Biomedical subjects
Publications and source records attributed to Y Becker.
Infection by mumps virus was found to lead to complications involving the CNS and heart in certain individuals. It is hypothesized that infection of the pancreas by mumps virus leads to the production of toxic peptides which damage the brain and heart.
Studies of the in vitro phenotype of a series of AT strains established in Israel revealed the following features: premature senescence and increased demands for growth factors, normal sensitivity to the cytotoxic effect of alkylating agents, hypersensitivity to agents that damage the deoxyribose moiety of DNA via a "targeted" free radical attack (this hypersensitivity is coupled with reduced inhibition of DNA synthesis compared to normal cells), varying degrees of intermediate hypersensitivity to the same agents in AT heterozygous cells, lack of potentially lethal damage repair and sublethal damage repair in AT homozygous cells following treatment with free radical-producing agents. We conclude that AT involves a DNA repair defect and that the AT DNA lesion is probably a gap with the 3'-phosphate or 3'-phosphoglycolate end left in the DNA following sugar destruction.
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Neocarzinostatin is a radiomimetic antibiotic with a potent cytotoxic effect which elicits a hypersensitive response in human cells homozygous or heterozygous for the gene for ataxia-telangiectasia. The extent and the time course of potentially lethal damage repair and sublethal damage repair following neocarzinostatin treatment were investigated in human skin fibroblast strains and were found to be remarkably similar to those obtained following X-irradiation. Ataxia-telangiectasia homozygous cells essentially lacked potentially lethal damage repair, but were able to perform some degree of sublethal damage repair following neocarzinostatin treatment. Ataxia-telangiectasia heterozygous cells which show an intermediate degree of neocarzinostatin sensitivity could perform both processes but with somewhat reduced efficiency as compared to normal cells. These observations provide further evidence for a DNA repair defect in ataxia-telangiectasia cells.
Abdominal adhesions were experimentally induced in rats by gentle scraping. Severe adhesions developed in 38.7% of the control animals. The flavonoid cianidanol ( [+]-cyanidanol-3), an in vitro inhibitor of procollagen production, was administered intragastrically or intraperitoneally in doses of 9 to 72 mg per rat. Administered intraperitoneally at a dose of 36 or 72 mg per rat, cianidanol substantially inhibited adhesion formulation, when given immediately, three days, or five days after surgery. Oral administration of the drug was less effective in preventing the formation of adhesions. Thimerosal, another connective-tissue inhibitor, was found to be toxic at doses of 2.5 or 5 mg per rat, and its effect on the prevention of adhesion formation was poor. The ability of cianidanol to inhibit the development of abdominal adhesions in rats suggests that a possible approach to treatment is to inhibit the production of procollagen and thus prevent the formation of the collagenous fibers that are the cause of adhesions.
The susceptibility of mice of different ages (from four to 28 days) to infection with herpes simplex virus type 1 (HSV-1) mutants inoculated onto scarified corneas was studied. The TK+ isolate from wild type virus was pathogenic in mice of all age groups. An HSV-1 mutant (designated TK1/4) with a less active thymidine kinase (TK) gene expressing 25 per cent of the TK activity of the TK+ isolate was pathogenic for mice up to 10 days of age. In older mice, virus pathogenicity was dependent on the inoculum dose: increasing the TK1/4 virus dose tenfold raised the level of TK activity and thus the virulence of the virus. A TK- mutant with no TK activity was pathogenic for four to eight day old mice that have TK activity in the brain, but not in older mice. Thus, resistance to HSV-1 that is age-dependent in mice can be determined by the extent to which the virus strain is liable to express its TK gene and by the amount of TK activity present in the brain.
The wild type NIH strain of herpes simplex virus type 1 (HSV-1) has a mixed plaque morphology of both large and small plaques. From this virus we selected a large plaque isolate that was a high producer of thymidine kinase (TK) activity (designated TK+) and a small plaque isolate that produced 25 per cent of the TK activity of the large plaque mutant (designated TK 1/4). A TK- mutant of the large plaque virus was obtained after passage of the virus in the presence of BUdR. The pathogenicity of the TK 1/4 virus strain in relation to the TK+ and TK- strains was investigated in mice after inoculation of the virus into the eyes by corneal scarification. The TK+ strain was highly pathogenic, caused encephalitis and killed most of the mice, whereas the TK- strain did not cause latent infections in the trigeminal ganglia or kill the mice. The TK 1/4 virus strain replicated in the eyes within 24 hours after inoculation and entered the trigeminal ganglia, establishing a latent infection in almost all of the mice. By increasing the infectious dose tenfold, the TK 1/4 virus caused an active infection in the trigeminal ganglia (ganglionitis), migrated to the brain, and killed the mice. The results indicate that not only is a low level of TK required to establish latent infections in mice, but also the degree of virulence is determined by the amount of TK produced by the infecting virus.
