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Biomedical subjects

M Chen

Publications and source records attributed to M Chen.

At least 379 records · Page 21Linked to original sources
PubMed1998

Effect of citrulline for arginine replacement on the structure and turnover of phosphopeptide substrates of protein phosphatase-1.

Phosphorylated and nonphosphorylated forms of a decapeptide corresponding to residues 9 to 18 of glycogen phosphorylase were compared using two-dimensional nuclear magnetic resonance with assignment of both peptides done by the sequential method. Both forms had little secondary structure, but there was evidence for an interaction between arginine-16 and phosphorylated serine at position 14. A change in the chemical shift for the epsilon-nitrogen hydrogen of arginine in position 16 was observed in the spectrum of the phosphorylated peptide and was not evident in a phosphopeptide having citrulline in place of arginine-16. Hydrolysis catalyzed by protein phosphatase-1 was decreased with the citrulline-containing phosphopeptide compared to the arginine-containing phosphopeptide with effects observed on both kcat and Km of the phosphatase reaction. Alkaline phosphatase hydrolyzed these peptides and a di-citrulline peptide equally well. These results are consistent with arginine being favorable in the recognition of substrates by phosphatase-1, possibly recognition as an arginine-phosphoserine complex. As a model study, arginine and two analogs, citrulline and canavanine, were examined for association with inorganic phosphate by nuclear magnetic resonance spectrometry. 31P-NMR measurements showed that arginine and canavanine caused a shift in the phosphate resonance at 20 degreesC. Citrulline caused no change. Changes in chemical shift were measured over the pH range 5-9 with arginine and canavanine both causing a slight decrease in the apparent pKa of inorganic phosphate (DeltapKa approximately 0.15). NaCl, NH4Cl, and guanidine hydrochloride showed little effect on the resonance signal position of inorganic phosphate at pH 6.5, consistent with selectivity for the guanidino group. Temperature (6 degrees, 20 degrees, and 37 degreesC) caused little change in the effect of arginine, but there was some dependency with canavanine, decreasing with temperature. Citrulline caused no change in the chemical shift of phosphate at any temperature. It was concluded that hydrogen bonded complexes were formed between the dianion of phosphate and the protonated form of arginine or canavanine with a bifurcated structure having preference for the omega-hydrogens.

Alkaline Phosphatase↗
PubMed1998

Molecular cloning and characterization of a cDNA encoding canine type VII collagen non-collagenous (NC1) domain, the target antigen of autoimmune disease epidermolysis bullosa acquisita (EBA).

Type VII collagen, the major component of anchoring fibrils, serves as tight adhesion of skin basement membrane zone (BMZ) through its amino-terminal non-collagenous (NC1) domain. The NC1 domain is targeted by autoantibodies of an acquired blistering skin disease termed epidermolysis bullosa acquisita (EBA) naturally occurring in humans and dogs. We cloned the full-length canine type VII collagen NC1 domain cDNA and delineated its molecular and immunological characteristics. The canine NC1 domain cDNA consists of 3759 nucleotides encoding for 1253 amino acids, with a molecular mass of approx. 134 kDa and a 17 amino acid signal peptide. The expression of canine type VII collagen was confirmed by Northern blot analysis and by a rabbit antibody raised against a 17 amino acid peptide deduced from canine NC1 sequence. Comparison of canine NC1 with the corresponding human sequence indicated 86.7% and 87.6% identity at the nucleotide and deduced amino acid levels respectively. The protein homology reached greater than 95% within two immunodominant epitope areas. Furthermore, human EBA autoantibodies and a rabbit anti-human NC1 cross-reacted with canine skin BMZ and the newly synthesized canine type VII collagen. The molecular and immunological identities between human and canine NC1 domains suggest that NC1 may be critical for the EBA development.

Amino Acid Sequence↗
PubMed1998

Ischemic colitis and sumatriptan use.

Sumatriptan succinate, a serotonin-1 (5-hydroxytryptamine-1) receptor agonist, is an antimigraine drug that is reported to act by selectively constricting intracranial arteries. Recently, vasopressor responses that are distinct from the cranial circulation have been demonstrated to occur in the systemic, pulmonary, and coronary circulations. Cases have been published of coronary vasospasm, myocardial ischemia, and myocardial infarction occurring after sumatriptan use. We report on the development of 8 serious cases of ischemic colitis in patients with migraine treated with sumatriptan.

