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W W Zhang

Publications and source records attributed to W W Zhang.

At least 19 recordsLinked to original sources
PubMed2001

Development of a p53 responsive GFP reporter; identification of live cells with p53 activity.

p53 is among the most intensely studied human proteins because of its vital role as the prototype tumor suppressor. As a result, there are widespread applications for p53 functional analysis in biotechnology as it relates to cancer research. p53 is a potent sequence specific transcription factor, which induces the expression of a number of genes whose products mediate cell cycle arrest and apoptosis. Because the tumor suppressor activity of p53 is dependent on its transcription regulatory function, we have undertaken to develop a p53-responsive green fluorescent protein reporter strategy to enable the identification of live cells containing p53 transcriptional transactivation activity. We demonstrate within the use of GFP fluorescence to monitor both endogenous and plasmid derived p53 biochemical and biological activity. Identifying live cells with p53 activity through GFP fluorescence will have wide application for both in vitro and in vivo studies of the p53 tumor suppressor protein.

Apoptosis↗
PubMed2001

Immunization with A2 protein results in a mixed Th1/Th2 and a humoral response which protects mice against Leishmania donovani infections.

The A2 genes of Leishmania donovani encode amastigote-specific A2 proteins, which are considered to be virulence factors required for the survival of this protozoan parasite in the mammalian host. The A2 genes are present within a multigene family and corresponding A2 proteins are composed predominantly of multiple copies of a 10 amino acid repeat sequences. A2-specific antibodies have been detected in the sera of patients suffering from visceral leishmaniasis (VL) and it has been shown that generation of A2 deficient L. donovani resulted in an avirulent phenotype. In this report, we show that immunization of mice with recombinant A2 protein conferred significant protection against challenge infection with L. donovani. The protection correlated with in vitro splenocyte proliferation, production of IFN-gamma in response to A2 protein and the presence of A2-specific antibodies in the sera of immunized mice. These data demonstrate that A2 represents a potential antigen for protection against infection with L. donovani and VL.

Adoptive Transfer↗
PubMed2001

Simvastatin inhibits noradrenaline-induced hypertrophy of cultured neonatal rat cardiomyocytes.

1. Oxidative mechanisms have been implicated in neonatal cardiomyocyte hypertrophy. We and others have shown that a HMG-CoA reductase inhibitor preserves endogenous antioxidant enzyme activity and inhibits cardiac hypertrophy in vivo. We therefore have examined whether noradrenaline (NA) induces the generation of reactive oxygen species (ROS) during its induction of neonatal cardiomyocyte hypertrophy and whether simvastatin, a HMG-CoA reductase inhibitor, attenuates ROS production and thus NA-induced hypertrophy of cardiomyocytes. 2. NA increased the intracellular ROS levels in a concentration-dependent manner. This increase of ROS was significantly inhibited by simvastatin and catalase. Prazosin partially suppressed NA-induced increase of ROS and beating, while preincubation with both prazosin and propranolol completely abolished NA-evoked increase of ROS and beating. Simvastatin did not affect NA-induced increase of beating. 3. The NA-induced increase of protein content was partially suppressed by prazosin and completely abolished by preincubation with both prazosin and propranolol. Simvastatin inhibited the increase of NA-induced increase of RNA content and [(3)H]-leucine incorporation in a concentration-dependent manner. Mevalonic acid (MVA) reversed the inhibition of NA-induced RNA and protein increase by simvastatin. Catalase also inhibited the NA-induced increase of RNA and protein. 4. We conclude that the inhibitory effects of simvastatin on myocyte hypertrophy were associated with its antioxidant effects and inhibition of MVA-metabolism pathway in neonatal rat cardiomyocytes. NA-induced increases of intracellular ROS and cardiomyocyte hypertrophy requires both alpha and beta adrenoceptors activation in neonatal rat cardiomyocytes. The increases of ROS induced by NA is required for hypertrophy.

Adrenergic alpha-Agonists↗
PubMed2001

Characterization of the A2-A2rel gene cluster in Leishmania donovani: involvement of A2 in visceralization during infection.

