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Biomedical subjects

Y Zhai

Publications and source records attributed to Y Zhai.

At least 19 recordsLinked to original sources
PubMed2006

FK330, a novel inducible nitric oxide synthase inhibitor, prevents ischemia and reperfusion injury in rat liver transplantation.

Nitric oxide (NO), produced via inducible NO synthase (iNOS), is implicated in the pathophysiology of liver ischemia/reperfusion injury (IRI). We examined the effects of a novel iNOS inhibitor, FK330 (FR260330), in well-defined rat liver IRI models. In a model of liver cold ischemia followed by ex vivo reperfusion, treatment with FK330 improved portal venous flow, increased bile production and decreased hepatocellular damage. FK330 prevented IRI in rat model of 40-h cold ischemia followed by syngeneic orthotopic liver transplantation (OLT), as evidenced by: (1) increased OLT survival (from 20% to 80%); (2) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic pyruvic transaminase levels); (3) improved histological features of IRI; (4) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin/intracellular adhesion molecule 1, ED-1/CD3 cells and neutrophils; (5) depressed lymphocyte activation, as evidenced by expression of pro-inflammatory cytokine (TNF-alpha, IL-1beta, IL-6) and chemokine (IP-10, MCP-1, MIP-2) programs; (6) prevented hepatic apoptosis and down-regulated Bax/Bcl-2 ratio. Thus, by modulating leukocyte trafficking and cell activation patterns, treatment of rats with FK330, a specific iNOS inhibitor, prevented liver IRI. These results provide the rationale for novel therapeutic approaches to maximize organ donor pool through the safer use of liver grafts despite prolonged periods of cold ischemia.

Animals↗
PubMed2005

HO-1 upregulation suppresses type 1 IFN pathway in hepatic ischemia/reperfusion injury.

Upregulation of heme oxygenase (HO)-1, a heat shock protein 32, protects against hepatic ischemia/reperfusion (I/R) injury. Activation of "innate" toll-like receptor (TLR) 4 system triggers the I/R injury cascade. This study explores cytoprotective functions of HO-1 overexpression following exogenous administration of cobalt protoporphyrin (CoPP), and its relationship with the TLR4 pathway in a model of mouse partial hepatic warm I/R injury. CoPP treatment markedly improved hepatic function and histology, and suppressed pro-inflammatory cytokine elaboration profile, as compared with untreated controls. Although administration of CoPP did not affect intrahepatic TLR4, it downregulated IFN-inducible protein 10 (IP-10) expression. As IP-10 is the major product of type-1 IFN pathway downstream of TLR4, we then infused recombinant IFN-beta (rIFN-beta) directly into mouse livers. Interestingly, infusion of rIFN-beta upregulated hepatic IP-10 expression. In contrast, adjunctive CoPP treatment decreased IP-10 levels in mouse livers infused with rIFN-beta. Thus, CoPP-induced HO-1 upregulation suppresses type-1 IFN pathway downstream of TLR4 system in hepatic warm I/R injury model.

Animals↗
PubMed2001

Diverse mechanisms of beta-catenin deregulation in ovarian endometrioid adenocarcinomas.

