PubMed Health⌕ Search

Biomedical subjects

S D Rabkin

Publications and source records attributed to S D Rabkin.

At least 55 records · Page 3Linked to original sources
PubMed1994

Treatment of malignant gliomas using ganciclovir-hypersensitive, ribonucleotide reductase-deficient herpes simplex viral mutant.

We have demonstrated that attenuated mutants of herpes simplex virus (HSV) have therapeutic potential for malignant brain tumors. In this report, we tested a ribonucleotide reductase-deficient (RR-) HSV mutant as an experimental treatment for malignant brain tumors. The HSV-RR- mutant hrR3, containing an Escherichia coli lacZ gene insertion in the ICP6 gene that encodes the large subunit of RR, was used in this study. We examined the cytopathic effect of hrR3 (0.1 plaque-forming unit/cell) on the U-87MG human glioblastoma cell line in vitro. Only 0.2% of U-87 cells were alive 67 h postinfection. Drug sensitivity assays demonstrated that hrR3 is hypersensitive to the antiherpetic agent ganciclovir. For in vivo studies, 10 animals harboring U-87MG tumors were randomly divided and treated intraneoplastically with either 5 x 10(6) plaque-forming units of hrR3 or medium alone. The viral treatment group showed significant inhibition of tumor growth (P < 0.01; one-sided Wilcoxon rank test). Expression of the lacZ gene in hrR3, visualized by 5-bromo-4-chrolo-3-indolyl-beta-D-galactopyranoside histochemistry, could be detected in treated tumors. The therapeutic potential of this HSV-RR- mutant for malignant gliomas is discussed.

Animals↗
PubMed1994

Specific patterns of defective HSV-1 gene transfer in the adult central nervous system: implications for gene targeting.

Viral vectors are a means by which genes can be delivered to specific sites in the adult central nervous system. Nevertheless, the interaction between the viral vector and cells of the nervous system, which forms the basis for specific gene transfer, is not well understood. In this study a nonreplicating defective herpes simplex virus type 1 vector, expressing the marker gene lacZ, was stereotaxically injected at varying titers into the rat central nervous system. Three sites were targeted: the caudate nucleus, dentate gyrus, and cerebellar cortex, and the resulting patterns of beta-galactosidase activity were examined. Many cells of neuronal and glial morphology, and of differing neuronal subtypes, expressed beta-galactosidase at each of the injection sites. However, beta-galactosidase activity was also detected in distant secondary brain areas, the neurons of which make afferent connections with the primary sites. This strongly suggested that the retrograde transport of defective virus was the basis for the enzyme activity observed at a distance. Moreover, retrograde transport to secondary sites was found to be highly selective and restricted to certain retrograde neuroanatomical pathways in a specific and titer dependent fashion. The pathways observed were predominantly, but not exclusively, monoaminergic in origin. This finding is supported by reports of specific tropism by HSV for monoaminergic circuits in experimental encephalitis and transneuronal tracing studies. Our observations suggest that certain functional neuronal populations, which are permissive for the retrograde transfer of defective HSV-1 vectors, might be specifically targeted for gene transfer using this approach. Conversely, a knowledge of the pathways permissive for viral uptake, retrograde transfer, and subsequent gene expression will be essential in order to predict the consequences of gene transfer using viral vectors.

Animals↗
PubMed1994

Mutant herpes simplex virus induced regression of tumors growing in immunocompetent rats.

Herpes simplex virus (HSV) mutants kill dividing tumor cells but spare non-proliferating, healthy brain tissue and may be useful in developing new treatment strategies for malignant brain tumors. Two HSV mutants, a thymidine kinase deficient virus (TK-) and a ribonucleotide reductase mutant (RR-), killed 7/7 human tumor cell lines in tissue culture. The TK-HSV killed Rat RG2 glioma and W256 carcinoma lines but not the rat C6 glioma in culture. TK-HSV replication (12 pfu/cell) was similar to wild-type HSV (10 pfu/cell) in rapidly dividing W256 cells in tissue culture, but was minimal (< 1 pfu/cell) in serum-starved cells, suggesting that the proliferative activity of tumor cells at the site and time of TK-HSV injection may influence efficacy in vivo. Subcutaneous W256 tumors in male Sprague-Dawley rats were injected with TK-HSV or free inoculum. A significant effect of TK-HSV therapy on W256 tumor growth was demonstrated compared to controls (p = 0.002). Complete regression was observed in 4/9 experimental tumors, with no recurrence over 6 months. Tumor growth in the remaining 5/9 animals was attenuated during the first 3 to 5 days after treatment, but not beyond 5 days compared to 9 matched control animals; no tumor regression was observed in any of the control animals. These results suggest that HSV mutants are potentially useful as novel therapeutic agents in the treatment of tumors in immunocompetent subjects.

