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At least 1,459 records · Page 81Linked to original sources
PubMed1984

Alteration by estrogen of the nucleoli in nerve cells of the rat hypothalamus.

Estrogen is accumulated from the blood by nerve cells in the ventromedial nucleus of the hypothalamus and can facilitate female reproductive behavior by acting on this region of the brain. This cell group was examined in ovariectomized female rats, given estrogen or control treatment, by use of light and electron microscopy. A significantly greater portion of the nerve cells in the estrogen-treated animals had protuberances on their nucleolar surfaces, apparent under the light microscope. The fine structure of such protuberances included dense, aggregated material, which is shown to contain DNA by the sodium tungstate staining technique. Because increased numbers of such protuberances were found in nuclei of cells of the experimental group where previous studies demonstrated a significant increase in ultrastructural signs of biosynthetic activity, they may be associated with increased RNA synthesis. Thus, they could indicate, ultrastructurally, increased synthetic rates for RNA in nerve cells through which estrogen promotes reproductive behavior.

Animals↗
PubMed1987

Organization of the Sindbis virus nucleocapsid as revealed by bifunctional cross-linking agents.

Purified Sindbis virus nucleocapsids were reacted with a variety of bifunctional protein-specific cross-linking agents. The products were analyzed in concentration-gradient polyacrylamide gels and amounts of various products determined. These studies indicated that available lysine residues within adjacent capsid proteins in purified intact nucleocapsids are separated by 6 A. The capsid proteins in intact nucleocapsids are cross-linked in a pattern predicted for discrete monomeric entities, rather than in dimeric or trimeric aggregates. Purified, soluble capsid protein exists in a conformation that differs from the arrangement of protein within nucleocapsids. These conformational differences suggest that topological changes may occur in the capsid protein during virus maturation. Cross-linked nucleocapsids that were treated with RNases resulted in the generation of RNA-free protein shells that retained hexagonal morphology, indicating that, together, the RNA and protein form the outer surface of the nucleocapsid. These data are used to produce a model of the Sindbis virus nucleocapsid in which the proteins are arranged quasi-equivalently in a T = 4 icosahedral shell.

Capsid↗
PubMed2003

DNA damage-mediated apoptosis induced by selenium compounds.

Selenium (Se) compounds, which are the most extensively studied cancer chemopreventive agents, induce apoptotic death of tumor cells. In the current study, we show that selenite-induced apoptosis involves DNA damage. We showed that selenite-induced apoptosis as evidenced by cleavage of poly(ADP-ribose) polymerase was reduced in NIH 3T3 cells treated with ATM small interfering RNA, suggesting the involvement of the DNA damage regulator ATM. Consistent with ATM/ATR involvement, selenite was also shown to stimulate Ser-139 phosphorylation of the ATM/ATR substrate H2AX. Selenite-induced apoptosis was shown to involve DNA topoisomerase II (Top II) as selenite-induced apoptosis was reduced in Top II-deficient HL-60/MX2 cells and in HL-60 cells co-treated with the Top II catalytic inhibitor ICRF-193. Using purified human recombinant Top II, selenite was shown to induce reversible Top II cleavage complexes in vitro. In the aggregate, these results suggest that selenite-induced apoptosis, which involves ATM/ATR and Top II, is likely to be because of DNA damage.

3T3 Cells↗
PubMed1987

Characterization of wheat germ initiation factor eIF-2.

The initiation factor eIF-2 that specifically binds Met-tRNAf and GTP in ternary complex (eIF-2. GTP. Met-tRNAf) has been purified to apparent homogeneity from wheat germ ribosomal salt wash. The purified factor exhibits a sedimentation coefficient of 5 X 5S and an aggregate molecular weight of 122,000-daltons for the native protein.

Centrifugation, Density Gradient↗
PubMed1986

Visualization of intermediary transcription states in the complex between Escherichia coli DNA-dependent RNA polymerases and a promoter-carrying DNA fragment using the gel retardation method.

