Biomedical subjects

Z Chen

Publications and source records attributed to Z Chen.

At least 145 records · Page 8Linked to original sources

PubMed2001

Chemically L-phenylalaninamide-modified monolithic silica column prepared by a sol-gel process for enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography.

A new type of chiral monolithic column was successfully developed for the enantioseparation of dansyl amino acids by ligand exchange-capillary electrochromatography (LE-CEC) in this work. The monolithic column matrix was prepared by a sol-gel process and then chemically modified with the spacer (3-glycidoxypropyl)trimethoxysilane and the chiral selector L-phenylalaninamide. After being conditioned with Cu(II) aqueous solution, the ligand exchange-chiral stationary phase (LE-CSP) possesses positive charges. When the external electric field was applied in CEC, electroosmotic flow (EOF) was generated on the surface of LE-CSP in the direction from the cathode to the anode. The EOF was found to be dependent on the applied electric field strength and the composition of the mobile phase. With the increase of pH of the mobile phase, the EOF showed a tendency to decrease. Scanning electron microscopy showed that the chiral monolithic column has a continuous skeleton and large through-pore structure. The separation efficiency (theoretic plate numbers) for the separation of Dns-DL-Leu reached up to 9.0 x 10(4) plates m(-1) for the D-enantiomer and 6.6 x 10(4) plates m(-1) for the L-enantiomer, by using pH 5.5, acetonitrile/0.50 mM Cu(Ac)2-50 mM NH4Ac (7:3) as mobile phase. The reproducibility and lifetime were satisfactory. CEC was carried out with conventional capillary electrophoresis equipment without pressurizing the ends of the capillary. No bubble was formed during the operation, after degassing the mobile phase and conditioning the column.

Amino Acids↗

PubMed2001

Mitochondria isolated from liver contain the essential factors required for RNA/DNA oligonucleotide-targeted gene repair.

Chimeric RNA/DNA oligonucleotides (ONs) have been used successfully for site-specific modifications of episomal and chromosomal DNA in eukaryotic cells. We explored the possibility of applying this technique to mitochondrial DNA, as single-nucleotide defects in this genome are associated with a series of human diseases. Therefore, we determined whether mitochondria possess the enzymatic machinery for chimeric ON-mediated DNA alterations. We utilized an in vitro DNA repair assay and an Escherichia coli readout system with mutagenized plasmids carrying point mutations in antibiotic resistance genes. RNA/DNA ONs were designed to correct the defects and restore kanamycin and tetracyclin resistance. Using this system, we demonstrated that extracts from highly purified rat liver mitochondria possess the essential enzymatic activity to mediate precise single-nucleotide changes. Interestingly, the frequency of gene conversion was similar in both mitochondrial and nuclear extracts, as well as from quiescent and regenerating liver. The results indicate that mitochondria contain the machinery required for repair of genomic single-point mutations, and suggest that RNA/DNA ONs may provide a novel approach to the treatment of certain mitochondrial-based diseases.

Animals↗

PubMed2001

Quantitative autoradiographic mapping of the ORL1, mu-, delta- and kappa-receptors in the brains of knockout mice lacking the ORL1 receptor gene.

Until recently the opioid receptor family was thought to consist of only the mu-, delta- and kappa-receptors. The cloning of opioid receptor like receptor (ORL1) and its endogenous ligand nociceptin/orphanin FQ, which displayed anti-opioid properties, has raised the issue of functional co-operativity of this system with the classical opioid system. ORL1 receptor knockout mice have been successfully developed by homologous recombination to allow the issue of potential heterogeneity of this receptor and also of compensatory changes in mu-, delta- or kappa-receptors in the absence of ORL1 to be addressed. We have carried out quantitative autoradiographic mapping of these receptors in the brains of mice that are wild-type, heterozygous and homozygous for the deletion of the ORL1 receptor. ORL1, mu-, delta- and kappa-receptors were labelled with [(3)H] leucyl-nociceptin (0.4 nM), [(3)H] DAMGO (4 nM), [(3)H] deltorphin-I (7 nM), and [(3)H] CI-977 (2.5 nM) respectively. An approximately 50% decrease in [(3)H] leucyl-nociceptin binding was seen in heterozygous ORL1 mutant mice and there was a complete absence of binding in homozygous brains indicating the single gene encodes for the ORL1 receptor and any putative subtypes. No significant gross changes in the binding to other opioid receptors were seen across genotypes in the ORL1 mutant mice demonstrating a lack of major compensation of classical opioid receptors in the absence of ORL1. There were a small number of region specific changes in the expression of classical opioid receptors that may relate to interdependent function with ORL1.