The A2 gene family is present in Leishmania donovani, which causes fatal visceral leishmaniasis in human patients, but is not present in Leishmania major, which causes cutaneous leishmaniasis infections. The A2 genes in L. donovani are stage specific and are expressed at high levels in the amastigote stage in the mammalian host, but are not expressed in the promastigote stage in the insect sandfly vector. The A2 genes are tandem repeated with a distinct gene family termed the A2rel genes. In order to characterize the structure and function of the A2-A2rel gene clusters, the 5' and 3' DNA sequences flanking the A2-A2rel cluster were isolated, sequenced and used to generate mutants through gene targeting. Although it was possible to generate partial A2-A2rel gene clusters knock-out mutants, it was not possible to delete all the A2-A2rel gene clusters completely from the L. donovani genome, suggesting that, within this cluster, there are genes that are essential for survival in culture. Characterization of these mutants revealed that A2 and A2rel gene expression was compensated by amplifying the remaining intact A2 and A2rel genes, and the proliferation of these mutants in culture and their virulence in BALB/c mice were compromised. In order to explore further the biological role of A2, the L. donovani A2 gene was introduced into L. major. In comparison with the control L. major, the A2-expressing L. major parasites demonstrated an increased ability to survive in the spleen of BALB/c mice. These data suggest that A2 plays a role in the visceralization of infection associated with L. donovani.

Animals↗
PubMed2000

Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector.

The successful prophylactic treatment of hemophilia A by frequent infusions of plasma concentrates or recombinant factor VIII (hFVIII) indicates that gene therapy may be a potential alternative for the treatment of the disease. For efficient delivery and long-term expression of the hFVIII gene, a novel minimal adenovirus (mini-Ad) vector, MiniAdFVIII, has been developed. The vector is devoid of all viral genes and carries the full-length hFVIII cDNA under the control of the human 12.5-kb albumin promoter. The MiniAdFVIII vector was propagated with the assistance of an ancillary vector in 293 cells and was purified by CsCl banding. Sustained expression of hFVIII at physiologic levels (100-800 ng/mL) was achieved in mice after a single intravenous injection of MiniAdFVIII. The expressed hFVIII had a structure identical to that of recombinant hFVIII, as determined by Western blot analysis. The functionality of the protein was confirmed by the restoration of blood coagulation capacity in MiniAdFVIII-treated hemophilic mice, as determined by tail clipping observations. Although antivector or antihuman FVIII antibodies at various levels were detected, long-term expression of the transgene was observed in the mice that did not generate antibodies against the transgene product. The vector DNA persisted in the liver tissues of the mice with long-term expression. No significant histopathologic findings or toxicities were observed to be associated with the vector in the MiniAdFVIII-treated C57BL/6 mice. These results support the further development of MiniAdFVIII for clinical trials toward the treatment of hemophilia A.

Adenoviridae↗
PubMed2000

Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32.

The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask-) and E. coli X6118 (asd-), respectively. The A. mediterranei U32 asparto-kinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has Km values of 1.25 mM for DL-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by L-threonine, L-lysine, and L-methionine, but not by L-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that asparto-kinase was subject to allosteric control by L-threonine. Repression of both aspartokinase and aspartate semi-aldehyde dehydrogenase gene transcription in A. mediterranei U32 by L-lysine, L-methionine, L-threonine, and L-isoleucine were found. The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.

Actinomycetales↗
PubMed2000

D-2-hydroxyglutaric aciduria with cerebral, vascular, and muscular abnormalities in a 14-year-old boy.

D-2-Hydroxyglutaric Aciduria is a rare metabolic disorder that can cause injury to the brain and other organs. This case report concerns a 14-year-old boy showing irritability and typical signs of pyloric stenosis early postnatally. From the age of 3 months he had epilepsy. He was mentally retarded, hypotonic with preserved reflexes, and dystonic. The features were dysmorphic with elongated head and high arched palate. Cardiomegaly with aortic insufficiency was diagnosed. Magnetic resonance imaging of the brain revealed atrophy, reduced periventricular white matter, and multiple bilateral aneurysms of the middle cerebral arteries. The boy died at the age of 14 years. Autopsy confirmed the white-matter reduction of the cerebral hemispheres as well as the arterial aneurysms of the middle cerebral arteries. Lesions of a few leptomeningeal and cerebral microvessels and of the renal and pulmonary arteries were also found. There were bilateral infarcts of the kidneys and signs of cardiomyopathy with noncompensated left ventricular failure. Signs of myopathy were evident. The clinical and postmortem findings imply a disseminated mesenchymal process.

Adolescent↗
PubMed1999

Simvastatin inhibits cardiac hypertrophy and angiotensin-converting enzyme activity in rats with aortic stenosis.

