Biomedical subjects
Publications and source records attributed to X Wu.
Total reflection X-ray fluorescence (TXRF) analysis technique and the instrument with a short path, high efficiency, low power and small volume are briefly presented. The detection limit of the system are at pg-level for Cu and Mo target excitation. Teeth of a marine mollusc were measured quantitatively and the spectrum and analysis results were given.
A spectrophotometric method for the quantitative determination of polyethylene glycol-600 (PEG-600) in urine is based on partitioning a chromophore present in ammonium ferrothiocyanate reagent into an aqueous phase and a chloroform phase in the presence of PEG. The method is simple and reproducible, and can detect PEG in a concentration of 0.25 g/L. It gives a linear response over a range of 0.25-2.5 g/L. The recovery of PEG-600 in urine is 107% and relative standard deviation is less than 5%.
The dimerization of Aluminum (III),Vanadium(lV), Gallium(III) tetrasulfonated phthalocyanine (AlTSPc, VOTSPc, GaTSPc) and Zinc(II) bisulfonated phthalocyanine (ZnBSPc) was studied by spectrophotometic method. The dimerization constants KD were calculated from absorbance measurements. The absorption spectra in water, aqueous alcoholic solutions and micelles of phthalocyanine sulfonates aluminum shows the "slow-forming dimer", and VOTSPc, GaTSPc, ZnBSPc show contact dimer formation.
The peroxisome proliferator activated receptors (PPARs) alpha, beta/delta, and gamma are novel nuclear hormone receptors activated by long chain fatty acids and synthetic ligands and which regulate lipid metabolism. Recent studies have detected PPARgamma mRNA in human mammary tumor cell lines. The current study examined the expression profile of PPAR mRNAs in normal and malignant rodent mammary tissues. Virgin murine mammary glands contained PPAR alpha, beta/delta, and gamma mRNAs based on northern blot analysis. The PPARgamma isoform was predominantly gamma2 based on quantitative PCR analysis. During pregnancy and lactation, the PPARalpha and gamma mRNAs decreased while the PPAR beta/delta mRNA remained relatively unchanged. NMuMG cells, an epithelial line derived from normal murine mammary gland, expressed PPAR alpha, beta/delta, and gamma mRNAs, independent of the presence or absence of compounds modifying PPAR activity. In rats, the physiologic expression pattern of PPARgamma mRNA paralleled the murine model; levels were detected in virgin but not lactating mammary glands. In addition, the PPARgamma mRNA was not detected in several histologically distinct 7,12-dimethylbenz(a)anthracene induced mammary tumors. These findings suggest that PPARs may regulate mammary epithelial and stromal cell function in response to physiologic or pathologic stimuli that profoundly alter lipid metabolism.
Unlike wild-type mouse melanocytes, where melanosomes are concentrated in dendrites and dendritic tips, melanosomes in dilute (myosin Va-) melanocytes are concentrated in the cell center. Here we sought to define the role that myosin Va plays in melanosome transport and distribution. Actin filaments that comprise a cortical shell running the length of the dendrite were found to exhibit a random orientation, suggesting that myosin Va could drive the outward spreading of melanosomes by catalyzing random walks. In contrast to this mechanism, time lapse video microscopy revealed that melanosomes undergo rapid ( approximately 1.5 microm/s) microtubule-dependent movements to the periphery and back again. This bidirectional traffic occurs in both wild-type and dilute melanocytes, but it is more obvious in dilute melanocytes because the only melanosomes in their periphery are those undergoing this movement. While providing an efficient means to transport melanosomes to the periphery, this component does not by itself result in their net accumulation there. These observations, together with previous studies showing extensive colocalization of myosin Va and melanosomes in the actin-rich periphery, suggest a mechanism in which a myosin Va-dependent interaction of melanosomes with F-actin in the periphery prevents these organelles from returning on microtubules to the cell center, causing their distal accumulation. This "capture" model is supported by the demonstration that (a) expression of the myosin Va tail domain within wild-type cells creates a dilute-like phenotype via a process involving initial colocalization of tail domains with melanosomes in the periphery, followed by an approximately 120-min, microtubule-based redistribution of melanosomes to the cell center; (b) microtubule-dependent melanosome movement appears to be damped by myosin Va; (c) intermittent, microtubule-independent, approximately 0.14 microm/s melanosome movements are seen only in wild-type melanocytes; and (d) these movements do not drive obvious spreading of melanosomes over 90 min. We conclude that long-range, bidirectional, microtubule-dependent melanosome movements, coupled with actomyosin Va-dependent capture of melanosomes in the periphery, is the predominant mechanism responsible for the centrifugal transport and peripheral accumulation of melanosomes in mouse melanocytes. This mechanism represents an alternative to straightforward transport models when interpreting other myosin V mutant phenotypes.
