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Biomedical subjects

J Bai

Publications and source records attributed to J Bai.

At least 19 recordsLinked to original sources
PubMed2001

Tumor suppression and potentiation by manipulation of pp32 expression.

Alternative use of genes of the closely-related pp32 family is a common occurrence in human prostate cancer. pp32r1 and pp32r2, the oncogenic members of the pp32 family, are expressed in prostatic adenocarcinoma, while adjacent benign prostate continues to express pp32. This study focuses upon the role of pp32 in tumor suppression. We demonstrate that antisense inhibition of pp32 in NIH3T3 cells leads to a variety of phenotypic changes associated with transformation including reduced serum dependence and loss of contact inhibition. NIH3T3 cells with antisense-inhibited pp32 are not tumorigenic, but are markedly more susceptible to oncogenic stimuli such as ras. In contrast, constitutive expression of pp32 abolishes ras mediated transformation in vitro and tumorigenesis in vivo. These data demonstrate, from the functional aspect, that pp32 acts as a tumor suppressor. Furthermore, inactivation of pp32 function through alternative gene use may be a critical event in tumor evolution and progression.

3T3 Cells↗
PubMed2001

Multivalent anti-CCR ribozymes for stem cell-based HIV type 1 gene therapy.

HIV-1 infection of susceptible cells is mediated by the specific interaction of viral envelope glycoproteins with the cell surface CD4 receptor and a chemokine coreceptor, CCR5 or CXCR4. Individuals with a CCR5 genetic defect show resistance to HIV-1 infection, indicating that downregulation of CCR5 expression on target cells can prevent viral infection. In previous studies we demonstrated the utility of an anti-CCR5 ribozyme targeted to a single cleavage site in downregulating CCR5 expression and consequently providing resistance to viral infection. To improve on the level of downregulation we designed a construct containing an anti-CCR5 ribozyme heterotrimer (R5RbzTM) targeted to three different cleavage sites in CCR5 mRNA. In vitro tests showed that the anti-CCR5 ribozyme heterotrimer could effectively cleave the CCR5 RNA substrates to yield products of the expected sizes. This construct was introduced into various retroviral vectors for stable gene transduction. HOS.CD4/R5 cells stably transduced with this anti-CCR5 heterotrimer showed a marked reduction in the surface expression of CCR5 and a concomitant 70% reduction in macrophage-tropic viral infection. In addition, a retroviral vector containing the anti-CCR5 ribozyme heterotrimer and an anti-HIV-1 tat-rev ribozyme heterodimer was constructed. This construct also showed a similar inhibition of CCR5 surface expression and reduced infectability by the macrophage-tropic HIV-1 vector in HOS.CD4/R5 cells. The trimeric and multimeric ribozyme constructs were transduced into CD34+ hematopoietic progenitor cells to determine their effects on lineage-specific differentiation. We show that multivalent ribozyme gene-transduced hematopoietic progenitors differentiated normally into mature macrophages that bear CD14 and CD4 surface markers. Macrophages containing the transgenes expressed ribozymes, and showed resistance to M-tropic HIV-1 infection. These results provide strong support for the use of the trimeric anti-CCR5 ribozyme approach in a gene therapy setting for the treatment of HIV infection.

CCR5 Receptor Antagonists↗
PubMed2001

synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.

Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

Adaptor Protein Complex alpha Subunits↗
PubMed2001

Diagnosis of primary HIV-1 infection. Los Angeles County Primary HIV Infection Recruitment Network.

BACKGROUND: The optimal approach for diagnosing primary HIV-1 infection has not been defined. OBJECTIVE: To determine the usefulness of symptoms and virologic tests for diagnosing primary HIV-1 infection. DESIGN: Prospective cohort study. SETTING: A teaching hospital in Los Angeles and a university research center in San Diego, California. PATIENTS: 436 patients who had symptoms consistent with primary HIV infection. MEASUREMENTS: Clinical information and levels of HIV antibody, HIV RNA, and p24 antigen. RESULTS: Primary infection was diagnosed in 54 patients (12.4%). The sensitivity and specificity of the p24 antigen assay were 88.7% (95% CI, 77.0% to 95.7%) and 100% (CI, 99.3% to 100%), respectively. For the HIV RNA assay, sensitivity was 100% and specificity was 97.4% (CI, 94.9% to 98.9%). Fever, myalgia, rash, night sweats, and arthralgia occurred more frequently in patients with primary infection (P < 0.05). CONCLUSIONS: No sign or symptom allows targeted screening for primary infection. Although assays for HIV RNA are more sensitive than those for p24 antigen in diagnosing primary infection, they are more expensive and are more likely to yield false-positive results.

