Antifungal effect of gamma irradiation and sodium dichloroisocyanurate against Penicillium expansum on pears.

UNLABELLED: Gamma irradiation (GI) was evaluated for its in vitro and in vivo antifungal activity against Penicillium expansum on pear fruits. GI showed a complete inhibition of spore germination, germ tube elongation and mycelial of P. expansum, especially 1·8 kGy. GI affected the membrane integrity and cellular leakage of conidia in a dose-dependent manner. Furthermore, the leakage of protein and sugar from mycelia increased along with the dose. GI was evaluated at lower doses in combination with a chlorine donor, sodium dichloro-s-triazinetrione (NaDCC), to examine the inhibition of P. expansum. Interestingly, only a combined treatment with 0·2 kGy of GI and 70 ppm of NaDCC exhibited significant synergistic antifungal activity. The mechanisms by which the combined treatment decreased the blue mould decay of pear fruits could directly associated with the disruption of the cell membrane of the fungal pathogen, resulting in a loss of cytoplasmic materials from the hyphae. SIGNIFICANCE AND IMPACT OF THE STUDY: Gamma irradiation (GI) is used as an effective nonchemical approach to inactive pathogens. This study investigated the antifungal effect of gamma irradiation and its combined treatment with a chlorine donor on this fungal pathogen, both in vitro and in vivo. This study emphasized that the integration of low-dose GI and a chlorine donor, NaDCC, exhibited a significant antifungal effect, and that its mechanisms are directly associated with membrane integrity of fungal spores, promising that GI has the potential to be an antifungal approach.

Alterations in damage processes in dense cancellous bone following gamma-radiation sterilization.

Structurally intact cancellous bone allograft is an attractive tissue form because its high porosity can provide space for delivery of osteogenic factors and also allows for more rapid and complete in-growth of host tissues. Gamma radiation sterilization is commonly used in cancellous bone allograft to prevent disease transmission. Commonly used doses of gamma radiation sterilization (25-35 kGy) have been shown to modify cortical bone post-yield properties and crack propagation but have not been associated with changes in cancellous bone material properties. The purpose of this study was to determine the effects of irradiation on the elastic and yield properties and microscopic tissue damage processes in dense cancellous bone. Cancellous bone specimens (13 control, 14 irradiated to 30 kGy) from bovine proximal tibiae were tested in compression to 1.3% apparent strain and examined for microscopic tissue damage. The yield strain in irradiated specimens (0.93+/-0.11%, mean+/-SD) did not differ from that in control specimens (0.90+/-0.11%, p=0.44). No differences in elastic modulus were observed between groups after accounting for differences in bone volume fraction. However, irradiated specimens showed greater residual strain (p=0.01), increased number of microfractures (p=0.02), and reduced amounts of cross-hatching type damage (p<0.01). Although gamma radiation sterilization at commonly used dosing (30 kGy) does not modify elastic or yield properties of dense cancellous bone, it does cause modifications in damage processes, resulting in increased permanent deformation following isolated overloading.

Expression of phosphorylated histone H2AX in cultured cell lines following exposure to X-rays.

PURPOSE: Exposure to ionizing radiation results in phosphorylation of histone H2AX (gammaH2AX) at sites of DNA double-strand breaks. To determine the relationship between gammaH2AX formation and radiosensitivity, the rate of formation and loss of gammaH2AX were examined in several cultured cell lines following exposure to 253 kV X-rays. MATERIALS AND METHODS: Flow and image cytometry were both performed using a mouse monoclonal antibody against gammaH2AX. Immunoblotting was used to confirm cell line-dependent differences in antibody staining. Cell lines examined included V79 and CHO-K1 hamster cells, the human tumour cell lines SiHa, WiDr, DU145, WIL-2NS, HT144, HCC1937 and U87, and the normal cell strain HFL1. Radiosensitivity was measured using a standard clonogenic assay. RESULTS: Using flow cytometry, gammaH2AX formation was detected 1 h after doses as low as 20 cGy. Peak levels of gammaH2AX were observed within 15-30 min after irradiation and both the rate of radiation-induced gammaH2AX formation and loss were cell type dependent. Maximum levels of gammaH2AX formation were lower for HT144 cells mutant for the ataxia telangiectasia gene. Half-times of loss after irradiation ranged from 1.6 to 7.2 h and were associated with a decrease in the total number of foci per cell. The half-time of loss of gammaH2AX was correlated with clonogenic survival for 10 cell lines (r2=0.66). CONCLUSIONS: GammaH2AX can be detected with excellent sensitivity using both flow and image analysis. The rate of gammaH2AX loss may be an important factor in the response of cells to ionizing radiation, with more rapid loss and less retention associated with more radioresistant cell lines.

