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Biomedical subjects

Z Wang

Publications and source records attributed to Z Wang.

At least 613 records · Page 34Linked to original sources
PubMed2000

Flow cytometry-based minisequencing: a new platform for high-throughput single-nucleotide polymorphism scoring.

Single-nucleotide polymorphisms (SNPs) are the most abundant type of human genetic variation. These variable sites are present at high density in the genome, making them powerful tools for mapping and diagnosing disease-related alleles. We have developed a sensitive and rapid flow cytometry-based assay for the multiplexed analysis of SNPs based on polymerase-mediated primer extension, or minisequencing, using microspheres as solid supports. The new method involves subnanomolar concentrations of sample in small volumes ( approximately 10 microl) which can be analyzed at rates of one sample per minute or faster, without a wash step. Further, genomic analysis using multiplexing microsphere arrays (GAMMArrays), enables the simultaneous analysis of dozens, and potentially hundreds of SNPs per sample. We have tested the new method by genotyping the Glu69 variant from the HLA DPB1 locus, a SNP associated with chronic beryllium disease, as well as HLA DPA1 alleles using the multiplexed method. The results demonstrate the sensitivity and accuracy of flow cytometry-based minisequencing, a powerful new tool for genome- and global-scale SNP analysis.

Base Sequence↗
PubMed2000

Changes in wheat germination following gamma-ray irradiation: an in vivo electronic paramagnetic resonance spin-probe study.

Embryos excised from wheat (Triticum aestivum) grains following gamma-ray irradiation at different doses were analyzed on membrane permeability by electron paramagnetic resonance (EPR) technique with 4-oxo-2, 2, 6, 6-tetramethyl-1-piperidinyloxy (TEMPONE) as spin probe to acquire an EPR spectrum. The broadening agent ferricyanide added leads to changes in the high-field region of the EPR spectrum, which reflects differences in membrane permeability. R-value, defined as the ratio of water (W) to lipid (L) component in height in the high-field region of the EPR spectrum, symbolizes membrane permeability for a given sample. The R-values corresponding to a certain dose treatment of grains displayed a definitive distribution pattern. A unit row vector with 20 components was used to describe the R-value distribution pattern for a given treatment. The transaction angle between vectors corresponding to grains irradiated and unirradiated, &theta;, was used as quantitative index for membrane permeability changes following gamma-ray irradiation. gamma-Ray irradiated grains germinated at low rates, and the regression equation of germination rate as a function of the irradiation dose is: Germination Rate (%)=94.8 exp[-0.264xIrradiation Dose (kGy)] (r(2)=0.991, P<0.001). Embryos excised from grains following gamma-ray irradiation show increases in &theta; values with irradiation dose. The &theta; value is negatively linearly correlated with the germination rate. It suggests that gamma-ray irradiation leading to increases in membrane permeability is consistent with that leading to low germination rate of grains. The introduction to vector analysis method on membrane permeability changes in this study is very practical.

Journal Article↗
PubMed2000

Microglia-like cells in rat organ of Corti following aminoglycoside ototoxicity.

The repair process of neomycin induced cochlear damage in the postnatal developing rat was investigated in the present study. The results showed that electron dense atypical cells with a cluster of microvilli on their apical surface, resembling early stage of embryonic hair cell, were observed in the former hair cell region. The striking finding was that microglia-like cells appeared and replaced OHCs in the injured auditory sensory epithelium. Using Brdu immunohistochemistry, cell proliferation was found in the area of inner and outer spiral sulcus but not in the hair cells and supporting cells. It is proposed that microglia-like cells play a role in eliminating waste products from the organ of Corti and may participate in direct structural repair.

Animals↗
PubMed2000

Reducing premature death and renal failure in Australian aboriginals. A community-based cardiovascular and renal protective program.

