Bicycle helmet protection in the Fox Point community.
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Adolescent↗
Biomedical subjects
M Chen
Publications and source records attributed to M Chen.
Apolipoprotein E2 (Arg136 --> Cys) mutation in the receptor binding domain of apoE is not associated with dominant type III hyperlipoproteinemia.
Using apoE phenotyping by immunoblotting and apoE genotyping we identified four heterozygous carriers of a rare apolipoprotein (apo) E2 variant, apoE2 (Arg136 --> Cys). ApoE2 (Arg136 --> Cys) was not distinct from apoE2 (Arg158 --> Cys) by phenotyping, but produced a unique pattern of bands on CfoI restriction typing of a 244 bp apoE gene fragment. Two of the four apoE2 (Arg136 --> Cys)/3 heterozygotes had elevated triglycerides, two were normolipidemic. The composition of very low density lipoproteins (VLDL) was normal in each of the four apoE2 (Arg136 --> Cys) carriers, regardless of the triglyceride concentrations. None of the apoE2 (Arg136 --> Cys) carriers displayed a broad beta-band and none revealed beta-migrating particles in the VLDL. The two hypertriglyceridemic carriers of apoE2 (Arg136 --> Cys) were, therefore, classified as having type IV rather than type III hyperlipoproteinemia. LDL receptor binding activities were studied using recombinant apoE loaded to dimyristoylphosphatidylcholine (DMPC) vesicles and to VLDL and from an apoE-deficient individual. LDL receptor binding of apoE2 (Arg136 --> Cys) was 14% of apoE3 and was thus higher than that of apoE2 (Arg158 --> Cys). Both apoE2 (Arg136 --> Cys) and apoE2 (Arg158 --> Cys) displayed substantial heparin binding (61 and 53% of apoE3, respectively). As the dominant apoE variants known so far are characterized by more pronounced reductions of heparin binding, we suggest that apoE2 (Arg136 --> Cys) is not associated with dominant expression of type III hyperlipoproteinemia. These findings lend support to the concept that apoE variants predisposing to dominant type III hyperlipoproteinemia differ from recessive mutations by a more severe defect in heparin binding.
Adult↗
Physical effects of cholesterol on arterial smooth muscle membranes: evidence of immiscible cholesterol domains and alterations in bilayer width during atherogenesis.
Small angle X-ray diffraction was used to examine arterial smooth muscle cell (SMC) plasma membranes isolated from control and cholesterol-fed (2%) atherosclerotic rabbits. A microsomal membrane enriched with plasma membrane obtained from animals fed cholesterol for up to 13 weeks showed a progressive elevation in the membrane unesterified (free) cholesterol:phospholipid (C/PL) mole ratio. Beyond 9 weeks of cholesterol feeding, X-ray diffraction patterns demonstrated a lateral immiscible cholesterol domain at 37 degrees C with a unit cell periodicity of 34 A coexisting within the liquid crystalline lipid bilayer. On warming, the immiscible cholesterol domain disappeared, and on cooling it reappeared, indicating that the immiscible cholesterol domain was fully reversible. These effects were reproduced in a model C/PL binary lipid system. In rabbits fed cholesterol for less than 9 weeks, lesser increases in membrane C/PL mole ratio were observed. X-ray diffraction analysis demonstrated an increase in membrane bilayer width that correlated with the C/PL mole ratio. This effect was also reproduced in a C/PL binary lipid system. Taken together, these findings demonstrate that in vivo, feeding of cholesterol causes cholesterol-phospholipid interactions in the membrane bilayer that alter bilayer structure and organization. This interaction results in an increase in bilayer width peaking at a saturating membrane cholesterol concentration, beyond which lateral phase separation occurs resulting in the formation of separate cholesterol bilayer domains. These alterations in structure and organization in SMC plasma membranes may have significance in phenotypic modulation or aortic SMC during early atherogenesis.
