Search PubMed⌕ Search

Biomedical subjects

J Wu

Publications and source records attributed to J Wu.

At least 793 records · Page 44Linked to original sources
PubMed1998

Abundant mitochondrial genome diversity, population differentiation and convergent evolution in pines.

We examined mitochondrial DNA polymorphisms via the analysis of restriction fragment length polymorphisms in three closely related species of pines from western North America: knobcone (Pinus attenuata Lemm.), Monterey (P. radiata D. Don), and bishop (P. muricata D. Don). A total of 343 trees derived from 13 populations were analyzed using 13 homologous mitochondrial gene probes amplified from three species by polymerase chain reaction. Twenty-eight distinct mitochondrial DNA haplotypes were detected and no common haplotypes were found among the species. All three species showed limited variability within populations, but strong differentiation among populations. Based on haplotype frequencies, genetic diversity within populations (HS) averaged 0.22, and population differentiation (GST and theta) exceeded 0.78. Analysis of molecular variance also revealed that >90% of the variation resided among populations. For the purposes of genetic conservation and breeding programs, species and populations could be readily distinguished by unique haplotypes, often using the combination of only a few probes. Neighbor-joining phenograms, however, strongly disagreed with those based on allozymes, chloroplast DNA, and morphological traits. Thus, despite its diagnostic haplotypes, the genome appears to evolve via the rearrangement of multiple, convergent subgenomic domains.

DNA, Mitochondrial↗
PubMed1998

A new source of human oocytes: preliminary report on the identification and maturation of human preantral follicles from follicular aspirates.

This study aimed to investigate the development of human preantral follicles and oocyte maturation in vitro. Preantral follicles were obtained from follicular aspirates during egg retrieval carried out during an in-vitro fertilization (IVF) programme. They were first incubated in Ham's F10 medium with 15% fetal cord serum (FCS). After 28 days, the medium was supplemented with different doses of human menopausal gonadotrophin (HMG), human follicular fluid (hFF) and epidermal growth factor (EGF) by orthogonal design. Promotion of final maturation was completed in the presence of HMG and hFF. Development from preantral to antral follicles was found within 6-12 days of culture. With time, the proportion of follicles with diameters of >300 microm increased at 21-28 days of culture (P < 0.005). The maximum number of oocytes extruded, and first polar body formation, occurred in the presence of 0.15 IU/ml HMG 40% (v/v) hFF and 6 ng/ml EGF. We conclude that follicular aspirates obtained during egg retrieval in an IVF programme contain many preantral follicles which could develop into antral follicles with extrusion of oocytes in culture, and that the oocytes can mature in vitro. Hence, a new source of human oocytes is available.

Cell Differentiation↗
PubMed1998

Rapid synthesis of IFN-gamma by T cells in skin may play a pivotal role in the human skin immune system.

This study has shown that T lymphocytes with alphabeta TCR present in normal human skin possess unique characteristics which may allow them to fulfil a unique role in skin. It disclosed that unlike T cells from blood, they are devoid of proliferative capacity in response to any ligand including lectins and phorbol esters, and show a diminished Ca2+ flux in response to phytohemagglutinin (PHA). However, despite this apparent lack of reactivity, these cells respond to stimulation by synthesis of IFN-gamma. Furthermore, this response by T cells from the skin differs from that of T cells in blood in its tempo. Whereas IFN-gamma is first detectable in peripheral blood lymphocytes (PBL) 42 h following stimulation, T cells from skin produce IFN-gamma 2.5 h after activation, or 17 times faster. Whereas synthesis persists in PBL for several days, it only lasts in skin T cells for 90 min. This difference in the kinetics is a consequence of differences in the regulation of IFN-gamma synthesis in PBL and skin-derived T cells. Thus, unlike PBL, T cells in intact unimmunized skin or ex vivo express mRNA for IFN-gamma, transcribed in a constitutive manner. Activation of these cells turns on translation of IFN-gamma synthesis directly and at the same time turns off further transcription of the IFN-gamma gene: cessation of IFN-gamma synthesis coincides with the disappearance of IFN-gamma mRNA within 4 h of activation. These observations suggest that this pre-formed mRNA may be solely responsible for the early synthesis of IFN-gamma in skin, determining both the amount and the duration of its synthesis. We propose that these cells may represent the antigen-specific component of a first-line host defence system in human skin. The reported absence of delayed-type hypersensitivity reactivity in patients with a selective deficit of skin T cells supports this conclusion.