OBJECTIVE: To describe results of a systematic treatment program to modify renal and cardiovascular disease in an Aboriginal community whose rates of renal failure and cardiovascular deaths are among the highest in Australia. DESIGN: Longitudinal survey of people during treatment, and comparison of rates of natural death and renal failure with those in a historical control group. SETTING: Tiwi Islands (population, about 1800), November 1995 to December 1998. PARTICIPANTS: All adults with blood pressure > or = 140/90, with diabetes and urinary albumin/creatinine ratio (ACR) > or = 3.4 g/mol (microalbuminuria threshold), or with progressive overt albuminuria (ACR > or = 34 g/mol) were eligible for treatment. The historical control group comprised 229 people who satisfied these criteria in the pretreatment period 1992-1995. INTERVENTIONS: Perindopril, combined with calcium-channel blockers and diuretics if needed to achieve blood pressure goals; attempts to improve control of blood glucose and lipid levels; health education. MAIN OUTCOME MEASURES: Blood pressure, ACR, serum creatinine level and glomerular filtration rate (GFR) over two years of treatment; rates of renal failure and natural death compared with control group (analysed on intention-to-treat basis). RESULTS: 258 people enrolled in the program, and 118 had complete data for two years of treatment. In these 118, blood pressures fell significantly, while ACR and GFR stabilised. Rates of the combined endpoints of renal failure and natural death per 100 person-years were 2.9 for the treatment group (95% CI, 1.7-4.6) and 4.8 for the control group (95% CI, 3.3-7.0). After adjustment for baseline ACR category, the relative risk of the treatment group versus the control group for these combined endpoints was 0.47 (95% CI, 0.25-0.86; P = 0.013). Treatment benefit was especially marked in people with overt albuminuria or hypertension and in non-diabetic people. The estimates of benefit were supported by a fall in community rates of death and renal failure. CONCLUSIONS: Aboriginal people can participate enthusiastically in chronic disease management, with rapid, dramatic improvement in clinical profiles and mortality. Similar programs should be introduced urgently into other Aboriginal communities nationwide.

Adult↗
PubMed2000

Absence of proximal duct apoptosis in the ventral prostate of transgenic mice carrying the C3(1)-TGF-beta type II dominant negative receptor.

BACKGROUND: Prostatic epithelial cells are sensitive to the inhibitory effects of TGF-beta. However, TGF-beta signaling in the prostate is dependent on androgenic status. Under the in vivo conditions, it is difficult to dissociate the effect of TGF-beta from that of androgen on the prostate. METHODS: The objective of the present study was to create and verify a transgenic mouse system in which epithelial cells of the ventral prostate are insensitive to the actions of TGF-beta. By using a modified prostate-specific promoter, C3(1), the TGF-beta dominant negative receptor is only expressed in the epithelial cells of the ventral prostate, and these cells are resistant to TGF-beta. Morphology of transgenic animal prostates was compared to wild-type animal prostates by immunohistochemistry and microscopy. RESULTS: The prostate of transgenic mice exhibited an abnormal morphology with multiple layers of epithelial cells lining the proximal ducts, in contrast to the simple cuboidal monolayer of cells seen in the normal prostate. This observation was accompanied by a loss of apoptosis in this region, as seen by TUNEL assay. There was no significant difference in serum levels of testosterone between the wild-type and transgenic animals. CONCLUSIONS: These results demonstrated that a loss of sensitivity to TGF-beta results in the accumulation of multiple layers of epithelial cells in the proximal region of the ventral prostate. This abnormal growth illustrates that TGF-beta plays an important role in regulating prostate growth. The current transgenic system can be used as an experimental model to study the functional role of TGF-beta in prostatic growth and function.

Activin Receptors, Type I↗
PubMed2000

Direct observation of calcium-independent intercellular ATP signaling in astrocytes.

Adenosine triphosphate (ATP) is assumed to be involved in the regulation of many extracellular signaling systems including calcium wave propagation. So far all supportive evidence is indirect, such as monitoring changes in intracellular calcium on application of extracellular ATP or off-site measurement of ATP from superfusates. Furthermore, the causal relationships among the various signaling agents are still unclear. A novel chemiluminescence dynamic imaging method was developed to monitor ATP release from living biological cells. The assay has linear response over 3 orders of magnitude for fixed concentrations of enzyme and cofactors, with a correlation coefficient of 0.999. The detectability of ATP is down to 10(-8) M at millisecond exposure times with an intensified charge-coupled device camera. The direct imaging of ATP waves in astrocyte cultures was performed together with Fluo-3-Ca imaging at millisecond temporal resolution and micrometer-scale spatial resolution. We discovered that extracellular ATP mediates intercellular calcium wave propagation, but surprisingly, release and propagation of ATP are not calcium dependent. Therefore, ATP rather than Ca or IP3 is the primary intercellular signaling messenger.

Adenosine Triphosphate↗
PubMed2000

Interaction of photosynthetic pigments with various organic solvents 2. Application of magnetic circular dichroism to bacteriochlorophyll a and light-harvesting complex 1.

