Biomedical subjects

X Zhang

Publications and source records attributed to X Zhang.

At least 19 recordsLinked to original sources

PubMed2000

Distinct role of follicular dendritic cells and T cells in the proliferation, differentiation, and apoptosis of a centroblast cell line, L3055.

Germinal center (GC) B cells undergo complex interactions with follicular dendritic cells (FDC) and T cells in the course of differentiation into memory B and plasma cells. To delineate the individual roles of FDC and T cells at each stage of GC B cell differentiation at the clonal level and to analyze the signals involved, we adopted a unique experimental model using an FDC line, HK, and a lymphoma cell line, L3055, that resembles centroblasts. A detailed phenotypic analysis revealed L3055 cells to be a clonal population originating from the GC. Like freshly isolated centroblasts, L3055 cells underwent spontaneous apoptosis when cultured in the absence of fresh FDC or HK cells. L3055 cells proliferated continuously in the presence of HK cells, while they differentiated into a population with the phenotype of centrocytes after stimulation with CD40 ligand (CD40L) and IL-4. The CD40L-stimulated L3055 cells underwent CD95-mediated apoptosis, which was reminiscent of the feature of CD40L-stimulated tonsillar GC B cells. In contrast to HK cells that did not protect L3055 cells from anti-Ig killing, CD40L plus IL-2, IL-4, and IL-10 prevented anti-Ig-induced apoptosis. These experimental results demonstrate a distinct function of FDC and activated T cells, in that FDC provide signals for rapid proliferation of centroblasts, whereas T cells confer signals for differentiation of centroblasts into centrocytes and resistance to B cell receptor-mediated apoptosis. T cells collaborate with FDC in the protection and expansion of the Ag-specific GC B cells.

Adolescent↗

PubMed2000

Decreased protein levels of nicotinic receptor subunits in the hippocampus and temporal cortex of patients with Alzheimer's disease.

Deficits of cortical nicotinic acetylcholine receptors (nAChRs) have been observed in Alzheimer's disease (AD) by receptor binding assays. Little is known about the receptor subunit specificity influenced by AD, and it might be of importance for therapeutic strategies. In the present study, the protein levels of nAChR alpha3, alpha4, alpha7, and beta2 subunits were investigated using western blot analysis on postmortem brains of patients with AD and age-matched controls. The results showed that in human postmortem brain samples, bands with molecular masses of 52, 42, and 50 kDa were detected by anti-alpha4, anti-alpha7, and anti-beta2 antibodies, respectively. When anti-alpha3 antibody was used, one major band of 49 kDa and two minor bands of 70 and 38 kDa were detected. In AD patients, as compared with age-matched controls, the alpha4 subunit was reduced significantly by approximately 35 and 47% in the hippocampus and temporal cortex, respectively. A significant reduction of 25% in the alpha3 subunit was also observed in the hippocampus and a 29% reduction in the temporal cortex. For the alpha7 subunit, the protein level was reduced significantly by 36% in the hippocampus of AD patients, but no significant change was detected in the temporal cortex. In neither the hippocampus nor the temporal cortex was a significant difference observed in the beta2 subunit between AD patients and controls. These results reveal brain region-specific changes in the protein levels of the nAChR alpha3, alpha4, and alpha7 subunits in AD.

Aged↗

PubMed1999

Recruitment of the protein tyrosine phosphatase CSW by DOS is an essential step during signaling by the sevenless receptor tyrosine kinase.

The pleckstrin homology (PH) domain-containing protein Daughter of Sevenless (DOS) is an essential component of the Sevenless receptor tyrosine kinase (SEV) signaling cascade, which specifies R7 photoreceptor development in the Drosophila eye. Previous results have suggested that DOS becomes tyrosine phosphorylated during SEV signaling and collaborates with the protein tyrosine phosphatase CSW. We have investigated this possibility by identifying tyrosine residues 801 and 854 of DOS as the phosphorylated binding sites for the CSW SH2 domains. We show that these sites become phosphorylated in response to SEV activation and that phosphorylation of both sites is required to allow CSW to bind DOS. Mutant DOS proteins in which either Y801 or Y854 of DOS has been changed to phenylalanine are unable to function during signaling by SEV and other receptor tyrosine kinases. In contrast, we find that a mutant DOS protein in which all tyrosine phosphorylation sites except Y801 and Y854 have been removed is able effectively to provide DOS function during SEV signaling and to rescue the lethality associated with dos loss-of-function mutations. These results indicate that a primary role for DOS during signaling by SEV and other receptor tyrosine kinases is to become phosphorylated at Y801 and Y854 and then recruit CSW.