Calcium↗
PubMed1998

Cys-scanning mutagenesis: a novel approach to structure function relationships in polytopic membrane proteins.

The entire lactose permease of Escherichia coli, a polytopic membrane transport protein that catalyzes beta-galactoside/H+ symport, has been subjected to Cys-scanning mutagenesis in order to determine which residues play an obligatory role in the mechanism and to create a library of mutants with a single-Cys residue at each position of the molecule for structure/function studies. Analysis of the mutants has led to the following: 1) only six amino acid side chains play an irreplaceable role in the transport mechanism; 2) positions where the reactivity of the Cys replacement is increased upon ligand binding are identified; 3) positions where the reactivity of the Cys replacement is decreased by ligand binding are identified; 4) helix packing, helix tilt, and ligand-induced conformational changes are determined by using the library of mutants in conjunction with a battery of site-directed techniques; 5) the permease is a highly flexible molecule; and 6) a working model that explains coupling between beta-galactoside and H+ translocation. structure-function relationships in polytopic membrane proteins.

Alkylation↗
PubMed1998

A software system for data analysis in automated DNA sequencing.

Software for gel image analysis and base-calling in fluorescence-based sequencing consisting of two primary programs, BaseFinder and GelImager, is described. BaseFinder is a framework for trace processing, analysis, and base-calling. BaseFinder is highly extensible, allowing the addition of trace analysis and processing modules without recompilation. Powerful scripting capabilities combined with modularity and multilane handling allow the user to customize BaseFinder to virtually any type of trace processing. We have developed an extensive set of data processing and analysis modules for use with the program in fluorescence-based sequencing. GelImager is a framework for gel image manipulation. It can be used for gel visualization, lane retracking, and as a front end to the Washington University Getlanes program. The programs were designed using a cross-platform development environment, currently allowing them to run in Windows NT, Windows 95, Openstep/Mach, and Rhapsody. Work is ongoing to deploy the software on additional platforms, including Solaris, Linux, and MacOS. This software has been thoroughly tested and debugged in the analysis of >2 million bp of raw sequence data from human chromosome 19 region q13. Overall sequencing accuracy was measured using a significant subset of these data, consisting of approximately 600 sequences, by comparing the individual shotgun sequences against the final assembled contigs. Also, results are reported from experiments that analyzed the accuracy of the software and two other well-known base-calling programs for sequencing the M13mp18 vector sequence. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF025422]

Algorithms↗
PubMed1998

Early phase tumor accumulation of macromolecules: a great difference in clearance rate between tumor and normal tissues.

The objective of this study was to investigate the molecular weight (MW) and time-dependence of the phenomenon termed "the enhanced permeability and retention" (EPR) effect in solid tumor, in particular to determine and define the early phase accumulation of macromolecules in tumor and normal tissues and the relationship between blood concentration and tissue clearance. As a model, radioiodinated N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers of MW ranging from 4.5 K to 800 K were administered i.v. to mice bearing sarcoma 180 tumor. Within 10 min all HPMA copolymers accumulated effectively in the tumor regardless of MW (1.0-1.5% of injected dose per g of tumor). However, higher MW copolymers (> 50 K) showed significantly increased tumor accumulation after 6 h, while the lower MW copolymers (< 40 K) were cleared rapidly from tumor tissue due to rapid diffusion back into the bloodstream. Blood clearance was also MW-dependent; the lower MW copolymers displayed rapid clearance, with kidney radioactivity of the copolymers of MW < 20 K representing 24% of injected dose per g kidney at 1 min after i.v. administration. Within 10 min these copolymers passed through the kidney and were excreted in the urine. Higher MW copolymers consistently showed kidney levels of 3-5% dose per g kidney in the early phase with no time-dependent accumulation in kidney. There was also no progressive accumulation in muscle or liver, regardless of polymer MW. These results suggest the "EPR effect" in solid tumor primarily arises from in the difference in clearance rate between the solid tumor and the normal tissues after initial penetration of the polymers into these tissues.

