Biomedical subjects

X Wu

Publications and source records attributed to X Wu.

At least 19 recordsLinked to original sources

PubMed1999

Identity of the F52F12.1 gene product in Caenorhabditis elegans as an organic cation transporter.

We describe here the cloning and functional characterization of an organic cation transporter from Caenorhabditis elegans (CeOCT1). The CeOCT1 cDNA is 1826 bp long and codes for a protein of 568 amino acids. The oct1 gene is approximately 3.2 kb in size and consists of 12 exons. The location of this gene corresponds to the F52F12.1 gene locus on chromosome I. The predicted protein contains 12 putative transmembrane domains. It exhibits significant homology to mammalian OCTs. When expressed in mammalian cells, CeOCT1 induces the transport of the prototypical organic cation tetraethylammonium. The Michaelis-Menten constant for this substrate is 80+/-16 microM. The substrate specificity of CeOCT1 is broad. This represents the first report on the cloning and functional characteristics of an organic cation transporter from C. elegans.

Amino Acid Sequence↗

PubMed1999

Cloning, expression and biochemical characterization of a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys pallas.

A cDNA encoding a basic-acidic hybrid phospholipase A2-II from Agkistrodon halys Pallas with an N-terminus highly homologous to that of BPLA2 and a C-terminus sequence almost the same as that of APLA2 was inserted into a bacterial expression vector and effectively expressed in Escherichia coli RR1. The protein was produced as insoluble inclusion bodies. After partial purification by washing, the inclusion bodies with Triton X-100, denaturing and refolding, the renatured recombinant protein was purified by FPLC column superose 12. The purified recombinant enzyme with an isoelectric point of pH 6.8 could cross-react with antiserum prepared against acidic phospholipase A2. The enzymatic activity of the expressed basic-acidic hybrid phospholipase A2-II is close to that of denatured-refolded native basic phospholipase A2, and has the same inhibiting effect on platelet aggregation as denatured-refolded acidic phospholipase A2, but lacks the hemolytic activity of denatured-refolded basic phospholipase A2. To study the structural relationships among basic phospholipase A2, acidic phospholipase A2 and basic-acidic hybrid phospholipase A2-II, molecular modeling of basic-acidic hybrid phospholipase A2-II was done. The roles of various amino acid residues in the enzymatic activity and pharmacological activities of phospholipase A2 are discussed.

Agkistrodon↗

PubMed1999

Recurrence of bronchioloalveolar carcinoma in transplanted lungs.

BACKGROUND: Bronchioloalveolar carcinoma is a distinctive subtype of typical adenocarcinoma of the lung that tends to metastasize widely throughout the lungs but less commonly elsewhere. Because conventional therapies for intrapulmonary metastatic bronchioloalveolar carcinoma are generally ineffective, we treated seven patients who had intrapulmonary metastatic bronchioloalveolar carcinoma with lung transplantation. METHODS: Seven patients with biopsy-proved bronchioloalveolar carcinoma and no evidence of extrapulmonary disease received transplants of either one or two cadaveric lungs. At transplantation, all native lung tissue was removed and replaced with a donor lung or lungs. The patients received the usual post-transplantation care given at the institution. RESULTS: Four of the seven patients had recurrent bronchioloalveolar carcinoma within the donor lungs; the recurrences appeared from 10 to 48 months after transplantation. All recurrences were limited to the donor lungs. Histologic and molecular analyses showed that the recurrent tumors in three patients originated from the recipients of the transplants. CONCLUSIONS: Lung transplantation for bronchioloalveolar carcinoma is technically feasible, but recurrence of the original tumor within the donor lungs up to four years after transplantation was common.

Adenocarcinoma, Bronchiolo-Alveolar↗

PubMed1999

A full-length cDNA of hREV3 is predicted to encode DNA polymerase zeta for damage-induced mutagenesis in humans.

