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Growth of SV 40, adeno 7 and SV 40-adeno 7 viruses in monkey and human cells at 29 degrees and 37 degrees C.

Adeno 7 virus replicated well in human diploid (LEP) cells but only to a low degree in green monkey kidney (GMK) cells at 37 degrees C; it did not replicate in either system at 29 degrees C. At 37 degrees C SV 40 virus replicated well in GMK cells but only moderately in LEP cells; at 29 degrees C it did not replicate in either system. SV 40-adeno 7 hybrid grew in both GMK cells and LEP cells at 37 degrees C. At 29 degrees C this virus replicated in GMK cells but not in LEP cells. While the formation of V-antigen generally corresponded to the infectious virus production in the respective system, considerable differences were encountered in the T-antigens production. Adeno 7 T-antigen was detected earlier and in a higher percentage of GMK cells than in the fully permissive LEP cells and its formation was only slightly influenced by the incubation temperature. SV 40 T-antigen was more efficiently formed in GMK cells than in LEP cells. At 29 degrees C SV 40 T-antigen was only found in GMK cells and was detected later than at 37 degrees C. The difference in the formation of SV 40 T-antigen in GMK cells infected with SV 40 and SV 40-adeno 7 hybrid virus was further analyzed. The results obtained suggest that an early step of the virus-cell interaction, but neither virus attachment nor penetration, was involved.

Adenoviridae

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.

Cells, Cultured

Expression of "early" and "late" viral functions in a somatic cell hybrid between a mouse cell and a spontaneous yielder SV 40-transformed Chinese hamster cell.

A somatic cell hybrid (Cl. 6d) was originated from the fusion of mouse 3T3-4E) and spontaneous yielder SV 40-transformed Chinese hamster (CHK/SVLP AG) cells. During the early stages of its history, the C1. 6d hybrid underwent a rapid chromosome loss, preferentially loosing hamster chromosomes. This was not a constant tendency of the hybrid cells. As the parental CHK)SVLP AG cells, the hybrid cells were always found 100 per cent SV40 T-antigen positive. While CHK/SVLP AG cells infectious SV 40 DNA, V-antigen and virus were regularly detected, in the hybrid cells only infectious DNA was occasionally detected. This was not due either to the loww of an essential Chinese hamster gene(s) or to the presence of an inhibiting mouse cell component(s); it was apparently the consequence of inability of the cells to properly activate the resident SV 40 genome(s). After superinfection with SV 40 DNA, the hybrid cells-though capable of synthesizing SV 40 V-antigen--were unable to ensure virus assembly. Experimental evidence was obtained suggesting that SV 40 maturation is dependent of a cellular function(s).

Animals

The existence of triphosphorylated 5'-ends in virus-specific RNA isolated from SV-40 transformed cells.

The question about the nature of promoters in the transcriptional units containing SV-40 sequences in transformed cells was analyzed. It was found that the pulse-labeled RNA hybridizing to SV-40 DNA contains small but significant amounts of triphosphorylated 5'-ends detected as pppGp in alkaline hydrolyzates of this RNA. In another series of experiments the fragments of RNA containing triphosphorylated 5'-ends about 100 nucleotides in length have been isolated by hydroxyapatite chromatography. Some of them form hybrids with SV-40 DNA. The conclusion is drawn that at least some of SV-40 promoters are used for transcription initiation in SV-40 transformed cells.

Alkaline Phosphatase

The interaction of rifamycin SV with hepatic transport of taurocholic acid in the isolated perfused rat liver.

The effect of rifamycin SV on hepatic transport of taurocholic acid was investigated using isolated perfused rat liver technique. In all experiments, the perfused liver was maintained at taurocholic acid steady state by infusing constant amount of taurocholic acid. Infusion of rifamycin SV at various rates decreased biliary secretion of bile acids in a dose-dependent manner. Replacement of rifamycin SV by perfusion medium reversed this effect. To determine the site of action of rifamycin SV, kinetic experiments with 14C-taurocholic acid were undertaken. Rifamycin SV elevated the half-life of the medium disappearance of 14C-taurocholic acid. Furthermore, the antibiotic delayed the biliary appearance of 14C-taurocholic acid. The analysis of the results gave indications that the antibiotic interferred with hepatic uptake as well as biliary secretion of taurocholic acid.

Animals

Size and distribution of SV 40 DNA Sequences covalently linked with the DNA of permissive mammalian cells.

CV-1 cells productively infected with SV 40 contain viral DNA which is covalently linked with the host cell DNA. These linear duplex viral-host DNA molecules are replicated during the infectious cycle. They can be selectively isolated and purified by two successive cycles of DNA-DNA hybridization and elution steps using first CV-1 cell and then SV 40-DNA immobilized on filters. In an attempt to clarify the nature of the host DNA sequences neighbouring the viral DNA it was found that reiterated host DNA must be within the range of 800 bases from the viral sequences. Reassociation kinetics and treatment of the reassociated viral-host DNA sequences with single strand-specific S1 nuclease have shown that unique host DNA sequences are always present in close neighbourhood of the viral DNA. Most of the SV 40 DNA sequences are probably intergrated as fragments of subgenomic length.

