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Biomedical subjects

J Wu

Publications and source records attributed to J Wu.

At least 1,027 records · Page 57Linked to original sources

Fas ligand mutation in a patient with systemic lupus erythematosus and lymphoproliferative disease.

The pathogenesis of systemic lupus erythematosus (SLE) is multifactorial and multigenetic. The apoptosis genes, fas and fas ligand (fasL), are candidate contributory genes in human SLE, as mutations of these genes result in autoimmunity in several murine models of this disease. In humans, fas mutations result in a familial autoimmune lymphoproliferative syndrome, but defects in FasL have not yet been identified. In this study, DNA from 75 patients with SLE was screened by single-stranded conformational polymorphism analysis for potential mutations of the extracellular domain of FasL. A heterozygous single-stranded conformational polymorphism for FasL, was identified in one SLE patient, who exhibited lymphadenopathy. Molecular cloning and sequencing indicated that the genomic DNA of this patient contained an 84-bp deletion within exon 4 of the fasL gene, resulting in a predicted 28 amino acid in-frame deletion. Analysis of PBMC from this patient revealed decreased FasL activity, decreased activation-induced cell death, and increased T cell proliferation after activation. This is the first report of defective FasL-mediated apoptosis related to a mutation of the human Fasl, gene in a patient with SLE and suggests that fasL mutations are an uncommon cause of the disease.

Amino Acid Sequence↗

Amygdala activity at encoding correlated with long-term, free recall of emotional information.

Positron emission tomography of cerebral glucose metabolism in adult human subjects was used to investigate amygdaloid complex (AC) activity associated with the storage of long-term memory for emotionally arousing events. Subjects viewed two videos (one in each of two separate positron emission tomography sessions, separated by 3-7 days) consisting either of 12 emotionally arousing film clips ("E" film session) or of 12 relatively emotionally neutral film clips ("N" film session), and rated their emotional reaction to each film clip immediately after viewing it. Three weeks after the second session, memory for the videos was assessed in a free recall test. As expected, the subjects' average emotional reaction to the E films was higher than that for the N films. In addition, the subjects recalled significantly more E films than N films. Glucose metabolic rate of the right AC while viewing the E films was highly correlated with the number of E films recalled. AC activity was not significantly correlated with the number of N films recalled. The findings support the view derived from both animal and human investigations that the AC is selectively involved with the formation of enhanced long-term memory associated with emotionally arousing events.

Adult↗

Thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns in single pancreatic acinar cells of mouse.

Modulation of the agonist-specific cytosolic Ca2+ oscillatory pattern by thimerosal has been investigated in single pancreatic acinar cells using patch-clamp perforated whole-cell recording to measure the calcium-dependent chloride current (I(C1)(Ca2+)). 1 microM thimerosal, which fails to evoke Ca2+ oscillation alone, clearly changed the pattern of Ca2+ oscillation from pulsatile spikes (evoked by low concentrations of activators) to sinusoidal or transient oscillations. The mimetic action of thimerosal was independent of extracellular Ca2+, was blocked by extracellular application of dithiothreitol or 10 mM caffeine, as well as by internal perfusion with heparin; but was unaffected by ruthenium red. We conclude that thimerosal modulates the agonist-specific cytosolic Ca2+ oscillatory patterns mediated by sensitizing the InsP3-induced Ca2+ release.

Acetylcholine↗

The human FGF-8 gene localizes on chromosome 10q24 and is subjected to induction by androgen in breast cancer cells.