Clinical and molecular findings suggest that the four major histological subtypes of ovarian carcinoma (serous, clear cell, mucinous, and endometrioid) likely represent distinct disease entities. Prior studies have shown that ovarian endometrioid adenocarcinomas (OEAs) often carry mutations in the CTNNB1 gene, which encodes beta-catenin, a critical component of the Wnt signaling pathway. However, the nature of other defects in the Wnt signaling pathway in ovarian carcinomas remains largely unknown. Thus, in 45 primary OEAs and two OEA-derived cell lines, we sought to comprehensively address the prevalence of and mechanisms underlying beta-catenin and Wnt pathway deregulation. CTNNB1 missense mutations were detected in 14 primary tumors. All mutations affected the NH(2)-terminal regulatory domain of beta-catenin, presumably rendering the mutant proteins resistant to degradation. Immunohistochemical studies revealed nuclear accumulation of beta-catenin in all but two tumors with CTNNB1 mutations. Two primary tumors lacking CTNNBI mutations showed strong nuclear immunoreactivity for beta-catenin. In one of the two tumors, biallelic inactivation of the APC gene was found. In the remaining 29 primary OEAs, unequivocal nuclear beta-catenin immunoreactivity was not observed, though a nonsense mutation in AXIN1 was observed in one tumor and a truncating frameshift mutation in AXIN2 was seen in another case. Both OEA-derived cell lines studied (TOV-112D and MDAH-2774) had elevated constitutive T-cell factor/lymphoid enhancer factor transcriptional activity. TOV-112D cells were shown to harbor mutant beta-catenin, whereas a missense AXIN1 sequence alteration was identified in MDAH-2774 cells. Collectively, our findings demonstrate frequent defects of the Wnt signaling pathway in a particular subtype of ovarian carcinomas, i.e., OEAs. Although mutations in the CTNNB1 gene are the most common mechanism of beta-catenin deregulation in OEAs, beta-catenin deregulation may also result from mutations in the APC, AXIN1, and AXIN2 genes.

Adult↗
PubMed2001

T cell subsets and in vitro immune regulation in "infectious" transplantation tolerance.

CD4-targeted mAb therapy results in permanent acceptance of cardiac allografts in rat recipients, in conjunction with features of the infectious tolerance pathway. Although CD4(+) T cells play a central role, the actual cellular and molecular tolerogenic mechanisms remain elusive. This study was designed to analyze in vitro alloimmune responses of T lymphocytes from CD4 mAb-treated engrafted hosts. Spleen, but not lymph node, cells lost proliferative response against donor alloantigen in MLR and suppressed test allograft rejection in adoptive transfer studies, suggesting compartmentalization of tolerogenic T cells in transplant recipients. A high dose of exogenous IL-2 restored the allogeneic response of tolerogenic T cells, indicating anergy as a putative mechanism. Vigorous proliferation of the tolerogenic T cells in in vivo MLR supports the existence of alloreactive lymphocytes in tolerogenic T cell repertoire and implies an active operational suppression mechanism. The tolerogenic splenocytes suppressed proliferation of naive splenocytes in vitro, consistent with their in vivo property of dominant immune regulation. Finally, CD45RC(+) but not CD45RC(-) T cells from tolerant hosts were hyporesponsive to alloantigen and suppressed the proliferation of normal T cells in the coculture assay. Thus, nondeletional, anergy-like regulatory mechanisms may operate via CD4(+)CD45RC(+) T cells in the infectious tolerance pathway in transplant recipients.

Animals↗
PubMed2001

Evidence of T cell clonality in the infectious tolerance pathway: implications toward identification of regulatory T cells.

We have shown that a rare population of regulatory CD4+ T cells plays a key role in the acquisition of infectious tolerance in rat sensitized recipients of cardiac allografts pretreated with nondepleting anti-CD4 mAb. This study was designed to analyze the TCR Vbeta expression patterns in this transplantation model. First, we used Vbeta-specific RT-PCR to show that there was no differential usage of TCR Vbeta genes by T cells mediating rejection or tolerance. Indeed, graft-infiltrating lymphocytes expressed most of the 22 known rat TCR Vbeta genes in both recipient groups, suggesting unrestricted TCR Vbeta repertoire in alloreactive T cells. Then, we applied CDR3 spectrotyping of TCR beta-chain to assess the clonality of T cells at different anatomic sites. CDR3 size restriction, indicative of the presence of T cell clones, was observed in graft-infiltrating lymphocytes but not in draining lymph nodes or spleen of tolerant hosts. Consisent with the clonal expansion, T cells in tolerated grafts exhibited the memory phenotype at a much higher percentage as compared with peripheral lymphoid organs. Moreover, in tolerated graft-infiltrating lymphocytes, the CD3 size restriction occurred in limited Vbeta gene families, with Vbeta8.1 and Vbeta18 most frequently detected. Hence, T cells at the graft site of tolerant recipients contain T cell clones expressing selective Vbeta genes. This phenotypic characteristics of the tolerogenic GILs may potentially be used as a novel marker to identify operational regulatory T cells in organ allograft recipients.