Animals

Increase in plasma membrane phosphodiesterase activity in contact-inhibited 3T3 cells and in phenotypically reverted SV-3T3 cells.

Contact-inhibited 3T3 mouse fibroblast cells, in contrast to logarithmically growing 3T3 cells and SV-3T3 transformed cells, have increased levels of plasma membrane-bound phosphodiesterase (oligonucleotidase, E.C.3.1.4.19; nucleotide pyrophosphatase, E.C. 3.6.1.9) activity. The increase in enzyme, recorded as increased specific activity, is reversible, as evidenced by the return to normal values following dilution of confluent 3T2 cells and re-initiation of growth. Increased enzyme activity is induced again when the cells regain the confluent state. Transformed SV-3T3 cells can be induced to mimic the contact inhibited state, including increased plasma membrane phosphodiesterase activity, by exposure to a combination of: (i) agents that are known to induce increased intracellular cAMP levels and (ii) additions of purified 3T3 or SV-3T3 plasma membranes. Additions of either alone fails to induce the increase in membrane phosphodiesterase activity, although each alone can significantly suppress cell growth, as measured by incorporation of 3H amino acids. We suggest that the elevation of plasma membrane phosphodiesterase activity may serve as a measure of conversion to the contact-inhibited state in both normal cells and phenotypically reverted transformed cells.

Animals

[Effectiveness of cystolusis with rifamycin SV in patients with permanent catheters].

It has tested the effect of Rifamicina SV in the cystolusis employed on patients with permanent catheter: compared to the treatment with physiological solution this one, with Rifamicina SV, has showed, in most cases, the lower incidence of urine bacteria and their quick negative answer. This by an only local theatment. During the experiment has also been showed the possibility for the Rifamicina SV of selecting the Citrobacter as a resistant bacterial species.

Bacterial Infections

Regulation of amino acid and glucose transport activity expressed in isolated membranes from untransformed and SV 40-transformed mouse fibroblasts.

Membrane vesicles isolated from untransformed Balb/c and Swiss mouse fibroblasts and their SV 40-transformed derivatives were shown to catalyze carrier-mediated, intravesicular uptake of alpha-aminoisobutyric acid and D-glucose. Concentrative uptake of alpha-aminoisobutyric acid required the presence of a Na+-gradient (external greater than internal) and could occur independently of endogenous (Na+ + K+)ATPase activity. A K+ diffusion gradient (internal greater than external) in the presence of valinomycin, or the addition of the Na+ salt of a highly permeant anion, conditions expected to create an interior-negative membrane potential stimulated Na+-gradient-dependent uptake, suggesting this process is electrogenic. D-Glucose uptake was nonconcentrative and did not require ion gradients or metabolic conversion. Na+ gradient-dependent transport of alpha-aminoisobutyric acid was reduced both in initial rate and extent of uptake in vesicles from confluent untransformed cells and increased in those from SV 40-transformed cells, compared with activities observed in vesicles from proliferating untransformed cells. No changes in D-glucose carrier activity were observed when assayed at low glucose concentrations.

Aminoisobutyric Acids

The control of human WI-38 cell proliferation by extracellular calcium and its elimination by SV-40 virus-induced proliferative transformation.

The proliferative activity of diploid human WI-38 cells in sparse cultures depends on the extracellular concentration of free (or physiologically available) calcium, and cultivation in a medium having a calcium concentration of 0.1 mM or less gradually, but reversibly, arrest their proliferative development in the prereplicative (G1) phase of the cell cycle. Calcium's proliferative control of this cell type is eliminated by proliferative and morphological transformation by the oncogenic SV-40 virus, and the proliferative activity of SV-WI-38 cells in sparse cultures is unaffected by variation of the extracellular free calcium concentration between 0.00 and 1.25 mM.

Calcium

SV 40 related papova-viruses in human meningiomas.

Human meningiomas show characteristic chromosome aberrations very similar to those reported in human cell cultures experimentally transformed by Simian virus 40 (SV 40). Three out of eight meningiomas tested showed SV 40 specific tumor antigen but no virus capsid antigen. Experiments to rescue virus were successfully performed. Electron microscopic studies of these cell cultures revealed cytoplasmic inclusions containing virus particles which corresponded in size and morphology to Papova viruses.

Antigens, Viral

3-N-Substituted aminomethyl derivatives of rifamycin SV. A convenient method of synthesis, cyclization of certain derivatives, and anticellular and antiviral activities of several derivatives.

