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At least 145 records · Page 8Linked to original sources
PubMed1993

Replication of the A7(74) strain of Semliki Forest virus is restricted in neurons.

In neonatal mice the A7(74) and L10 strains of Semliki Forest virus (SFV) are virulent. In 3- to 4-week-old mice the L10 strain is virulent, the A7(74) strain is avirulent. Following intraperitoneal inoculation of 3- to 4-week-old mice both strains produce a transient plasma viremia. This is cleared by IgM antibodies. IgG antibodies of all subclasses are produced. The distribution of viral RNA in the brain as determined by autoradiographic analysis of in situ hybridizations shows that in all cases virus is first apparent as small foci of infected cells around cerebral capillaries. In both neonatal and 3- to 4-week-old mice infected with L10 or neonatal mice infected with A7(74), infection spreads rapidly from the original foci to infect large areas throughout the brain. Both neurons and glial cells are infected resulting in pycnosis and death of the animals. In the brains of 3- to 4-week-old mice infected with A7(74) virus there is little spread from the original perivascular foci. Again neurons and oligodendrocytes are infected but cellular destruction is minimal. The same pattern of A7(74) infection is observed in 3- to 4-week-old athymic nu/nu mice and mice with severe combined immunodeficiency, indicating that failure to spread is not related to specific immune responses. Furthermore, in nu/nu and SCID mice the small restricted foci of A7(74) infection persist. Comparison of the replication of these two viruses by electronmicroscopy shows that although A7(74) virus replicates completely in the neurons of neonatal mice, the virus is unable to bud from the neurons of 3- to 4-week-old mice and aggregates of viral RNA and capsid accumulate. We conclude that there is an age-related restriction of A7(74) replication in mouse neurons and that this restriction is not associated with the maturity of virus-specific immune responses but probably reflects age-related changes in neurons.

Animals↗
PubMed1986

Regulation by calcium of parathyroid hormone mRNA in cultured parathyroid tissue.

We have examined the effect of changes in the concentration of extracellular calcium on parathyroid hormone mRNA in both short-term (hours) and long-term (days) cultures of bovine parathyroid tissue. Using a 32P-labeled PreProPTH cDNA probe, PTH mRNA was measured by gel blot hybridization of total RNA from tissue slices incubated for 4 h in low (0.5 mM) or high (5 mM) calcium concentrations and also by dot blot hybridization of cytoplasmic RNA extracted from aggregates of partially dispersed cells cultured up to 72 h in low (0.4 mM), normal (1 mM), or high (3 mM) calcium concentrations. PTH mRNA was unchanged over 4 h while high calcium had suppressed PTH secretion. However PTH mRNA did respond during long-term culture. By 24 h in high calcium there was a 50% suppression which was maintained for a further 48 h. PTH mRNA in normal calcium remained unchanged over 72 h while in low calcium it had increased slightly by 48 h. In contrast to the effect seen in cultured bovine parathyroid cells, PTH mRNA in human parathyroid adenoma cells cultured for 48 h in high calcium was decreased by only 10%.

Adenoma↗
PubMed2005

RASA1: variable phenotype with capillary and arteriovenous malformations.

Capillary malformation-arteriovenous malformation (CM-AVM) is a newly discovered hereditary disorder. Its defining features are atypical cutaneous multifocal capillary malformations often in association with high-flow lesions: cutaneous, subcutaneous, intramuscular, intraosseous and cerebral arteriovenous malformations and arteriovenous fistulas. Some patients have Parkes Weber syndrome - a large congenital cutaneous vascular stain in an extremity, with bony and soft tissue hypertrophy and microscopic arteriovenous shunting. In the past, arteriovenous malformations and arteriovenous fistulas had been considered non-hereditary. A classical genetic approach was used to identify the locus. Candidate gene screening pinpointed mutations in RASA1 (p120-RASGAP) - a RasGTPase. RASA1 reverts active GTP-bound Ras into inactive GDP-bound form. Murine Rasa1 knockout and tetraploid-aggregated embryos with RNA interference exhibited abnormal vascular development. Lack of RASA1 activity caused inhibition of cell motility, possibly through p190-RhoGAP. Thus, RASA1 defects probably cause abnormal angiogenic remodeling of the primary capillary plexus that cannot be compensated for by other RasGAPs: RASA2, RASAL and NF1. Signaling pathways involving RASA1 might offer novel targets for treatment of high-flow vascular anomalies.

Animals↗
PubMed1973

The nature of polydisperse ribonucleic acid in plants.

