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Biomedical subjects

J Ma

Publications and source records attributed to J Ma.

At least 469 records · Page 26Linked to original sources
PubMed1995

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Animals↗
PubMed1995

Identification and expression analysis of a potential familial Alzheimer disease gene on chromosome 1 related to AD3.

The inheritance of much early-onset Alzheimer disease (AD) has been linked to a dominant-acting locus on chromosome 14. Recently, the gene likely responsible for this genetic linkage has been identified and termed AD3. Five mutations have been found in AD3 that segregate with the disease phenotype in seven AD families and are not present in unaffected individuals. Here we report the existence of a gene encoding a seven transmembrane domain protein very similar to that encoded by AD3 in structure and sequence. This gene is located on chromosome 1, is expressed in a variety of tissues, including brain, and is predicted to harbor mutations causing nonchromosome 14 familial AD. The presence of several S/TPXX DNA binding motifs in both the AD3 protein and the AD3-like protein /AD4 protein suggests a possible role in intracellular signaling and gene expression or in linking chromatin to the nuclear membrane. Ways in which mutations in either gene could lead to AD are discussed.

Alzheimer Disease↗
PubMed1995

Intracellular loop between transmembrane segments IV and V of cystic fibrosis transmembrane conductance regulator is involved in regulation of chloride channel conductance state.

The cystic fibrosis transmembrane conductance regulator (CFTR) contains two membrane-spanning domains; each consists of six transmembrane segments joined by three extracellular and two intracellular loops of different length. To examine the role of intracellular loops in CFTR channel function, we studied a deletion mutant of CFTR (delta 19 CFTR) in which 19 amino acids were removed from the intracellular loop joining transmembrane segments IV and V. This mutant protein was expressed in a human embryonic kidney cell line (293 HEK). Fully mature glycosylated CFTR (approximately 170 kDa) was immunoprecipitated from cells transfected with wild-type CFTR cDNA, while cells transfected with the mutant gene expressed only a core-glycosylated form (approximately 140 kDa). The chloride efflux rate (measured by 6-methoxyl-N-(3-sulfopropyl) quinolinium SPQ fluorescence) from cells expressing wild-type CFTR increased 600% in response to forskolin. In contrast, delta 19 CFTR-expressing cells had no significant response to forskolin. Western blotting performed on subcellular membrane fractions showed that delta 19 CFTR was located in the same fractions as delta F508 CFTR, a processing mutant of CFTR. These results suggest that delta 19 CFTR is located in the intracellular membranes, without reaching the cell surface. Upon reconstitution into lipid bilayer membranes, delta 19 CFTR formed a functional Cl- channel with gating properties nearly identical to those of the wild-type CFTR channel. However, delta 19 CFTR channels exhibited frequent transitions to a 6-picosiemens subconductance state, whereas wild-type CFTR channels rarely exist in this subconductance state. These data suggest that the intracellular loop is involved in stabilizing the full conductance state of the CFTR Cl- channel.

Amino Acid Sequence↗
PubMed1995

Detection of high density lipoprotein binding proteins with a new method using apolipoprotein AI-DNP as a ligand.

To detect high density lipoprotein binding proteins, a new ligand blotting method that uses apolipoprotein AI-dinitrophenol as a ligand was established. Apolipoprotein AI-dinitrophenol was prepared by incubating the mixture of apolipoprotein AI and 2,4-dinitrobenzene sulfonic acid sodium salt at pH 8.5. Four to six dinitrophenol groups were found to be conjugated with an apolipoprotein AI molecule without any obvious impairment of its binding activity to the binding proteins. By this detection procedure, we detected two apolipoprotein AI binding proteins in plasma membrane fractions from bovine liver and adrenal cortex with molecular masses of 120 kDa and 95 kDa. Since this new ligand blotting method is simple and time-saving, it is expected to become a useful tool in the studies on high density lipoprotein binding proteins.

Adrenal Cortex↗
PubMed1995

Retinal degeneration slow (rds) in mouse results from simple insertion of a t haplotype-specific element into protein-coding exon II.

Retinal degeneration slow (rds) is a semidominant mutation of mice that causes dysplasia and degeneration of rod and cone photoreceptors. Mutations in RDS, the human ortholog of the rds gene, are responsible for several inherited retinal dystrophies including a subset of retinitis pigmentosa. The normal rds locus encodes rds/peripherin, an integral membrane glycoprotein present in outer segment discs. Genomic libraries from wildtype and rds/rds mice were screened with an rds cDNA, and phage lambda clones that span the normal and mutant loci were mapped. We show that in mice, rds is caused by the insertion into exon II of a 9.2-kb repetitive genomic element that is very similar to the t haplotype-specific element in the H-2 complex. The entire element is included in the RNA products of the mutant locus. We present evidence that rds in mice represents a null allele.