[Cloning and sequencing of CTP synthetase cDNA from Chinese hamster cells].

The CTP synthetase cDNA was cloned from Chinese hamster lung cells V79. By comparison of the amino acid sequence of CTP synthetase with the counterpart of other species published, it was show that the CTP synthetase was a highly conserved and stable gene among the mammalian cells, but more differences of the CTP synthetase sequence could be seen between procaryotic and eukaryotic cells. This phenomenon suggested that the catalysing model of the enzyme might have some small differences in the organisms.

Perspectives on genetics in china.

The long civilization of China traces its origin to the development of agriculture. Early records and archaeological findings demonstrate the success of plant and animal breeding in contributing to human livelihood and the national economy. Teaching and research in modern genetics began in China in the 1920s with individuals who received advanced training in the West. Over the following 30 years, notable contributions to genetics, both basic and applied, were made by Chinese scientists in universities and research institutions. Unfortunately, these activities were brought to a virtual standstill first by the official advocacy of Lysenkoism, followed by ten years of Cultural Revolution. Schools were closed and scientific research was interrupted. Not until 1978 did the universities begin to enroll new students and scientific research, including genetics, again receive endorsement. Since then, Chinese scientists have resumed contacts with colleagues in other countries and publication of their research findings in national and international journals. The material summarized in this article covers a long time span and a broad spectrum of scientific subjects and events. Representative examples of Chinese contributions to various areas of genetics are cited to depict the history, current status, and prospects for the development of genetics in China. After many ups and downs and despite limitations in material resources, the study of genetics in China seems to be on a sound footing, with a relatively complete system in place for training workers in this discipline.

Chromosomal aberrations in mouse lymphocytes exposed in vitro and in vivo to benzidine and 5 related aromatic amines.

Mouse lymphocytes were exposed in vitro for 2 h or in vivo for 24 h to benzidine and related aromatic amines to test for chromosome aberrations (CA) and mitotic indices. Uninduced mouse S9 was used to activate the amines for the in vitro tests to be consistent with the in vivo tests. Contrary to a previous report, no difference could be established in the genotoxicity of benzidine following activation with uninduced S9 compared to induced S9. There were concentration related increases in CA for benzidine and all the amines in vitro except for 4,4'-diaminostilbene which exhibited the greatest cellular toxicity towards cultured lymphocytes. Benzidine and its derivatives showed significant increases in CA in vivo compared to its negative control. The CA values for 4-aminostilbene were significantly higher than the other amines in both in vivo and in vitro studies. These genotoxicity results for 4-aminostilbene are consistent with our previous report of the pronounced CA effects in murine bone-marrow cells but would not be predicted from Salmonella mutagenicity tests.

Chromosomal aberrations in mouse lymphocytes exposed in vivo and in vitro to aliphatic epoxides.

Mouse lymphocytes in vivo or in vitro were exposed for 24 h to 4 aliphatic epoxides, glycidyl 1-naphthyl ether, glycidyl 4-nitrophenyl ether, 1-naphthyl-propylene oxide and trichloropropylene oxide (TCPO), and tested for the induction of chromosomal aberrations (CA). These epoxides were among the most genotoxic aliphatic epoxides in our previous studies. With the exception of TCPO, the test epoxides caused significant increases in CA in vivo compared to a negative control. There were concentration related increases in CA for all 4 epoxides in vitro and TCPO produced the greatest cellular toxicity and genotoxic effects towards cultured lymphocytes. The difference in the order of genotoxicity for the two test systems can be explained on the basis of a much shorter half-life for TCPO than for the other epoxides.

