. UVC-induced phosphorylation of p53 at serine 389 was markedly impaired by either pretreatment of cells with p38 kinase inhibitor, SB202190, or stable expression of a dominant negative mutant of p38 kinase. In contrast, there was no inhibition observed in cells treated with specific MEK1 inhibitor, PD98059, or with stable expression of a dominant negative mutant of ERK2 or JNK1. Most importantly, p38 kinase could be co-immunoprecipitated with p53 by using antibodies against p53. Incubation of active p38 kinase with p53 protein caused the phosphorylation of p53 protein at serine 389 in vitro, while no phosphorylation of p53 at serine 389 was observed when p53 was incubated with activated JNK2 or ERK2. Furthermore, pretreatment of cells with SB202190 blocked the p53 DNA binding activity and p53-dependent transcription. These results strongly suggest that the p38 kinase is at least one of the most important mediators of p53 phosphorylation at serine 389 induced by UVC radiation.

Antibodies, Monoclonal↗
PubMed1999

p53 down-regulates human matrix metalloproteinase-1 (Collagenase-1) gene expression.

Recent studies show that the p53 tumor suppressor protein is overexpressed in rheumatoid arthritis (RA) synovium and that somatic mutations previously identified in human tumors are present in RA synovium (Firestein, G. S., Echeverri, F., Yeo, M., Zvaifler, N. J., and Green, D. R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10895-10900; Firestein, G. S., Nguyen, K., Aupperle, K. R., Yeo, M., Boyle, D. L., and Zvaifler, N. J. (1996) Am. J. Pathol. 149, 2143-2151; Reme, T., Travaglio, A., Gueydon, E., Adla, L., Jorgensen, C., and Sany, J. (1998) Clin. Exp. Immunol. 111, 353-3581). We hypothesize that the abnormality of p53 seen in RA synovium may contribute to joint degeneration through the regulation of human matrix metalloproteinase-1 (hMMP-1, collagenase-1) gene expression. Transcription assays were performed with luciferase reporters driven by the promoter of the hMMP-1 gene or by a minimal promoter containing tandem repeats of the consensus binding sequence for activator protein-1, cotransfected with p53-expressing plasmids. The results revealed that (i) wild-type (wt) p53 down-regulated the promoter activity of hMMP-1 in a dose-dependent fashion; (ii) four of six p53 mutants (commonly found in human cancers) lost this repression activity; and (iii) this p53 repression activity was mediated at least in part by the activator protein-1 sites found in the hMMP-1 promoter. These findings were further confirmed by Northern analysis. The down-regulation of hMMP-1 gene expression by endogenous wt-p53 was shown by treatment of U2-OS cells, a wt-p53-containing osteogenic sarcoma line, and Saos-2 cells, a p53-negative osteogenic sarcoma line, with etoposide, a potent inducer of p53 expression. p53, activated by etoposide, appears to block hMMP-1 promoter activity induced by etoposide in U2-OS cells. In summary, we have shown for the first time that the hMMP-1 gene is a p53 target gene, subject to p53 repression. Because MMP-1 is principally responsible for the irreversible destruction of collagen in articular tissue in RA, abnormality of p53 may contribute to joint degeneration through the regulation of MMP-1 expression.

Binding Sites↗
PubMed1999

Transcriptional activation of the human glutathione peroxidase promoter by p53.

Glutathione peroxidase (GPX) is a primary antioxidant enzyme that scavenges hydrogen peroxide or organic hydroperoxides. We have recently found that GPX is induced by etoposide, a topoisomerase II inhibitor and a p53 activator. In a search for a cis-element that confers potential p53 regulation of GPX, we identified a p53 binding site in the promoter of the GPX gene. This site bound to purified p53 as well as p53 in nuclear extract activated by etoposide. A luciferase reporter driven by a 262-base pair GPX promoter fragment was transcriptionally activated by wild type p53 in a p53 binding site-dependent manner. The same reporter was also activated in a p53 binding site-independent manner by several p53 mutants. The p53 binding and transactivation of the GPX promoter were enhanced by etoposide in p53-positive U2-OS cells. Etoposide-induced transactivation was blocked by a dominant negative p53 mutant, indicating that endogenous wild type p53, upon activation by etoposide, transactivated the GPX promoter. Furthermore, expression of endogenous GPX was induced significantly at both mRNA and enzyme activity levels by etoposide in U2-OS cells but not in p53-negative Saos-2 cells. This is the first report demonstrating that GPX is a novel p53 target gene. The finding links the p53 tumor suppressor to an antioxidant enzyme and will facilitate study of the p53 signaling pathway and antioxidant enzyme regulation.

Base Sequence↗