Amino Acid Sequence↗

PubMed1999

The nucleocapsid protein of coronavirus mouse hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein A1 in vitro and in vivo.

The nucleocapsid (N) protein of mouse hepatitis virus (MHV) and the cellular heterogeneous nuclear ribonucleoprotein A1 (hnRNP-A1) are RNA-binding proteins, binding to the leader RNA and the intergenic sequence of MHV negative-strand template RNAs, respectively. Previous studies have suggested a role for both N and hnRNP-A1 proteins in MHV RNA synthesis. However, it is not known whether the two proteins can interact with each other. Here we employed a series of methods to determine their interactions both in vitro and in vivo. Both N and hnRNP-A1 genes were cloned and expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins, and their interactions were determined with a GST-binding assay. Results showed that N protein directly and specifically interacted with hnRNP-A1 in vitro. To dissect the protein-binding domain on the N protein, 15 deletion constructs were made by PCR and expressed as GST fusion proteins. Two hnRNP-A1-binding sites were identified on N protein: site A is located at amino acids 1 to 292 and site B at amino acids 392 to 455. In addition, we found that N protein interacted with itself and that the self-interacting domain coincided with site A but not with site B. Using a fluorescence double-staining technique, we showed that N protein colocalized with hnRNP-A1 in the cytoplasm, particularly in the perinuclear region, of MHV-infected cells, where viral RNA replication/transcription occurs. The N protein and hnRNP-A1 were coimmunoprecipitated from the lysates of MHV-infected cells either by an N- or by an hnRNP-A1-specific monoclonal antibody, indicating a physical interaction between N and hnRNP-A1 proteins. Furthermore, using the yeast two-hybrid system, we showed that N protein interacted with hnRNP-A1 in vivo. These results thus establish that MHV N protein interacts with hnRNP-A1 both in vitro and in vivo.

Animals↗

PubMed1999

Development of New Chiral P,N Ligands and Their Application in the Cu-Catalyzed Enantioselective Conjugate Addition of Diethylzinc to Enones.

High enantioselectivity (up to 98 % ee) has been achieved for the conjugate addition of diethylzinc to acyclic enones utilizing Cu(I) complexes of the novel chiral P,N ligands 1. The high enantioselectivities are best achieved using [Cu(OTf)](2) small middle dotC(6)H(6) as the copper catalyst precursor in nonpolar solvents such as toluene or Cl(CH(2))(2)Cl. R=H, CH(3); Tf=F(3)CSO(2).

Journal Article↗

PubMed1999

Cloning and mapping of a novel human serum/glucocorticoid regulated kinase-like gene, SGKL, to chromosome 8q12.3-q13.1.

Serum/glucocorticoid regulated kinase (sgk) belongs to a newly emerging subfamily of the serine/threonine protein kinase family. Although human SGK shares 98% amino acid identity with rat sgk, their expression levels are regulated differently, which indicates the existence of other SGKs in humans. In this paper, we reported the cloning of human SGKL, which encodes a protein sharing 67 and 66% amino acid identity with rat sgk and human SGK, respectively. A 4.4-kb transcript of human SGKL was detected in 16 human tissues examined and was found to be most abundant in lung. By radiation hybrid mapping, the SGKL gene was located to human chromosome 8q12. 1-q13.1 between markers D8S510 and D8S1797.

Adrenocorticotropic Hormone↗

PubMed1999

Inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity by dopachrome analogs.

A macrophage migration inhibitory factor (MIF) dopachrome tautomerization assay was employed for identification of MIF inhibitors. One group of dopachrome analogs was identified which inhibits MIF tautomerase activity. In particular, the analogs with a leaving group at beta position displayed activity at a concentration of tenfold less than that of the MIF-substrate. These findings could lead to a better understanding of MIF biological activities and the development of agents for the treatment of MIF related diseases.