Molecular analysis of hypoxanthine phosphoribosyl transferase mutants induced by glycidyl 1-naphthyl ether in mouse spleen cells in vivo.

Treatment of C57BL/6J mice with an epoxide, glycidyl 1-naphthyl ether (GNE), resulted in an average of a 3.4-fold increase in frequency of 6-thioguanine-resistant mutants of mouse spleen T-lymphocytes. In similar experiments with the epoxide trichloropropylene oxide, no increase in mutant frequency was found. To determine the kind and location of mutations in the coding region of the hypoxanthine phosphoribosyl transferase (HPRT) gene, 26 GNE-induced mutants and 17 spontaneous mutants were analyzed by direct sequencing of polymerase chain reaction amplified cDNA. Among the GNE-induced mutants, HPRT cDNA was present in 22, while that from 4 could not be detected. Among the spontaneous mutants, HPRT cDNA was present in 15 and absent in 2. Among GNE-induced mutants, base substitution in HPRT occurred in 15 of 22 mutants analyzed. Nine of 15 base substitutions involved TA base pairs, primarily TA-->CG transitions. Base substitutions were found throughout exons 3-7 but 46% of substitutions were located in exon 3 and one frameshift mutation involving a GC base pair in exon 3 was also observed. Among the spontaneous mutants, base substitutions of HPRT occurred in 7 of 15 mutants analyzed with 6 of 7 base substitutions involving a TA base pair and another 2 of the 15 mutants showed a 4 base pair deletion. The base substitution spectrum in GNE-induced mutants was different from that of the spontaneous mutants.

Similarity of spontaneous germinal and in vitro somatic cell mutation rates in humans: implications for carcinogenesis and for the role of exogenous factors in "spontaneous" germinal mutagenesis.

The rate of spontaneous mutation resulting in electrophoretic variants per cell generation in a human lymphoblastoid cell line, on the basis of experiments described in this paper, is found to be 7.2 x 10(-8) per locus. A review of similar data on electrophoretic variants resulting from spontaneous mutation in the human germ line leads to an estimate of 3.3 x 10(-8) per locus per cell generation. It is argued that the similarity of these two estimates, despite an average cell generation time of 18.5 hr for the cultured somatic cells but about 26 days in the germ line, suggests that spontaneous mutation involving nucleotide substitutions is much more dependent on cell generation than on time. This finding permits the inference that environmental (exogenous) variables make a relatively small contribution to the rate of this type of human germinal spontaneous mutation. While in vitro somatic-cell mutation rates, such as derived in this study, provide a basis for modeling the contribution of nucleotide substitutions in multihit/clonal theories of carcinogenesis, it is also argued that the complex of events involved in carcinogenesis, including chromosomal rearrangements and mitotic recombination, could have very different individual probabilities. Estimates for the rates of these other types of mutation are needed to provide a better understanding of the manner in which multiple mutations accumulate in malignant cells.

Single-step selection of mammalian cell mutants deficient in CTP synthetase.

A single-step selection of Chinese hamster V79 cells deficient in CTP synthetase (CTPS-) is presented. The underlying principle of the direct selection is the differential and efficient killing of synchronized wild-type cells through incorporation of [3H]uridine and [3H]thymidine. The CTPS- mutant cells were recovered by virtue of their not engaging in DNA synthesis, because (1) CTPS- cells are deficient in CTP synthetase and thus are unable to convert [3H]UTP into [3H]CTP, which eventually is converted into [3H]dCTP and incorporated into DNA; (2) the growth of CTPS- mutant cells was arrested as a result of cytidine deprivation, thus escaping the killing by the incorporation of [3H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 x 10(-7). The mutant cells have no or very low CTP synthetase activity when tested by in vitro CTP synthetase assay or by whole-cell [3H]uridine labeling assay. This modified "tritium suicide" method combined with the S-phase cell synchronization could provide a powerful means for the recovery from the cell population of nondividing mutant cells that are auxotrophic for some special nutrient requirement.

Identification of a polypeptide associated with the malignant phenotype in acute leukemia.