Animals↗
PubMed1994

Expression of the growth-associated protein B-50/GAP43 via a defective herpes-simplex virus vector results in profound morphological changes in non-neuronal cells.

This study describes the creation and application of a defective herpes simplex viral (HSV) vector for B-50/GAP-43, a neural growth-associated phosphoprotein. We demonstrate abundant expression of B-50/GAP-43 in cultured non-neuronal cells (African green monkey kidney cells [vero cells] and Rabbit skin cells) via this HSV vector. When B-50/GAP-43 was expressed in non-neuronal cells major morphological changes occurred that included extensive membrane ruffling, the formation of filopodia and long thin extensions reminiscent of neurites. These extensions often terminated in growth cone-like structures. Quantitation of these morphological changes at different times following infection demonstrates that the surface area of the B-50/GAP-43-expressing cells started to increase between 6 and 10 h post-infection. At 72 h, B-50/GAP-43-positive cells were 3.0 times larger in size and one third of the cells expressed long processes with a mean length of 165 +/- 14.5 microns. Ultrastructural studies of cells 48 h after infection revealed that B-50/GAP-43 is predominantly localized at the plasma membrane of the elaborated processes. Some immunoreactivity was associated with vesicular structures that appear to be in-transit in the processes. These observations suggest that B-50/GAP-43 acts at the plasmamembrane to induce a neuron-like morphology in non-neuronal cells persisting for several days in culture. In the future the defective viral vector will enable gene transfer to express B-50/GAP-43 in neurons in vivo in order to study its involvement in regenerative sprouting and neuroplasticity.

Animals↗
PubMed1994

Inflammatory effects of gene transfer into the CNS with defective HSV-1 vectors.

The use of viral vectors which infect and express genes in post-mitotic neurons is a potential strategy for the treatment of disorders affecting the central nervous system (CNS). However, the inflammatory consequences of such strategies have yet to be systematically examined. Preparations of non-replicating defective herpes simplex virus type 1 (HSV-1) amplicon vectors containing the lacZ gene were obtained by standard methods and stereotaxically injected into the adult rat dentate gyrus (DG). The consequent gene expression and inflammatory effects following microinjection were investigated. beta-Galactosidase activity was detected in neurons of the DG from 24 h to at least 12 days after vector injection. A strong inflammatory response developed within 2 days, characterized by diffuse up-regulation of major histocompatibility complex (MHC) class I antigens and the activation of microglia. After 4 days the recruitment of MHC class II+ cells, activated T lymphocytes and macrophages was detected. These features persisted for at least 31 days. Of importance was the finding of beta-galactosidase activity in a bilateral group of neurons in the supramammillary nuclei (SMN) of the posterior hypothalamus, known to send afferent projections to the DG. The onset of inflammation at this secondary site was delayed, but its cellular characteristics resembled those found at the primary site of injection. Thus, the use of preparations of defective HSV-1 vectors for gene transfer in the CNS has immunological implications both at primary and secondary sites within the CNS.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Immunological consequences of HSV-1-mediated gene transfer into the CNS.

Defective HSV-1 viral vectors were prepared using amplicon methods. The amplicon contained the cytomegalovirus immediate-early promoter and the lacZ gene as a reporter in addition to the HSV elements required for replication and packaging in vitro. Viral vectors were stereotaxically injected into the rat dentate gyrus and the resulting expression and immune response were investigated. Beta-galactosidase activity was detected in several thousand neurons from as early as 24 hours to as late as 10 days after injection. A significant immune response to the vector inoculation developed, which was characterised by diffuse MHC class I up-regulation from 48 hours and the infiltration of MHC class II+ cells and activated T lymphocytes and macrophages from day 4. These features persisted for at least 31 days. Of particular interest was a small group of neurons in the posterior hypothalamus which were found bilaterally to express beta-galactosidase. The immune response at this distant uninjected site was delayed in onset but its features were similar to that found at the primary site of inoculation.

