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At least 559 records · Page 31Linked to original sources
PubMed1991

Platelet-derived growth factor and transforming growth factor-beta regulate plasminogen activator inhibitor-1 synthesis in vascular smooth muscle cells.

Bovine vascular smooth muscle cells (SMC) were examined for production of plasminogen activator inhibitor-1 (PAI-1) which may play a key role in regulating the fibrinolytic system. Growth-arrested SMC released active PAI (101 arbitrary units (AU)/10(6) cells/24 h) and a latent form of PAI (880 AU/10(6) cells/24 h) into the conditioned medium (CM). The levels of PAI were significant since 880 AU of PAI could inhibit approximately 1 microgram of tissue plasminogen activator. The extracellular matrix of SMC also contained PAI activity; however, the level was 17-fold less than that observed in the CM. SMC-PAI was a rapid inhibitor of tissue plasminogen activator (kass greater than 10(7) M-1 S-1) and was identified as a 45-kDa protein immunologically related to endothelial cell PAI-1. PAI-1 comprised 20 and 30%, respectively, of the newly synthesized protein detected in the CM and extracellular matrix of SMC. The SMC growth modulators, platelet-derived growth factor and transforming growth factor-beta, induced PAI-1 activity and protein synthesis by 2- and 3-fold, respectively, in a dose- and time-dependent manner. The increases in PAI-1 activity and protein synthesis were ascribed to elevated levels of PAI-1 mRNA as judged by Northern blot analysis of total RNA prepared from control and platelet-derived growth factor- and transforming growth factor-beta-treated cells. Increases in PAI-1 mRNA levels were evident 1 h after growth factor treatment and were maximal after 4 h. PAI-1 mRNA levels were unaffected by cycloheximide treatment. The results indicate that SMC synthesize and release PAI-1 which could regulate the normal fibrinolytic environment of the arterial wall. During atherosclerosis or after vascular injury increases in platelet-derived or locally produced mitogens may stimulate further PAI-1 synthesis and generate a prothrombotic state.

Animals↗
PubMed2006

The interaction between tylophorine B and TMV RNA.

Tylophorine B exhibits 60% inhibition against tobacco mosaic virus (TMV) at a concentration of 1.0 x 10(-6) g/ml. In our study, high affinity for TMV RNA and assembly origin of TMV RNA (oriRNA) was revealed, accompanied by the conformational change of RNA. Considering that TMV assembly begins with the specific recognition by the coat protein aggregate of oriRNA, and that tylophorine B has favorable interaction with oriRNA, we speculate that tylophorine B likely exerts its virus inhibition by binding to oriRNA and interfering with virus assembly initiation. This work may shed light on the possible molecular inhibition mechanism against TMV by tylophorine B, and provide clues in rational design of sequence-specific RNA binding antivirus drugs.

Alkaloids↗
PubMed1988

Signal transduction, chemotaxis, and cell aggregation in Dictyostelium discoideum cells without myosin heavy chain.

Dictyostelium discoideum cells have been generated that lack myosin heavy chain (MHC) due to antisense RNA inactivation of the endogenous mRNA or to insertional mutagenesis of the myosin gene. These cells retain chemotactic movement in gradients of the chemoattractant cAMP. Furthermore, cAMP does induce many biochemical and physiological responses in aggregative cells, including binding of cAMP to surface receptors, modification, and down-regulation of the receptor; activation of adenylate and guanylate cyclase, secretion of cAMP; and the association of actin to the Triton-insoluble cytoskeleton. Cells lacking MHC were found to have a requirement for bivalent cations in the medium for optimal chemotaxis and cell aggregation.

Actins↗
PubMed2005

Involvement of the mouse Prp19 gene in neuronal/astroglial cell fate decisions.

The molecular mechanisms involved in neuronal/astroglial cell fate decisions during the development of the mammalian central nervous system are poorly understood. Here, we report that PRP19beta, a splice variant of mouse PRP19alpha corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The beta-variant possesses an additional 19 amino acid residues in-frame in the N-terminal region of the alpha-variant. The forced expression of the alpha-variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the beta-variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the beta-variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the beta-variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the alpha-variant gene plays a pivotal role in neural differentiation and that the beta-variant participates in neuronal/astroglial cell fate decisions.