Androgen-induced growth factor (AIGF or FGF-8) was originally isolated from the conditioned medium of an androgen-dependent Shionogi carcinoma, SC-3, cell line. It shares structural similarity with other members of the FGF family. The temporal and spatial expression patterns of the FGF-8 gene suggest its involvement in gastrulation, regionalization of the brain, and organogenesis of the limb and face as an embryonic epithelial factor. In the adult, expression of FGF-8 is restricted to gonads including testes and ovaries. Since FGF-8 is identified as a corroborating gene in MMTV-induced mammary tumors in Wnt-1 transgenic mice and because FGF-8 manifested its autocrine mitogenic activity in SC-3 cells, it is possible that aberrant expression of FGF-8 may be present in human cancers which are hormone dependent. However, very little is known about human FGF-8. To determine whether FGF-8 plays a role in human breast cancer, we have isolated the full-length cDNA from SK-BR-3 breast cancer cells. We have also isolated the corresponding genomic DNA in a P1 cloning vector. The FGF-8 gene has been mapped to chromosome 1Oq24 using both somatic cell hybrid genetic analysis and fluorescence in situ hybridization. Finally, we show that FGF-8 gene expression in a human breast cancer cell line, MDA-MB-231, is inducible by androgen. The findings presented here will facilitate our understanding of the molecular mechanism underlying hormone-responsive breast and prostate cancers.

Adenocarcinoma↗

The novel catenin p120cas binds classical cadherins and induces an unusual morphological phenotype in NIH3T3 fibroblasts.

p120cas (CAS) is a tyrosine kinase substrate whose phosphorylation has been implicated in cell transformation by Src and in ligand-induced signaling through the EGF, PDGF, and CSF-1 receptors. More recently, CAS has been shown to associate with E-cadherin and its cofactors (catenins), molecules that are involved in cell adhesion. Although both CAS and beta-catenin contain armadillo repeat domains (Arm domains), the amino acid identity between these proteins in this region is only 22%, and it is not yet clear whether CAS will emulate other catenins by associating with other members of the cadherin family. Here we report that in addition to binding E-cadherin, wild-type CAS associated with N-cadherin and P-cadherin. Transient transfection of cloned CAS isoforms into MDCK epithelial cells indicated that CAS1 and CAS2 isoforms are equally capable of binding to E-cadherin even though these cells preferentially express CAS2 isoforms. In addition, CAS colocalized with N-cadherin in NIH3T3 cells and analysis of CAS mutants in vivo indicated that the CAS-N-cadherin interaction requires an intact CAS Arm domain. The data suggest that CAS-cadherin interactions in general are dictated by the conserved armadillo repeats and are not heavily influenced by sequences added outside the Arm domain by alternative splicing. Interestingly, overexpression of CAS in NIH3T3 cells induced a striking morphological phenotype characterized by the presence of long dendrite-like processes. This branching phenotype was specific for CAS, since (i) overexpression of the structurally similar beta-catenin had little effect on cell morphology, and (ii) the branching was abolished by deletions in the CAS Arm domain. Our data indicate that, like other catenins, CAS is a cofactor for multiple members of the cadherin family. However, the dramatically distinct phenotype exhibited by fibroblasts overexpressing CAS, versus beta-catenin, support recent data suggesting that these catenins have fundamentally different and possibly opposing roles in cadherin complexes.

3T3 Cells↗

Specification of the wing by localized expression of wingless protein.

Limb development in Drosophila depends on subdivision of the limb primordia into functional units called compartments. Cell interactions across compartment boundaries establish pattern-organizing centres that control growth and specify cell fates along the anteroposterior (AP) and dorsoventral (DV) axes of the limbs. AP subdivision of the disc primordia is inherited from the embryonic ectoderm. DV subdivision of the wing disc occurs during the second larval instar through localized expression of the apterous protein (Apterous) in dorsal cells. A third major subdivision of the wing disc into wing and body-wall compartments also occurs in the second instar. Here we show that specification of the wing primordium in early second instar depends on activity of the AP patterning system but not the DV system. These results define two distinct roles for the wingless gene: a primary role in specifying the wing primordium, and a subsequent role mediating the patterning activities of the DV compartment boundary.

Animals↗

A dual vacuum chamber fourier transform mass spectrometer with rapidly interchangeable LSIMS, MALDI, and ESI sources: initial results with LSIMS and MALDI.

