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Biomedical subjects

Xudong Zhu

Publications and source records attributed to Xudong Zhu.

At least 19 recordsLinked to original sources

SUG2 controls grain size and weight by influencing GS2 transcription level in rice.

Grain size is a critical yield determinant and a complex quantitative trait in rice. The major quantitative trait locus GS2, which encodes the transcriptional regulator OsGRF4, has been characterized as a key controller of grain size and grain weight in rice. Building upon existing germplasm resources, exploring the upstream and downstream genes of known grain size regulators is an effective approach to gradually refine and expand the molecular regulatory network underlying grain size. Here, we report that a suppressor of the gain-of-function allele GS2AA, SUG2, which encodes an importin β nuclear transport protein. Phenotypic characterization reveals that the sug2 GS2AA mutant exhibits small grains by inhibiting cell expansion in the spikelet hull. Protein interaction analyses demonstrate that SUG2 physically interacts with GS2 both in vivo and in vitro, and the SUG2A mutation reduces SUG2 expression levels and inhibits GS2 transcriptional activation activity, resulting in decreased GS2 expression levels and GS2 protein abundance in sug2 GS2AA. Genetic analyses indicate that SUG2 and GS2 may be partially involved in a common pathway regulating grain size and weight in rice. These findings elucidate the grain size regulatory relationship between SUG2 and GS2 and provide novel insights into the precision breeding of yield optimization in rice.

Oryza↗

Stimulatory cross-talk between NFAT3 and estrogen receptor in breast cancer cells.

Estrogen receptors (ERalpha and ERbeta) are ligand-regulated transcription factors that play critical roles in the development and progression of breast cancer by regulating target genes involved in cellular proliferation. The transcriptional activity of ERalpha and ERbeta is known to be modulated by cofactor proteins. We used a yeast two-hybrid system and identified NFAT3 as a novel ERbeta-binding protein. NFAT3 interacted with ERalpha and ERbeta both in vitro and in mammalian cells in a ligand-independent fashion. NFAT3 bound specifically to the ERbeta region containing the activation function-1 domain, a ligand-independent transactivation domain. Overexpression of NFAT3 enhanced both ERalpha and ERbeta transcriptional activities in a ligand-independent manner and up-regulated downstream estrogen-responsive genes including pS2 and cathepsin D. Reduction of endogenous NFAT3 with NFAT3 small interfering RNA or overexpression of NFAT3 deletion mutants that lack the ER-binding sites reduced the NFAT3 coactivation of ERalpha and ERbeta. NFAT3 increased binding of ERalpha to the estrogen-responsive element and was recruited to endogenous estrogen-responsive promoters. NFAT3 was expressed differentially in many breast cancer cell lines and overexpressed in a subset of breast cancer patients. Knockdown of endogenous NFAT3 reduced the growth of human breast cancer ZR75-1 cells in a ligand-independent manner. Taken together, these results suggest that NFAT3 may play important roles in ER signaling and represent a novel target for breast cancer therapy.

Binding Sites↗

Conserved and specific functions of mammalian ssu72.

We describe the cloning and characterization of a human homolog of the yeast transcription/RNA-processing factor Ssu72, following a yeast two-hybrid screen for pRb-binding factors in the prostate gland. Interaction between hSsu72 and pRb was observed in transfected mammalian cells and involved multiple domains in pRb; however, so far, mutual effects of these two factors could not be demonstrated. Like the yeast counterpart, mammalian Ssu72 associates with TFIIB and the yeast cleavage/polyadenylation factor Pta1, and exhibits intrinsic phosphatase activity. Mammals contain a single ssu72 gene and a few pseudogenes. During mouse embryogenesis, ssu72 was highly expressed in the nervous system and intestine; high expression in the nervous system persisted in adult mice and was also readily observed in multiple human tumor cell lines. Both endogenous and ectopically expressed mammalian Ssu72 proteins resided primarily in the cytoplasm and only partly in the nucleus. Interestingly, fusion to a strong nuclear localization signal conferred nuclear localization only in a fraction of transfected cells, suggesting active tethering in the cytoplasm. Suppression of ssu72 expression in mammalian cells by siRNA did not reduce proliferation/survival, and its over-expression did not affect transcription of candidate genes in transient reporter assays. Despite high conservation, hssu72 was unable to rescue an ssu72 lethal mutation in yeast. Together, our results highlight conserved and mammalian specific characteristics of mammalian ssu72.