DNA damage can cause mutations which in turn may lead to carcinogenesis. In the yeast Saccharomyces cerevisiae, DNA damage-induced mutagenesis pathway requires the REV3 gene. It encodes the catalytic subunit of DNA polymerase zeta that specifically functions in translesion DNA synthesis. We have cloned a cDNA of the human homologue of REV3 (hREV3), which consists of 10,716 bp and codes for a protein of 3130 amino acid residues (352,737 Da). Its C-terminal 755 amino acids show extensive homology with the yeast protein at the C-terminus: 43% identity and 74% similarity. This region contains the six highly conserved DNA polymerase motifs. Furthermore, we have identified four sequence motifs in the N-terminal region outside the polymerase domain that are conserved in DNA polymerase delta from various sources. Three of which are present in DNA polymerase zeta encoded by human, yeast, and plant REV3 genes, indicating that this protein is a member of the DNA polymerase delta family. DNA polymerases delta and zeta are structurally distinguished by the presence of a specific delta IV motif in the former and motifs zeta I and zeta II in the latter, respectively. Human DNA polymerase zeta is ubiquitously expressed in various tissues, consistent with the notion that the hREV3 pathway may be a fundamental mechanism of damage-induced mutagenesis in humans.

Amino Acid Sequence↗

PubMed1999

Repair of DNA lesions: mechanisms and relative repair efficiencies.

DNA is frequently damaged by endogenous agents inside the cells. Some exogenous agents such as polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment and may thus contribute to the 'background' DNA damage in humans. DNA lesions are normally removed by various repair mechanisms. The major repair mechanisms for various DNA lesions are summarized. In contrast to the extensively studied repair mechanisms, much less is known about the relative repair efficiencies of various DNA lesions. Since DNA repair is a crucial defense against carcinogenesis, it may constitute an important factor affecting the carcinogenicity of DNA damaging agents. We have adopted a human cell-free system for measuring relative DNA repair efficiencies based on the concept of repair competition between acetylaminofluorene adducts and other DNA lesions of interest. Using this in vitro system, we determined the relative repair efficiencies of PAH adducts induced by: anti-(+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE), anti-(+/-)-benz[a]anthracene-trans-3,4-dihydrodiol-1,2-epoxide (BADE-I), anti-(+/-)-benz[a]anthracene-trans-8,9-dihydrodiol-10, 11-epoxide (BADE-II), anti-(+/-)-benzo[b]fluoranthene-trans-9, 10-dihydrodiol-11,12-epoxide (BFDE), anti-(+/-)-chrysene-trans-1, 2-dihydrodiol-3,4-epoxide (CDE), and anti-(+/-)-dibenzo[a, l]pyrene-trans-11,12-dihydrodiol-13,14-epoxide (DBPDE). While damage by BPDE, DBPDE, CDE, and BFDE were repaired by nucleotide excision repair as efficiently as AAF adducts, the repair of BADE-I and BADE-II adducts were significantly slower in human cell extracts. Damage by DBPDE at 3 microM in vitro yielded approximately 5-fold higher DNA adducts than BPDE as determined by quantitative PCR. This potent DNA reactivity may account in part for the potent carcinogenicity of dibenzo[a,l]pyrene. The correlation of these results to the carcinogenic properties of the PAH compounds is discussed. Furthermore, we show that NER plays a role in AP site repair in vivo in the eukaryotic model organism yeast.

Carcinogens↗

PubMed1999

Losartan and enalapril therapies enhance vasodilatation in the mesenteric artery of spontaneously hypertensive rats.

