Biomedical subjects

Jun Zhang

Publications and source records attributed to Jun Zhang.

At least 37 records · Page 2Linked to original sources

PubMed2006

Altered expression of FANCL confers mitomycin C sensitivity in Calu-6 lung cancer cells.

Fanconi anemia (FA) results from mutations in a group of genes whose products, including BRCA2 and BACH1/BRIP1, are known to function in one common pathway (the FA-BRCA pathway) to guard genome integrity, especially when challenged by DNA crosslinking agents, such as Cisplatin and mitomycin C (MMC). The extremely high incidence of cancer in FA patients reveals the essentialness of this pathway in tumor suppression. However, this pathway's involvement in nonFA cancers is not well understood. To evaluate the contribution of the FA-BRCA pathway to cancer, we investigated the integrity of the FA-BRCA pathway in ten human cancer cell lines. We found that the Calu-6 lung cancer cell line carries a defective FA-BRCA pathway. In this cell line, the examination of six FA proteins, essential for the activation of the FA-BRCA pathway, detected substantially reduced expression of FANCL, a catalytic subunit of the ubiquitin ligase/E3-complex. Reconstitution of FANCL in these cells restored the activation of the FA-BRCA pathway, but MMC sensitivity of the cells with a complemented FA-BRCA pathway was decreased as compared to the cells with an impaired FA-BRCA pathway. Collectively, the abnormal FANCL expression is the cause leading to a defective FA-BRCA pathway, which confers the sensitivity of Calu-6 cells to MMC. This suggests that the correlation of an intact FA-BRCA pathway with MMC resistance may emerge as a common mechanism underlying resistance to DNA crosslinking agents in cancer patients.

Alternative Splicing↗

PubMed2006

Effect of genetic variants of the human flavin-containing monooxygenase 3 on N- and S-oxygenation activities.

The decreased capacity of the flavin-containing monooxygenase 3 (FMO3) to oxygenate xenobiotics including trimethylamine is believed to contribute to metabolic disorders. The aim of this study was to functionally characterize FMO3 variants recently found in a Japanese population and compare them with selective functional activity of other FMO3 variants. Recombinant Glu158Lys and Glu158Lys-Glu308Gly FMO3 expressed in Escherichia coli membranes showed slightly decreased N-oxygenation of benzydamine and trimethylamine. Selective functional S-oxygenation of these variants by methyl p-tolyl sulfide or sulindac sulfide was comparable to that of wild-type FMO3. The Glu158Lys-Thr201Lys-Glu308Gly and Val257Met-Met260Val variants showed significantly decreased oxygenation of typical FMO3 substrates (i.e., approximately one-tenth of the V(max)/K(m) values). Val257Met FMO3 had a lower catalytic efficiency for methyl p-tolyl sulfide and sulindac sulfide S-oxygenation. However, compared with wild-type FMO3, Val257Met FMO3 showed a similar catalytic efficiency for N-oxygenation of benzydamine and trimethylamine. The catalytic efficiency for benzydamine and trimethylamine N-oxygenation by Arg205Cys FMO3 was only moderately decreased, but it possessed decreased sulindac sulfide S-oxygenation activity. Kinetic analysis showed that Arg205Cys FMO3 was inhibited by sulindac in a substrate-dependent manner, presumably because of selective interaction between the variant enzyme and the substrate. The results suggest that the effects of genetic variation of human FMO3 could operate at the functional level for N- and S-oxygenation for typical FMO3 substrates. Genetic polymorphism in the human FMO3 gene might lead to unexpected changes of catalytic efficiency for N- and S-oxygenation of xenobiotics and endogenous materials.

Benzydamine↗

PubMed2006

Template-free preparation of bunches of aligned boehmite nanowires.

A simple method based on a hydrothermal process using alkali salts as mineralizers is proposed for the synthesis of aligned bunches of boehmite (gamma-AlOOH) nanowires without a template's assistance. Most bunches of aligned boehmite nanowires are constructed by two separated shorter bundles with widths of 700 to approximately 800 nm and lengths of about 1 microm. XRD patterns, FTIR spectra, and SEM and TEM images were used to characterize the products. The specific surface area and pore-size distribution of the obtained product as determined by gas-sorption measurements show that the boehmite bundles possess a high BET surface area and porosity properties. The importance of adding Na2B4O7 salts for the formation of bundle morphologies has been discussed.

