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At least 685 records · Page 38Linked to original sources
PubMed1987

The two segments of the infectious bursal disease virus genome are circularized by a 90,000-Da protein.

The genome of infectious bursal disease virus (IBDV) consists of two segments of double-stranded (ds)RNA with molecular weights of 2.2 X 10(6) and 1.9 X 10(6) Da, respectively. After treatment of IBDV particles with proteinase K in the presence of sodium dodecyl sulfate (SDS), linear dsRNA molecules are released from the virus particles. However, after heating of virus particles at 100 degrees for 3 min in 1.5% SDS, without the protease, dsRNA-protein complexes can be seen under the electron microscope: Knob-like proteinaceous structures are linked to the ends of the dsRNA molecules of either size class which are circularized to form individual rings. A 90,000-Da IBDV structural polypeptide, the only protein encoded by the smaller genome segment, has been demonstrated to remain firmly linked to the IBDV genome under these conditions. No functional data exist about this circularizing protein; it is a probable candidate for an RNA-dependent RNA polymerase or an assembly protein for the two dsRNA segments. At high particle concentrations, or when the preparations are allowed to stand for several hours before spreading, these complexes tend to aggregate to form flower-like structures.

Infectious bursal disease virus↗
PubMed1977

Rates of aggregation, loss of anchorage dependence, and tumorigenicity of cultured cells.

The net rate of spontaneous aggregation of cells suspended with EDTA was measured for various cell types including spontaneous transformants and cells transformed with DNA and RNA viruses. The anchorage dependence as determined by growth in methyl cellulose and the tumorigenicity in vivo were also determined. All cells that had lost their anchorage dependency and were tumorigenic showed a high net rate of spontaneous adhesion. A31 was the only nontransformed cell line to have a high net rate of adhesion. The net rate of spontaneous aggregation of cells is a quick and reliable index of tumorigenicity and offers a new approach to understanding the mechanisms of cell surface changes associated with transformation.

Cell Adhesion↗
PubMed1996

Antisense RNA inactivation of gene expression of a cell-cell adhesion protein (gp64) in the cellular slime mold Polysphondylium pallidum.

The gp64 protein of Polysphondylium pallidum has been shown to mediate EDTA-stable cell-cell adhesion. To explore the functional role of gp64, we made an antisense RNA expression construct designed to prevent the gene expression of gp64; the construct was introduced into P. pallidum cells and the transformants were characterised. The antisense RNA-expressing clone L3mc2 which had just been harvested at the growth phase tended to re-form in aggregates smaller in size than did the parental cells in either the presence or absence of 10 mM EDTA. In contrast, 6.5-hour starved L3mc2 cells remained considerably dissociated from each other after 5 minutes gyrating, although aggregation gradually increased by 50% during a further 55 minutes gyrating in the presence of 10 mM EDTA. Correspondingly, L3mc2 lacked specifically the cell-cell adhesion protein, gp64. We therefore conclude that the gp64 protein is involved in forming the EDTA-resistant cell-cell contact. In spite of the absence of gp64, L3mc2 exhibited normal developmental processes, a fact which demonstrates that another cell-cell adhesion system exists in the development of Polysphondylium. This is the first report in which an antisense RNA technique was successfully applied to Polysphondylium.

Cell Adhesion Molecules↗
PubMed1990

Polarity of binding of monoclonal antibodies to tobacco mosaic virus rods and stacked disks.

Monoclonal antibodies to tobacco mosaic virus that bind only to one end of the viral rods have been shown to recognize the surface of the protein subunit designated as the bottom, which contains the right radial and left radial alpha-helices. The specificity of the antibody binding was established by immunoelectron microscopy of complexes in which the 5' end of the RNA had been exposed at the bottom of the helical virus particle. These antibodies have been shown to bind to both ends of the stacked disk aggregate of TMV protein, which is therefore bipolar. The observations on the bipolarity of this structure are inconsistent with the presumption that stacked disks are formed by aggregation of polar two-layer disks.

