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M Dinowitz

Publications and source records attributed to M Dinowitz.

16 recordsLinked to original sources
PubMed1992

Recent studies on retrovirus-like particles in Chinese hamster ovary cells.

Highly concentrated (4000-7000-fold) culture fluids from CHO cells were analysed for the presence of retrovirus-like activity. Concentrates containing reverse transcriptase activity were detected and further purified by sucrose density gradient centrifugation. Particles banding at 1.13-1.16 g/ml were found to contain nucleic acid sequences and structural proteins related to those found in murine and other retroviruses. Analysis of the endonuclease gene has shown no intact open reading frames. Approximately 100-300 copies of the C-type sequences were present in the genome of CHO cell lines as well as in the DNA extracted from Chinese hamster liver, indicating that these sequences are present in the germ line of the species. Concentrates were analysed for infectivity by direct inoculation and co-cultivation with a series of detector cells. No evidence of infectivity was detected by reverse transcriptase, mink cell S+L- focus assay or by electron microscopic analysis of the inoculated detector cells after at least four passages in culture.

Animals↗
PubMed1991

Endogenous origin of defective retroviruslike particles from a recombinant Chinese hamster ovary cell line.

The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. Continuous-flow ultracentrifugation has been used to concentrate extracellular particles from culture fluid of a recombinant CHO cell subclone for molecular characterization. Particles exhibiting reverse transcriptase activity and associated with mammalian C-type retrovirus structural proteins banded in sucrose gradients at a density characteristic of retroviruses. Examination of gradient-purified particles by electron microscopy revealed morphology and size similar to other retroviruses. Double-gradient-purified particles contained RNA which hybridized to probes for murine leukemia virus, and endogenous Chinese hamster intracisternal A-particle elements. DNA sequence analysis of a cDNA clone isolated from purified particles revealed multiple interruptions of the endonuclease reading frame, providing one possible explanation for the noninfectious nature of the observed particles. Sequences present as RNA in purified particles were also present as conserved, repetitive, provirus sequences in genomic DNA of all CHO cell lines examined and in Chinese hamster liver DNA. The observed particles are therefore likely to be the products of endogenous retroviruslike elements present in the germline of Chinese hamsters.

Animals↗
PubMed1991

Defective endogenous retrovirus-like sequences and particles of Chinese hamster ovary cells.

The presence of budding C-type and intracytoplasmic A-type particles in Chinese hamster ovary (CHO) cells is well documented. However, extensive screening has failed to detect any evidence of infectivity. To investigate the origin and expression of these particles, retrovirus-like sequences which are actively transcribed in CHO cells have been cloned and characterized. Two families of sequences related to intracisternal A-particle (IAP) genomes of mice and Syrian hamsters were identified in cytoplasmic RNA from CHO cells (CHO IAP family I and family II). None of the four clones which were sequenced exhibited intact gag, pol, or env open reading frames. Only IAP family II sequences were present in purified extracellular particles of CHO cells. Several cDNA sequences related to mammalian C-type retrovirus genomes were isolated and cloned from gradient-purified, extracellular particles of recombinant CHO cells. All were homologous to the conserved endonuclease domain of murine leukemia virus. Nucleotide sequence analysis of the largest cDNA revealed multiple interruptions of the endonuclease encoding reading frame providing one possible explanation for the non-infectious nature of the particles observed in CHO cells. Both types of retrovirus-like sequences identified in purified extracellular particles of CHO cells (CHO IAP family II and C-type) were present as conserved, moderately repetitive sequences in DNA of all CHO cell lines examined, as well as in DNA from a Chinese hamster liver. It is therefore likely that the extracellular retrovirus-like particles of CHO cells are the products of endogenous provirus elements present in the germline of Chinese hamsters.

Animals↗
PubMed1979

Naturally occurring lymphocyte-mediated immunity to endogenous type-C virus in the mouse. Blocking of the lymphocyte reactivity with antisera to the virus.

The natural immune response in mice to their endogenous type-C viruses involves a complex interaction between cellular and humoral immune mechanisms. The virus-specific immune reactivities are a function of age and appear only subsequent to endogenous virus expression. Cellular immune activity was found to reside in a population of lymphocytes that were characterized as natural killer cells based on their absence of theta surface antigens or immunoglobulin or complement receptors. Cellular and humoral virus-specific immune responses co-occur in the same animal and pretreatment of virus-positive target cells with sera from virus-positive aging mice is capable of partially blocking the cytotoxic activity of reactive lymphocytes. The blocking activity of sera from individual mice increases as a function of age and endogenous virus expression and is highly correlated with the virus-specific complement-dependent cytotoxic activity of these sera. Mouse sera, whether naturally immune or immune as a result of hyperimmunization with type-C virus, exhibit blocking activity that can be removed by absorption with purified type-C virus or purified viral glycoprotein (gp 70) but not by absorption with noninfected syngeneic cells. High-titered and highly specific antisera directed against certain individual R-MuLV structural proteins reveal blocking activity. Monospecific antisera to gp 70 and p 12 exhibited high-titered blocking reactivities which are absorbable by the respective purified proteins. Blocking activity of antisera directed against other viral structural proteins could not be excluded with certainty. These findings raise the possibility that immunity in the mouse to endogenous type-C virus or virus-infected cells involves competition between serum-blocking activity and natural-killer cell activity and further provides a unique model system for studying the mechanism of action of blocking antisera known to have monospecific reactivity against defined and purifiable transplantation antigens.

