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Biomedical subjects

X Zhu

Publications and source records attributed to X Zhu.

At least 91 records · Page 5Linked to original sources

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of cytosolic phospholipase A2 is essential for human eosinophil adhesion to fibronectin.

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A(2) (cPLA(2)) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 microg/ml FN at 30 min, and decreased after 60-90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA(2) were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA(2) inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA(2) phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA(2) phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA(2) activation is essential for eosinophil adhesion to FN.

Actins↗

Redox-active iron mediates amyloid-beta toxicity.

While amyloid-beta toxicity is mediated by oxidative stress and can be attenuated by antioxidants, the actual biochemical mechanism underlying neurotoxicity remains to be established. However, since aggregated amyloid-beta can interact with transition metals, such as iron, both in vitro and in vivo, we suspected that bound iron might be the mediator of toxicity such that holo- and apo-amyloid would have differential effects on cellular viability. Here we demonstrate that when amyloid-beta is pretreated with the iron chelator deferoxamine, neuronal toxicity is significantly attenuated while conversely, incubation of holo-amyloid-beta with excess free iron restores toxicity to original levels. These data, taken together with the known sequelae of amyloid-beta, suggest that the toxicity of amyloid-beta is mediated, at least in part, via redox-active iron that precipitates lipid peroxidation and cellular oxidative stress.

Amyloid beta-Peptides↗

Interactions among subunits of human Arp2/3 complex: p20-Arc as the hub.

The Arp2/3 complex is critical for nucleation and crosslinking of actin filaments. To gain insight into its subunit topology and assembly pathway, we systematically examined interactions among subunits of human Arp2/3 complex by yeast two-hybrid assays. It was shown that p20-Arc was able to interact with p21-Arc, p34-Arc, and p16-Arc, respectively. In contrast, p41-Arc only interacted with p20-Arc/p16-Arc heterodimer. In addition, we found that structural integrity was important for association between p20-Arc and p21-Arc, while the N-terminal half of p34-Arc was dispensable for its binding to p20-Arc. Our data suggest a key role of p20-Arc and a multistep pathway for the complex formation.

Actin-Related Protein 2↗

Transmission/disequilibrium tests for quantitative traits.

Spielman et al. [1993] proposed a transmission-disequilibrium test (TDT), based on marker data collected on affected offspring and their parents, to test for linkage between a genetic marker and a binary trait provided there is allelic association. It has been shown that this TDT is powerful and is not affected by allelic association due to population stratification in the absence of linkage. For quantitative traits, George and Elston [1987] proposed a likelihood method to detect the effect of a candidate gene in pedigree data when familial correlations are present. This test will detect allelic association but will do so in the absence of linkage. In this paper, we investigate two new likelihood-ratio test statistics for multi-generational quantitative traits to test either for linkage in the presence of allelic association or for allelic association in the presence of linkage, such as may be due to linkage disequilibrium. We compare these two tests analytically and by simulation with respect to 1) the sample size required for the asymptotic null distributions to be valid and 2) their power to detect association in those cases in which they are not sensitive to population stratification unless linkage is present. In general, 80 nuclear families with two children each and at least one heterozygous parent, or the equivalent number of children in large pedigrees, are enough for the asymptotic null distribution of the proposed conditional and TDT methods to be valid. The theoretical power is close to the simulated power except for the case of a recessive allele with low frequency. A sampling strategy is proposed that dramatically improves power.

Alleles↗

Mutation scanning for sequence variation in three mitochondrial DNA regions for members of the Contracaecum osculatum (Nematoda: Ascaridoidea) complex.

Anisakid nematodes of seals from different geographical origins, previously identified by multilocus enzyme electrophoresis as Contracaecum osculatum A (CoA), C. osculatum B (CoB), C. osculatum C (CoC), C. osculatum D (CoD), C. osculatum E (CoE) and C. osculatum baicalensis (Cob), were characterised genetically using a mutation scanning approach, in order to define genetic markers for their specific identification and differentiation. Three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit I (COI), and the small and large subunits of rRNA (ssrRNA and IsrRNA, respectively) were amplified separately from individual nematodes by polymerase chain reaction (PCR), analysed by single-strand conformation polymorphism (SSCP), and samples displaying sequence variability were subjected to sequencing. Forty-six haplotypes were defined for 62-66 individuals (representing the six members of C. osculatum). All taxa except CoD and CoE could be identified, or delineated from one another, by nucleotide differences in the COI, ssrRNA and/or IsrRNA sequences. For all three mtDNA regions, 4 (10.5%), 7 (18.4%), 15 (39.5%) and 11 (28.9%) of 38 nucleotide positions were considered diagnostic (fixed) and could thus unequivocally delineate CoA, CoB, CoC and Cob. The lack of an unequivocal nucleotide difference in any of the three mtDNA sequences between CoD and CoE was in accordance with previous ribosomal DNA sequence data but inconsistent with multilocus enzyme electrophoretic data. Using all fixed nucleotide positions, CoA, CoD/E and CoB were genetically more similar to Cob than each was to CoC, similar to previous findings. In spite of not being able to distinguish among all six taxa of C. osculatum, the present study demonstrated clearly the usefulness and attributes of the mutation scanning approach for investigating population genetic structures of species of parasitic nematodes.