Alternative Splicing↗
PubMed1999

Efficient formation of influenza virus-like particles: dependence on the expression levels of viral proteins.

It has previously been demonstrated in this laboratory that an influenza virus-like chloramphenicol acetyltransferase (CAT) RNA could be expressed in COS-1 cells that synthesized all ten influenza A virus-encoded proteins from recombinant plasmids. It was also shown that supernatant fluids harvested from these cultures contained virus-like particles (VLPs) that could deliver an enclosed CAT RNA to MDCK cells. Here, it is shown that the levels of expression of the reporter gene in the COS-1 and/or MDCK cells can be altered drastically by modifying the concentrations of the recombinant plasmids transfected in the COS-1 cells. Thus, it was observed that overexpression of NS2 reduced CAT expression in COS-1 cells, whereas overexpression of M2 and NS1 proteins dramatically decreased transmission of the CAT RNA to the MDCK cultures. These results are discussed with reference to the roles of these proteins during virus replication. From these experiments, a ratio of transfected plasmids was found that increased the efficiency of the previously described system by 50-100-fold. Under these optimized conditions, it was demonstrated that VLPs can be formed in the absence of neuraminidase expression and that these VLPs remained aggregated to each other and to cell membranes. Moreover, it is shown that CAT RNA transmission was dependent on specific interactions of the ribonucleoprotein complex with other viral structural polypeptides. These data demonstrate the usefulness of this encapsidation-packaging system for the study of different aspects of the influenza virus life-cycle.

Animals↗
PubMed1997

Tumour necrosis factor-alpha expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A.

1. Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-alpha is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-alpha expression influences recruitment of specific inflammatory cell types. 2. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24-72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3. Messenger RNA encoding TNF-alpha was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-alpha protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. 4. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-alpha mRNA expression by BAL cells. 5. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-alpha, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-alpha expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.

Animals↗
PubMed1992

Procedures for the efficient purification of pea seed-borne mosaic virus and its genomic RNA.

An efficient protocol for the purification of pea seed-borne mosaic potyvirus (PSbMV) particles was developed. This led to the purification of 10 PSbMV isolates by a single procedure. Virus aggregation during purification did not occur and consequently, high virus yield was consistently obtained. The virus thus purified was suitable for preparing viral genomic RNA, although conventional methods for RNA extraction resulted in RNA degradation. An alternative method was adopted which yielded reproducibly full length and infectious RNA. This was applied to three isolates of PSbMV and the RNA used to direct complementary DNA synthesis which in turn yielded nearly full length cDNA products.

Fabaceae↗
PubMed1986

Structure of tobacco mosaic virus at 3.6 A resolution: implications for assembly.

X-ray fiber diffraction analysis of tobacco mosaic virus (TMV) has led to the building of a molecular model of the intact virus, based on a map at 3.6 A resolution derived from five separated Bessel orders. This has been made possible by advances in the solution of the fiber diffraction phase problem. It is now possible to understand much of the chemical basis of TMV assembly, particularly in terms of intersubunit electrostatic interactions and RNA binding. Consideration of the molecular structure in conjunction with physical chemical studies by several groups of investigators suggests that the nucleating aggregate for initiation of TMV assembly is a short (about two turns) helix of protein subunits, probably inhibited from further polymerization in the absence of RNA by the disordering of peptide loop near the inner surface of the virus.

RNA, Viral↗
PubMed1993

In borna disease virus infected rabbit neurons 100 nm particle structures accumulate at areas of Joest-Degen inclusion bodies.

Borna disease virus infected rabbits were chosen to search for electronmicroscopic structures. Intensively investigated hippocampal neurons showed intranuclear inclusions; 100 nm particle-like structures surrounded by 20 nm granular forms were prominent. In connection with elsewhere reported in situ hybridization studies of virus-specific RNA to areas of the Joest-Degen inclusions we suggest that these particle structures may represent Borna virus. Jost-Degen (8) found intranuclear inclusion bodies in neurons to be pathognomonic for Borna disease (BD) in the horse. Half a century later these structures were suggested to represent BD virus (BDV)-specific antigen aggregates (15). A century later we characterized the virus to contain a single and negative stranded RNA of 8.5 kb, which transcribes in the nucleus (5) and could show that virus complementary RNA seems to hybridize spot-like to nuclear areas, probably representing the Jost-Degen inclusions (7). Electron microscopic (EM) findings on structures in BDV infected brain cells and about particle-like structures obtained from infected tissue culture cells have been reported by different groups (1, 2, 3, 4, 5, 6, 9, 11, 12, 13). The demonstration of crystalline aggregates and filament bundles in the cytoplasma and karyospheridia (nuclear bodies) were prominent. Such structures were seen in infected rabbits, hamsters, rats mice and naturally infected horses. The phenotypic description of filamentous structures, crystalline aggregates in the cytoplasma and large karyospheridia were prominent. Based on our experience with BDV infections in rabbits we selected this species for ultrastructural studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