Animals↗
Detection of Rickettsia sibirica in ticks and small mammals collected in three different regions of China.
The primers Rr 190.70p and Rr 190.602n were used to detect spotted fever group (SFG) rickettsiae by polymerase chain reaction (PCR) in ticks and small mammals collected in three different regions of China. The obtained results indicated that specific DNA fragments of SFG rickettsiae were amplified from Dermacentor silvarum, D. sinicus, D. auratus, Haemaphysalis concinna, H. wellingtoni, H. yeni, Apodemus agrarius, Microtus fortis. Clethrionomys rufocanus, Ondatra zibethica, Rattus flavipectus and hedgehog. The PCR product were digested with restriction endonucleases PstI and RsaI and the obtained electrophoretic profiles were compared with those of the prototype strains of SFG rickettsiae by the restriction fragment length polymorphism (RFLP) technique. The comparisons showed that the profiles were identical to those of Rickettsia sibirica. In addition, three new isolates of R. sibirica were obtained from H. yeni, D. sinicus and hedgehog, and designated NH-95, BJ-95 and BHJ-95, respectively. These results not only demonstrated a horizontal transmission of the rickettsiae between ticks and hosts but also suggested that R. sibirica is widely distributed in China and its hosts and vectors are various, all that indicating the existence of natural foci of North Asia tick-borne spotted fever specific to China.
Animals↗
Sequence analysis of a fragment of rOmpA gene of several isolates of spotted fever group rickettsiae from China.
The nucleotide sequence of rOmpA gene fragment of three Chinese isolates of spotted fever group rickettsiae (SFGR) (BJ-90, Ha-91 and HLJ-054) was determined. The obtained nucleotide and putative amino acid sequences were compared with those of another Chinese SFGR isolate (HL-93) and several prototype SFGR strains. This comparison showed that the isolates BJ-90 and Ha-91 are closely related to each other and R. sibirica but different from the isolates HLJ-054 and HL-93. We assume that with exception of the isolates HLJ-054 and HL-93 that represent new, unique members of SFGR, the isolates BJ-90 and Ha-91 are closely related to R. sibirica, one of the prototype SFGR strains.
Amino Acid Sequence↗
Synapse-specific, long-term facilitation of aplysia sensory to motor synapses: a function for local protein synthesis in memory storage.
The requirement for transcription during long-lasting synaptic plasticity has raised the question of whether the cellular unit of synaptic plasticity is the soma and its nucleus or the synapse. To address this question, we cultured a single bifurcated Aplysia sensory neuron making synapses with two spatially separated motor neurons. By perfusing serotonin onto the synapses made onto one motor neuron, we found that a single axonal branch can undergo long-term branch-specific facilitation. This branch-specific facilitation depends on CREB-mediated transcription and involves the growth of new synaptic connections exclusively at the treated branch. Branch-specific long-term facilitation requires local protein synthesis in the presynaptic but not the postsynaptic cell. In fact, presynaptic sensory neuron axons deprived of their cell bodies are capable of protein synthesis, and this protein synthesis is stimulated 3-fold by exposure to serotonin.
Animals↗
Role of B cells in antigen presentation of the hepatitis B core.
The hepatitis B virus (HBV) nucleocapsid or core antigen (HBcAg) is extremely immunogenic during infection and after immunization. For example, during many chronic infections, HBcAg is the only antigen capable of eliciting an immune response, and nanogram amounts of HBcAg elicit antibody production in mice. Recent structural analysis has revealed a number of characteristics that may help explain this potent immunogenicity. Our analysis of how the HBcAg is presented to the immune system revealed that the HBcAg binds to specific membrane Ig (mIg) antigen receptors on a high frequency of resting, murine B cells sufficiently to induce B7.1 and B7.2 costimulatory molecules. This enables HBcAg-specific B cells from unprimed mice to take up, process, and present HBcAg to naive Th cells in vivo and to T cell hybridomas in vitro approximately 10(5) times more efficiently than classical macrophage or dendritic antigen-presenting cells (APC). These results reveal a structure-function relation for the HBcAg, confirm that B cells can function as primary APC, explain the enhanced immunogenicity of HBcAg, and may have relevance for the induction and/or maintenance of chronic HBV infection.