Journal Article↗

PubMed2006

RUNX3 inhibits the expression of vascular endothelial growth factor and reduces the angiogenesis, growth, and metastasis of human gastric cancer.

PURPOSE: Recent studies indicated that RUNX3 exhibits potent antitumor activity. However, the underlying molecular mechanisms of this activity remain unclear. In the present study, we used a gastric cancer model to determine the effect of RUNX3 expression on tumor angiogenesis. EXPERIMENTAL DESIGN: The effects of increased RUNX3 expression on vascular endothelial growth factor (VEGF) expression in and angiogenic potential of human gastric cancer cells were determined in vitro and in animal models. RUNX3 and VEGF expression was determined in 120 human gastric cancer specimens and their relationship was analyzed. RESULTS: RUNX3 gene transfer suppressed VEGF expression in human gastric cancer cells. Down-regulation of VEGF expression correlated with a significantly impaired angiogenic potential of human gastric cancer cells. Furthermore, RUNX3 restoration inhibited tumor growth and metastasis in animal models, which was consistent with inhibition of angiogenesis as determined by evaluating VEGF expression and tumor microvessel formation. In gastric cancer specimens, loss or decrease in RUNX3 expression inversely associated with increased VEGF expression and elevated microvessel formation. CONCLUSIONS: Our clinical and experimental data provide a novel molecular mechanism for the antitumor activity of RUNX3 and may help design effective therapy targeting RUNX3 pathway to control gastric cancer growth and metastasis.

Adenocarcinoma↗

PubMed2006

Histone deacetylation is involved in activation of CXCL10 upon IFNgamma stimulation.

Histone deacetylase (HDAC) activity is commonly associated with transcriptional repression. However, there is also evidence for a function in transcriptional activation. Previous studies have demonstrated a fundamental role of deacetylase activity in IFNa-responsive gene transcription. In the case of type II IFN (IFNgamma) results are controversial: some genes require HDAC activity, while transcription of others is repressed by HDAC. To investigate the effect of HDAC on transcription of an IFNgamma-activated gene, real-time PCR was used to measure CXCL10 mRNA in Hela cells stimulated with IFNgamma in the presence or absence of the HDAC inhibitor TSA. Chromatin imunoprecipitation combined with real-time PCR was used to check acetylation of histone H4 and recruitment of the STAT1 complex to the ISRE locus of the CXCL10 gene. Activation of CXCL10 transcription in response to IFNgamma was paralleled by a decrease in histone H4 acetylation and an increase in recruitment of the STAT1 complex to the CXCL10 ISRE locus. The transcription of CXCL10 and histone H4 deacetylation were blocked by TSA, but the latter had no obvious affect on recruitment of the STAT1 complex. Our data indicate that IFNgamma and STAT-dependent gene transcription requires the participation of HDAC, as does the IFNalpha-STAT pathway.

Acetylation↗

PubMed2006

Distinct perisynaptic and synaptic localization of NMDA and AMPA receptors on ganglion cells in rat retina.

At most excitatory synapses, AMPA and NMDA receptors (AMPARs and NMDARs) occupy the postsynaptic density (PSD) and contribute to miniature excitatory postsynaptic currents (mEPSCs) elicited by single transmitter quanta. Juxtaposition of AMPARs and NMDARs may be crucial for certain types of synaptic plasticity, although extrasynaptic NMDARs may also contribute. AMPARs and NMDARs also contribute to evoked EPSCs in retinal ganglion cells (RGCs), but mEPSCs are mediated solely by AMPARs. Previous work indicates that an NMDAR component emerges in mEPSCs when glutamate uptake is reduced, suggesting that NMDARs are located near the release site but perhaps not directly beneath in the PSD. Consistent with this idea, NMDARs on RGCs encounter a lower glutamate concentration during synaptic transmission than do AMPARs. To understand better the roles of NMDARs in RGC function, we used immunohistochemical and electron microscopic techniques to determine the precise subsynaptic localization of NMDARs in RGC dendrites. RGC dendrites were labeled retrogradely with cholera toxin B subunit (CTB) injected into the superior colliculus (SC) and identified using postembedding immunogold methods. Colabeling with antibodies directed toward AMPARs and/or NMDARs, we found that nearly all AMPARs are located within the PSD, while most NMDARs are located perisynaptically, 100-300 nm from the PSD. This morphological evidence for exclusively perisynaptic NMDARs localizations suggests a distinct role for NMDARs in RGC function.