Animals↗

Mutation scanning analysis of sequence heterogeneity in the second internal transcribed spacer (rDNA) within some members of the Hypodontus macropi (Nematoda: Strongyloidea) complex.

Single-strand conformation polymorphism analysis was employed to investigate sequence variation in the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA within and among individuals representing three operational taxonomic units (OTUs) of Hypodontus macropi from different species of Australian macropodid marsupials. Of the 96 nematodes analysed, totals of 3 (OTU1 from Petrogale persephone), 10 (OTU2 from Macropus robustus) and 7 (OTU9 from Macropus rufus) representative individuals were selected for DNA sequencing to characterise and estimate the magnitude of nucleotide variation in the ITS-2. While no unequivocal nucleotide difference in the ITS-2 was detectable within OTU1, most sequence variation (3/44.7%) detected within OTU2 and OTU9 was related chiefly to dinucleotide (CA, TA, or a combination of both) differences. This microsatellite variability in some H. macropi OTUs suggests that the ITS-2 rDNA may be subjected to slippage events during DNA replication, resulting in short dinucleotide repeat tracts being dispersed throughout the ITS-2 lineages, or possibly transposition and/or crossing-over events. Nucleotide variation in the ITS-2 of individual OTUs was related to the proposed secondary structure for the precursor ribosomal RNAs. Most of the sequence heterogeneity or polymorphism within OTU2 and OTU9 occurred in loops or bulges of the predicted secondary structure, which appear not to be under functional constraint. The findings of this study have implications for investigating speciation events and population differentiation in nematodes at the molecular level.

Animals↗

Genotyping Cryptosporidium parvum by single-strand conformation polymorphism analysis of ribosomal and heat shock gene regions.

Polymerase chain reaction (PCR)-coupled single-strand conformation polymorphism (SSCP) approaches utilizing nuclear DNA regions of the small subunit (SSU) of ribosomal RNA and heat shock protein 70 gene (HSP70) were established for genotyping Cryptosporidium parvum. The regions were amplified (individually or in a multiplex reaction) by PCR from DNA extracted from oocysts from ruminant or human hosts, then denatured and subjected to electrophoresis in a mutation detection enhancement (nondenaturing) gel matrix. Single-strand profiles produced in SSCP allowed the unequivocal identification/differentiation of the two common (human, 1 and cattle, 2) genotypes of C. parvum and the direct display of sequence variability within some samples, reflecting population variation. As these are considered among the most closely related genotypes (based on SSU and HSP70 sequence data), these findings and other preliminary results for C. felis (from cat) C. serpentis (from snake) and C. baileyi (from bird) indicate that the SSCP approaches established could be employed to identify any of the currently recognised genotypes and species of Cryptosporidium.

Animals↗

Electrophoretic detection of population variation within Contracaecum ogmorhini (Nematoda: Ascaridoidea: Anisakidae).

This study examined genetic variation among specimens of Contracaecum ogmorhini from different otariid hosts and geographical origins using a polymerase chain reaction (PCR)-based mutation detection approach. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified individually by PCR, scanned for sequence variation by single-strand conformation polymorphism (SSCP), and samples displaying variable SSCP profiles were subjected to cycle sequencing. While C. ogmorhini individuals from Arctocephalus pusillus pusillus (CoAPP) from South Africa and those from Arctocephalus pusillus doriferus (CoAPD) from Australia had very similar SSCP profiles for both ITS-1 and ITS-2, individuals of C. ogmorhini from Zalophus californianus (CoZC) from Pacific Canada could be unequivocally distinguished based on their profiles. In accordance with SSCP results, both CoAPP and CoAPD had identical ITS consensus sequences, whereas CoZC differed in sequence from both CoAPP and CoAPD populations by 0.2% (one base in the ITS-1) and 0.7% (two bases in the ITS-2). Based on the nucleotide difference in the ITS-2 sequence, a PCR-linked restriction fragment length polymorphism (RFLP) could be employed to distinguish individuals representing CoZC from those of both CoAPP and CoAPD. The findings suggest that C. ogmorhini may represent a complex of at least two species.

