Biomedical subjects
Publications and source records attributed to X Wu.
Two compounds containing cyanide [Ni(1,10-phen)2O2MoS2(CuCN)]n and [ZnCu4 (CN)6(dmf)4]n have been synthesized. The FTIR spectra and the crystal structure of those clusters have also been deal with. The stretching frequency of cyanide is alterable with the different coordinated environments.
OBJECTIVE: To investigate the relationship between the expression of matrix metalloproteinase-2 (MMP-2) and clinical characteristics of patients with epithelial ovarian tumor. METHODS: Reverse transcriptase-ploymerase chain reaction and immunohistochemistry were used to detect the expression of MMP-2 in 41 epithelial ovarian cancer and 26 ovarian benign tumors. Western blot analysis was used to examine proMMP-2 and active MMP-2 in these tumors. RESULTS: The expression of MMP-2 gene and protein was not significantly higher in malignant tissues than that in benign ones. The positive percentage of active MMP-2 expression in epithelial ovarian cancer was 71% and 42% in ovarian benign tumor (P < 0.05). The positive percentage of active MMP-2 in stage IlI and IV epithelial ovarian cancer was significantly higher than in stage I and II epithelial ovarian cancer (P < 0.05). The positive percentage of active MMP-2 in progressive patients with epithelial ovarian cancer was significantly higher than in patients in remission (P < 0.05). CONCLUSIONS: MMP-2 gene and protein were generally expressed in epithelial ovarian tumor. There was a significant correlation between active MMP-2 and the invasion and metastasis of epithelial ovarian cancer. Active MMP-2 is a prognostic indicator in patients with epithelial ovarian cancer.
We performed visible/near-infrared optical measurements on the forearm of human subjects. We conducted four studies: one study using a commercial diffuse reflectance spectrometer, and three studies using a breadboard temperature-controlled localized reflectance tissue photometer. Calibration relationships were established between skin reflectance signal and either the reference blood hemoglobin (Hb) concentration or the hematocrit values (Hct). Prediction results were expressed as the prediction correlation coefficient (r(p)) and the standard error for cross-validation prediction (CV-SEP). Using diffuse reflectance measurement, r(p) = 0. 8, CV-SEP = 0.9 g/dL for Hb and r(p) = 0.7, CV-SEP = 3.3% for Hct (n = 40). In a localized reflectance study involving calculating the absorption and scattering coefficients and including effect of change of skin temperature in the calibration model, the best prediction results were r(p) = 0.9, CV-SEP = 0.8 g/dL for Hb and r(p) = 0.8, CV-SEP = 3% for Hct (n = 26). In a second localized reflectance study on a population having diverse skin colors at 34 degrees C, the optimal model led to r(p) = 0.8, CV-SEP = 0.9 g/dL for Hb and r(p) = 0.9, CV-SEP = 2.1% for Hct (n = 28) using the localized reflectance values without deducing the absorption and scattering coefficients. Improvement in the correlation was more noticeable for Hct than for the case of Hb. The photometer was used to screen prospective blood donors with low Hb concentration. It was possible to predict anemic subjects in the limited prospective blood donor population.
The human endogenous retrovirus, type K (HERV-K) represents the most biologically active form of known retroelements present in the human genome. Several HERV-K genomes have transcriptionally active open reading frames and encode their own protease (PR). The HERV-K PR has been shown to authentically cleave human immunodeficiency virus type 1 (HIV-1) matrix-capsid peptide in the presence of HIV-1 PR inhibitors. This raised the possibility that HERV-K PR could complement HIV-1 PR function in HIV-1-infected individuals. To investigate this possibility, we fused the HIV-1 vpr gene to the HERV-K PR gene (vpr-PR). The vpr-PR expression plasmid and a PR-defective HIV-1 clone were cotransfected into 293T cells. Progeny virions were assayed for processing of the HIV-1 polyproteins by Western blot and for changes in infectivity. HERV-K PR fused to Vpr was incorporated into HIV-1 virions at a high concentration and cleaved the Gag and Pol precursor proteins. However, neither Gag nor Pol polyproteins were correctly processed. Moreover, the HERV-K PR did not restore virus infectivity. While these results do not exclude the possibility that the HERV-K PR could complement an HIV-1 PR whose function is impaired due to drugs or drug-resistant mutations, they clearly demonstrate that the HERV-K PR cannot substitute for the function of the wild-type HIV-1 PR.
The synthesis of (25R)-ruscogenin-1-yl beta-D-xylopyranosyl-(1-->3)-[beta-D-glucopyranosyl-(1-->2)]-beta-D-fucopyranoside, a saponin from the tuber of Liriope muscari (Decne.) Bailey, is described.