Adult↗
PubMed1998

Identification of Nck family genes, chromosomal localization, expression, and signaling specificity.

Already a dozen molecules share binding to the Src homology (SH) 3 domains of human Nck, an SH3-SH3-SH3-SH2 adapter protein. We reason that there may be multiple gene members of Nck to accommodate the large binding repertoires. Here we report identification of novel human and mouse Nck genes and rename them as the Nckalpha and Nckbeta genes (including the human Nckalpha, human Nckbeta, mouse Nckalpha, and mouse Nckbeta genes). Nckalpha and Nckbeta share 68% amino acid identity, whereas the two Nckalpha and two Nckbeta across the species show 96% identity to each other. The human Nckbeta gene is mapped to 2q12, whereas the human Nckalpha gene has previously been mapped at 3q21. Antibodies specifically against Nckalpha and Nckbeta detect Nckalpha and Nckbeta with an identical molecular mass in the same cells of various origins. Ectopically expressed Nckbeta, but not its SH2 domain mutant, strongly inhibits epidermal growth factor- and platelet-derived growth factor-stimulated DNA synthesis. Consistently, epidermal growth factor receptor and platelet-derived growth factor receptor preferentially interact with Nckbeta over Nckalpha in vitro. This study indicates that Nck is a multiple gene family and that each gene may have its own signaling specificity. Because previous anti-Nck (human Nckalpha) antibodies cross-react with Nckbeta, reassessment of those studies with specific Nck genes would be necessary.

Adaptor Proteins, Signal Transducing↗
PubMed1998

High-density perfusion culture of insect cells with a biosep ultrasonic filter.

The baculovirus/insect cell expression system has provided a vital tool to produce a high level of active proteins for many applications. We have developed a very high-density insect cell perfusion process with an ultrasonic filter as a cell retention device. The separation efficiency of the filter was studied under various operating conditions. A cell density of over 30 million cells/mL was achieved in a controlled perfusion bioreactor and cell viability remained greater than 90%. Sf9 cells from a high-density culture and a spinner culture were infected with two recombinant baculoviruses expressing genes for the production of human chitinase and monocyte-colony inhibition factor. The protein yield on a cell basis from infecting high-density Sf9 cells was the same as or higher than that from the spinner Sf9 culture. Virus production from the high-density culture was similar to that from the spinner culture. The results show that the ultrasonic filter did not affect insect cells' ability to support protein expression and virus production following infection with baculovirus. The potential applications of the high-density perfusion culture for large-scale protein expression from Sf9 cells are also highlighted.

Animals↗
PubMed1998

Pleiotropic effects of HIV-1 protein R (Vpr) on morphogenesis and cell survival in fission yeast and antagonism by pentoxifylline.

Expression of HIV-1 Vpr causes cell cycle G2 arrest, change in cell shape, and cell death over a large evolutionary distance ranging from human to yeast cells. As a step toward understanding these highly conserved Vpr functions, we have examined the effect of Vpr on cytoskeletal elements and the viability of fission yeast. We demonstrate that the changes in cell morphology induced by Vpr in fission yeast are caused by several underlying cellular abnormalities, including increased biosynthesis of chitin in the cell wall, disruption of the actin cytoskeleton, and altered polarity for cell growth. The extent of these cellular alterations and cell survival correlates with the level of vpr expression. Accompanying cell death, Vpr induces aberrant nuclear morphologies in fission yeast which are similar to those found during the apoptosis induced by Vpr in mammalian cells. The Vpr-induced cytopathic effects and cell death can be suppressed by treatment with pentoxifylline, a compound that inhibits HIV-1 viral replication and suppresses Vpr-induced cell cycle G2 arrest in human and fission yeast cells. The results presented here suggest that pentoxifylline suppresses the effects of Vpr by blocking interactions of Vpr with cellular proteins. Given that pentoxifylline has potential therapeutic value in blocking the effects of Vpr in HIV-infected patients, understanding the molecular mechanisms by which pentoxifylline antagonizes Vpr may have general implications for HIV therapy.

Anti-HIV Agents↗
PubMed1998

The Alzheimer's plaques, tangles and memory deficits may have a common origin. Part III: animal model.