Animals↗

Localization of the Q1 mutation by cladistic analysis.

The completion of the human genome project appears to be in sight and enormous new information resources will be available. The challenge now facing the scientific community is to use this information. In this paper, we applied cladistic analysis to the sequenced data in a Genetic Analysis Workshop 12 simulation problem. The Q1 mutation was localized to a 5-kb segment, in which the true mutation is located. Our analysis shows that cladistic analysis, which summarizes evolutionary history, can be a powerful tool in gene mapping.

Alleles↗

Cloning and sequencing of cDNA encoding for a novel human testis-specific contraceptive vaccinogen: role in immunocontraception.

Sperm-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the subtractive cDNA hybridization technology, the present study was conducted to obtain a human sperm-specific antigen. The 32P-labeled single stranded cDNA of human testis, subtracted with poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis cDNA-ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 10(7) colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of approximately 1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full-length sequence was obtained by using the 5' rapid amplification of 5' -cDNA ends (5'-RACE) procedure. The full-length CV cDNA has an ORF of 312 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 35 and the stop codon TAA, at nt 959. The translated protein has a calculated molecular mass of 35.3 kD and four potential N-linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane-anchored peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis-specific expression of CV antigen. The coding region of CV cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV protein had a molecular size of approximately 44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of approximately 64 kD in the human sperm extract. The antibodies showed binding with the live and methanol-fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of zona-free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm- specific CV antigen has a role in human sperm function and may find clinical applications in the contraceptive vaccine development and in the specific diagnosis and treatment of male infertility.

Adult↗

Coexpression of normal and mutated CD40 ligand with deletion of a putative RNA lariat branchpoint sequence in X-linked hyper-IgM syndrome.

We describe a novel CD40 ligand (CD40L) splicing mutation in a patient with X-linked hyper-IgM syndrome (X-HIM) associated with alternate splicing of exon 3, resulting in the expression of both full-length and exon-3-skipped CD40L mRNA. The mutation is an 8-bp deletion 25 bp upstream of the intron 2/exon 3 junction which overlaps a putative RNA branchpoint, suggesting that it may impair RNA lariat formation. The exon-3-skipped CD40L transcript encodes a truncated protein (CD40LDeltaE3) encompassing the cytoplasmic, transmembrane, and extracellular stalk domains, but lacking the CD40L receptor binding domain. CD40LDeltaE3 protein expression was readily detectable in transfected Cos cells by immunofluorescence. In cells cotransfected with CD40LDeltaE3 and wild-type CD40L, expression of CD40LDeltaE3 did not inhibit the expression of wild-type CD40L monomers, but strongly inhibited staining by the conformationally sensitive anti-CD40L mAb 5c8, suggesting that CD40LDeltaE3 acts in a dominant negative manner to inhibit the assembly of functional CD40L trimers. This mechanism may contribute to the pathophysiology of CD40L deficiency in X-HIM patients with leaky splice site mutations.

CD40 Ligand↗

Measurement of urinary methoxyphenols and their use for biological monitoring of wood smoke exposure.

A urinary assay for methoxyphenols was developed for the biological monitoring of wood smoke exposure. Methoxyphenols in 10-ml samples of urine were extracted after acid hydrolysis using XAD in a solid-phase extraction cartridge. The methoxyphenols were eluted with ethyl acetate and then analyzed by gas chromatography/mass spectrometry. Specific chemicals quantified were guaiacol, 4-methylguaiacol, 4-ethylguaiacol, 4-propylguaiacol, syringol, 4-methylsyringol, 4-ethylsyringol, vanillin, eugenol, and syringaldehyde. Recoveries ranged from 60 to 90%, with coefficients of variation of < or =20%. Background levels of the compounds were measured in 21 nonsmoking adults. Guaiacol, 4-methylguaiacol, eugenol, and vanillin were detected in all subjects. An experimental feeding of a commercial wood smoke flavoring demonstrated that methoxyphenols were rapidly and efficiently eliminated in urine. Preliminary field studies demonstrated that urinary excretion rates of some methoxyphenols increased after inhalation exposure to wood smoke.

Adult↗

Role of opioid delta1 receptors, mitochondrial K(ATP) channels, and protein kinase C during cardiocyte apoptosis.