Alleles↗
PubMed1995

Cerebrovascular alterations in mice lacking neuronal nitric oxide synthase gene expression.

Nitric oxide (NO) is known to mediate increases in regional cerebral blood flow elicited by CO2 inhalation. In mice with deletion of the gene for neuronal NO synthase (NOS), CO2 inhalation augments cerebral blood flow to the same extent as in wild-type mice. However, unlike wild-type mice, the increased flow in mutants is not blocked by the NOS inhibition, N omega-nitro-L-arginine, and CO2 exposure fails to increase brain levels of cGMP. Topical acetylcholine elicits vasodilation in the mutants which is blocked by N omega-nitro-L-arginine, indicating normal functioning of endothelial NOS. Moreover, immunohistochemical staining for endothelial NOS is normal in the mutants. Thus, following loss of neuronal NOS, the cerebral circulatory response is maintained by a compensatory system not involving NO.

Acetylcholine↗
PubMed1995

Properties and roles of an intramembranous charge mobilized at high voltages in frog skeletal muscle.

1. Membrane Ca2+ currents (ICa), intramembranous charge movement currents and changes in intracellular Ca2+ concentrations were recorded in voltage clamped cut skeletal muscle fibres of the frog. Intra- and extracellular solutions, designed to prevent ionic current, and use of the saponin-permeabilization procedure made possible the measurement of transfer of intramembranous charge up to high positive potentials. 2. Substantial charge moved at positive potentials. This charge was shown to be intramembranous in four tests of charge conservation, demonstrating that the total displacement of charge depended only on the initial and final voltages, and not on the history or pathway of intermediate voltages. 3. On average, in twenty-three cells, the charge moved at 50 mV was 31 +/- 1.9 nC microF-1 (mean +/- S.E.M.), and at 0 mV was 25 +/- 1.5 nC microF-1. Approximately one-fifth of the total charge moved above 0 mV. 4. The charge that moved at high voltage could be fitted, in most cases, with a Boltzmann distribution function. In twenty of twenty-three cells, the total charge distribution could be fitted as the sum of two Boltzmann terms; the high voltage term was centred at 11 +/- 3.9 mV, with a steepness factor of 12 +/- 1.6 mV and a magnitude of 8.6 +/- 1.1 nC microF-1. The low voltage term was centered at -43 +/- 2.1 mV, with a steepness factor of 7.7 +/- 0.6 mV and a magnitude of 22 +/- 1.8 nC microF-1. Thus, the high voltage component comprised about one-quarter of the mobile charge. In four cells it was possible to fit the sum of three Boltzmann terms to the distribution of mobile charge; the parameters of the high voltage term then were similar to those found by fitting the sum of two Boltzmann terms to the same data. 5. The voltage dependence of activation of ICa was determined in a buffered 2 mM Ca2+ external solution, from the tails of ionic current at -30 mV, after activating pulses to various voltages, the duration of which was sufficient to reach the peak of inward current. The voltage dependence was described by a Boltzmann function centred at 2.6 +/- 6.9 mV (n = 6), with a steepness factor of 20 +/- 1.4 mV. The voltages at which the high voltage charge moved were roughly the same as those at which ICa was activated. 6. Calcium release from the sarcoplasmic reticulum was determined from the Ca2+ transients. Calcium release continued to increase at potentials above 0 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Cloning and sequence analysis of the cDNA for bovine mitochondrial translational initiation factor 2.

The complete sequence of the cDNA encoding bovine mitochondrial translational initiation factor 2 (IF-2mt) has been obtained by library screening followed by 3'-RACE PCR. The open reading frame for bovine IF-2mt encodes a protein of 727 amino acids. The sequence of bovine IF-2mt exhibits 85% identity to human IF-2mt, but only 38% identity to yeast IF-2mt and 39% identity to Escherichia coli IF-2 alpha.

Animals↗
PubMed1995

Interleukin-1-induced calcium flux in human fibroblasts is mediated through focal adhesions.