Antibodies, Monoclonal↗
PubMed1995

Mitochondrial encephalomyopathy with A to G transition of mitochondrial transfer RNA(Leu(UUR)) 3,243 presenting hypertrophic cardiomyopathy.

In a 24-year-old woman with mitochondrial encephalomyopathy presenting hypertrophic cardiomyopathy, microscopical examination of myocardial biopsy specimen disclosed severe vacuolar degeneration of myocardium and aggregates of enlarged mitochondria with proliferated cristae. Limb muscle biopsy specimen showed "ragged-red fibers" light microscopically and enlarged abnormal mitochondria with markedly increased cristae ultrastructurally. Mitochondrial DNA analysis by polymerase chain reaction (PCR) revealed an A-to-G transition in the mitochondrial transfer RNA(Leu)(UUR) gene at nucleotide position 3,243 which is reported to be associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). However, the clinical features of this case, presenting mainly cardiac abnormalities, were not consistent with the typical MELAS.

Adult↗
PubMed1999

Self-assembly of tobacco mosaic virus: the role of an intermediate aggregate in generating both specificity and speed.

The tobacco mosaic virus (TMV) particle was the first macromolecular structure to be shown to self-assemble in vitro, allowing detailed studies of the mechanism. Nucleation of TMV self-assembly is by the binding of a specific stem-loop of the single-stranded viral RNA into the central hole of a two-ring sub-assembly of the coat protein, known as the 'disk'. Binding of the loop onto its specific binding site, between the two rings of the disk, leads to melting of the stem so more RNA is available to bind. The interaction of the RNA with the protein subunits in the disk cause this to dislocate into a proto-helix, rearranging the protein subunits in such a way that the axial gap between the rings at inner radii closes, entrapping the RNA. Assembly starts at an internal site on TMV RNA, about 1 kb from its 3'-terminus, and the elongation in the two directions is different. Elongation of the nucleated rods towards the 5'-terminus occurs on a 'travelling loop' of the RNA and, predominantly, still uses the disk sub-assembly of protein subunits, consequently incorporating approximately 100 further nucleotides as each disk is added, while elongation towards the 3'-terminus uses smaller protein aggregates and does not show this 'quantized' incorporation.

Base Sequence↗
PubMed2026

Symmetric and near-symmetric cyanine probes for G-quadruplexes: molecular recognition, signal transduction, and biological applications.

G-quadruplexes (G4s) are dynamic noncanonical nucleic-acid structures involved in genome maintenance, transcription, RNA metabolism, and mitochondrial function, and are implicated in disease-associated processes. Symmetric and near-symmetric cyanines are versatile platforms for G4 recognition because their polymethine length, terminal heterocycles, charge distribution, conformational freedom, and supramolecular organization can be systematically tuned within related scaffolds. This review discusses how these structural features control G4 recognition and optical signal transduction through terminal G-tetrad stacking, loop and groove contacts, restriction of molecular motion, and aggregate reorganization. We first summarize in vitro recognition, structural discrimination, and G4-mediated sensing, and then discuss DNA and RNA G4 imaging, G4-associated biological processes, and emerging in vivo applications. Particular attention is given to several distinctions that are essential for interpreting probe performance: binding affinity versus fluorescence activation, topology preference versus DNA/RNA selectivity, organelle accumulation versus molecular targeting, and imaging contrast versus biological validation. Overall, molecular symmetry is considered a tunable design variable rather than a direct predictor of performance. Future studies should emphasize matched structural series, reversible and minimally perturbing probes, optical readouts that are less dependent on probe concentration, clear separation of DNA and RNA contributions, and standardized validation across solution, cellular, and whole-organism studies.

Journal Article↗
PubMed2000

Smoking decreases alveolar macrophage function during anesthesia and surgery.

