Biomedical subjects

Hua Zhu

Publications and source records attributed to Hua Zhu.

At least 37 records · Page 2Linked to original sources

PubMed2005

Ab initio intermolecular potential-energy surface and microwave spectra for the Ne-OCS complex.

An ab initio potential-energy surface for the Ne-OCS complex was calculated using the coupled-cluster singles and doubles with noniterative inclusion of connected triples [CCSD(T)] with a large basis set containing bond functions. The interaction energies were obtained by the supermolecular approach with the full counterpoise correction for the basis set superposition error. The CCSD(T) potential was found to have three minima corresponding to the T-shaped and the linear Ne-SCO and Ne-OCS structures. The two-dimensional discrete variable representation method was employed to calculate the rovibrational energy levels for five isotopomers Ne-OCS, (22)Ne-OCS, Ne-OC(34)S, Ne-O(13)CS, and Ne-(18)OCS. The calculated pure rotational transition frequencies for the vibrational ground state of the five isotopomers are in good agreement with the observed values. The corresponding microwave spectra show that the b-type transitions (deltaK(a)=+/-1) are significantly stronger than the a-type transitions (deltaK(a)=0).

Journal Article↗

PubMed2005

The characterization of plasma membrane Ca2+-ATPase in rich sphingomyelin-cholesterol domains.

According to the raft hypothesis, sphingolipid-cholesterol (CHOL) microdomains are involved in numerous cellular functions. Here, we have prepared liposomes to simulate the lipid composition of rafts/caveolae using phosphatidylchone, sphingomyelin (SPM)-CHOL in vitro. Experiments of both 1,6-diphenyl-1,3,5-hexatriene and merocyanine-540 fluorescence showed that a phase transition from l(d) to l(o) can be observed clearly. In particular, we investigated the behavior of a membrane protein, plasma membrane Ca(2+)-ATPase (PMCA), in lipid rafts (l(o) phase). Three complementary approaches to characterize the physical appearance of PMCA were employed in the present study. Tryptophan intrinsic fluorescence increase, fluorescence quenching by both acrylamid and hypocrellin B decrease, and MIANS fluorescence decrease, indicate that the conformation of PMCA embedded in lipid l(o) phase is more compact than in lipid l(d) phase. Also, our results showed that PMCA activity decreased with the increase of SPM-CHOL content, in other words, with the increase of l(o) phase. This suggests that the specific domains containing high SPM-CHOL concentration are not a favorable place for PMCA activity. Finally, a possible explanation about PMCA molecules concentrated in caveolae/rafts was discussed.

Acrylamide↗

PubMed2005

An animal model of SARS produced by infection of Macaca mulatta with SARS coronavirus.

A new SARS animal model was established by inoculating SARS coronavirus (SARS-CoV) into rhesus macaques (Macaca mulatta) through the nasal cavity. Pathological pulmonary changes were successively detected on days 5-60 after virus inoculation. All eight animals showed a transient fever 2-3 days after inoculation. Immunological, molecular biological, and pathological studies support the establishment of this SARS animal model. Firstly, SARS-CoV-specific IgGs were detected in the sera of macaques from 11 to 60 days after inoculation. Secondly, SARS-CoV RNA could be detected in pharyngeal swab samples using nested RT-PCR in all infected animals from 5 days after virus inoculation. Finally, histopathological changes of interstitial pneumonia were found in the lungs during the 60 days after viral inoculation: these changes were less marked at later time points, indicating that an active healing process together with resolution of an acute inflammatory response was taking place in these animals. This animal model should provide insight into the mechanisms of SARS-CoV-related pulmonary disease and greatly facilitate the development of vaccines and therapeutics against SARS.

Animals↗

PubMed2005

Increased lungkine and chitinase levels in allergic airway inflammation: a proteomics approach.

Asthma is a chronic inflammatory disease characterized by pulmonary eosinophilia and airway hyperresponsiveness. Mechanisms underlying the pathogenesis of asthma are still not fully understood. The present study investigated alterations in global protein expression in bronchoalveolar lavage fluid in allergic airway inflammation using a proteomics approach. BALB/c mice sensitized and challenged with ovalbumin developed airway eosinophilia, mucus hypersecretion, elevation of immunoglobulin E, and airway hyperresponsiveness. Lavage fluid proteins from normal and asthmatic mice were resolved by two-dimensional gel electrophoresis, and identified by peptide mass fingerprinting matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A total of 28 protein spots were significantly altered. Several of these proteins were undetectable or at very low levels in normal mice but were significantly increased in airway inflammation. These include lungkine, a recently described chemokine, a family of chitinases including Ym1, Ym2, and acidic mammalian chitinase, gob-5, a protein that mediates mucus secretion, and surfactant protein-D, a C-type lectin capable of modulating inflammatory responses. Overall, proteomics is a powerful tool in unraveling protein expression changes in allergic airway inflammation. The proteins identified in this study may be associated with the pathogenesis of allergic airway inflammation and may also be found useful as surrogate biomarkers for asthma.