Animals↗
PubMed2001

Homologues of archaeal rhodopsins in plants, animals and fungi: structural and functional predications for a putative fungal chaperone protein.

The microbial rhodopsins (MR) are homologous to putative chaperone and retinal-binding proteins of fungi. These proteins comprise a coherent family that we have termed the MR family. We have used modeling techniques to predict the structure of one of the putative yeast chaperone proteins, YRO2, based on homology with bacteriorhodopsins (BR). Availability of the structure allowed depiction of conserved residues that are likely to be of functional significance. The results lead us to predict an extracellular protein folding function and a transmembrane proton transport pathway. We suggest that protein folding is energized by a novel mechanism involving the proton motive force. We further show that MR family proteins are distantly related to a family of fungal, animal and plant proteins that include the human lysosomal cystine transporter (LCT) of man (cystinosin), mutations in which cause cystinosis. Sequence and phylogenetic analyses of both the MR family and the LCT family are reported. Proteins in both families are of the same approximate size, exhibit seven putative transmembrane alpha-helical spanners (TMSs) and show limited sequence similarity. We show that the LCT family arose by an internal gene duplication event and that TMSs 1-3 are homologous to TMSs 5-7. Although the same could not be demonstrated statistically for MR family members, homology with the LCT family suggests (but does not prove) a common evolutionary pathway. Thus, TMSs 1-3 and 5-7 in both LCT and MR family members may share a common origin, accounting for their shared structural features.

Amino Acid Sequence↗
PubMed2001

Characterization and tissue expression of a novel human gene npdc1.

We report the molecular characterization of a novel human homologue of mouse npdc1 (neural proliferation, differentiation and control, 1) gene, designated human npdc1 (hnpdc1). hnpdc1 was identified by large-scale sequencing of fetal liver cDNA libraries and the full-length cDNA was obtained by PCR amplification. The hnpdc1 gene, which contains nine exons, was mapped to human chromosome 15. It encodes a polypeptide of 325 amino acids, which shows high homology (77% identity) to the mouse NPDC1. Sequence analysis has shown that hNPDC1 protein contains a putative signal peptide of 34 amino acids, a transmembrane segment, and a typical bipartite nuclear localization signal. Northern blot and dot blot hybridization indicates that, just like mnpdc1, hnpdc1 mRNA is strongly expressed in adult brain (especially in hippocampus, frontal lobe and temporal lobe) and about 1.82-fold higher in adult brain than that in fetal brain. Unlike mnpdc1, however, hnpdc1 contains two transcripts instead of only one (1.5 kb), and has high expression levels in prostate, pituitary gland, and mammary glands. These results support that hNPDC1 plays a role in the control of neural cell proliferation and differentiation, and suggest that it may be involved in the development of several secretion glands.

Amino Acid Sequence↗
PubMed2001

Homing of in vitro-generated donor antigen-reactive CD4+ T lymphocytes to renal allografts is alpha 4 beta 1 but not alpha L beta 2 integrin dependent.