Indolequinones↗

PubMed1999

NK cell-deficient mice develop a Th1-like response but fail to mount an efficient antigen-specific IgG2a antibody response.

NK cells have been shown to play a role in the modulation of B cell differentiation and Ab production. Using a novel murine model of NK cell deficiency, we analyzed the in vivo role of NK cells in the regulation of Ag-specific Ab production. After immunization with OVA or keyhole limpet hemocyanin in CFA, NK cell-deficient (NK-T+) mice developed an efficient Th1 response and produced significant levels of IFN-gamma but displayed markedly reduced or absent Ag-specific IgG2a production. There were no differences in the levels of Ag-specific IgG, IgG1, and IgG2b between NK-T+ and NK+T+ mice. Furthermore, NK cell-reconstituted, NK+T+ (tgepsilon26Y) mice produced significant amounts of Ag-specific IgG2a after immunization with OVA. These results indicate that NK cells are involved in the induction of Ag-specific IgG2a production in vivo. Moreover, they also demonstrate that the lack of Ag-specific IgG2a Ab production in NK-T+ mice is not associated with the impaired Th1 response and IFN-gamma production.

Animals↗

PubMed1999

Expression of the simian virus 40 large tumor antigen (Tag) and formation of Tag-p53 and Tag-pRb complexes in human brain tumors.

BACKGROUND: The presence of simian virus 40 (SV40) in human brain tumors remains a controversial issue. Even if SV40 does exist in brain tumors, the questions of whether it is associated with brain tumorigenesis and by what mechanisms are unknown. METHODS: SV40 large tumor antigen (Tag) was investigated by immunoprecipitation, silver staining, and Western blot analysis in 65 brain tumor cases and 8 cases of normal brain tissue. Tag-p53 and Tag-pRb complexes were screened by immunoprecipitation and Western blot analysis in 18 and 15 Tag positive tumor tissues, respectively. RESULTS: Tag was found in all 8 cases of ependymoma and 2 cases of choroid plexus papilloma, 90% of pituitary adenoma cases (9 of 10), 73% of astrocytoma cases (11 of 15), 70% of meningioma cases (7 of 10), 50% of glioblastoma multiforme cases (4 of 8), and 33% of medulloblastoma cases (2 of 6). Five oligodendroglioma cases, 1 pineocytoma case, and 8 cases of normal brain tissue were negative for Tag. The Tag-p53 complex was detected in all 18 Tag positive tumors tested and the Tag-pRb complex was detected in all 15 Tag positive tumors tested. CONCLUSIONS: SV40 Tag not only is expressed in brain tumors; it also can form specific complexes with tumor suppressors p53 and pRb. SV40 is correlated with brain tumorigenesis. The inactivation of p53 and pRb due to the formation of Tag-p53 and Tag-pRb complexes possibly is a significant mechanism in the etiopathogenesis of brain tumors.

Adolescent↗

PubMed1999

Formation of a ribonucleoprotein complex of mouse hepatitis virus involving heterogeneous nuclear ribonucleoprotein A1 and transcription-regulatory elements of viral RNA.

The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) specifically binds to two transcription-regulatory elements, i.e., the leader and intergenic sequence, of the negative-strand (template-strand) RNA of mouse hepatitis virus (MHV) and may play a role in viral RNA transcription. Previous studies based on the defective-interfering RNAs of MHV suggested that these two RNA elements may interact with each other during transcription, although they do not have complementary sequences. In this study, we showed by an in vitro reconstitution assay that hnRNP A1 could mediate the formation of an RNP complex involving these two RNA elements. Both the RNA-binding domains and protein-interacting domain of hnRNP A1 contributed to the efficient formation of the RNP complex; however, the presence of the two RNA-binding domains alone, without the protein-interacting domain, also resulted in some RNP formation. Omission of hnRNP A1 in the reconstitution reaction abolished the RNP formation, and mutations of the IG sequences significantly inhibited the RNP formation. These findings suggest that the two cis-acting transcription-regulatory sequences of MHV RNA can interact with each other through the formation of an RNP complex involving a cellular protein hnRNP A1. This RNP complex may participate in MHV RNA transcription.

Animals↗

PubMed1999

Independent optimization of capillary electrophoresis separation and native fluorescence detection conditions for indolamine and catecholamine measurements.