Animals↗
PubMed1998

Cytochalasin D as excitation-contraction uncoupler for optically mapping action potentials in wedges of ventricular myocardium.

INTRODUCTION: Cytochalasin D in tissue bath superfusate inhibits the contraction of isolated thin trabeculae from canine right ventricle without affecting the intracellular action potential recorded with glass microelectrode. The purpose of this study was to test whether cytochalasin D could also be used to immobilize perfused wedges of ventricular muscle without affecting the action potential duration or propagation, and also to determine the optimal concentration and time duration of drug in the perfusate. METHODS AND RESULTS: Using a membrane potential sensitive dye, di-4-ANEPPS, and a high-resolution photodiode optical mapping system at a rate of 1,000 frames/sec, we recorded action potentials on the transmural surface of arterially perfused wedges of muscle from the canine left ventricular free wall. We also recorded arterial pulse pressure as a surrogate for tissue contraction. Cytochalasin D at > or = 20 micromol/L in the perfusate for > or = 6 minutes reduced the arterial pulse pressure to approximately one tenth of its initial value and significantly reduced or eliminated motion artifacts in the action potentials. A sustained concentration of 10 micromol/L cytochalasin D in the perfusate prevented contraction from recurring after the tissue was immobilized with an initial concentration of 25 micromol/L. Cytochalasin D had little effect on the action potential duration and on its transmural gradient, and did not slow the transmural velocity of excitation propagation. CONCLUSION: Cytochalasin D can be used to uncouple excitation and contraction in perfused canine cardiac muscle for the fluorescent-optical mapping of action potentials without affecting action potential duration or slowing transmural propagation.

Action Potentials↗
PubMed1998

Differential effects of cytochalasin D and 2,3 butanedione monoxime on isometric twitch force and transmembrane action potential in isolated ventricular muscle: implications for optical measurements of cardiac repolarization.

INTRODUCTION: 2,3-Butanedione monoxime (BDM) has been widely used to inhibit contraction during optical recordings of cardiac membrane voltage changes, even though it markedly abbreviates cardiac action potentials. METHODS AND RESULTS: We compared the effects of BDM and of the F-actin disrupter cytochalasin D (cyto D) on isometric twitch force and transmembrane action potentials in isolated canine right ventricular trabeculae superfused with Tyrode's solution (2 mmol/L CaCl2, 37 degrees C) and stimulated at 0.5 Hz. BDM at 10 mmol/L and cyto D at 80 micromol/L were equally effective in reducing peak isometric force to 10%+/-3% (n = 6; mean+/-SEM) and 8%+/-1% (n = 8), respectively. Neither agent significantly altered resting tension. While 10 mmol/L BDM markedly shortened the action potential duration at 90% repolarization (APD90) from 198+/-7 msec to 146+/-9 msec (P < 0.001), 80 micromol/L cyto D had no significant effects on APD90 or on any other action potential parameter. The effects of BDM on peak isometric force and APD were completely reversible after 15 minutes of washout, whereas in the cyto D group contractile force continued to be reduced (13%+/-3%) and action potential characteristics did not show significant changes from control values after a 60-minute period of superfusion with cyto D-free Tyrode's solution. CONCLUSION: We conclude that cyto D should be considered an alternative excitation-contraction uncoupler for optical mapping studies of cardiac repolarization.

Action Potentials↗
PubMed1998

Transcription factor Sp1 mediates cell-specific trans-activation of the human cytomegalovirus DNA polymerase gene promoter by immediate-early protein IE86 in glioblastoma U373MG cells.