Interleukin-1 (IL-1) is an important mediator of inflammation and also modulates fibroblast metabolism. To assess mechanisms of IL-1-induced signal transduction and calcium flux, early passage human fibroblasts were loaded with fura2/AM. Cells grown on coverslips exhibited dose-dependent [Ca2+]i responses that were maximal at 10(-8) M IL-1 beta with time to maximum flux of 50 s. Cells incubated with anti-Type 1-IL-1 receptor antibody exhibited a 45 nM increase in [Ca2+]i above baseline but demonstrated no calcium response after IL-1 beta treatment. Incubation with EGTA (5 mM) or thapsigargin (1 microM) caused 75% and 37% reductions, respectively, in the IL-1-induced [Ca2+]i increase, suggesting that extracellular Ca2+ predominates in IL-1-stimulated calcium flux. Cells in suspension did not exhibit [Ca2+]i responses to IL-1 beta. The relationship between [Ca2+]i signaling and focal adhesions was examined by plating cells on fibronectin or poly-L-lysine, conditions that either permitted or blocked the formation of focal adhesions. Cells on fibronectin exhibited co-distribution of immunostaining for talin, vinculin, IL-1 receptor, and focal adhesion kinase (pp125fak) in focal adhesions and demonstrated [Ca2+]i responses with 10(-8) M IL-1 beta. Cells on poly-L-lysine or cells in suspension did not exhibit co-distribution of pp125fak, IL-1 receptor, and focal adhesion proteins and did not exhibit calcium flux. The dependence of IL-1-stimulated [Ca2+]i responses on tyrosine kinases was examined first by treating cells with genistein, a selective inhibitor of tyrosine kinases. Genistein (100 microM) completely blocked [Ca2+]i responses to 10(-8) M IL-1, whereas its inactive analogue genistin was not inhibitory. Second, fibroblasts lysates were immunoprecipitated with an antiphosphotyrosine antibody and the lysates were Western-blotted with an anti-pp125fak antibody. Cells grown on fibronectin and stimulated with IL-1 exhibited tyrosine phosphorylation of pp125fak whereas untreated cells or cells grown on poly-L-lysine and treated with IL-1 showed no reaction. Fibroblasts electroinjected with anti-pp125fak monoclonal antibody showed no [Ca2+], response, whereas cells treated with an irrelevant antibody exhibited a normal [Ca2+]i response. Collectively, these data indicate that fibroblasts require substrate attachment and clustering of IL-1 receptors to focal adhesions for IL-1-induced [Ca2+]i responses. Calcium fluxes are mediated through tyrosine kinases whose substrates include pp125fak. These studies therefore demonstrate that activation of intracellular signaling pathways by IL-1 is dependent on IL-1 receptor-cytoskeletal protein interactions.

Calcium↗
PubMed1995

Effective water model for Monte Carlo simulations of proteins.

We present an effective theory for water. Our goal is to formulate an accurate model for the effects of solvation on protein dynamics, without incurring the huge computational cost and the slow temporal evolution typical of molecular dynamics simulations of liquids. We replace the individual water molecules in an all-atom potential with a local dielectric density field, with self-interactions given by the Landau-Ginzburg free energy and external interactions by Lennard-Jones forces at the surface of the protein atoms. We explore conformational space with finite temperature Monte Carlo dynamics, using parallel Langevin and Fourier acceleration algorithms well suited to data-parallel computer architectures such as the Connection Machine. To establish the validity of our approximations, we compare our electrostatic contribution to the solvation energy with the results of Lim, Bashford, and Karplus using a conventional static continuum dielectric cavity model, and the nonelectrostatic contributions with estimates of hydrophobic surface free energy. Our model can also accommodate ionic charges and temperature fluctuations. We propose future investigations extending our effective theory of solvation to include explicit orientational entropy and hydrogen-bonding terms.

Models, Molecular↗
PubMed1995

Increase of cyclin B by overexpression of cystatin alpha.

Degradation of cyclin B was effectively suppressed when cells were treated with ALLN (N-acetylleucylleucylnorleucinal) which inhibits proteasome, calpain and cysteine proteinase cathepsins. In order to examine which protease degrades cyclin B, the effect of a cathepsin inhibitor, cystatin alpha, was investigated. The cystatin alpha gene was inserted into an inducible expression vector, pMSG, and transfected into NIH3T3 mouse fibroblasts. The expression of cystatin alpha was induced effectively in the transfected cells after treatment with dexamethasone. Overexpression of cystatin alpha resulted in an increase of the amount of cyclin B, suggesting that cysteine proteinase cathepsins might be involved in the degradation of cyclin B.

