Biomedical subjects

X Wu

Publications and source records attributed to X Wu.

At least 91 records · Page 5Linked to original sources

PubMed2001

Threonine 68 of Chk2 is phosphorylated at sites of DNA strand breaks.

The protein kinase Chk2 has been implicated in signaling DNA damage to cell cycle checkpoints. In response to ionizing radiation, Chk2 becomes rapidly phosphorylated at threonine 68 by ataxia-telangiectasia mutated (ATM). Here we show that the Thr(68)-phosphorylated form of Chk2 forms distinct nuclear foci in response to ionizing radiation. Only this activated form of Chk2 localizes at sites of DNA strand breaks. The kinase activity of Chk2 and the number of Chk2 foci formed depend on the severity of DNA damage and gradually decline correlating with the predicted value of slowly re-joining double strand breaks. These results suggest that Chk2 is regulated at the sites of DNA strand breaks in response to ionizing radiation.

Amino Acid Sequence↗

PubMed2001

Clathrin exchange during clathrin-mediated endocytosis.

During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein-clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.

Adenosine Triphosphate↗

PubMed2001

Hydrogen peroxide generated during cellular insulin stimulation is integral to activation of the distal insulin signaling cascade in 3T3-L1 adipocytes.

In a variety of cell types, insulin stimulation elicits the rapid production of H(2)O(2), which causes the oxidative inhibition of protein-tyrosine phosphatases and enhances the tyrosine phosphorylation of proteins in the early insulin action cascade (Mahadev, K., Zilbering, A., Zhu, L., and Goldstein, B. J. (2001) J. Biol. Chem. 276, 21938-21942). In the present work, we explored the potential role of insulin-induced H(2)O(2) generation on downstream insulin signaling using diphenyleneiodonium (DPI), an inhibitor of cellular NADPH oxidase that blocks insulin-stimulated cellular H(2)O(2) production. DPI completely inhibited the activation of phosphatidylinositol (PI) 3'-kinase activity by insulin and reduced the insulin-induced activation of the serine kinase Akt by up to 49%; these activities were restored when H(2)O(2) was added back to cells that had been pretreated with DPI. Interestingly, the H(2)O(2)-induced activation of Akt was entirely mediated by upstream stimulation of PI 3'-kinase activity, since treatment of 3T3-L1 adipocytes with the PI 3'-kinase inhibitors wortmannin or LY294002 completely blocked the subsequent activation of Akt by exogenous H(2)O(2). Preventing oxidant generation with DPI also blocked insulin-stimulated glucose uptake and GLUT4 translocation to the plasma membrane, providing further evidence for an oxidant signal in the regulation of the distal insulin-signaling cascade. Finally, in contrast to the cellular mechanism of H(2)O(2) generation by other growth factors, such as platelet-derived growth factor, we also found that insulin-stimulated cellular production of H(2)O(2) may occur through a unique pathway, independent of cellular PI 3'-kinase activity. Overall, these data provide insight into the physiological role of insulin-dependent H(2)O(2) generation, which is not only involved in the regulation of tyrosine phosphorylation events in the early insulin signaling cascade but also has important effects on the regulation of downstream insulin signaling, involving the activation of PI 3'-kinase, Akt, and ultimately cellular glucose transport in response to insulin.

3T3 Cells↗

PubMed2001

Five unique compounds: xyloketals from mangrove fungus Xylaria sp. from the South China Sea coast.

Five unique metabolites, xyloketals A (1), B (2), C (3), D (4), and E (5), and the known 6 were isolated from mangrove fungus Xylaria sp. (no. 2508), obtained from the South China Sea. The structures of these compounds were elucidated by spectroscopic and X-ray diffraction experiments. Xyloketal A is a ketal compound with a C(3) symmetry and xyloketals B-E are its analogues. It was found that xytoketal C slowly rearranged to xytoketal B in DMSO-d(6)() solution at room temperature. Xyloketal A exhibited the activity of inhibiting acetylcholine esterase.