Apolipoprotein (apo) B100 is an atypical secretory protein in that its translocation across the endoplasmic reticulum membrane is inefficient, resulting in the partial translocation and exposure of apoB100 on the cytoplasmic surface of the endoplasmic reticulum. Cytosolic exposure leads to the association of nascent apoB with heat shock protein 70 and to its predisposition to ubiquitination and proteasomal degradation. The basis for the inefficient translocation of apoB100 remains unclear and controversial. To test the hypothesis that beta sheet domains present in apoB100 contribute to its inefficient translocation, we created human apoB chimeric constructs apoB13,16 and apoB13,13,16, which contain amino-terminal alpha globular domains but no beta sheet domains, and apoB13,16,beta, which has an amphipathic beta sheet domain of apoB100 inserted into apoB13,16. These constructs, along with carboxyl-terminal truncations of apoB100, apoB34 and apoB42, were used to transfect HepG2 and Chinese hamster ovary cells. In contrast to the lack of effect of proteinase K on apoB13,16 and apoB13,13,16, the levels of apoB34, apoB42, and apoB13,16,beta were decreased by 70-85% after proteinase K-induced proteolysis in both HepG2 and Chinese hamster ovary cells. Either oleic acid or proteasomal inhibitors (N-acetyl-leucinyl-leucinyl-norleucinal and lactacystin) significantly increased the cell levels of apoB13,16,beta, apoB34, apoB42, and full-length apoB100 but had no effect on the cell levels of apoB13,16 and apoB13,13,16. When HepG2 cells were incubated with a microsomal triglyceride transfer protein inhibitor, the cellular levels of apoB13,16,beta, apoB34, and apoB42 were decreased by 70-80%, whereas the levels of apoB13,16 and apoB13,13,16 were unaffected. The effects of microsomal triglyceride transfer protein inhibition were reversed by lactacystin. Our results clearly demonstrate that the translocation efficiency, susceptibility to proteasomal degradation, and lipid responsiveness of apoB were determined by the presence of a lipid binding beta sheet domain. It is possible that beta sheet domains may at least transiently facilitate the interaction of apoB with the lipid bilayer surrounding the translocation channel.
The PTEN/MMAC1 phosphatase is a tumor suppressor gene implicated in a wide range of human cancers. Here we provide biochemical and functional evidence that PTEN/MMAC1 acts a negative regulator of the phosphoinositide 3-kinase (PI3-kinase)/Akt pathway. PTEN/MMAC1 impairs activation of endogenous Akt in cells and inhibits phosphorylation of 4E-BP1, a downstream target of the PI3-kinase/Akt pathway involved in protein translation, whereas a catalytically inactive, dominant negative PTEN/MMAC1 mutant enhances 4E-BP1 phosphorylation. In addition, PTEN/MMAC1 represses gene expression in a manner that is rescued by Akt but not PI3-kinase. Finally, higher levels of Akt activation are observed in human prostate cancer cell lines and xenografts lacking PTEN/MMAC1 expression when compared with PTEN/MMAC1-positive prostate tumors or normal prostate tissue. Because constitutive activation of either PI3-kinase or Akt is known to induce cellular transformation, an increase in the activation of this pathway caused by mutations in PTEN/MMAC1 provides a potential mechanism for its tumor suppressor function.