Animals↗

PubMed2005

Tissue factor-dependent coagulation is preferentially up-regulated within arterial branching areas in a baboon model of Escherichia coli sepsis.

Endothelium plays a critical role in the pathobiology of sepsis by integrating systemic host responses and local rheological stimuli. We studied the differential expression and activation of tissue factor (TF)-dependent coagulation on linear versus branched arterial segments in a baboon sepsis model. Animals were injected intravenously with lethal doses of Escherichia coli or saline and sacrificed after 2 to 8 hours. Whole-mount arterial segments were stained for TF, TF-pathway inhibitor (TFPI), factor VII (FVII), and markers for endothelial cells (ECs), leukocytes, and platelets, followed by confocal microscopy and image analysis. In septic animals, TF localized preferentially at branches, EC surface, leukocytes, and platelet aggregates and accumulated in large amounts in the subendothelial space. FVII strongly co-localized with TF on ECs and leukocytes but less so with subendothelial TF. TFPI co-localized with TF and FVII on endothelium and leukocytes but not in the subendothelial space. Focal TF increases correlated with fibrin deposition and increased endothelial permeability to plasma proteins. Biochemical analysis confirmed that aortas of septic baboons expressed more TF mRNA and protein than controls. Branched segments contained higher TF protein levels and coagulant activity than equivalent linear areas. These data suggest that site-dependent endothelial heterogeneity and rheological factors contribute to focal procoagulant responses to E. coli.

Animals↗

PubMed2005

Effects of alpha-toxin-deficient Staphylococcus aureus on the production of peripheral corneal ulceration in an animal model.

PURPOSE: To examine the role of Staphylococcus aureus alpha-toxin in contact lens-induced peripheral ulceration (CLPU). MATERIALS AND METHODS: Proteolytic enzyme, hyaluronidase, alpha-toxin, and beta-toxin production by S. aureus 8325-4 and its alpha-toxin-deficient mutant (S. aureus DU1090) were examined. Using a rabbit model of CLPU, animals were fitted with hydrogel contact lenses colonized by either S. aureus 8325-4 or the mutant strain. The clinical presentation, bacterial cultures, and histology of the ulceration were examined. RESULTS: Both strains of S. aureus produced similar levels of caseinase, gelatinase, elastase, hyaluronidase, and beta-toxin. S. aureus DU1090 induced weaker haemolysis of rabbit blood cells than S. aureus 8325-4. Ulceration in the S. aureus DU1090 eye was less frequent and less severe than that caused by S. aureus 8325-4. CONCLUSIONS: The enzyme production profile of S. aureus DU1090 was similar to the parent strain. S. aureus strains may produce CLPU-like lesions irrespective of alpha-toxin production, but severe infectious lesions are produced only in the presence of alpha-toxin.

Animals↗

PubMed2005

Human cytomegalovirus genes in the 15-kilobase region are required for viral replication in implanted human tissues in SCID mice.

Since animal models for studying human cytomegalovirus (HCMV) replication in vivo and pathogenesis are not available, severe combined immunodeficiency mice into which human tissues were implanted (SCID-hu mice) provide an alternative and valuable model for such studies. The HCMV clinical isolates, including those of the Toledo strain, replicate to high titers in human tissue implanted into SCID mice; however, the attenuated AD169 strain has completely lost this ability. The major difference between Toledo and AD169 is a 15-kb segment, encoding 19 open reading frames, which is present in all virulent strains but deleted from attenuated strains. This fact suggests that crucial genes required for HCMV replication in vivo are localized to this region. In this study, the importance of this 15-kb segment for HCMV replication in vivo was determined. First, Toledo(BAC) virus (produced from a Toledo bacterial artificial chromosome) and AD169 virus were tested for growth in SCID-hu mice. Toledo(BAC), like Toledo, grew to high titers in implanted human thymus and liver tissues, while AD169 did not. This outcome showed that the Toledo genome propagated in bacteria (Toledo(BAC)) retained its virulence. The 15-kb segment was then deleted from Toledo(BAC), and the resulting virus, Toledo(Delta15kb), was tested for growth in both human foreskin fibroblast (HFF) cells and SCID-hu mice. Toledo(Delta15kb) had a minor growth defect in HFF but completely failed to replicate in human thymus and liver implants. This failure to grow was rescued when the 15-kb region was inserted back into the Toledo(Delta15kb) genome. These results directly demonstrated that the genes located in the 15-kb segment are crucial for HCMV replication in vivo.