Antibodies, Viral↗
PubMed2001

Comparison of image reconstruction algorithms in myocardial perfusion scintigraphy.

The purpose of this study was to compare the clinical utility of two image reconstruction algorithms in myocardial perfusion SPECT (single-photon emission computed tomography): filtered back-projection (FBP) and ordered subset expectation maximization (OSEM). A rest/stress one-day protocol with 99mTc-MIBI or tetrofosmin was performed on 102 consecutive patients who underwent coronary angiography. After SPECT data acquisition, images were reconstructed with FBP and OSEM algorithms. We assessed diagnostic performance (sensitivity, specificity and accuracy) in detecting coronary artery stenosis and evaluated regional tracer uptake with a 4-point scoring system. Although there were no significant differences in diagnostic performance between FBP and OSEM reconstruction, the OSEM method yielded higher uptake in the RCA area than the FBP method by reducing the count-loss artifact due to hepatic uptake of the tracers. In addition, regional uptake in the LCX area was significantly lower in the OSEM image than in the FBP image; this phenomenon was observed mainly in patients with coronary stenosis and/or infarction in the LCX territory. In conclusion, OSEM and FBP offered comparable diagnostic performance in stress myocardial perfusion SPECT. The OSEM method contributed to reduction of the count-loss artifact in inferior and posterior walls and to easy recognition of hypoperfusion in the LCX area.

Algorithms↗
PubMed2001

Multiple modeling in the study of interaction of hemodynamics and gas exchange.

Circulation plays an important rule in gas exchange. Therefore, there is an interaction between circulation and gas exchange. To understand the dynamic effect of these two physiological systems, a computer simulation model of hemodynamics and gas exchange is established in this work. This model includes two physiological systems, namely the respiratory and circulatory systems. It consists of five parts: the model of gas transport, exchange and storage within the body, the multi-element nonlinear mathematical model of human circulatory system, an alveolar ventilation controller, a cardiac output controller, and a controller of breathing frequency. Model simulations provide results consistent with both dynamic and steady-state responses under hypoxia. Simulation results can reflect the interaction of hemodynamics and gas exchange. Using this model, the changes of pulmonary arterial pressure and right ventricular pressure in high altitude are studied. The optimal mode of breathing extra oxygen using nasal prongs or a facial mask is studied. This model may provide a useful tool to study reaction of hypoxia and the oxygen inhalation mode under hypoxia environments.

Carbon Dioxide↗
PubMed2001

The incidence of tuberculosis in a cohort of South-East Asian refugees arriving in Australia 1984-94.

We have used record linkage analysis to describe the incidence of tuberculosis in a cohort of 24 652 predominantly south-east Asian refugees who arrived in Sydney, Australia during the period 1984 to 1994. Cases that had been registered with the State Department of Health were confirmed by examination of case records. After an average follow-up interval of 10.3 years there were 189 cases of tuberculosis, equivalent to an average incidence rate of 74.9 cases per 100 000 person-years. The highest incidence rate was in 40-49 year olds and 47% of cases were in women. One hundred and twenty seven cases (67%) were pulmonary and, of these, 64 (50%) were direct smear positive. The incidence of tuberculosis in this cohort is similar to that observed among Vietnamese migrants to Australia and the USA and substantially higher than the incidence among people born in Australia. It is important to maintain awareness of the diagnosis of tuberculosis, especially in countries such as Australia, where the incidence in the general population is low but where there are large populations of migrants and refugees in whom a higher incidence is expected.

Adolescent↗
PubMed2001

Proliferation and differentiation of prostatic stromal cells.