Human cytomegalovirus (HCMV) gene expression is highly cell and tissue specific. Cell factor-mediated regulatory interactions are involved in regulating the restricted expression of the HCMV major immediate-early (IE) gene (J. F. Baskar, P. P. Smith, G. Nilaver, R. A. Jupp, S. Hoffmann, N. J. Peffer, D. J. Tenney, A. M. Colberg-Poley, P. Ghazal, and J. A. Nelson, 70:3207-3213, 1996). To gain an understanding of HCMV early gene activation, we studied the effect of each of the three major IE proteins, IE72, IE86, and IE55, on the HCMV DNA polymerase gene (pol; UL54) promoter. In transient-expression assays, the IE86 protein alone was able to transactivate the pol promoter, but IE72 and IE55 were not, in permissive U373MG cells. However, we were unable to detect IE86-mediated transactivation in nonpermissive HeLa or C33-A cells. Using electrophoretic mobility shift assays (EMSAs), we found that expression of the IE86 protein in U373MG cells resulted in specific binding of a DNA complex to an inverted-repeat element, IR1, of the pol promoter. Antibody supershifting and EMSA-Western blotting experiments further showed that IE86 and the cellular transcription factor Sp1 were components of the IR1 DNA-binding complex. Furthermore, we found that binding of DNA by Sp1 was dramatically increased in the presence of IE86. Interestingly, this IE86-induced DNA-binding activity of Sp1 was inhibited by a repressor activity presented in HeLa cells. In summary, our study suggests that a viral regulatory protein can modulate the DNA binding activity of a cellular transcription factor, resulting in cell-specific transactivation of viral genes.

Base Sequence↗
PubMed1998

Impaired motion sensitivity as a predictor of subsequent field loss in glaucoma suspects: the Roscommon Glaucoma Study.

AIM: To determine if impaired motion sensitivity is a significant predictor of subsequent field loss in glaucoma suspects. METHOD: A population based prospective study; a 5 year follow up of all glaucoma suspects who had been identified from a population based random sample survey in the west of Ireland. 78 glaucoma suspects whose visual field function was annually measured by Henson CFS 2000 and for whom data on family history of glaucoma, ocular status, and motion impairment had been recorded. Visual field loss was defined as Henson visual field survival score of 94 or less. RESULTS: 18 people developed visual field loss in at least one eye. Motion impairment at baseline was associated with a 2-18 times greater risk of development of the visual field loss (p < 0.001). This association was independent of sex, family history of glaucoma, intraocular pressure, and C/D ratio at baseline. The Cox's proportional hazards regression analysis confirmed the above results after adjustment for age and the C/D ratio. CONCLUSION: Motion impairment is an independent predictor of visual field loss in glaucoma suspects, although it is not clear how long motion impairment precedes visual field loss.

Age Factors↗
PubMed1998

Evidence of Trolox and some gallates as synergistic protectors of erythrocytes against peroxyl radicals.

The peroxidation of human erythrocytes induced by peroxyl radical initiator and its inhibition by several gallate esters (e.g., propyl, methyl, ethyl) and Trolox (a more polar analogue of vitamin E) have been studied. The antioxidant activity was determined on erythrocytes against hemolysis generated by a thermal activator, 2,2'-azobis-(2-amidinopropane)dihydrogenchloride. It was found that propyl gallate and its two analogues were more effective than Trolox in preventing cell lysis. However, the combination of gallate esters and Trolox produced a protective effect exceeding the arithmetic sum of their individual contributions. These perceived synergisms occur at more than one level of Trolox at a given level of a gallate ester.

Antioxidants↗
PubMed1998

Nerve-derived trophic factors and DNA elements controlling expression of genes encoding synaptic proteins in skeletal muscle fibers.