Animals↗

PubMed2006

Impaired regulation of NF-kappaB and increased susceptibility to colitis-associated tumorigenesis in CYLD-deficient mice.

Cylindromatosis (CYLD) is a deubiquitinating enzyme that is altered in patients with familial cylindromatosis, a condition characterized by numerous benign adnexal tumors. However, the regulatory function of CYLD remains unsettled. Here we show that the development of B cells, T cells, and myeloid cells was unaffected in CYLD-deficient mice, but that the activation of these cells with mediators of innate and adaptive immunity resulted in enhanced NF-kappaB and JNK activity associated with increased TNF receptor-associated factor 2 (TRAF2) and NF-kappaB essential modulator (NEMO) ubiquitination. CYLD-deficient mice were more susceptible to induced colonic inflammation and showed a dramatic increase in the incidence of tumors compared with controls in a colitis-associated cancer model. These results suggest that CYLD limits inflammation and tumorigenesis by regulating ubiquitination in vivo.

Animals↗

PubMed2006

Mechanism of template-independent nucleotide incorporation catalyzed by a template-dependent DNA polymerase.

Numerous template-dependent DNA polymerases are capable of catalyzing template-independent nucleotide additions onto blunt-end DNA. Such non-canonical activity has been hypothesized to increase the genomic hypermutability of retroviruses including human immunodeficiency viruses. Here, we employed pre-steady state kinetics and X-ray crystallography to establish a mechanism for blunt-end additions catalyzed by Sulfolobus solfataricus Dpo4. Our kinetic studies indicated that the first blunt-end dATP incorporation was 80-fold more efficient than the second, and among natural deoxynucleotides, dATP was the preferred substrate due to its stronger intrahelical base-stacking ability. Such base-stacking contributions are supported by the 41-fold higher ground-state binding affinity of a nucleotide analog, pyrene nucleoside 5'-triphosphate, which lacks hydrogen bonding ability but possesses four conjugated aromatic rings. A 2.05 A resolution structure of Dpo4*(blunt-end DNA)*ddATP revealed that the base and sugar of the incoming ddATP, respectively, stack against the 5'-base of the opposite strand and the 3'-base of the elongating strand. This unprecedented base-stacking pattern can be applied to subsequent blunt-end additions only if all incorporated dAMPs are extrahelical, leading to predominantly single non-templated dATP incorporation.

Binding Sites↗

PubMed2006

Neogenesis and proliferation of beta-cells induced by human betacellulin gene transduction via retrograde pancreatic duct injection of an adenovirus vector.

Betacellulin (BTC) has been shown to have a role in the differentiation and proliferation of beta-cells both in vitro and in vivo. We administered a human betacellulin (hBTC) adenovirus vector to male ICR mice via retrograde pancreatic duct injection. As a control, we administered a beta-galactosidase adenovirus vector. In the mice, hBTC protein was mainly overexpressed by pancreatic duct cells. On immunohistochemical analysis, we observed features of beta-cell neogenesis as newly formed insulin-positive cells in the duct cell lining or islet-like cell clusters (ICCs) closely associated with the ducts. The BrdU labeling index of beta-cells was also increased by the betacellulin vector compared with that of control mice. These results indicate that hBTC gene transduction into adult pancreatic duct cells promoted beta-cell differentiation (mainly from duct cells) and proliferation of pre-existing beta-cells, resulting in an increase of the beta-cell mass that improved glucose tolerance in diabetic mice.

Adenoviridae↗

PubMed2006

Dynamics of a deformable body in a fast flowing soap film.