Opioids attenuate cardiac injury after ischemia and reperfusion. We wanted to determine whether the protection of opioids is mediated by blocking cardiocyte apoptosis, and if so, to describe the role of opioid delta1 receptors and protein kinase C (PKC) in this effect. Chick embryonic cardiomyocytes were subjected to 12 h of simulated ischemia and then 12 h of re-oxygenation, which resulted in 54+/-3% (n=6) of cell apoptosis (n=6) as measured by flow cytometry. This result was consistent with DNA laddering and TUNEL assay. Preconditioning, elicited with three cycles of 1 min of simulated ischemia separated by 5 min of reoxygenation before prolonged simulated ischemia, reduced apoptosis (36+/-4%, n=6*). Pretreatment with BNTX (0.1 micromol/l), a selective opioid delta1 receptor blocker, abolished the effects of preconditioning (57+/-5%, n=6). The selective opioid delta receptor agonist BW373U86 (20 pmol/l) also attenuated apoptosis (39+/-3%, n=6* v control). These effects were abolished by 5-hydroxydecanoate (100 microm), a selective mitochondrial K(ATP) channel blocker (50+/-5%, n=6) and by Go-6976 (0.1 micromol/l), a specific PKC inhibitor. Both preconditioning and BW373U86 activated the PKC delta isoform of particulate fraction before simulated ischemia without effect on total and cytosolic fractions. Stimulation of opioid delta1 receptors activates mitochondrial K(ATP) channels and the PKC delta isoform in cultured ventricular myocytes. This is one important signal transduction pathway through which ischemic preconditioning blocks apoptosis and preserves cardiac function.

Adenosine Triphosphate↗

In vivo surgical resection plus adjuvant gene therapy in the treatment of mammary and prostate cancer.

Adenoviral-mediated gene therapy delivery, combining the herpes simplex virus thymidine kinase gene (Ad-tk) with gancyclovir, has been evaluated as a treatment modality for numerous tumors in the laboratory and in the clinics. As a single modality, gene therapy has shown some promising local and systemic results but no curative success. Surgery is the standard of care for many solid tumors. However, minor residual tumor following surgical resection can lead to local recurrence, and surgery is neither efficient nor plausible for metastatic disease. In this study, two tumor models were used to evaluate the effects of Ad-tk gene therapy as an adjuvant to surgery. Subcutaneous mammary- and prostate-derived tumors were produced in syngeneic mice. To evaluate systemic effects, tumor cells were injected intravenously, with subsequent formation of lung nodules. The subcutaneous tumors were surgically resected and the tumor bed was bathed with saline or Ad-tk. The animals were evaluated for toxicity, local tumor recurrence, survival, and lung nodule formation. No evidence of additional toxicity was observed. In the less aggressive mammary model, the time to recurrence was increased from 11.7 (+/-1.0) days to 22.7 (+/-5.5) days. In the prostate model, recurrence went from a mean of 17.3 (+/-5.6) to 22.6 (+/-6.8) days. Survival was also improved from a mean of 19.7 (+/-1.1) to 32.3 (+/-4.8) and 26.1 (+/-5.0) to 34.1 (+/-6.1) days in the mammary and prostate models, respectively. Evidence of systemic benefits from the use of adjuvant Ad-tk therapy was demonstrated by a significant reduction in lung nodules from a mean of 17 to 3.5. These results suggest that Ad-tk gene therapy may be a useful adjuvant for patients undergoing surgery for treatment of cancer.

Adenoviridae↗

Enhanced therapeutic effect of HSV-tk+GCV gene therapy and ionizing radiation for prostate cancer.

Standard therapies for prostate cancer including radiation, prostatectomy, and hormone ablation have significant toxicities and recurrence risk. HSV-tk gene therapy may be effective in combination with radiation therapy due to complementary mechanisms and distinct toxicity profiles. Mouse prostate tumors transplanted subcutaneously were treated by either gene therapy involving intratumoral injection of AdV-tk followed by systemic ganciclovir or local radiation therapy or the combination of gene and radiation therapy. Both single-therapy modalities showed a 38% decrease in tumor growth compared to controls. The combined treatment resulted in a decrease of 61%. In addition the combined-therapy group had a mean survival of 22 days versus 16.6 days for single therapy and 13.8 days for nontreated controls. To analyze systemic anti-tumor activity, lung metastases were generated by tail vein injection of RM-1 prostate cancer cells on the same day that they were injected subcutaneously. The primary tumors were treated as before with AdV-tk followed by ganciclovir, radiation, or the combination. The number of lung nodules was reduced by 37% following treatment with AdV-tk, whereas radiotherapy alone had no effect on metastatic growth. The combination led to an additional 50% reduction in lung colonization. Primary tumors that received the combination therapy had a marked increase in CD4 T cell infiltrate. This is the first report showing a dramatic systemic effect following the local combination treatment of radiation and AdV-tk. A clinical study using this strategy has been initiated and patient accrual is ongoing.