Herpesvirus type 1 thymidine-kinase-negative mutants are readily selected for in tissue culture and in humans by acyclovir, a promising antiviral agent. We investigated the ocular pathogenicity of thymidine-kinase-negative mutants in the rabbit. The natural course of untreated keratitis induced by the herpesvirus type 1 thymidine-kinase-negative strain was characterized by superficial dendrites and geographic ulcers that healed spontaneously without loss of corneal clarity. We also studied the relationship between herpesvirus type 1 thymidine-kinase activity and virulence in the rabbit with three strains of herpesvirus type 1: NIH thymidine-kinase-positive (100% thymidine-kinase activity), NIH thymidine-kinase-intermediate (25% thymidine-kinase activity), and NIH thymidine-kinase-negative (0% thymidine-kinase activity). Despite comparable ocular titers, the NIH thymidine-kinase-positive strain proved to be the most virulent, causing significantly (P less than .002) more keratitis, encephalitis, and death than the other strains.
The ability of human fibroblast strains to repair the mutagenic DNA adduct O6-methylguanine (O6-MeG) induced by brief exposure to N-methyl-N'-nitroso-N-nitrosoguanidine (MNNG) was investigated. The repair reaction proceeded rapidly during the first hour after alkylation, followed by a slow, continuous phase of repair, and both processes were saturated by low doses of carcinogen. This was similar to what had previously been found in human lymphoblastoid lines. Three fibroblast strains from healthy donors and six strains from patients with ataxia telangiectasia were all proficient in their capacity to repair O6-MeG and had the same sensitivity to the cytotoxicity of MNNG and methyl methanesulphonate as normal cells. Three of these cell strains were derived from individuals whose lymphoblastoid lines were deficient in their ability to repair O6-MeG. These lymphoblastoid lines were also extremely hypersensitive to killing by methylating carcinogens. Because non-transformed cells from the same donors behaved normally with regard to both parameters, we concluded that the repair deficiency accompanied by carcinogen hypersensitivity of the lymphoblastoid lines does not indicate a genetic deficiency in the donor. These findings imply that lymphoblastoid lines may not always be the appropriate cell type for investigating genetic susceptibility to chemical mutagens.
A defect in DNA repair coupled to anomalous DNA synthesis after induction of certain radiogenic DNA damage is suspected to underlie the radiosensitivity of cells from patients with ataxia-telangiectasia (A-T). The response of cultured skin fibroblasts from A-T patients and A-T heterozygotes to six agents inducing various levels of DNA strand breakage by different mechanisms was studied to obtain further information on the nature of the 'A-T critical DNA lesion'. The A-T cells showed varying degrees of hypersensitivity to the cytotoxic action of the quinone-containing anti-tumor antibiotics streptonigrin and adriamycin and to hydrogen peroxide. This hypersensitivity was accompanied by reduced inhibition of DNA synthesis compared to normal cells after treatment with these agents. A limited degree of cellular hypersensitivity that was not sufficient to allow for definition of a separate sensitivity range was shown by A-T heterozygous cells. On the other hand, the A-T cells showed a normal response to paraquat, saframycin A and ellipticine. Taken together with previous results showing hypersensitivity of A-T cells to ionizing radiation, bleomycin and neocarzinostatin, these data indicate that the critical DNA lesion in A-T cells is a strand break caused by deoxyribose destruction following the action of free radicals targeted into the DNA.