We study the behavior of an elastic loop embedded in a flowing soap film. This deformable loop is wetted into the film and is held fixed at a single point against the oncoming flow. We interpret this system as a two-dimensional flexible body interacting in a two-dimensional flow. This coupled fluid-structure system shows bistability, with both stationary and oscillatory states. In its stationary state, the loop remains essentially motionless and its wake is a von Kármán vortex street. In its oscillatory state, the loop sheds two vortex dipoles, or more complicated vortical structures, within each oscillation period. We find that the oscillation frequency of the loop is linearly proportional to the flow velocity, and that the measured Strouhal numbers can be separated based on wake structure.

Journal Article↗

PubMed2006

Stop codon mutations in the flavin-containing monooxygenase 3 (FMO3) gene responsible for trimethylaminuria in a Japanese population.

The reduced capacity of flavin-containing monooxygenase 3 (FMO3) to N-oxidize trimethylamine (TMA) is believed to cause a metabolic disorder. The aim of this study was to investigate the inter-individual variations of FMO3. Genomic DNA of case subjects that showed only 10-20% of FMO3 metabolic capacity among self-reported trimethylaminuria Japanese volunteers was sequenced. Functional analysis of recombinant FMO3 proteins was also performed. One homozygote for a novel single nucleotide substitution causing a stop codon at Arg500 was observed. The biological parents of this Proband A were heterozygous and showed >90% TMA N-oxygenation metabolic capacity. Another Proband B had the Arg500Stop and Cys197Stop codons. The TMA N-oxygenation metabolic capacities of the father and brother of this Proband B were apparently observed by possessing Arg205Cys mutant that coded for decreased TMA N-oxygenase. Recombinant Arg500Stop FMO3 cDNA expressed in Escherichia coli membranes and a series of highly purified truncation mutants at different positions of the C-terminus of FMO3 showed no detectable functional activity toward typical FMO3 substrates. The results suggest that individuals homozygous for either of the nonsense mutations, Arg500Stop and/or Cys197Stop alleles, in the FMO3 gene can possess abnormal TMA N-oxygenation.

Adolescent↗

PubMed2006

Investigation of structure and function of a catalytically efficient variant of the human flavin-containing monooxygenase form 3.

To characterize the contribution of amino acid 360 to the functional activity of the human flavin-containing monooxygenase form 3 (FMO3) and form 1 (FMO1) in the oxygenation of drugs and chemicals, we expressed four FMO3 variants (i.e., Ala360-FMO3, His360-FMO3, Gln360-FMO3, and Pro360-FMO3) and one FMO1 variant (i.e., Pro360-FMO1) and compared them to wild-type enzymes (Leu360-FMO3 and His360-FMO1, respectively). The amino acid substitutions were introduced into wild-type FMO3 or FMO1 cDNA by site-directed mutagenesis. The thermal stability of variants of Leu360 FMO3 was also studied, and the thermal stability was significantly different from that of wild-type FMO3. The influence of different substrates to modulate the catalytic activity of FMO3 variants was also examined. Selective functional substrate activity was determined with mercaptoimidazole, chlorpromazine, and 10-[(N,N-dimethylaminopentyl)-2-(trifluoromethyl)]phenothiazine. Compared with wild-type FMO3, the Ala360-FMO3 and His360-FMO3 variants were less catalytically efficient for mercaptoimidazole S-oxygenation. N-Oxygenation of chlorpromazine was significantly less catalytically efficient for His360-FMO3 compared with wild-type FMO3. Human Pro360-FMO1 was significantly more catalytically efficient at S-oxygenating mercaptoimidazole and chlorpromazine compared with wild-type FMO1. The data support the mechanism that the Pro360 loci affect thermal stability of FMO3. Because different amino acids at position 360 affect substrate oxygenation in a unique fashion compared with that of FMO3 stimulation, we conclude that the mechanism of stimulation of FMO3 is distinct from that of enzyme catalysis. A molecular model of human FMO3 was also constructed to help explain the results. The increase in catalytic efficiency observed for Pro360 in human FMO3 was also observed when the His of FMO1 was replaced by Pro at loci 360.

Amino Acid Substitution↗

PubMed2006

Recruitment of the de novo DNA methyltransferase Dnmt3a by Kaposi's sarcoma-associated herpesvirus LANA.