DNA-dependent RNA polymerase in complex with a DNA fragment was analyzed by electrophoresis in non-denaturing gels as core enzyme, holoenzyme, during initiation and elongation. The DNA fragment carried the promoter A1 of the phage T7. The stoichiometry between holoenzyme and promoter and between sigma and core enzyme in complex with DNA was determined. Holoenzyme bound as a monomer to the DNA, whereas core enzyme formed aggregates before binding to the DNA. If the molar ratio of holoenzyme to DNA exceeded 0.5:1 a second holoenzyme molecule interacted with the DNA fragment with diminished affinity. A large difference in the frictional coefficient of the holoenzyme-promoter and the core enzyme-DNA complex indicated a drastic conformational difference between the two types of complexes. The stability of the holoenzyme-promoter complex decreased with decreasing temperature, accompanied by at least partial dissociation of holoenzyme into core enzyme and sigma factor. Addition of nucleoside triphosphates did not change the electrophoretic mobility of the complex if abortive transcription only was allowed, but increased it after addition of all four nucleoside triphosphates owing to release of the sigma factor.

DNA, Bacterial↗
PubMed1992

Production of endothelin-1 in vascular endothelial cells is regulated by factors associated with vascular injury.

We studied the effects of factors associated with vascular injury on the production of endothelin-1 (ET-1) in cultured porcine aortic endothelial cells. ET-1 mRNA expression and peptide production in endothelial cells were increased by thrombin, transforming growth factor beta 1 (TGF beta 1), interleukin-1 (IL-1) and tumor necrotizing factor alpha (TNF alpha). The analysis of the enhancer/promoter region of the human ET-1 gene showed that the ET-1 gene transcription is regulated in a cell-specific manner and is activated by thrombin, TGF beta 1 and IL-1. These results suggest that the production of ET-1 in endothelial cells is regulated by factors associated with platelet aggregation, macrophage infiltration and the formation of atherosclerotic lesions.

Animals↗
PubMed1976

[Enzyme induction in Streptomyces hydrogenans, VI. Studies on the induction of 20beta-hydroxysteroid dehydrogenase, using immunological methods].

Antiserum against crystallized 20beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans was used for various immunodiffusion and immunoprecipitation tests to show an increase of the de novo synthesis of 20beta-hydroxysteroid dehydrogenase by Streptomyces hydrogenans after cultivation of the cells in the presence of 11beta,21-dihydroxy-4,17(20)-pregnadien-3-one. Half lives of mRNA for 20beta-hydroxysteroid dehydrogenase in induced cells and of total mRNA in non-induced cells were calculated to be 126 s and 66 s, respectively. In vivo, 20beta-hydroxysteroid dehydrogenase appears to consist of four equal subunits. The monomers, molecular weight of 27 300, show a high tendency to form dimers and tetramers in the absence of dissociating agents. The aggregation is completely reversible in the presence of increasing concentrations of sodium dodecylsulfate.

Animals↗
PubMed2006

SiRNA-mediated selective inhibition of mutant keratin mRNAs responsible for the skin disorder pachyonychia congenita.

RNA interference offers a novel approach for treating genetic disorders including the rare monogenic skin disorder pachyonychia congenita (PC). PC is caused by mutations in keratin 6a (K6a), K6b, K16, and K17 genes, including small deletions and single nucleotide changes. Transfection experiments of a fusion gene consisting of K6a and a yellow fluorescent reporter (YFP) resulted in normal keratin filament formation in transfected cells as assayed by fluorescence microscopy. Similar constructs containing a single nucleotide change (N171K) or a three-nucleotide deletion (N171del) showed keratin aggregate formation. Mutant-specific small inhibitory RNAs (siRNAs) effectively targeted these sites. These studies suggest that siRNAs can discriminate single nucleotide mutations and further suggest that "designer siRNAs" may allow effective treatment of a host of genetic disorders including PC.