The neuromuscular junction represents an excellent model system for studying various critical issues in neurobiology at the molecular, cellular, and physiological levels. Our understanding of the basic events underlying synpase formation, maintenance, and plasticity has progressed considerably over the last few years primarily because of the numerous studies that have focused on this synapse and used sophisticated recombinant DNA technology. Recent data indicate that myonuclei located in the vicinity of the postsynaptic membrane are in a differential state of transcription compared to nuclei of the extrasynaptic sarcoplasm. Thus, renewal of postsynaptic membrane proteins appears to occur via a mechanism involving the local transcriptional activation of genes encoding these specialized proteins and extracellular cues originating from motoneurons. Such interaction between presynaptic nerve terminals and the postsynaptic sarcoplasm indicates that the entire signal transduction pathway is compartmentalized at the level of the neuromuscular junction. Expression of these genes appears less coregulated than originally anticipated, indicating that maintenance of the postsynaptic membrane requires the contribution of multiple extracellular signals, which ultimately urge target transcription factors to distinct DNA regulatory elements via various second messenger systems.

Cell Nucleus↗
PubMed1998

Suppression of ANP gene transcription by liganded vitamin D receptor: involvement of specific receptor domains.

We showed previously that liganded vitamin D receptor (VDR) effects a suppression of human atrial natriuretic peptide (hANP) gene-promoter activity in cultured neonatal rat atrial myocytes. In the present study, we have attempted to identify the structural domains of the VDR that are involved in mediating this suppression. We examined the effects of a series of VDR mutants on a cotransfected hANP promoter-driven chloramphenicol acetyltransferase (CAT) reporter. Neither the native VDR nor any of the mutants tested displayed inhibitory activity in the absence of the 1,25-dihydroxyvitamin D3 (VD3) ligand. Delta134, a deletant harboring solely the DNA binding region of the VDR, and L254G, a mutant shown to be defective in retinoid X receptor (RXR) heterodimer formation in other systems, were as effective as the native VDR in reducing promoter activity. HBD, a deletant containing only the hormone-binding domain of the VDR, and K246G, a point mutant that is defective in the activation function of the receptor, did not attenuate reporter activity. A similar activity profile was displayed when a positively regulated promoter containing a direct-repeat vitamin D responsive element (DR3-CAT) was examined in these cells. Liganded VDR, the delta134 mutant, and liganded L254G effected increases in DR3-CAT activity of 2.5-, 2-, and 4-fold, respectively. Two nonhypercalcemic analogues of VD3 (RO 23-7553 and RO 25-6760) displayed the same inhibitory activity as VD3. These studies suggest that the inhibition of hANP promoter activity requires both the DNA binding and activation functions of the receptor but does not appear to require formation of a classic RXR alpha-VDR heterodimer.

Analysis of Variance↗
PubMed1998

The effect of chloride concentration on human neutrophil functions: potential relevance to cystic fibrosis.

Recently, some investigators have observed elevated concentrations of chloride in the airway surface fluid (ASF) overlying respiratory epithelia from cystic fibrosis (CF) patients compared with ASF overlying non-CF epithelia. Others have shown that this elevated ASF salt concentration can inactivate human beta-defensin-1, an antimicrobial peptide secreted by respiratory epithelia. This could impair the primary epithelial defense against bacteria in the CF airway, thereby forcing a greater reliance on polymorphonuclear leukocyte (PMN)-mediated defenses. Pseudomonas aeruginosa (Psa) flourishes in the CF airway despite the presence of abundant PMN. We therefore investigated whether elevated ASF chloride concentration in CF might also compromise PMN function. We employed a cell-culture model in which halide concentrations and osmolarity were varied independently. We examined the effects of chloride concentration on three aspects of PMN function: recruitment of PMN to the airway (production of interleukin-8 [IL-8]), PMN antimicrobial activity (killing of Psa), and PMN clearance from the airways (apoptosis and lysis). We found that exposure to elevated chloride concentration increased PMN synthesis of IL-8, decreased PMN killing of Psa, and accelerated PMN apoptosis and lysis. In CF airways, elevated chloride therefore could contribute to the increased number of PMN recruited into the airways, the increased survival of Psa, and the increased quantity of toxic mediators released by PMN into the airways. These effects of elevated chloride on PMN function may provide another causal link between loss of cystic fibrosis transmembrane conductance regulator function and CF lung disease.

Adult↗
PubMed1998

Expression of the human integrin beta6 subunit in alveolar type II cells and bronchiolar epithelial cells reverses lung inflammation in beta6 knockout mice.