Animals↗
PubMed1992

Molecular analysis of simian varicella virus DNA.

Clinical and pathological studies indicate that simian varicella virus (SVV) infection in primates is the counterpart of human varicella zoster virus (VZV) infection. The SVV and VZV genomes are also similar in size and structure. To extend studies of SVV DNA, we analyzed virus DNA from African green monkey kidney cells infected with the Delta-herpes-virus strain of SVV. The infectivity of SVV DNA was 88 PFU/micrograms. The buoyant density of SVV DNA, determined by isopycnic banding in CsCl gradients, was 1.700 +/- 0.002 g/ml, corresponding to a G + C molar ratio of 40.8%. The size of SVV DNA, estimated by analysis of restriction endonuclease digestion products and pulsed-field gel electrophoresis was 125.1 and 124.9 kbp, respectively. Electron microscopy of SVV DNA revealed a long region of 110.0 kbp, a unique short (Us) region of 5.1 kbp, and inverted repeat regions of 7.5 kbp flanking the Us. An EcoRI map of SVV DNA revealed two fragments not previously reported; our complete Pstl map also shows some differences. Mapping of SVV DNA with an additional restriction enzyme, measurement of full-length SVV DNA molecules, and the first use of pulsed-field electrophoresis to size SVV DNA, confirm and extend Gray's recent finding that SVV DNA has the same size and molecular configuration as VZV. We also show for the first time that the density of SVV DNA is similar to that of VZV DNA and that SVV DNA is infectious.

Animals↗
PubMed1991

Nucleoprotein complex formed between herpes simplex virus UL9 protein and the origin of DNA replication: inter- and intramolecular interactions.

The UL9 gene of herpes simplex virus type 1 encodes an origin-binding protein. UL9 protein purified from baculovirus vector-infected insect cells forms a stable complex with DNA containing the herpes simplex virus origin of DNA replication, oriS. Contained within oriS are two UL9 protein-binding sites, I and II, bracketing an (A + T)-rich region. UL9 protein, visualized by electron microscopy, binds selectively at the site of the origin and covers approximately 120 base pairs. Upon formation of the nucleoprotein complex, the apparent contour length of the DNA is shortened, suggesting that this amount of DNA is wrapped or condensed by the protein. A nucleoprotein complex of similar size and structure forms on an inactive origin deleted for binding site II. Multiple intermolecular interactions occur. In particular, UL9 nucleoprotein complexes interact in trans with other UL9 nucleoprotein complexes such that dimer DNA molecules are formed with a junction at the position of protein binding. The DNA molecules in these intermolecular complexes are aligned predominantly in a parallel orientation.

Animals↗
PubMed1990

In vivo analysis of the initiation of bacteriophage T7 DNA replication.

We have examined the initiation of bacteriophage T7 DNA replication in vivo using a pulse-labeling technique. The pulse-labeling technique permits the rapid identification of initiation sites on the T7 chromosome and a determination of the rate of movement of the replication fork. This technique has been used to analyze a number of phage mutants having alterations in the nucleotide sequence of the primary origin. The experiments confirm the results obtained by electron microscope analysis on the mapping of the primary origin region and demonstrate the requirement for a T7 promoter in the primary origin. The secondary origins were found to be located near the center and at the right end of the genome. Analysis of T7 phage harboring mutations in the essential replication genes of T7 shows that they fell into three classes. The first, including those mutated in genes 4 and 5, do not initiate DNA synthesis. The second, in genes 3, 6, and 1.2, initiate and elongate as wild-type phage, albeit some with lower rates of synthesis, during the first round of replication and then cease DNA synthesis. Mutations in gene 2 have no apparent effect on initiation or elongation.

DNA Replication↗
PubMed1990

Herpes simplex virus DNA synthesis at a preformed replication fork in vitro.