Animals↗
Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity.
Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, gammac and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.
Animals↗
Oncogenic Ki-ras but not oncogenic Ha-ras blocks integrin beta1-chain maturation in colon epithelial cells.
Human colorectal tumors commonly contain mutations in Ki-ras but rarely, if ever, in Ha-ras. The selectivity for Ki-ras mutations in this tumor was explored using the HD6-4 colon epithelial cell line which contains no ras mutations. After adhesion to an extracellular matrix, HD6-4 cells polarize into columnar goblet cells with distinct apical and basal regions. Stable HD6-4 transfectants were made with mini-gene constructs of the oncogenic cellular Ki-ras4BG12V gene, the oncogenic Ha-rasG12V gene, or mini-gene constructs of wild-type Ki-ras4B as a control. Ki-ras mutations, but not Ha-ras mutations, disrupted colon epithelial cell apicobasal polarity and adhesion to collagen I and laminin. Three Ha-ras transfectants and three Ki-ras transfectants exhibited Ras proteins expressing the Val-12 mutation by Western blotting with pan-rasG12V antibody. Only wild-type Ki-ras transfectant cells and oncogenic Ha-ras transfectant cells synthesized the mature, fully glycosylated forms of beta1 integrin. Instead of the mature integrin beta1-chain, a faster migrating beta1-chain intermediate was detected on the cell surface and in the cytoplasm of the oncogenic Ki-ras transfectants. Expression of the oncogenic Ki-ras gene caused the altered beta1 integrin maturation because phosphorothiolated antisense oligonucleotides to Ki-ras reduced expression of both the mutant Ki-Ras protein and the aberrant integrin beta1-chain and increased expression of the mature integrin beta1-chain. Altered glycosylation generated the new beta1 integrin form since integrin core beta1-chain proteins of the same molecular weight were yielded in Ki-ras, Ha-ras, and control transfectants after removal of sugar residues with endoglycosidase F or following tunicamycin treatment to inhibit glycosylation. The selective effect of oncogenic Ki-ras on beta1 integrin glycosylation was not due to selective activation of mitogen-activated protein kinases because both mutated Ki- and Ha-ras genes activated this pathway and increased cell proliferation. Since blocking the glycosylation of integrin beta1-chain inhibited the adherence, polarization, and subsequent differentiation of colon epithelial cells, the selective effects of the oncogenic cellular Ki-ras gene on integrin beta1-chain glycosylation may account, at least in part, for the selection of Ki-ras mutations in human colon tumors.
Calcium-Calmodulin-Dependent Protein Kinases↗
Apoptosis-suppressor gene bcl-2 expression after traumatic brain injury in rats.
Neuronal death after experimental traumatic brain injury (TBI) has features of both apoptosis and necrosis. Neurons in the peritrauma cortex, hippocampus, and dentate gyrus are particularly vulnerable. The apoptosis-suppressor gene bcl-2 is induced in brain after ischemia and epilepsy-induced injury and may serve to regulate neuronal death. We studied expression of bcl-2 mRNA and protein after experimental TBI in rats. To determine whether bcl-2 protein expression occurred in cells with evidence of apoptosis, triple-labeling studies were performed using (1) antibody against bcl-2, (2) bis-benzimide dye to examine gross nuclear morphology, and (3) terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling (TUNEL) to assess for DNA fragmentation. At 6 and 24 hr, bcl-2 mRNA was induced in ipsilateral peritrauma cortex, hippocampus, and dentate gyrus. By 72 hr the increase in bcl-2 mRNA was detected only in cortex. bcl-2 protein was induced at 8, 24, 72, and 168 hr in ipsilateral cortex and hippocampus. Cells expressing bcl-2 protein included neurons in the peritrauma cortex, hippocampus, hilus, and dentate gyrus. The gross nuclear morphology of neurons expressing bcl-2 appeared normal. Furthermore, biochemical evidence of DNA fragmentation, in a pattern characteristic of either apoptosis or necrosis, was seldom seen in neurons expressing bcl-2 protein (bcl-2 colocalized with TUNEL in 0-2% of TUNEL-positive cells observed). These data suggest that bcl-2 may play an important role in the regulation of neuronal death after TBI, and they support a role for bcl-2 as an inducible neuroprotective gene.
