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Biomedical subjects

X Yang

Publications and source records attributed to X Yang.

At least 127 records · Page 7Linked to original sources
PubMed2001

Invasiveness and MMP expression in pancreatic carcinoma.

BACKGROUND: Previous investigations have suggested that expression of matrix metalloproteinases (MMPs) may be related to increased invasiveness of various tumors. This study evaluated a possible relation between pancreatic tumor cell invasiveness and MMPs. METHODS: A Matrigel invasion assay was performed with pancreatic tumor cell line SUIT-2 and its sublines S2-007, S2-013, S2-020, and S2-028. The degree of invasiveness of stimulated and unstimulated cell lines was correlated with MMP gene expression measured by RT-PCR and MMP protein product measured by gelatin zymography. Cell lines were stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA), concanavalin (Con-A), and polymerized collagen type I gel (Vitrogen). RESULTS: For SUIT-2, S2-007, S2-013, S2-020, and S2-028, 3.2, 1.0, 4.1, 6.4, and 0.4%, respectively, of the cells invaded the Matrigel membrane. TPA, Con-A, and Vitrogen resulted in the up-regulation of MMP-2 in S2-020. TPA and Vitrogen resulted in up-regulation of MMP-9 in each of the cell lines, while Con-A could up-regulate MMP-9 expression only in SUIT-2. There was no constitutive expression of either MMP-2 or MMP-9 in SUIT-2 or its sublines. There was a positive relationship between Matrigel invasiveness and up-regulation of MMP-2 and MMP-9 expression. CONCLUSION: These data suggest that, while MMP-2 and MMP-9 are not constitutively expressed in pancreatic carcinoma cell lines, they may be up-regulated by TPA, Con-A, and Vitrogen. Since MMP-2 and MMP-9 expression correlated with degree of tumor cell invasiveness, the ability to up-regulate MMP-2 and MMP-9 expression may play a role in facilitating pancreatic tumor cell invasion.

Biocompatible Materials↗
PubMed2001

Discordance between the binding affinity of mitogen-activated protein kinase subfamily members for MAP kinase phosphatase-2 and their ability to activate the phosphatase catalytically.

MKP-2 is a member of the mitogen-activated protein (MAP) kinase phosphatase family which has been suggested to play an important role in the feedback control of MAP kinase-mediated gene expression. Although MKP-2 preferentially inactivates extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) MAP kinase subfamilies, the mechanisms underlying its own regulation remain unclear. In this report, we have examined the MKP-2 interaction with and catalytic activation by distinct MAP kinase subfamilies. We found that the catalytic activity of MKP-2 was enhanced dramatically by ERK and JNK but was affected only minimally by p38. By contrast, p38 and ERK bound MKP-2 with comparably strong affinities, whereas JNK and MKP-2 interacted very weakly. Through site-directed mutagenesis, we defined the ERK/p38-binding site as a cluster of arginine residues in the NH(2)-terminal domain of MKP-2. Mutation of the basic motif abrogated its interaction with both ERK and p38 and severely compromised the catalytic activation of MKP-2 by these kinases. Unexpectedly, such mutations had little effect on JNK-triggered catalytic activation. Both in vitro and in vivo, wild type MKP-2 effectively inactivated ERK2 whereas MKP-2 mutants incapable of binding to ERK/p38 did not. Finally, in addition to its role as a docking site for ERK and p38, the MKP-2 basic motif plays a role in regulating its nuclear localization. Our studies provided a mechanistic explanation for the substrate preference of MKP-2 and suggest that catalytic activation of MKP-2 upon binding to its substrates is crucial for its function.

Amino Acid Substitution↗
PubMed2001

O-linkage of N-acetylglucosamine to Sp1 activation domain inhibits its transcriptional capability.

