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Biomedical subjects

X Zhan

Publications and source records attributed to X Zhan.

At least 37 records · Page 2Linked to original sources
PubMed2000

In vitro and in vivo characterization of a murine cytomegalovirus with a transposon insertional mutation at open reading frame M43.

We have recently generated a pool of murine cytomegalovirus (MCMV) mutants by using a Tn3-based transposon mutagenesis approach. In this study, one of the MCMV mutants, RvM43, which contained the transposon inserted in open reading frame M43, was characterized. Our results provide the first direct evidence to suggest that M43 is not essential for viral replication in vitro in NIH 3T3 cells. Moreover, RvM43 exhibited a titer similar to that of the wild-type virus in the lungs, livers, spleens, and kidneys of both BALB/c and SCID mice and was as virulent as the wild-type virus in killing SCID mice that had been intraperitoneally infected with the viruses. In contrast, titers of the mutant virus in the salivary glands of the infected animals at 21 days postinfection were significantly (100 to 1,000-fold) lower than those of the wild-type virus and a rescued virus that restored the M43 region and its expression. Thus, M43 appears to be not essential for viral growth in vivo in the lungs, livers, spleens, and kidneys of infected animals and is also dispensable for virulence in killing SCID mice. Moreover, our results suggest that M43 is an MCMV determinant for growth in the salivary glands. Studies of viral genes required for replication in the salivary glands are important in understanding the mechanism of viral tropism for the salivary glands and shedding in saliva, which is believed to be one of the major routes of CMV transmission among healthy human populations.

3T3 Cells↗
PubMed2000

Murine cytomegalovirus containing a mutation at open reading frame M37 is severely attenuated in growth and virulence in vivo.

A pool of murine cytomegalovirus (MCMV) mutants was generated by using a Tn3-based transposon mutagenesis procedure. One of the mutants, RvM37, which contained the transposon sequence at open reading frame M37, was characterized both in tissue culture and in immunocompetent BALB/c and immunodeficient SCID mice. Our results provide the first direct evidence to suggest that M37 is not essential for viral replication in vitro in NIH 3T3 cells. Compared to the wild-type strain and a rescued virus that restored the M37 region, the viral mutant was severely attenuated in growth in both BALB/c and SCID mice after intraperitoneal infection. Specifically, titers of the Smith strain and rescued virus in the salivary glands, lungs, spleens, livers, and kidneys of the SCID mice at 21 days postinfection were about 5 x 10(5), 2 x 10(5), 5 x 10(4), 5 x 10(3), and 1 x 10(4) PFU/ml of organ homogenate, respectively; in contrast, titers of RvM37 in these organs were less than 10(2) PFU/ml of organ homogenate. Moreover, the virulence of the mutant virus appeared to be significantly attenuated because none of the SCID mice infected with RvM37 had died by 120 days postinfection, while all animals infected with the wild-type and rescued viruses had died by 26 days postinfection. Our results suggest that M37 probably encodes a virulence factor and is required for MCMV virulence in SCID mice and for optimal viral growth in vivo.

3T3 Cells↗
PubMed2000

Regulation of ciliary beat frequency by the nitric oxide-cyclic guanosine monophosphate signaling pathway in rat airway epithelial cells.

Nitric oxide (NO) upregulates ciliary beat frequency (CBF). The present study evaluates mechanisms of the NO-cyclic guanosine monophosphate (cGMP) pathway regulation of CBF. Rat tracheal explants were loaded with 4,5-diaminofluorescein diacetate for the demonstration of NO production by ciliated epithelial cells after L-arginine (L-Arg) stimulation. CBF was measured using phase contrast microscopy and videotape analysis. The roles of NO, soluble guanylate cyclase (sGC), cGMP-dependent protein kinase (PK) G, and phosphodiesterase (PDE) V in regulation of CBF were evaluated. NO synthase (NOS) was activated with L-Arg or inhibited with N(G)-monomethyl-L-Arg. sGC was stimulated with NO donors 1-hydroxy-2-oxo-3- (N-ethyl-2-aminoethyl)-3-ethyl-1-triazene and S-nitroso-L-glutathione or mimicked by 8-bromo-guanosine 3', 5'-cyclic monophosphate (8-Br-cGMP) and inhibited with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one. The effects of the PKG inhibition with KT5823 and PDE V inhibition with Zaprinast were also examined. The studies demonstrate that ciliated epithelial cells produce NO, which is correlated with CBF stimulation. L-Arg dose- and time-dependently increases CBF, and NO donors, 8-Br-cGMP, and Zaprinast also enhance CBF. Inhibitors of NOS, sGC, and PKG can block the stimulant effect of L-Arg on CBF. Thus, NO is a regulator of CBF acting via sGC and PKG. The NO-cGMP signaling pathway regulates CBF in an autocrine manner in cultured rat ciliated airway epithelium.