Animals↗
Protective antibodies develop, and murine Lyme arthritis regresses, in the absence of MHC class II and CD4+ T cells.
Murine Lyme borreliosis is characterized by arthritis and carditis that are most severe at 2 to 3 wk, then regress during the course of persistent infection. Borrelia burgdorferi-specific Abs and CD4+ T cells have been implicated in the resolution phase of arthritis. Therefore, MHC class II transactivator (CIITA)-deficient mice that do not express conventional class II molecules and lack the normal CD4 repertoire were used to investigate the role of MHC class II-mediated responses in Lyme disease. The development of arthritis and carditis, and the resolution of arthritis, were similar in CIITA-deficient and control C57/BL6 mice. In contrast, the resolution of carditis was delayed in CIITA-deficient animals compared with controls. Moreover, CIITA-deficient mice developed B. burgdorferi-specific IgG2b Abs, and sera from these animals passively protected naive C3H/HeN mice from challenge inoculation and cleared B. burgdorferi from 2 day-infected C.B.17 SCID mice. These data suggest that CD4+ T cells and MHC class II-mediated responses are not required for the generation of protective Abs or the regression of arthritis, but may be important in the resolution of Lyme carditis in mice.
Animals↗
Alzheimer's alpha-secretase may be a calcium-dependent protease.
Proteolytic processing of beta-amyloid precursor protein (APP) is believed to be fundamental to the understanding of Alzheimer's disease. The identities and the regulatory elements of the proteases involved in the process, known as alpha/beta/gamma secretases, are unclear. In this study, by examining reported data, we found some indications suggesting that the putative alpha-secretase may be a calcium-dependent protease, and that this enzyme may play a primary role in the regulation of APP processing. Based on this, we proposed a model for the membrane orientations of the secretases for further discussions.
Alzheimer Disease↗
Transient gene expression from yeast artificial chromosome DNA in mammalian cells is enhanced by adenovirus.
The introduction of high molecular weight DNA into mammalian cells is useful for gene expression studies. However, current transfection strategies are inefficient, necessitating propagation of stable DNA transformants prior to analysis of gene expression. Here we demonstrate that transient lipid-mediated DNA transfection can be used to assess gene expression from yeast artificial chromosomes (YACs) containing the 230 kb cystic fibrosis transmembrane conductance regulator gene ( CFTR ) and Escherichia coli lacZ . We also show that psoralen-UV inactivated adenovirus significantly enhances transfection efficiency. The ability to deliver high molecular weight DNA using lipid-mediated transfection should expedite the analysis of large human genes contained within artificial chromosome vectors.
Adenoviridae↗
Oncogenic c-Ki-ras but not oncogenic c-Ha-ras up-regulates CEA expression and disrupts basolateral polarity in colon epithelial cells.