The extravasation and sequestration of Ag-reactive T lymphocytes into vascularized organ allografts depend on a cascade of complex interactions among circulating lymphocytes, endothelial cells, and extracellular matrix proteins. Ag-activated donor-specific CD4 T cells are major initiators and effectors in the allograft rejection response. Interfering with the intragraft homing of activated CD4 T cells may represent a novel therapeutic approach in transplant recipients. We have developed a FACS-based short-term homing assay that allows tracing in vitro-generated Ag-reactive CD4 T cells after adoptive transfer in test rat recipients. Allospecific cell lines were preincubated with anti-alpha(4)beta(1) or anti-alpha(L)beta(2) mAb, because of enhanced expression of both integrin receptors after alloactivation. The pretreated Lewis(BN) lymphocytes were carboxyfluorescein diacetate succinimidyl ester labeled and adoptively transferred into Lewis rat recipients of Brown Norway kidney allografts. The injection of equal numbers of PKH-26-labeled untreated cells allowed quantitative comparison of both populations in the same animal. Ex vivo treatment with anti-alpha(4)beta(1) mAb diminished intragraft infiltration of adoptively transferred T cells by 85% in a donor-specific fashion. In contrast, treatment with anti-alpha(L)beta(2) mAb did not affect intragraft cell sequestration. Hence, blocking alpha(4)beta(1) integrin interactions represents a novel strategy in preventing local intragraft recruitment of Ag-reactive CD4 T cells in transplant recipients.

Adoptive Transfer↗
PubMed2001

Cellular expression of retinal dehydrogenase types 1 and 2: effects of vitamin A status on testis mRNA.

We examined expression of retinal dehydrogenase (RALDH) types 1 and 2 in liver and lung, and the effect of vitamin A status on testis expression by in situ hybridization. Liver expressed RALDH1 and RALDH2 only in stellate cells and hepatocytes, respectively. Lung expressed RALDH1 and RALDH2 throughout the epithelia of the airways, from the principal bronchi to the respiratory bronchiole. Vitamin A-sufficient rats expressed RALDH1 in spermatocytes, with less intense expression in spermatogonia and spermatids, and expressed RALDH2 in interstitial cells, spermatogonia, and spermatocytes. Neither Sertoli nor peritubular cells showed detectable RALDH1 or RALDH2 mRNA. Vitamin A deficiency produced a sevenfold increase in RALDH1 and a 70-fold decrease in RALDH2 mRNA in testis. In each case, the net change reflected extensive loss of germ cells, increased intensity of expression in residual germ cells, and expression in Sertoli and peritubular cells. Low-dose RA relatively early during vitamin A depletion supported spermatogenesis and affected expression of both RALDHs, but did not reinstate "vitamin A normal" expression patterns. These results show that: RALDH1 and RALDH2 have distinct mRNA expression patterns in multiple cell types in three vitamin A target tissues; RALDH expression occurs in cell types that express cellular retinol-binding protein and retinol dehydrogenase isozymes (except stellate cells, for which retinol dehydrogenase expression remains unknown); vitamin A deficiency and RA supplementation affects the loci and intensity of RALDH mRNAs in testis; and low-dose RA does not substitute completely for retinol. Overall, these data provide insight into the unique functions of RALDH1 and RALDH2 in retinoid metabolism.

Aldehyde Oxidoreductases↗
PubMed2001

Gene expression profiling in human fetal liver and identification of tissue- and developmental-stage-specific genes through compiled expression profiles and efficient cloning of full-length cDNAs.

Fetal liver intriguingly consists of hepatic parenchymal cells and hematopoietic stem/progenitor cells. Human fetal liver aged 22 wk of gestation (HFL22w) corresponds to the turning point between immigration and emigration of the hematopoietic system. To gain further molecular insight into its developmental and functional characteristics, HFL22w was studied by generating expressed sequence tags (ESTs) and by analyzing the compiled expression profiles of liver at different developmental stages. A total of 13,077 ESTs were sequenced from a 3'-directed cDNA library of HFL22w, and classified as follows: 5819 (44.5%) matched to known genes; 5460 (41.8%) exhibited no significant homology to known genes; and the remaining 1798 (13.7%) were genomic sequences of unknown function, mitochondrial genomic sequences, or repetitive sequences. Integration of ESTs of known human genes generated a profile including 1660 genes that could be divided into 15 gene categories according to their functions. Genes related to general housekeeping, ESTs associated with hematopoiesis, and liver-specific genes were highly expressed. Genes for signal transduction and those associated with diseases, abnormalities, or transcription regulation were also noticeably active. By comparing the expression profiles, we identified six gene groups that were associated with different developmental stages of human fetal liver, tumorigenesis, different physiological functions of Itoh cells against the other types of hepatic cells, and fetal hematopoiesis. The gene expression profile therefore reflected the unique functional characteristics of HFL22w remarkably. Meanwhile, 110 full-length cDNAs of novel genes were cloned and sequenced. These novel genes might contribute to our understanding of the unique functional characteristics of the human fetal liver at 22 wk.