1. In the present study, we tested the hypothesis that long-term administration of the hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor simvastatin may regress hypertrophy and the possible effect of simvastatin on angiotensin-converting enzyme (ACE) activity in rats with pressure-overload cardiac hypertrophy. 2. Pressure-overload left ventricular hypertrophy (LVH) of rats was induced by part coarctation of the abdominal aorta; a sham-operated group served as the control. Six weeks after operation, animals were divided into three groups and an 8 week treatment period was insitgated as follows: (i) the simvastatin treatment group received simvastatin at 3.6 mg/kg per day, p.o.; (ii) the ACE inhibitor group received captopril at 50 mg/kg per day, p.o.; and (iii) the LVH control group received no drug treatment. 3. At the end of the treatment period, left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP) were monitored in vivo. Diastolic pressure-volume relationships were evaluated in a Langendorff preparation with a balloon-in-left ventricle (LV) heart. Myocyte cell width was measured. Angiotensin-converting enzyme activity and angiotensin (Ang)II and hydroxyproline contents of the LV were determined. 4. At the end of the experiments, LVH was established in the LVH control group by increases in LV weight, LV weight/body-weight ratio, LV weight/right ventricle weight ratio, LV myocyte cell width, LVSP and LVEDP by 40, 26, 19, 61, 56 and 59%, respectively (all P < 0.01), compared with the sham-operated group. In the simvastatin-treated and ACE inhibitor groups all these parameters were significantly reduced compared with sham-operated controls. In the LVH control group, ACE activity and AngII and hydroxyproline contents of LV tissue increased by 180, 123 and 70, respectively (all P < 0.01), compared with the sham-operated group. Compared with the LVH group, in the simvastatin-treated and ACE inhibitor groups ACE activity was reduced by 36 (P < 0.05) and 48% (P < 0.01), respectively, AngII content was reduced by 11 (P < 0.05) and 43% (P < 0.01), respectively, and hydroxyproline content was reduced by 23 (P < 0.01) and 10% (P < 0.05), respectively. 5. For the first time, the results of the present study demonstrate that simvastatin significantly reduces LVH, cardiac tissue ACE activity and improves LV performance in pressure-overloaded rats. Because, compared with captopril, simvastatin is more potent in its reduction of LVH and less potent in its inhibition of ACE activity, the mechanism of its antihypertrophic action, in addition to ACE inhibition, may involve inhibition of the mevalonic acid pathway, the main target of action of statins. Thus, HMG-CoA reductase inhibitors may be beneficial for the clinical treatment of cardiac hypertrophy.

Angiotensin II↗
PubMed1999

Effects of simvastatin on left ventricular hypertrophy and function in rats with aortic stenosis.

AIM: To investigate the effects of simvastatin (Sim) on left ventricular hypertrophy in rats with pressure-overload cardiac hypertrophy. METHODS: The left ventricular hypertrophy (LVH) of rats was induced by partly occluding abdominal aorta below right renal artery. The rats were given i.g. Sim (1.8 and 3.6 mg.kg-1.d-1) for 8 wk. Three days after operation, left ventricular function was measured. Then the left ventricle (LV) + septum and the right ventricle (RV) were weighed. Hydroxyproline content of LV was measured. RESULTS: Eight weeks later, in the LVH group, LV weight (LVW), LVW/body weight (BW), LVW/RV weight (RVW), LV ending diastolic pressure (LVEDP), and hydroxyproline content increased by 36%, 51%, 28%, 92%, and 23%, respectively (all P < 0.01) compared with the sham group. LV + dp/dtmax and -dp/dtmax decreased by 39.2% and 39.4% (all P < 0.01). After the rats were given i.g. Sim 3.6 mg.kg-1.d-1, LVW, LVW/BW, LVW/RVW, left ventricle ending diastolic pressure (LVEDP), and hydroxyproline content decreased by 22%, 21%, 23%, 24%, and 11% compared with LVH group (all P < 0.01), LV + dp/dtmax and -dp/dtmax increased by 60% and 32% (all P < 0.01). CONCLUSION: Sim inhibited development of LV hypertrophy and improved LV function in rats with aortic stenosis.

Animals↗
PubMed1999

Development and application of a minimal-adenoviral vector system for gene therapy of hemophilia A.