Alkaloids↗
PubMed2000

[Effect of chrysotile on nitric oxide production and anti-oxidasic activity in rabbit alveolar macrophages].

This study aimed to explore the role of nitric oxide (NO) and nitric oxide synthase (NOS) in chrysotile asbestos-caused diseases. The production of NO2-/NO3- (the final product of NO) and the alterations of NOS, SOD and GSH-Px activity were investigated when rabbit alveolar macrophages (AM) were stimulated by UICC chrysotile. The results revealed that with the dosage elevation of UICC chrysotile, the rabbit alveolar macrophages showed: (1) increased mortality, decreased survival activity; (2) increased product of NO2-/NO3-, decreased SOD and GSH-Px activity; (3) increased NOS activity in the lower dosage groups, but decreased NOS activity in the higher dosage groups. There was significantly negative correlation between NO release and SOD (or GSH-Px) activity (r1 = -0.7125, P < 0.05; r2 = -0.8496, P < 0.05 respectively). Moreover, the significantly positive correlation between NO release and NOS activity was found in the lower dosage groups, but significantly negative correlation in the higher dosage groups. These findings suggest that chrysotile could induce the alveolar macrophages to increase NO release and to decrease SOD and GSH-Px activity, which may play a role in the process of asbestos-caused diseases.

Animals↗
PubMed2000

[Discussions on some problems related to fluid inclusion analysis by SXRF].

X-ray fluorescence experiments were carried out at Beijing Synchrotron Radiation Facilities (BSRF) using synchrotron radiation as excitation source. The samples were made by mixing national reference standard GBW07106 and certain amount of reagent NaCl and KCl, and pressed into pellets. The purpose of the experiment is to verify the detection ability of the XRF setup at BSRF for elements in geological samples, especially for light elements. The beam size was 20 x 20 microns2, and the current in the storage ring was about 40 mA. The gap between the sample and the Si(Li) detector was 2 cm. With measuring time of 200 sec., The relative and absolute detection limits for 10 elements have been calculated. The sampling depth and sampling weight, which are critical in individual fluid inclusion analysis, have also been estimated. Some problems concerning the individual fluid inclusion analysis by SXRF, such as inclusion depth determination, effect of the inclusion depth on XRF intensity etc., are discussed based on the experiment results.

English Abstract↗
PubMed1999

Src is required for cell migration and shape changes induced by fibroblast growth factor 1.

Fibroblast growth factor 1 (FGF-1) is a potent chemotactic factor and induces tyrosine phosphorylation of a cortical actin-associated protein (cortactin). The tyrosine phosphorylation of cortactin induced by FGF-1 requires the tyrosine residues 421, 482 and 466, which are targeted by the protein tyrosine kinase Src in vitro. Furthermore, FGF-1 is unable to induce tyrosine phosphorylation of cortactin within the cells derived from Src knockout mice (Src-/-), indicating that Src is required for the tyrosine phosphorylation of cortactin induced by FGF-1. Although Src-/- cells are able to undergo rapid proliferation, they are impaired to respond to FGF-1 for the shape change and cell migration. Morphological analysis further reveals that FGF-1 fails to induce the formation of polarized lamellipodia and the translocation of cortactin into the leading edge of Src-/- cells. Consistent with the mitogenic response to FGF-1, the lack of Src does not affect the tyrosine phosphorylation of Snt (or Frs2), a FGF-1 early signaling protein that links to Ras. Therefore, our data support the notion that Src and cortactin participate in a FGF signal pathway for cell migration and shape change rather than mitogenesis.

3T3 Cells↗
PubMed1999

Immunohistochemical evidence for the NO cGMP signaling pathway in respiratory ciliated epithelia of rat.