Colon carcinomas commonly contain mutations in Ki-ras4B, but very rarely in Ha-ras, suggesting that different Ras isoforms may have distinct functions in colon epithelial cell biology. In an earlier study we had demonstrated that oncogenic Ki-ras4BVal-12, but not oncogenic Ha-rasVal-12, blocks the apicobasal polarization of colon epithelial cells by preventing normal glycosylation of the integrin beta1 chain of the collagen receptor. As a result, only the Ki-ras mutated cells exhibited altered cell to substratum attachment, whereas mutation of either Ras isoform activated mitogen-activated protein kinases. We have now asked whether intercellular adhesion proteins implicated in establishing basolateral polarity in colon epithelial cells are modulated by oncogenic Ki-Ras4BVal-12 proteins but not oncogenic Ha-RasVal-12 proteins. The embryonic adhesion protein carcinoembryonic antigen (CEA) was up-regulated on the mRNA and protein levels in each of three stable Ki-rasVal-12 transfectant lines but in none of three stable Ha-rasVal-12 transfectant lines. The elevated protein levels of CEA in Ki-ras4BVal-12 transfectant cells were decreased by blocking expression of Ki-ras4BVal-12 with antisense oligonucleotides. N-cadherin levels were decreased in only the Ki-ras transfectants, whereas E-cadherin levels were unchanged. Immunohistochemical analysis demonstrated that Ki-ras4BVal-12 transfectant cells did not polarize into cells with discrete apical and basal regions and so could not restrict expression of CEA to the apical region. These unpolarized cells displayed elevated levels of CEA all along their surface membrane where CEA mediated random, multilayered associations of tumor cells. This aggregation was both calcium-independent and blocked by Fab' fragments of anti-CEA monoclonal antibody col-1. Trafficking of the lysosomal cysteine protease cathepsin B may also be altered when cell polarity cannot be established. Ki-ras4BVal-12 transfectant cells expressed 2-fold elevated protein levels of the lysosomal cysteine protease cathepsin B but did not up-regulate cathepsin B mRNA expression. One function of oncogenic c-Ki-Ras proteins in colon cancer progression may be to up-regulate CEA and thus to prevent the lateral adhesion of adjacent colon epithelial cells that normally form a monolayer in vivo.
Carcinoembryonic Antigen↗
Role of ribosomes in reinitiation of membrane insertion of internal transmembrane segments in a polytopic membrane protein.
The topogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the topogenesis and membrane assembly of membrane proteins with multiple TM segments (polytopic) is still incomplete. Recently, several studies on P-glycoprotein (Pgp) suggested that the topogenesis of polytopic membrane proteins is likely more complicated than anticipated. While studying the mechanism by which Pgp topogenesis is determined, we unexpectedly found that ribosomes or proteins associated with ribosomes are involved in regulating the membrane insertion and folding of Pgp during its translation. We discovered that when Pgp was translated by wheat germ ribosomes in vitro, TM3 could not reinitiate the insertion of the protein into microsomal membranes following the membrane insertion of TM1 and TM2. In contrast, TM3 could reinitiate membrane insertion when the protein was translated by rabbit reticulocyte ribosomes. These findings suggest that ribosomes or proteins associated with ribosomes play an important role in membrane insertion and folding of TM segments of Pgp and that rabbit reticulocyte and wheat germ ribosomes may use different mechanisms to control the membrane insertion of the same nascent peptide. We propose that ribosomes or proteins associated with ribosomes help reinitiate insertion of internal TM segments into the membrane by dissociation and reassociation with the protein-conducting channel in ER membranes.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Differential effects of Ca2+ channel blockers on Ca2+ overload induced by lysophosphatidylcholine in cardiomyocytes.