Amino Acid Sequence↗

Characterization of a monoclonal antibody that antagonizes the function of human endostatin.

Endostatin, a 20-kDa proteolytic fragment of collagen XVIII, is a potent inhibitor of angiogenesis and tumor growth. The anti-angiogenic effects of endostatin include inhibition of endothelial cell migration and proliferation, and inhibition of the activity of MMP2. Structure-function analysis of endostatin that implies this contravention function buried in separate fragments of endostatin introduces new issues into the understanding of the structure-function relationship of endostatin. We developed and characterized a novel murine MAb, 4E7, to human endostatin, which antagonizes the function of endostatin. As we show here, MAb 4E7 blocks the anti-migration/adhesion effects of endostatin in vitro and the anti-angiogenesis effect of endostatin in vivo, but the inhibition effect of endostatin on endothelial cell proliferation is not affected by MAb4E7. These results suggest that the anti-migration and anti-proliferation functions of endostatin may have distinct structural foundations.

Animals↗

The DEAD-box RNA helicase Vad1 regulates multiple virulence-associated genes in Cryptococcus neoformans.

The study of fungal regulatory networks is essential to the understanding of how these pathogens respond to host environmental signals with effective virulence-associated traits. In this study, a virulence-associated DEAD-box RNA helicase-encoding gene (VAD1) was isolated from a mutant defective in the virulence factor laccase. A Deltavad1 mutant exhibited a profound reduction in virulence in a mouse model that was restored after reconstitution with WT VAD1. Loss of VAD1 resulted in upregulation of NOT1, a gene encoding a global repressor of transcription. NOT1 was found to act as an intermediary transcriptional repressor of laccase. Vad1 was located within macromolecular complexes that formed cytoplasmic granular bodies in mature cells and during infection of mouse brain. In addition, VAD1 was shown by in situ hybridization to be expressed in the brain of an AIDS patient coinfected with C. neoformans. To understand the role of VAD1 in virulence, a functional genomics approach was used to identify 3 additional virulence determinants dependent on VAD1: PCK1, TUF1, and MPF3, involved in gluconeogenesis, mitochondrial protein synthesis, and cell wall integrity, respectively. These data show that fungal virulence-associated genes are coordinately regulated and that an analysis of such transcriptomes allows for the identification of important new genes involved in the normal growth and virulence of fungal pathogens.

Amino Acid Motifs↗

A novel in vitro transcription method for producing siRNAs without specific sequence requirements.

RNA interference is a powerful genetic approach for silencing of target genes in which 21-23 nucleotide small interfering RNAs (siRNAs) work as sequence-specific RNA interference (RNAi) mediators. Production of siRNAs by in vitro transcription is a useful method that it is simple, effective, and inexpensive. Commonly used in vitro transcription promoters use guanine as the transcription start nucleotide. This method is, therefore, restricted to generating siRNAs with target sequences of 5'-NNGN17C. Here we report a modified in vitro transcription method, in which pre-siRNAs containing the 5' overhanging single-stranded leader sequence are first synthesized, then a DNA oligonucleotide complementary to the leader sequence is added to form a RNA-DNA hybrid, which is removed using RNase H to obtain desired siRNAs. Using siRNAs prepared with this method, we successfully inhibited the expression of both exogenous and endogenous genes.

Base Sequence↗

Alkali burn causes aldehyde dehydrogenase 3A1 (ALDH3A1) decrease in mouse cornea.