OBJECTIVES: To investigate the effects of androgen, transforming growth factor beta1 (TGF-beta) and basic fibroblast growth factor (bFGF) on the proliferation and differentiation of prostatic stromal cells of the dog in vivo and human stromal cells in vitro. MATERIALS AND METHODS: Twenty-two dogs had their serum concentration of testosterone and oestradiol determined by radioimmunoassay before and after castration. Light microscopy, transmission electron microscopy and an in situ cell-death assay were carried out successively before and after castration to evaluate prostatic histomorphology. A semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used to evaluate the expression of TGF-beta, bFGF and myosin in the canine prostate tissue after castration. In vitro serum-free cell cultures from human prostatic stroma were established and exposed to dihydrotestosterone (DHT), TGF-beta and bFGF in various concentrations. The proliferation of the cell cultures was detected by the tetrazolium assay. The differentiation from fibroblasts to smooth muscle cells (SMCs) was deduced by measuring the expression of SMC-specific proteins (myosin and smoothelin) using immunohistochemistry and RT-PCR. RESULTS: Castration resulted in a significant decrease in circulating testosterone levels (P < 0.01), but did not affect the circulating oestradiol levels (P > 0.05). The prostatic stromal cells, including SMCs and fibroblasts, diminished and underwent a serial pathological change of atrophy and apoptosis after castration. The atrophic cells were filled with intracellular lipofuscin. The expression of SMC myosin declined after castration, coincident with the increase in TGF-beta mRNA level and decline in bFGF mRNA. In vitro, TGF-beta inhibited the growth of human prostatic stromal cells during exponential growth, while enhancing myosin staining and stimulating the expression of smoothelin in confluent cultured stromal cells. bFGF stimulated the growth of the culture and inhibited the expression of smoothelin. DHT caused a weak increase in the proliferation and expression of SMC-specific proteins (P < 0.05). However, DHT and bFGF together stimulated the proliferation of stromal cells significantly more than either agent alone (P < 0.01). The combination of DHT and TGF-beta greatly enhanced the expression of SMC-specific proteins (P < 0.01), more strongly than either alone (P < 0.01). CONCLUSIONS: The whole prostate gland is an androgen-sensitive organ, with both the epithelium and stroma under the control of androgen. Androgen may direct the proliferation and differentiation of prostatic stromal cells by regulating the expression of TGF-beta and bFGF. Thus DHT, TGF-beta and bFGF may have important roles in regulating stromal cell homeostasis.

Animals↗
PubMed2001

The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway.

Photoreceptor and cone cells in the Drosophila eye are recruited following activation of the epidermal growth factor receptor (EGFR) pathway. We have identified echinoid (ed) as a novel putative cell adhesion molecule that negatively regulates EGFR signaling. The ed mutant phenotype is associated with extra photoreceptor and cone cells. Conversely, ectopic expression of ed in the eye leads to a reduction in the number of photoreceptor cells. ed expression is independent of EGFR signaling and ED is localized to the plasma membrane of every cells throughout the eye disc. We present evidence that ed acts nonautonomously to generate extra R7 cells by a mechanism that is sina-independent but upstream of Tramtrack (TTK88). Together, our results support a model whereby ED defines an independent pathway that antagonizes EGFR signaling by regulating the activity, but not the level, of the TTK88 transcriptional repressor.

Alleles↗
PubMed2000

Regulation of invasive cell behavior by taiman, a Drosophila protein related to AIB1, a steroid receptor coactivator amplified in breast cancer.

Steroid hormones are key regulators of numerous physiological and developmental processes, including metastasis of breast and ovarian cancer. Here we report the identification of a Drosophila gene, named taiman, which encodes a steroid hormone receptor coactivator related to AIB1. Mutations in tai caused defects in the migration of specific follicle cells, the border cells, in the Drosophila ovary. Mutant cells exhibited abnormal accumulation of E-cadherin, beta-catenin, and focal adhesion kinase. TAI protein colocalized with the ecdysone receptor in vivo and augmented transcriptional activation by the ecdysone receptor in cultured cells. The finding of this type of coactivator required for cell motility suggests a novel role for steroid hormones, in stimulating invasive cell behavior, independent of effects on proliferation.

Animals↗
PubMed2000

Predicting durability of a disease resistance gene based on an assessment of the fitness loss and epidemiological consequences of avirulence gene mutation.

Durability of plant disease resistance (R) genes may be predicted if the cost of pathogen adaptation to overcome resistance is understood. Adaptation of the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo), to virulence in rice is the result of the loss of pathogen avirulence gene function, but little is known about its effect on aggressiveness under field conditions. We evaluated the cost in pathogenic fitness (aggressiveness and persistence) associated with adaptation of Xoo to virulence on near-isogenic rice lines with single R genes (Xa7, Xa10, and Xa4) at two field sites endemic for bacterial blight. Disease severity was high in all 3 years on all lines except the line with Xa7. Of two Xoo lineages (groups of strains inferred to be clonally related based on DNA fingerprinting) detected, one, lineage C, dominated the pathogen population at both sites. All Xoo strains were virulent to Xa4, whereas only lineage C strains were virulent to Xa10. Only a few strains of lineage C were virulent to Xa7. Adaptation to virulence on Xa7 occurred through at least four different pathways and was associated with a reduction in aggressiveness. Loss of avirulence and reduced aggressiveness were associated with mutations at the 3' terminus of the avrXa7 allele. Strains most aggressive to Xa7 were not detected after the second year, suggesting they were less persistent than less aggressive strains. These experiments support the prediction that Xa7 would be a durable R gene because of a fitness penalty in Xoo associated with adaptation to Xa7.

