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Biomedical subjects

W Xiao

Publications and source records attributed to W Xiao.

At least 55 records · Page 3Linked to original sources
PubMed2000

UBC13, a DNA-damage-inducible gene, is a member of the error-free postreplication repair pathway in Saccharomyces cerevisiae.

The Ubc13 protein was recently identified for its unique role in ubiquitin (Ub) chain assembly at the Ub Lys-63 residue instead of the conventional Lys-48 residue. This activity requires Ubc13 to form a complex with Mms2 and indeed ubc13 and mms2 mutations have been shown elsewhere to be epistatic with respect to UV sensitivity. The MMS2 gene is known to be a member of the error-free DNA postreplication repair (PRR) pathway. By contrast, the Ub Lys-63 residue has been previously implicated in the error-prone PRR pathway, since yeast cells carrying the ubiK63R mutation are defective in UV-induced mutagenesis. In the present study, we attempted to define the role of UBC13 within the PRR pathway. We found that the ubc13 mutation is epistatic to mms2 and rad6, confirming that UBC13 belongs to the PRR-pathway. We also found that ubc13 is synergistic to the error-prone PRR pathway mutation rev3, indicating that UBC13 is in a pathway alternative to REV3 mutagenesis. The ubc13 mutant displays up to a 30-fold increase in the spontaneous mutation rate, and this increase is largely REV3 dependent. In addition, UV-induced mutagenesis is fully functional in the ubc13 mutant. These results together demonstrate that UBC13 is a member of the error-free PRR pathway. The involvement of UBC13 in cellular tolerance to DNA-damage is further implicated by our finding that the UBC13 transcript level is increased up to 6-fold in response to DNA-damage.

Blotting, Northern↗
PubMed2000

The role of hexokinase in plant sugar signal transduction and growth and development.

Previous studies have revealed a central role of Arabidopsis thaliana hexokinases (AtHXK1 and AtHXK2) in the glucose repression of photosynthetic genes and early seedling development. However, it remains unclear whether HXK can modulate the expression of diverse sugar-regulated genes. On the basis of the results of analyses of gene expression in HXK transgenic plants, we suggest that three distinct glucose signal transduction pathways exist in plants. The first is an AtHXK1-dependent pathway in which gene expression is correlated with the AtHXK1-mediated signaling function. The second is a glycolysis-dependent pathway that is influenced by the catalytic activity of both AtHXK1 and the heterologous yeast Hxk2. The last is an AtHXK1-independent pathway in which gene expression is independent of AtHXK1. Further investigation of HXK transgenic Arabidopsis discloses a role of HXK in glucose-dependent growth and senescence. In the absence of exogenous glucose, plant growth is limited to the seedling stage with restricted true leaf development even after a 3-week culture on MS medium. In the presence of glucose, however, over-expressing Arabidopsis or yeast HXK in plants results in the repression of growth and true leaf development, and early senescence, while under-expressing AtHXK1 delays the senescence process. These studies reveal multiple glucose signal transduction pathways that control diverse genes and processes that are intimately linked to developmental stages and environmental conditions.

Arabidopsis↗
PubMed2000

The Saccharomyces cerevisiae mre11(ts) allele confers a separation of DNA repair and telomere maintenance functions.

The yeast Mre11 protein participates in important cellular functions such as DNA repair and telomere maintenance. Analysis of structure-function relationships of Mre11 has led to identification of several separation-of-function mutations as well as N- and C-terminal domains essential for Mre11 meiotic and mitotic activities. Previous studies have established that there is a strong correlation between Mre11 DNA repair and telomere maintenance functions and that Mre11-Rad50-Xrs2 complex formation appears to be essential for both of these activities. Here we report that the mre11(ts) allele, previously shown to cause temperature-dependent defects in DNA repair and meiosis, confers a temperature-independent telomere shortening, indicating that mre11(ts) is a separation-of-function mutation with respect to DNA repair and telomere maintenance. In a yeast two-hybrid system, Mre11(ts) fails to form a homodimer or interact with Rad50 and Xrs2 irrespective of experimental temperatures. These observations collectively suggest that the Pro(162)Ser substitution in Mre11(ts) confers a novel separation of Mre11 mitotic functions. Moreover, we observed that while overexpression of the 5'-3' exonuclease gene EXO1 partially complements the MMS sensitivity of mre11, rad50, and xrs2 null mutants, it has no effect on telomere shortening in these strains. This result provides additional evidence on possible involvement of distinctive mechanisms in DNA repair and telomere maintenance by the Mre11-Rad50-Xrs2 complex.