The Kaposi's sarcoma-associated herpesvirus LANA protein is expressed in all Kaposi's sarcoma-associated herpesvirus-infected cells, including the tumor cells of endemic and AIDS-associated Kaposi sarcoma, primary effusion lymphoma, and Castleman disease. LANA modulates cell gene expression, but the mechanisms of LANA-mediated transcriptional reprogramming are poorly understood. LANA-repressed cell genes were identified by using retroviral-transduced telomerase-immortalized microvascular endothelial cells. Transciptional repression of targeted genes was relieved by treatment with the methyltransferase inhibitor 5-aza-2'-deoxycytidine, suggesting a role for DNA methylation in repression. We found that LANA coprecipitated with DNA methyltransferases (Dnmts) and recruited endogenous DNA methyltransferase activity from the cell extract. LANA preferentially relocalized Dnmt3a from the nuclear matrix into the chromatin fraction. Further, LANA associated with repressed cellular promoters, recruited Dnmt3a to DNA, and facilitated de novo promoter methylation of a down-regulated gene, cadherin 13 (H-cadherin). The data provide an example of promoter-specific epigenetic DNA modification through viral protein recruitment of de novo Dnmt activity.

Antigens, Viral↗

PubMed2006

Molecular oxygen dianions trapped in lithium cluster cages.

In an important recent report, Coles and Hitchcock (Chem. Commun. 2005, 3165) have synthesized a unique compound of the form Li9O2(hpp)7, with an O2 unit encapsulated in the Li cluster. The group hpp is the bicyclic guanidinate 1,3,4,6,7,8-hexahydropyrimido[1,2-a]pyrimidinate. Coles and Hitchcock raise the interesting possibility that the trapped species is the O2 dianion. We have theoretically investigated (with full geometry optimization) the structural, vibrational, and electronic properties of this fascinating system with 165 atoms. Every indication supports the qualitative conclusion that this system is best represented as an O2 dianion embedded in a Li cluster. Several smaller model systems, including replacement of the hpp ligands by Cl atoms, are also examined and provide some interesting comparisons.

Journal Article↗

PubMed2006

Self-assembly of flowerlike AlOOH boehmite 3D nanoarchitectures.

In this work, a hydrothermal route using an ethanol-water solution to progressively synthesize a sequence of flowerlike three-dimensional gamma-AlOOH boehmite nanostructures without employing templates or matrixes for self-assembly is presented. The flowerlike boehmite nanoarchitectures exhibit three hierarchies of self-organization, i.e., single-crystalline nanorods, nanostrips, and bundles, which are characterized by scanning and transmission electron microscopy. The sequence of products obtained after different processing times indicates a self-assembly mechanism. The hydrogen bonding on the surface of nanorods or nanostrips possibly plays a key role, as identified by FTIR spectra of the products after they had been heated to 1000 degrees C. The specific surface area and pore-size distribution of the obtained product as determined by gas-sorption measurements show that the boehmite nanoarchitectures exhibit high BET surface area and porosity properties.

Journal Article↗

PubMed2006

The Caenorhabditis elegans CPI-2a cystatin-like inhibitor has an essential regulatory role during oogenesis and fertilization.

In the present study, we characterized a sterile cpi-2a(ok1256) deletion mutant in Caenorhabditis elegans and showed that CPI-2a has an essential regulatory role during oogenesis and fertilization. We have also shown that the CPI2a inhibitor and both Ce-CPL-1 and Ce-CPZ-1 enzymes are present in the myoepithelial sheath surrounding germ cells, oocytes, and embryos as well as in the yolk granules within normal oocytes. Staining of mutant worms with anti-yolk protein antibodies has indicted that the proteins are not present in the mature oocytes. Moreover, green fluorescent protein expression was absence or reduced in cpi-2a/yp170:gfp mutant oocytes, although it was expressed in one of the successfully developed embryos. Based on these results, we hypothesize that the sterility in cpi-2a(ok1256) mutant worms is potentially caused by two possible mechanisms: 1) defects in the uptake and/or processing of yolk proteins by the growing oocytes and 2) indirect induction of defects in cell-cell signaling that is critical for promoting germ line development, oocyte maturation, ovulation, and fertilization. A defect in any of these processes would have detrimental effects on the development of normal embryos and consequently normal production of progenies as we observed in cpi-2a mutant worms. This is the first study that demonstrates the expression of cysteine proteases and their endogenous inhibitor in the gonadal sheath cells surrounding germ cells and oocytes, which indirectly have established their potential involvement in proteolytic processing of molecules within the gonadal sheath cells, such as components of the extracellular matrix or the cytoskeletal proteins, which are essential for proper cell-cell signaling activities of the gonadal sheath cells during normal maturation and ovulation processes.