PURPOSE: Aldehyde dehydrogenase 3A1 (ALDH3A1) is the most abundant soluble protein component in the mouse cornea, produced mainly by corneal epithelial cells. High levels of ALDH3A1 in cornea contribute to maintenance of a stable an d transparent corneal structure. Alkali burn is a common damage to the corneal surface, which produces an alkaline hydrolysis of matrix proteins and induces an inflammatory reaction. Our study was intended to detect changes in ALDH3A1 expression after corneal alkaline burn. METHODS: To address this issue we employed RTQ-PCR to monitor the transcriptional change of ALDH3A1 after alkali burn. We used zymography to test enzyme activity changes of ALDH3A1 in the alkali burn cornea; And SDS-PAGE and mass spectrometry technology were used to verify protein content changes and to identify ALDH3A1 protein. RESULTS: Using zymography, ALDH3A1 enzymic activity was observed to decrease immediately after corneal alkali burn and the levels recovered following healing. Proteins extracted from alkali burned corneas, when run on SDS-PAGE, showed the same sized band (about 54 kDa, which is the molecular weight of ALDH3A1) but in much smaller quantity, compared to normal corneas. This result was further verified by mass spectrometry fingerprinting of the in-gel lysis product. An immediate decrease of ALDH3A1 transcription after alkali burning of the cornea was also found using RTQ-PCR. This level of transcription was gradually restored during healing. CONCLUSIONS: Alkali burn of the corneal surface caused a rapid decrease of ALDH3A1 in the corneal at both the RNA and protein levels, which leads to the loses of the protective component of the corneal surface and makes it vulnerable to further damage. The ALDH3A1 level in the cornea gradually recovered during the healing process. Use of an anti-oxidation reagent as a treatment ingredient for alkali burn of the corneal surface could compensate for the decrease of anti-oxidation protection potential caused by ALDH3A1 loss.

Aldehyde Dehydrogenase↗

NADPH oxidase mediates vascular endothelial cadherin phosphorylation and endothelial dysfunction.

Vascular endothelial activation is an early step during leukocyte/endothelial adhesion and transendothelial leukocyte migration in inflammatory states. Leukocyte transmigration occurs through intercellular gaps between endothelial cells. Vascular endothelial cadherin (VE-cadherin) is a predominant component of endothelial adherens junctions that regulates intercellular gap formation. We found that tumor necrosis factor (TNF) caused tyrosine phosphorylation of VE-cadherin, separation of lateral cell-cell junctions, and intercellular gap formation in human umbilical vein endothelial cell (HUVEC) monolayers. These events appear to be regulated by intracellular oxidant production through endothelial NAD(P)H (nicotinamide adenine dinucleotide phosphate) oxidase because antioxidants and expression of a transdominant inhibitor of the NADPH oxidase, p67(V204A), effectively blocked the effects of TNF on all 3 parameters of junctional integrity. Antioxidants and p67(V204A) also decreased TNF-induced JNK activation. Dominant-negative JNK abrogated VE-cadherin phosphorylation and junctional separation, suggesting a downstream role for JNK. Finally, adenoviral delivery of the kinase dead PAK1(K298A) decreased TNF-induced JNK activation, VE-cadherin phosphorylation, and lateral junctional separation, consistent with the proposed involvement of PAK1 upstream of the NADPH oxidase. Thus, PAK-1 acts in concert with oxidase during TNF-induced oxidant production and loss of endothelial cell junctional integrity.

Antigens, CD↗

Role of laccase in the biology and virulence of Cryptococcus neoformans.

Laccase is an important virulence factor for the human pathogen, Cryptococcus neoformans. In this review, we examine the structural, biological and genetic features of the enzyme and its role in the pathogenesis of cryptococcosis. Laccase is expressed in C. neoformans as a cell wall enzyme that possesses a broad spectrum of activity oxidizing both polyphenolic compounds and iron. Two paralogs, CNLAC1 and CNLAC2, are present in the fungus, of which the first one expresses the dominant enzyme activity under glucose starvation conditions. Regulation of the enzyme is in response to various environmental signals including nutrient starvation, the presence of multivalent cations and temperature stress, and is mediated through multiple signal transduction pathways. Study of the function and regulation of this important virulence factor has led to further understanding of mechanisms of fungal pathogenesis and the regulation of stress response in the host cell environment.