Acetylcholinesterase↗

PubMed2001

Thoracic patterning by the Drosophila gap gene hunchback.

Localized gene expression patterns are critical for establishing body plans in all multicellular animals. In Drosophila, the gap gene hunchback (hb) is expressed in a dynamic pattern in anterior regions of the embryo. Hb protein is first detected as a shallow maternal gradient that prevents expression of posterior gap genes in anterior regions. hb mRNA is also expressed zygotically, first as a broad anterior domain controlled by the Bicoid (Bcd) morphogen, and then in a stripe at the position of parasegment 4 (PS4). Here, we show that the PS4-hb stripe changes the profile of the anterior Hb gradient by generating a localized peak of protein that persists until after the broad domain has started to decline. This peak is required specifically for the formation of the mesothoracic (T2) segment. At the molecular level, the PS4-hb stripe is critical for activation of the homeotic gene Antennapedia, but does not affect a gradient of Hb repressive activity formed by the combination of maternal and Bcd-dependent Hb. The repressive gradient is critical for establishing the positions of several target genes, including the gap genes Kruppel (Kr), knirps (kni), and giant (gt), and the homeotic gene Ultrabithorax (Ubx). Different Hb concentrations are sufficient for repression of gt, kni, and Ubx, but a very high level of Hb, or a combinatorial mechanism, is required for repression of Kr. These results suggest that the individual phases of hb transcription, which overlap temporally and spatially, contribute specific patterning functions in early embryogenesis.

Animals↗

PubMed2001

TGF-alpha induces a stationary, radial-glia like phenotype in cultured astrocytes.

Transgenic mice studies have suggested that transforming growth factor-alpha (TGF-alpha) influences the postnatal differentiation of astrocytes. To understand the role of TGF-alpha during astrocytic differentiation, it is important to determine how this factor affects astrocytes in the absence of other influences. We have thus examined in vitro under serum-free medium conditions the effect of TGF-alpha on the properties of astrocytes derived from the cerebral cortex of newborn rats. When TGF-alpha is added to serum-free medium, most astrocytes lose their polygonal shape and extend two long processes running in opposite directions. This bipolar morphology strikingly resembles that of radial glial cells. Intriguingly, serum inhibits this morphological transformation. TGF-alpha also triggers an increase in glial fibrillary acidic protein (GFAP) expression and a decrease in nestin expression. Another major effect of TGF-alpha is to practically abolish the motility of astrocytes. TGF-alpha, however, does not appear to influence the proliferation and apoptosis of astrocytes. These results suggest that polygonal astrocytes are derived primarily from radial glial cells, and that in vivo TGF-alpha may be instrumental in determining the shape and migratory potential of radial glial cells.

Aging↗

PubMed2001

Loss of presenilin 1 is associated with enhanced beta-catenin signaling and skin tumorigenesis.

Presenilin 1 (PS1) is required for the proteolytic processing of Notch and the beta-amyloid precursor protein (APP), molecules that play pivotal roles in cell-fate determination during development and Alzheimer's disease pathogenesis, respectively. In addition, PS1 interacts with beta-catenin and promotes its turnover through independent mechanisms. Consistent with this activity, we report here that PS1 is important in controlling epidermal cell proliferation in vivo. PS1 knockout mice that are rescued through neuronal expression of human PS1 transgene develop spontaneous skin cancers. PS1-null keratinocytes exhibit higher cytosolic beta-catenin and beta-catenin/lymphoid enhancer factor-1/T cell factor (beta-catenin/LEF)-mediated signaling. This effect can be reversed by reintroducing wild-type PS1, but not a PS1 mutant active in Notch processing but defective in beta-catenin binding. Nuclear beta-catenin protein can be detected in tumors. Elevated beta-catenin/LEF signaling is correlated with activation of its downstream target cyclin D1 and accelerated entry from G(1) into S phase of the cell cycle. This report demonstrates a function of PS1 in adult tissues, and our analysis suggests that deregulation of beta-catenin pathway contributes to the skin tumor phenotype.