Animals↗

PubMed2006

Mapping the murine cardiac 26S proteasome complexes.

The importance of proteasomes in governing the intracellular protein degradation process has been increasingly recognized. Recent investigations indicate that proteasome complexes may exist in a species- and cell-type-specific fashion. To date, despite evidence linking impaired protein degradation to cardiac disease phenotypes, virtually nothing is known regarding the molecular composition, function, or regulation of cardiac proteasomes. We have taken a functional proteomic approach to characterize 26S proteasomes in the murine heart. Multidimensional chromatography was used to obtain highly purified and functionally viable cardiac 20S and 19S proteasome complexes, which were subjected to electrophoresis and tandem mass spectrometry analyses. Our data revealed complex molecular organization of cardiac 26S proteasomes, some of which are similar to what were reported in yeast, whereas others exhibit contrasting features that have not been previously identified in other species or cell types. At least 36 distinct subunits (17 of 20S and 19 of 19S) are coexpressed and assembled as 26S proteasomes in this vital cardiac organelle, whereas the expression of PA200 and 11S subunits were detected with limited participation in the 26S complexes. The 19S subunits included a new alternatively spliced isoform of Rpn10 (Rpn10b) along with its primary isoform (Rpn10a). Immunoblotting and immunocytochemistry verified the expression of key alpha and beta subunits in cardiomyocytes. The expression of 14 constitutive alpha and beta subunits in parallel with their three inducible subunits (beta1i, beta2i, and beta5i) in the normal heart was not expected; these findings represent a distinct level of structural complexity of cardiac proteasomes, significantly different from that of yeast and human erythrocytes. Furthermore, liquid chromatography/tandem mass spectroscopy characterized 3 distinct types of post-translational modifications including (1) N-terminal acetylation of 19S subunits (Rpn1, Rpn5, Rpn6, Rpt3, and Rpt6) and 20S subunits (alpha2, alpha5, alpha7, beta3, and beta4); (2) N-terminal myristoylation of a 19S subunit (Rpt2); and (3) phosphorylation of 20S subunits (eg, alpha7)). Taken together, this report presents the first comprehensive characterization of cardiac 26S proteasomes, providing critical structural and proteomic information fundamental to our future understanding of this essential protein degradation system in the normal and diseased myocardium.

Amino Acid Sequence↗

PubMed2006

Narrowband Bragg reflectors in Ti:LiNbO3 optical waveguides.

Bragg grating reflectors etched in amorphous silicon overlay films have been integrated with Ti:LiNbO3 optical waveguides to obtain a narrow (0.05 nm) reflectance spectrum with a > 20 dB dip in the transmittance spectrum. These results were realized at a wavelength of 1542.7 nm for TE polarization on an x-cut, y-propagating substrate with gratings etched to a depth of approximately 93 nm in a 105 nm thick silicon film over a length of 12.5 mm. The reflectance in the channel waveguides is found to be strongly dependent on the depth of the etched grating. The effect of the Bragg waveguide loss factor on the transmittance and reflectance spectra is investigated by using a model for contradirectional coupling that includes an attenuation coefficient. The values for coupling constants kappa and amplitude attenuation constants alpha of samples etched for different time durations to control the grating depths are obtained from the model through the use of the depth of the dips in the transmittance spectra and the spectral widths of the reflectance peaks. It is concluded that the corrugated Si overlay film increases the insertion loss by approximately 2.7 dB, and the loss is not significantly affected by the grating depth.

Journal Article↗