Alleles↗
PubMed2000

The Saccharomyces cerevisiae RAD6 group is composed of an error-prone and two error-free postreplication repair pathways.

The RAD6 postreplication repair and mutagenesis pathway is the only major radiation repair pathway yet to be extensively characterized. It has been previously speculated that the RAD6 pathway consists of two parallel subpathways, one error free and another error prone (mutagenic). Here we show that the RAD6 group genes can be exclusively divided into three rather than two independent subpathways represented by the RAD5, POL30, and REV3 genes; the REV3 pathway is largely mutagenic, whereas the RAD5 and the POL30 pathways are deemed error free. Mutants carrying characteristic mutations in each of the three subpathways are phenotypically indistinguishable from a single mutant such as rad18, which is defective in the entire RAD6 postreplication repair/tolerance pathway. Furthermore, the rad18 mutation is epistatic to all single or combined mutations in any of the above three subpathways. Our data also suggest that MMS2 and UBC13 play a key role in coordinating the response of the error-free subpathways; Mms2 and Ubc13 form a complex required for a novel polyubiquitin chain assembly, which probably serves as a signal transducer to promote both RAD5 and POL30 error-free postreplication repair pathways. The model established by this study will facilitate further research into the molecular mechanisms of postreplication repair and translesion DNA synthesis. In view of the high degree of sequence conservation of the RAD6 pathway genes among all eukaryotes, the model presented in this study may also apply to mammalian cells and predicts links to human diseases.

Adenosine Triphosphatases↗
PubMed2000

4,5-Diazafluoren-9-one benzoylhydrazone monohydrate.

The title compound, C(18)H(12)N(4)O.H(2)O, adopts the keto tautomeric form and the azomethine C=N double bond is in the E configuration. The dihedral angle between the planes of the diazafluorene moiety and the phenyl ring is 11.3 (1) degrees. In the solid state, the molecules form infinite chain-like structures via O-H.N hydrogen bonds involving the water molecules and diazafluorene moieties.

Crystallography, X-Ray↗
PubMed2000

p-methoxybenzaldehyde benzoylhydrazone monohydrate

The crystal structure of the title compound, C(15)H(14)N(2)O(2). H(2)O, is in the keto tautomeric form and the configuration at the azomethine C=N double bond is E. The molecule is non-planar, with a dihedral angle of 27.3 (1) degrees between the aromatic rings. The crystal structure is stabilized by extensive hydrogen bonding involving the water molecule and hydrazone moiety.

Journal Article↗
PubMed2000

Mutational analysis of the adeno-associated virus type 2 (AAV2) capsid gene and construction of AAV2 vectors with altered tropism.