Animals↗

PubMed2001

The analysis of heparin-protein interactions using evanescent wave biosensor with regioselectively desulfated heparins as the ligands.

Evanescent wave biosensor has been recently employed as a powerful tool for analyses of macromolecular interactions. In the present study, evanescent wave biosensor analysis was developed to analyze the heparin-protein interaction using as ligands a series of heparin derivatives regioselectively desulfated by chemical methods, particularly to evaluate the effect of each sulfate group of heparin. The method for immobilizing heparin on the cuvette of the evanescent wave biosensor equipment was optimized to obtain the high response required for accurate measurement. The best result was achieved when the amino group introduced at the reducing end of heparin was coupled with carboxymethyl dextran on the surface of the cuvette using glycolchitosan as a multivalent linker. The established system appeared to describe well the interactions of heparin with such proteins as acidic and basic fibroblast growth factors and tissue factor pathway inhibitor.

Biosensing Techniques↗

PubMed2001

Induction of apoptosis by cyclo-oxygenase-2 inhibitor NS398 through a cytochrome C-dependent pathway in esophageal cancer cells.

Non-steroidal anti-inflammatory drugs (NSAIDs) can induce tumor cells to undergo apoptosis in vitro. They have also shown cancer-preventive activity in vivo. The mechanism of their effects is, however, not well defined. We investigated the mechanism by which a new NSAID, NS398, induces apoptosis in esophageal cancer cell lines. NS398 decreased cell viability in 2 cyclo-oxygenase-2-positive (COX-2(+)) esophageal cancer cell lines but not in a COX-2(-) cell line. DNA fragmentation and TUNEL assays demonstrated that NS398 induced the 2 COX-2(+) cancer cell lines to undergo apoptosis. The percentage of apoptosis induced by NS398 was associated with the level of COX-2 expression. Further investigation showed that the cytochrome c pathway was responsible for NS398-induced apoptosis; i.e., cytochrome c was released from mitochondria, caspase-9 and caspase-3 were activated and finally poly(ADP-ribose)polymerase (PARP) was cleaved. Furthermore, the effect of NS398 was inhibited by the caspase inhibitor Z-DEVD-FMK and prostaglandin E(2). In contrast, bcl-2, bax, c-myc, Fas and Fas-ligand showed minor changes. Altogether, our data suggest that induction of apoptosis by NS398 is associated with COX-2 expression and occurs through the cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3 and cleaves PARP.

Apoptosis↗

PubMed2001

Accessibility of DNA polymerases to repair synthesis during nucleotide excision repair in yeast cell-free extracts.

Nucleotide excision repair (NER) removes a variety of DNA lesions. Using a yeast cell-free repair system, we have analyzed the repair synthesis step of NER. NER was proficient in yeast mutant cell-free extracts lacking DNA polymerases (Pol) beta, zeta or eta. Base excision repair was also proficient without Polbeta. Repair synthesis of NER was not affected by thermal inactivation of the temperature-sensitive mutant Polalpha (pol1-17), but was reduced after thermal inactivation of the temperature-sensitive mutant Poldelta (pol3-1) or Polvarepsilon (pol2-18). Residual repair synthesis was observed in pol3-1 and pol2-18 mutant extracts, suggesting a repair deficiency rather than a complete repair defect. Deficient NER in pol3-1 and pol2-18 mutant extracts was specifically complemented by purified yeast Poldelta and Polvarepsilon, respectively. Deleting the polymerase catalytic domain of Polvarepsilon (pol2-16) also led to a deficient repair synthesis during NER, which was complemented by purified yeast Polvarepsilon, but not by purified yeast Poleta. These results suggest that efficient repair synthesis of yeast NER requires both Poldelta and Polvarepsilon in vitro, and that the low fidelity Poleta is not accessible to repair synthesis during NER.

Catalytic Domain↗

PubMed2001

Translesion synthesis by yeast DNA polymerase zeta from templates containing lesions of ultraviolet radiation and acetylaminofluorene.