We have hypothesized that an intracellular calcium deficit may occur in the early phase of Alzheimer's disease (AD). This hypothesis has two important corollaries. First, it predicts that elevation of calcium levels by many calcium agonists, in principle, would have protective effects in the individuals at-risk to AD. Second, it implies that an artificial decrease of the calcium levels by the use of calcium antagonists might mimic the AD pathologies in the experimental animals. Obviously, the latter prediction not only would allow a direct testing of the hypothesis, but also might offer a "new" route for developing an animal model for sporadic AD. In fact, a number of the existing models that target various neurotransmitter receptors and calcium channels has manifested memory deficits. This suggests that a fully successful animal model for AD might be developed by improving the current paradigms. Furthermore, we discuss a potential relationship between AD and schizophrenia in terms of intracellular calcium imbalance.

Alzheimer Disease↗
PubMed1998

The Alzheimer's plaques, tangles and memory deficits may have a common origin. Part II: therapeutic rationale.

In the previous papers, we hypothesized that there could be an intracellular calcium deficit in the early phase of Alzheimer's disease (AD). In the present study, we consider, from this point of view, several long-lasting questions about AD, which include: why plaques and tangles are only found in the brain; why aging is the primary risk factor; why presenilins are so vulnerable in the disease; and why AD only affects the most delicate function of the brain. Although discrepancies and alternative views exist in many aspects of AD, it came to our attention that our hypothesis perhaps could offer a reasonable and coherent explanation, at least in part, to several such questions by the known biochemical principles. Based on the analyses, we discuss some controversies in therapeutic strategies.

Alzheimer Disease↗
PubMed1998

The Alzheimer's plaques, tangles and memory deficits may have a common origin; part I; a calcium deficit hypothesis.

Review of the literature reveals that several biochemical events implicated in the pathology of Alzheimer's disease (AD) are calcium dependent processes. These processes include normal processing of beta-amyloid precursor protein, dephosphorylation and degradation of tau, neurotransmitter release and memory formation. Since all of these processes appear to be inactivated during progression of AD, we propose that a "deficit" of intracellular calcium levels may occur in the early phase of the disease. We also propose several experiments to test this hypothesis. The hypothesis predicts that presenilins most likely act as calcium channels in vivo and that their gene mutations may cause the disease by diminishing the Ca2+ channeling function.

Alzheimer Disease↗
PubMed1998

Grass genomes.

For the most part, studies of grass genome structure have been limited to the generation of whole-genome genetic maps or the fine structure and sequence analysis of single genes or gene clusters. We have investigated large contiguous segments of the genomes of maize, sorghum, and rice, primarily focusing on intergenic spaces. Our data indicate that much (>50%) of the maize genome is composed of interspersed repetitive DNAs, primarily nested retrotransposons that insert between genes. These retroelements are less abundant in smaller genome plants, including rice and sorghum. Although 5- to 200-kb blocks of methylated, presumably heterochromatic, retrotransposons flank most maize genes, rice and sorghum genes are often adjacent. Similar genes are commonly found in the same relative chromosomal locations and orientations in each of these three species, although there are numerous exceptions to this collinearity (i.e., rearrangements) that can be detected at the levels of both the recombinational map and cloned DNA. Evolutionarily conserved sequences are largely confined to genes and their regulatory elements. Our results indicate that a knowledge of grass genome structure will be a useful tool for gene discovery and isolation, but the general rules and biological significance of grass genome organization remain to be determined. Moreover, the nature and frequency of exceptions to the general patterns of grass genome structure and collinearity are still largely unknown and will require extensive further investigation.

Journal Article↗
PubMed1998

Matrix attachment regions and structural colinearity in the genomes of two grass species.

In order to gain insights into the relationship between spatial organization of the genome and genome function we have initiated studies of the co-linear Sh2/A1- homologous regions of rice (30 kb) and sorghum (50 kb). We have identified the locations of matrix attachment regions (MARs) in these homologous chromosome segments, which could serve as anchors for individual structural units or loops. Despite the fact that the nucleotide sequences serving as MARs were not detectably conserved, the general organizational patterns of MARs relative to the neighboring genes were preserved. All identified genes were placed in individual loops that were of comparable size for homologous genes. Hence, gene composition, gene orientation, gene order and the placement of genes into structural units has been evolutionarily conserved in this region. Our analysis demonstrated that the occurrence of various 'MAR motifs' is not indicative of MAR location. However, most of the MARs discovered in the two genomic regions were found to co-localize with miniature inverted repeat transposable elements (MITEs), suggesting that MITEs preferentially insert near MARs and/or that they can serve as MARs.