The posttranslational modification of eukaryotic intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc) monosaccharides is essential for cell viability, yet its precise functional roles are largely unknown. O-GlcNAc transferase utilizes UDP-GlcNAc, the end product of hexosamine biosynthesis, to catalyze this modification. The availability of UDP-GlcNAc correlates with glycosylation levels of intracellular proteins as well as with transcriptional levels of some genes. Meanwhile, transcription factors and RNA polymerase II can be modified by O-GlcNAc. A linkage between transcription factor O-GlcNAcylation and transcriptional regulation therefore has been postulated. Here, we show that O-GlcNAcylation of a chimeric transcriptional activator containing the second activation domain of Sp1 decreases its transcriptional activity both in an in vitro transcription system and in living cells, which is in concert with our observation that O-GlcNAcylation of Sp1 activation domain blocks its in vitro and in vivo interactions with other Sp1 molecules and TATA-binding protein-associated factor II 110. Furthermore, overexpression of O-GlcNAc transferase specifically inhibits transcriptional activation by native Sp1 in cells. Thus, our studies provide direct evidence that O-GlcNAcylation of transcription factors is involved in transcriptional regulation.

Acetylglucosamine↗
PubMed2001

Distinct function of Th1 and Th2 type delayed type hypersensitivity: protective and pathological reactions to chlamydial infection.

The role of delayed-type hypersensitivity (DTH) to chlamydial infection has been shown to be a double-edged sword to the host. Reported animal and human studies have, on the one hand, shown that DTH is associated with protective immunity against chlamydial infection and, on the other hand, shown links to immunopathology. Using a murine lung infection model, we recently demonstrated that there might be two different functional types of DTH induced by chlamydial infection based on its association with cytokine patterns. Th1 type DTH is associated with protection while Th2 type DTH is associated with immunopathology. The Th2 type DTH demonstrated in IFNgamma gene knockout (KO) mice is characterized by eosinophil infiltration in addition to mononuclear cell infiltration that exists in Th1 DTH, observed in wild-type C57BL/6 mice and IL-10 KO mice. In addition, the inflammatory cells in IFNgamma KO mice fail to target the cellular sites of chlamydial inclusions in infected tissues and fail to clear the infection. The functional differences in Th1 and Th2 type DTH responses may account for the dual role DTH plays in chlamydial protective immunity and immunopathology.

Animals↗
PubMed2001

Crystallographic analysis of a novel complex of actinomycin D bound to the DNA decamer CGATCGATCG.

The potent anticancer drug actinomycin D (ActD) acts by binding to DNA, thereby interfering with replication and transcription. ActD inhibits RNA polymerase far more specifically than DNA polymerase. Such discrimination is not easily understood by the conventional DNA binding mode of ActD. We have solved and refined at 1.7 A resolution the crystal structure of ActD complexed to CGATCGATCG, which contains no canonical GpC binding sequence. The crystal data are space group P4(3)2(1)2, a = b = 47.01 A, and c = 160.37 A. The structure was solved by the multiple wavelength anomalous diffraction method using a 5-bromo-U DNA. The asymmetric unit of the unit cell contains two independent dimers of a novel slipped duplex complex consisting of two decamer DNA strands bound with two ActD drug molecules. (The DNA in one dimer is numbered C1 to G10 in one strand and C11 to G20 in the complementary strand and in the second dimer, C101 to G110 and C111 to G120, respectively.) The structure reveals a highly unusual ActD binding mode in which the DNA adopts a slipped duplex with the A3-T4/A13-T14 dinucleotides looped out. ActD intercalates between G2-C11* (C11* being from a symmetry-related molecule) and C5-G20 base pairs. Two such slipped duplex-ActD complexes bound to each other by mutually intercalating their T4/T14 bases into the helix cavities (located between C5-G20 and G6-C19 base pairs) of neighboring complexes, forming a dimer of drug-DNA complexes. The binding site mimics the drug binding at the elongation point during transcription. Modeling studies show that the ActD-DNA complex fits snugly in the active site cavity in RNA polymerase but not in DNA polymerase. This may explain the strong preference of ActD inhibition toward transcription.

Antibiotics, Antineoplastic↗
PubMed2001

CCAAT/enhancer-binding protein beta mediates interferon-gamma-induced p48 (ISGF3-gamma ) gene transcription in human monocytic cells.