We studied the effects of 10-week long enalapril and losartan treatments (4 and 15 mg kg(-1) day(-1), respectively) on mesenteric arterial function in vitro in spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY). The relaxations of noradrenaline-precontracted rings to acetylcholine, nitroprusside and cromakalim were similar in WKY and enalapril- and losartan-treated SHR, and more pronounced than in untreated SHR. The responses to acetylcholine were attenuated by N(G)-nitro-L-arginine methyl ester in WKY and drug-treated SHR, but were completely inhibited in untreated SHR. When hyperpolarization of smooth muscle was prevented by KCl-induced precontractions, no differences were found in the relaxations to acetylcholine and nitroprusside between the groups, and the dilatations to cromakalim were abolished. Moreover, in noradrenaline-precontracted rings of drug-treated SHR, the addition of tetraethylammonium attenuated the nitric oxide synthase and cyclooxygenase-resistant relaxations to acetylcholine and abolished the enhanced dilatations to nitroprusside. In conclusion, since the enhancement of vasorelaxation in enalapril- and losartan-treated SHR was abolished by conditions preventing hyperpolarization, the improved vasodilatation following these therapies could be attributed to enhanced vasodilatation via K+ channels in this model of hypertension.

Animals↗

PubMed1999

DNA ligation during excision repair in yeast cell-free extracts is specifically catalyzed by the CDC9 gene product.

Excision repair of DNA is an important cellular response to DNA damage induced by radiation and many chemicals. In eukaryotes, base excision repair (BER) and nucleotide excision repair (NER) are two major excision repair pathways which are completed by a DNA ligation step. Using a cell-free system, we have determined the DNA ligase requirement during BER and NER of the yeast S. cerevisiae. Under nonpermissive conditions in extracts of the cdc9-2 temperature-sensitive mutant, DNA ligation in both BER and NER pathways was defective, and the repair patches were enlarged. At the permissive temperature (23 degrees C), DNA ligation during excision repair was only partially functional in the mutant extracts. In contrast, deleting the DNA ligase IV gene did not affect DNA ligation of BER or NER. Defective DNA ligation of BER and NER in cdc9-2 mutant extracts was complemented in vitro by purified yeast Cdc9 protein, but not by DNA ligase IV even when overexpressed. These results demonstrate that the ligation step of excision repair in yeast cell-free extracts is catalyzed specifically by the Cdc9 protein, the homologue of mammalian DNA ligase I.

Catalysis↗

PubMed1999

cDNA cloning and expression of acutin.

Acutin, a thrombin-like enzyme was purified from Agkistrodon acutus venom in three steps by DEAE-Sepharose CL-6B, Superose 12 column on FPLC and Mono-Q column chromatographies. Its first 15 N-terminal amino acid residues sequence was then determined and the acutin cDNA was isolated from venom gland total RNA using RT-PCR. Determination of its nucleotide sequence allowed elucidation of the amino acid sequence of mature peptide for the first time. The mature acutin has 233 amino acids and its amino acid sequence exhibits significant homology with those of thrombin-like enzymes from crotaline snakes venoms. Based on the homology, the catalytic residues and disulfide bridges of acutin were deduced to be as follows: catalytic residues, His41, Asp84 and Ser179; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys231, Cys118-Cys185, Cys150-Cys164, Cys175-Cys200. The recombinant acutin has been expressed in E. coli and purified by affinity column. The renatured recombinant acutin is reported for the first time to have the activity of clotting fibrinogen and arginine-esterase.

Agkistrodon↗

PubMed1999

A role for FEN-1 in nonhomologous DNA end joining: the order of strand annealing and nucleolytic processing events.

Eukaryotic repair of double-strand DNA breaks can occur either by homologous recombination or by nonhomologous DNA end joining (NHEJ). NHEJ relies on Ku70/86, XRCC4, DNA ligase IV, and DNA-dependent protein kinase. NHEJ involves a synapsis step in which the two ends are maintained in proximity, processing steps in which nucleases and polymerases act on the ends, an alignment step in which a few nucleotides of terminal homology guide the ends into preferred alignments, and a ligation step. Some of the steps, such as ligation, rely on a single enzymatic component. However, the processing steps begin and end with a wide array of alternative substrates and products, respectively, and likely involve multiple nucleases and polymerases. Given the alternative pathways that can be catalyzed by the remaining nucleases and polymerases, no one of these processing enzymes is likely to be essential. The only requirement for the processing enzymes, as a collective, is to generate a ligatable configuration, namely a ligatable nick on each strand. Here, we have tested the two major known 5'-specific nucleases of Saccharomyces cerevisiae for involvement in NHEJ. Whereas EXO1 does not appear to be involved to any detectable level, deleting RAD27 (FEN-1 of yeast) leads to a 4.4-fold reduction specifically of those NHEJ events predicted to proceed by means of 5' flap intermediates. Because Rad27/FEN-1 acts specifically at 5' flap structures, these results suggest that the NHEJ alignment step precedes nucleolytic processing steps in a significant fraction of NHEJ events.