BACKGROUND: Smoking changes numerous alveolar macrophage functions and is one of the most important risk factors for postoperative pulmonary complications. The current study tested the hypothesis that smoking impairs antimicrobial and proinflammatory responses in alveolar macrophages during anesthesia and surgery. METHOD: The authors studied 30 smoking and 30 nonsmoking patients during propofol-fentanyl general anesthesia. Alveolar immune cells were harvested by bronchoalveolar lavage immediately and 2, 4, and 6 h after induction of anesthesia and at the end of surgery. The types of alveolar immune cell and macrophage aggregation were determined. The authors measured opsonized and unopsonized phagocytosis. Microbicidal activity was determined as the ability of the macrophages to kill Listeriamonocytogenes directly. Finally, RNA was extracted from harvested cells and cDNA was synthesized by reverse transcription. The expression of interleukin 1beta, 6, and 8, interferon gamma, and tumor necrosis factor alpha were measured by semiquantitative polymerase chain reaction using beta-actin as an internal standard. RESULTS: The fraction of aggregated macrophages increased significantly over time in both groups, whereas phagocytosis of opsonized and nonopsonized particles and microbicidal activity of alveolar macrophages decreased significantly. The changes, though, were nearly twice as great as in patients who smoked. Gene expression of all proinflammatory cytokines in alveolar immune cells except interleukin 6 increased 2- to 20-fold over time in both groups. The expression of interleukin 1beta, interferon gamma, and tumor necrosis factor alpha, however, increased only half as much in smokers as in nonsmokers. CONCLUSION: Smoking was associated with macrophage aggregation but markedly reduced phagocytic and microbicidal activity-possibly because expression of proinflammatory cytokines was reduced in these patients. Our data thus suggest that smokers may have a limited ability to mount an effective pulmonary immune defense after anesthesia and surgery.

Anesthesia, General↗
PubMed2005

Suppression of MUC1 synthesis downregulates expression of the epidermal growth factor receptor.

The transmembrane mucin, MUC1, is overexpressed on many human carcinoma cells, increasing their metastatic potential through decreased cell-cell and cell-matrix adhesion. These cellular changes are mediated both through the altered physical properties of the mucin itself and through the role of the MUC1 cytoplasmic domain as a signaling molecule. The epidermal growth factor receptor (EGFR) is also overexpressed in many cancers and both it and MUC1 constitute important therapeutic targets. In the present study, expression of MUC1 was downregulated by treatment of KB carcinoma cells with a MUC1 small interfering RNA resulting in an inhibition of cell proliferation and colony formation and an increase in cell-cell aggregation. Surprisingly, suppression of MUC1 also inhibited expression of EGFR at both the mRNA and protein levels whereas the reciprocal effect was not observed. These results demonstrate a role for MUC1 in the regulation of EGFR expression and suggest that MUC1 gene silencing may represent a novel therapeutic approach in the treatment of a variety of human cancers.

Antigens, Neoplasm↗
PubMed1998

A comparative differential scanning calorimetric study of tobacco mosaic virus and of its coat protein ts mutant.

The differential scanning calorimetry (DSC) 'melting curves' for virions and coat proteins (CP) of wild-type tobacco mosaic virus (strain U1) and for its CP ts mutant ts21-66 were measured. Strain U1 and ts21-66 mutant (two amino acid substitutions in CP: 121 --> T and D66 --> G) differ in the type of symptoms they induce on some host plants. It was observed that CP subunits of both U1 and ts21-66 at pH 8.0, in the form of small (3-4S) aggregates, possess much lower thermal stability than in the virions. Assembly into the virus particles resulted in a DSC melting temperature increase from 41 to 72 degrees C for U1 and from 38 to 72 degrees C for ts21-66 CP. In the RNA-free helical virus-like protein assemblies U1 and ts21-66 CP subunits had a thermal stability intermediate between those in 3-4S aggregates and in the virions. ts21-66 helical protein displayed a somewhat lower thermal stability than U1.

Amino Acid Substitution↗
PubMed2000

Hepatitis G virus coinfection influences the liver histology of patients with chronic hepatitis C.