A new synthesis of Mannich bases of rifamycin SV using the Borch2 procedure with rifaldehyde is described. This new synthesis offers two advantages over the previously published method. It provides a route to monoalkyl-aminomethylrifamycins (le-h) and to unsubstituted aminomethylrifamycins that were not accessible by the old procedure. The new method also offers a preparative route to Mannich bases 1a and 1b were needed in multigram quantities for biological testing. In addition, the cyclization of certain of the monoalkylaminomethylrifamycins to the novel N,15-didehydro-15-epi[methano(alkylimino)]rifamycin SV derivatives (2) is described. The anticellular and antiviral effects of representatives of both series of compounds against cultured mouse cells and murine oncornavirus are are discussed.

Animals

Estimation of tubule or cylinder LV, SV and VV on thick sections.

The estimators of tubule or cylinder LV = 2NA, SV = 2IL, and VV = PP are considered in terms of bias due to section thickness, diameter, overlap, and grazing or fuzzy profiles. The superiority in a number of cases of the indirect estimation of SV and VV as functions of LV and profile diameter is discussed. Statistical aspects are dealt with in a practical example.

Animals

Immunoglobulin synthesis by an SV-40-induced hamster lymphoma.

A transplantable lymphoid tumour induced in Syrian hamsters by SV-40 was shown to synthesize a 7Sgamma2 monoclonal immunoglobulin of the same idiotype was also found in the membrane of the tumour cell. The induction of immunoglobulin synthesizing tumours by SV-40 should be useful for studies on immunoglobulin synthesis, lymphoid cell receptors and tumour immunity.

Animals

Dependence of intracellular alkali-ion concentrations of 3T3 and SV 40-3T3 cells on growth density.

Intracellular contents of potassium and of sodium are determined for 3T3 and SV 40-3T3 cells in dependence of growth density. In parallel, total cell volume and volume of intracellular water is determined for these cells suspended in physiological buffer. Intracellular potassium concentration thus evaluated for suspended 3T3 cells exhibits a sharp decrease at cellular growth densities which lead to density dependent inhibition of cell proliferation. In the case of SV 40-3T3 cells, this drop of potassium concentration with increasing cellular growth density is not observed, which correlates well with the absence of cell density dependent inhibition of cell growth in the transformed cell line. These results support the notion that processes of stimulation of quiescent 3T3 cells or of cell density dependent inhibition of their proliferation are mediated by processes including changes of potassium transport characteristics leading to increase or decrease respectively of their intracellular potassium concentration. Furthermore, these and other results suggest, that a difference between normal and transformed cells most relevant to their different proliferation behaviour might reside in different transport characteristics for potassium of the plasma membranes of these cells.

Animals

[Influence of allopurinol on the half-life of tolbutamide and rifamycin-SV in blood of healthy volunteers].

The influence of treatment with allopurinol (5 mg/kg/die for 15 days) on T/2 of tolbutamide and rifomycin-SV intravenously administered, has been studied in 10 healthy volunteers. We have observed reduction of T/2 of tolbutamide and, on the contrary, prolongation of T/2 of rifamycin-SV. Tolbutamide behaviour was unexpected, considering that other Authors had previously found inhibition of metabolic degradation of other drugs metabolized by the microsomal enzymes. We conclude that data concerning the influence of a drug (in our case, allopurinol) on the metabolism of another drug cannot always authorize general deduction and previsions regarding the metabolic interferences on the pharmacokinetics of other substances.

Adult

Ultrastructural and cytochemical features of SV 40 transformed hypothalamic cell lines.

A continuous cell line was previously obtained by Simian Virus (SV) 40 transformation of primary cultures of dissociated mouse fetal hypothalami. One clone from this cell line has been previously shown to possess some of the ultrastructural features, immunological properties and synthesizing capacities of magnocellular hypothalamic neurons which secrete vasopressin and neurophysins. The present paper reports on the morphological characterization of 14 other clones or subclones of the original cell line, using the following criteria: phase contrast microscopy, electron microscopy, Gomori's aldehyde fuchsin staining, cytochemical detection of beta-glucuronidase, immunochemical staining with antisera against bovine neurophysin I, bovine neurophysin II, lys-vasopressin, oxytocin, LH-RH and TRH. The results allowed the conclusion that the clones as well as the subclones can be distributed into two groups: 1) neurosecretory neurons which all possess several of the ultrastructural and cytochemical features of the neurophysin-vasopressin synthesizing clone, and 2) primitive nerve cells which are devoid of such features but display numerous bundles of filaments. In addition some clones were found to display intermediate features between groups 1 and 2. A similar diversity was observed within clones of the original strain and subclones of a neurosecretory clone. It is suggested that the primitive clones could represent precursors of the neurosecretory clones.

Animals