DNA lesion bypass is an important cellular response to genomic damage during replication. Human DNA polymerase eta (Pol(eta)), encoded by the Xeroderma pigmentosum variant (XPV) gene, is known for its activity of error-free translesion synthesis opposite a TT cis-syn cyclobutane dimer. Using purified human Pol(eta), we have examined bypass activities of this polymerase opposite several other DNA lesions. Human Pol(eta) efficiently bypassed a template 8-oxoguanine, incorporating an A or a C opposite the lesion with similar efficiencies. Human Pol(eta) effectively bypassed a template abasic site, incorporating an A and less frequently a G opposite the lesion. Significant -1 deletion was also observed when the template base 5' to the abasic site is a T. Human Pol(eta) partially bypassed a template (+)-trans-anti-benzo[a]pyrene-N:(2)-dG and predominantly incorporated an A, less frequently a T, and least frequently a G or a C opposite the lesion. This specificity of nucleotide incorporation correlates well with the known mutation spectrum of (+)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in mammalian cells. These results show that human Pol(eta) is capable of error-prone translesion DNA syntheses in vitro and suggest that Pol(eta) may bypass certain lesions with a mutagenic consequence in humans.
TNAs [(L)-alpha-threofuranosyl oligonucleotides] containing vicinally connected (3'-->2') phosphodiester bridges undergo informational base pairing in antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. Being derived from a sugar containing only four carbons, TNA is structurally the simplest of all potentially natural oligonucleotide-type nucleic acid alternatives studied thus far. This, along with the base-pairing properties of TNA, warrants close scrutiny of the system in the context of the problem of RNA's origin.
Vascular endothelial growth factor (VEGF), through activation of its endothelial receptors VEGFR-1 and VEGFR-2, is an important positive modulator of tumor angiogenesis and edema in solid tumors such as malignant astrocytomas. Neuropilin-1 (Npn-1) is a transmembrane receptor expressed by both endothelial and non-endothelial cells, including tumor cells. Npn-1 has been postulated to function as a co-factor in activation of the biologically relevant VEGFR-2, by the most abundant VEGF165 isoform. However, the function of Npn-1 in normal and pathological angiogenesis, its expression pattern in relation to VEGF in tumors such as astrocytomas and whether it is similarly or differentially regulated compared to VEGF remain unknown. In our study, the expression pattern of Npn-1 and VEGF by human astrocytoma cell lines and specimens was closely correlated and associated with malignant astrocytomas. Mitogens, such as epidermal growth factor and activation of p21-Ras, previously demonstrated to be relevant in astrocytoma proliferation and induction of VEGF, also induce Npn-1 expression. Hypoxia, the main physiological inducer of VEGF expression, decreased Npn-1 expression. Increased Npn-1 expression was also demonstrated in a transgenic mouse astrocytoma model. Astrocytomas are an ideal system for furthering our understanding of the functional relevance, if any, of Npn-1 in tumor angiogenesis.
Injury to the central nervous system (CNS) invokes a reparative response known as astrogliosis, characterized largely by hypertrophy, proliferation and increased expression of glial fibrillary acidic protein (GFAP), resulting in reactive astrocytosis. Based on our prior observation that peritumoral reactive astrocytes express Vascular Endothelial Growth Factor (VEGF), a highly potent and specific angiogenic growth factor, we have hypothesized that reactive astrocytosis also contributes to the neovascularization associated with astrogliosis. To evaluate this hypothesis we evaluated human surgical/autopsy specimens from a variety of CNS disorders that induce astrogliosis and an experimental CNS needle injury model in wild type and GFAP:Green Fluorescent Protein (GFP) transgenic mice. Using computer image semi-quantitative analysis we evaluated the number of GFAP-positive reactive astrocytes, degree of VEGF expression by these astrocytes, associated Factor VIII-positive microvascular density (MVD) and Ki-67 proliferating endothelial cells. The degree of reactive astrocytosis correlated to levels of VEGF immunoreactivity and MVD in the neuropathological specimens. The mouse-needle-stick brain injury model demonstrated this correlation was temporally and spatially related and maximal after 1 week. These results, involving both human pathology specimens augmented by experimental animal data, supports our hypothesis that the neoangiogenesis associated with reactive astrogliosis is correlated to increased reactive astrocytosis and associated VEGF expression.