RNA replication by a respiratory syncytial virus RNA analog does not obey the rule of six and retains a nonviral trinucleotide extension at the leader end.

Genome analogs ("minigenomes") of Sendai and measles viruses replicate efficiently only if their nucleotide length is an even multiple of six, a requirement called the rule of six (P. Calain and L. Roux, J. Virol. 67:4822-4830, 1993; M. S. Sidhu, J. Chan, K. Kaelin, P. Spielhofer, F. Radecke, H. Schneider, M. Masurekar, P. C. Dowling, M. A. Billeter, and S. A. Udem, Virology 208:800-807, 1995). The existence of a comparable requirement was tested for respiratory syncytial virus (RSV), which also is a member of family Paramyxoviridae and whose natural genome length also is a multiple of six. An internally truncated analog of RSV positive-sense replicative intermediate RNA (antigenome) bearing the chloramphenicol acetyltransferase gene as a reporter was synthesized from cDNA in vitro. This RNA was transfected into cells which were infected with RSV as a helper. Miniantigenomic RNA was indistinguishable from previously studied negative-sense minigenome RNA in its ability to participate in transcription, RNA replication, and incorporation into transmissible particles. Sixteen miniantigenomes which were of slightly different lengths and which in aggregate represented multiples of a wide range of integers including 1 to 15 were constructed. During transfection and two serial passages, the various miniantigenomes were essentially indistinguishable with regard to the efficiency of transcription, RNA replication, and packaging into transmissible particles. Progeny minigenomes of six different mutants were recovered postpassage, copied into cDNA, cloned, and sequenced completely. The length of each of these RNAs was found to have remained unchanged during replication and passage. Thus, RSV transcription and replication appear to lack the requirement that the template length be an even multiple of an integer such as six, which for Sendai and measles viruses is obligatory for nucleocapsid function. Each of the in vitro-synthesized miniantigenomes used in transfection contained a nonviral extension of three nucleotides, GGG, on the 5' (leader) end contributed by the T7 promoter. The termini of the recovered minigenomes were examined for five mutants by RNA circularization followed by cDNA synthesis, amplification, cloning, and sequencing. Unexpectedly, each recovered minigenome contained the complement of this nonviral extension on the 3' (leader) end, showing that it had been faithfully copied and maintained during RNA replication and passage. The nonviral trinucleotide did not appear to affect the activity of the template.

DNA, Complementary↗
PubMed1974

Properties of intracytoplasmic A particles isolated from oncornavirus-producing human cells.

A particles with the diameter of 70 to 80 nm were isolated from the cytoplasm of HEp-2, HeLa, and AO cells producing oncornavirus of Mason-Pfizer-like type. Most of the A particles banded at 1.23 to 1.24 g/ml, whereas 3 to 10% banded at 1.29 g/ml in equilibrium sucrose gradients. They banded at 1.30 g/ml in CsCl gradients suggesting that they contained 8% RNA. Individual A particles sedimented at 200 to 250S in velocity sucrose gradients, but their significant part was found aggregated and sedimented at more than 300S. They were resistant to RNase digestion. A particles possessed polymerase activity which was preferentially activated by Mn(2+) rather than by Mg(2+), the RNA template being 60S RNA. Cross-hybridization with two DNA products and immunoassay showed that A particles and Mason-Pfizer-like oncornavirus produced by the same cells contained neither homological RNA sequences nor common antigens, suggesting that A particles are not intracellular precursors of Mason-Pfizer-like oncornavirus but represent an independent oncornavirus. Hybridization of A particle RNA with excess of cellular DNA revealed about 20 proviral copies per HEp-2 cell genome and no proviral copies in human embryo and placenta cell genomes.