The effects of Ca2+ channel blockers (verapamil, diltiazem, nicardipine, bepridil and flunarizine) on Ca2+ overload induced by lysophosphatidylcholine were examined in rat isolated cardiomyocytes. Addition of lysophosphatidylcholine (15 microM) produced Ca2+ overload as evidenced by a marked increase in the concentration of intracellular Ca2+ and hypercontracture of cells. Verapamil, flunarizine and bepridil concentration dependently inhibited the lysophosphatidylcholine-induced Ca2+ overload, whereas diltiazem and nicardipine did not. Lysophosphatidylcholine increased the release of creatine kinase, which was significantly attenuated by verapamil, flunarizine or bepridil (5 microM for each), but not by diltiazem or nicardipine (20 microM for each). Verapamil, flunarizine, bepridil (which attenuated the lysophosphatidylcholine-induced Ca2+ overload) and nicardipine (which did not) inhibited the veratridine-induced increase in the concentration of intracellular Na+ (indicated by the increase in fluorescence ratio of Na(+)-binding benzofuran isophthalate) and cell contracture, whereas diltiazem did not. These results suggest that verapamil, bepridil and flunarizine attenuate the Ca2+ overload induced by lysophosphatidylcholine, and that the Ca2+ channel blocking action of these drugs does not contribute substantially to this effect. The Na+ channel inhibition together with high lipophilicity of these drugs may be important for the attenuation of the lysophosphatidylcholine-induced Ca2+ overload.
Animals↗
The beta subunit of CKII negatively regulates Xenopus oocyte maturation.
CKII (formerly known as casein kinase II) is a ubiquitously expressed enzyme that plays an important role in regulating cell growth and differentiation. The beta subunit of CKII (CKIIbeta) is not catalytic but forms heterotetramers with the catalytic subunit alpha to generate an alpha2beta2 holoenzyme. In Xenopus oocytes, CKIIbeta also associates with another serine/threonine kinase, Mos. As a key regulator of meiosis, Mos is necessary and sufficient to initiate oocyte maturation. We have previously shown that the binding of CKIIbeta to Mos represses Mos-mediated mitogen-activated protein kinase (MAPK) activation and that the ectopic expression of CKIIbeta inhibits progesterone-induced Xenopus oocyte maturation. We have now used an antisense oligonucleotide technique to reduce the endogenous CKIIbeta protein level in Xenopus oocytes, and we find that oocytes with a reduced content of CKIIbeta are more sensitive to low doses of progesterone and show accelerated MAPK activation and germinal vesicle breakdown. Furthermore, ectopic expression of a Mos-binding fragment of CKIIbeta suppressed the effect of antisense oligonucleotide. These results suggest that the endogenous CKIIbeta normally sets a threshold level for Mos protein, which must be exceeded for Mos to activate the MAPK signaling pathway and induce oocyte maturation.
Animals↗
Temporal trends and factors associated with survival after Pneumocystis carinii pneumonia in California, 1983-1992.
The authors investigated quarterly trends in survival after the diagnosis of Pneumocystis carinii pneumonia for 19,607 patients in California in the decade from January 1, 1983, through December 31, 1992. Subjects included all cases for whom P. carinii pneumonia was the initial (and only) acquired immunodeficiency syndrome (AIDS)-defining diagnosis as reported to the California human immunodeficiency virus/AIDS surveillance registry. There was a period of rapidly improving survival from approximately June 1986 until April 1988, coincident with the widespread introduction of antiretroviral therapy (zidovudine) and the institution of P. carinii pneumonia prophylaxis (with cotrimoxazole and pentamidine). There was no evidence, however, of meaningful improvements in survival for these patients after that period. The association of several covariates (risk transmission group, gender, race/ethnicity, certainty of P. carinii pneumonia diagnosis, age, region of residence, availability of CD4 count, and level of CD4 count) were also studied both by proportional hazards regression and by recursive partitioning (i.e., tree-based) survival analysis. The availability of a CD4 count (regardless of its level) was the single factor most strongly associated with survival (median survival 36 months among those with and 14 months among those without reported CD4 counts, p < 0.05). Data from this large, population-based surveillance registry of AIDS in California suggest that, despite earlier improvements in survival after the diagnosis of P. carinii pneumonia, the long-term survival of these patients remains poor (39% alive 2 years after diagnosis) and that no improvement in survival has occurred since 1988.
AIDS-Related Opportunistic Infections↗