Amino Acid Sequence↗

Antiproliferative effects of Src inhibition on medullary thyroid cancer.

There is no effective treatment for recurrent or metastatic medullary thyroid cancer (MTC). Hereditary MTC is associated with mutations in the RET protooncogene, which encodes for a tyrosine kinase. We postulated that Src tyrosine kinases regulate MTC proliferation. Proliferation of the human MTC cell line, TT, was examined in the presence of a Src-specific tyrosine kinase inhibitor, PP2, or genistein. Cell counts were performed with a Coulter counter or by flow cytometry. DNA synthesis was evaluated by bromodeoxyuridine incorporation. A cell death ELISA was used to assess apoptosis. Akt phosphorylation was determined by Western immunoblot. MAPK activity was measured using an immunoprecipitation kinase assay, and MAPK inhibition was achieved with SB202190 (p38 MAPK) and PD098059 (MAPK kinase). Data were analyzed by ANOVA. Compared with controls, PP2 reduced DNA synthesis, abolished Akt phosphorylation, and increased apoptosis. The MAPK kinase inhibitor, PD098059, attenuated DNA synthesis, whereas genistein caused modest declines in cell count and DNA synthesis and minimal changes in apoptosis. We conclude that Src-dependent MTC proliferation occurs via increased DNA synthesis and reduced apoptosis. The latter effect may be mediated by Akt survival signals. Modulation of Src activity is a potential therapeutic target in MTC.

Apoptosis↗

A fission yeast homologue of the human uracil-DNA-glycosylase and their roles in causing DNA damage after overexpression.

A functional homologue (ung1) of the human uracil-DNA-glycosylase (UNG) gene was characterized from fission yeast (Schizosaccharomyces pombe). The ung1 gene is highly conserved and encodes a protein with uracil-DNA-glycosylase activity similar to human UNG. The Ung1 protein localizes predominantly to the nucleus, suggesting that it is more similar to the nuclear form (UNG2) than the mitochondrial form (UNG1) of human UNG. Even though deletion of ung1 does not cause any obvious defects, overexpression of ung1 increases the mutation frequency. Overexpression of ung1 or human UNG2 induces a DNA checkpoint-dependent cell cycle delay and causes cell death which is enhanced when the checkpoints are inactive. In addition, the steady-state level of AP (apurinic/apyrimidinic) sites increases after ung1 overexpression, indicating that AP sites are likely to be the DNA damage caused by overexpression. Analysis of mutant ung indicates that catalytic activity is not required for the effects of overexpression, but that binding of Ung1 or UNG2 to AP sites may be important.

Cell Cycle↗

Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability.

Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R. Shock 18: 82-85, 2002). Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability. Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance. MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK. MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator. Rho-associated coiled-coil kinase was inhibited with Y-27632. TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity. Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability. C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability. Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance. Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak.

ADP Ribose Transferases↗

Expression of thrombospondin-1 is correlated with microvessel density in gastric carcinoma.

Thrombospondin-1 (TSP-1) has been shown to play a role in angiogenesis in a variety of cancers, but some studies indicated a difference in the mechanism of TSP-1 on neovascularization according to organ or histological type. Wild-type p53 protein has been shown to induce TSP-1 expression. We examined the expression of TSP-1 protein in 80 gastric carcinomas using immunohistochemistry and studied the relationship with microvessel counts, p53 expression and clinicopathological factors. We also performed reverse-transcriptase polymerase chain reaction analysis for the TSP-1 mRNA expression in gastric carcinoma cell lines and gastric cancer tissue after laser capture microdissection. Strong expression of TSP-1 protein was detected in 30 (38%) of the 80 cases. Positive staining for TSP-1 was seen in the cytoplasm of the cancer cells. TSP-1 mRNA expression was confirmed in a majority of gastric carcinoma cell lines and carcinoma tissues. Microvessel counts were significantly higher in tumors with strong TSP-1 protein expression than in those without expression or weak expression of TSP-1 ( P=0.011). No significant correlation was found between TSP-1 expression and p53 staining and clinicopathological factors. Our results support an idea that increased TSP-1 expression may be associated with an angiogenic phenotype in gastric carcinoma and suggest that TSP-1 may play diverse roles in each organ.