Analgesics, Opioid↗

PubMed2001

Gene expression analysis of an H(2)O(2)-resistant lens epithelial cell line.

Gene expression patterns were examined in lens epithelial cells conditioned to grow in 125 microM hydrogen peroxide in order to define the protective mechanisms that may be involved in survival during oxidative stress. RNA was extracted from normal and hydrogen peroxide-resistant alphaTN4 mouse lens epithelial cells. Gene expression was evaluated using Differential Display (DD) and RT-PCR. Upregulation of mRNAs for antioxidant and cellular defense enzymes was observed. The highest elevation detected was a 14-fold increase in catalase in the hydrogen peroxide-resistant cells. Glutathione peroxidase, ferritin, and alphaB-crystallin were upregulated 2-fold, and reticulocalbin was upregulated 6-fold in the resistant cells. alphaA-crystallin was downregulated 5-fold, while aldose reductase and mitochondrial gene products were unchanged. Thus, in the alphaTN4 mouse lens cell line, long-term exposure to high levels of hydrogen peroxide elicited an upregulation of transcripts for enzymes involved in hydrogen peroxide degradation, metal binding, and chaperone function. Since mitochondrial gene transcription is sensitive to hydrogen peroxide, the presence of normal levels of mitochondrial transcripts, in this study, demonstrates the effectiveness of the antioxidant defense systems.

Animals↗

PubMed2001

Effect of trends on detrended fluctuation analysis.

Detrended fluctuation analysis (DFA) is a scaling analysis method used to estimate long-range power-law correlation exponents in noisy signals. Many noisy signals in real systems display trends, so that the scaling results obtained from the DFA method become difficult to analyze. We systematically study the effects of three types of trends--linear, periodic, and power-law trends, and offer examples where these trends are likely to occur in real data. We compare the difference between the scaling results for artificially generated correlated noise and correlated noise with a trend, and study how trends lead to the appearance of crossovers in the scaling behavior. We find that crossovers result from the competition between the scaling of the noise and the "apparent" scaling of the trend. We study how the characteristics of these crossovers depend on (i) the slope of the linear trend; (ii) the amplitude and period of the periodic trend; (iii) the amplitude and power of the power-law trend, and (iv) the length as well as the correlation properties of the noise. Surprisingly, we find that the crossovers in the scaling of noisy signals with trends also follow scaling laws--i.e., long-range power-law dependence of the position of the crossover on the parameters of the trends. We show that the DFA result of noise with a trend can be exactly determined by the superposition of the separate results of the DFA on the noise and on the trend, assuming that the noise and the trend are not correlated. If this superposition rule is not followed, this is an indication that the noise and the superposed trend are not independent, so that removing the trend could lead to changes in the correlation properties of the noise. In addition, we show how to use DFA appropriately to minimize the effects of trends, how to recognize if a crossover indicates indeed a transition from one type to a different type of underlying correlation, or if the crossover is due to a trend without any transition in the dynamical properties of the noise.

Analysis of Variance↗

PubMed2001

Electroresistance and electronic phase separation in mixed-valent manganites.