To achieve efficient delivery and sustained expression of the human factor VIII cDNA in vivo, a minimal-adenoviral (mini-Ad) vector system was developed. The system is composed of a mini-Ad vector with essential cis-elements (less than 1 kb) of the viral genome, an E1-deleted ancillary Ad with packaging attenuation, and an E1-complementing production cell line. Based on this system, MiniAdFVIII was generated to deliver a 27 kb expression cassette consisting of a full-length human factor VIII cDNA flanked by human albumin promoter and genomic sequences. The MiniAdFVIII vector mediated expression of functional human factor VIII in HepG2 and 293 cells. A single-dose intravenous injection of 10(11) viral particles in hemophilic mice of MiniAdFVIII produced a sustained high-level expression of human factor VIII (at 100-800 ng/ml up to 369 days) which corrected the FVIII-deficient phenotype. Safety studies of MiniAdFVIII showed that there were no significant toxic effects in mice and dogs after single intravessel doses of up to 3 x 10(11) and 6 x 10(12) viral particles, respectively. Studies for developing the MiniAdFVIII vector with a site-specific integration mechanism and progress in the development of a human factor VIII-tolerized mouse model for pre-clinical studies of MiniAdFVIII are reported. Further pre-clinical studies and product development of MiniAdFVIII for clinical trials are also discussed.

Adenoviridae↗
PubMed1998

Inducible expression of suicide genes in Leishmania donovani amastigotes.

This study tests the feasibility of using the A2 gene regulatory system to create a Leishmania cell line in which attenuation is developmentally regulated when the parasite differentiates from promastigotes to amastigotes. The Leishmania donovani- inducible A2 gene regulatory system was used to differentially express in amastigotes two potential suicide genes: a truncated version of the L. donovani 3'-nucleotidase/nuclease expressed in the cytoplasm and the herpes simplex virus thymidine kinase gene. These genes were inserted between A2 noncoding regulatory sequences for up-regulation of expression in amastigotes. The accumulation of toxic products affected L. donovani cell replication and viability both in vitro and in vivo. The inducible expression of toxic gene products represents a valuable tool for the development of safe and effective vaccines.

Animals↗
PubMed1998

Amyloid angiopathy of the human brain: immunohistochemical studies using markers for components of extracellular matrix, smooth muscle actin and endothelial cells.

Cerebral amyloid angiopathies comprise a heterogeneous group of conditions characterised by amyloid deposition in leptomeningeal and cortical vessels. We have studied the deposition of extracellular matrix components in such vessels from controls and ten cases with marked amyloid angiopathy. Arterial vessels which were heavily loaded with amyloid often showed lack of immunostaining to collagen type I, III, V and VI in the amyloid-containing parts of the vessel wall but some immunoreactivity remained in the adventitia. The subintimal region of some arterioles presented a faint staining with collagen V and collagen VI antisera. Immunostaining to collagen IV and laminin revealed normal reactivity in the vascular basal lamina and frequently remaining activity in the media. Immunostaining for actin showed a complete or partial loss of reactivity in the amyloid-containing parts of the media but often there was a thin line of staining at the position of pericytes. The endothelial markers did not reveal any changes compared with controls. In other cerebral microangiopathies, for instance Binswanger's leukoencephalopathy, CADASIL and cases presenting hyalinosis there is a deposition of fibrillary collagens in the wall of afflicted microvessels. Degeneration of smooth muscle cells and absence of marked fibrosis in some of the arterial vessels in cases of amyloid angiopathy may explain why such vessels are susceptible to ruptures and haemorrhages.

Actins↗
PubMed1997

Cell-cycle regulation of DNA damage-induced expression of the suppressor gene PML.

The promyelocytic leukemia (PML) gene, which encodes a growth- and transformation-suppressor, has been identified at the non-random chromosomal translocation break point t(15; 17)(q22; q12) of acute promyelocytic leukemia. To elucidate if PML may play a role in cellular response to DNA damage, PML expression was analyzed by immunofluorescence staining in HeLa cells treated with ionizing radiation (IR) and cisplatin. Our studies demonstrated IR at 20Gy, and cisplatin at 6 micrograms/ml caused more than 5-10 fold increases in PML protein expression in the PML Oncogenic Domain (POD) by immunofluorescent staining. Northern blotting showed that there was no gross increase in mRNA levels indicating that the induction is a post-transcriptional event. Flow cytometry showed that HeLa cells treated with IR were progressively arrested in G1, which correlates with the optimal expression of PML in the cell cycle. To determine if PML expression was under the control of the tumor suppressor p53, which is known to arrest cells in G1, HeLa cells were transfected with the wild-type p53 gene. PML expression in p53 transduced cells were 5-10 fold higher than the control, indicating that the enhanced expression of PML is apparently dependent on the p53 pathway. These data also indicate that PML may play an important role in cellular response to DNA damage such as DNA repair or apoptosis during G1 arrest.

Blotting, Northern↗
PubMed1997

p53 expression overcomes p21WAF1/CIP1-mediated G1 arrest and induces apoptosis in human cancer cells.