Aging↗
PubMed1978

Cytoplasmic DNA synthesis in rhinovirus type 14-inoculated KB cells.

Rhinovirus type 14 (RV14) incuced a transient statistically significant stimulation in synthesis of DNA which appeared between 0 and 3 h post-inoculation in the cytoplasm of high density monolayer cultures of KB cells. Newly synthesized DNA was measured by incorporation of [3H] thymidine into acid-insoluble DNAase-sensitive material and the cytoplasmic location established by cell fractionation and electron microscope radioautographic methods. A minimum of 10 plaque-forming units per cell of RV14 was required to stimulate DNA synthesis which did not occur above 34.5 degrees C, a temperature optimal for virus replication. Cytoplasmic DNA taken from RV14-infected or control cells could be differentiated from the bulk of cell (nuclear) DNA by several criteria, including: (1) RV14 induction of synthesis; (2) lower buoyant density and greater heterogeneity in CsCl and ethidium bromide/CsCl gradients; and (3) a different kinetic complexity upon reannealing. The Cot 1/2 value of cytoplasmic DNA, calclated as 50--100 from reassociation profiles, was about 10-fold less complex than the Cot 1/2 value of nuclear DNA (800-1000). These data rule out the possibility that cytoplasmic DNA arises by random breakage of nuclear DNA during cell disruption and extraction and are compatible with the hypothesis that inoculation of KB cells with RV14 results in stimulation of synthesis of a specific class of cell DNA which is detected in the cytoplasm.

Cell Line↗
PubMed1977

Particles resembling oncornaviruses. Spontaneous release from cultured meningioma cells.

A cell culture from thoracic cord meningioma tissue obtained at surgery was maintained for several passages in cell culture. These cells spontaneously released particles with an RNA of high-molecular weight (90S to 95S) and a density similar to that of type C oncornaviruses. The implications of these results and similar findings by others are discussed.

Adolescent↗
PubMed1977

Altered sensitivity of Rous sarcoma virus transformed cells to inhibition of RNA synthesis by alpha-amanitin.

Chick embryo cells transformed by Rous sarcoma virus (RSV) continue to synthesize 40--50 percent of control amounts of RNA following 12--24 hour exposure to 2 microng/ml of the toxin whereas normal chick embryo cells similarly treated synthesize less than 5 percent of control amounts of RNA. Analysis of cells treated with alpha-amanitin, or the resistant forms I and III polymerase, do not increase in infected cells over the levels found in uninfected control cells during the first 24 hours following infection indicating that increase in polymerase levels in infected cells does not account for the observed resistance. No significant difference was detected in the sensitivity to alpha-amanitin in the form II polymerase isolated from normal and transformed cells; The greater sensitivity of normal cells to alpha-amanitin can be reduced by growing the cells at low cell density but the resistance of RSV transformed cells is not significantly altered by changes in cell density. The results suggest that the resistance of RSV transformed cells may be related to altered control of density dependent contact-inhibition of the transformed cells.

Amanitins↗
PubMed1977

A continuous line of Rous sarcoma virus-transformed chick embryo cells.

A continuous line of Rous sarcoma virus (RSV)-transformed chick embryo cells was established. The cells, designated RTAZ-1, which constitute the only known line of continuously growing RSV-transformed cells of chick embryo origin, grow rapidly, display uniform morphology, and perpetually release large amounts of RSV (Rous-associated virus, type 1). RTAZ-1 cells display a heteroploid chromosome complement with 92-94 chromosomes characteristic of chicken cells. Molecular hybridization studies demonstrated that DNA from these cells hybridized with normal chick DNA but not with human (KB) cell DNA, thus providing additional evidence of the chicken origin of RTAZ-1.

Animals↗
PubMed1977

Transfer of delayed hypersensitivity in mice to microbial antigens with dialyzable transfer factor.

Dialyzable Lawrence-type transfer factor was prepared from the spleen cells of CF1 mice inoculated with Coccidioides immitis- and Candida albicans-killed vaccines and with live Mycobacterium tuberculosis vaccine (BCG). These preparations were shown to transfer antigen-specific cell-mediated immunity to naive mice, as measured by the delayed skin test and footpad-swelling methods. Reactivity could be demonstrated when the test antigens were given 24 h after the transfer factor, but not when they were given simultaneously. Coccidioides-specific transfer factor was shown to be sensitive to Pronase and resistant to trypsin and ribonuclease. A preparation of BCG transfer factor was sensitive to snake venom phosphodiesterase.