Animals↗

PubMed2005

Characterization of the elements and proteins responsible for interferon-stimulated gene induction by human cytomegalovirus.

Human cytomegalovirus (HCMV) infection of human fibroblast cells activates a large number of interferon-stimulated genes (ISGs) in a viral envelope-cell membrane fusion-dependent mechanism. In this study, we identified two interferon response elements, the interferon-stimulated response element (ISRE) and the gamma interferon-activated site (GAS), which act as HCMV response sites (VRS). Gel mobility shift assays showed that cellular proteins form specific and identical complexes with ISRE and GAS elements, and the binding of these complexes to ISRE and GAS is stimulated by HCMV infection. Point mutations in the consensus sequences of ISRE and GAS completely abolished their activities in response to HCMV-mediated transactivation, as well as their abilities to interact with HCMV-activated VRS-binding proteins. Interferon regulatory factor 3 does not appear to be present in the VRS-binding complexes or to be involved directly in HCMV-mediated ISG activation. Using ProteinChip technology, four potential proteins were identified, ranging from 20 to 42 kDa, in the VRS-binding complexes. The data suggest that HCMV infection activates VRS-binding proteins, which then bind to the VRS and stimulate ISG expression.

Base Sequence↗

PubMed2005

Two gamma interferon-activated site-like elements in the human cytomegalovirus major immediate-early promoter/enhancer are important for viral replication.

Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.

Base Sequence↗

PubMed2004

A theoretical study of solvent effects on tautomerism and electronic absorption spectra of 3-hydroxy-2-mercaptopyridine and 2,3-dihydroxypyridine.

The tautomerization equilibria of 3-hydroxy-2-mercaptopyridine (HMP) and 2,3-dihydroxypyridine (DHP) in vacuo and in ethanol solution have been studied using the density functional theory (DFT) at B3LYP/6-31Gd level. The results indicate that the thione form of HMP and the keto form of DHP are the most stable tautomers in the equilibrium, and the energy barrier for the thiol-thione and enol-keto proton transfer decreases significantly when the tautomerism is mediated by a specific ethanol molecule in solution. The time-dependent density functional theory--polarizable continuum model (TDDFT-PCM) calculations on all tautomers of HMP and DHP in vacuo and in ethanol have assigned the lowest pi --> pi* excitations of thione and keto tautomers to the observed absorption bands of HMP and DHP in solutions. The solvation is predicted to have relatively small effect on these pi --> pi* excitations in ethanol.

Journal Article↗

PubMed2004

Suppression of P-glycoprotein gene expression in Hs578T/Dox by the overexpression of caveolin-1.

Caveolin-1, the principal component of caveolae, is a 21-24 kDa integral membrane protein. The interaction of the caveolin-1 scaffolding domain with signaling molecules can functionally inhibit the activity of these signaling proteins. Little is known about how caveolin-1 influences the expression of P-glycoprotein (P-gp), an ABC transporter encoded by multi-drug resistance (MDR1) gene. To elucidate the possible mechanism between caveolin-1 and P-gp expression, in the present study, we overexpressed caveolin-1 in the Hs578T/Dox breast adenocarcinoma cells, a multidrug resistant line, and then selected single clone cells highly expressing caveolin-1 level. Both Western blot and confocal microscopy analyses showed that caveolin-1 was markedly overexpressed in the transfectants, while P-gp protein was almost abolished. Reverse transcription polymerase chain reaction also showed that the expression of P-gp mRNA was significantly suppressed in the transfectants. It was confirmed further by Northern blot analysis. Moreover, through measuring the changes of drug resistance and P-gp transport activity in the transfectants, we found that overexpression of caveolin-1 reversed drug resistance of transfectants and lowered their P-gp transport activity to the level of Hs578T/S. Taken together, our results indicate that such suppression of P-gp in the transfectants overexpressing caveolin-1 may occur at the transcriptional level.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Overexpression of caveolin-1 increases plasma membrane fluidity and reduces P-glycoprotein function in Hs578T/Dox.