Separation conditions in capillary electrophoresis with native fluorescence detection often represent a compromise in terms of the separation and detection figures of merit. As both the separation and fluorescence properties greatly depend on pH, the ability to independently optimize pH in the separation capillary and the detection region can improve many complex separations. When using a sheath flow cell, the pH at the detection zone can be adjusted independently of the electrophoresis buffer pH. Using capillary electrophoresis with 257-nm excitation and native fluorescence detection, more than an order of magnitude improvement in the limits of detection for dopamine (from 1400 to 120 nM) and epinephrine (from 850 to 60 nM) is achieved by maintaining the basic separation conditions and an acidified sheath buffer. The detection of dopamine in an individual Aplysia californica cerebral ganglion neuron is demonstrated.

Animals↗

PubMed1999

Differential effect of retinoic acid on growth regulation by phorbol ester in human cancer cell lines.

Phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and all-trans-retinoic acid (trans-RA) are potent regulators of growth of cancer cells. In this study, we investigated the effect of TPA and trans-RA alone or their combination on proliferation of human breast cancer ZR75-1 and T47D and lung cancer H460 and H292 cell lines. trans-RA caused various degrees of growth inhibition of these cell lines. However, TPA showed inhibition of proliferation of H460 and H292 cells and induction of ZR75-1 cell growth. Although trans-RA did not significantly regulate the growth inhibitory effect of TPA, it completely prevented its growth stimulating function. The divergent effects of TPA were associated with specific disruption of cell cycle events, an induction of G(0)/G(1) arrest in H460 and H292 cells and inhibition of G(0)/G(1) arrest with increase of S phase in ZR75-1 cells. Induction of G(0)/G(1) arrest was accompanied by induction of p21(WAF1) and ERK activity, whereas inhibition of G(0)/G(1) arrest was associated with enhanced activity of JNK and AP-1 but not ERK. trans-RA did not affect TPA-induced p21(WAF1) expression. However, it inhibited TPA-induced AP-1 activity in ZR75-1 cells and the constitutive AP-1 activity in H460 and H292 cells. Thus, trans-RA modulates TPA activity through its interaction through TPA-induced JNK/AP-1 pathway but not TPA-induced ERK/p21(WAF1) pathway.

Breast Neoplasms↗

PubMed1999

High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H.

Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.

Animals↗

PubMed1999

Mouse suppressor of fused is a negative regulator of sonic hedgehog signaling and alters the subcellular distribution of Gli1.

The Hedgehog (Hh) signaling pathway has critical functions during embryogenesis of both invertebrate and vertebrate species [1]; defects in this pathway in humans can cause developmental disorders as well as neoplasia [2]. Although the Gli1, Gli2, and Gli3 zinc finger proteins are known to be effectors of Hh signaling in vertebrates, the mechanisms regulating activity of these transcription factors remain poorly understood [3] [4]. In Drosophila, activity of the Gli homolog Cubitus interruptus (Ci) is likely to be modulated by its interaction with a cytoplasmic complex containing several other proteins [5] [6], including Costal2, Fused (Fu), and Suppressor of fused (Su(fu)), the last of which has been shown to interact directly with Ci [7]. We have cloned mouse Suppressor of fused (mSu(fu)) and detected its 4.5 kb transcript throughout embryogenesis and in several adult tissues. In cultured cells, mSu(fu) overexpression inhibited transcriptional activation mediated by Sonic hedgehog (Shh), Gli1 and Gli2. Co-immunoprecipitation of epitope-tagged proteins indicated that mSu(fu) interacts with Gli1, Gli2, and Gli3, and that the inhibitory effects of mSu(fu) on Gli1's transcriptional activity were mediated through interactions with both amino- and carboxy-terminal regions of Gli1. Gli1 was localized primarily to the nucleus of both HeLa cells and the Shh-responsive cell line MNS-70; co-expression with mSu(fu) resulted in a striking increase in cytoplasmic Gli1 immunostaining. Our findings indicate that mSu(fu) can function as a negative regulator of Shh signaling and suggest that this effect is mediated by interaction with Gli transcription factors.

Amino Acid Sequence↗

PubMed1999

Molecular cloning of the cDNA and promoter sequences for the mouse sodium-hydrogen exchanger regulatory factor.

