Biomedical subjects

J Ma

Publications and source records attributed to J Ma.

At least 361 records · Page 20Linked to original sources

PubMed1997

Function of the R domain in the cystic fibrosis transmembrane conductance regulator chloride channel.

For a cystic fibrosis transmembrane conductance regulator (CFTR) channel to enter its open state, serine residues in the R domain must be phosphorylated by cAMP-dependent protein kinase, and intracellular ATP must bind to the nucleotide-binding folds and subsequently be hydrolyzed. CFTR with its R domain partially removed, DeltaR(708-835)-CFTR, forms a chloride channel that opens independently of protein kinase A phosphorylation, with open probability approximately one-third that of the wild type CFTR channel. Deletion of this portion of the R domain from CFTR alters the response of the channel to 5'-adenylylimidodiphosphate, pyrophosphate, and vanadate, compounds that prolong burst duration of the wild type CFTR channel but fail to do so in the DeltaR-CFTR. In addition, the addition of exogenous unphosphorylated R domain protein, which blocks the wild type CFTR channel, has no effect on the DeltaR-CFTR channel. However, when the exogenous R domain is phosphorylated, significant stimulation of the DeltaR-CFTR channel results; Po increases from 0.10 to 0.22. These data are consistent with a model for CFTR function in which the R domain in the unphosphorylated state interacts with the first nucleotide binding fold to inhibit either binding or hydrolysis of ATP or transduction of the effect to open the pore, but when the R domain is phosphorylated, it undergoes conformational change and interacts at a separate site in the first nucleotide binding fold to stimulate either binding or hydrolysis of ATP or transduction of the effect to open the pore.

Adenosine Triphosphate↗

PubMed1997

Molecular switch in signal transduction: reaction paths of the conformational changes in ras p21.

Conformational changes in ras p21 triggered by the hydrolysis of GTP play an essential role in the signal transduction pathway. The path for the conformational change is determined by molecular dynamics simulation with a holonomic constraint directing the system from the known GTP-bound structure (with the gamma-phosphate removed) to the GDP-bound structure. The simulation is done with a shell of water molecules surrounding the protein. In the switch I region, the side chain of Tyr-32, which undergoes a large displacement, moves through the space between loop 2 and the rest of the protein, rather than on the outside of the protein. As a result, the charged residues Glu-31 and Asp-33, which interact with Raf in the homologous RafRBD-Raps complex, remain exposed during the transition. In the switch II region, the conformational changes of alpha2 and loop 4 are strongly coupled. A transient hydrogen bonding complex between Arg-68 and Tyr-71 in the switch II region and Glu-37 in switch I region stabilizes the intermediate conformation of alpha2 and facilitates the unwinding of a helical turn of alpha2 (residues 66-69), which in turn permits the larger scale motion of loop 4. Hydrogen bond exchange between the protein and solvent molecules is found to be important in the transition. Possible functional implications of the results are discussed.

Computer Simulation↗

PubMed1997

Subunit II of the cytochrome bo3 ubiquinol oxidase from Escherichia coli is a lipoprotein.

The purified Escherichia coli cytochrome bo3 ubiquinol oxidase contains four subunits that are each integral components of the cytoplasmic membrane. The molecular weight of each of the subunits has been determined by matrix-assisted laser desorption ionization mass spectrometry (MALDI). The observed molecular weight of subunit II (CyoA) is considerably less than the calculated value from the deduced amino acid sequence, indicating possible posttranslational processing. The similarity of a portion of the sequence near the N-terminus of CyoA with the sequences of known prokaryotic membrane-bound lipoproteins suggested that CyoA is proteolytically processed to generate an N-terminus at Cys25, and that Cys25 is covalently modified by the addition of lipids. This would be consistent with the observed molecular mass, and was confirmed by demonstrating the incorporation of radioactive palmitic acid into subunit II of the cytochrome bo3 oxidase. Site-directed mutagenesis replacing Cys25 by alanine prevents the processing, generating a precursor form of CyoA with a higher molecular mass. The C25A mutant of CyoA still assembles as an active quinol oxidase capable of supporting growth of the cells by aerobic respiration. Hence, this unusual processing of a cytoplasmic membrane protein, which is already anchored to the membrane by two transmembrane helices, is not essential for either assembly or function.