Bacterial Proteins↗
PubMed2000

Adenovirus-mediated overexpression of catalase in the cytosolic or mitochondrial compartment protects against cytochrome P450 2E1-dependent toxicity in HepG2 cells.

Cytochrome P450 2E1 (CYP2E1) is an effective producer of reactive oxygen species such as superoxide radical and hydrogen peroxide, which may contribute to the development of alcohol liver disease or cytotoxicity. To investigate the protective role of catalase against CYP2E1-dependent cytotoxicity, E47 cells, a transfected HepG2 cell line overexpressing CYP2E1, were infected with adenoviral vectors containing human catalase cDNA (AdCat) and catalase cDNA with a mitochondrial leader sequence (AdmCat). Forty-eight hours after infection with AdCat or AdmCat at a multiplicity of infection of 100, intracellular catalase protein was increased >2-fold compared with uninfected E47 cells and E47 cells infected with empty adenoviral vector (AdNull) as determined by Western blotting and catalase activity measurements. Overexpression of catalase in the cytosol (AdCat) and in mitochondria (AdmCat) was confirmed by confocal microscopy. Cell death caused by arachidonic acid plus iron was considerably suppressed in both AdCat- and AdmCat-infected E47 cells as determined by assays of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide absorbance, lactate dehydrogenase release, and morphology changes. AdCat- and AdmCat-infected cells were also more resistant to the loss of mitochondrial membrane potential and to the increase in lipid peroxidation induced by arachidonic acid and iron. This study indicates that catalase in the cytosol and catalase in mitochondria are capable of protecting HepG2 cells expressing CYP2E1 against cytotoxicity induced by oxidants that promote lipid peroxidation and suggests the possibility that such agents may be useful in protecting against the development of alcohol liver injury.

Adenoviridae↗
PubMed2000

Geranylgeranylacetone enhances expression of thioredoxin and suppresses ethanol-induced cytotoxicity in cultured hepatocytes.

Geranylgeranylacetone (GGA) has been introduced into the clinical field as an anti-ulcer drug. In addition to protective effects on gastric mucosal cells, GGA also has anti-apoptotic effects against ischemia and reperfusion injury in hepatocytes and intestinal cells. However, the molecular mechanisms of the cytoprotective or anti-apoptotic effect of GGA are largely unknown. To explore the molecular mechanism of GGA action, we focused on thioredoxin (TRX), an endogenous-redox-acting molecule. We have demonstrated that GGA induces the messenger RNA and protein of TRX and affects the activation of transcription factors, AP-1 and NF-kappaB, and that GGA blunted ethanol-induced cytotoxicity of cultured hepatocytes. These results provide evidence suggesting that a possible novel molecular mechanism of GGA is to protect cells via the induction of TRX and the activation of transcription factors such as NF-kappaB and AP-1.

Animals↗
PubMed2000

Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism.

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a cytoplasmic domain largely comprised of two C2 domains designated C2A and C2B. We have determined how deep the Ca(2+)-binding loops of Ca(2+).C2A penetrate into the lipid bilayer and report mutations in synaptotagmin that can uncouple membrane penetration from Ca(2+)-triggered interactions with the SNARE complex. To determine whether C2A penetrates into the vesicle ("cis") or plasma ("trans") membrane, we reconstituted a fragment of synaptotagmin that includes the membrane-spanning and C2A domain (C2A-TMR) into proteoliposomes. Kinetics experiments revealed that cis interactions are rapid (< or =500 micros). Binding in the trans mode was distinguished by the slow diffusion of trans target vesicles. Both modes of binding were observed, indicating that the linker between the membrane anchor and C2A domain functions as a flexible tether. C2A-TMR assembled into oligomers via a novel N-terminal oligomerization domain suggesting that synaptotagmin may form clusters on the surface of synaptic vesicles. This novel mode of clustering may allow for rapid Ca(2+)-triggered oligomerization of the protein via the membrane distal C2B domain.

Acrylamide↗
PubMed2000

Approaches to functional genomics: potential of matrix-assisted laser desorption ionization--time of flight mass spectrometry combined with separation methods for the analysis of DNA in biological samples.