Cells from patients with the hereditary multisystem disorder ataxia-telangiectasia (A-T) are hypersensitive to the cytotoxic action of DNA-breaking agents, such as X-rays, bleomycin and neocarzinostatin (NCS). A defect in the repair of a certain DNA lesion induced by all three agents may underlie this hypersensitivity. This DNA lesion may be a certain type of DNA strand break. Most of the previous experiments done with X-rays and bleomycin failed to show any retardation in the rejoining of DNA strand breaks in A-T cells. However, since both A-T homozygous and heterozygous cells are particularly hypersensitive to NCS, we studied the time course of strand breakage induction and repair in A-T skin fibroblast strains treated with NCS, using the sensitive method of alkaline or neutral elution. A linear dose response was obtained for the induction by NCS of single-strand breaks and double-strand breaks. A-T cells did not respond with a higher initial extent of strand breakage compared with normal cells. NCS is an appropriate agent for studying the kinetics of rejoining strand breaks, due to its rapid action in the cells; this action, which is completed within 2--4 min, was studied by monitoring strand break induction, inhibition of DNA synthesis and decrease in cellular survival. The time course of strand break rejoining found after NCS treatment was very similar to that found following X-irradiation: with both single- and double-strand breaks, a rapid phase of rejoining was first noticed (t 1/2 approximately 5 min for single-strand breaks and 20--25 min for double-strand breaks). This was followed by a second, slow phase that continued for several hours. No difference could be detected between normal and A-T cells either with regard to the time course of rejoining or the fraction of non-rejoined breaks remaining several hours after treatment.
Ataxia telangiectasia (AT) is an autosomal recessive disorder in which increased level of chromosome breakage and specific sensitivity to radiation and carcinogens have been reported. The effect of the radiomimetic drug bleomycin on chromosome breakage has been tested in skin fibroblasts of three patients with AT, two AT obligate heterozygotes, two normal human controls, and one normal amniotic fluid cell culture. Bleomycin in two concentrations (1 and 5 micrograms/ml) was added for 1 hr and cultures were harvested 4 hr later. A significant increase in chromosome damage was found in AT fibroblasts: a higher number of total breaks per cell, affected cells, and breaks per affected cell was found. The heterozygotes did not differ significantly from the controls. Chromosome breakage in skin fibroblasts of AT patients after bleomycin treatment has not been reported before.
The relationship between thymidine kinase (TK) activity and virulence was studied in the mouse using three HSV-1 strains: (1) NIH TK+ (100% activity), (2) NIH TK+/- (25% TK activity), and (3) NIH TK- (0% TK activity). Following corneal inoculation, keratitis, virus titers (eye, trigeminal ganglia brain), survival, and latency were determined for each strain. The most virulent strain, NIH TK+ (30% survival) produced the worst keratitis, highest CNS titers, and established latency in 78% of surviving mice. NIH TK+/- demonstrated dose-dependent intermediate virulence (57-90% survival) and established latency in 80% of mice. NIH TK-, the most avirulent strain (93-100% survival) produced eye virus titers equal to the other strains but did not appear to invade the CNS or establish latency. These results indicate that TK gene activity is essential for HSV-1 murine neurovirulence (ie, efficient CNS invasion, replication and establishment of latency), but not for ocular replication.
Cells from patients with the genetic disease ataxia telangiectasia are hypersensitive to the DNA-breaking agents X-rays, bleomycin and neocarzinostatin, and show reduced inhibition of DNA synthesis after treatment with these agents, as compared to normal cells. The rate of replicon initiation and chain elongation was measured shortly after brief exposure of two normal and two ataxia telangiectasia fibroblast strains to low doses (0.10-0.30 microgram/ml) of neocarzinostatin, by means of alkaline sucrose gradient analysis. Neocarzinostatin was found to inhibit both initiation and elongation, and both components of DNA synthesis were more resistant to this inhibition in the A-T strains.
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Varicella-zoster virus (VZV) DNA was extracted from infected cells with 0.25% Triton X-100-0.2 M NaCl and purified by isopycnic centrifugation in CsCl. In each of eight experiments, 1.8-9.8 micrograms VZV DNA was obtained from 107 infected cells. The VZV DNA obtained by this procedure had a molecular weight of 88-100 x 106 as determined by sucrose gradient sedimentation and electron microscopy, and cleavage patterns after digestion with four restriction enzymes that corresponded to patterns previously described with six strains of VZV; the pattern of BamHI-cleaved Triton-NaCl-extracted VZV DNA was identical to the pattern seen after DNA extraction from virions. These studies expand the usefulness of Triton X-100-NaCl for extraction of large molecular weight viral DNA from a system where considerable cell-free virus is produced (Pignatti et al., 1979, Virology 93, 260) to a system known for its marked cell association.