Base Composition↗
PubMed1998

Human and murine antibody recognition is focused on the ATPase/helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein.

The hepatitis C virus (HCV) nonstructural (NS) 3 protein has been shown to possess at least two enzymatic domains. The amino terminal third contains a serine-protease domain, whereas the carboxy terminal two thirds is comprised of an adenosine triphosphatase (ATPase)/helicase domain. These domains are essential for the maturation of the carboxy-terminal portion of the HCV polyprotein and catalyze the cap synthesis of the RNA genome. In this report, human and murine antibody responses induced by NS3 were characterized using a recombinant full-length NS3 (NS3-FL) protein, or the isolated protease or ATPase/ helicase domains, expressed and purified from Escherichia coli. Sera from 40 patients with chronic HCV infection were assayed in enzyme-linked immunoassays (EIAs) for antibody binding to the panel of NS3 proteins. Virtually all patient sera contained antibodies specific for NS3-FL and the ATPase/helicase domain, whereas only 10% of sera reacted with the protease domain of NS3. Human antibodies reactive with NS3-FL were highly restricted to the immunoglobulin G1 (IgG1) isotype and were inhibited by soluble ATPase/helicase, but not by the protease domain. The anti-NS3 (ATPase/helicase) reactivity decreased on denaturation by sodium dodecyl sulfate (SDS) and beta-mercaptoethanol (2ME), suggesting the recognition of nonlinear or conformational B-cell determinants. Similar to infected humans, mice immunized with NS3-FL developed high-titered primary antibody responses to the NS3 ATPase/ helicase domain, whereas an anti-NS3 protease response was not observed after primary or secondary immunizations. Thus, the human and murine humoral immune responses to the HCV NS3 protein are focused on the ATPase/helicase domain, are restricted to the IgG1 isotype in humans, and are conformationally dependent. Unexpectedly, in both species, the NS3 protease domain, present in the context of the full-length NS3, appears to possess low intrinsic immunogenicity in terms of antibody production.

Adenosine Triphosphatases↗
PubMed1998

Endothelial cell integrin laminin receptor expression in multiple sclerosis lesions.

Laminin, a major glycoprotein component of vessel basement membranes, is recognized by beta1- and beta3-integrins expressed on endothelial cells. To determine how endothelial cell integrins might function in multiple sclerosis (MS) lesions, integrin laminin receptors and laminin were analyzed in central nervous system samples from MS patients and controls by immunohistochemistry. In active MS lesions, endothelial cell VLA-6 and beta1 subunits were decreased compared to controls whereas alpha(v) subunit and VLA-1 were increased. In chronic inactive lesions beta1, VLA-6 and alpha(v) were the same as controls but VLA-1 remained increased. Alpha3 subunit was constant in all samples. By immunoelectron microscopy VLA-1, VLA-6, beta1, and laminin were distributed throughout endothelial cells; alpha(v) was adjacent to and on luminal surfaces; alpha(v) and VLA-1 were on intercellular junctions. These results indicate distinct regulation and functions of these integrins in different lesion stages. In active lesions decreased endothelial cell beta1/VLA-6 could result in their detachment from laminin thereby facilitating leukocyte transvascular migration and blood-brain barrier breakdown. Alpha(v) and VLA-1 on intercellular junctions may participate in re-establishing vessel integrity after leukocyte migration. Luminal surface alpha(v) also likely binds intraluminal ligands and cells. In chronic inactive plaques persistently elevated endothelial cell VLA-1 correlates with long-standing endothelial cell and blood-brain barrier dysfunction.

Adolescent↗
PubMed1998

A new approach to the development of anti-ischemic drugs: protective drugs against cell injury induced by lysophosphatidylcholine.

Recent studies have revealed that lysophosphatidylcholine (LPC) produces mechanical and metabolic derangements in perfused working rat hearts and Ca2+-overload in isolated cardiac myocytes. Thus, LPC possesses an ischemia-like effect on the heart. Therefore, a drug that possesses an anti-LPC action would protect or improve ischemia/reperfusion damage. We examined the effects of various anti-ischemic drugs on the Ca2+ overload induced by LPC. Our data suggest that a drug with high lipophilicity possesses a protective effect on cell injury induced by LPC, probably because of preservation of membrane integrity.