The aggregation of ribosomal RNA species during chromatography on methylated albumin on kieselguhr was decreased from 50 to 15% by the lower salt concentrations made possible by the use of higher pH values. The polydisperse RNA was resolved into two fractions. About 50% was eluted with the rRNA whereas the remainder was bound to the column, and was recovered only by solubilization of the methylated albumin. Both fractions of polydisperse RNA were similar in size range, but the bound fraction was considerably richer in AMP. No D-RNA (DNA-like RNA) peak was resolved under these conditions of column fractionation. However, the properties of the bound RNA were consistent with it containing both D-RNA and TB-RNA (tenaciously bound RNA). The relationship between these two fractions of AMP-rich RNA was considered. The bound RNA and ribosomal RNA responded differently to various treatments. The salt concentration required to elute ribosomal RNA was halved by increasing the pH of the fractionation, but the amount of bound RNA was in fact increased. Denaturation by hot urea decreased the binding of ribosomal RNA to the methylated albumin, but did not facilitate elution of bound RNA. The high affinity between the AMP-rich polydisperse RNA and the methylated albumin does not therefore appear to arise for the secondary structure conferred by the high AMP content.

Adenosine Monophosphate↗
PubMed1976

Assembly of a spherical plant virus.

The conditions previously reported as necessary for the reassembly of spherical viruses have been distinctly unphysiological and such reassembly cannot be related directly to the in vivo reaction. Mild conditions for the in vitro reassembly of cowpea chlorotic mottle virus (CCMV) from its isolated components have now been described (Adolph & Butler 1975) and the reassembled virus characterized. This reassembly involved the co-aggregation of the RNA and protein around neutrality and at ionic strength 0.2, giving yields of 70% encapsidation at pH 6.0. The reaction was independent of temperature over the range 5-25 degrees C and did not require the presence of Mg2+ ions. The reassembled virus shows a stability similar to that of native CCMV, with the same change in sedimentation coefficient around pH 6.5. The molecular mass and buoyant density in CsCl are also the same as those of native CCMV, while the electron microscope reveals a surface morphology on the reassembled particles like that on native CCMV. Analysis of the number-average, mass-average, and Z-average molecular masses of the purified protein at both pH 6.0 and pH 7.5 suggests that the active unit for reassembly is a dimer of the protein subunit.

Capsid↗
PubMed1983

An extended secondary structure model for the TMV assembly origin, and its correlation with protection studies and an assembly defective mutant.

Recognition of the unique internal assembly origin on tobacco mosaic virus (TMV) RNA by the disk aggregate of the viral coat protein probably involves an extended region of the RNA (larger than that coated by a single disk) folded into a specific conformation. A secondary structure model is proposed for the RNA preferentially coated by limiting amounts of coat protein disks on the basis of partial nuclease digestion data. Part of this sequence can form three symmetrically spaced hairpins with marginally stable base paired sequences at the tips of the stems. The pattern of progressive protection of the RNA from nuclease attack during assembly suggests that these three hairpins are successively coated by the first three disks to add. The spacing of these hairpins is identical to that of three hairpins in the pseudo assembly origin (part of the coat protein gene homologous to the assembly origin). In Ni 2519, a TMV mutant whose assembly is defective at high temperature because it can no longer discriminate between the true and pseudo assembly origins, a point mutation has occurred near the tip of the third metastably base paired stem of the true assembly origin which would disrupt its structure and alter one copy of a repeated heptanucleotide. This suggests an important role for the ordered and cooperative recognition of successive loops in determining the specificity of assembly.

Journal Article↗
PubMed2005

Messenger RNA degradation may be inhibited in sporadic inclusion body myositis.

OBJECTIVE: To integrate an immune-mediated mechanism and the disturbed protein expression in sporadic inclusion body myositis (IBM). BACKGROUND: In IBM, abnormal fibers harbor inclusions of some proteins found in the brains of patients with Alzheimer disease (AD). Poly(A)-binding protein 1 (PABP1) is the RNA binding protein that attaches to the poly(A) tail of mRNA and is involved in translation and mRNA degradation. Under stresses, mRNA combined with PABP1 forms cytoplasmic granules called stress granules. METHODS: Using 12 muscle biopsies with sporadic IBM and 46 controls, the authors localized PABP1 by immunohistochemistry, and poly(A)-containing RNA (poly(A)+ RNA) using the in situ hybridization method. They also immuno-localized HuR, one of the components of stress granules. RESULTS: In IBM, a proportion of fibers, including those vacuolated, showed an abnormal accumulation of PABP1 immuno-positive deposits. An immunofluorescence study indicated that large PABP1 positive deposits formed conglomerates with poly(A)+ RNA and PABP1 colocalized with HuR. Although PABP1-positive cytoplasmic inclusions were found in disease controls, their aggregates combined with poly(A)+ RNA were only detected in IBM. CONCLUSIONS: The localization of PABP1 positive deposits in inclusion body myositis (IBM) and other diseases may correspond to the stress granules that are formed under exposure to cellular stresses and the sites of mRNA turnover. The concomitant aggregation of poly(A)+ RNA that is specifically found in IBM may be due to the inhibition of mRNA degradation, which may affect translation. The authors speculate that an autoantibody against mRNA degradation machinery could play a role in this inhibition.