MALDI-TOF MS has potential as a valuable technique in DNA mapping studies and may well be complementary to other approaches to DNA analysis such as gel electrophoresis and sequencing. This study used 2,6-dihydroxyacetophenone (DHAP) mixed with diammonium hydrogen citrate (DAHC) as the matrix. In addition, recent technical advances such as time lag focussing (TLF) and better selection of matrices (such as 3-hydroxypicolinic acid (3 HPA) and picolinic acid (PA)) extended the range of DNA fragments that can be studied by this approach. The following samples were investigated: Poly-T mixture (dT 15, 19, 20, 25, 74 and 75), plasmid pBR322 derived oligonucleotides (10, 11, 12, 13, 14, 15, 19, 20 and 50 nucleotides long) and DNA fragments of 25, 36 and 37 base pairs corresponding to a fragment in the restriction map for the gene corresponding to the hexon protein of Adenovirus 2 and 5. The results were contrasted with similar analyses performed by ion-paired reversed-phase HPLC coupled to on-line electrospray mass spectrometry.

Chromatography, High Pressure Liquid↗
PubMed2000

Overexpression of catalase in the mitochondrial or cytosolic compartment increases sensitivity of HepG2 cells to tumor necrosis factor-alpha-induced apoptosis.

The sensitivity of HepG2 cells overexpressing catalase in either the cytosolic or mitochondrial compartment to tumor necrosis factor-alpha (TNF-alpha) and cycloheximide was studied. Cells overexpressing catalase in the cytosol (C33 cells) and especially in mitochondria (mC5 cells) were more sensitive to TNF-alpha-induced apoptosis than were control cells (Hp cells). The activities of caspase-3 and -8 were increased by TNF-alpha, with the highest activities found in mC5 cells. Sodium azide, an inhibitor of catalase, reduced the increased sensitivity of mC5 and C33 cells to TNF-alpha to the level of toxicity found with control Hp cells. Azide also decreased the elevated caspase-3 activity of mC5 cells. A pan-caspase inhibitor prevented the TNF-alpha-induced apoptosis and toxicity produced by catalase overexpression. Addition of H(2)O(2) prevented TNF-alpha-induced apoptosis and caspase activation, an effect prevented by simultaneous addition of catalase. TNF-alpha plus cycloheximide increased ATP levels, with higher levels in C33 and mC5 cells compared with Hp cells. TNF-alpha did not produce apoptosis in mC5 cells maintained in a low energy state. TNF-alpha signaling was not altered by the overexpression of catalase, as activation of nuclear factor kappaB and AP-1 by TNF-alpha was similar in the three cell lines. These results suggest that catalase, overexpressed in the cytosolic or especially the mitochondrial compartment, potentiates TNF-alpha-induced apoptosis and activation of caspases by removal of H(2)O(2).

Adenosine Triphosphate↗
PubMed2000

Characterization of anti-CCR5 ribozyme-transduced CD34+ hematopoietic progenitor cells in vitro and in a SCID-hu mouse model in vivo.

The cellular entry of HIV is mediated by the specific interaction of viral envelope glycoproteins with the cell-surface marker CD4 and a chemokine receptor (CCR5 or CXCR4). Individuals with a 32-base-pair (bp) deletion in the CCR5 coding region, which results in a truncated peptide, show resistance to HIV-1 infection. This suggests that the downregulation of CCR5 expression on target cells may prevent HIV infection. Therefore, ribozymes that inhibit the CCR5 expression offer a novel approach for anti-HIV gene therapy. To assess the effect of an anti-CCR5 ribozyme (R5Rbz) on macrophage differentiation, CD34+ hematopoietic progenitor cells were transduced with a retroviral vector carrying RSRbz and allowed to differentiate in the presence of appropriate cytokines. R5Rbz-transduced CD34+ cells differentiated normally into mature macrophages that carried CD14 and CD4 surface markers, expressed the anti-CCR5 ribozyme, and showed significant resistance to viral infection upon challenge with the HIV-1 BaL strain. Using an in vivo thymopoiesis model, the effect of RSRbz on stem cell differentiation into thymocytes was evaluated by reconstituting SCID-hu mice thymic grafts with ribozyme-transduced CD34+ cells. FACS analysis of cell biopsies at 4 and 6 weeks postengraftment for HLA, CD4, and CD8 markers showed comparable levels of reconstitution and similar percentages of subpopulations of thymocytes between grafts receiving R5Rbz-transduced and control CD34+ cells. RT-PCR assays demonstrated the expression of the anti-CCR5 ribozyme in CD4+, CD8+, and CD4+/CD8+ thymocyte subsets derived from RSRbz-transduced CD34+ cells. These results indicate that anti-CCR5 ribozyme can be introduced into hematopoietic stem cells without adverse effects on their subsequent lineage-specific differentiation and maturation. The expression of anti-CCR5 ribozymes in HIV-1 target cells offers a novel gene therapy strategy to control HIV infection.

Animals↗