Adenoviridae↗

In-situ separation of the matrix for the direct determination of traces of chromium, cobalt, and nickel in titanium dioxide powder by electrothermal atomic-absorption spectrometry with slurry sampling.

A novel method has been developed for the direct determination of traces of chromium, cobalt, and nickel in TiO2 powder; it entails slurry sampling and electrothermal atomic-absorption spectrometry (ETAAS) with a polytetrafluoroethylene (PTFE) slurry (6% m/v) as fluorinating reagent. The factors which could affect the vaporization of the matrix and analytes were studied in detail; the fluorinating vaporization behavior of the analyte both in the slurry and in solution were also investigated. Owing to the in-situ separation of the matrix, the matrix influences were reduced significantly. The proposed method has been applied to the direct determination of traces of chromium, cobalt, and nickel in high-purity TiO2 powder without chemical pretreatment. Under the optimum experimental conditions the detection limits of the analytes (Cr, Co, and Ni) were 1.9 ng g(-1), 2.4 ng g(-1) and 5.4 ng g(-1), respectively, the relative standard deviations (RSD) were 3.4% (n=6, c=7.0 ng mL(-1)), 2.9% (n=6, c=0.70 ng mL(-1)), and 7.6% (n=6, c=4.0 ng mL(-1)), again respectively, and the characteristic masses for Cr, Co, and Ni were 8.4 pg/ 0.0044A, 9.3 pg/0.0044A, and 40.0 pg/0.0044A, respectively.

Alloys↗

Abortive apoptosis in Alzheimer's disease.

Multiple studies suggest that neuronal death in Alzheimer's disease (AD) is the result of an apoptotic mechanism. However, the stereotypical manifestations that define the terminal phases of apoptosis, such as chromatin condensation, apoptotic bodies, and blebbing, are not seen in AD. In this study, we show that the caspases, such as caspase 6, which cleave amyloid-beta protein precursor (A beta PP) and presenilins, are localized to the pathological lesions associated with AD. However, while upstream caspases such as 8 and 9 are clearly found in association with the intraneuronal pathology in AD, downstream caspases such as 3, 6 and 7 are present only at control levels. Given that execution of apoptosis requires amplification of the caspase-mediated apoptotic signal, our results indicate that in AD there is a lack of effective apoptotic signal propagation to downstream caspase effectors. Therefore, while the presence of caspases, especially caspase 6, in association with extracellular deposits of amyloid-beta, could obviously have important ramifications on the proteolytic processing of A beta PP and, thereby, on disease pathogenesis, it seems that AD represents the first in vivo situation reported in which the initiation of apoptosis does not proceed to caspase-dependent cell death. This novel phenomenon of apoptotic avoidance, which we term abortive apoptosis, or abortosis, may represent an exit from the caspase-induced apoptotic program that leads to neuronal survival in AD.

Adult↗

Over-expression of beta-1,4-galactosyltransferase I, II, and V in human astrocytoma.

PURPOSE: beta-1,4-Galactosyltransferase (beta-1,4-GalT) I, II, and V are the enzymes responsible for the biosynthesis of N-acetyllactosamine on N-glycans by transferring UDP-galactose to the terminal N-acetylglucosamine (N-GlcNAc) residues with the formation of a beta-1,4-linkage. Neoplasms undergo various changes in the carbohydrate of their glycoconjugates, indicating the possible changes in glycosyltransferases themselves. METHOD: Therefore, we compared the expression of beta-1,4-GalTs between astrocytoma and normal brain tissues. RESULTS: Our reverse-transcription polymerase chain reaction (RT-PCR) results showed that beta-1,4-GalT I transcript was absent in normal adult brain but detectable in grade II, III, and IV astrocytomas; the level of beta-1,4-GalT II transcript was increased in grade II, III, and IV astrocytomas while only a trace amount was found in normal brain; beta-1,4-GalT V transcript existed in normal brain and increased in the process of astrocytoma progress, with the highest level in grade IV astrocytoma. By Ricinus communis agglutinin-1 (RCA-1) lectin blot assay, we also found the more extensive galactosylated bands in astrocytomas compared with normal brain. A major 61kD protein was galactosylated in astrocytoma but not in normal brain tissues. CONCLUSION: These results indicate that the increase of galactosylation in astrocytomas may be caused by the alterations of gene expression of beta-1,4-GalT I, II, and V and that the malignant degree of astrocytoma is correlated with the expression of beta-1,4-GalT V.

Astrocytoma↗