Antigens, Viral↗
PubMed1992

DNA packaging induced by micellar aggregates: a novel in vitro DNA condensation system.

Evidence for a conceptually novel DNA packaging process is presented. X-ray scattering, electron microscopy, and circular dichroism measurements indicate that in the presence of positively charged micellar aggregates and flexible anionic polymers, such as negatively charged polypeptides or single-stranded RNA species, a complex is formed in which DNA molecules are partially embedded within a micellar scaffold and partially condensed into highly packed chiral structures. Based on studies of micelle-DNA and micelle-flexible anionic polymer systems, as well as on the known effects of a high charge density upon the micellar organization, a DNA packaging model is proposed. According to this model, the DNA induces the elongation of the micelles into rodlike aggregates, forming a closely packed matrix in which the DNA molecules are immobilized. In contrast, the flexible anionic polymers stabilize clusters of spherical micelles which are proposed to effect a capping of the rodlike micelles, thus arresting their elongation and creating surfactant-free segments of the DNA that are able to converge and collapse. Thus, unlike other in vitro DNA packaging systems, in which condensation follows encounters between charge-neutralized DNA molecules, a prepackaging phase where the DNA is immobilized within a matrix is proposed in this case. Cellular and nuclear membranes have been implicated in DNA packaging processes in vivo, and negatively charged polyelectrolytes were shown to be involved in the processes. These observations, combined with the basic tenets of the DNA condensation system described here, allow for the progression to the study of more elaborate model systems and thus might lead to insights into the nature and roles of the intricate in vivo DNA-membrane complexes.

Circular Dichroism↗
PubMed1992

Direct visualization of the structure of the "20 S" aggregate of coat protein of tobacco mosaic virus. The "disk" is the major structure at pH 7.0 and the Proto-helix at lower pH.

We have employed the rapid-freeze technique to prepare specimens for electron microscopy of a coat protein solution of tobacco mosaic virus at equilibrium at pH 7.0 and 6.8, ionic strength 0.1 M and 20 degrees C. The former are the conditions for the most rapid assembly of the virus from its isolated protein and RNA. At both pH values, the equilibrium mixture contains approximately 80% of a "20 S" aggregate and 20% of a "4 S" aggregate (the so-called A-protein). The specimens were prepared either totally unstained or positively stained with methyl mercury nitrate, which binds to an amino acid residue (Cys27) internally located within the subunit, which we show not to affect the virus assembly. The images in the electron microscope are compatible only with the major structure for the "20 S" aggregate at pH 7.0 containing two rings of subunits and these aggregates display the same binding contacts as those seen between the aggregate that forms the asymmetric unit in the crystal, which has been shown by X-ray crystallography to be a disk containing two rings, each of 17 subunits, oriented in the same direction. In contrast, the images from specimens prepared at pH 6.8 show the major structure to be a proto-helix at this slightly lower pH, demonstrating that the technique of cryo-electron microscopy is capable of distinguishing between these aggregates of tobacco mosaic virus coat protein. The main structure in solution at pH 7.0 must therefore be very similar to that in the crystal, although slight differences could occur and there are probably other, minor, components in a mixture of species sedimenting around 20 S under these conditions. The equilibrium between aggregates is extremely sensitive to conditions, with a drop of 0.2 pH unit tipping the disk to proto-helix ratio from approximately 10:1 at pH 7.0 to 1:10 at pH 6.8. This direct determination of the structure of the "20 S" aggregate in solution, under conditions for virus assembly, contradicts some recent speculation that it must be helical, and establishes that, at pH 7.0, it is in fact predominantly a two-layer disk as it had been modelled before.

Capsid↗
PubMed1990

Substrate sequence effects on "hammerhead" RNA catalytic efficiency.

The "hammerhead" RNA self-cleaving domain can be assembled from two RNA molecules: a large (approximately 34 nucleotide) ribozyme RNA containing most of the catalytically essential nucleotides and a small (approximately 13 nucleotide) substrate RNA containing the cleavage site. Four such hammerheads that contained identical catalytic core sequences but differed in the base composition of the helices that are involved in substrate binding had been reported to vary in cleavage rates by more than 70-fold under similar reaction conditions. Steady-state kinetic analyses reveal that kcat values are nearly the same for these hammerheads but Km values vary nearly 60-fold. The substrates for reactions having high Km values form aggregates that are virtually nonreactive. These observations demonstrate that the secondary structure of substrate RNA can be a major determinant of hammerhead catalytic efficiency.