14-3-3 Proteins↗

PubMed1999

Differential regulation of maternal vs. paternal centrosomes.

Centrosomes are the main microtubule-organizing centers in animal cells. During meiosis and mitosis, two centrosomes form the poles that direct the assembly of a bipolar spindle, thus ensuring the accurate segregation of chromosomes. Cells cannot tolerate the presence of more than two active centrosomes during meiosis or mitosis because doing so results in the formation of multipolar spindles, infidelity in chromosome segregation, and aneuploidy. Here, we show that fertilization of Spisula solidissima oocytes results in cells that contain three active centrosomes, two maternal and one paternal. During meiosis I, the paternal centrosome's ability to nucleate microtubules is selectively shut off while maternal centrosomes remain competent to nucleate microtubules and assemble asters in the same cytoplasm. We propose that embryos can identify paternal vs. maternal centrosomes and can control them differentially.

Animals↗

PubMed1999

Relationships between yeast Rad27 and Apn1 in response to apurinic/apyrimidinic (AP) sites in DNA.

Yeast Rad27 is a 5'-->3' exonuclease and a flap endo-nuclease. Apn1 is the major apurinic/apyrimidinic (AP) endonuclease in yeast. The rad27 deletion mutants are highly sensitive to methylmethane sulfonate (MMS). By examining the role of Rad27 in different modes of DNA excision repair, we wish to understand why the cytotoxic effect of MMS is dramatically enhanced in the absence of Rad27. Base excision repair (BER) of uracil-containing DNA was deficient in rad27 mutant extracts in that (i) the Apn1 activity was reduced, and (ii) after DNA incision by Apn1, hydrolysis of 1-5 nucleotides 3' to the baseless sugar phosphate was deficient. Thus, some AP sites may lead to unprocessed DNA strand breaks in rad27 mutant cells. The severe MMS sensitivity of rad27 mutants is not caused by a reduction of the Apn1 activity. Surprisingly, we found that Apn1 endonuclease sensitizes rad27 mutant cells to MMS. Deleting the APN1 gene largely restored the resistance of rad27 mutants to MMS. These results suggest that unprocessed DNA strand breaks at AP sites are mainly responsible for the MMS sensitivity of rad27 mutants. In contrast, nucleotide excision repair and BER of oxidative damage were not affected in rad27 mutant extracts, indicating that Rad27 is specifically required for BER of AP sites in DNA.

Apurinic Acid↗

PubMed1999

[Presinilin 1 gene polymorphism and Alzheimer's disease in Chinese].

OBJECTIVE: To detect the relationship between Presinilin 1(PS1) intronic polymorphism and Alzheimer's disease(AD) in Chinese. METHODS: PS1 intronic polymorphism was genotyped in 58 early-onset AD cases(EOAD), 65 late-onset AD cases (LOAD) and 157 age-matched controls by using PCR methods and RFLP typing. Then the association between PS1 polymorphism and AD was analyzed. RESULTS: (1) In EOAD cases, the frequency of allele 1 increased and the frequencies of allele 2 and genotype 2/2 decreased markedly, but in LOAD cases, no differences were observed. (2) EOAD was significantly associated with allele 1 (RR=2.29), P< 0.05), allele 2 (RR=0.44) and genotype 2/2(RR=0.23, P<0.05) of PS1 polymorphism, while LOAD showed no association with PS1 polymorphism. (3)PS1 polymorphism was associated with AD predominantly in non-ApoE epsilon4, female, early-onset cases. CONCLUSION: PS1 polymorphism was only associated with EOAD in Chinese, and this may be influenced by age of onset and sex.