Adeno-associated virus type 2 (AAV2) has proven to be a valuable vector for gene therapy. Characterization of the functional domains of the AAV capsid proteins can facilitate our understanding of viral tissue tropism, immunoreactivity, viral entry, and DNA packaging, all of which are important issues for generating improved vectors. To obtain a comprehensive genetic map of the AAV capsid gene, we have constructed 93 mutants at 59 different positions in the AAV capsid gene by site-directed mutagenesis. Several types of mutants were studied, including epitope tag or ligand insertion mutants, alanine scanning mutants, and epitope substitution mutants. Analysis of these mutants revealed eight separate phenotypes. Infectious titers of the mutants revealed four classes. Class 1 mutants were viable, class 2 mutants were partially defective, class 3 mutants were temperature sensitive, and class 4 mutants were noninfectious. Further analysis revealed some of the defects in the class 2, 3, and 4 mutants. Among the class 4 mutants, a subset completely abolished capsid formation. These mutants were located predominantly, but not exclusively, in what are likely to be beta-barrel structures in the capsid protein VP3. Two of these mutants were insertions at the N and C termini of VP3, suggesting that both ends of VP3 play a role that is important for capsid assembly or stability. Several class 2 and 3 mutants produced capsids that were unstable during purification of viral particles. One mutant, R432A, made only empty capsids, presumably due to a defect in packaging viral DNA. Additionally, five mutants were defective in heparan binding, a step that is believed to be essential for viral entry. These were distributed into two amino acid clusters in what is likely to be a cell surface loop in the capsid protein VP3. The first cluster spanned amino acids 509 to 522; the second was between amino acids 561 and 591. In addition to the heparan binding clusters, hemagglutinin epitope tag insertions identified several other regions that were on the surface of the capsid. These included insertions at amino acids 1, 34, 138, 266, 447, 591, and 664. Positions 1 and 138 were the N termini of VP1 and VP2, respectively; position 34 was exclusively in VP1; the remaining surface positions were located in putative loop regions of VP3. The remaining mutants, most of them partially defective, were presumably defective in steps of viral entry that were not tested in the preliminary screening, including intracellular trafficking, viral uncoating, or coreceptor binding. Finally, in vitro experiments showed that insertion of the serpin receptor ligand in the N-terminal regions of VP1 or VP2 can change the tropism of AAV. Our results provide information on AAV capsid functional domains and are useful for future design of AAV vectors for targeting of specific tissues.

Alanine↗
PubMed2000

Granulocyte-macrophage colony-stimulating factor priming plus papillomavirus E6 DNA vaccination: effects on papilloma formation and regression in the cottontail rabbit papillomavirus--rabbit model.

A cottontail rabbit papillomavirus (CRPV) E6 DNA vaccine that induces significant protection against CRPV challenge was used in a superior vaccination regimen in which the cutaneous sites of vaccination were primed with an expression vector encoding granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that induces differentiation and local recruitment of professional antigen-presenting cells. This treatment induced a massive influx of major histocompatibility complex class II-positive cells. In a vaccination-challenge experiment, rabbit groups were treated by E6 DNA vaccination, GM-CSF DNA inoculation, or a combination of both treatments. After two immunizations, rabbits were challenged with CRPV at low, moderate, and high stringencies and monitored for papilloma formation. As expected, all clinical outcomes were monotonically related to the stringency of the viral challenge. The results demonstrate that GM-CSF priming greatly augmented the effects of CRPV E6 vaccination. First, challenge sites in control rabbits (at the moderate challenge stringency) had a 0% probability of remaining disease free, versus a 50% probability in E6-vaccinated rabbits, and whereas GM-CSF alone had no effect, the interaction between GM-CSF priming and E6 vaccination increased disease-free survival to 67%. Second, the incubation period before papilloma onset was lengthened by E6 DNA vaccination alone or to some extent by GM-CSF DNA inoculation alone, and the combination of treatments induced additive effects. Third, the rate of papilloma growth was reduced by E6 vaccination and, to a lesser extent, by GM-CSF treatment. In addition, the interaction between the E6 and GM-CSF treatments was synergistic and yielded more than a 99% reduction in papilloma volume. Finally, regression occurred among the papillomas that formed in rabbits treated with the E6 vaccine and/or with GM-CSF, with the highest regression frequency occurring in rabbits that received the combination treatment.

Animals↗
PubMed2000

High-titer, wild-type free recombinant adeno-associated virus vector production using intron-containing helper plasmids.

Recombinant adeno-associated virus (rAAV) is capable of directing long-term, high-level transgene expression without destructive cell-mediated immune responses. However, traditional packaging methods for rAAV vectors are generally inefficient and contaminated with replication-competent AAV (rcAAV) particles. Although wild-type AAV is not associated with any known human diseases, contaminating rcAAV particles may affect rAAV gene expression and are an uncontrolled variable in many AAV gene transfer studies. In the current study, a novel strategy was designed to both optimize AAV rep gene expression and increase vector yield, as well as simultaneously to diminish the potential of generating rcAAV particles from the helper plasmid. The strategy is based on the insertion of an additional intron in the AAV genome. In the AAV infectious clone, the intron insertion had no effects on the properties of Rep proteins expressed. Normal levels of both Rep and Cap proteins were expressed, and the replication of the AAV genome was not impaired. However, the generation of infectious rcAAV particles using intronized AAV helper was greatly diminished, which was due to the oversized AAV genome caused by the insertion of the artificial introns. Moreover, the rAAV packaging was significantly improved with the appropriate choice of intron and insertion position. The intron is another element that can regulate the rep and cap gene expression from the helper plasmid. This study provides for a novel AAV packaging system which is highly versatile and efficient. It can not only be combined with other AAV packaging systems, including rep-containing cell lines and herpes simplex virus hybrid packaging methods, but also be used in other vector systems as well.