Cloning, Molecular↗
PubMed2001

Molecular identification and characterization of the platelet ADP receptor targeted by thienopyridine antithrombotic drugs.

ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.

Adenosine Diphosphate↗
PubMed2001

A web-based program for the prediction of average hydropathy, average amphipathicity and average similarity of multiply aligned homologous proteins.

We designed a web-based program, AveHAS, to determine and plot the average hydropathy, average amphipathicity and average similarity for a clustal X-derived multiple alignment of homologous protein sequences. This method is based on the TREEMOMENT and Hydro programs. It has a user-friendly interface, a convenient input format and an improved algorithm.

Algorithms↗
PubMed2001

A web-based program (WHAT) for the simultaneous prediction of hydropathy, amphipathicity, secondary structure and transmembrane topology for a single protein sequence.

We designed a web-based program, WHAT, which uses a sliding window to determine and plot the hydropathy, amphipathicity, secondary structure and transmembrane topology along the length of any protein sequence. This method is based on programs designed by us for hydropathy and amphipathicity but on JNET and MEMSAT for secondary structure and transmembrane topology predictions, respectively. It has a user-friendly interface and a convenient input format. It is available at our website http://www.biology.ucsd.edu/~yzhai/biotools.html.

Internet↗
PubMed2001

[A clinical analysis of 798 cases of M protein positive patients].

OBJECTIVE: To improve the understanding of the diseases with positive M protein by analyzing 798 cases, so as to reduce the failure and error of diagnosis. METHODS: The relations of diseases with the types of the disease and age and sex of the patients were analyzed. The presenting symptoms of multiple myeloma(MM) and non-MM were compared. The difference of Ig content among each type of MM and the characteristics of M protein in non-MM were also studied. RESULTS: In 798 cases with positive M protein, there were 648 cases of MM (81.2%) and 34 cases of Waldenstrom's macroglobulinemia (WM) (4.3%). The remaining 116 cases (14.5%) were benign or secondary monoclonal gammopathies. The average age of the patients was 58.3 years, and there was no difference of disease incidence between men and women. There was a marked difference between MM and non-MM in the presenting symptoms which were mainly lumbago and bone pain in MM but fever, weight loss and fatigue in non-MM. 648 cases of MM included 290 cases of IgG type (44.8%), 143 of IgA type (22.1%), 138 of light chain type (21.3%), 45 of IgD type (6.9%), 30 of non-secretion type (4.6%) and 2 of double-clonal type (0.3%). There was no marked distinction between kappa and lambda chain except in IgD type. The average M protein content of 116 cases of benign or secondary monoclonal gammopathies was (11.3 +/- 6.5) g/L. 55 of the 116 cases were monoclonal gammopathy of undetermined significance (MGUS), which accounted for 6.9% of all the cases. 35 cases were non-Hodgkin's lymphoma (NHL) (4.4%). CONCLUSION: M protein was mainly discovered in MM; it may also be discovered in MGUS, NHL, WM and other diseases. It is essential to have a better understanding of these diseases.

Adult↗
PubMed2001

[Expression of D-animo acid oxidase gene in K562 cells and the cytotoxicity of D-alanine to the cells].