Previous studies have identified a novel interferon-stimulated response element-like element, termed gamma-interferon-activating transcription element, within the interferon-stimulating gene factor-3gamma (p48) promoter region that is bound by novel transcription factors in response to stimulation with interferons (IFNs) (Weihua, X., Kolla, V., and Kalvakolanu, D. V. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 103-108). In the present study, we have identified CCAAT/enhancer-binding protein beta (C/EBP-beta) as one of the gamma-interferon-activating transcription element cognate transcription factors by screening a human monophage-derived cDNA library in a yeast one-hybrid system. Electrophoretic mobility shift assay studies suggest that C/EBP-beta dynamically regulates p48 gene expression upon IFN-gamma stimulation by undergoing changes in its heterodimerization partners. Transient transfection studies demonstrate that overexpression of C/EBP-beta strongly enhanced IFN-gamma-induced transcription from the p48 promoter. However, deletion mutants of C/EBP-beta that lack the N-terminal transactivation domain were unable to stimulate the p48 promoter. Western blotting revealed that C/EBP-beta is induced by IFN-gamma stimulation in THP-1-derived macrophages. Collectively, these results suggest that C/EBP-beta plays an important role in the human IFN-gamma signaling pathway by transcriptional regulation of p48 gene expression, an essential component in the IFN signaling pathway.

Base Sequence↗
PubMed2001

BAG-1 p50 isoform interacts with the vitamin D receptor and its cellular overexpression inhibits the vitamin D pathway.

Human BAG-1 is an anti-apoptotic protein with four protein isoforms (BAG-1 p50, p46, p33, and p29). BAG-1 p46 was originally isolated in a screen for proteins binding to the glucocorticoid receptor; it binds and modulates the action of several members of the nuclear steroid hormone receptor superfamily. The vitamin D receptor (VDR) is another member of this superfamily, and the vitamin D pathway is important for prevention and therapy of osteoporosis, renal failure, cancer, and psoriasis. Therefore, we investigated the effect of the recently isolated BAG-1 p50 on the vitamin D pathway. By use of Far Western blot analysis and glutathione S-transferase BAG-1 p50 binding assays, BAG-1 p50 was demonstrated to interact with the VDR, and the BAG-1 p50 N-terminus was required. In U87 cells that were stably transfected with BAG-1 p50, binding of the VDR to its response element in electrophoretic mobility shift assays was blocked, enhancement of transcriptional activation was inhibited, cell growth rate was enhanced, cell growth inhibition induced by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was blocked, and 1,25(OH)2D3-mediated VDR induction was inhibited. These results suggest that BAG-1 p50 is a novel regulator of the vitamin D signaling pathway, and its overexpression may lead to cellular resistance to 1,25(OH)2D3 therapy.

Calcitriol↗
PubMed2001

TGF-beta/Smad3 signals repress chondrocyte hypertrophic differentiation and are required for maintaining articular cartilage.

Endochondral ossification begins from the condensation and differentiation of mesenchymal cells into cartilage. The cartilage then goes through a program of cell proliferation, hypertrophic differentiation, calcification, apoptosis, and eventually is replaced by bone. Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic differentiation. In this study, we showed that TGF-beta/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and are essential for maintaining articular cartilage. Mutant mice homozygous for a targeted disruption of Smad3 exon 8 (Smad3(ex8/ex8)) developed degenerative joint disease resembling human osteoarthritis, as characterized by progressive loss of articular cartilage, formation of large osteophytes, decreased production of proteoglycans, and abnormally increased number of type X collagen-expressing chondrocytes in synovial joints. Enhanced terminal differentiation of epiphyseal growth plate chondrocytes was also observed in mutant mice shortly after weaning. In an in vitro embryonic metatarsal rudiment culture system, we found that TGF-beta1 significantly inhibits chondrocyte differentiation of wild-type metatarsal rudiments. However, this inhibition is diminished in metatarsal bones isolated from Smad3(ex8/ex8) mice. These data suggest that TGF-beta/Smad3 signals are essential for repressing articular chondrocyte differentiation. Without these inhibition signals, chondrocytes break quiescent state and undergo abnormal terminal differentiation, ultimately leading to osteoarthritis.

Animals↗
PubMed2001

A family of snail-related zinc finger proteins regulates two distinct and parallel mechanisms that mediate Drosophila neuroblast asymmetric divisions.