A design is presented involving two separate vacuum chambers to provide nearly simultaneous capabilities of liquid secondary ion mass spectrometry (LSIMS), matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) in an external source Fourier transform mass spectrometer. The instrument consists of two vacuum chambers, one with five stages of differential pumping for a combined LSIMS/MALDI source. The chamber dedicated to ESI was formerly a three-stage chamber with LSIMS and electron ionization. Two additional stages were added with the ESI source. LSIMS and MALDI have similar vacuum requirements and were moved to a newly built chamber with two stages of pumping. We present our first results obtained on the new vacuum chamber. Data presented for the MALDI source show that, with only two stages of pumping, and with shorter radio frequency-only quadrupole rods for ion injection, spectra comparable to those obtained on the formerly three-stage instrument can be obtained. Characterization of the MALDI source and data on linear, cyclic, and branched oligosaccharides are given. Finally, the design of the secon chamber is proposed as a low-cost prototype for an external source FTMS instrument.

Carbohydrate Sequence↗

High-Spatial-Resolution Medical-Imaging System Using a HARPICON Camera Coupled with a Fluorescent Screen.

A high-sensitivity HARPICON camera was developed for medical X-ray imaging using a fluorescent screen. It is an avalanche-multiplication-type image pick-up tube and is 32 times more sensitive than conventional tubes. The camera also has a wider dynamic range than conventional medical-imaging cameras because a maximum output signal current of 2.3 muA is obtained and, in high-illumination-intensity regions, photocurrent is not proportional to illumination intensity. The fluorescent screen is an intensifying screen of the type used for radiographic screen-film combinations in medical examination. An X-ray image on the screen is focused on the photoconductive layer of the pick-up tube using a coupling lens with f/0.65. Experiments were performed using monochromated X-rays at the Photon Factory. An image of a spatial resolution test chart was taken in a 525 scanning-line mode of the camera. The chart pattern of 5 line-pairs mm(-1 )(spatial resolution of 100 mum) was observed at an X-ray input field of 50 x 50 mm. Real-time digital images of the heart of a 12 kg dog were obtained at a frame rate of 60 images s(-1) after injection of a contrast medium into an artery. The images were stored in digital format at 512 x 480 pixels with 12 bits pixel(-1). High-spatial-resolution and high-contrast images of coronary arteries were obtained in aortography using X-rays with energy above that of the iodine K edge; the image quality was comparable with that of conventional selective coronary angiography.

Journal Article↗

Purification and functional characterization of the C-terminal half of the lactose permease of Escherichia coli.

The lactose permease has been expressed in contiguous, non-overlapping polypeptide fragments containing the N-terminal (N6) and C-terminal (C6) transmembrane domains of the protein [Bibi, E., & Kaback, H. R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 4325; Zen, K., et al. (1994) Biochemistry 33, 8198]. When expressed individually, N6 and C6 are unstable and do not catalyze active transport. However, when expressed simultaneously, the polypeptides stabilize each other and form a complex that catalyzes active lactose transport. Moreover, a deletion construct containing the first transmembrane domain and the six C-terminal transmembrane domains mediates downhill lactose translocation [Bibi et al. (1991) proc. Natl. Acad. Sci. U.S.A. 88, 7271]. Here we report that C6 can be expressed independently in a relatively stable form that binds monoclonal antibodies 4B1 and 4B11, which interact with conformationally dependent epitopes on the periplasmic and cytoplasmic surfaces of the membrane, respectively. In addition, C6 retains the ability to catalyze lactose translocation down a concentration gradient in a specific manner. Finally, as observed with full-length Val331Cys permease, beta-D-galactopyranosyl 10thio-beta-D-galactopyranoside quenches the fluorescence of 2-(4'-maleimidylanilino)naphthalene- 6-sulfonic acid (MIANS)- labeled C6 with a single-Cys residue in place of Val331, exhibiting as apparent Kd of 0.2 mM. Unlike full-length Val331Cys permease, however, ligand does not induce a chance in the position of the emission maximum of MIANS-labeled C6(Val331Cys) permease not in the reactivity of C6 (Val331Cys) permease with MINAS. the results indicate that C6 retains a conformation similar to that on the native permease and that most of the structure required of high-affinity binding and substrate translocation is located in the C-terminal half of the molecule.