OBJECTIVE: To explore the feasibility of expression of R. gracilis D-amino acid oxidase(DAAO) gene in leukemia cell line K562 and the cytotoxicity of D-Alanine to the cells. METHODS: DAAO cDNA was cloned into retroviral vector pLSN and pLDAAOSN was generated. The vector was then packaged with phiNXA and the virus titer was measured with NIH3T3 cells. Leukemia cell line K562 was infected with the viral supernatant. The positive clones were obtained by G418 selection and named KDAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in KDAAO. KDAAO was treated with different concentrations of D-Alanine. RESULTS: pLDAAOSN was confirmed containing the full-length of DAAO cDNA. Infectious titer generated by the packaging cells was 5.2 x 10(6) cfu/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in KDAAO and expression of DAAO mRNA. Preliminary observation suggested that D-Ala could effectively kill KDAAO. CONCLUSION: DAAO/D-Ala suicide gene system might be useful in cancer gene therapy.

Alanine↗
PubMed2000

The amoebapore superfamily.

Amoebapores, synthesized by human protozoan parasites, form ion channels in target cells and artificial lipid membranes. The major pathogenic effect of these proteins is due to their cytolytic capability which results in target cell death. They comprise a coherent family and are homologous to other proteins and protein domains found in eight families. These families include in addition to the amoebapores (1) the saposins, (2) the NK-lysins and granulysins, (3) the pulmonary surfactant proteins B, (4) the acid sphingomyelinases, (5) acyloxyacyl hydrolases and (6) the aspartic proteases. These amoebapore homologues have many properties in common including membrane binding and stability. We note for the first time that a new protein, countin, from the cellular slime mold, Dictyostelium discoideum, comprises the eighth family within this superfamily. All currently sequenced members of these eight families are identified, and the structural, functional and phylogenetic properties of these proteins are discussed.

Amino Acid Sequence↗
PubMed2000

Regulatory cells potentiate the efficacy of IL-4 gene transfer by up-regulating Th2-dependent expression of protective molecules in the infectious tolerance pathway in transplant recipients.

We have previously shown that the tolerant state in allograft recipients can be maintained and perpetuated by an "infectious" T cell-dependent regulatory mechanism. Hence, 1) treatment of LEW rats with RIB-5/2, a CD4 nondepleting mAb, produces indefinite survival of LBNF1 cardiac allografts; 2) donor-specific tolerance can be then transferred by spleen cells into new cohorts of test allograft recipients; and 3) putative regulatory CD4+ Th2-like cells are instrumental in this tolerance model. We now report on studies aimed at exposing mechanisms underlying the infectious tolerance pathway, with emphasis on the interactions between intragraft adenovirus-IL-4 gene transfer and systemic infusion of regulatory cells from tolerant hosts. Unlike individual treatment regimens, adjunctive therapy with adenovirus-IL-4 and suboptimal doses of regulatory spleen cells was strongly synergistic and extended donor-type test cardiac allograft survival to about 2 mo. RT-PCR-based expression of intragraft mRNA coding for IL-2 and IFN-gamma remained depressed, whereas that of IL-4 and IL-10 reciprocally increased selectively in the combined treatment group, data supported by ELISA studies. In parallel, only adjunctive treatment triggered intragraft induction of molecules with anti-oxidant (HO-1) and anti-apoptotic (Bcl-xL/Bag-1) but not with pro-apoptotic (CPP-32) functions, both in the early and late posttransplant phases. Hence, systemic infusion of regulatory cells potentiates the effects of local adenovirus-IL-4 gene transfer in transplant recipients. Th2-driven up-regulation of protective molecule programs at the graft site, such as of anti-oxidant HO-1 and/or anti-apoptotic Bcl-xL and Bag-1, may contribute, at least in part, to the maintenance of the infectious tolerance pathway in transplant recipients.

Adenoviridae↗