AIMS/BACKGROUND: The present investigation compared the histological features of the liver of chronic hepatitis C patients who are or are not coinfected with hepatitis G virus (HGV) to determine the histological and clinical characteristics of HGV infection. SUBJECTS AND METHODS: This study included 194 patients with chronic hepatitis C who visited our institution between 1993 and 1995. Detection of serum HGV RNA was performed by nested reverse transcription-polymerase chain reaction. Scores were assigned to indicate the severity of each of the following features on the liver biopsy of a patient: inflammatory cell infiltration in the periportal, parenchymal, and portal area; fibrous stage; lymphoid aggregates in the portal area; portal sclerotic change; perivenular fibrosis; pericellular fibrosis; bile duct damage; bridging necrosis; and irregular regeneration of hepatocytes (IR). RESULTS: HGV RNA was detected in the sera of 18 (9.3%) of the 194 patients. The histological features of the HGV RNA-positive patients show significantly more severe bile duct damage, perivenular fibrosis, pericellular fibrosis and IR than the liver of the HGV RNA-negative patients. The biochemical results in the two groups did not significantly differ. CONCLUSION: Our data suggest that chronic HGV coinfection worsens the histological features of liver disease.

Bile Ducts↗
PubMed1997

Stoichiometries of AMPA receptor subunit mRNAs in rat brain fall into discrete categories.

In situ hybridization was used to estimate the relative concentrations of mRNAs encoding different subunits (GluR1-4) of alpha-amino 3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors in rat brain and to test the hypothesis that within-region expression profiles reflect a limited number of recurring patterns. Fractional subunit mRNA concentrations were calculated for 33 brain regions, and cluster analysis methods were applied to test for statistically meaningful groupings in the data. Four relatively homogeneous classes were identified and designated as AMPA receptor (AR) categories, numbered according to dominant subunit mRNAs. The AR-1 class (47% GluR1 mRNA) was expressed by structures near the mesodiencephalic border, including basal ganglia-related areas. The AR-2 class (57% GluR2 mRNA) was expressed in cortex and tectum. The AR-1,2 class (31% GluR1, 45% GluR2) was found in the largest number of regions, including such dissimilar cell fields as hippocampus and substantia nigra pars compacta. The AR-2,3 grouping (33% GluR2, 31% GluR3) was associated with the sensory relay and reticular thalamic nuclei. It is suggested that AR-1,2 and AR-2, the most closely related categories in clustering space, are largely telencephalic receptors with the former predominant in the subcortex and the latter in the cortex. The AR-2,3 class is associated with ascending sensory stations, whereas AR-1 appears to include several smaller categories expressed by specialized systems. If the balance of subunit mRNAs is reflected at the protein level, then the present data suggest that forebrain AMPA-type glutamate receptors can be classified into a limited number of recurring types.

Animals↗
PubMed1994

VASE exon expression alters NCAM-mediated cell-cell interactions.

The neural cell adhesion molecule (NCAM) is found on cells as several related polypeptides formed by alternative splicing of the single NCAM gene. The alternatively spliced 30-bp VASE exon in the fourth immunoglobulin-like domain is the structural variation nearest those portions of the polypeptide proposed to mediate cell-cell adhesion. To test the ability of distinct forms of the NCAM molecules to mediate cell adhesion, L cells were transfected with expression vectors encoding rat 140 kD NCAM +/- the VASE exon. L cell lines which expressed these polypeptides were isolated and tested for self-aggregation in a low shear, rapid aggregation assay. Increased cellular aggregation of the transfectants was observed to be a function of the NCAM molecule expressed. These transfected cells showed segregation in a long term co-aggregation assay: cells expressing NCAM--VASE formed aggregates which tended to exclude cells expressing NCAM+VASE and vice versa. These results provide direct evidence that this small difference in NCAM structure is sufficient to allow segregation of cells.