Aged↗

PubMed1999

No association between the intronic presenilin 1 polymorphism and Alzheimer's disease in the Chinese population.

Wragg et al. [1996: Lancet 347:509-512] recorded an association between the intron-based presenilin 1 (PS1) genotype 1/1 and late-onset Alzheimer's disease (AD). This study was performed to determine if there is a similar association in the Chinese population. Ninety-one AD cases, 50 multiinfarct dementia (MID) patients, and 73 age-matched normal controls were recruited. Genotyping of PS1 and apolipoprotein E (APOE) was performed by the methods of polymerase chain reaction and restriction fragment length polymorphism. In AD, MID, and normal controls PS1 allele 1 frequency was 0.6703, 0.5600, and 0.6301, respectively; PS1 allele 2 frequency was 0.3297, 0.4400, and 0.3699, respectively. No association was detected between these diseases and any PS1 allele or genotype. There was only a nearly significant negative association between MID and PS1 genotype 1/1 in the subgroup population bearing APOE allele E4 (odds ratio = 0.2753, P = 0.0776). Our results do not support the conclusion that the intronic PS1 polymorphism is associated with Alzheimer's disease.

Aged↗

PubMed1999

Plasma levels of insulin-like growth factor-I and lung cancer risk: a case-control analysis.

BACKGROUND: Insulin-like growth factors (IGFs), in particular IGF-I and IGF-II, strongly stimulate the proliferation of a variety of cancer cells, including those from lung cancer. To examine the possible causal role of IGFs in lung cancer development, we compared plasma levels of IGF-I, IGF-II, and an IGF-binding protein (IGFBP-3) in patients with newly diagnosed lung cancer and in control subjects. METHODS: From an ongoing hospital-based, case-control study, we selected 204 consecutive patients with histologically confirmed, primary lung cancer and 218 control subjects who were matched to the case patients by age, sex, race, and smoking status. IGF-I, IGF-II, and IGFBP-3 plasma levels were measured by enzyme-linked immunosorbent assay and then divided into quartiles, based on their distribution in the control subjects. Associations between the IGF variables and lung cancer risk were estimated by use of odds ratios (ORs). Reported P values are two-sided. RESULTS: IGF and IGFBP-3 levels were positively correlated (all r>.27; all P<.001). High plasma levels of IGF-I were associated with an increased risk of lung cancer (OR = 2.06; 95% confidence interval [CI] = 1.19-3.56; P = .01), and this association was dose dependent in both univariate and multivariate analyses. Plasma IGFBP-3 showed no association with lung cancer risk unless adjusted for IGF-I level; when both of these variables were analyzed together, high plasma levels of IGFBP-3 were associated with reduced risk of lung cancer (OR = 0.48; 95% CI = 0.25-0.92; P = .03). IGF-II was not associated with lung cancer risk. CONCLUSIONS: Plasma levels of IGF-I are higher and plasma levels of IGFBP-3 are lower in patients with lung cancer than in control subjects. If these findings can be confirmed in prospective studies, measuring levels of IGF-I and IGFBP-3 in blood may prove useful in assessing lung cancer risk.

Analysis of Variance↗

PubMed1999

Expression of dominant-negative and mutant isoforms of the antileukemic transcription factor Ikaros in infant acute lymphoblastic leukemia.