Aerobiosis↗

PubMed1997

Alzheimer presenilins in the nuclear membrane, interphase kinetochores, and centrosomes suggest a role in chromosome segregation.

Mutations in two related genes, presenilin 1 and 2, account for most early-onset familial Alzheimer's disease. Although structural features indicate that the presenilins are membrane proteins, their function(s) is unknown. We have localized the presenilins to the nuclear membrane, its associated interphase kinetochores, and the centrosomes-all subcellular structures involved in cell cycle regulation and mitosis. The colocalization of the presenilins with kinetochores on the nucleoplasmic surface of the inner nuclear membrane, together with other results, suggests that they may play a role in chromosome organization and segregation, perhaps as kinetochore binding proteins/receptors. We discuss a pathogenic pathway for familial Alzheimer's disease in which defective presenilin function causes chromosome missegregation during mitosis, resulting in apoptosis and/or trisomy 21 mosaicism.

Alzheimer Disease↗

PubMed1997

Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation.

Chemokines are small secreted proteins that stimulate the directional migration of leukocytes and mediate inflammation. During screening of a murine choroid plexus complementary DNA library, we identified a new chemokine, designated neurotactin. Unlike other chemokines, neurotactin has a unique cysteine pattern, Cys-X-X-X-Cys, and is predicted to be a type 1 membrane protein. Full-length recombinant neurotactin is localized on the surface of transfected 293 cells. Recombinant neurotactin containing the chemokine domain is chemotactic for neutrophils both in vitro and in vivo. Neurotactin messenger RNA is predominantly expressed in normal murine brain and its protein expression in activated brain microglia is upregulated in mice with experimental autoimmune encephalomyelitis, as well as in mice treated with lipopolysaccharide. Distinct from all other chemokine genes, the neurotactin gene is localized to human chromosome 16q. Consequently we propose that neurotactin represents a new delta-chemokine family and that it may play a role in brain inflammation processes.

Animals↗

PubMed1997

Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): I. As2O3 exerts dose-dependent dual effects on APL cells.

Recent clinical studies in China showed that As2O3 is an effective and relatively safe drug in the treatment of acute promyelocytic leukemia (APL). We found previously that As2O3 can trigger apoptosis of APL cell line NB4 cells, which is associated with downregulation of bcl-2 gene expression and modulation of PML-RAR alpha chimeric protein. To further understand the mechanisms of this alternative therapy for APL, we investigated in this report the effects of a wide range of concentrations of As2O2 on cultured primary APL cells, all-trans retinoic acid (ATRA)-susceptible (NB4 cells) and ATRA-resistant (MR2 subclone) APL cell lines. The results indicated that As2O3 had dose-dependent dual effects on APL cells: inducing preferentially apoptosis at relatively high concentrations (0.5 to 2 micromol/L) and inducing partial differentiation at low concentrations (0.1 to 0.5 micromol/L). The rapid modulation and degradation of PML-RAR alpha proteins, which was induced by As2O3 at 0.1 to 2 micromol/L, could contribute to these two effects. Bone marrow and peripheral blood examination showed that myelocyte-like cells, probably as a result of partial in vivo differentiation, and degenerative cells increased after 2 to 3 weeks of continuous in vivo As2O3 treatment when leukemic promyelocytes decreased. In conclusion, combination of induction of apoptosis and partial differention could be the main cellular mechanisms of As2O3 in the treatment of APL, and PML-RAR alpha could play an important role in determining the specific effects of As2O3 on APL cells.

Antineoplastic Agents↗

PubMed1997

Use of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL): II. Clinical efficacy and pharmacokinetics in relapsed patients.