Airway epithelia play a crucial role in protecting the lung from the external environment. Ciliated airway epithelial cells contribute to mucociliary transport systems via ciliary beating and electrolyte transport mechanisms to defend against respiratory tract infection. Both of these activities are regulated by nitric oxide (NO)-dependent mechanisms. To better understand the role of the NO-cGMP signal transduction cascade in these responses, we investigated the localization of endothelial nitric oxide synthase (eNOS), soluble guanylyl cyclase (sGC), cGMP-dependent protein kinase (PKG) I-alpha, and PKG I-beta in the tracheas and lungs of normal rats by immunohistochemistry. Mouse anti-eNOS, rabbit anti-sGC, PKG I-alpha, and PKG I-beta antibodies were used. Strong immunostaining for eNOS was detected in ciliated tracheal, bronchial, and bronchiolar epithelia, in Clara cells, and in Type II alveolar cells. The pattern of sGC and PKG I-beta immunostaining showed striking parallels with that of eNOS staining. No staining was detectable in ciliated epithelium with the anti-PKG I-alpha antibody. Taken together, these observations suggest that PKG I-beta might transduce NO-sGC signaling into biological responses in ciliated respiratory epithelia.(J Histochem Cytochem 47:1369-1374, 1999)

Animals↗
PubMed1998

The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells.

Cortactin, a filamentous actin cross-linking protein and a substrate of Src protein tyrosine kinase, is phosphorylated at tyrosine residues upon stimulation by extracellular signals. We have previously demonstrated that the filamentous actin cross-linking activity of cortactin is attenuated by Src (Huang, C., Ni, Y., Gao, Y., Haudenschild, C. C., and Zhan, X. (1997) J. Biol. Chem. 272, 13911-13915). In vitro, tyrosine phosphorylation of cortactin occurs specifically within the region between the proline-rich sequence and the Src homology 3 domain. Among the nine tyrosine residues in this region, mutations at Tyr421, Tyr466, and Tyr482 significantly reduced Src-meditated tyrosine phosphorylation both in vitro and in vivo. Ectopic expression of wild-type cortactin in ECV304, a spontaneously transformed human umbilical endothelial cell line, resulted in an enhanced cell migration. In contrast, overexpression of a cortactin mutant deficient in tyrosine phosphorylation impaired the migration of endothelial cells. These findings reveal an intracellular signaling mechanism whereby the motility of endothelial cells is regulated by a Src-mediated tyrosine phosphorylation of cortactin.

Actins↗
PubMed1998

Role of phosphatidylinositol 4,5-bisphosphate in Ras/Rac-induced disruption of the cortactin-actomyosin II complex and malignant transformation.

Oncogenic Ras mutants such as v-Ha-Ras cause a rapid rearrangement of actin cytoskeleton during malignant transformation of fibroblasts or epithelial cells. Both PI-3 kinase and Rac are required for Ras-induced malignant transformation and membrane ruffling. However, the signal transduction pathway(s) downstream of Rac that leads to membrane ruffling and other cytoskeletal change(s) as well as the exact biochemical nature of the cytoskeletal change remain unknown. Cortactin/EMS1 is the first identified molecule that is dissociated in a Rac-phosphatidylinositol 4,5-biphosphate (PIP2)-dependent manner from the actin-myosin II complex during Ras-induced malignant transformation; either the PIP2 binder HS1 or the Rac blocker SCH51344 restores the ability of EMS1 to bind the complex and suppresses the oncogenicity of Ras. Furthermore, while PIP2 inhibits the actin-EMS1 interaction, HS1 reverses the PIP2 effect. Thus, we propose that PIP2, an end-product of the oncogenic Ras/PI-3 kinase/Rac pathway, serves as a second messenger in the Ras/Rac-induced disruption of the actin cytoskeleton and discuss the anticancer drug potential of PIP2-binding molecules.

3T3 Cells↗
PubMed1998

[Clinical significance of cytokine and eosinophil cationic protein concentrations in sputum of asthmatic patients].

OBJECTIVE: To examine whether levels of inflammatory cytokines and eosinophil cationic protein (ECP) in the sputum reflect the severity of bronchial asthma. METHOD: We collected sputum expectorated spontaneously from 15 asthmatics with acute attacks of moderate to severe degree (MS group) and 10 subjects with acute attacks of mild asthma (M group). The interleukin (IL)-5(35 ng/L) tumor necrosis factor (TNF)-alpha(M 149 +/- 59 ng/L, MS 267 +/- 147 ng/L), soluble IL-2 receptor (sIL-2R) (M 348 +/- 107 kU/L, MS 488 +/- 127 kU/L) levels in the sputum were measured with enzyme-linked immunosorbent assay, and sputum ECP (M 127 +/- 95 micrograms/L, MS 278 +/- 150 micrograms/L) concentration were measured by Immuno-CAP System. RESULT: Sputum IL-5, TNF-alpha, sIL-2R, ECP concentrations in moderate to severe patients were significantly higher than in mild subjects. CONCLUSION: These findings suggest that inflammatory cytokines and mediator levels are detectable in the sputum from asthmatics and they might participate in the exacerbation of asthma.