The sensitivity of transport in colossal magnetoresistance (CMR) manganites to external electric and magnetic fields is examined using field effect configurations with La(0.7)Ca(0.3)MnO(3) (LCMO), Na(0.7)Sr(0.3)MnO(3), La(0.7)Ba(0.3)MnO(3), and La(0.5)Ca(0.5)MnO(3) (0.5-doped LCMO) channels, and ferroelectric PbZr(0.2)Ti(0.8)O(3) (PZT) or dielectric (SrTiO(3)) gates. A large electroresistance (ER) of approximately 76% at 4 x 10(5) V/cm is found in LCMO with PZT-ferroelectric gate, but the magnitude of the effect is much smaller (a few percent) in the other three channels. The ER and CMR effects are remarkably complimentary. The size and systematics of the effect strongly favor a percolative phase separation picture.

Journal Article↗

PubMed2001

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina.

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.

Acetylcholinesterase↗

PubMed2001

Cloning, expression patterns, and chromosome localization of three human and two mouse homologues of GABA(A) receptor-associated protein.

Type A receptors of gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter, contain alpha, beta, delta, gamma, and rho subunits. The gamma subunit has four subtypes: gamma1, gamma2, gamma3, andgamma4. GABA(A) receptor-associated protein (GABARAP) was previously demonstrated to act as a linker protein between microtubules and the gamma2 subunit of GABA(A) receptors. However, no other linker proteins have been identified as mediating the linkage of microtubules and the remaining subunits of GABA(A) receptors. In this study we identified three human paralogues (GABARAPL1, GABARAPL2, and GABARAPL3) and two mouse orthologues (Gabarapl1 and Gabarapl2) of human GABARAP, all of which encoded 117 amino acids, as does Gabarapl. The expression patterns of GABARAPL1, GABARAPL2, and GABARAP in 16 adult tissues showed that they were expressed ubiquitously. The expression levels of GABARAPL1 as a 2.3-kb transcript were very high in brain, heart, peripheral blood leukocytes, liver, kidney, placenta, and skeletal muscle, very low in thymus and small intestine, and moderate in other tissues tested. The unique 1.35-kb transcript of GABARAPL2 was expressed at high levels in heart, brain, testis, prostate, ovary, spleen, and skeletal muscle, at very low levels in lung, thymus, and small intestine, and moderately in other tissues tested. For GABARAP, a 1.3-kb transcript was abundantly expressed in all tested tissues with small variation. The expression patterns of Gabarapl1 and Gabarapl2 were similar to those of their counterparts in human. In addition, GABARAPL1 was localized to human chromosome 12p12.3 and GABARAPL2 to 16q22.3-q24.1 by RH mapping, while GABARAP and GABARAPL3 were found to be localized at chromosomes 17p13.2 and 15q25.1, respectively, by searching the related databases. Sequence comparison of the cDNAs and their corresponding genomic sequences shows that GABARAP, GABARAPL1, and GABARAPL2 are composed of four exons each, while GABARAPL3 is distributed only at one exon.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Liver-derived DEC205+B220+CD19- dendritic cells regulate T cell responses.

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205(bright)B220+CD11c-CD19-, and negative for T (CD3, CD4, CD8alpha), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19- cells release both IL-10 and IFN-gamma, small amounts of TGF-beta, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-gamma, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.

Animals↗

PubMed2001

Molecular profiling of transformed and metastatic murine squamous carcinoma cells by differential display and cDNA microarray reveals altered expression of multiple genes related to growth, apoptosis, angiogenesis, and the NF-kappaB signal pathway.