We investigated the transport of cationic neurotoxins and neurotransmitters by the potential-sensitive organic transporter OCT3 and its steroid sensitivity using heterologous expression systems and also analyzed the expression of OCT3 in the brain. When expressed in mammalian cells, OCT3 mediates the uptake of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and the neurotransmitter dopamine. Competition experiments show that several cationic neuroactive agents including amphetamines interact with OCT3. When expressed in Xenopus laevis oocytes, OCT3-mediated MPP+ uptake is associated with inward currents under voltage-clamp conditions. The MPP+-induced currents are saturable with respect to MPP+ concentration, and half-maximal saturation (K0.5) occurs at about 25 microM MPP+ with membrane potential clamped at -50 mV. The K0.5 for MPP+ is markedly influenced by membrane potential. OCT3 is inhibited by several steroids, and beta-estradiol is the most potent inhibitor (Ki approximately 1 microM). The pattern of steroid sensitivity of OCT3 is different from that of OCT1 and OCT2 but correlates significantly with that of the extraneuronal monoamine transporter (uptake2). The transport characteristics and steroid sensitivity provide strong evidence for the molecular identity of OCT3 as uptake2. OCT3 is expressed in the brain as evidenced from Northern blot analysis, reverse transcription-polymerase chain reaction, and in situ hybridization using OCT3-specific probes. The molecular identity of the transcript hybridizing to the probe has been established by sequencing the reverse transcription-polymerase chain reaction product and also by the isolation of the OCT3 cDNA from a brain cDNA library. Regional distribution studies with in situ hybridization show that OCT3 is expressed widely in different brain regions, especially in the hippocampus, cerebellum, and cerebral cortex. OCT3 is likely to play a significant role in the disposition of cationic neurotoxins and neurotransmitters in the brain.
The interest in the applications of biocatalysis in organic syntheses has rapidly increased. In this context, lipases have recently become one of the most studied groups of enzymes. We have demonstrated that lipases can be used as biocatalyst in the production of useful biodegradable compounds. A number of examples are given. 1-Butyl oleate was produced by direct esterification of butanol and oleic acid to decrease the viscosity of biodiesel in winter use. Enzymic alcoholysis of vegetable oils without additional organic solvent has been little investigated. We have shown that a mixture of 2-ethyl-1-hexyl esters can be obtained in a good yield by enzymic transesterification from rapeseed oil fatty acids for use as a solvent. Trimethylolpropane esters were also similarly synthesized as lubricants. Finally, the discovery that lipases can also catalyze ester syntheses and transesterification reactions in organic solvent systems has opened up the possibility of enzyme catalyzed production of biodegradable polyesters. In direct polyesterification of 1,4-butanediol and sebacic acid, polyesters with a mass average molar mass of the order of 56,000 g mol-1 or higher, and a maximum molar mass of about 130,000 g mol-1 were also obtained by using lipase as biocatalyst. Finally, we have demonstrated that also aromatic polyesters can be synthesized by lipase biocatalysis, a higher than 50,000 g mol-1 mass average molar mass of poly(1,6-hexanediyl isophthalate) as an example.
Key to p53 ability to mediate its multiple cellular functions lies in its stability. In the present study we have elucidated the mechanism by which Mdm2 regulates p53 degradation. Using in vitro and in vivo ubiquitination assays we demonstrate that Mdm2 association with p53 targets p53 ubiquitination. Exposure of cells to UV-irradiation inhibits this targeting. Mdm2 which is deficient in p53 binding failed to target p53 ubiquitination, suggesting that the association is essential for Mdm2 targeting ability. While mdm2-p53 complex is found in non-stressed cells, the amount of p53-bound mdm2 is decreased after UV-irradiation, further pointing to the relationship between mdm2 binding and p53 level. Similar to Swiss 3T3 cells, the dissociation of mdm2-p53 complex was also found in UV-treated Scid cells, lacking functional DNA-PK, suggesting that DNA-PK is not sufficient for dissociating mdm2 from p53. Together our studies point to the role of Mdm2, as one of p53-associated proteins, in targeting p53 ubiquitination.