Aged↗
PubMed1992

Acquisition of thermotolerance induced by heat and arsenite in HeLa S3 cells: multiple pathways to induce tolerance?

Recent data indicate that cells may acquire thermotolerance via more than one route. In this study, we observed differences in thermotolerance development in HeLa S3 cells induced by prior heating (15 minutes at 44 degrees C) or pretreatment with sodium-arsenite (1 hour at 37 degrees C, 100 microM). Inhibition of overall protein and heat shock protein (HSP) synthesis (greater than 95%) by cycloheximide (25 micrograms/ml) during tolerance development nearly completely abolished thermotolerance induced by arsenite, while significant levels of heat-induced thermotolerance were still apparent. The same dependence of protein synthesis was found for resistance against sodium-arsenite toxicity. Toxic heat, but not toxic arsenite treatments caused heat damage in the cell nucleus, measured as an increase in the protein mass of nuclei isolated from treated cells (intranuclear protein aggregation). Recovery from this intranuclear protein aggregation was observed during post-heat incubations of the cells at 37 degrees C. The rate of recovery was faster in heat-induced tolerant cells than in nontolerant cells. Arsenite-induced tolerant cells did not show an enhanced rate of recovery from the heat-induced intranuclear protein aggregation. In parallel, hyperthermic inhibition of RNA synthesis was the same in tolerant and nontolerant cells, whereas post-heat recovery was enhanced in heat-induced, but not arsenite-induced thermotolerant cells. The more rapid recovery from heat damage in the nucleus (protein aggregation and RNA synthesis) in cells made tolerant by a prior heat treatment seemed related to the ability of heat (but not arsenite) to induce HSP translocations to the nucleus.

Arsenic↗
PubMed2006

Purification of granulosa cells from human ovarian follicular fluid using granulosa cell aggregates.

Human follicular fluid can provide a source of human granulosa cells for scientific study. However, removing potentially contaminating cells, such as white and red blood cells, is important for molecular and in vitro studies. We have developed a purification technique for human granulosa cells based on the selection of cellular aggregates. Human granulosa cells from 21 IVF patients were collected. A 50% Percoll gradient was used to remove red blood cells, and granulosa cell aggregates were collected, washed and processed for histology, electron microscopy, flow cytometry analysis, cell culture and RNA extraction. Granulosa cell aggregates were found to be homogeneous and free of white blood cells after histological and electron microscopic analysis. White blood cell contamination, measured by flow cytometry, was found to be between 2 and 4%. Polymerase chain reaction analysis revealed expression of known human granulosa cell genes and a white blood cell marker. Human granulosa cells grown in vitro showed flattened fibroblast-like morphology with lipid droplets consistent with previous reports. Cultured cells expressed the FSH receptor. Selection of human granulosa cell aggregates following centrifugation through a Percoll gradient provides an efficient method of selecting granulosa cells, suitable for both molecular and in vitro studies.

Adult↗
PubMed1976

The region of tobacco mosaic virus RNA involved in the nucleation of assembly.

The interaction of TMV RNA with the disk aggregate of TMV protein at the initiation of assembly has been studied by using the techniques of RNA sequencing. The 5' end group has been identified, and shown not to be protected in the early stages of assembly from accessibility to nuclease digestion. A population of RNA fragments of average length 250 nucleotides, originating from a unique region of TMV RNA, is encapsidated by limited assembly, and sufficient sequence information is available to identify certain unusual features. The protected region does not contain highly reiterated simple repeating sequences, but may contain more complicated repeats. The length and complexity of the nucleation region may reflect adaptation to the efficient mediation of the conformational change from disk to helix of TMV protein, besides a requirement for binding to the disk, and this may be an important part of the mechanism of specificity in the nucleation of assembly.

Electrophoresis↗
PubMed1977

Assembly of tobacco mosaic virus in vitro: elongation of partially reconstituted RNA.

It was reported that the rate of elongation of partially reconstituted RNA, if prepared without centrifugation, showed dependency upon the state of protein aggregation like that found when free RNA was used as the nucleic acid source [Butler, P. J. G. (1974) J. Mol. Biol. 82, 333-341]. Butler concluded from this result that disks are the kinetically favored protein source for the elongation of the tobacco mosaic virus rod. We show that partially reconstituted RNA prepared without centrifugation always contains a significant amount of free RNA and conclude that the dependency of its elongation rate upon the state of protein aggregation is due to contamination of his partially reconstituted RNA preparation by free RNA. The elongation rate of partially reconstituted RNA, which had been pelleted and contained no free RNA, was found to be about the same regardless of the state of aggregation of the added protein.

Centrifugation, Density Gradient↗
PubMed1978

Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor.

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.