In the yeast Saccharomyces cerevisiae, DNA polymerase zeta (Polzeta) is required in a major lesion bypass pathway. To help understand the role of Polzeta in lesion bypass, we have performed in vitro biochemical analyses of this polymerase in response to several DNA lesions. Purified yeast Polzeta performed limited translesion synthesis opposite a template TT (6-4) photoproduct, incorporating A or T with similar efficiencies (and less frequently G) opposite the 3' T, and predominantly A opposite the 5' T. Purified yeast Polzeta predominantly incorporated a G opposite an acetylaminofluorene (AAF)-adducted guanine. The lesion, however, significantly inhibited subsequent extension. Furthermore, yeast Polzeta catalyzed extension DNA synthesis from primers annealed opposite the AAF-guanine and the 3' T of the TT (6-4) photoproduct with varying efficiencies. Extension synthesis was more efficient when A or C was opposite the AAF-guanine, and when G was opposite the 3' T of the TT (6-4) photoproduct. In contrast, the 3' T of a cis-syn TT dimer completely blocked purified yeast Polzeta, whereas the 5' T was readily bypassed. These results support the following dual-function model of Polzeta. First, Polzeta catalyzes nucleotide incorporation opposite AAF-guanine and TT (6-4) photoproduct with a limited efficiency. Secondly, more efficient bypass of these lesions may require nucleotide incorporation by other DNA polymerases followed by extension DNA synthesis by Polzeta.

2-Acetylaminofluorene↗

PubMed2001

A rare premalignant prostate tumor epithelial cell syndecan-1 forms a fibroblast growth factor-binding complex with progression-promoting ectopic fibroblast growth factor receptor 1.

The abnormal appearance and age-dependent loss of resident fibroblast growth factor receptor-2 (FGFR2) and gain of activity of FGFR1 in epithelial cells is a hallmark of the slow progression to malignancy in some models of prostate cancer. Pericellular matrix heparan sulfate (HS) is an integral subunit of the FGFR tyrosine kinase complex that restricts activity in absence of FGF, facilitates binding of an activating FGF, and confers specificity for FGF isoforms. In this report, we isolated and purified HS proteoglycan (HSPG) from premalignant prostate tumor epithelial cells based on the ability of the HS chains to form a binary complex with immunoglobulin module II of the ectopic and progression-promoting FGFR1 that was competent to bind FGF. The FGFR1 affinity-purified product exhibited a specific activity of over 600 times that of crude cellular HSPG enriched from cell lysates by ion exchange chromatography. The purified preparation exhibited a single NH(2)-terminal sequence with 11 of 13 residues identical to syndecan-1. The activity of purified recombinant glutathione S-transferase-tagged syndecan-1 expressed in premalignant epithelial cells confirmed that syndecan-1 bears HS chains that exhibit the rare motif that forms the FGF-binding complex with ectopic FGFR1. These results are the first to identify by affinity purification a specific HSPG core protein, the HS chains of which act as an integral subunit of the FGFR complex. The results suggest that syndecan-1 provides HS chains in premalignant epithelial cells to both the FGFR2- and FGFR1-signaling complexes that are integral to their dual roles in progression to malignancy.

Amino Acid Sequence↗

PubMed2001

Rescue of a telomere length defect of Nijmegen breakage syndrome cells requires NBS and telomerase catalytic subunit.

Nijmegen breakage syndrome (NBS) is a rare human disease displaying chromosome instability, radiosensitivity, cancer predisposition, immunodeficiency, and other defects [1, 2]. NBS is complexed with MRE11 and RAD50 in a DNA repair complex [3-5] and is localized to telomere ends in association with TRF proteins [6, 7]. We show that blood cells from NBS patients have shortened telomere DNA ends. Likewise, cultured NBS fibroblasts that exhibit a premature growth cessation were observed with correspondingly shortened telomeres. Introduction of the catalytic subunit of telomerase, TERT, was alone sufficient to increase the proliferative capacity of NBS fibroblasts. However, NBS, but not TERT, restores the capacity of NBS cells to survive gamma irradiation damage. Strikingly, NBS promotes telomere elongation in conjunction with TERT in NBS fibroblasts. These results suggest that NBS is a required accessory protein for telomere extension. Since NBS patients have shortened telomeres, these defects may contribute to the chromosome instability and disease associated with NBS patients.