The therapeutic effect of arsenic trioxide (As2O3) in the treatment of acute promyelocytic leukemia (APL) was evaluated among 15 APL patients at relapse after all-trans retinoic acid (ATRA) induced and chemotherapy maintained complete remission (CR). As2O3 was administered intravenously at the dose of 10 mg/d. Clinical CR was achieved in nine of 10 (90%) patients treated with As2O3 alone and in the remaining five patients treated by the combination of As2O3 and low-dose chemotherapeutic drugs or ATRA. During the treatment with As2O3, there was no bone marrow depression and only limited side effects were encountered. Pharmacokinetic studies, which were performed in eight patients, showed that after a peak level of 5.54 micromol/L to 7.30 micromol/L, plasma arsenic was rapidly eliminated, and the continuous administration of As2O3 did not alter its pharmacokinetic behaviors. In addition, increased amounts of arsenic appeared in the urine, with a daily excretion accounting for approximately 1% to 8% of the total daily dose administered. Arsenic contents in hair and nail were increased, and the peak content of arsenic could reach 2.5 to 2.7 microg/g tissue at CR. On the other hand, a decline of the arsenic content in hair and nail was observed after withdrawal of the drug. We conclude that As2O3 treatment is an effective and relatively safe drug in APL patients refractory to ATRA and conventional chemotherapy.

Adolescent↗

PubMed1997

Mutational studies of yeast transcription factor IIB in vivo reveal a functional surface important for gene activation.

Recent experiments in yeast (Saccharomyces cerevisiae) cells have identified a species-specific region of yeast transcription factor IIB (TFIIB) located at residues 144-157. According to the human TFIIB structure, this region is part of a solvent-exposed helix in the first repeat of the carboxyl-terminal core domain. In this report, we systematically analyze four positions in this region (Lys-147, Cys-149, Lys-151, and Glu-152) that together have been shown previously to be important for yeast TFIIB's function in vivo. Our experiments suggest that all of these four positions, and in particular positions 151, 149, and 152, are critical for yeast TFIIB's ability to support cell growth. In addition, we describe an intragenic suppressor screening experiment to identify mutations that reverse, or partially reverse, the temperature-sensitive phenotype of a yeast TFIIB derivative bearing amino acid changes at these four positions to human residues. The suppressor mutations reveal changes at positions 115, 117, and 182 that are located outside the species-specific region of yeast TFIIB, suggesting an extended surface available to interact with other proteins. Finally, we demonstrate that the suppressor mutations restore gene activation in vivo, further supporting the idea that one important function of yeast TFIIB in living cells is to mediate gene activation.

Amino Acid Sequence↗

PubMed1997

Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer.

Folate derivatives are important in experimental colorectal carcinogenesis; low folate intake, particularly with substantial alcohol intake, is associated with increased risk. The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methylenetetrahydrofolate, required for purine and thymidine syntheses, to 5-methyltetrahydrofolate, the primary circulatory form of folate necessary for methionine synthesis. A common mutation (677C-->T) in MTHFR reduces enzyme activity, leading to lower levels of 5-methyltetrahydrofolate. To evaluate the role of folate metabolism in human carcinogenesis, we examined the associations of MTHFR mutation, plasma folate levels, and their interaction with risk of colon cancer. We also examined the interaction between genotype and alcohol intake. We used a nested case-control design within the Physicians' Health Study. Participants were ages 40-84 at baseline when alcohol intake was ascertained and blood samples were drawn. During 12 years of follow-up, we identified 202 colorectal cancer cases and matched them to 326 cancer-free controls by age and smoking status. We genotyped for the MTHFR polymorphism and measured plasma folate levels. Men with the homozygous mutation (15% in controls) had half the risk of colorectal cancer [odds ratio (OR), 0.49; 95% confidence interval (CI), 0.27-0.87] compared with the homozygous normal or heterozygous genotypes. Overall, we observed a marginal significant increased risk of colorectal cancer (OR, 1.78; 95% CI, 0.93-3.42) among those whose plasma folate levels indicated deficiency (<3 ng/ml) compared with men with adequate folate levels. Among men with adequate folate levels, we observed a 3-fold decrease in risk (OR, 0.32; 95% CI, 0.15-0.68) among men with the homozygous mutation compared with those with the homozygous normal or heterozygous genotypes. However, the protection due to the mutation was absent in men with folate deficiency. In men with the homozygous normal genotype who drank little or no alcohol as reference, those with the homozygous mutation who drank little or no alcohol had an 8-fold decrease in risk (OR, 0.12; 95% CI, 0.03-0.57), and for moderate drinkers, a 2-fold decrease in risk (OR, 0.42; 95% CI, 0.15-1.20); no decrease in risk was seen in those drinking 1 or more drinks/day. Our findings provide support for an important role of folate metabolism in colon carcinogenesis. In particular, these results suggest that the 677C-->IT mutation in MTHFR reduces colon cancer risk, perhaps by increasing 5,10-methylenetetrahydrofolate levels for DNA synthesis, but that low folate intake or high alcohol consumption may negate some of the protective effect.