Amino Acid Sequence↗

The receptor binding site for the methyltransferase of bacterial chemotaxis is distinct from the sites of methylation.

The principal locus for binding interactions between the aspartate and serine receptors of escherichia coli and the methyltransferase was found to be in the last five amino acids of the receptor. The thermodynamic parameters of transferase-receptor interactions were determined by isothermal titration calorimetry. the serine receptor and three C-terminal fragments (C-fragments) of the aspartate receptor consisting of ether the last 297, 88, or 38 amino acids gave comparable values for binding (n=1, deltaH approximately 13 kcal/mol, and Ka approximately 4 x 10(5)M-1). Truncating either 16 or 36 amino acids form the C-terminus eliminated observable interactions. Finally the pentapeptide Asn-Trp-Glu-Thr-Phe, which corresponds to the last five amino acids of the receptor and is strictly conserved among E. coli serine amd aspartate receptors and the Salmonella typhimurium aspartate receptor, was found to have all the binding activity of the full-length receptor and the C-fragments. An in vitro methylation assay was used to obtain evidence for the physiological significance of this interaction in which excess peptide was able to completely block receptor methylation. The location of the binding site far from the methylation sites in the primary structure of the receptor suggests that the principle role of this interaction may be to hold the transferase in close proximity to all the methylation sites. Intersubunit methylation implication is proposed as plausible consequence of this "controlled proximity" mechanism since the ribose-galactose and dipeptide receptors lack the transferase binding sequence, and appear unable to bind transferase. Intersubunit methylation implies that transferase bound to eother the serine or aspartate receptor subunit may catalyze methylation of receptor subunits in a neighboring dimer, including those that have ligand specificity.

Base Sequence↗

Nitric oxide inhibits viral replication in murine myocarditis.

Nitric oxide (NO) is a radical molecule that not only serves as a vasodilator and neurotransmitter but also acts as a cytotoxic effector molecule of the immune system. The inducible enzyme making NO, inducible NO synthase (iNOS), is transcriptionally activated by IFN-gamma and TNF-alpha, cytokines which are produced during viral infection. We show that iNOS is induced in mice infected with the Coxsackie B3 virus. Macrophages expressing iNOS are identified in the hearts and spleens of infected animals with an antibody raised against iNOS. Infected mice have increased titers of virus and a higher mortality when fed NOS inhibitors. Thus, viral infection induces iNOS in vivo, and NO inhibits viral replication. NO is a novel, nonspecific immune defense against viruses in vivo.

Amino Acid Sequence↗

Tetrahydroberberine suppresses dopamine-induced potassium current in acutely dissociated CA1 pyramidal neurons from rat hippocampus.

Effects of tetrahydroberberine (THB) on dopamine (DA)-induced response have been investigated in single pyramidal neurons freshly dissociated from CA1 area of the hippocampus using the nystatin perforated patch-clamp, whole-cell recording technique under voltage-clamp configuration. At a holding potential (VH) of -20mV, DA-induced responses included a transient outward current, a slow inward current and a combination of the two. The outward current had a reversal potential of -83.5 +/- 8.0 mV which was close to K+ equilibrium potential and was sensitive to TEA, suggesting that this outward current was carried by K+. Application of THB reversibly suppressed three type responses induced by DA with different degrees of inhibitory ratio. THB inhibited the DA-induced outward current in a concentration-dependent manner with an IC50 of 13 microM. The maximal value of the concentration-response curve for DA-induced outward current was suppressed by THB, suggesting a non-competitive inhibition. The results support the hypothesis that THB acts as a novel dopaminergic system antagonist. Furthermore, THB inhibits the DA-induced K+ current non-competitively in single dissociated cells, implying that THB may exhibit other pharmacological action on the central nervous system.