Dependovirus↗
PubMed2000

Humoral immunity to adeno-associated virus type 2 vectors following administration to murine and nonhuman primate muscle.

Adeno-associated virus (AAV) is being developed as a vector capable of conferring long-term gene expression, which is useful in the treatment of chronic diseases. In most therapeutic applications, it is necessary to readminister the vector. This study characterizes the humoral immune response to AAV capsid proteins following intramuscular injection and its impact on vector readministration. Studies of mice and rhesus monkeys demonstrated the formation of neutralizing antibodies to AAV capsid proteins that persisted for over 1 year and then diminished, but this did not prevent the efficacy of vector readministration. More-detailed studies strongly suggested that the B-cell response was T cell dependent. This was further evaluated with a blocking antibody to human CD4, primatized for clinical trials, in a biologically compatible mouse in which the endogenous murine CD4 gene was functionally replaced with the human counterpart. Transient pharmacologic inhibition of CD4 T cells with CD4 antibody prevented an antivector response long after the effects of the CD4 antibody diminished; readministration of vector without diminution of gene expression was possible. Our studies suggest that truly durable transgene expression (i.e., prolonged genetic engraftment together with vector readministration) is possible with AAV in skeletal muscle, although it will be necessary to transiently inhibit CD4 T-cell function to avoid the activation of memory B cells.

Animals↗
PubMed2000

Improving synthetic lethal screens by regulating the yeast centromere sequence.

The synthetic lethal screen is a useful method in identifying novel genes functioning in an alternative pathway to the gene of interest. The current synthetic lethal screen protocol in yeast is based on a colony-sectoring assay that allows direct visualization of mutant colonies among a large population by their inability to afford plasmid loss. This method demands an appropriate level of stability of the plasmid carrying the gene of interest. YRp-based plasmids are extremely unstable and complete plasmid loss occurs within a few generations. Consequently, YCp plasmids are the vector of choice for synthetic lethal screens. However, we found that the high-level stability of YCp plasmids resulted in a large number of false positives that must be further characterized. In this study, we attempt to improve the existing synthetic lethal screen protocol by regulating the plasmid stability and copy number. It was found that by placing a yeast centromere sequence under the control of either inducible or constitutive promoters, plasmid stability can be significantly decreased. Hence, altering the conditions under which yeast cells carrying the plasmid PGAL1-CEN4 were cultivated allowed us to develop a method that eliminated virtually 100% of false positives and drastically reduced the time required to carry out a synthetic lethal screen.

Aminohydrolases↗
PubMed2000

Bench-scale anaerobic bioconversion of newsprint and office paper.

Anaerobic bioconversion of newsprint and waste office paper was performed in bench-scale reactors with three inocula sources: landfill, rumen, and anaerobic digester. Office paper bioconversion was nearly complete within 20 days but continued for about 165 days with methane yield efficiencies ranging from 71-85% of potential chemical oxygen demand (COD) conversion. Average newsprint methane conversion efficiencies ranged from 32-41% of total COD under strictly anaerobic conditions for 300 days. Mass balance calculations revealed that more than 80% of newsprint cellulose was biodegraded. The apparent limiting factor for anaerobic bioconversion of newsprint was the physical association between lignin and cellulose. After proper acclimation, the three inocula tested equally well for methane production under strictly anaerobic conditions. Testing of ground, shredded strips, and whole paper pieces showed no effects of feedstock size on bioconversion rate or extent. Alkali pretreatment with NaOH concentration up to 10% significantly improved newsprint biodegradability. Treatment for longer duration or at elevated temperatures increased the solubilization of lignin, but did not improve bioconversion of newsprint to methane. Neutralizing treated samples with carbon dioxide gave higher methane yields compared to sulfuric or hydrochloric acids, suggesting that digester neutralization could be combined with biogas scrubbing.