Three snail family genes snail, escargot and worniu, encode related zinc finger transcription factors that mediate Drosophila central nervous system (CNS) development. We show that simultaneous removal of all three genes causes defective neuroblast asymmetric divisions; inscuteable transcription/translation is delayed/suppressed in the segmented CNS. Further more, defects in localization of cell fate determinants and orientation of the mitotic spindle in dividing neuroblasts are much stronger than those associated with inscuteable loss of function. In inscuteable neuroblasts, cell fate determinants are mislocalized during prophase and metaphase, yet during anaphase and telophase the great majority of mutant neuroblasts localize these determinants as cortical crescents overlying one of the spindle poles. This phenomenon, known as 'telophase rescue', does not occur in the absence of the snail family genes; moreover, in contrast to inscuteable mutants, mitotic spindle orientation is completely randomized. Our data provide further evidence for the existence of two distinct asymmetry-controlling mechanisms in neuroblasts both of which require snail family gene function: an inscuteable-dependent mechanism that functions throughout mitosis and an inscuteable-independent mechanism that acts during anaphase/telophase.

Animals↗
PubMed2001

Human and mouse ABCA1 comparative sequencing and transgenesis studies revealing novel regulatory sequences.

The expression of ABCA1, a major participant in apolipoprotein-mediated cholesterol efflux, is regulated by a variety of factors, including intracellular cholesterol concentration. To identify sequences involved in its regulation, we sequenced and compared approximately 200 kb of mouse and human DNA containing the ABCA1 gene. Furthermore, expression of the human gene containing different 5' ends was examined in transgenic mice. Sequence comparison revealed multiple conserved noncoding sequences. The two most highly conserved noncoding elements (CNS1, 88% identity over 498 bp; CNS2, 81% identity over 214 bp) were also highly conserved in other organisms. Mice containing the human ABCA1 gene, 70 kb of upstream DNA, and 35 kb of downstream DNA expressed the transgene similarly to endogenous Abca1. A second transgene beginning 3' to exon 1 was expressed only in liver, providing strong evidence of an unsuspected liver-specific promoter. The identified conserved noncoding sequences invite further investigation to elucidate ABCA1 regulation.

ATP Binding Cassette Transporter 1↗
PubMed2001

Mitogen-activated protein kinase kinase 4 metastasis suppressor gene expression is inversely related to histological pattern in advancing human prostatic cancers.

We have shown recently (B. A. Yoshida et al., Cancer Res., 59: 5483-5487) that mitogen-activated protein kinase kinase 4 (MKK4) can suppress AT6.1 rat prostate cancer metastases in vivo. Evaluation of the expression of components of the MKK4 signaling cascade showed a loss or down-regulation of expression of MKK4 or c-Jun, a downstream mediator of MKK4, in six of eight human prostate cancer cell lines. Given these findings, we next assessed whether MKK4 dysregulation occurs during the development of clinical prostate cancer. Immunohistochemical studies showed high levels of MKK4 expression in the epithelial but not the stromal compartment of normal prostatic tissues. In neoplastic tissues, a statistically significant, direct, inverse relationship between Gleason pattern and MKK4 was established. These results demonstrate that MKK4 protein is consistently down-regulated during prostate cancer progression and support a role for dysregulation of its signaling cascade in clinical disease. To test the possibility that down-regulation of MKK4 protein is the result of allelic loss, metastatic prostate cancer lesions were examined for loss of heterozygosity (LOH) within the MKK4 locus (D17S969). These studies showed a 31% (5 of 16) LOH of MKK4 that is not associated with coding region mutations, which suggests that the nucleotide sequence of the gene in the remaining allele is infrequently mutated.

Enzyme Activation↗
PubMed2001

Role of retinoid receptor coactivator pockets in cofactor recruitment and transcriptional regulation.