Cell Line, Tumor↗
PubMed2006

Correction of a murine model of von Willebrand disease by gene transfer.

von Willebrand disease (VWD), the most common inherited bleeding disorder in the U.S. population, is caused by defects in the expression and processing of von Willebrand factor (VWF), a blood glycoprotein required for normal hemostasis that mediates the adhesion of platelets to sites of vascular damage by binding to specific platelet glycoproteins and to constituents of exposed connective tissue. To assess whether VWF deficiency can be corrected by gene transfer, a plasmid expressing the intact 8.4-kb murine VWF coding sequence, directed by the cyto-megalovirus immediate/early promoter/enhancer, was delivered through hydrodynamic tail vein injection into VWF knockout mice (VWF(-/-)) that exhibit defects in hemostasis, including highly prolonged bleeding time and spontaneous bleeding events, closely mimicking severe human VWD. VWF antigen levels in plasma from animals receiving VWF cDNA, but not control animals, revealed normalized levels of circulating VWF that persisted for at least 1 week after injection. Western blot analysis of plasma from animals receiving VWF cDNA, but not control animals, revealed high molecular-weight multimers with patterns similar to those observed in wild-type mice. Reverse transcription-polymerase chain reaction (RT-PCR) on RNA isolated from the livers of animals receiving VWF cDNA, but not control animals, demonstrated that VWF was expressed in the liver, and immunohistochemical analysis of the livers of treated VWF(-/-) mice revealed VWF-specific staining throughout the liver parenchyma but not in endothelial cells. Plasma from treated VWF(-/-) mice, but not control VWF(-/-) mice, supported the hypothesis that murine platelets aggregate in the presence of botrocetin. Although levels of circulating factor VIII in untreated VWF(-/-) mice were less than 10% those in wild-type mice, levels of factor VIII in VWF(-/-) animals treated with VWF cDNA, but not in control animals, were normalized to values in wild-type mice, indicating the restoration of factor VIII carrier function for VWF in treated mice that persisted for at least 1 week at higher doses of VWF cDNA. Most important, bleeding time was normalized by 48 hours after the delivery of VWF cDNA, but not by the control plasmid. These data suggest that with the use of gene transfer of VWF cDNA, VWF protein can be expressed, processed, and secreted in a physiologically active form; thus, it may be possible to correct VWD using gene transfer.

Animals↗
PubMed1998

Spermatid perinuclear ribonucleic acid-binding protein binds microtubules in vitro and associates with abnormal manchettes in vivo in mice.

Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The RNA target of SPNR in vivo is unknown, although we have previously suggested the possibility that SPNR is involved in the translational activation of the protamine 1 mRNA in elongated spermatids. To increase our understanding of SPNR's association with the manchette, we sought to determine SPNR's subcellular localization in several mouse mutants that show reduced fertility or sterility and that have structurally abnormal manchettes. We show here that despite the highly abnormal manchettes and microtubule aggregates formed in azh, hop-sterile, tw2, and tw8 mutants, SPNR remains associated with the manchettes. Localization of SPNR to the abnormal manchettes suggests that SPNR is tightly bound to the manchette. SPNR could bind manchette microtubules directly, or it could bind indirectly via an interaction with a microtubule-associated protein (MAP). We sought to determine whether SPNR binds microtubules in vitro, and if so, whether it requires a MAP. We show by Western analysis that the endogenous SPNR protein can be pelleted with murine testis microtubules in a taxol-dependent manner in vitro. A recombinant version of SPNR produced in bacteria can also be pelleted with testis microtubules, as well as microtubules polymerized from purified bovine brain tubulin, an association that is salt-sensitive. These results suggest that SPNR, in addition to its function as an RNA-binding protein, is also a bona fide MAP.

Animals↗
PubMed1994

Heterogeneous response to the growth factors [EGF, PDGF (bb), TGF-alpha, bFGF, IL-2] on glioma spheroid growth, migration and invasion.