Alkalies↗
PubMed2000

[The imbalance of Th1/Th2 cytokine expression in peripheral blood mononuclear cell from asthmatic patients and the effect of erythromycin on these cytokines].

OBJECTIVE: To observe the state of Th1/Th2 cytokine expression in peripheral blood mononuclear cells (PBMCs) from asthmatic patients and the effect of erythromycin (Ery) on it. METHODS: 23 asthmatic patients and 10 healthy volunteers were included. The mRNA expression for Th1/Th2 cytokines in PBMC before and after the culture with Ery were detected by RT-PCR, considering IL-2 and IFN-gamma as Th1 cytokines, and IL-4, IL-6, IL-10 as Th2 ones. RESULTS: The positive rate of mRNA expression for Th2-derived IL-4, IL-6, IL-10 in the study group were 18/23, 19/23 and 10/23 respectively, and those for IFN-gamma and IL-2 produced by Th1 cells were 1/23 and 13/23, respectively. In the control group, the positive rate for IL-4, IL-6, IL-10 were 0/10, 2/10 and 1/10, respectively, and those for IL-2 and IFN-gamma were both 1/10. Comparing with the control group, the positive rate of mRNA expression for IL-4 and IL-6 in PBMC from asthmatic patients were significantly higher (P = 0.00003, 0.001 respectively). After the culture with Ery for 48 h, the positive rate for IL-4 expression in the study group decreased from 18/23 to 5/23 (P = 0.0006), and IL-6 from 19/23 to 9/23 (P = 0.006). The positive rate for IFN-gamma expression was elevated while IL-2 decreased(But both had no statistical significance). There was no difference observed before and after the culture with Ery in the control group. CONCLUSIONS: There is a predominant expression of Th2-type cytokines in PBMC from asthmatic patients. Ery can down-regulate the mRNA expression for IL-4 and IL-6 in asthmatic patients, and might convert the imbalance of Th1/Th2 cytokine expression in asthmatic patients.

Adult↗
PubMed2000

[Rapid detection of alpha-thalassemia of Southeast Asian deletion by polymerase chain reaction and its application to prenatal diagnosis].

OBJECTIVE: To establish a rapid and simple polymerase chain reaction (PCR) method for detecting alpha-thalassemia of Southeast Asia deletion, and apply it to the prenatal diagnosis for high risk fetuses. METHODS: Two pairs of primers were designed: one pair bridging the breakpoints to identify the specific deletion, the other located in the common deletion region of --(SEA), -alpha(3.7) and -alpha(4.2) gene to detect the normal chromosomes. In this system, the two amplifications ran in the same PCR tube under identical condition. RESULTS: A 740 bp fragment was amplified in chromosomes with --(SEA) determinant and a 1,052 bp fragment in normal chromosomes. For prenatal diagnosis, 3 of 8 at-risk cases were diagnosed as normal, 3 as heterozygotes, and 2 as homozygotes of --(SEA) deletion. CONCLUSION: This detection method is rapid and accurate and can be used as a routine method for carrier detection and prenatal diagnosis.

Female↗
PubMed2000

[Evaluation of polymerase chain reaction for detection of chlamydia trachomatis in eye swabs].

OBJECTIVE: To investigate the clinical value of Polymerase Chain Reaction (PCR) in the diagnosis of trachoma. METHODS: Direct amplication of 16S rRNA gene which is chlamydia-specific by PCR was used to detect chlamydia trachomatis (CT). RESULTS: Of 93 subjects who conformed to the diagnosis criteria of trachoma, 69 (74.2%) was PCR-Positive; Of 178 subjects who did not comform to the criteia, 6(3.4%) was PCR-positive. PCR had a sensitivity of 74.2% and a speciticity of 96.7% for the clinical diagnosis of trachoma. The difference between trachoma group and nontrachoma group is significant(P < 0.001). CONCLUSION: The PCR may be a good method in the diagnosis of ocular chlamydia trachomatis infection because of its excellent sensitivity and specificity.

Adolescent↗