Adult↗
PubMed1998

[Preliminary observation of immunotherapy on asthmatic guinea pig].

OBJECTIVE: To observe the effect of the anti-IL-5 monoclonal antibody(McAb) on eosinophils of the guinea pigs with asthma. METHOD: Sensitized guinea pigs by OVA were divided into two groups randomly, which were treated with the McAb and normal saline (NS), respectively. Difference of peripheral blood eosinophil count (PBEC) among basal pre-treatment and post-treatment, and the difference of bronchoalveolar lavage fluid eosinophil count (BALFEC) between two groups above were compared. RESULT: The PBEC after treatment with the McAb [(1.63 +/- 0.26) x 10(9)/L] was lower than that before treatment [(1.76 +/- 0.27) x 10(9)/L] (P < 0.05) and the BALFEC of treatment group with the McAb was lower than that with NS [(0.41 +/- 0.06) x 10(9)/L vs. (0.46 +/- 0.07) x 10(9)/L] (P < 0.05). CONCLUSION: The anti-IL-5 McAb has the ability to reduce the PBEC and the BALFEC.

Animals↗
PubMed1997

The isolated RNase H domain of murine leukemia virus reverse transcriptase. Retention of activity with concomitant loss of specificity.

Retroviral RNases H are similar in sequence and structure to Escherichia coli RNase HI and yet have differences in substrate specificities, metal ion requirements, and specific activities. Separation of reverse transcriptase (RT) into polymerase and RNase H domains yields an active RNase H from murine leukemia virus (MuLV) but an inactive human immunodeficiency virus (HIV) RNase H. The "handle region" present in E. coli RNase HI but absent in HIV RNase H contributes to the binding to its substrate and when inserted into HIV RNase H results in an active enzyme retaining some degree of specificity. Here, we show MuLV protein containing the C-terminal 175 amino acids with its own handle region or that of E. coli RNase HI has the same specific activity as the RNase H of RT, retains a preference for Mn2+ as the cation required for activity, and has association rate (KA) 10% that of E. coli RNase HI. However, with model substrates, specificities for removal of the tRNAPro primer and polypurine tract stability are lost, indicating specificity of RNase H of MuLV requires the remainder of the RT. Differences in KA, while significant, appear insufficient to account for the differences in specific activities of the bacterial and viral RNases H.

Base Sequence↗
PubMed1997

Proteolysis of platelet cortactin by calpain.

Cortactin, a substrate of pp60(c-)src and a potent filamentous actin binding and cross-linking protein, is abundant in circulating platelets. After stimulation of platelet aggregation with collagen, cortactin undergoes a dramatic increase in tyrosine phosphorylation followed by a rapid degradation. The cleavage of platelet cortactin was detected in lysates prepared using either Triton-containing buffer or SDS-sample buffer. However, the degradation of cortactin was not observed in platelets derived from a Glanzmann's patient, who lacked functional integrin alphaIIbbeta3 (GPIIb-IIIa). In addition, the proteolysis of cortactin was abolished by treating platelets before but not after collagen stimulation with EGTA or calpeptin. Furthermore, recombinant cortactin was digested by mu-calpain in vitro in a dose-dependent manner, indicating that cortactin is a substrate for calpain. We also observed that the calpain-mediated digestion in vitro is dependent on the presence of a sequence containing a proline-rich region and multiple tyrosine residues that are phosphorylated by pp60(c-)src. Tyrosine phosphorylation by pp60(c-)src up-regulates the activity of calpain toward cortactin. Our data suggest that the calpain-mediated proteolysis of tyrosine-phosphorylated cortactin may provide a mechanism to remodel irreversibly the cytoskeleton in response to platelet agonists.

Blood Platelets↗
PubMed1997

Down-regulation of the filamentous actin cross-linking activity of cortactin by Src-mediated tyrosine phosphorylation.