The effects of 5 different growth factors [EGF, PDGF(bb), TGF-alpha, bFGF and IL-2] were studied on tumour spheroids obtained from 5 different human glioma cell lines (U-251MG, D-263MG, D-37MG, D-54MG, GaMG). The expression of EGF and PDGF receptors as well as the endogenous production of TGF-alpha and PDGF were studied by Northern blot analyses. After growth-factor-exposure, tumour spheroid volume growth, and directional cell migration from the spheroids were studied. In addition, tumour-cell invasion was studied in vitro, where foetal rat-brain aggregates were used as a target for the tumour cells. In all the assays a common stimulator for most of the cell lines was EGF. The other growth factors had a more heterogeneous stimulatory effect. Tumour-cell invasion, cell growth and cell migration are biological properties which are not necessarily related to each other. This may explain why the tumours often responded differently to the growth factors in the various assay systems. Two of the cell lines studied were non-invasive (U-251MG, D-263MG). It is shown that these were stimulated both in the directional migration assay and in the spheroid-volume-growth assay. However, their non-invasive behaviour was not influenced by the growth factors studied.

Animals↗
PubMed1993

Cellular interactions implicated in the mechanism of photoreceptor degeneration in transgenic mice expressing a mutant rhodopsin gene.

Photoreceptors of transgenic mice expressing a mutant rhodopsin gene (Pro347-->Ser) slowly degenerate. The mechanism of degeneration was studied by aggregation of embryos of normal and transgenic mice to form chimeras. In these chimeras, mosaicism was observed in the coat color, retinal pigment epithelium, and retina. In the retina, the genotype of adjacent patches of normal and transgenic photoreceptors was determined by in situ hybridization with a transgene-specific RNA probe. Photoreceptors in the chimeric retina degenerated uniformly, independent of the genotype and similar to the photoreceptors in transgenic mice. However, the chimeric retinas showed varying proportions of normal and transgenic cells. The chimeric retina with a nearly even proportion of normal and transgenic photoreceptors displayed uniform but slower degeneration than that observed in a transgenic mouse of the same age. Our results demonstrate non-autonomy of gene action for the mutated rhodopsin gene and imply that cellular interactions between photoreceptors in the retina probably play a role in degeneration.

Amino Acid Sequence↗
PubMed2003

P58IPK, a novel endoplasmic reticulum stress-inducible protein and potential negative regulator of eIF2alpha signaling.

The unfolded protein response, which is activated in response to the loss of endoplasmic reticulum (ER) Ca(2+) homeostasis and/or the accumulation of misfolded, unassembled, or aggregated proteins in the ER lumen, involves both transcriptional and translational regulation. In the current studies we sought to identify novel ER stress-induced genes by conducting microarray analysis on tunicamycin-treated cells. We identified P58(IPK), an inhibitor of the interferon-induced double-stranded RNA-activated protein kinase, as induced during ER stress. Additional studies suggested that p58(IPK) induction was mediated via ATF6 and that P58(IPK) played a role in down-regulating the activity of the pancreatic eIF2 kinase/eukaryotic initiation factor 2alpha (eIF2alpha)-like ER kinase/activation transcription factor (ATF) 4 pathway. Modulation of P58(IPK) levels altered the phosphorylation status of eIF2alpha, and thereby affected expression of its downstream targets, ATF4 and Gadd153. Overexpression of P58(IPK) inhibited eIF2alpha phosphorylation and reduced ATF4 and Gadd153 protein accumulation, whereas silencing of P58(IPK) expression enhanced pancreatic eIF2alpha-like ER kinase and eIF2alpha phosphorylation and increased ATF4 and Gadd153 accumulation. These findings implicate P58(IPK) as an important component of a negative feedback loop used by the cell to inhibit eIF2alpha signaling, and thus attenuate the unfolded protein response.

Activating Transcription Factor 6↗
PubMed1990

Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte function-associated antigen-1 expression and function.

Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.

Animals↗
PubMed2000

Dictyostelium myosin IK is involved in the maintenance of cortical tension and affects motility and phagocytosis.