The Na/H exchanger regulatory factor (NHE-RF) was first identified as a co-factor for cAMP dependent protein kinase regulation of the rabbit epithelial Na/H exchanger. Subsequently, this protein which contains two PDZ motifs, was shown to interact with multiple cellular targets. To understand more fully the function of NHE-RF and its regulation, we have cloned the full-length cDNA for mouse NHE-RF and a portion of the mouse gene containing the promoter elements. NHE-RF cDNA, isolated from a mouse kidney cDNA library, predicted a polypeptide of 356 amino acids that shares striking sequence conservation within the two PDZ domains and in-vitro phosphorylation sites with the human and rat homologs. The nucleotide sequence 5' of the transcription start site, identified by primer extension analysis, was highly 'GC' rich and lacked canonical TATA or CAAT sequences. Using a luciferase reporter construct, deletion analyses localized the critical segment for gene expression in mouse medullary thick ascending limb cells to 114 bp 5' of the transcription start site. Although NHE-RF has been recently identified as an estrogen-inducible gene, the lack of an estrogen-response element in the mouse NHE-RF 5'-non-coding-sequence and the inability to demonstrate estrogen stimulation of reporter gene expression in MCF-7 cells suggests a non-conventional or indirect mechanism for NHE-RF regulation by estrogen.

Amino Acid Sequence↗

PubMed1999

Ultra-short columns for low-pressure ion chromatography.

Since the development of low-pressure ion chromatography (LPIC), many inorganic cations and anions as well as organic acids could be analyzed at a low-pressure of 1.96 x 10(5)-2.94 x 10(5) Pa. And the ultra-short columns took the place of the common long chromatographic columns. Furthermore, the ultra-short columns of LPIC not only reduced the system pressure appreciably, but also achieved high sensitivity and precision. In order to assess the characteristics of the super-short columns, much analysis, i.e., the analysis of alkali metals, alkaline earth metals, transition-metals, rare-earth elements, inorganic anions and organic acids as well as the common amino acids, were conducted. Moreover, excellent results were obtained from the LPIC columns.

Chromatography, Liquid↗

PubMed1999

Inhibition of infection of incoming HIV-1 virus by RNA-cleaving DNA enzyme.

Nine different DNA enzymes (DzV3-n, n=1-9) targeting the V3 loop region of HIV-1 HXB2 were synthesized. One of those, DzV3-9, efficiently cleaved the target in the conserved sequence in the RNA transcript in vitro. DzV3-9 was stable in the cells and inhibited replication of both NL432 and SF162 strains in U87 cells expressing CD4 and co-receptors. The inhibitory effect of DNAzyme on incoming HIV-1 was also demonstrated with pseudotype virions generated by NL432-based luciferase reporter genes. Thus, an efficient, stable DNAzyme against a functionally important region of HIV-1 was identified, and it may be useful for prevention of HIV-1 infection.

Antiviral Agents↗

PubMed1999

Regulation of a cell type-specific silencer in the human interleukin-3 gene promoter by the transcription factor YY1 and an AP2 sequence-recognizing factor.

Negative regulation of cytokine gene transcription is an important mechanism in maintaining homeostasis of immune function. In this study, we characterized a silencer element in the human interleukin-3 gene promoter that is responsible for the cell-specific expression of interleukin-3. This silencer activity was proposed to be mediated by an unidentified nuclear inhibitory protein (NIP). In this study, we have identified two nuclear factors that are responsible for the silencer activity in T cells. The NIP element forms four specific DNA-protein complexes (designated as complexes A-D) with the Jurkat nuclear proteins. Complex A contains a nuclear protein that shares DNA-binding specificity with the transcription factor AP2 (designated as an AP2 sequence-recognizing factor (ASRF)). Formation of this ASRF complex is required for the NIP silencer function, as mutation of the ASRF-binding site abrogated the silencer activity. Complex B contains the nuclear factor YY1 (Yin-Yang 1), whose function is to down-regulate ASRF activity in the silencer. YY1 activity is supported by data from mutation and cotransfection analyses. Complexes C and D are formed by nonspecific binding proteins and do not express any regulatory activity in the NIP element. These data indicate that a cell type-specific silencer activity might be determined by a unique profile of ubiquitous transcription factors.

DNA-Binding Proteins↗