Biomedical subjects
Publications and source records attributed to T Xu.
For individual cell, calcium is a signal of life and death and many functions are mediated by it. The development of new fluorescence dye and imaging technique have greatly promoted the research in the field of cellular calcium signal transduction path.
In chromaffin cells, an increase in intracellular Ca2+ leads to an exocytotic burst followed by sustained secretion. The burst can be further resolved into two kinetically distinct components, which suggests the presence of two separate pools of vesicles. To investigate how these components relate to SNARE complex formation, we introduced an antibody that blocks SNARE assembly but not disassembly. In the presence of the antibody, the sustained component was largely blocked, the burst was slightly reduced, and one of its kinetic components was eliminated. We conclude that SNARE complexes form before Ca(2+)-triggered membrane fusion and exist in a dynamic equilibrium between a loose and a tight state, both of which support exocytosis. Interaction of the antibody with preformed SNARE complexes favors the loose state.
With T(4)-bovine IgG as a solid-phase antigen, we have developed a direct competitive-type immunoassay for serum total thyroxine (TT(4)), which depends on the competitive distribution of europium-labeled anti-T(4) monoclonal antibody between solid-phase-bound T(4) and the T(4) in the sample or standard. The captured fraction of the tracer was measured after a dissociation-enhancement step. Four different T(4) protein conjugates were synthesized, of which T(4)-bovine IgG was selected as the most favorable for the preparation of solid-phase antigen. The sensitivity was 3.5 ng/ml with a sample volume of 20 microl. T(4) values obtained by this procedure agreed well with those obtained by RIA (r = 0.967, n = 38) and EG&G Wallac TRFIA (r = 0.926, n = 64). All other quality criteria was also fulfilled with respect to precision, accuracy, and dynamic range.
Members of the ADAM family (type I integral membrane protein with a disintegrin and metalloprotease domain) have been implicated in many important biological processes involving cell-cell and cell-matrix interactions, such as fertilization and myoblast fusion. We report here the cDNA sequence of a novel human ADAM gene (ADAM29) that contains a putative fusion peptide. Northern blot analysis revealed that the mRNA of ADAM29 is highly expressed in the testis. By radiation hybrid panel mapping, the ADAM29 gene was assigned to human chromosome 4q34.2-qter.
A hospital case-control study of meningioma was conducted in Heilongjiang Province in northeast China between September 1989 and December 1996. It included 183 cases of newly diagnosed primary meningioma and 366 individually matched hospital controls with non-neoplastic and non-neurological disease selected from six major hospitals. Cases and controls were matched by sex, age and area of residence and interviewed in the hospital wards to obtain information on medical history, occupation and lifestyle. No association with liquor or beer consumption was apparent. Cigarette smoking was positively associated with meningioma risk in women but not in men. In women, compared with non-smokers, the adjusted OR for pack-years of smoking above the median (124) was 6.2 (CI 2.04-18.87). Both of these observations contrast with the results of a study of glioma in the same population, using similar methods. The risk of meningioma was positively associated with reported occupational exposure to lead, tin, cadmium and ionising radiation in both genders.
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The vacuolar (H(+))-ATPases (or V-ATPases) are structurally related to the F(1)F(0) ATP synthases of mitochondria, chloroplasts and bacteria, being composed of a peripheral (V(1)) and an integral (V(0)) domain. To further investigate the arrangement of subunits in the V-ATPase complex, covalent cross-linking has been carried out on the V-ATPase from clathrin-coated vesicles using three different cross-linking reagents. Cross-linked products were identified by molecular weight and by Western blot analysis using polyclonal antibodies raised against individual V-ATPase subunits. In the intact V(1)V(0) complex, evidence for cross-linking of subunits C and E, D and F, as well as E and G by disuccinimidyl glutarate was obtained, while in the free V(1) domain, cross-linking of subunits H and E was also observed. Subunits C and E as well as D and E could be cross-linked by 1-ethyl-3-(dimethylaminopropyl)carbodiimide, while subunits a and E could be cross-linked by 4-(N-maleimido)benzophenone. It was further demonstrated that it is possible to treat the V-ATPase with potassium iodide and MgATP in such a way that while subunits A, B, and H are nearly quantitatively removed, significant amounts of subunits C, D, E, and F remain attached to the membrane, suggesting that one or more of these latter subunits are in contact with the V(0) domain. In addition, treatment of the V-ATPase with cystine, which modifies Cys-254 of the catalytic A subunit, results in dissociation of subunit H, suggesting communication between the catalytic nucleotide binding site and subunit H. Finally, the stoichiometry of subunits F, G, and H were determined by quantitative amino acid analysis. Based on these and previous observations, a new structural model of the V-ATPase from clathrin-coated vesicles is proposed.