Inactivation of the integrin beta6 subunit gene in mice resulted in an unexpected phenotype-functionally significant inflammation of the skin and lungs. These findings suggested a role for ligation of the alphav beta6 integrin on epithelial cells in downregulating epithelial inflammation. However, the results of gene inactivation could have been due to inactivation of adjacent genes and provided no information about the role of this integrin in specific populations of epithelial cells. In the current study, we used transgenic mice constitutively expressing the human beta6 subunit in alveolar type II cells and bronchiolar epithelial cells to examine directly the significance of alphav beta6 in these cells. Expression of this transgene largely inhibited the increases in airspace lymphocytes and macrophages and the lymphocyte and macrophage activation caused by inactivation of the beta6 subunit gene, and reduced the peribronchial and perivascular accumulations of lymphocytes. In the genetically mixed mice used for this study, we identified airway eosinophilia as an additional effect of beta6 inactivation. This effect was also partially inhibited by limited expression of the human transgene. These results definitively identify a role for distal lung epithelial alphav beta6 in downregulating pulmonary inflammation and suggest that interventions augmenting beta6 expression or function in these cells could influence the course of inflammatory lung diseases.

Animals↗
PubMed1998

The integrin alphavbeta6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin.

The integrin alphavbeta6 is expressed on a variety of epithelial cells during dynamic processes including organogenesis, tissue injury and malignant transformation. However, because of the lack of tools to specifically inhibit the function of this integrin, little is known about its effects on cell behavior. To directly examine the role of this integrin in cell migration, we used keratinocytes derived from wild-type mice or mice expressing a null mutation in the beta6 subunit (beta6-/-) to perform migration assays in vitro. Migration on the known alphavbeta6 ligand, fibronectin was reduced in keratinocytes from beta6-/- mice. Interestingly, keratinocytes from beta6-/- mice also demonstrated markedly reduced migration on vitronectin, a protein not previously known to be a ligand for alphavbeta6. An anti-alphavbeta6 monoclonal antibody 10D5, generated by immunization of beta6-/- mice with murine keratinocytes, inhibited adhesion and migration of wild-type keratinocyte on both vitronectin and fibronectin to levels similar to those seen with keratinocytes from beta6-/- mice. alphavbeta6-mediated migration on both ligands was dramatically augmented by treatment with phorbol myrisate acetate (PMA) or with hepatocyte growth factor, and augmentation of migration by either stimulus could be abolished by the PKC inhibitor GF109203X, suggesting a critical role for PKC in enhancement of alphavbeta6-mediated cell migration.

Animals↗
PubMed1998

Cepharanthine inhibits proliferation of cancer cells by inducing apoptosis.

Cepharanthine, a biscoclaurine alkaloid extracted from Stephania cepharantha Hayata was examined for a possible apoptosis-inducing effect in murine P388 doxorubicin-sensitive (P388/S) and -resistant (P388/DOX) cells. A significant increase in LDH release from cells was observed after P388/S and P388/DOX cells had been exposed to cepharanthine for 24 h. Cepharanthine (10 micrograms/ml) markedly induced apoptosis in resistant cells after 6 h and 24 h. By the means of agarose electrophoresis the DNA ladder was detected in cell lines treated with cepharanthine for 24 h. Cepharanthine (1-10 micrograms/ml) also induced the production of reactive oxygen species in P388/DOX cells, while no such cepharanthine-induced increase in reactive oxygen species was observed in P388/S cells. Flow cytometry analysis showed a high level of Fas-antigen expression in P388/DOX cells treated with cepharanthine. Furthermore, we found that the inhibitition of DNA and protein synthesis caused by cepharanthine (10 micrograms/ml) was more significant in resistant cells than in sensitive cells. Cepharanthine had no effect on the GSH content of P388/S and P388/DOX cells. Our experimental results suggest that cepharanthin can induce apoptosis both in P388/S and P388/DOX cells, especially in the latter. Apoptosis induced by cepharanthine may be implicated in the production of reactive oxygen species and Fas-antigen expression in tumor cells.

Alkaloids↗