Animals↗
PubMed1996

Up-regulation of the intracellular Ca2+ signaling and mRNA expression of platelet-activating factor receptor by estradiol in human uterine endometrial cells.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), a potent chemical mediator in inflammation, plays a role in reproduction. Using primary culture of human uterine endometrial cells, we investigated the effect of sex steroid hormones on the PAF-induced signal and its receptor mRNA expression. After a 24 hr treatment with estradiol, PAF increased the intracellular calcium ion ([Ca2+]i) in the glandular cells, but not in the stromal cells. This response was not observed in the non-treated cells, and was blocked by a PAF antagonist, WEB2086. Two types of mRNA (transcript 1 and transcript 2) occurred for PAF receptor by alternative splicing, which are under control of two distinct promoters. Using RT-PCR analysis, it was shown that both transcripts existed in endometrial cells and that estradiol alone or a combination of estradiol and progesterone induced the accumulation of transcript 2, the promoter of which responded to estrogen in our previous studies. The regulation of PAF receptor by sex steroid hormones in human uterine endometrial cells suggests that PAF is involved in the physiological process of reproduction.

Alternative Splicing↗
PubMed1980

De novo construction of cell-to-cell channels.

Nexus (gap junctions), which are considered to contain cell-to-cell channels, are newly formed in uterine smooth muscle during parturition or in response to estrogen treatment of virginal animals. A mRNA preparation was isolated from estrogen-dominated rat myometria and was encapsulated into liposomes. Subsequently the liposomes were fused with cultured cells of a mouse cell line CL-1D. It is established that these tumor cells normally are neither electrically coupled nor do they contain nexus. The cells, however, become electrically coupled a few hours after being loaded with the mRNA preparation. This de novo expression of cell coupling persisted for a litte more than 24 hr after a single loading procedure. Freeze-fracture electron microscopy revealed small nexus-like particle aggregates at the time coupling was present. In control experiments the cells remained noncoupling when the RNA preparation was pretreated with ribonuclease, when cycloheximide was applied to the cells, or when liposomes filled with buffer solution only were used. These data suggest that the de novo expression of cell-to-cell coupling is accomplished by mRNA-induced protein biosynthesis resulting in the formation of cell-to-cell channels.

Animals↗
PubMed2006

NMDA receptors mediate calcium-dependent, bidirectional changes in dendritic PICK1 clustering.

AMPA receptor (AMPAR) trafficking at CNS synapses is regulated by several receptor-binding proteins. One model of AMPAR endocytosis entails the cotargeting of the GluR2-interacting protein PICK1 and activated PKC to synapses. We demonstrate that NMDA receptor (NMDAR) activation mediates bidirectional changes in surface AMPARs through two additional forms of PICK1 redistribution. In neurons, NMDAR activation, which induces AMPAR endocytosis, increases endosomal PICK1 clustering. In contrast, stronger NMDAR activation rapidly reduces PICK1 clustering accompanied by decreases in PICK1/GluR2 association and increases in surface AMPAR levels. PICK1-siRNA similarly increases surface AMPARs and occludes the NMDAR-mediated effect, demonstrating the role of PICK1 in this process. Bidirectional NMDAR-mediated changes in PICK1 localization are determined by the magnitude of receptor-activated dendritic calcium signals. Our results show that PICK1 localization in dendrites is subject to multiple forms of regulation that contribute to surface AMPAR expression, likely by modulating the numbers of AMPARs maintained in intracellular compartments.

Animals↗
PubMed1997

Multiple female reproductive failures in cyclooxygenase 2-deficient mice.

Cyclooxygenase (COX) is the rate-limiting enzyme in the synthesis of prostaglandins (PGs) and exists in two isoforms, COX-1 and COX-2. In spite of long-standing speculation, definitive roles of PGs in various events of early pregnancy remain elusive. We demonstrate herein that the targeted disruption of COX-2, but not COX-1, in mice produces multiple failures in female reproductive processes that include ovulation, fertilization, implantation, and decidualization. Using multiple approaches, we conclude that these defects are the direct result of target organ-specific COX-2 deficiency but are not the result of deficiency of pituitary gonadotropins or ovarian steroid hormones, or reduced responsiveness of the target organs to their respective hormones.

Animals↗