Seedlings grown in darkness, i.e., etiolated seedlings, lack chlorophyll and most other components of the photosynthetic apparatus. On illumination, the plastids become photosynthetically competent through the production of chlorophylls and proteins encoded by certain chloroplast and nuclear genes. There are two types of photosynthetic cells in leaves of the C4 plant maize: bundle sheath cells (BSC) and adjacent mesophyll cells (MC). Some proteins of the maize photosynthetic machinery are solely or preferentially localized in MC and others in BSC. A particular gene may be photoregulated up in one cell type and down in the other. Transcripts of the nuclear gene rpl29, encoding the chloroplast ribosomal protein L29, increase in abundance about 17-fold during light-induced maturation of plastids. There is about 1.5 times more L29 protein in ribosomes of greening leaves than in ribosomes of unilluminated leaves; the L29 contents of MC and BSC are about the same. However, L21 is present about equally in plastid ribosomes of unilluminated and illuminated seedlings. In contrast to both L29 and L21, the fraction of the ribosome population containing L2 is about the same in MC and BSC of etiolated leaves but, on illumination, the proportion of the ribosome population with L2 increases in BSC but not in MC. The existence of different subpopulations of plastid ribosomes-e.g., those with and without L21 and/or L29 during development-evokes interesting, but as yet unanswered, questions about the roles of different types of ribosomes in differentiation.
NSF and alpha-SNAP have been shown to be required for SNARE complex disassembly and exocytosis. However, the exact requirement for NSF and alpha-SNAP in vesicular traffic through the secretory pathway remains controversial. We performed a study on the kinetics of exocytosis from bovine chromaffin cells using high time resolution capacitance measurement and electrochemical amperometry, combined with flash photolysis of caged Ca2+ as a fast stimulus. alpha-SNAP, a C-terminal mutant of alpha-SNAP, and NEM were assayed for their effects on secretion kinetics. Two kinetically distinct components of catecholamine release can be observed upon fast step-like elevation of [Ca2+]i. One is the exocytotic burst, thought to represent the readily releasable pool of vesicles. Following the exocytotic burst, secretion proceeds slowly at maintained high [Ca2+]i, which may represent vesicle maturation/recruitment, i.e. some priming steps after docking. alpha-SNAP increased the amplitude of both the exocytotic burst and the slow component but did not change their kinetics, which we examined with millisecond time resolution. In addition, NEM only partially inhibited the slow component without altering the exocytotic burst, fusion kinetics and the rate of endocytosis. These results suggest a role for alpha-SNAP/NSF in priming granules for release at an early step, but not modifying the fusion of readily releasable granules.
The effects of acute and chronic nicotine treatment on activator protein-1 (AP-1) gene transcription factor binding activity in the rat cortex were investigated. AP-1 DNA binding activity was determined by the electrophoretic gel mobility shift assay. It was observed that 1 h after acute nicotine treatment (single injection) AP-1 DNA binding activity was significantly increased in the rat cortex. On the other hand, AP-1 DNA binding activity in the rat cortex was not altered at 1 and 8 h of nicotine withdrawal after repeated nicotine treatment (10 days). However, at 18 and 24 h of nicotine withdrawal after 10 days of nicotine treatment, AP-1 DNA binding activity was significantly decreased in the rat cortex. Thus, these findings suggest that desensitization of cortical AP-1 DNA binding activity may be involved in the neuroadaptational mechanisms to nicotine dependence.