Magnetic circular dichroism (MCD) and absorption spectra of metal bacteriochlorin complexes have been measured on bacteriochlorophyll (BChl) a in various solvents and different forms of light-harvesting complexes 1 (LH1 complexes). In hydrophilic organic solvents, the MCD intensity of the Q(y)(0-0) transition of BChl a was sensitive to the wavelength of absorption maximum of Q(x)(0-0), and the ratio of MCD Q(y)(0-0) intensity to the dipole strength (B/D) was inversely proportional to the difference in energy between the Q(x)(0-0) and Q(y)(0-0). The similar correlation has been observed in metal chlorin derivatives as previously reported. The correlation depends on the coordination number of the Mg atom in BChl a and the molecules ligating to it. In a hydrophobic solvent such as carbon tetrachloride (CCl(4)), however, the correlation did not hold because of the existence of aggregates. Hence, the correlation between the values of B/D and the energy difference can be used to estimate the type and number of the molecules ligated to the Mg atom and to disclose the existence of aggregated pigments. We further apply the correlation to the LH 1 complex treated with n-octyl beta-D-glucopyranoside.

Bacterial Proteins↗
PubMed2000

Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of nuclear retinoid receptors.

Amino Acid Motifs↗
PubMed2000

Ligand-dependent interactions of coactivators steroid receptor coactivator-1 and peroxisome proliferator-activated receptor binding protein with nuclear hormone receptors can be imaged in live cells and are required for transcription.

Members of the nuclear receptor superfamily are thought to activate transcription by recruitment of one or more recently identified coactivator complexes. Here we demonstrate that both peroxisome proliferator-activated receptor binding protein (PBP) and steroid receptor coactivator-1 (SRC-1) are required for ligand-dependent transcription of transiently transfected and chromosomally integrated reporter genes by the estrogen receptor (ER) and retinoic acid receptor (RAR). To examine ligand-dependent interactions between nuclear receptors and specific coactivators in living cells, these proteins were tagged with cyan (CFP) and yellow (YFP) mutants of the green fluorescent protein. Fluorescence resonance energy transfer (FRET) from the CFP to the YFP indicated interaction between the receptor and coactivator. CFP fusions to RAR or its ligand-binding domain exhibited rapid ligand-dependent FRET to YFP-tagged nuclear receptor interaction domains of the coactivators SRC-1 and PBP. The ER-ligand-binding domain, unlike RAR, also exhibited some basal interaction with coactivators in unstimulated cells that was abolished by the receptor antagonists tamoxifen or ICI182,780. Inhibition of FRET by tamoxifen but not ICI182,780 could be reversed by estradiol, whereas estradiol-enhanced FRET could not be inhibited by either antagonist, indicating that ligand effects can show varying degrees of hysteresis. These findings suggest that ligand-dependent transcriptional activities of the RAR and ER require concurrent or sequential recruitment of SRC-1 and PBP-containing coactivator complexes.

Carrier Proteins↗
PubMed2000

Direct block of inward rectifier potassium channels by nicotine.

Nicotine has been shown to depolarize membrane potential and to lengthen action potential duration in isolated cardiac preparations. To investigate whether this is a consequence of direct interaction of nicotine with inward rectifier K(+) channels which are a key determinant of membrane potentials, we assessed the effects of nicotine on two cloned human inward rectifier K(+) channels, Kir2.1 and Kir2.2, expressed in Xenopus oocytes and the native inward rectifier K(+) current I(K1) in canine ventricular myocytes. Nicotine suppressed Kir2.1-expressed currents at varying potentials negative to -20 mV, with more pronounced effects on the outward current between -70 and -20 mV relative to the inward current at hyperpolarized potentials (below -70 mV). The inhibition was concentration dependent. For the outward currents recorded at -50 mV, the IC50 was 165 +/- 18 microM. Similar effects of nicotine were observed for Kir2.2. A more potent effect was seen with I(K1) in canine myocytes. Significant blockade ( approximately 60%) was found at a concentration as low as 0.5 microM and the IC50 was 4.0 +/- 0.4 microM. The effects in both oocytes and myocytes were partially reversible upon washout of nicotine. Antagonists of nicotinic receptors (mecamylamine, 100 microM), muscarinic receptors (atropine, 1 microM), and beta-adrenergic receptors (propranolol, 1 microM) all failed to restore the depressed currents, suggesting that nicotine acted directly on Kir channels, independent of catecholamine release. This property of nicotine may explain its membrane-depolarizing and action potential duration-prolonging effects in cardiac cells and may contribute in part to its ability to promote propensity for cardiac arrhythmias.