Enhancer II (ENII) of hepatitis B virus (HBV) is one of the essential cis-elements for the transcriptional regulation of HBV gene expression. Its function is highly liver-specific, suggesting that liver-enriched transcriptional factors play critical roles in regulating the activity of ENII. In this report, a novel hepatocyte transcription factor, which binds specifically to the B1 region (AACGACCGACCTTGAG) within the major functional unit (B unit) of ENII, has been cloned from a human liver cDNA library by yeast one-hybrid screening, and demonstrated to trans-activate ENII via the B1 region. We named this factor hB1F, for human B1-binding factor. Amino acid analysis revealed this factor structurally belongs to nuclear receptor superfamily. Based on the sequence similarities, hB1F is characterized to be a novel human homolog of the orphan receptor fushi tarazu factor I (FTZ-F1). Using reverse transcription-polymerase chain reaction, a splicing isoform of hB1F (hB1F-2) was identified, which has an extra 46 amino acid residues in the A/B region. Examination of the tissue distribution has revealed an abundant 5.2-kilobase transcript of hB1F is present specifically in human pancreas and liver. Interestingly, an additional transcript of 3.8 kilobases was found to be present in hepatoma cells HepG2. Fluorescent in situ hybridization has mapped the gene locus of hB1F to the region q31-32.1 of human chromosome 1. Altogether, this study provides the first report that a novel human homolog of FTZ-F1 binds and regulates ENII of HBV. The potential roles of this FTZ-F1 homolog in tissue-specific gene regulation, in embryonic development, as well as in liver carcinogenesis are discussed.
In this study, we aimed to confirm the finding that chromosome 5 aberrations are predisposing factors for lung cancer. The study population consisted of 118 previously untreated lung cancer patients and 101 healthy controls. Lymphocytes were treated with bleomycin for 5 hr and then allowed to recover in a drug-free medium for 48 hr. The mean number of cells with chromosome 5 abnormalities among 100 cells examined was significantly higher in patients (9.12) than in controls (4.69) (p < 0.001). The most frequent aberration was a 5q deletion and the breakpoints clustered at the 5q13-5q31 region. We then dichotomized the number of induced chromosome 5 abnormalities in peripheral blood lymphocytes by the 75th percentile in that of the controls. 103 (87.3%), of the 118 patients, but only 31 (30.7%) of the 101 controls, exhibited induced breaks above this point. After adjustment for age, sex, ethnicity and smoking status, we found that the sensitive group was at 14.4-fold increased risk for lung cancer. There was also a significant (p < 0.01) gradient of increased risk for lung cancer with an increasing number of chromosome 5 lesions. Therefore, chromosome 5 lesions, especially those at 5q, may be a molecular target of carcinogens in the development of lung cancer.
A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.
Productive infection by human papillomaviruses (HPVs) occurs only in differentiated squamous epithelial cells in papillomas, condylomata, and low grade intraepithelial neoplasias. Host DNA replication is reactivated in a fraction of terminally differentiated keratinocytes in benign human lesions and in organotypic raft cultures of primary human keratinocytes (PHKs) transduced with retroviruses expressing HPV-18 E7 oncogene from its native upstream regulatory region (URR). Thus the natural function of E7 protein, which inactivates pRB family proteins, is to induce host genes essential to support viral DNA replication in post-mitotic cells. Using this raft culture model system, we show that HPV-18 URR-E7 induces the universal cyclin-dependent kinase inhibitor p21cip1 protein in a fraction of differentiated PHKs. Induction is mediated by posttranscriptional mechanisms independent of p53. Double immunofluorescence studies demonstrate that, in raft cultures and in laryngeal papillomas, p21cip1 induction and reactivated host DNA synthesis take place in a mutually exclusive manner in PCNA-positive, differentiated keratinocytes. We suggest that p21cip1 induction effectively blocks unscheduled DNA synthesis reactivated by E7. These results begin to explain the inverse relationship between p21cip1 induction and HPV activities previously observed in a spectrum of benign lesions regardless of HPV types present.
Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.