Organisms, across the animal kingdom, vary in their tolerance or susceptibility to cell injury from O2 deprivation. In this study we have taken advantage of the genetically well studied fruit fly to dissect basic mechanisms underlying their ability to tolerate lack of O2. Using differential display and molecular techniques, we cloned and characterized a novel gene, named fau, which is up-regulated considerably following anoxia in Drosophila melanogaster. Northern blot analysis revealed that the transcript of this gene is approximately 0.9 kb in length with an open reading frame encoding a small hydrophilic protein ( approximately 14.4 kDa). This protein has no homology to previously described gene products but has many potential phosphorylation sites. In situ hybridization showed that this gene is located in region 7C-D on the Drosophila X-chromosome and its transcript concentrated in the lamina and cortical neurons of the Drosophila central nervous system (CNS). Transgenic flies showed that over-expression of fau significantly reduced the recovery time of the flies from anoxia. We conclude that (1) this study provided a framework on which the mechanisms underlying anoxia tolerance can be dissected in the fruit fly and (2) fau gene plays an important role in the regulation of tissue responsiveness to O2 deprivation.
Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues. This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required for this processing event. These observations raise the possibility that Notch signaling in vivo is modulated by soluble forms of the Notch ligands.
Human bone marrow stromal cells (hBMSC) are pluripotent cells that have the ability to differentiate into bone, cartilage, hematopoietic-supportive stroma, and adipocytes in a process modulated by dexamethasone (DEX). To characterize changes in hBMSC in response to DEX, we carried out differential display experiments using hBMSC cultured for 1 week in the presence or absence of 10(-8) M DEX. When RNA from these cells was used for differential display, numerous cDNA bands were identified that were up-regulated and down-regulated by DEX. The cDNA bands were reamplified by PCR and directly used to screen an hBMSC cDNA library. Seven clones were isolated and characterized by DNA sequencing and found to encode the following genes: transforming growth factor-beta-induced gene product ((beta)ig-h3), calphobindin II, cytosolic thyroid-binding protein, 22-kDA smooth muscle protein (SM22), and the extracellular matrix proteins osteonectin/SPARC, type III collagen, and fibronectin. To confirm that these genes were regulated by DEX, the cells were treated continuously with this hormone for periods ranging from 2 to 30 days, and steady-state mRNA levels were measured by Northern blot analysis. All genes showed some level of regulation by DEX. The most profound regulation by DEX was observed in the (beta)ig-h3 gene, which showed a relative 10-fold decrease in mRNA levels after 6 days of treatment. Interestingly, (beta)ig-h3 expression was not altered by DEX in fibroblasts from other human tissues, including thymus stromal fibroblasts, spleen stromal fibroblasts, and foreskin fibroblasts. In summary, differential display of DEX-treated hBMSC revealed unique patterns of gene expression and has provided new information about phenotypic changes that accompany the differentiation of hBMSC toward osteogenesis. J. Cell. Biochem. 76:231-243, 1999. Published 1999 Wiley-Liss, Inc.
We have expanded the use of the Semliki Forest virus (SFV) by infecting chromaffin cells with synaptic proteins at high efficiency. Using the SFV gene expression system, up to 40% of cultured bovine chromaffin cells express the protein of interest within 12-48 h after infection. In order to learn about the basic physiological properties of infected cells, we performed membrane capacitance measurements using the whole-cell patch-clamp technique and monitored catecholamine release with amperometry. We found that chromaffin cells infected with green fluorescent protein (GFP) were comparable to control cells in intracellular calcium concentrations ([Ca2+]i), leak currents and cell sizes. In response to depolarization, calcium currents were elicited and the cells secreted catecholamine. Comparison of the calcium current amplitude and the size of the readily releasable pool of vesicles revealed a small decrease in these parameters compared to control cells. The refilling kinetics after pool depletion, however, were not altered. Overexpressed munc13-1 translocates to the plasma membrane in response to phorbol esters, an effect that is also observed in fibroblasts transfected with conventional methods. Thus, the use of the SFV gene expression system to infect chromaffin cells represents a major improvement in infection efficiency compared to other methods. It opens up new opportunities to introduce synaptic proteins into chromaffin cells and study their role in secretion.