Adult↗

PubMed1997

alpha beta T cell regulation and CD40 ligand dependence in murine systemic autoimmunity.

To explore the mechanisms by which alpha beta T cells and gamma delta T cells regulate systemic autoimmunity, lupus-prone mice were rendered deficient in CD40 ligand and/or alpha beta T cells by intercrossing CD40L -/- and TCR-alpha -/- knockouts, generating CD40L-intact or -deficient (CD40L+ or CD40L-), alpha beta T cell-intact or -deficient (alpha beta+ or alpha beta-) MRL-lpr/lpr animals. As expected, CD40L+ alpha beta+ mice developed high titer autoantibodies along with severe renal and cutaneous disease. CD40L+ alpha beta- animals developed lower levels of autoantibodies, accompanied by less severe or delayed renal and cutaneous disease. CD40L- alpha beta+ mice developed even lower titers of autoantibodies and less severe renal disease yet developed cutaneous lesions indistinguishable from those of CD40L+ alpha beta+ disease. Most surprisingly, CD40L- alpha beta- animals developed higher levels of some autoantibodies than did CD40L- alpha beta+ mice and developed renal disease similar in severity to CD40L+ alpha beta- counterparts; however, they failed to develop skin disease. Thus, disruption of CD40L and alpha beta T cells provides a novel dissection of the physiology and pathology of murine lupus; while these data confirm previous findings demonstrating a role for CD40L-dependent, alpha beta T cell-dependent mechanisms in autoantibody production and renal disease in murine lupus, they also: 1) establish that alpha beta T cells may drive autoimmune skin disease by a CD40L-independent mechanism; 2) identify a role for CD40L in non-alpha beta T cell-dependent autoantibody production and autoimmune skin disease; and 3) suggest a role for alpha beta T cells in the down-regulation of autoimmunity driven by other T cells. Thus, both alpha beta and non-alpha beta T cells, such as gamma delta T cells, regulate systemic autoimmunity by CD40L-dependent and -independent mechanisms.

Animals↗

PubMed1997

Synthesis of Copper Nanoparticles in Nonionic Water-in-Oil Microemulsions

Synthesis of well-dispersed copper nanoparticles was achieved by reduction of aqueous copper chloride solution using NaBH4 in the nonionic water-in-oil (w/o) microemulsions formed by Triton X-100, n-hexanol, cyclohexane, and water. It has been shown that instead of copper oxide produced in aqueous solution, metallic copper particles are formed in w/o microemulsions because of the high local copper concentration in water pools of the microemulsions, indicating the advantage offered by w/o microemulsions over aqueous phases. The absorption spectrum of the colloidal copper particles obtained in microemulsions does not exhibit the plasmon peak characteristic of the Cu surface. It is conceivable that the lack of the plasmon absorption band is attributed to the formation of a CuCl monolayer on the copper particles.

Journal Article↗

PubMed1997

Two naturally occurring alpha2,6-sialyltransferase forms with a single amino acid change in the catalytic domain differ in their catalytic activity and proteolytic processing.

The alpha2,6-sialyltransferase (ST) is a Golgi glycosyltransferase that adds sialic acid residues to glycoprotein N-linked oligosaccharides. Here we show that two forms of alpha2,6-sialyltransferase are expressed by the liver and are encoded by two different RNAs that differ by a single nucleotide. The ST tyr possesses a Tyr at amino acid 123, whereas the ST cys possesses a Cys at this position. The ST tyr is more catalytically active than the ST cys; however, both are functional when introduced into tissue culture cells. The proteolytic processing and turnover of the ST tyr and ST cys proteins differ dramatically. The ST cys is retained intact in COS-1 cells, whereas the ST tyr is rapidly cleaved and secreted. Analysis of the N-linked oligosaccharides of these proteins demonstrates that both proteins enter the late Golgi. However, differences in ST tyr and ST cys proteolytic processing may be related to differences in their localization, because ST tyr but not ST cys is expressed at low levels on the cell surface. The possibility that the ST tyr is cleaved in a post-Golgi compartment is supported by the observation that a 20 degrees C temperature block, which stops protein transport in the trans Golgi network, blocks both cleavage and secretion of the ST tyr.