The nuclear receptor for retinoic acid (RAR) forms a heterodimeric complex with the retinoid X receptor (RXR). This RXR/RAR heterodimer binds to the promoter of retinoic acid target genes and recruits coactivators and corepressors to regulate gene expression. Currently, the relative role of each receptor monomer in regulating coactivator and corepressor recruitment remains unclear. Here we show that the receptor-associated coactivator 3 (RAC3) uses two separate LXXLL motifs to bind RAR and RXR. The mutation of the coactivator-binding pockets of RAR and RXR abolishes RAC3 binding. Although the coactivator pocket of RXR is essential for the function of the RXR homodimer, it has a minor role for the recruitment of RAC3 and trans-activation by the RXR/RAR heterodimer. Consistently, deletion of the activation helix of RXR enhances binding of RAC3 to the heterodimer, and mutation of the coactivator pocket of RXR had little effect on RXR/RAR activity. In contrast, the coactivator pocket and the activation helix of RAR are absolutely required. We also show that different residues of the RAR coactivator pocket are used differently for interactions with the corepressor silencing mediator for retinoid and thyroid hormone receptor (SMRT) and coactivator. These results indicate a differential role for each retinoid receptor to the overall binding of cofactors and regulation of transcription by the retinoid receptor heterodimer.

Amino Acid Motifs↗
PubMed2001

Analytical method for yrast line states in interacting Bose-Eeinstein condensates.

We propose a method to investigate the energy eigenvalue problem and corresponding yrast line for harmonically trapped interacting N-boson systems. This method is particularly simple and effective to obtain the explicit analytical expressions of low-L energy eigenstates with L denoting systems' total angular momentum but with an arbitrarily large N. We have derived the explicit analytical results for L = 0,1,....,9 and discussed the yrast line for these low- L energy eigenstates.

Journal Article↗
PubMed2001

Inducible nitric oxide synthase deficiency does not affect the susceptibility of mice to atherosclerosis but increases collagen content in lesions.

BACKGROUND: Although endothelial nitric oxide synthase (NOS) is antiatherogenic, the role of inducible NOS (iNOS) in the development of atherosclerosis is not established. METHODS AND RESULTS: We compared the susceptibility of iNOS knockout (iNOS(-/-)) and wild-type (iNOS(+/+)) mice to the development of atherosclerosis induced by feeding an atherogenic diet for 15 weeks. Plasma lipid level, atherosclerotic lesion size, and cellular density in the lesions were all similar in the 2 strains (lesion size: iNOS(+/+) 285+/-73x10(3) microm(2), iNOS(-/-) 293+/-82x10(3) microm(2), n=10). iNOS mRNA was detected in the lesions of iNOS(+/+) but not iNOS(-/-) mice through RT-PCR. Immunohistochemically, iNOS(+/+) mice showed iNOS staining in macrophages and medial smooth muscle cells in the lesions. Nitrotyrosine staining showed a similar distribution, whereas it was absent in iNOS(-/-) mice. There was no apparent difference in the intensity or distribution of vascular cell adhesion molecule-1 staining in the lesions of the 2 strains. However, the lesions of iNOS(+/+) mice showed a markedly decreased extracellular collagen content compared with those of iNOS(-/-) mice CONCLUSIONS: iNOS induction does not affect the development of atherosclerosis in mice fed an atherogenic diet, but the resulting lesions show decreased levels of extracellular collagen and may be more fragile.

Animals↗
PubMed2001

[Effective suppression of the infection and pathogenesis of simian immunodeficiency virus in transgenic lymphocytes mediated by self-cleavagble ribozyme].