Adenocarcinoma↗

Copper-mediated reversal of defective laccase in a Deltavph1 avirulent mutant of Cryptococcus neoformans.

Previous studies have shown that a Deltavph1 Cryptococcus neoformans mutant defective in vesicular acidification lacked several important virulence factors including a copper-containing laccase and was avirulent in a mouse model. In the present studies, we characterized laccase transcription and protein production to obtain insights into the mechanism of the vph1 mutation in this pathogen. Although transcription and protein expression were somewhat reduced, laccase protein was found to be successfully translated and correctly targeted to the cell wall in the Deltavph1 mutant as shown by Western blot and immuno-electron microscopy, despite a complete lack of laccase activity. Laccase activity was substantially restored in metabolically active Deltavph1 cells at 30 degrees C by addition of 100 micro M copper sulphate. This restoration by copper was found to occur through both transcriptional and post-translational mechanisms. Laccase transcriptional induction by copper was found to be dependent on enhancer region II within the 5'-untranslated region of CNLAC1. Copper was also found to restore partial activity to Deltavph1 cells at 0 degrees C, suggesting that cell wall laccase was expressed in the mutant as an apo-enzyme. Apo-laccase restoration by copper was found to be facilitated by an acidic environment, consistent with a role for the vacuolar (H+)-ATPase proton pump in copper assembly of laccase in C. neoformans.

Animals↗

A CLC-type chloride channel gene is required for laccase activity and virulence in Cryptococcus neoformans.

Laccase is a major virulence factor required for infection caused by the human pathogenic yeast Cryptococcus neoformans. However, cellular processes involved in the regulation and expression of laccase remain largely unknown in C. neoformans. Here we report the identification of a chloride channel gene CLC-A which is essential for laccase activity in C. neoformans. CLC-A shares homology to CLC-type voltage-gated chloride channels from other organisms; for example, 63% homology to GEF1, a chloride channel gene from Saccharomyces cerevisiae. A clc-a mutant, Mlac3, generated by insertional mutagenesis as well as a targeted Deltaclc-a mutant produced undetectable laccase in a liquid assay and produced no melanin on asparagine agar containing norepinephrine. Mlac3 was complemented with wild-type CLC-A which restored laccase activity and melanin biosynthesis. The clc-a mutants also showed reduced synthesis of another important virulence factor, capsule, and showed reduced growth at elevated pH. In addition, the clc-a mutation resulted in attenuated virulence in a mouse cryptococcosis model that was restored by complementation with wild-type CLC-A, indicating that the chloride channel plays an important role in the virulence of the organism. Further analysis revealed that the basis for absent laccase expression in the clc-a mutant was a laccase transcriptional defect that could be restored by adding exogenous copper. In conclusion, our findings show that CLC-A plays a role in the expression of two important virulence factors, capsule and laccase expression, which are required for virulence of the fungal pathogen.

Animals↗

Expression of integrin-linked kinase is closely correlated with invasion and metastasis of gastric carcinoma.