Base Composition↗
PubMed1979

L-Tryptophan action on hepatic RNA synthesis and enzyme induction.

L-Tryptophan increases the activity of hepatic amino acid metabolizing enzymes, affects gluconeogenesis and displays a modulatory effect on several enzymes connected with RNA synthesis. The underlying mechanism differ in individual cases and result in both an increase of enzyme synthesis de novo and a decrease of enzyme degradation. Tryptophan displays a unique effect causing aggregation of hepatic polyribosomes connected with enhanced protein synthesis and preceded by a higher transport of poly (A) messenger RNA from the nucleus to the cytoplasm. The variety of rather specific effects mediated by tryptophan brings to mind hormonal action and the existence of specific tryptophan receptors is predicted.

Amino Acids↗
PubMed1998

Histological changes of concurrent hepatitis C virus infection in asymptomatic hepatitis B virus patients.

In chimpanzees and in vitro cell culture studies, hepatitis C infection has been shown to suppress hepatitis B virus expression. In addition, hepatitis C infection can cause much more severe liver disease in patients chronically infected with hepatitis B virus. The aims of the present study were to determine the prevalence of hepatitis C infection in asymptomatic chronic hepatitis B Hong Kong Chinese patients and the histological changes and hepatic expression of hepatitis B virus and hepatitis C virus. Five hundred and seventy-one Hong Kong Chinese asymptomatic chronic hepatitis B patients were studied. Only four (0.7%) were hepatitis C virus antibody positive; they were also all positive for hepatitis C viral RNA in serum by reverse transcription-polymerase chain reaction (RT-PCR). Portal lymphoid aggregates and bile duct damage was noted in the liver sections of three of the four patients. Hepatic expression of hepatitis B surface antigen was detected in three patients; none had detectable hepatitis B core antigen. By branched DNA assay, serum hepatitis B DNA could not be detected in any of the four patients, but three had hepatitis C RNA. By in situ RT-PCR, hepatitis C RNA was detected in the cytoplasm of three of the four patients. These findings suggest that hepatitis C coinfection in asymptomatic chronic hepatitis B patients is uncommon in Hong Kong Chinese and active hepatitis B viral replication is absent in these patients.

Adolescent↗
PubMed2003

Clustering of nucleosides in the presence of alkali metals: Biologically relevant quartets of guanosine, deoxyguanosine and uridine observed by ESI-MS/MS.

Electrospray ionization (ESI) mass spectra of nucleosides, recorded in the presence of alkali metals, display alkali metal ion-bound quartets and other clusters that may have implications for understanding non-covalent interactions in DNA and RNA. The tetramers of guanosine and deoxyguanosine and also their metaclusters (clusters of clusters), cationized by alkali metals, were observed as unusually abundant magic number clusters. The observation of these species in the gas phase parallels previous condensed-phase studies, which show that guanine derivatives can form quartets and metaclusters of quartets in solution in the presence of metal cations. This parallel behavior and also internal evidence suggest that bonding in the guanosine tetramers involves the bases rather than the sugar units. The nucleobases thymine and uracil are known to form magic number pentameric adducts with K+, Cs+ and NH4+ in the gas phase. In sharp contrast, we now show that the nucleosides uridine and deoxythymidine do not form the pentameric clusters characteristic of the corresponding bases. More subtle effects of the sugars are evident in the fact that adenosine and cytidine form numerous higher order clusters with alkali metals, whereas deoxyadenosine and deoxycytidine show no clustering. It is suggested that hydrogen bonding between the bases in the tetramers of dG and rG are the dominant interactions in the clusters, hence changing the ribose group to deoxyribose (and vice versa) generally has little effect. However, the additional hydroxyl group of RNA nucleosides enhances the non-selective formation of higher-order aggregates for adenosine and cytidine and results in the lack of highly stable magic number clusters. Some clusters are the result of aggregation in the course of ionization (ESI) whereas others appear to be intrinsic to the solution being examined.

Deoxyguanosine↗