OBJECTIVE: To obtain transgenic lymphocytes that are resistant to the infection and pathogenesis of simian immunodeficincy virus (SIV). METHODS: A self-cleaving ribozyme was formed by introducing a fragment of ribozyme target sequence cleft by hammerhead ribozyme to the ribozyme downstream. Self-cleavable ribozyme gene was synthesized, amplified and cloned at the XhoI SalI site of pBluescript SK. The construct bearing 10 copies of ribozyme was obtained through successively cloning four times. The in vitro activity of self-cleavable ribozyme was tested, and then the self-cleaving ribozyme gene was transferred into mammalian expression vector pCI before transfection. The expression vectors harboring self-cleavable ribozyme gene were transfected into lymphocytes with liposome-mediated transfection method. The transfected cell clones were selected in the culturing medium containing 400 mg/L G418. The resistance of transgenic cell to simian immunodeficency virus infection was evaluated by immunofluorescence assays. RESULTS: more than 85% of decameric ribozyme self-cleaved into monomeric ribozymes after incubation for 15 minutes at 37 degrees C. The number of cleavage products of decameric ribozyme during the first 25 minutes exceeds those of the monomeric ribozyme by 13.31% though the molar mass average of decameric ribozyme is less than 1/10 of that of the monomer ribozyme. The Northern dot blot results demonstrated that the monomer ribozyme gene and the decameric ribozyme gene had been transfected and expressed in the cells. SIV challenge trials showed that after 6 days after inoculation of SIV the cells transfected with the monomer ribozyme gene grew normally, no fused cell was observed; and most of the cells transfected with decameric gene grew well, but a few fused cells were observed; The inhibition rate was 100% for the cells transfected with monomer ribozyme gene to the SIV antigen positive cells, 91.2% for the cells transfected with decameric ribozyme gene, and 48% for the cells transfected with the empty expression vector. CONCLUSION: The lymphocytes transfected with self-cleavable ribozyme gene make a strong resistance to SIV infection and pathogenesis. The cells transfected with monomeric ribozyme gene show a better inhibiting effect on SIV infection and pathogenesis in comparison with the cells transfected with decameric ribozyme gene.

Animals↗
PubMed2001

cdc2 links the Drosophila cell cycle and asymmetric division machineries.

Asymmetric cell divisions can be mediated by the preferential segregation of cell-fate determinants into one of two sibling daughters. In Drosophila neural progenitors, Inscuteable, Partner of Inscuteable and Bazooka localize as an apical cortical complex at interphase, which directs the apical-basal orientation of the mitotic spindle as well as the basal/cortical localization of the cell-fate determinants Numb and/or Prospero during mitosis. Although localization of these proteins shows dependence on the cell cycle, the involvement of cell-cycle components in asymmetric divisions has not been demonstrated. Here we show that neural progenitor asymmetric divisions require the cell-cycle regulator cdc2. By attenuating Drosophila cdc2 function without blocking mitosis, normally asymmetric progenitor divisions become defective, failing to correctly localize asymmetric components during mitosis and/or to resolve distinct sibling fates. cdc2 is not necessary for initiating apical complex formation during interphase; however, maintaining the asymmetric localization of the apical components during mitosis requires Cdc2/B-type cyclin complexes. Our findings link cdc2 with asymmetric divisions, and explain why the asymmetric localization of molecules like Inscuteable show cell-cycle dependence.

Animals↗
PubMed2001

IL-12-dependent vascular cell adhesion molecule-1 expression contributes to airway eosinophilic inflammation in a mouse model of asthma-like reaction.

Bronchial-alveolar eosinophilic inflammation is among the characteristic pathological changes in asthma, which has been shown to be correlated with type 2 cytokine and chemokine production. Exogenous IL-12 has been found to be inhibitory for pulmonary eosinophilia in reported studies. Using a murine asthma-like model induced by OVA, we found in the present study that IL-12 gene knockout (KO) mice showed substantially reduced airway recruitment of eosinophils compared with wild-type control mice following OVA sensitization/challenge, although the levels of circulating eosinophils were comparable in these two groups of mice. Cytokine analysis showed Ag-driven Th1 (IFN-gamma) and Th2 (IL-4, IL-5, IL-10, and IL-13) cytokine production by CD4 T cells from local draining lymph nodes and spleen. Similarly, local eotaxin production was comparable in wild-type and IL-12 KO mice. In contrast, immunohistochemical analysis showed that the expression of VCAM-1 on the lung endothelium of IL-12 KO mice was dramatically less than that in wild-type mice. Furthermore, administration of rIL-12 at the stage of sensitization and challenge with OVA restored airway eosinophilia and VCAM-1 expression in IL-12 KO mice. The results suggest that endogenous IL-12 contributes to the recruitment of eosinophils into airways observed in asthma, possibly via enhancement of the expression of VCAM-1 on local vascular endothelial cells.

Administration, Intranasal↗