Animals↗
PubMed2000

[The protective effects of zinc metallothionein against erythrocyte membrane damage induced by methylmercury].

The influences of zinc metallothionein(ZnMT) induced by Zn on mice erythrocyte membrane configuration, membrane sulfhydryl content, membrane fluidity, membrane permeability and so on after methylmercury (MeHg) treatment were studied, the protection of ZnMT against the membrane damage of MeHg was studied. The results showed that Zn could induce the production of ZnMT in the liver of mice. With the increase of Zn content, the level of ZnMT increased and had dose-effect relationship between them (r = 0.996). ZnMT could protect membrane configuration and -SH and prevent the damage of MeHg on the membrane fluidity and permeability. The results also showed that the dose of Zn administered to mice should be appropriate. High doses of Zn could cause damages.

Animals↗
PubMed2000

Pituitary tumor transforming gene (PTTG) transforming and transactivation activity.

Pituitary tumor transforming gene (PTTG) is a newly identified transforming gene, the functional mechanism of which is little understood. Computational analysis reveals a C terminus rich in Glu and Pro, a known characteristic of transcriptional activation domains. We report here that murine PTTG indeed possesses transactivation ability, which correlates highly with its transforming properties. Pro(139), Ser(159), Pro(157)-Pro(158)-Ser(159)-Pro(160) (PPXP motif), and Leu(120)-Asp(121)-Phe(122)-Asp(123)-Leu(124) were found to be important for transactivation. Mutation to Ala at a key Pro(139) residue not only disrupted the transactivation function but also resulted in the loss of transforming ability in NIH3T3 cells. A murine PTTG cDNA that encodes a variant C-terminal tail (Gly-Lys-Gly-Val-Arg-Ser-Asn-Gly-Cys-Lys-Asp-Leu-Val-Thr) was cloned. This novel PTTG is devoid of transactivation and transforming ability; deletion of its variant C-terminal tail restores both transactivation and transforming ability. These results show a high correlation between the transforming and transactivation functions of PTTG and also indicate that the novel PTTG variant may function as an endogenous competitor to wild-type PTTG.

Amino Acid Sequence↗
PubMed2000

Specificity of DNA lesion bypass by the yeast DNA polymerase eta.

DNA polymerase eta (Pol(eta), xeroderma pigmentosum variant, or Rad30) plays an important role in an error-free response to unrepaired UV damage during replication. It faithfully synthesizes DNA opposite a thymine-thymine cis-syn-cyclobutane dimer. We have purified the yeast Pol(eta) and studied its lesion bypass activity in vitro with various types of DNA damage. The yeast Pol(eta) lacked a nuclease or a proofreading activity. It efficiently bypassed 8-oxoguanine, incorporating C, A, and G opposite the lesion with a relative efficiency of approximately 100:56:14, respectively. The yeast Pol(eta) efficiently incorporated a C opposite an acetylaminofluorene-modified G, and efficiently inserted a G or less frequently an A opposite an apurinic/apyrimidinic (AP) site but was unable to extend the DNA synthesis further in both cases. However, some continued DNA synthesis was observed in the presence of the yeast Pol(zeta) following the Pol(eta) action opposite an AP site, achieving true lesion bypass. In contrast, the yeast Pol(alpha) was able to bypass efficiently a template AP site, predominantly incorporating an A residue opposite the lesion. These results suggest that other than UV damage, Pol(eta) may also play a role in bypassing additional DNA lesions, some of which can be error-prone.

2-Acetylaminofluorene↗
PubMed2000

Regulation of transient Na+ conductance by intra- and extracellular K+ in the human delayed rectifier K+ channel Kv1.5.