Catalytic Domain↗

PubMed2001

Mutations in yeast protein phosphatase type 1 that affect targeting subunit binding.

Protein phosphatase type 1 (PP1) is a major Ser/Thr protein phosphatase that is involved in many cellular processes. The activity of PP1 is controlled by regulatory subunits, many of which are thought to bind to a hydrophobic groove in PP1 via a short consensus sequence termed the V/IXF motif. To test this hypothesis, 11 variants of yeast PP1 (Glc7) were constructed in which one or more of the residues comprising the groove were changed to alanine. These variants were tested for their biological activity in vivo, for their biochemical activity in vitro, and for their ability to associate with three PP1 binding proteins. Five variants are unable to complement the essential function of PP1 in vivo although they are catalytically active in vitro. Many of the mutants are deficient in binding two V/IXF-containing subunits, Gac1 and Reg1, which regulate glycogen accumulation and glucose repression, respectively, but all retain the ability to associate with Sds22, a regulatory subunit that lacks this motif. The subcellular locations at which PP1 normally accumulates (bud neck, nucleolus, spindle pole body) were not occupied by one PP1 variant. Additionally, we provide evidence that mutations in the hydrophobic groove of PP1 affect substrate specificity. Together, these results demonstrate the importance of the hydrophobic groove for the interaction with regulatory subunits, for the proper subcellular localization of PP1 and for the substrate specificity of PP1.

Amino Acid Substitution↗

PubMed2001

The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains.

Fusion proteins containing the Src homology (SH)3 domains of Dictyostelium myosin IB (myoB) and IC (myoC) bind a 116-kD protein (p116), plus nine other proteins identified as the seven member Arp2/3 complex, and the alpha and beta subunits of capping protein. Immunoprecipitation reactions indicate that myoB and myoC form a complex with p116, Arp2/3, and capping protein in vivo, that the myosins bind to p116 through their SH3 domains, and that capping protein and the Arp2/3 complex in turn bind to p116. Cloning of p116 reveals a protein dominated by leucine-rich repeats and proline-rich sequences, and indicates that it is a homologue of Acan 125. Studies using p116 fusion proteins confirm the location of the myosin I SH3 domain binding site, implicate NH(2)-terminal sequences in binding capping protein, and show that a region containing a short sequence found in several G-actin binding proteins, as well as an acidic stretch, can activate Arp2/3-dependent actin nucleation. p116 localizes along with the Arp2/3 complex, myoB, and myoC in dynamic actin-rich cellular extensions, including the leading edge of cells undergoing chemotactic migration, and dorsal, cup-like, macropinocytic extensions. Cells lacking p116 exhibit a striking defect in the formation of these macropinocytic structures, a concomitant reduction in the rate of fluid phase pinocytosis, a significant decrease in the efficiency of chemotactic aggregation, and a decrease in cellular F-actin content. These results identify a complex that links key players in the nucleation and termination of actin filament assembly with a ubiquitous barbed end-directed motor, indicate that the protein responsible for the formation of this complex is physiologically important, and suggest that previously reported myosin I mutant phenotypes in Dictyostelium may be due, at least in part, to defects in the assembly state of actin. We propose that p116 and Acan 125, along with homologues identified in Caenorhabditis elegans, Drosophila, mouse, and man, be named CARMIL proteins, for capping protein, Arp2/3, and myosin I linker.

Actin Depolymerizing Factors↗

PubMed2001

A combination of weakly stabilizing mutations with a disulfide bridge in the alpha-helix region of Trichoderma reesei endo-1,4-beta-xylanase II increases the thermal stability through synergism.