To identify changes in gene expression with transformation and metastasis, we investigated differential gene expression in a squamous carcinoma model established in syngeneic mice. We used mRNA differential display (DD) to detect global differences and cDNA arrays enriched for cancer-associated genes using mRNA from primary keratinocytes, transformed Pam 212 squamous carcinoma cells, and metastases of Pam 212. After DD, 72 candidate cDNAs expressed primarily in transformed and metastatic cells were selected and cloned. Fifty-seven were detected, and 32 were confirmed to be differentially expressed by Northern blot analysis. mRNA expression profiles were also generated using a mouse cDNA array composed of 4000 elements representing known genes and expressed sequence tags plus the 57 DD candidate cDNAs detected by Northern analysis to facilitate data validation. cDNA array detected 76.9% of the differentially expressed mRNAs selected from DD and confirmed by Northern blot, whereas low-abundance mRNAs did not reach the threshold for detection by the lower-sensitivity array method. Clustering analysis of DD and array results from transformed and metastatic cells identified genes that exhibited decreased or increased expression with transformation and metastasis. Alterations in the expression of several genes detected during tumor progression were consistent with their functional activities involving growth (p21, p27, and cyclin D1), resistance and apoptosis (glutathione-S-transferase, cIAP-1, PEA-15, and Fas ligand), inflammation and angiogenesis [chemokine growth-regulated oncogene 1 (also called KC)], and signal transduction (c-Met, yes-associated protein, and syk). Strikingly, 10 of 22 genes in the cluster expressed in metastases have been associated with activation of the nuclear factor (NF)-kappaB signal pathway. The NF-kappaB-inducible cytokine Gro-1 was recently shown to promote tumor growth, metastasis, and angiogenesis of squamous cell carcinomas in vivo (Loukinova et al., Oncogene, 19: 3477-3486, 2000). The results demonstrate that early response genes related to NF-kappaB contribute to metastatic tumor progression. Comparison of cell lines and tumor tissue revealed a concordance of approximately 50% by array, and 70% for Northern-confirmed, metastasis-related genes. Functional genomic approaches comparing expression among cell lines and tumor tissue may promote a better understanding of the genes expressed by malignant and host cells during tumor progression and metastasis.

Animals↗

PubMed2001

Isolation and purification of the ovulation-inducing factor from seminal plasma in the bactrian camel (Camelus bactrianus).

The purpose of this study was to extract, identify and partially characterize a newly found ovulation-inducing factor, and thus gain our understanding of induced ovulation in biology. In our preliminary research, an ovulation-inducing factor (OIF) was isolated and purified from seminal plasma of the bactrian camel by ion-exchange chromatography on DEAE-cellulose, HPLC and reverse-phase HPLC. The OIF is a peptide with 74 residues and GnRH-like bioactivity, which is heat-stable in camel seminal plasma because the OIF is wrapped up in several protein layers with different properties. However, purified OIF is degraded when it is exposed to oxygen or is heated in water. According to the analysis of amino acid components and partial amino acid sequence aminated N-terminus, and its molecular weight, the OIF is completely different from the native-LHRH, LH, HCG, PMSG and PGF-2alpha. The OIF is a novel ovulation hormone in the bactrian camel, but is similar to that reported in the bull.

Amino Acid Sequence↗

PubMed2001

Preparation and functional properties of polyclonal and monoclonal antibodies to murine MD-1.

Rabbits, rats and hamsters were immunized with KLH-coupled synthetic peptide sequences of the murine MD-1 molecule. Serum from immunized animals bound in Western gels to a 25 KDa protein extracted from LPS stimulated mouse spleen cells, as did a rat hybridoma (SH1.2.47) prepared from peptide-immunized rats. CHO cells transfected with a plasmid cDNA construct encoding murine MD-1, the target antigen for the antibodies in question, were also stained (in FACS) by the same antibodies. Patching and capping of the antigen(s) detected by any one of these sera abolished binding of all antibodies in subsequent FACS analysis, consistent with the hypothesis that they all detected the same antigen. In a final study to assess the possible involvement of MD-1 in regulation of cell activation for cytokine production following allostimulation, we found that all of the antibodies inhibited IL-2 and IFNgamma production, while enhancing IL-4 and IL-10 production, in mixed leukocyte reactions (MLR) in vitro.

Amino Acid Sequence↗

PubMed2001

Functional recovery after brain lesion--contralateral neuromodulation: an fMRI study.

Behavioral recovery takes place even after permanent damage to the entire brain region normally controlling sensorimotor hind limb function in the rat. In our study, 2 weeks after full behavioral recovery from an experimental unilateral permanent brain damage, the topographic representation of the previous paretic hindlimb was investigated by fMRI. The analysis showed that during electrical stimulation of the previously paretic hindlimb, two normally inactive brain regions were now being activated. One region was the non-damaged contralateral sensori-motor cortex and the other region was located lateral to the lesion. These results suggest that behavioral recovery can be explained by functional reorganization and neuromodulation of the brain.