Error-free lesion bypass and error-prone lesion bypass are important cellular responses to DNA damage during replication, both of which require a DNA polymerase (Pol). To identify lesion bypass DNA polymerases, we have purified human Polkappa encoded by the DINB1 gene and examined its response to damaged DNA templates. Here, we show that human Polkappa is a novel lesion bypass polymerase in vitro. Purified human Polkappa efficiently bypassed a template 8-oxoguanine, incorporating mainly A and less frequently C opposite the lesion. Human Polkappa most frequently incorporated A opposite a template abasic site. Efficient further extension required T as the next template base, and was mediated mainly by a one-nucleotide deletion mechanism. Human Polkappa was able to bypass an acetylaminofluorene-modified G in DNA, incorporating either C or T, and less efficiently A opposite the lesion. Furthermore, human Polkappa effectively bypassed a template (-)-trans-anti-benzo[a]pyrene-N:(2)-dG lesion in an error-free manner by incorporating a C opposite the bulky adduct. In contrast, human Polkappa was unable to bypass a template TT dimer or a TT (6-4) photoproduct, two of the major UV lesions. These results suggest that Polkappa plays an important role in both error-free and error-prone lesion bypass in humans.
Escherichia coli DNA polymerase IV encoded by the dinB gene is involved in untargeted mutagenesis. Its human homologue is DNA polymerase kappa (Polkappa) encoded by the DINB1 gene. Our recent studies have indicated that human Polkappa is capable of both error-free and error-prone translesion DNA synthesis in vitro. However, it is not known whether human Polkappa also plays a role in untargeted mutagenesis. To examine this possibility, we have measured the fidelity of human Polkappa during DNA synthesis from undamaged templates. Using kinetic measurements of nucleotide incorporations and a fidelity assay with gapped M13mp2 DNA, we show that human Polkappa synthesizes DNA with extraordinarily low fidelity. At the lacZalpha target gene, human Polkappa made on average one error for every 200 nucleotides synthesized, with a predominant T-->G transversion mutation at a rate of 1/147. The overall error rate of human Polkappa is 1.7-fold lower than human Poleta, but 33-fold higher than human Polbeta, a DNA polymerase with very low fidelity. Thus, human Polkappa is one of the most inaccurate DNA polymerases known. These results support a role for human Polkappa in untargeted mutagenesis surrounding a DNA lesion and in DNA regions without damage.
The integrity of the DNA damage response pathway is essential for prevention of neoplastic transformation. Several proteins involved in this pathway including p53, BRCA1, and ATM are frequently mutated in human cancer. Checkpoint kinase 2 (Chk2) is a DNA damage-activated protein kinase that lies downstream of ATM in this pathway. Recently, heterozygous germline mutations in Chk2 have been identified in a subset of patients with Li-Fraumeni syndrome, a highly penetrant familial cancer phenotype, suggesting that Chk2 is a tumor suppressor gene. In this study, we have reported the biochemical characterization of the four tumor-associated Chk2 mutants. Two of the reported Chk2 mutations identified in Li-Fraumeni syndrome result in loss of Chk2 kinase activity. Whereas one mutation within the Chk2 forkhead homology-associated (FHA) domain, R145W, retains some basal kinase activity, this mutant cannot be phosphorylated at an ATM-dependent phosphorylation site (Thr-68) and cannot be activated following gamma radiation. Wild-type Chk2 exists mainly in a protein complex of M(r) approximately 200,000 whereas the R145W mutant forms a larger, presumably inactive complex in the cell. The other FHA domain mutant, I157T, behaves as wild-type Chk2 in all the assays used here. Because the FHA domain is involved in protein-protein interactions, this mutation may affect associations of Chk2 with other proteins. Additionally, we have shown that Chk2 can also be inactivated by down-regulation of its expression in cancer cells. Thus, Chk2 may be inactivated by multiple mechanisms in the cell.
Mitochondria is believed to play a central role in p53-mediated apoptosis. However, the signal transduction pathways leading to mitochondria remain unclear. Here, we report that translocation of Bax protein from cytosol to mitochondria is required for p53-induced apoptosis. Cytosolic Bax is unable to induce apoptosis, and blocking Bax translocation inhibits cell death. Expression of Bcl-2 blocks cytochrome c release and apoptosis but has no effect on Bax translocation, suggesting that Bax translocation acts upstream of Bcl-2. We further demonstrate that Peg3/Pw1, a protein up-regulated in p53-mediated cell death process, induces Bax translocation independent of apoptosis. The results suggest that Bax translocation represents an important regulatory step in p53-mediated apoptosis, and Peg3/Pw1 functions as a modulator downstream of p53 to regulate Bax redistribution in the cells, thus favoring the cellular decision toward apoptosis over growth arrest following p53 induction.
The solid-phase syntheses of two deglycobleomycin A(5) analogues were achieved using a commercially available polystyrene resin containing triphenylmethyl-linked spermidine. The final products were deblocked and released from the resin, analyzed, and purified by C(18) reversed phase HPLC and characterized by high-field (1)H NMR spectroscopy and mass spectrometry. The purified products relaxed supercoiled plasmid DNA in a concentration-dependent fashion and to the same extent as authentic material derived from natural BLM A(5).