Amanitins↗
PubMed1991

Scanning tunnelling microscopy of 16S ribosomal RNA in water.

The scanning tunnelling microscope has been used to image 16S ribosomal RNA molecules in water electrophoretically deposited on graphite surface. Two kinds of images have been obtained: images showing aggregates of 16S ribosomal RNA molecules similar to those obtained from DNA solutions and others showing individual 16S ribosomal RNA molecules. An interesting characteristic of these images, recorded in constant current mode, is that the 16S ribosomal RNA molecules appear to be located below the graphite surface. The morphology and several structural parameters of the molecules were consistent with the data obtained from electron microscopy.

Centrifugation, Zonal↗
PubMed1990

Switching in the self-assembly of tobacco mosaic virus.

Experimental observations on the structure and physicochemical properties of TMV protein assemblies have led to a fundamental switch in the model of the self-assembly process: rather than being nucleated by the hypothetical two-layer disk, virus assembly appears to be initiated by interaction of the specific RNA sequence with a short helical aggregate of the coat protein arranged as in the virus. Formation of the 20s nucleating aggregate involves the binding of an average of half a proton per protein subunit. This proton-binding site can be identified with the carboxyl-carboxylate pair that is formed between top and bottom protein surfaces at a radius of 58 A in the virus helix. Because the 20s aggregate consists of about two helical turns, only one carboxyl-carboxylate pair will be formed between each top-bottom pair of protein subunits. Limitation of the length of the 20s helical aggregate at neutral pH can be accounted for by disorder of the inner loop of the protein chain, due to electrostatic repulsion among the carboxyl groups that form the anomalous proton-binding site at 25 A radius in the ordered virus structure. To grow beyond two to three turns, inner loops of the protein at the interior of the helix must be ordered in the close-packed arrangement. The electrostatic repulsion opposing this ordering can be overcome by binding of the viral RNA at neutral pH, by calcium binding, or by proton binding in slightly acid solution. Virus disassembly upon infection appears to result from the low intracellular calcium and proton concentration compared to the extracellular environment, which increases the electrostatic repulsion among the negatively charged groups involved in calcium and proton binding, thereby allowing cellular ribosomes to competitively bind the viral RNA. Disk aggregates of TMV protein, which form at high ionic strength in alkaline solution, do not appear to be involved in virus assembly. The stacked-disc aggregate, which was previously presumed to be built of a polar stack of the hypothetical polar two-layer aggregate, is, in fact, a bipolar structure. Because the bonding between turns of the disc structures is different from that of the virus helix, direct switching between these structures by the postulated dislocation does not occur. TMV assembly does appear to involve conservation of bonding specificity, as initially presumed, but only in helical packing arrangements of the protein subunits. Switching from disordered to ordered conformations of the protein, dependent on changes in the electrostatic interactions among the protein subunits, appears to be critical in controlling the assembly process.

Microscopy, Electron↗
PubMed2004

The Ca2+/calcineurin-regulated cup gene family in Dictyostelium discoideum and its possible involvement in development.

Changes in free intracellular Ca2+ are thought to regulate several major processes during Dictyostelium development, including cell aggregation and cell type-specific gene expression, but the mechanisms involved are unclear. To learn more about Ca2+ signaling and Ca2+ homeostasis in this organism, we used suppression subtractive hybridization to identify genes up-regulated by high extracellular Ca2+. Unexpectedly, many of the genes identified belong to a novel gene family (termed cup) with seven members. In vegetative cells, the cup genes were up-regulated by high Ca2+ but not by other ions or by heat, oxidative, or osmotic stress. cup induction by Ca2+ was blocked completely by inhibitors of calcineurin and protein synthesis. In developing cells, cup expression was high during aggregation and late development but low during the slug stage. This pattern correlates closely with reported levels of free intracellular Ca2+ during development. The cup gene products are highly homologous, acidic proteins possessing putative ricin domains. BLAST searches failed to reveal homologs in other organisms, but Western analyses suggested that Cup-like proteins might exist in certain other cellular slime mold species. Localization experiments indicated that Cup proteins are primarily cytoplasmic but become cell membrane-associated during Ca2+ stress and cell aggregation. When cup expression was down-regulated by antisense RNA, the cells failed to aggregate. However, this developmental block was overcome by partially up-regulating cup expression. Together, these results suggest that the Cup proteins in Dictyostelium might play an important role in stabilizing and/or regulating the cell membrane during Ca2+ stress and/or certain stages of development.

Amino Acid Sequence↗
PubMed1987

Prokaryotic and eukaryotic RNA polymerases have homologous core subunits.

Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined. The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function. This gene, RPB2, exists in a single copy in the haploid genome. Disruption of the gene is lethal to the yeast cell. RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase. These observations suggest that the yeast and the E. coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis. The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases.

Amino Acid Sequence↗