A novel polypeptide, designated p18, was detected in a variety of hematopoietic cells by two-dimensional polyacrylamide gel electrophoresis. Quantitative analysis of p18 indicated its occurrence in a substantially greater amount in acute leukemia relative to nonleukemic cells. The increased amount of p18 in leukemia could not be explained on the basis of specific lineage, differentiation stage, or cell proliferation and thus appears to be a part of the malignant phenotype of the leukemic cells.

Human repair gene restores normal pattern of preferential DNA repair in repair defective CHO cells.

The pattern of preferential DNA repair of UV-induced pyrimidine dimers was studied in repair-deficient Chinese hamster ovary (CHO) cells transfected with the human excision repair gene, ERCC-1. Repair efficiency was measured in the active dihydrofolate reductase (DHFR) gene and in its flanking, non-transcribed sequences in three cell lines: Wild type CHO cells, a UV-sensitive excision deficient CHO mutant, and the transfected line of the mutant carrying the expressed ERCC-1 gene. The CHO cells transformed with the human ERCC-1 gene repaired the active DHFR gene much more efficiently than the non-transcribed sequences, a pattern similar to that seen in wild type CHO cells. This pattern differs from that previously reported in CHO cells transfected with the denV gene of bacteriophage T4, in which both active and non-transcribed DNA sequences were efficiently repaired (Bohr and Hanawalt, Carcinogenesis 8: 1333-1336, 1987). The ERCC-1 gene product may specifically substitute for the repair enzyme present in normal hamster cells while the denV product, T4 endonuclease V, does not be appear to be constrained in its access to inactive chromatin.

Estimation of mutation rates based on the analysis of polypeptide constituents of cultured human lymphoblastoid cells.

A subclone of a human diploid lymphoblastoid cell line, TK-6, with consistently high cloning efficiency has been used to estimate the rates of somatic mutations on the basis of protein variation detected by two-dimensional polyacrylamide gel electrophoresis. A panel of 267 polypeptide spots per gel was screened, representing the products of approximately 263 unselected loci. The rate of human somatic mutation in vitro was estimated by measuring the proportion of protein variants among cell clones isolated at various times during continuous exponential growth of a TK-6 cell population. Three mutants of spontaneous origin were observed, giving an estimated spontaneous rate of 6 x 10(-8) electrophoretic mutations per allele per cell generation (i.e., 1.2 x 10(-7) per locus per cell generation). Following treatment of cells with N-ethyl-N-nitrosourea, a total of 74 confirmed variants at 54 loci were identified among 1143 clones analyzed (approximately 601,000 allele tests). The induced variants include 65 electromorphs which exhibit altered isoelectric charge and/or apparent molecular weight and nine nullimorphs for each of which a gene product was not detected at its usual location on the gel. The induced frequency for these 65 structural gene mutants is 1.1 x 10(-4) per allele. An excess of structural gene mutations at ten known polymorphic loci and repeat mutations at these and other loci suggest nonrandomness of mutation in human somatic cells. Nullimorphs occurring at three heterozygous loci in TK-6 cells may be caused by genetic processes other than structural gene mutation.

Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer.

Cytidine-triphosphate synthetase (UTP: ammonia ligase (ADP-forming), EC 6.3.4.2) has been purified over 31,000-fold to homogeneity with 17% recovery from rat liver cytosol, using high-performance liquid chromatography (HPLC) techniques. The presence of CTP synthetase monomer, dimer and tetramer has been demonstrated in the ammonium sulfate fraction of rat liver cytosol. By gel-permeation HPLC, the molecular weights of the three molecular forms of the enzyme have been estimated as 240,000 (tetramer), 120,000 (dimer) and 60,000 (monomer). By gel-permeation chromatography on Bio-Gel A-1.5m column, the molecular weights of dimer and monomer were estimated as 100,000 and 50,000, respectively. The molecular weight of the monomeric subunit is determined to be 66,000 by SDS-polyacrylamide gel electrophoresis. Monomers isolated fresh from 0-30 (NH4)2SO4 fraction of rat liver cytosol are enzymatically active. Purified rat liver CTP synthetase exhibited sigmoidal kinetic plots as a function of the substrate UTP in the presence of the end-product, CTP. Partially purified CTP synthetase usually forms an inactive coagulum on freezing and subsequent thawing. Incubation of CTP synthetase dimer at 25 degrees C for 1 h in the presence of UTP, ATP and Mg2+ resulted in optimum conversion to tetramer with least inactivation. The purified tetramer dissociates to dimers when UTP, ATP and Mg2+ are removed by dialysis.