Proteins from herpes simplex virus (HSV)-infected cells were used to reconstitute DNA synthesis in vitro on a preformed replication fork. The preformed replication fork consisted of a nicked, double-stranded, circular DNA molecule with a 5' single-strand tail that was noncomplementary to the template. The products of DNA synthesis on this substrate were rolling-circle molecules, as demonstrated by electron microscopy and alkaline agarose gel electrophoresis. The tails contained double-stranded regions, indicating that both leading- and lagging-strand DNA syntheses occurred. Rolling-circle DNA replication was dependent upon HSV DNA polymerase and ATP and was stimulated by a crude fraction containing ICP8 (HSV DNA-binding protein). Similar protein fractions from mock-infected cells were unable to support rolling-circle DNA replication. This in vitro DNA replication system should prove useful in the identification and characterization of the enzymatic activities required at the HSV replication fork.

Acyclovir↗
PubMed1988

Initiation of DNA replication at cloned origins of bacteriophage T7.

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region. When the primary origin region is deleted replication initiates at secondary origins. We have analyzed the ability of plasmids containing cloned fragments of T7 to replicate after infection of Escherichia coli with bacteriophage T7. All cloned T7 fragments that support plasmid replication contain a T7 promoter but a T7 promoter alone is not sufficient for replication. Replication of plasmids containing the primary origin is dependent on T7 DNA polymerase and gene 4 protein (helicase/primase) and a portion of the A + T-rich region. The other T7 fragments that support plasmid replication after T7 infection are promoter regions phi OR, phi 13 and phi 6.5 (secondary origins). When both the primary and secondary origins are present simultaneously on compatible plasmids, replication of each is temporally regulated. Such regulation may play a role during T7 DNA replication.

Base Sequence↗
PubMed1984

A role for DNA polymerase in the specificity of nucleotide incorporation opposite N-acetyl-2-aminofluorene adducts.

Escherichia coli DNA polymerase I (Klenow fragment), DNA polymerase alpha from both calf thymus and human lymphoma cells and DNA polymerase beta from calf thymus and Novikoff hepatoma cells can incorporate nucleotides opposite N-guanin-8-yl-acetyl-2-aminofluorene in DNA. The polymerases incorporate dCTP opposite some AAF-dG lesions when Mg2+ is the divalent cation. Substitution of Mn2+ for Mg2+ broadens the specificity of insertion: E. coli DNA polymerase I (Klenow fragment) also inserts A, and at specific sites G or T; DNA polymerase alpha inserts any of the four dNTPs with A and C incorporated preferentially to G and T. Polymerase beta is specific, inserting mainly C even in the presence of Mn2+. The Km for addition of dATP opposite a lesion by E. coli polymerase I (Klenow fragment) in the presence of Mn2+ is about 0.5 mM. dNMPs increase the insertion of nucleotides opposite AAF-dG in the presence of Mg2+ and increase both the rate and number of sites at which incorporation occurs in the presence of Mn2+. dNTP alpha S and recA protein increase only the insertion of C. We suppose that the incorporation of dCTP reflects normal base-pairing with the AAF-deoxyguanine in the anti conformation, whereas insertion of the other nucleotides (including some of the C) reflects insertion opposite the AAF adduct in its preferred syn conformation. The fact that the DNA polymerase plays a role in determining the specificity of insertion opposite a lesion terminating DNA synthesis suggests that the spectrum of base substitution mutagenesis seen in vivo may reflect the properties of the protein components, including the polymerase, involved in bypass synthesis.

2-Acetylaminofluorene↗
PubMed1983

In vitro bypass of UV-induced lesions by Escherichia coli DNA polymerase I: specificity of nucleotide incorporation.

A variety of DNA polymerases, synthesizing in vitro on an UV-irradiated phi X174 DNA template, terminate synthesis one nucleotide before the 3' pyrimidines of putative dimers on the template. We have devised a system using Escherichia coli DNA polymerase I (Klenow fragment) that can synthesize past at least some of these dimers. The bypass is carried out in a multistep process--first, the incorporation of nucleotides opposite the pyrimidines in the dimer and, then, the addition of nucleotides complementary to the bases distal to the dimer. The insertion of a nucleotide opposite the first (3') pyrimidine of a putative dimer in the presence of Mn2+ occurs in a concentration-dependent fashion with a 3- to 4-fold preference for purine nucleotides over pyrimidine nucleotides. In the presence of Mg2+, insertion is less frequent. Correlation of these results with in vivo mutation data suggests a role for the polymerase in determining the spectrum of base substitution mutagenesis in SOS induced cells.