Afferent Pathways↗

PubMed2001

BN-doped fullerenes: an NICS characterization.

Heterofullerenes C(58)(BN), C(54)(BN)(3), C(48)(BN)(6), and C(12)(BN)(24) and their hexaanions as well as the C(58)(BN) dimer have been investigated by ab initio calculations. On the basis of the computed nucleus independent chemical shifts (NICS) at the cage center and also at the center of individual rings, BN-doped fullerenes C(58)(BN), C(54)(BN)(3), and C(48)(BN)(6) are slightly more aromatic than C(60), whereas the corresponding hexaanions are significantly less aromatic than C(60)(6)(-). The predicted NICS values may be useful for the identification of the heterofullerenes through their endohedral (3)He NMR chemical shifts. Compared to C(60), the dimerization of C(58)(BN) is calculated to be more exothermic by 16 kcal/mol.

Journal Article↗

PubMed2001

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense.

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

Cloning, Molecular↗

PubMed2001

African origin of modern humans in East Asia: a tale of 12,000 Y chromosomes.

To test the hypotheses of modern human origin in East Asia, we sampled 12,127 male individuals from 163 populations and typed for three Y chromosome biallelic markers (YAP, M89, and M130). All the individuals carried a mutation at one of the three sites. These three mutations (YAP+, M89T, and M130T) coalesce to another mutation (M168T), which originated in Africa about 35,000 to 89,000 years ago. Therefore, the data do not support even a minimal in situ hominid contribution in the origin of anatomically modern humans in East Asia.

Africa↗

PubMed2001

Optimal design features of camelized human single-domain antibody libraries.

We have constructed a human V(H) library based on a camelized V(H) sequence. The library was constructed with complete randomization of 19 of the 23 CDR3 residues and was panned against two monoclonal antibody targets to generate V(H) sequences for determination of the antigen contact residue positions. Furthermore, the feasibility and desirability of introducing a disulfide bridge between CDR1 and CDR3 was investigated. Sequences derived from the library showed a bias toward the use of C-terminal CDR3 residues as antigen contact residues. Mass spectrometric analyses indicated that CDR1-CDR3 disulfide formation was universal. However, surface plasmon resonance and NMR data showed that the CDR3 constraint imposed by the disulfide bridge was not always desirable. Very high yields of soluble protein products and lack of protein aggregation, as demonstrated by the quality of the (1)H-(15)N HSQC spectra, indicated that the V(H) sequence for library construction was a good choice. These results should be useful in the design of V(H) libraries with optimal features.

Amino Acid Sequence↗

PubMed2001

DNA sequencing by capillary electrophoresis using mixtures of polyacrylamide and poly(N,N-dimethylacrylamide).

The possibility of using polymer mixtures with different chemical compositions as a DNA sequencing matrix by capillary electrophoresis (CE) has been exploited. Polyacrylamide (PAM, 2.5%, w/v) having a molecular mass of 2.2 x 10(6) has been mixed with poly(N,N-dimethylacrylamide) (PDMA) having molecular masses of 8000, 470000 and 2.1 x 10(6) at concentrations of 0.2, 0.5 and 1% (w/v). Unlike polymer mixtures of the same polymer with different molecular masses, the use of polymer mixtures with different chemical compositions encounters an incompatibility problem. It was found that the incompatibility increased with increasing PDMA molecular mass and PDMA concentration, which resulted in decreased efficiency in DNA sequencing. Also, the incompatibility had a more pronounced effect on the efficiency as the base number was increased. However, by choosing a low-molecular-mass PDMA of 8000 and a low concentration of 0.2% (w/v), the incompatibility of PAM and PDMA has been alleviated. At the same time, the advantage of using polymer mixtures revealed a higher efficiency for such a polymer mixture when compared with PAM. The mixture also endowed the separation medium with a dynamic coating ability. An efficiency of over 10 x 10(6) theoretical plates per meter has been achieved by using the bare capillaries without the additional chemical coating step.

Acrylamides↗