DNA damage can lead to mutations during replication. The damage-induced mutagenesis pathway is an important mechanism that fixes DNA lesions into mutations. DNA polymerase zeta (Pol zeta), formed by Rev3 and Rev7 protein complex, and Rev1 are components of the damage-induced mutagenesis pathway. Since mutagenesis is an important factor during the initiation and progression of human cancer, we postulate that this mutagenesis pathway may provide an inhibiting target for cancer prevention and therapy. In this study, we tested if UV-induced mutagenesis can be altered by molecular modulation of Rev3 enzyme levels using the yeast Saccharomyces cerevisiae as a eukaryotic model system. Reducing the REV3 expression in yeast cells through molecular techniques was employed to mimic Pol zeta inhibition. Lower levels of Pol zeta significantly decreased UV-induced mutation frequency, thus achieving inhibition of mutagenesis. In contrast, elevating the Pol zeta level by enhanced expression of both REV3 and REV7 genes led to a approximately 3-fold increase in UV-induced mutagenesis as determined by the arg4-17 mutation reversion assays. In vivo, UV lesion bypass by Pol zeta requires the Rev1 protein. Even overexpression of Pol zeta could not alleviate the defective UV mutagenesis in the rev1 mutant cells. These observations provide evidence that the mutagenesis pathway could be used as a target for inhibiting damage-induced mutations.
A novel nucleotide inhibitor (ADP-S-HBES-S-dGTP) of mouse ribonucleotide reductase was designed to span the active site and the allosteric specificity site of the enzyme. The inhibitor contains ADP and dGTP moieties which are linked by 1,6-hexane-(bis-ethylenesulfone), and has a Ki value of 12 microM.
We studied the sequential topology of the NH(2) and COOH termini of apoB during translocation by expressing, in Chinese hamster ovary (CHO) and HepG2 cells, an apoB42 construct with c-Myc and hemagglutinin (HA) tags at 2 and 41% (relative to apoB100) of its amino acid sequence. We conducted similar studies using monoclonal antibodies against the NH(2) and COOH termini of apoB100 in HepG2 cells. After radiolabeling, microsomes were immunoisolated from transfected CHO cells using anti-c-Myc or anti-HA antibodies. Throughout a 60-min chase in the presence of N-acetyl-leucyl-norleucinal, more than 90% of microsomes were isolated by anti-HA antibodies, whereas less than 10% were isolated by anti-c-Myc antibodies. Proteinase K digestion of total microsomes consistently generated two fragments ( approximately 70 and approximately 120 kDa) of apoB42 containing the NH(2) terminus throughout the chase; no fragments containing the COOH terminus were detected. Immunofluorescent studies of transfected CHO cells were consistent with results from the labeling studies. Essentially identical results were obtained from pulse-chase studies in both native and apoB42-transfected HepG2 cells. The present studies support a model in which, in the absence of adequate core lipid synthesis, there is partial translocation of apoB leading to cytosolic exposure, ubiquitination, and proteasomal degradation directly from the original translocation channel.
GIRK (G protein-gated inward rectifier K(+) channel) proteins play critical functional roles in heart and brain physiology. Using antibodies directed to either GIRK1 or GIRK4, site-directed mutagenesis, and specific glycosidases, we have investigated the effects of glycosylation in the biosynthesis and heteromerization of these proteins expressed in oocytes. Both GIRK1 and GIRK4 have one extracellular consensus N-glycosylation site. Using chimeras between GIRK1 and GIRK4 as well as a GIRK1 N-glycosylation mutant, we report that GIRK1 was glycosylated at Asn(119), whereas GIRK4 was not glycosylated at Asn(132). GIRK1 membrane-spanning domain 1 was required for optimal glycosylation at Asn(119) because a chimera that contained GIRK4 membrane-spanning domain 1 significantly reduced the addition of a carbohydrate structure at this site. This finding may partly account for the reason that GIRK4 is not glycosylated at Asn(132), either as a homomer or when coexpressed with GIRK1. When the GIRK1(N119Q) mutant was coexpressed with GIRK4, the biophysical properties of the heteromeric channel and the magnitude of the agonist-induced currents were similar to those of controls. Thus, N-glycosylation of GIRK1 at Asn(119) does not appear to affect its physical association with GIRK4, the routing of the heteromer to the cell surface, or heteromeric channel function, unlike the dramatic functional effects of N-glycosylation of ROMK1 at Asn(117) (Schwalbe, R. A., Wang, Z., Wible, B. A., and Brown, A. M. (1995) J. Biol. Chem. 270, 15336-15340).