Animals↗

PubMed1997

In vivo MR microscopy of the human skin.

The requirements for imaging the skin are dictated by the organ's layered structure, which extends only a few millimeters from the surface and thus demands extremely high resolution in this direction. While less critical, resolution in the remaining two dimensions determines whether the skin's accessory structures can be resolved. The problem is compounded by short transverse relaxation times, in particular of the dermis, the structure of most clinical interest. In this work images of the normal human skin were obtained in vivo at voxel sizes as small as 19 x 78 x 800 microm3, by means of customized 3D gradient and partial flip-angle spin-echo pulse sequences and very small transmit/receive coils on a 1.5T clinical imager equipped with high-power whole-body gradients. Structures resolved include hair follicles and the sublayers of the dermis. The very short time constant for the major component (91%) for transverse relaxation in the dermis (T2* approximately 10 ms) suggests the potential of substantial gains in achievable signal-to-noise ratio by shortening the echo time.

Adipose Tissue↗

PubMed1997

A single-scan imaging technique for measurement of the relative concentrations of fat and water protons and their transverse relaxation times.

A two-component chemical-shift-imaging technique is described from which fat and water images can be obtained in a single scan and in the presence of an inhomogeneous field. In addition, the method provides transverse relaxation rates R2 and R2' separately for each of the spectral components. The method is a combination and extension of the GESFIDE [gradient echo sampling of FID and echo, J. Ma and F. W. Wehrli, J. Magn. Reson. B 111, 61 (1996)] and the multipoint Dixon techniques. It is based on sampling the descending and ascending portions of a Hahn spin echo with a train of gradient echoes which are spaced at one-half of the chemical-shift modulation period. Processing of the complex echo data, involving an automated phase unwrapping algorithm, affords relative amplitudes and transverse relaxation rates of the two spectral components. An additional benefit of the method is its superior signal-to-noise ratio resulting from echo summation. Applications targeted and illustrated involve MRI osteodensitometry of trabecular bone in the presence of varying fractions of hematopoietic and fatty bone marrow.

Adult↗

PubMed1997

Alteration of substrate specificity of Zymomonas mobilis alcohol dehydrogenase-2 using in vitro random mutagenesis.

Random mutagenesis of the gene encoding Zymomonas mobilis alcohol dehydrogenase-2 has enabled isolation of variants of the enzyme that have substrate specificities different from that of the wild-type enzyme. After amino acids responsible for the changes were identified, directed mutation at these sites was also carried out. Variants that are active on butanol have been investigated in detail. Changes at residue 161 and other changes at residues 155 and 165 cause enhanced activity with longer-chain alcohols. The 165 change also induces a marked alcohol-activation phenomenon that is observed not only with ethanol, but also with a nonsubstrate alcohol, 2-propanol, and with low concentrations of Triton X-100. These alterations to the alcohol binding pocket mainly introduce larger, more hydrophobic residues, suggesting that it is not the size but the hydrophobicity of the pocket that affects the substrate specificity. Variants active with NADP were isolated, and, as with similar variants of the yeast enzyme, they were found to have an Asp residue replaced by a neutral amino acid. However, unlike the yeast examples in which the affinity was substantially reduced, the affinity for NAD+ in these variants was little changed, and the affinity for NADP+ was higher than that for NAD+. As this enzyme is naturally ferrous ion-activated, and inactive with zinc, attempts were made to find variants that had activity with zinc. One was found, but the screening method also isolated other variants with altered metal ion preferences due to a mutation affecting amino acid 330.

1-Butanol↗

PubMed1997

Tissue-specific bioenergetic effects and increased enzymatic activities following acute sublethal peroral exposure to cyanide in the mallard duck.