The p21WAF1/CIP1 gene, which encodes a cyclin-dependent kinase inhibitor, may be critical for tumor suppressor gene p53-induced cell cycle arrest. The p53 gene is known to regulate G1 checkpoint, which can either induce G1 arrest or initiate apoptosis. To directly examine the role of p21WAF1/CIP1 in the control of p53 function, we have introduced human p21WAF1/CIP1 gene into a p53-deficient human non-small cell lung cancer cell line H1299 using a p21WAF1/CIP1-expressing adenoviral vector (AdCMVp21). Infection with AdCMVp21 resulted in high levels of p21WAF1/CIP1 expression and significantly suppressed the growth of H1299 cells through the G1 arrest of the cell cycle. In contrast, transient expression of the wild-type p53 gene by a recombinant adenoviral vector (AdCMVp53) in H1299 cells induced apoptotic cell death and resulted in a rapid loss of cell viability. We then examined the effects of combined infection with AdCMVp21 and AdCMVp53 on H1299 cells to explore the dominant function of these molecules. Interestingly, introduction of exogenous p53 overcame p21WAF1/CIP1-mediated cell cycle arrest at G1 and induced apoptosis, although viral-transduced p21WAF1/CIP1 expression level was unaffected. These observations suggest that p53 expression converts a p21WAF1/CIP1-mediated growth arrest into apoptosis. The result was repeated with two additional human colon adenocarcinoma cell lines with the different p53 status, mutant p53-expressing DLD-1 and wild-type p53-expressing LoVo, suggesting that this phemonenon is a general event among human cancer cells. Thus, p53-mediated apoptotic pathway is dominant over the growth arrest pathway, indicating that p53 may be an essential upstream mediator of p21WAF1/CIP1 in the regulation of a cell process leading either to growth arrest or to apoptotic suicide.

Apoptosis↗
PubMed1997

Loss of virulence in Leishmania donovani deficient in an amastigote-specific protein, A2.

Leishmania donovani is the etiologic agent of fatal visceral leishmaniasis in man. During their life cycle, Leishmania exist as flagellated promastigotes within the sandfly vector and as nonflagellated amastigotes in the macrophage phagolysosomal compartment of the mammalian host. The transformation from promastigotes to amastigotes is a critical step for the establishment of infection, and the molecular basis for this transformation is poorly understood. To define the molecular basis for amastigote survival in the mammalian host, we previously identified an amastigote stage-specific gene family termed "A2." In the present study, we have inhibited the expression of A2 mRNA and A2 protein in amastigotes using antisense RNA and show that the resulting A2-deficient amastigotes are severely compromised with respect to virulence in mice. Amastigotes that did survive in the mice had restored A2 protein expression. These data demonstrate that A2 protein is required for L. donovani survival in a mammalian host, and this represents the first identified amastigote-specific virulence factor identified in Leishmania. This study also reveals that it is possible to study gene function in Leishmania through the expression of antisense RNA.

Animals↗
PubMed1997

The angiopathy of subcortical arteriosclerotic encephalopathy (Binswanger's disease): immunohistochemical studies using markers for components of extracellular matrix, smooth muscle actin and endothelial cells.

A pronounced obliterative microangiopathy of the deep cerebral white matter is one of the cardinal features in classical cases of Binswanger's encephalopathy. We have characterised the alterations taking place in the intima, media and adventitia of obliterated arterial vessels in seven autopsy cases of this encephalopathy. The adventitia of fibrosed vessels showed immunoreactive material indicating a marked deposition of normally occurring collagen types, i.e. I, III and V. Similar deposits occurred in degenerated parts of the media. Two of the cases had, in addition, signs of collagen type VI-immunoreactive material in the adventitia and media. The elastica of arteries was often split and formed multiple layers. The inner part of the blood vessel walls contained immunoreactivity to collagen type IV and laminin, indicating increased amounts of basal lamina components. Using actin immunostaining the fibrosed arterial vessels showed a severe reduction of smooth muscle cells of the media. However, many terminal arterioles presented a marked actin immunostaining, possibly indicating hypertrophy of smooth muscle cells. The endothelial cell layer did not show any changes with regard to expression of glucose transporter 1, factor VIII, Ulex europaeus agglutinin I and CD34. Degeneration of the media associated with depositions of collagens type I, III, IV, V and possibly type VI, as well as other components of extracellular matrix, will jeopardize the regulatory functions of the afflicted vessels. The maintenance of the endothelial lining of the obliterated vessels probably counteracts thrombosis in the vessels.

Adult↗