Cholesterol is a key lipid in mediating the enzyme activity or signaling pathway of many proteins on the plasma membrane in mammalian cells. In this report, we demonstrate for the first time that after overexpressing caveolin-1, the plasma membrane cholesterol level was decreased by about 12% and 30% for doxorubicin-sensitive and doxorubicin-resistant Hs578T breast cancer cells, respectively. However, the total cholesterol level in both cell lines was increased by about 10%. By measuring fluorescence and flow cytometry using the fluorescence dyes 1,6-diphenyl-1,3,5-hexatriene and Merocyanine 540, we found that overexpressing caveolin-1 resulted in a similar increase in membrane fluidity and loosening of lipid packing density as cholesterol depletion by 1 mM methyl-beta-cyclodextrin (MbetaCD) or 2-hydroxypropyl-beta-cyclodextrin (HbetaCD). Moreover, we found that the transport activity of P-gp was significantly inhibited by 1 mM MbetaCD or HbetaCD, which is also similar to the inhibitory effect of caveolin-1 overexpression. Our data demonstrate for the first time that the reduction of the plasma membrane cholesterol level induced by overexpressing caveolin-1 may indirectly inhibit P-gp transport activity by increasing plasma membrane fluidity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Coupling generation of cytomegalovirus deletion mutants and amplification of viral BAC clones.

Human cytomegalovirus (HCMV) genome manipulation has always been difficult. Recently, the introduction of full-length HCMV DNA into Escherichia coli as an artificial bacterial chromosome (BAC) clone has allowed reliable targeted mutagenesis. Here, we show the next generation of improvement in designing recombinant HCMV, which will also be applicable to other viral BAC clones. An inducible origin of replication linked with an antibiotic resistance marker was used as a cassette for targeted replacement of sequences within a HCMV BAC clone, TowneBAC. The origin of replication allowed for the induction of increased amounts BAC DNA that improved recovery, ease of use and transfections for mutant viruses. By specific deletion of UL147 and the recombinant GFP gene, we have shown that targeted deletion of a gene and selection for a recombinant genome are coupled with the ability to amplify the BAC clone DNA. These HCMV BAC clones were amplified approximately 10-fold. In the case of the removal of GFP from the clone TowneBAC shown in this study, the resulting BAC DNA preparation following amplification was used for successful primary cell transfection. Both parental and deletion BAC clone transfections gave similar levels of recombinant HCMV, and the GFP deletion virus replicated the same as the TowneBAC in a multi-step growth curve analysis.

Cells, Cultured↗

PubMed2004

Effect of interleukin-7 gene transfection into ovarian carcinoma cell line SKOV3 in vitro and in vivo.

OBJECTIVE: To investigate the effect of interleukin-7 (IL-7) gene transfection into an established ovarian carcinoma cell line (SKOV3) in vitro and evaluate the tumorigenicity of SKOV3-IL-7 in severe combined immunodeficient (SCID) mice. METHODS: IL-7 gene was transfected into SKOV3 cells by liposome. IL-7 mRNA and protein of SKOV3-IL-7 and their parental control cells were detected by reverse transcriptive-polymerase chain reaction (RT-PCR) and Western blot, respectively. The levels of IL-7, IL-2, TNFalpha and TGFbeta1 in the supernatant were detected by ELISA. The cell cycle, HLA-ABC, HLA-DR and ICAM-1 expressions were assayed by flow cytometry. The sensitivities of tumor cells to lymphokine-activated killer (LAK) cells were measured by lactate dehydrogenase (LDH) release assay. Tumorigenicities of SKOV3-IL-7 and their parental cells in SCID mice were evaluated by macro- and histological examination, while IL-7 expression and secretion were detected by immunohistochemistry and ELISA. RESULTS: IL-7 mRNA and protein were detectable in SKOV3-IL-7 only. ICAM-1 expression was significantly higher and TGFbeta1 level in culture supernatants was significantly decreased in SKOV-IL-7, all other variables remained unchanged. The proliferative activity and cell cycle of SKOV3-IL-7 were unchanged. The cytotoxic sensitivity of SKOV3-IL-7 to LAK cells was significantly higher. Both gene-transfected and nontransfected SKOV3 cells were successfully inoculated into the peritoneal cavities of SCID mice. IL-7 proteins in plasmas and tumor tissues were detectable only in SCID mice inoculated with SKOV3-IL-7. The IL-7 engineered murine tumor models revealed better general aspects, reduced tumor development and dissemination. CONCLUSIONS: IL-7 gene transfection into SKOV3 cells downregulates TGFbeta1 secretion, upregulates ICAM-1 expression and enhances sensitivity to LAK cells in vitro. The tumorigenicity of IL-7 engineered cells in SCID mice is reduced. These findings may offer support to the development of cytokine gene therapy for ovarian carcinoma.