Protection of wildlife and in particular migratory birds, which are protected by the Migratory Bird Treaty Act, from cyanide waste in and around gold mining operations is an important environmental issue. We have investigated the bioenergetic effects of sublethal peroral cyanide exposure using the mallard duck (Anus platyrhynchos) as a model migratory bird. At cyanide concentrations well below levels considered safe by the mining industry and some regulatory agencies (50 ppm weak acid dissociable (WAD) cyanide) significant depletions of heart, liver, and brain tissue ATP levels were observed. Tissue ATP levels were restored to normal by 24 hr postexposure. Rhodanese and 3-mercaptopyruvate sulfurtransferase activities were determined in these tissues both for basal activity and post-cyanide exposure. Only brain tissue showed increased enzymatic activity following cyanide exposure, suggesting tissue-specific regulation of these enzymatic activities. These studies suggest that 50 ppm WAD cyanide is not a safe level of cyanide in water where avian wildlife exposure can occur.

Adenosine Triphosphate↗

PubMed1997

The organic component of diesel exhaust particles and phenanthrene, a major polyaromatic hydrocarbon constituent, enhances IgE production by IgE-secreting EBV-transformed human B cells in vitro.

Suspended airborne particulate matter such as diesel exhaust particles (DEP) have been implicated in the increased incidence of respiratory allergic diseases that has occurred over the past century. Studies in vitro and in vivo have shown that DEP may enhance allergic antibody (IgE) expression. DEP contain a wide spectrum of polycyclic aromatic hydrocarbons (PAH) that have been reported to have direct effects on the immune system, including the modulation of IgE production using various human and murine cell populations. We investigated the effects of the organic extract of DEP (PAH-DEP) and particularly, phenanthrene, a major component of DEP, in vitro on IgE production by 2C4/F3, a human Epstein-Barr virus transformed isotype switched, IgE producing B cell line. Phenanthrene consistently enhanced 2C4/F3 IgE production two- to threefold. This in vitro enhancement was associated with an increased expression of total IgE mRNA. Furthermore, the pattern of mRNA's coding for distinct isoforms of the epsilon chain was altered by both DEP-PAH and phenanthrene. While phenanthrene increased the level of productive epsilon transcripts, it did not increase epsilon germ line transcription. These effects were not due to an alteration of the cell cycle. Unstimulated 2C4/F3 cells contained detectable mRNA for IL6, IL10, TNF-alpha, and interestingly IL4; however, addition of PAH-DEP or phenanthrene did not significantly alter the level of these cytokines and thus did not appear to account for our findings. Thus, we have used our in vitro model to dissect the mechanism of DEP-PAH on IgE production in postswitch IgE producing cells and shown that phenanthrene, an important component in DEP and other pollutants, can act in a similar manner.

B-Lymphocytes↗

PubMed1997

Deletion of amino acids 1641-2437 from the foot region of skeletal muscle ryanodine receptor alters the conduction properties of the Ca release channel.

The ryanodine receptor (RyR) of skeletal muscle contains two functional domains: a carboxyl-terminal hydrophobic domain that forms the putative conduction pore of the calcium release channel, and a large cytoplasmic domain that corresponds to the "foot structure." To understand the contribution of the foot structure to the function of the calcium release channel, we studied a RyR deletion mutant, delta(1641-2437)-RyR, in which a region that is rich in glutamate and aspartate residues (a.a. 1641-2437) was removed. The wild-type and delta(1641-2437)-RyR proteins were expressed in a Chinese hamster ovary (CHO) cell line, and functions of single calcium release channels were measured in the lipid bilayer membrane. The wild-type RyR forms functional calcium release channels with a linear current-voltage relationship similar to that of the native channel identified in the sarcoplasmic reticulum membrane of skeletal muscle, whereas the channels formed by delta(1641-2437)-RyR exhibit significant inward rectification, i.e., currents moving from cytoplasm into SR lumen were approximately 20% less than that in the opposite direction. As in to the wt-RyR channel, opening of the delta(1641-2437)-RyR channel has a bell-shaped dependence on the cytoplasmic calcium, but the calcium-dependent activation and inactivation processes of the delta(1641-2437)-RyR channel are shifted to higher calcium concentrations. Our data show that deletion of a.a. 1641-2437 from the foot region of the skeletal muscle RyR results in changes in both ion conduction and calcium-dependent regulation of the calcium release channel.

Animals↗