Integrin-linked kinase (ILK), a serine/threonine protein kinase, implicates in cellular control of cell-matrix interactions and cell proliferation that is associated with a highly invasive phenotype of certain tumors. To study whether ILK is involved in the development and progression of gastric carcinoma, we examined the expression of ILK in gastric carcinoma cell lines, primary gastric carcinomas and corresponding non-neoplastic mucosa using reverse-transcription polymerase chain reaction (RT-PCR) and immunohistochemistry and analyzed the relationship with clinicopathological parameters. The expression of ILK mRNA was detected in 4 of 5 gastric carcinoma cell lines and 22 of 35 (63%) microdissected tumor samples of primary gastric carcinoma using RT-PCR. The incidence of cases with ILK mRNA expression was significantly higher in scirrhous and intermediate type (82%) than in medullary type (44%) (P=0.0204). Significant association was detected between ILK mRNA expression and presence of nodal metastasis (P=0.0388). Immunohistochemically, strong expression of ILK protein (over 50% of tumor cells were positive) was detected in 69% (84/122) of primary gastric carcinomas, whereas no ILK expression was found in non-neoplastic gastric epithelia. Strong expression of ILK protein was significantly associated with scirrhous and intermediate type (P=0.0217), deep invasion of tumor cells in gastric wall (P=0.0006), and presence of nodal metastasis (P=0.0176). These results strongly suggest that ILK may participate not only in stomach carcinogenesis especially of scirrhous and intermediate types but also in invasion and metastasis of gastric carcinoma. ILK might be a novel molecular marker for aggressive gastric cancer.

Adenocarcinoma↗

Tyrosine phosphorylation of vascular endothelial cadherin and the regulation of microvascular permeability.

BACKGROUND: Adherens junctions (AJ), by their association with the endothelial cytoskeleton, maintain microvascular barrier integrity. Phosphorylation states of AJ proteins, such as vascular endothelial (VE) cadherin, can potentially alter the interactions between component AJ proteins. Furthermore, AJ protein phosphorylation is susceptible to regulation by inflammatory mediators. We previously demonstrated the importance of VE cadherin in tumor necrosis factor (TNF)-mediated endothelial permeability. We now postulate that TNF-induced endothelial permeability is associated with tyrosine phosphorylation of VE cadherin. METHODS: Confluent monolayers of human umbilical vein endothelial cells were exposed to saline solution, TNF-alpha (100 U/mL) or TNF and the Src-tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Permeability was assessed by fluorescein isothiocyanate-dextran flux. VE-cadherin phosphorylation was determined by immunoprecipitation and immunoblotting with antiphosphotyrosine antibody. Data are expressed as mean +/- SEM and analyzed by analysis of variance. RESULTS: TNF-alpha-induced tyrosine phosphorylation of VE cadherin and increased intercellular gap formation. These changes were associated with increased endothelial-cell monolayer permeability, all of which were prevented by 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Exposure to an inactive tyrphostin, AG9 (negative control), did not prevent TNF-induced endothelial permeability. CONCLUSIONS: We conclude that tyrosine phosphorylation of VE cadherin is an important regulatory pathway associated with TNF-induced endothelial barrier dysfunction. Modulating AJ protein phosphorylation may provide targets for therapy during inflammation.

Antigens, CD↗

The role of p38 map kinase in tumor necrosis factor-induced redistribution of vascular endothelial cadherin and increased endothelial permeability.

The mechanism by which tumor necrosis factor alpha (TNFalpha) increases endothelial permeability is unclear. Vascular endothelial (VE) cadherin (cadherin 5) is an important contributor to endothelial monolayer integrity. The purpose of our study was to determine the effect of TNFalpha on VE-cadherin cell-surface expression and to identify the signaling pathways involved in TNF-induced changes in cadherin expression. Human umbilical vein endothelial cell monolayer permeability was measured by enzyme-linked immunosorbent assay for biotin-labeled albumin. Immunofluorescence, laser confocal microscopy, and Western immunobloting were used to assess VE cadherin distribution. Mitogen-activated protein kinase (MAPK) activity was determined using functional kinase assays and was inhibited with the compounds SB202190 and PD98059. TNFalpha significantly increased permeability and induced p38 and ERK MAPK activation compared with controls (P < 0.05). These changes were associated with a loss of membrane-associated VE cadherin. Inhibition of p38 but not ERK MAPK significantly reduced the effect of TNFalpha on endothelial permeability and cell-surface VE cadherin expression. p38 MAP kinase activation appears to be an important upstream signaling event associated with increased endothelial permeability and vascular endothelial cadherin redistribution.

Antigens, CD↗