1. Significant Na+ conductance has been described in only a few native and cloned K+ channels, but has been used to characterize inactivation and K+ binding within the permeation pathway, and to refine models of K+ flux through multi-ion pores. Here we use Na+ permeation of the delayed rectifier K+ channel Kv1.5 to study extra- and intracellular K+ (K+o and K+i, respectively) regulation of conductance and inactivation, using whole-cell recording from human embryonic kidney (HEK)-293 cells. 2. Kv1.5 Na+ currents in the absence of K+o and K+i were confirmed by: (i) resistance of outward Na+ currents to dialysis by K+-free solutions; (ii) tail current reversal potential changes with Na+o with a slope of 55.8 mV per decade; (iii) block by 4-aminopyridine (50 % at 50 microM), and resistance to Cl- channel inhibition. 3. Na+ currents were transient followed by a small sustained current. An envelope test confirmed that activated Kv1.5 channels conducted Na+, and that rapid current decay reflected C-type inactivation. Sustained currents ( approximately 13 % of peak) represented Na+ flux through inactivated Kv1.5 channels. 4. K+o could modulate the maximum available Na+ conductance in the stable cell line while channels were closed. Before the first pulse of a train, increasing K+o concentration increased the subsequent Na+ conductance from approximately 15 (0 mM K+o) to 30 nS (5 mM K+o), with a Kd of 23 microM. Repeated low rate depolarizations in Na+i/Na+o solutions induced a use-dependent loss of Kv1.5 channel Na+ conductance, distinct from that caused by C-type inactivation. K+o binding that sensed little of the electric field could prevent this secondary loss of available Kv1.5 channels with a Kd of 230 microM. These two effects on conductance were both voltage independent, and had no effect on channel inactivation rate. 5. K+o concentrations >= 0.3 mM slowed the inactivation rate in a strongly voltage-dependent manner. This suggested it could compete for binding at a K+ site or sites deeper in the pore, as well as restoring the Na+ conductance. K+i was able to modulate the inactivation rate but was unable to affect conductance. 6. Mutation of arginine 487 in the outer pore region of the channel to valine (R487V) greatly reduced C-type inactivation in Na+ solutions, caused loss of channel use dependence, and prevented any conductance increase upon the addition of 0.1 mM K+o. Our results confirm the existence of a high affinity binding site at the selectivity filter that regulates inactivation, and also reveals the presence of at least one additional high affinity outer mouth site that predominantly regulates conductance of resting channels, and protects channels activated by depolarization when they conduct Na+.

Amino Acid Substitution↗
PubMed2000

Fullerene-sensitized

[reaction: see text] Fullerene catalyzes the cycloaddition of dimethyliminodiacetate to maleimides under photolysis to form 2,5-dimethoxycarbonylpyrrolidine derivatives.

Journal Article↗
PubMed2000

Influences of amino acid sequences in FR1 region on binding activity of the scFv and Fab of an antibody to human gastric cancer cells.

Murine anti-gastric cancer mAb 3H11 has promising clinical applications. In this work, engineering of 3H11 scFv and Fab was conducted to increase its usefulness. 3H11 scFv and Fab were constructed by PCR amplification of the V-domains with degenerate primers for FR1. The bacterially expressed 3H11 Ab fragments showed no antigen binding activity. Then the N-terminal sequences of V regions were mutated to the 3H11 original sequence. The expressed scFv and Fab in bacterial culture supernatant showed binding activity to gastric cancer cells. Comparing the expression of unmutated and mutated 3H11 Fab, we found that the sequence changes of the V region N terminus introduced by PCR may seriously affect antigen binding but not the expression of antibody. Correction of either VL or VH N-terminal sequences can partially restore the antigen binding activity (61% for VL and 73% for VH).

Amino Acid Sequence↗
PubMed2000

gamma-Aminobutyric acid infusion in substantia nigra pars reticulata in rats inhibits ascorbate release in ipsilateral striatum.

A relatively high level of extracellular ascorbate in the striatum, which is known to modulate impulse flow in striatal neurons, originates primarily from glutamate-containing corticostriatal afferents. Increasing evidence suggests that ascorbate release from these fibers is regulated by a multisynaptic loop that includes gamma-aminobutyric acid (GABA) mechanisms in the substantia nigra. To assess the role that nigral GABA plays in striatal ascorbate release, extracellular ascorbate was monitored voltammetrically in the striatum during infusions of GABA into the substantia nigra pars reticulata (SNr) of awake, unrestrained rats. Compared to vehicle infusions, intranigral GABA lowered striatal ascorbate by >50%. In contrast, intranigral application of picrotoxin, a GABA antagonist, had the opposite effect. Neither GABA nor picrotoxin altered striatal 3,4-dihydroxyphenylacetic acid (DOPAC), a major dopamine metabolite. Collectively, these results indicate that intranigral GABA exerts a tonic inhibitory influence on ascorbate release in the striatum.

3,4-Dihydroxyphenylacetic Acid↗