Base Sequence↗
PubMed1982

In vitro replication of mutagen-damaged DNA: sites of termination.

We have examined the effect of DNA lesions, which in vivo are potentially mutagenic, on in vitro DNA synthesis carried out by a number of purified DNA polymerases using a 0X174 template. Both acetyl aminofluorene (AAF) adducts and UV-induced pyrimidine dimers are blocks to elongation by DNA polymerases. On UV-irradiated DNA templates synthesis terminates one nucleotide before the sites of pyrimidine dimers with all of the enzymes tested: Pol I and Pol III holoenzyme from Escherichia coli, T4 DNA polymerase, avian myeloblastosis virus reverse transcriptase and a mammalian DNA polymerase alpha. With AAF, which reacts at the C-8 position of guanine, differences are observed between the above enzymes, with the latter two inserting a nucleotide opposite the site of the lesion. Substitution of Mn2+ for Mg2+ as the cation in the Pol I reactions causes changes in the termination pattern on both UV-irradiated and AAF-reacted templates. The significance of these results to the process of inducible error-prone repair and the possible bypass of lesions in the DNA is discussed.

Bacteriophage phi X 174↗
PubMed1982

Effect of acetylated and deacetylated 2-aminofluorene adducts on in vitro DNA synthesis.

We have constructed primed phi X174 DNA templates containing either acetylated or deacetylated aminofluorene adducts at the C-8 position of guanine. T4 DNA polymerase terminates synthesis one nucleotide before the acetylated adducts but incorporates an additional nucleotide opposite the deacetylated guanylaminofluorene. These observations can be explained by the known preferred conformations of the acetylated and deacetylated guanosinylaminofluorene nucleosides--the former favoring the syn conformation (so that in DNA the guanine is displaced from the helix by the fluorene ring) and the latter preferring the anti conformation (which allows normal base pairing of the guanine with cytosine). A similar differentiation between the two adducts was found with Escherichia coli DNA polymerase I. In contrast, avian myeloblastosis virus (AMV) reverse transcriptase, which terminated with a nucleotide inserted opposite the acetylated adducts, was less able to do so at the deacetylated adducts. The nucleoside incorporated by AMV reverse transcriptase opposite the acetylated adduct was exclusively cytidine, which suggests regular base pairing with the reacted guanosine nucleoside in the anti conformation; however, synthesis was completely blocked and unable to continue beyond this point. The differences between the termination patterns of the prokaryotic enzymes and AMV reverse transcriptase indicates that specific properties of a replicating polymerase can influence the conformation of a reacted nucleoside in the DNA, thus altering its recognition and possibly its mutagenic activity.

2-Acetylaminofluorene↗
PubMed1981

Sites of termination of in vitro DNA synthesis on ultraviolet- and N-acetylaminofluorene-treated phi X174 templates by prokaryotic and eukaryotic DNA polymerases.

In vitro DNA synthesis on a phi X174 template primed with a restriction fragment and catalyzed by the Escherichia coli DNA polymerase I large (Klenow) fragment (pol I) terminates at the nucleotide preceding a site that has been altered by ultraviolet irradiation or treatment with N-acetylaminofluorene. Termination on ultraviolet-irradiated templates is similar when synthesis is catalyzed by E. coli DNA polymerase III holoenzyme (pol III), phage T4 DNA polymerase, a polymerase alpha from human lymphoma cells, or avian myeloblastosis virus reverse transcriptase. 3' leads to 5' exonuclease activity cannot be detected in the reverse transcriptase and DNA polymerase alpha preparations. On N-acetylaminofluorene templates, pol I, pol III, and T4 polymerase reactions terminate immediately preceding the lesion, whereas reverse transcriptase-catalyzed reactions and, at some positions in the sequence, polymerase alpha-catalyzed reactions terminate at the site of the lesion. Substitution of Mn2+ for Mg2+ changes the pattern of pol I-catalyzed termination sites. The data suggest that termination is a complicated process that does not depend exclusively on the 3' leads to 5' exonuclease activity associated with many polymerases.

2-Acetylaminofluorene↗