The transcription rates of glycolytic enzyme genes are coordinately induced when cells are exposed to low oxygen tension. This effect has been described in many cell types and is not restricted to species or phyla. In mammalian cells, there are 11 distinct glycolytic enzymes, at least 9 of which are induced by hypoxia. Recent reports described a role for the hypoxia-inducible factor-1 (HIF-1) in the transcriptional activation of lactate dehydrogenase A, aldolase-A, phosphoglycerate kinase, and enolase-1 genes. It is not known whether the HIF-1 factor acts exclusively to regulate these genes during hypoxia, or how the other genes of the pathway are regulated. In this paper, we describe analyses of the muscle-specific pyruvate kinase-M and beta-enolase promoters that implicate additional mechanisms for the regulation of glycolytic enzyme gene transcription by hypoxia. Transient transcription of a reporter gene directed by either promoter was activated when transfected muscle cells were exposed to hypoxia. Neither of these promoters contain HIF-1 binding sites. Instead, the hypoxia response was localized to a conserved GC-rich element positioned immediately upstream of a GATAA site in the proximal promoter regions of both genes. The GC element was essential for both basal and hypoxia-induced expression and bound the transcription factors Sp1 and Sp3. Hypoxia caused the progressive depletion of Sp3 determined by DNA binding studies and Western analyses, whereas Sp1 protein levels remained unchanged. Overexpression of Sp3 repressed expression of beta-enolase promoters. It is concluded that hypoxia activates these glycolytic enzyme gene promoters by down-regulating Sp3, thereby removing the associated transcriptional repression.
A method for the facile preparation of oligoribonucleotide analogues containing beta-d-allofuranosyl nucleosides with additional functional groups tethered to the 6'-O positions is presented. It is based on the synthesis of two protected nucleosides carrying a 6'-O -bromopentyl and a 6'-O -methylaminopentyl substituent. By a simple two-step procedure, these key intermediates were transformed into two phosphoramidites carrying a 1-aza-18-crown-6 and a triethyleneglycol group, respectively, each capable of complexing metal ions. By automated synthesis, these functionalized nucleoside analogues were efficiently incorporated into short oligoribonucleotides. Under physiological conditions (150 mM NaCl, 2 mM MgCl2, pH 7.4), incorporation of a single allofuranosyl cytosine substituted with a triethyleneglycol moiety led to a significant enthalpic stabilization of an A-type RNA duplex. This observation is in agreement with a metal ion-mediated stabilizing interaction between the two pairing strands.
BACKGROUND: Tobacco smoking is an established risk factor for cancers of the upper aerodigestive tract, and measurement of chromosomal aberrations, i.e., chromatid breaks, induced in lymphocytes in vitro by bleomycin has been shown to be a predictor of risk for these cancers. In a case-control study, we recruited case subjects who were previously treated with surgery and/or radiotherapy for stage I or stage II squamous cell carcinoma of the head and neck to test the hypothesis that lymphocytic chromatid breaks induced by benzo[a]pyrene diol epoxide (BPDE), a tobacco mutagen, may also be associated with risk of developing cancers of the upper aerodigestive tract. METHODS: Case subjects were matched to control subjects on the basis of age, sex, ethnicity, and smoking status. Primary lymphocytes from 67 case subjects and 81 control subjects were treated with 2 microM BPDE for 24 hours, and the frequency of induced chromatid breaks was determined. All statistical tests were two-sided. RESULTS: Lymphocytes from case subjects compared with lymphocytes from control subjects showed significantly more breaks per cell induced by BPDE (mean+/-standard deviation, 0.77+/-0.38 versus 0.49+/-0.25; P<.001). Lymphocytes from 64.2% of case subjects were sensitive to BPDE (using a cutoff value of > or =0.60 break per cell). Subjects in the highest quartile of chromatid breaks had an approximately 20-fold increased risk of cancer compared with those in the lowest quartile after adjustment for age, sex, ethnicity, and smoking status. The association between BPDE sensitivity and cancer risk was higher in former smokers than in current smokers and higher in younger patients than in older patients. Subjects with sensitivity to both BPDE and bleomycin were at a 19.2-fold increased risk of cancer compared with those who were not sensitive to either agent. CONCLUSIONS: Mutagen sensitivity assays may aid in identifying individuals at risk of cancer, and use of parallel assays with two mutagens may improve risk predictability.