Adrenergic beta-Antagonists↗
PubMed1998

Amino acid sequence of a Ca(2+)-transporting ATPase from the sarcoplasmic reticulum of the cross-striated part of the adductor muscle of the deep sea scallop: comparison to serca enzymes of other animals.

The RT PCR approach was used to obtain the nucleotide sequence of the mRNA of a sarco/endoplasmic reticulum calcium transporting ATPase (SERCA) from the cross-striated (phasic) part of the adductor muscle of the deep sea scallop. Initially, degenerate primers based on consensus sequences among SERCAs and tryptic fragments of the scallop Ca-ATPase were used. The sequence was then extended using homologous primers and the 5' and 3' ends of the transcript determined by 5' and 3' RACE. The mRNA codes for a polypeptide chain 994 amino acid residues long (coded for by 2982 nucleotides) and has a 195 bp 5' untranslated region, with a 697 bp 3' untranslated region. The scallop enzyme shows strongest amino acid similarity to the SERCA enzyme of Loligo, followed by those of Drosophila and Artemia. It resembles the vertebrate SERCA3 in that it does not possess the phospholamban binding motif and so is unlikely to be regulated by protein kinase A mediated signals.

Amino Acid Sequence↗
PubMed1998

Tissue plasminogen activator contributes to the late phase of LTP and to synaptic growth in the hippocampal mossy fiber pathway.

The expression of tissue plasminogen activator (tPA) is increased during activity-dependent forms of synaptic plasticity. We have found that inhibitors of tPA inhibit the late phase of long-term potentiation (L-LTP) induced by either forskolin or tetanic stimulation in the hippocampal mossy fiber and Schaffer collateral pathways. Moreover, application of tPA enhances L-LTP induced by a single tetanus. Exposure of granule cells in culture to forskolin results in secretion of tPA, elongation of mossy fiber axons, and formation of new, active presynaptic varicosities contiguous to dendritic clusters of the glutamate receptor R1. These structural changes are blocked by tPA inhibitors and induced by application of tPA. Thus, tPA may be critically involved in the production of L-LTP and specifically in synaptic growth.

Animals↗
PubMed1998

Mechanisms of spontaneous cytosolic Ca2+ transients in differentiated human neuronal cells.

We have studied Ca2+ homeostasis in a unique model of human neurons, the NT2N cell, which differentiates from a human teratocarcinoma cell line, NTera2/C1.D1 by retinoic acid treatment. When perifused with Krebs-HEPES buffer containing 2.5 mM CaCl2, fura-2 loaded NT2N cells produced spontaneous cytosolic Ca2+ oscillations, or Ca2+ transients. These cytosolic Ca2+ transients were not blocked by antagonists of glutamate (6-cyano-7-nitroquinoxaline-2,3-dione and D(-)-2-amino-5-phosphonopentanoic acid) or muscarinic (atropine) receptors. Omission of extracellular Ca2+ completely abolished Ca2+ oscillations and decreased the average Ca2+ level from 106 +/- 14 nM to 59 +/- 8 nM. Addition of the L-type Ca2+ channel blocker nifedipine (1 or 10 microM) or of the N-type inhibitor omega-conotoxin GVIA (5 microM) significantly, although incompletely, suppressed Ca2+ oscillations, while omega-conotoxin MVIIC (5 microM), a selective antagonist of P- and Q-channels, had no effect. Ni2+, at 100 microM, a concentration selective for T-type channels, did not inhibit Ca2+ transients. Non-specific blockage of Ca2+ channels by higher concentrations of Ni2+ (2-5 mM) or Co2+ (1 mM) abolished Ca2+ oscillations completely. The endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin (1 microM), slightly decreased Ca2+ oscillation frequency, and induced a small transitory increase in the average cytosolic Ca2+ concentration. The mRNAs of L- (alpha1D subunit) and N-type (alpha1B subunit) Ca2+ channel were present in NT2N cells, while that of a T-type Ca2+ channel (alpha1-subunit) was not present in the NT2N cells as shown by reverse transcription-polymerase chain reaction. In conclusion, NT2N neuronal cells generate cytosolic Ca2+ oscillations mainly by influx of extracellular Ca2+ through multiple channels, which include L- and N-type channels, and do not require activation of glutamate or muscarinic receptors.

Calcium↗