Animals↗

PubMed2004

Introns and splicing elements of five diverse fungi.

Genomic sequences and expressed sequence tag data for a diverse group of fungi (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Aspergillus nidulans, Neurospora crassa, and Cryptococcus neoformans) provided the opportunity to accurately characterize conserved intronic elements. An examination of large intron data sets revealed that fungal introns in general are short, that 98% or more of them belong to the canonical splice site (ss) class (5'GU...AG3'), and that they have polypyrimidine tracts predominantly in the region between the 5' ss and the branch point. Information content is high in the 5' ss, branch site, and 3' ss regions of the introns but low in the exon regions adjacent to the introns in the fungi examined. The two yeasts have broader intron length ranges and correspondingly higher intron information content than the other fungi. Generally, as intron length increases in the fungi, so does intron information content. Homologs of U2AF spliceosomal proteins were found in all species except for S. cerevisiae, suggesting a nonconventional role for U2AF in the absence of canonical polypyrimidine tracts in the majority of introns. Our observations imply that splicing in fungi may be different from that in vertebrates and may require additional proteins that interact with polypyrimidine tracts upstream of the branch point. Theoretical protein homologs for Nam8p and TIA-1, two proteins that require U-rich regions upstream of the branch point to function, were found. There appear to be sufficient differences between S. cerevisiae and S. pombe introns and the introns of two filamentous members of the Ascomycota and one member of the Basidiomycota to warrant the development of new model organisms for studying the splicing mechanisms of fungi.

Aspergillus nidulans↗

PubMed2004

Postattachment events associated with viral entry are necessary for induction of interferon-stimulated genes by human cytomegalovirus.

Utilizing a human cytomegalovirus-specific fusion inhibitor and an antiglycoprotein H antibody, we studied the role of virion fusion in interferon-stimulated gene (ISG) induction. Our results indicate that ISG induction does not occur when virion-mediated, post-high-affinity attachment events are inhibited by either reagent. Thus, virion-mediated postattachment events, such as fusion, are required for ISG induction.

Cells, Cultured↗

PubMed2004

Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection.

PURPOSE: To understand the importance of Pseudomonas aeruginosa quorum-sensing systems in the development of corneal infection, the genotypic characteristics and pathogenesis of seven ocular isolates with low-protease and acyl homoserine lactone (AHL) activity and quorum-sensing mutants of PAO1 deficient in lasI, lasR, or rhlR were investigated in the study. METHODS: The possession of the quorum-sensing genes lasI, lasR, rhlI, rhlR, and the quorum-sensing controlled genes lasB, aprA, and rhlAB in the clinical isolates were determined by polymerase chain reaction and Southern blot hybridization. Elastinolytic activity, controlled by the las system, was assayed using elastin Congo red and rhamnolipid production controlled by the rhl system was assessed using agar plates containing methylene blue/cetyltrimethyl ammonium bromide. Induction of keratitis was examined in a scarified inbred BALB/c mouse model. RESULTS: The clinical isolates Paer1 and -3 were lasI and lasR negative, and the isolates Paer2 and -4 were rhlR and rhlAB negative. The isolates Paer17, Paer26, 6294 and 6206 possessed all the genes examined. There was no rhamnolipid production in clinical isolates Paer2 and -4. The isolates Paer1 and -3 were virtually avirulent in the scarified mouse corneas. Using isogenic PAO1 mutants, strain lasI showed a markedly reduced virulence in the corneal infection model. The remainder of the clinical isolates and the lasR or rhlR mutant strains caused severe keratitis. CONCLUSIONS: These results indicate that quorum-sensing deficiency may occur naturally in clinical isolates, and the possession of lasI and hence a functional Las quorum-sensing system may be important in development of corneal infection.

Animals↗

PubMed2004

Experimental study on transient behavior of semi-open two-phase thermosyphon.

An experimental system was set up to measure the temperature, pressure, heat transfer rate and mass flow rate in a semi-open two-phase thermosyphon. The behaviors of a semi-open two-phase thermosyphon during startup, shutdown and lack of water were studied to get complete understanding of its thermal characteristics. The variation of wall temperature, heat-exchange condition and pressure fluctuations of semi-open two-phase thermosyphons showed that the startup of SOTPT needs about 60-70 min; the startup speed of SOTPT is determined by the startup speed of the condensation section; the average pressure in the heat pipe is equal to the environmental pressure usually; the shutdown of SOTPT needs about 30-50 min; a semi-open two-phase thermosyphon has good response to lack of water accident.

Energy Transfer↗