Ikaros, a zinc finger-containing DNA-binding protein, is required for normal lymphocyte development, and germline mutant mice that express only non-DNA binding dominant-negative "leukemogenic" Ikaros isoforms lacking critical N-terminal zinc fingers develop an aggressive form of lymphoblastic leukemia 3-6 months after birth. Therefore, we sought to determine whether molecular abnormalities involving the Ikaros gene could contribute to the development of acute lymphoblastic leukemia (ALL) in infants. Primary leukemic cells were freshly obtained from 12 infants (<1 year of age) with newly diagnosed ALL. In leukemic cells from each of the 12 infants with ALL, we found high level expression of dominant-negative isoforms of Ikaros with abnormal subcellular compartmentalization patterns. PCR cloning and nucleotide sequencing were used to identify the specific Ikaros isoforms and detect Ikaros gene mutations in these cells. Leukemic cells from seven of seven infants with ALL, including five of five MLL-AF4(+) infants, expressed dominant-negative Ikaros isoforms Ik-4, Ik-7, and Ik-8 that lack critical N-terminal zinc fingers. In six of seven patients, we detected a specific mutation leading to an in-frame deletion of 10 amino acids (Delta KSSMPQKFLG) upstream of the transcription activation domain adjacent to the C-terminal zinc fingers of Ik-2, Ik-4, Ik-7, and Ik-8. In contrast, only wild-type Ik-1 and Ik-2 isoforms with normal nuclear localization were found in normal infant bone marrow cells and infant thymocytes. These results implicate the expression of dominant-negative Ikaros isoforms and the disruption of normal Ikaros function in the leukemogenesis of ALL in infants.

Amino Acid Sequence↗

PubMed1999

Hydration and stability of sulfatide-containing phosphatidylethanolamine small unilamellar vesicles.

The effect of sulfatide on membrane hydration of 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) small unilamellar vesicles (SUVs) was investigated using steady-state and time-resolved fluorescence spectroscopy. The degree of hydration in the headgroup region of the bilayer lipids was assessed with the fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine along with the ratio of its fluorescence intensities measured in samples prepared either in D2O- or in H2O-based buffers. Similarly, hydration of acyl chains near the headgroup region and that close to the bilayer center were studied using 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3, 5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine as probes. Increasing sulfatide concentration up to 30 mol% resulted in an increase in surface hydration and a decrease in interchain hydration. These were correlated with an increase in bilayer stability of the DOPE/sulfatide SUVs. Moreover, variation of pH was found to affect the hydration and stability of the bilayer vesicles. No further change in headgroup hydration and interchain hydration near the bilayer center was observed at sulfatide concentrations >/=30 mol%. At such high sulfatide concentrations, bilayer hydration and stability were no longer pH-sensitive. The effects of sulfatide on hydration and stability of DOPE bilayer vesicles are discussed by taking into account the electrostatic and geometrical properties of the sulfated galactosyl headgroups.

Dansyl Compounds↗

PubMed1999

Processing of the notch ligand delta by the metalloprotease Kuzbanian.

Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required for this processing event. These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands.

Amino Acid Sequence↗

PubMed1999

Complement-mediated anti-HIV-1 effect induced by human IgM monoclonal antibody against ganglioside GM2.

HIV-infected cells aberrantly express a high level of antigenic glycosidic structures such as GM2 and Gg4. Some normal sera containing natural IgM Abs to GM2 and/or Gg4 cause C-mediated cytolysis of HIV-infected cells. In the present study we demonstrated that a human IgM anti-GM2 mAb (L55 Ab) can induce cytolysis of HIV-infected cells. Increased GM2 expression by HIV-1 infection of a human T cell line (MOLT4), a human monocyte cell line (U937), and human lymphoblastoid cells was confirmed by immunofluorescence staining with L55 Ab. These infected cells were readily lysed by L55 Ab in the presence of fresh human serum as a C source that alone did not cause cytolysis. L55 Ab also had the ability to destroy HIV-1 particles via C-mediated lysis. By adding L55 Ab together with human C to mixed culture of HIV-infected cells and naive cells, HIV-1 replication was significantly suppressed, and this effect was synergistic when L55 Ab was combined with a reverse transcriptase inhibitor and a proteinase inhibitor. Therefore, a human IgM anti-GM2 mAb may be effective in treating HIV-infected patients, especially when used together with chemotherapeutic agents.

Anti-HIV Agents↗