Cortactin, a prominent substrate for pp60(c-src), is a filamentous actin (F-actin) binding protein. We show here that cortactin can promote sedimentation of F-actin at centrifugation forces under which F-actin is otherwise not able to be precipitated. Electron microscopic analysis after negative staining further revealed that actin filaments in the presence of cortactin are cross-linked into bundles of various degrees of thickness. Hence, cortactin is also an F-actin cross-linking protein. We also demonstrate that the optimal F-actin cross-linking activity of cortactin requires a physiological pH in a range of 7.3-7.5. Furthermore, pp60(c-src) phosphorylates cortactin in vitro, resulting in a dramatic reduction of its F-actin cross-linking activity in a manner depending on levels of tyrosine phosphorylation. In addition, pp60(c-src) moderately inhibits the F-actin binding activity of cortactin. This study presents the first evidence that pp60(c-src) can directly regulate the activity of its substrate toward the cytoskeleton and implies a role of cortactin as an F-actin modulator in tyrosine kinase-regulated cytoskeleton reorganization.

Actins↗
PubMed1997

Upregulation of cortactin expression during the maturation of megakaryocytes.

Cortactin is a potent filamentous actin-binding protein acting as a prominent substrate of Src tyrosine kinases. We have evaluated cortactin expression in a series of murine tissues and shown an abundant expression of cortactin in megakaryocytes and platelets. Cortactin, but not its related protein HS1, is upregulated during the phorbol 12-myristate 13-acetate (PMA)-mediated maturation of a human megakaryoblastic cell line CMK. Although the expression of Src-related kinases is also upregulated more rapidly than cortactin in PMA-treated CMK cells, tyrosine phosphorylation of cortactin appears to be only transiently elevated 4 days after PMA stimulation. In addition, cortactin expression is induced by thrombopoietin and interleukin-3 in megakaryocytes derived from murine bone marrow cells. Thus, cortactin represents a megakaryocyte-specific gene in bone marrow and the interaction of Src kinases with cortactin may be involved in the maturation of megakaryocytes.

Animals↗
PubMed1997

Exponential heating in drug stability experiment and statistical evaluation of nonisothermal and isothermal prediction.

A new nonisothermal heating model (exponential heating model) for drug stability experiments, based on a theoretical study of simulated nonisothermal data, is presented. In the model, the heating rate dT/dt is increased by 2-4 times at every increase of 10 degrees C in temperature: dT/dt = a(T-T0)/10.(dT/dt)0 where a is the times by which the heating rate is increased at every increase of 10 degrees C. A computation method with optimization and Simpson integration for the experiment was also introduced. The estimates for the shelf-life and activation energy obtained by the exponential and other nonisothermal heating models were statistically evaluated and were compared with those obtained by the isothermal method under various conditions. The results indicated that under the same experimental conditions, the estimates obtained by the exponential heating model were significantly more accurate and precise than those obtained by the linear, reciprocal, and logarithmic heating models. The accuracy and precision of the estimates were independent of the shelf-life of drugs and experimental period. The estimates obtained by the isothermal method were somewhat more accurate and precise than those obtained by the exponential heating model. However, the experimental period needed by the isothermal method was about five times longer than that needed by the exponential heating model. The results also showed that in each of the heating models, the estimates are more accurate and precise by increasing the extent of drug degradation, changing temperature range or sampling frequency, or by having the mean temperature closer to room temperature. To demonstrate its applicability, the exponential heating model was used to study the stability of vitamin C tablets and predict their shelf-life.

Drug Stability↗
PubMed1997

Determination of rate order for degradation of drugs with nonisothermal stability experiment.

The inability of ordinary nonisothermal experiments to determine the rate order (n) of drug degradation is discussed on the basis of a theoretical study of simulated nonisothermal data. In ordinary nonisothermal experiments, using either r or sigma(C[experiment] C[compute])2 as the measure of goodness of fit, the rate order cannot be assessed because the same set of c-t data can be well fitted by different combinations of estimates for n and the activation energy (E). Hence, a new nonisothermal heating-and-cooling model is introduced as a revision, in which sigma(C[experiment] C[compute])2 changes significantly with various kinetic models. Therefore, all the kinetic parameters, including the rate order, can be obtained in one nonisothermal stability experiment. Furthermore, the ability to determine rate order is significantly affected by the extent of drug degradation and experimental error, though not by sampling frequency or temperature change. To demonstrate the applicability of the heating-and-cooling model, the stability and rate order of degradation of vitamin C tablets was studied by this method.

Ascorbic Acid↗