Animals↗
PubMed1977

Susceptibility of murine transfer factor to dimerized ribonuclease A.

Dialyzable transfer factor was prepared from the spleens of CF1 mice actively sensitized with killed Coccidioides immitis antigen. The transfer factor was administered to normal mice either intraperitoneally or into the hind footpads. The recipient mice were tested for reactivity to the coccidioides antigen and to Candida albicans antigen by means of the footpad swelling test. The transfer factor conferred antigen-specific reactivity upon normal recipient mice when given by the intraperitoneal and footpad routes. This capacity of the transfer factor was destroyed by in vitro pretreatment with dimerized ribonuclease A, an enzyme active against double-stranded, as well as single-stranded, ribonucleic acid. In contrast, monomeric ribonuclease A, which is active against only single-stranded ribonucleic acid under the conditions used here, was without effect upon the transfer factor. These data provide evidence that murine transfer factor contains ribonucleotides that are essential for immunological activity. In addition, the data are consistent with the hypothesis, advanced by others, that the ribonucleotides may be double-stranded or uniquely looped configurations.

Animals↗
PubMed1976

Delayed hypersensitivity to fungal antigens in mice. I. Use of the intradermal skin and footpad swelling tests as assays of active and passive sensitization.

Mice were sensitized to Coccidioides immitis and Candida albicans antigens and tested for sensitivity by the intradermal and footpad swelling methods. In mice actively sensitized with killed antigen, antigen-specific intradermal and footpad induration responses occurred 24 and 48 hr after sensitization. Antigen-specific intradermal and footpad responses were transferred to normal mice with spleen cells from immune animals. Such responses were also transferred with normal spleen cells that had been incubated in vitro with immune RNA preparations. Histologic studies of intradermal reactions showed a mixed response of neutrophilic and mononuclear leukocytes, with slight vascular involvement compatible with delayed hypersensitivity. No intradermal or footpad responses were observed 4, 24, or 48 hr after injection in recipients of serum from actively sensitized mice. Histologic examination of skin sites in these mice revealed only a polymorphonuclear response. It is concluded that these intradermal and footpad responses are the result of delayed hypersensitivity and can be used as assays for this type of immunity in mice.

Animals↗
PubMed1976

Delayed hypersensitivity to fungal antigens in mice. II. Molecular classes in immunogenic RNA extracts that transfer delayed hypersensitivity.

The transfer of delayed hypersensitivity to Coccidioides immitis and Candida albicans antigens with immunogenic RNA extracts was studied in a mouse model. Sensitivity was measured by skin tests and footpad swelling responses. Immunogenic RNA converted normal spleen cells in vitro so that they produced antigen-specific delayed hypersensitivity in mice that were given injections of the cells. RNase reduced the rate of, but did not abolish, in vitro interaction of immunogenic RNA extracts with lymphocytes. Immunogenic RNA transferred sensitivity on direct intraperitoneal inoculation into mice. The transfer ability was resistant to RNase preparations active against both single- and double-stranded RNA. Sedimentation gradient fractions of the immunogenic RNA were assayed by intraperitoneal injection, and converting activity was found in two fractions, greater than 33S and 6S-13S. After treatment with RNase, all activity was shifted to the less than 6S fraction. Two fractions of the immunogenic RNA in its native state (greater than 33S and 6S-13S) were also able to convert spleen cells. The data indicate that the transfer of delayed hypersensitivity by immunogenic RNA preparations is associated with RNA but may not require the intact RNA molecule.

Animals↗
PubMed1976

Delayed hypersensitivity to fungal antigens in mice. II. Characterization of the active component in immunogenic RNA extracts.

In a mouse model, cell-mediated immunity to Coccidioides immitis, as assayed by the delayed hypersensitivity skin test, was transferred with whole immunogenic RNA extract and its greater than 33S and 6S-13S sedimentation fractions. Both fractions were cleaved by RNase, but the products retained their transfer activity. The greater than 33S fraction of immunogenic RNA extract was inactivated by pronase, whereas the 6S-13S fraction was resistant to the proteolytic enzyme; however, after RNase treatment the latter fraction was sensitive to pronase. This finding suggests a protective role for RNA. Dialysis of immunogenic RNA extract yielded a dialysate with a ratio of absorbance at 260 nm to that at 280 nm (A260:A280) of 1.02. Similarly, the dialysis product of RNase-treated RNA is active and has an A260:A280 ratio of 1.34. The data indicate that at least part of the active moiety of immunogenic RNA extracts is an RNA-associated, pronase-labile peptide or nucleopeptide. Furthermore, it is possible that the dialyzable transfer factor may be the same peptide or nucleopeptide cleaved from immunogenic RNA during preparation of the transfer factor.

Animals↗