Animals↗

Site-directed mutagenesis of the CP 47 protein of photosystem II: alteration of conserved charged residues in the domain 364E-444R.

The intrinsic chlorophyll-protein CP 47 is a component of photosystem II in higher plants, green algae and cyanobacteria. We had shown previously by biochemical methods that the domain 364E-440D of CP 47 interacts with the 33 kDa extrinsic protein of photosystem II [Odom, W. R., & Bricker, T. M. (1992) Biochemistry 31, 5616-5620]. In this study, using oligonucleotide-directed mutagenesis in the cyanobacterium Synechocystis 6803, mutations at 17 conserved charged residues were introduced into the domain 364E-444R of the CP 47 protein. Only mutations introduced at positions 384R and 385R led to a modified PS II phenotype. We previously described a mutation at (RR384385GG) which resulted in a mutant with a defective oxygen-evolving complex [Putnam-Evans, C., & Bricker, T. M. (1992) Biochemistry 31, 11482-11488]. An additional set of mutations, 384R to 384G, 385R to 385G, and 384,385RR to 384,385EE has now been introduced at this site yielding the mutants R384G, R385G, and RR384385EE, respectively. Steady state oxygen evolution measurements and quantum yield measurements demonstrated that these mutants exhibited significant alterations in their ability to evolve oxygen. Total fluorescence yield measurements indicated that all of these mutants contained about 85%-90% of the PS II reaction centers found in the control strain. This decrease was insufficient to explain the oxygen evolution results. Analysis of oxygen flash yield parameters indicated that there was little change in the S-state parameters alpha, beta, gamma, or delta. Measurement of the S2 lifetime, however, demonstrated that the S2 lifetime of the mutants was 2-3 times longer than that of the control. Additionally, examination of the risetime of the oxygen signal indicated that there was a significant retardation (6-7-fold) in the rate of oxygen release, suggesting a retarded S3-[S4]-S0 transition. These data reinforce our hypothesis that the positive charge density at positions 384R and 385R in the large extrinsic loop of CP 47 is necessary for its function in water oxidation. We speculate that this positive charge density may be an important factor in establishing the proper interaction between CP 47 and the 33kDa extrinsic protein.

Amino Acid Sequence↗

Biochemical analysis of collagens at the ligament-bone interface reveals presence of cartilage-specific collagens.

The collagen fibrils of some ligaments attach to bone by passing through a zone that consists of nonmineralized and mineralized fibrocartilages. Very little, however, is known about the cells, the biochemical composition, the extracellular matrix organization, and function of these fibrocartilages. In this study, the collagens present in the fibrocartilages of the bovine medical collateral and anterior cruciate ligaments femoral attachments to bone were isolated, characterized, and their distribution at these sites was assessed by laser confocal microscopy. Types II, IX, and XI collagens were identified after pepsin digestion of the tissues in addition to the types I and V collagens. Immunoblotting using specific polyclonal antibodies confirmed the presence of types II and IX collagens at these sites. Immunofluorescence using confocal microscopy showed that type II collagen was prominent in the nonmineralized area and to a lesser extent in the mineralized zone of the insertion. Type IX collagen showed similar distribution as type II collagen. Type II collagen isolated from the ligament-bone interface contained half hydroxypyridinium cross-linking residues when compared to type II collagen isolated from articular cartilage of the same animals. These data indicate that the fibrocartilaginous zones at the ligament-bone interface are cartilaginous in nature. The cartilage collagens may play a role of anchoring the ligament to bone or the cartilage-like tissue may participate in the modulation of the mechanical stresses which are known to exist at the soft tissue-hard tissue interface.

Animals↗

Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production.

Recently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62- and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production.

Adaptor Proteins, Signal Transducing↗

Translational control of poly(A)-binding protein expression.