Dictyostelium discoideum myosin Ik (MyoK) is a novel type of myosin distinguished by a remarkable architecture. MyoK is related to class I myosins but lacks a cargo-binding tail domain and carries an insertion in a surface loop suggested to modulate motor velocity. This insertion shows similarity to a secondary actin-binding site present in the tail of some class I myosins, and indeed a GST-loop construct binds actin. Probably as a consequence, binding of MyoK to actin was not only ATP- but also salt-dependent. Moreover, as both binding sites reside within its motor domain and carry potential sites of regulation, MyoK might represent a new form of actin crosslinker. MyoK was distributed in the cytoplasm with a significant enrichment in dynamic regions of the cortex. Absence of MyoK resulted in a drop of cortical tension whereas overexpression led to significantly increased tension. Absence and overexpression of MyoK dramatically affected the cortical actin cytoskeleton and resulted in reduced initial rates of phagocytosis. Cells lacking MyoK showed excessive ruffling, mostly in the form of large lamellipodia, accompanied by a thicker basal actin cortex. At early stages of development, aggregation of myoK null cells was slowed due to reduced motility. Altogether, the data indicate a distinctive role for MyoK in the maintenance and dynamics of the cell cortex.

Actins↗
PubMed2004

HIV-1 does not provoke alteration of cytokine gene expression in lymphoid tissue after acute infection ex vivo.

The cytokine response to invading microorganisms is critical for priming the adaptive immune response. During acute HIV infection, the response is disrupted, but the mechanism is poorly understood. We examined the cytokine response in human lymphoid tissue, acutely infected ex vivo with HIV. Lymphoid tissue was cultured either as blocks or as human lymphocyte aggregate cultures (HLAC) of tonsils and lymph nodes. This approach allowed us to examine the effects of HIV on cytokines using distinct culture techniques. In contrast to HLAC, mock-infected tissue blocks displayed a 50- to 100-fold up-regulation of mRNAs for IL-1beta, -6, and -8 in the first 6 days of culture. Parallel increases were also noted at the protein level in the supernatants. Although IL-1beta, -6, and -8 are known to synergistically enhance HIV replication, peak HIV replication (measured as p24 Ag) was similar in tissue blocks and HLAC. Surprisingly, vigorous HIV replication of CXCR4- and CCR5-tropic HIV strains did not result in characteristic mRNA profiles for IL-1beta, -2, -4, -6, -8, -10, -12, -15, IFN-gamma, TNF-alpha, TGF-beta, and beta-chemokines in tissue blocks or HLAC. The increased expression of IL-1beta, -6, and -8 in tissue blocks may approximate clinical situations with heightened immune activation; neutralization of these cytokines resulted in inhibition of HIV replication, suggesting that these cytokines may contribute to HIV replication in certain clinical settings. These results also indicate that different molecular mechanisms govern HIV replication in tissue blocks and HLAC. Prevention of effective cytokine responses may be an important mechanism that HIV uses during acute infection.

Adolescent↗
PubMed2005

Opposing effects of CXCR3 and CCR5 deficiency on CD8+ T cell-mediated inflammation in the central nervous system of virus-infected mice.

T cells play a key role in the control of viral infection in the CNS but may also contribute to immune-mediated cell damage. To study the redundancy of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8+ T cell-mediated inflammation in the brain, CXCR3/CCR5 double-deficient mice were generated and infected intracerebrally with noncytolytic lymphocytic choriomeningitis virus. Because these chemokine receptors are mostly expressed by overlapping subsets of activated CD8+ T cells, it was expected that absence of both receptors would synergistically impair effector T cell invasion and therefore protect mice against the otherwise fatal CD8+ T cell-mediated immune attack. Contrary to expectations, the accumulation of mononuclear cells in cerebrospinal fluid was only slightly delayed compared with mice with normal expression of both receptors. Even more surprising, CXCR3/CCR5 double-deficient mice were more susceptible to intracerebral infection than CXCR3-deficient mice. Analysis of effector T cell generation revealed an accelerated antiviral CD8+ T cell response in CXCR3/CCR5 double-deficient mice. Furthermore, while the accumulation of CD8+ T cells in the neural parenchyma was significantly delayed in both CXCR3- and CXCR3/CCR5-deficient mice, more CD8+ T cells were found in the parenchyma of double-deficient mice when these were analyzed around the time when the difference in clinical outcome becomes manifest. Taken together, these results indicate that while CXCR3 plays an important role in controlling CNS inflammation, other receptors but not CCR5 also contribute significantly. Additionally, our results suggest that CCR5 primarily functions as a negative regulator of the antiviral CD8+ T cell response.

Animals↗