Nonrandom distribution of structural mutants in ethylnitrosourea-treated cultured human lymphoblastoid cells.

Two-dimensional polyacrylamide gel electrophoresis has been used to detect somatic cell gene mutations altering protein structure, following ethylnitrosourea treatment of cultured human lymphoblastoid cells. A total of 267 polypeptides encoded by 263 loci were scored in a series of 1143 lymphoblastoid clones. Sixty-five electrophoretic mutants were detected at a total of 49 loci. Sixteen of the 65 mutations were phenotypically repeat mutations, occurring at 11 loci. Furthermore, structural mutations occurred more frequently at loci known to be polymorphic. These results provide evidence that the mutations that are detectable at the protein level by two-dimensional polyacrylamide gel electrophoresis do not occur at random and that their frequency is greater among polymorphic loci.

Evaluation of methods for the estimation of mutation rates in cultured mammalian cell populations.

A systematic comparison of 5 different statistical methods for the estimation of mutation rate (mu) in cultured Chinese hamster V79 cells is presented. Fluctuation tests were performed with several large batches of parallel cell cultures each allowed to grow for a different length of time in order to reach different population size (Nt). Based on Lea and Coulson's theoretical distribution, a comparison has been made between the experimental data and the expected distribution of the number of ouabain-resistant mutants per culture in these hamster cell populations. The sum of squared deviation between the observed and expected values, or SSD, was used as a means of the adequacy of the estimation method; the method which gives the smallest SSD is regarded as the best one for the estimation of mu. Our results show that when Nt is small, the occurrence of mutation is infrequent, and SSDs from different methods are similar. However, when Nt is large, there is a great discrepancy of the SSD values, suggesting a preference of using the maximum likelihood method, the Po method, the median method, the upper quartile method and the mean method, in that order, for the estimation of mu. The order of preference is correlated with estimation efficiencies. Depending on the size of Nt and the method used, the estimated mu may vary up to more than 3-fold. At a large Nt, the mu obtained from the maximum likelihood method is very precise. This suggests the importance of choosing an appropriate Nt as well as method for the estimation of mu.

Chromosomal location of human genes encoding major heat-shock protein HSP70.

The HSP70 family of heat-shock proteins constitutes the major proteins synthesized in response to elevated temperatures and other forms of stress. In eukaryotes members of the HSP70 family also include a protein similar if not identical to bovine brain uncoating ATPase and glucose-regulated proteins. An intriguing relation has been established between expression of heat-shock proteins and transformation in mammalian cells. Elevated levels of HSP70 are found in some transformed cell lines, and viral and cellular gene products that are capable of transforming cells in vitro can also stimulate transcription of HSP70 genes. To determine the organization of this complex multigene family in the human genome, we used complementary approaches: Southern analysis and protein gels of Chinese hamster-human somatic cell hybrids, and in situ hybridization to human chromosomes. We demonstrate that functional genes encoding HSP70 proteins map to human chromosomes 6, 14, 21, and at least one other chromosome.

Detection of human somatic cell structural gene mutations by two-dimensional electrophoresis.

The feasibility of detecting human somatic structural gene mutations by two dimensional electrophoresis has been investigated. A lymphoblastoid cell line was grown as a mass culture in the presence of ethylnitrosourea, after which cells were regrown as single cell clones. A total of 257 polypeptide spots were analyzed in gels derived from 186 clones. Four structural mutations were detected by visual analysis of the gels. Computer analysis of gels corresponding to the mutant clones was also undertaken. At a spot size threshold of 200 spots to be matched using a computer algorithm, all four mutant polypeptides were detected. These results indicate the usefulness of the two-dimensional approach for mutagenesis studies at the protein level.