A detailed investigation of the tribological behaviour of vacuum arc diamond-like carbon coated Ti-6Al-4V against a medical grade ultra-high molecular weight polyethylene is conducted in this work in order to investigate the potential use of diamond-like carbon coatings for orthopaedic appplications. Lubricated and non-lubricated wear experiments are performed using a standard pin-on-disc wear tester. The coefficient of friction is monitored continuously during testing and wear rate calculations are performed using surface profilometry measurements of worn disc surfaces. Sliding wear tests show the existence of two distinct friction and wear regimes distinguished by physically different mechanisms. In the first stages of wear, adhesion and abrasion are the dominant mechanisms of wear while fatigue processes are activated later in the tests. The effects of diamond-like carbon coating structure, surface roughness and lubrication on tribological behaviour are presented. Optimal process-structure-property design for vacuum arc plasma deposition is utilized in order to obtain strong adhesion to the titanium alloy substrate. Diamond-like carbon coatings significantly improve the friction and wear performance of the orthopaedic bearing pair and show exceptional promise for biomedical applications.
We have previously used mosaic flies to screen for tumour suppressors or negative regulators of cell proliferation. The cellular composition of these flies resembles that of cancer patients who are chimaeric individuals carrying a small number of mutated somatic cells. One of the genes we identified is the large tumour suppressor gene, lats (also known as wts), which encodes a putative serine/threonine kinase. Somatic cells mutant for lats undergo extensive proliferation and form large tumours in many tissues in mosaic adults. Homozygous mutants for various lats alleles display a range of developmental defects including embryonic lethality. Although many tumour suppressors have been identified in Drosophila melanogaster, it is not clear whether these fly genes are directly relevant to tumorigenesis in mammals. Here, we have isolated mammalian homologues of Drosophila lats. Human LATS1 suppresses tumour growth and rescues all developmental defects, including embryonic lethality in flies. In mammalian cells, LATS1 is phosphorylated in a cell-cycle-dependent manner and complexes with CDC2 in early mitosis. LATS1-associated CDC2 has no mitotic cyclin partner and no kinase activity for histone H1. Furthermore, lats mutant cells in Drosophila abnormally accumulate cyclin A. These biochemical observations indicate that LATS is a novel negative regulator of CDC2/cyclin A, a finding supported by genetic data in Drosophila demonstrating that lats specifically interacts with cdc2 and cyclin A.
The lats gene has been identified as a tumour suppressor in Drosophila melanogaster using mosaic screens. Mosaic flies carrying somatic cells that are mutant for lats develop large tumours in many organs. The human LATS1 homologue rescues embryonic lethality and inhibits tumour growth in lats mutant flies, demonstrating the functional conservation of this gene. Biochemical and genetic analyses have revealed that LATS1 functions as a negative regulator of CDC2 (ref. 3). These data suggest that mammalian LATS1 may have a role in tumorigenesis. To elucidate the function of mammalian LATS1, we have generated Lats1-/- mice. Lats1-/- animals exhibit a lack of mammary gland development, infertility and growth retardation. Accompanying these defects are hyperplastic changes in the pituitary and decreased serum hormone levels. The reproductive hormone defects of Lats1-/- mice are reminiscent of isolated LH-hypogonadotropic hypogonadism and corpus luteum insufficiency in humans. Furthermore, Lats1-/- mice develop soft-tissue sarcomas and ovarian stromal cell tumours and are highly sensitive to carcinogenic treatments. Our data demonstrate a role for Lats1 in mammalian tumorigenesis and specific endocrine dysfunction.
Mutations in the tumor suppressor gene PTEN (MMAC1/TEP1) are associated with a large number of human cancers and several autosomal-dominant disorders. Mice mutant for PTEN die at early embryonic stages and the mutant embryonic fibroblasts display decreased sensitivity to cell death. Overexpression of PTEN in different mammalian tissue culture cells affects various processes including cell proliferation, cell death and cell migration. We have characterized the Drosophila PTEN gene and present evidence that both inactivation and overexpression of PTEN affect cell size, while overexpression of PTEN also inhibits cell cycle progression at early mitosis and promotes cell death during eye development in a context-dependent manner. Furthermore, we have shown that PTEN acts in the insulin signaling pathway and all signals from the insulin receptor can be antagonized by either Drosophila or human PTEN, suggesting a potential means for alleviating symptoms associated with altered insulin signaling.