3T3 Cells↗
PubMed1995

Expression of the Epstein-Barr virus DNA polymerase in Escherichia coli for use as antigen for the diagnosis of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) encoded DNA polymerase (POL) was cloned and over-expressed in Escherichia coli. Western blot analysis confirmed the presence of antibody to this POL protein in sera from nasopharyngeal carcinoma (NPC) patients. By Western blot analysis, moderate to high concentration of IgG POL-specific antibodies were present in 43 of 48 NPC sera and only 4 of 48 healthy, seropositive controls. The POL-specific IgG antibodies appear as early as stage I of NPC, suggesting that the recombinant POL protein can be a useful diagnostic marker for early diagnosis of the disease. It was also found that human sera containing high titer of cytomegalovirus (CMV) antibodies or herpes simplex virus type 1 (HSV-1) antibodies did not cross-react with the recombinant EBV POL, despite the homology shared by DNA polymerase proteins of these viruses.

Amino Acid Sequence↗
PubMed1995

The location of untranscribed DNA sequences within ras genes essential for eliciting plant growth suppression.

Three heterologous ras DNA-coding sequences and their deletion derivatives were introduced into plant cells to investigate the role of the ras-coding sequences, especially conserved regions, in eliciting growth inhibition. All three ras-coding sequences caused a similar inhibition of plant cell growth, and it was the conserved coding regions which were responsible for this inhibitory effect. The 493 bp conserved region within the v-Ha-ras-coding sequence was studied further, and was shown to be responsible for the inhibitory effect. This region is conserved (over 44%) among the three ras genes studied and encodes a catalytic region of the Ras protein. Small deletions at either the 5' or 3' end of this 493 bp sequence could abolish or dramatically reduce the inhibitory effect. A 36 bp region at the 5' end of the 493 bp region was found to be highly conserved between v-Ha-ras and eight different plant ras or ras-related genes based upon analysis of published sequences. Small deletions affecting this highly conserved 36 bp region completely abolished the inhibitory effect, while deletion of a similar number of base pairs in adjacent regions did not. These results indicate that plant growth inhibition by ras DNA requires small regions at both ends of the 493 bp conserved region.

Base Sequence↗
PubMed1995

Sequence analysis of the human hTg737 gene and its polymorphic sites in patients with autosomal recessive polycystic kidney disease.

DNA sequence analysis of the human Tg737 gene was performed in 36 patients with the autosomal recessive form of polycystic kidney disease (ARPKD). Coding exons and their adjacent splice sites were screened for mutations. Pathogenic exon or splice region mutations were not identified although one exonic and two intronic polymorphic sites were discovered. These results are in agreement with another study that has recently reported linkage to Chromosome (Chr) 6p21-cen in a set of 16 ARPKD families. STS mapping has localized the gene to a YAC contig that includes D13S175 on chromosome 13q12.1. The polymorphisms found in the htG737 gene will permit its future evaluation as a candidate gene for other recessive cystic renal diseases and as a modifier gene in human PKD.

Base Sequence↗
PubMed1995

Tumor necrosis factor-alpha responses are depressed and interleukin-6 responses unaltered in feline immunodeficiency virus infected cats.

Feline immunodeficiency virus (FIV), a lentivirus similar to HIV, causes an acquired immunodeficiency syndrome in cats. Similar to human immunodeficiency virus (HIV), the pathogenesis of FIV is associated with dysregulation of the cytokine network. While alterations in tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) expression have been reported in HIV-infected patients, changes attributable to HIV and those caused by cofactors such as secondary infections cannot always be readily distinguished. This study evaluated the effect of FIV infection on TNF-alpha and IL-6 production in cats not exposed to other potential cofactors such as secondary infections. TNF-alpha and IL-6 activities were evaluated in bronchoalveolar lavage (BAL) cells from FIV-infected and uninfected specific pathogen free (SPF) cats. Supernatants from lipopolysaccharide (LPS)-stimulated BAL cells from uninfected SPF cats had high levels of TNF-alpha and IL-6 activity, while stimulated BAL cell supernatants from FIV-infected SPF cats had significantly lower levels of TNF-alpha but unaltered IL-6 activity. Similarly, Con A/phorbol myristate acetate (PMA) stimulated non-adherent (NA-) peripheral blood mononuclear cells (PBMC) from FIV infected cats synthesized less TNF-alpha than similarly treated NA-PBMC from uninfected cats. Feline immunodeficiency virus could be recovered from the culture supernatants of BAL cells from infected cats by co-cultivation with susceptible lymphocytes. In situ hybridization identified FIV mRNA in a small fraction of alveolar macrophages in the BAL cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