Poly(A)-binding protein (PABP) is important for translation of eukaryotic mRNA and may be involved in shortening of its poly(A) tract. In many eukaryotic cells, this mRNA is inefficiently translated. The 5' untranslated region (UTR) of PABP mRNA has several adenine-rich regions which may serve as the PABP-binding sites to control its translation by a feed-back mechanism. This postulate was tested by using in vitro transcribed PABP mRNA and a rabbit reticulocyte lysate cell-free system. Results of our studies show that removal of the putative PABP-binding sites from the 5' UTR of this mRNA enhances its translation in the rabbit reticulocyte cell-free system. Furthermore, in vitro translation of the full-length PABP mRNA was inhibited by addition of purified PABP to the cell-free system. In contrast, translation of truncated mRNA lacking the putative PABP-binding sites at the 5' UTR was not inhibited by exogenous PABP. We have also tested the ability of purified PABP to bind to the 5' UTR of PABP mRNA using ultraviolet-mediated covalent cross-linking of RNA and proteins in vitro. Our results show that exogenous PABP binds to the 5' UTR of its full-length mRNA. Furthermore, incubation of PABP mRNA in rabbit reticulocyte lysate also led to binding of the endogenous PABP within the first 223 nucleotides of the 5' UTR. The adenine-rich regions are located within this segment of PABP mRNA. Following incubation of PABP mRNA in the reticulocyte lysate cell-free system under conditions of mRNA translation, the polysomal and non-translated free mRNA fractions were separated by centrifugation. Analysis of free and polysomal mRNA-protein (mRNP) complexes following ultraviolet-induced cross-linking showed that the free mRNP population was preferentially enriched in PABP. Results of our studies, therefore, suggest that PABP mRNA translation may be repressed by a unique feed-back mechanism.

DNA, Complementary↗

1,25(OH)2 vitamin D3, and retinoic acid antagonize endothelin-stimulated hypertrophy of neonatal rat cardiac myocytes.

1,25(OH)2 Vitamin D3 (VD3) and retinoic acid (RA) function as ligands for nuclear receptors which regulate transcription. Though the cardiovascular system is not thought to represent a classical target for these ligands, it is clear that both cardiac myocytes and vascular smooth muscle cells respond to these agents with changes in growth characteristics and gene expression. In this study we demonstrate that each of these ligands suppresses many of the phenotypic correlates of endothelin-induced hypertrophy in a cultured neonatal rat cardiac ventriculocyte model. Each of these agents reduced endothelin-stimulated ANP secretion in a dose-dependent fashion and the two in combination proved to be more effective than either agent used alone (VD3: 49%; RA:52%; VD3 + RA:80% inhibition). RA, at concentrations known to activate the retinoid X receptor, and, to a lesser extent, VD3 effected a reduction in atrial natriuretic peptide, brain natriuretic peptide, and alpha-skeletal actin mRNA levels. Similar inhibition (VD3:30%; RA:33%; VD3 + RA:59% inhibition) was demonstrated when cells transfected with reporter constructs harboring the relevant promoter sequences were treated with VD3 and/or RA for 48 h. These effects were not accompanied by alterations in endothelin-induced c-fos, c-jun, or c-myc gene expression, suggesting either that the inhibitory locus responsible for the reduction in the mRNA levels lies distal to the activation of the immediate early gene response or that the two are not mechanistically coupled. Both VD3 and RA also reduced [3H]leucine incorporation (VD3:30%; RA:33%; VD3 + RA:45% inhibition) in endothelin-stimulated ventriculocytes and, once again, the combination of the two was more effective than either agent used in isolation. Finally, 1,25(OH)2 vitamin D3 abrogated the increase in cell size seen after endothelin treatment. These findings suggest that the liganded vitamin D and retinoid receptors are capable of modulating the hypertrophic process in vitro and that agents acting through these or similar signaling pathways may be of value in probing the molecular mechanisms underlying hypertrophy.

Animals↗