Thermal stability and other functional properties of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII; family 11) were studied by designed mutations. Mutations at three positions were introduced to the XYNII mutant containing a disulfide bridge (S110C-N154C) in the alpha-helix. The disulfide bridge increased the half-life of XYNII from less than 1 min to 14 min at 65 degrees C. An additional mutation at the C-terminus of the alpha-helix (Q162H or Q162Y) increased the half-life to 63 min. Mutations Q162H and Q162Y alone had a stabilizing effect at 55 degrees C but not at 65 degrees C. The mutations N11D and N38E increased the half-life to about 100 min. Due to the stabilizing mutations the pH stability increased in a wide pH range, but at the same time the activity decreased both in acidic and neutral-alkaline pH, the pH optimum being at pH region 5-6. There was no essential difference between the specific activities of the mutants and the wild-type XYNII.

Catalytic Domain↗

PubMed2001

Regulation of the L-type calcium channel by alpha 5beta 1 integrin requires signaling between focal adhesion proteins.

The L-type calcium channel is the major calcium influx pathway in vascular smooth muscle and is regulated by integrin ligands, suggesting an important link between extracellular matrix and vascular tone regulation in tissue injury and remodeling. We examined the role of integrin-linked tyrosine kinases and focal adhesion proteins in regulation of L-type calcium current in single vascular myocytes. Soluble tyrosine kinase inhibitors blocked the increase in current produced by alpha(5) integrin antibody or fibronectin, whereas tyrosine phosphatase inhibition enhanced the effect. Cell dialysis with an antibody to focal adhesion kinase or with FRNK, the C-terminal noncatalytic domain of focal adhesion kinase, produced moderate (24 or 18%, respectively) inhibition of basal current but much greater inhibition (63 or 68%, respectively) of integrin-enhanced current. A c-Src antibody and peptide inhibitors of the Src homology-2 domain or a putative Src tyrosine phosphorylation site on the channel produced similar inhibition. Antibodies to the cytoskeletal proteins paxillin and vinculin, but not alpha-actinin, inhibited integrin-dependent current by 65-80%. Therefore, alpha(5)beta(1) integrin appears to regulate a tyrosine phosphorylation cascade involving Src and various focal adhesion proteins that control the function of the L-type calcium channel. This interaction may represent a novel mechanism for control of calcium influx in vascular smooth muscle and other cell types.

Actinin↗

PubMed2001

Astrocyte-specific expression of activated p21-ras results in malignant astrocytoma formation in a transgenic mouse model of human gliomas.

Activation of the p21-ras signaling pathway from aberrantly expressed receptors promotes the growth of malignant human astrocytomas. We developed a transgenic mouse astrocytoma model using the glial fibrillary acidic protein (GFAP) promoter to express oncogenic V(12)Ha-ras, specifically in astrocytes. The development of GFAP-immunoreactive astrocytomas was directly proportional to the level of V(12)Ha-ras transgene expression. Chimeras expressing high levels of V(12)Ha-ras in astrocytes died from multifocal malignant astrocytomas within 2 weeks, whereas those with moderate levels went to germ-line transmission. Ninety-five percent of these mice died from solitary or multifocal low- and high-grade astrocytomas within 2-6 months. These transgenic astrocytomas are pathologically similar to human astrocytomas, with a high mitotic index, nuclear pleomorphism, infiltration, necrosis, and increased vascularity. Derivative astrocytoma cells are tumorigenic upon inoculation in another host. The transgenic astrocytomas exhibit additional molecular alterations associated with human astrocytomas, including a decreased or absent expression of p16, p19, and PTEN as well as overexpression of EGFR, MDM2, and CDK4. Cytogenetic analysis revealed consistent clonal aneuploidies of chromosomal regions syntenic with comparable loci altered in human astrocytomas. Therefore, this transgenic mouse astrocytoma model recapitulates many of the molecular histopathological and growth characteristics of human malignant astrocytomas in a reproducible, germ-line-transmitted, and high-penetrance manner.

Animals↗