Mutagenic effect of freezing on nuclear DNA of Saccharomyces cerevisiae.

Although fragmentation of DNA has been observed in cells undergoing freezing procedures, a mutagenic effect of sub-zero temperature treatment has not been proved by induction and isolation of mutants in nuclear DNA (nDNA). In this communication we supply evidence for mutagenicity of freezing on nDNA of Saccharomyces cerevisiae cells. In the absence of cryoprotectors, cooling for 2 h at +4°C and freezing for 1 h at -10°C and 16 h at -20°C, with a cooling rate of 3°C/min, resulted in induction of frame-shift and reverse mutations in microsatellite and coding regions of nDNA. The sub-zero temperature exposure also has a strong recombinogenic effect, evidenced by induction of gene-conversion and crossing-over events. Freezing induces mutations and enhances recombination with a frequency equal to or higher than that of methylmethanesulphonate at comparable survival rates. The signals for the appearance of nDNA lesions induced by freezing are detected and transduced by the DNA damage pathway. Extracellular cryoprotectors did not prevent the mutagenic effect of freezing, while accumulation of trehalose inside cells reduced nDNA cryodamage. Freezing of cells is accompanied by generation of high ROS levels, and the oxidative stress raised during the freeze-thaw process is the most likely reason for the DNA damaging effect. Experiments with mitochondrial rho⁻ mutants or scavengers of ROS indicated that mutagenic and recombinogenic effects of sub-zero temperatures can be decreased but not eliminated by reduction of ROS level. The complete protection against cryodamage in nDNA required simultaneous usage of intracellular cryoprotector and ROS scavenger during the freeze-thaw process.

Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation.

Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 degrees C followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 degrees C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 degrees C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho-) and in cells with disrupted SCO1 gene (Deltasco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O2-) into the cells. Accumulation of O2- and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho-, Deltasco1), or in cells pretreated with O2- scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures.

The response of Ty1 test to genotoxins.

The Ty1 assay is a short-term test for detection of genotoxins based on induction of the transposition of a gene-engineered Ty1 retrotransposon in Saccharomyces cerevisiae cells. Here, we provide evidence that the Ty1 test responds positively in concentration-dependent manner to the carcinogenic genotoxins benz(a)anthracene, benzo(a)pyrene, chenodeoxycholic and taurodeoxycholic free bile acids and to environmental soil samples polluted with carcinogenic substances. The Ty1 test gives negative results with the noncarcinogenic mutagens benz(b)anthracene, benzo(e)pyrene, lithocholic and taurodeoxycholic conjugated bile acids and to soil samples not polluted with carcinogens. Presence or absence of genotoxins in soil samples was evidenced by chemical analysis. Several explanations for the sensitive differential test's response to genotoxins are proposed and discussed. It is concluded that the Ty1 test can complement existing assays in laboratory and environmental studies showing high sensitivity to a wider spectrum of carcinogenic genotoxins.

Enhanced protein export in Saccharomyces cerevisiae nud1 mutants is an active process.

Saccharomyces cerevisiae NUD1 gene codes for a spindle pole body component and nud1 temperature-sensitive mutants arrest at 38 degrees C in late anaphase with a tendency for lysis. We found that addition of 10% sorbitol to the medium complemented the lytic phenotype, and determination of colony-forming units evidenced the viability of nud1 cells for at least 48 hours at 38 degrees C. The protein amount in cell-free medium increased at 38 degrees C, and evidence is presented that intact nud1 cells exported proteins in amounts 10-fold higher compared wild type strains. The observed high amounts of extracellular acid phosphatase, invertase, and bacterial beta-galactosidase suggested the export of secretory proteins. This was evidenced by construction of nudlsec mutants and the observation that interruption of the secretory pathway resulted in absence of protein export at 38 degrees C. Proteins were exported through a cell wall showing increased porosity at 38 degrees C. The extracellular release of Gas1p and the facilitated transformability with plasmid DNA of nud1 cells indicated alternations of their cell walls at 38 degrees C. The export of proteins depends on oxidative phosphorylation as evidenced by disruption of the COX10 gene. Experiments with inhibitors of mitochondrial functions showed that the synthesis of adenosine triphosphate, but not the electron transport along the respiratory chain, has a key role in the export of proteins. The data show that the phenotype of S. cerevisiae nud1 mutants is characterized by enhanced export of secretory proteins and that the passage of proteins through the walls of nud1 cells is an active process.

Zinc(II) Complexes - A New Group of Non-Mutagenic Herpes Simplex Virus Inhibitors.

Former studies showed that complexes of Zn(II) with picolinic and with aspartic acids, Zn(pic)(2) and Zn(asp)(2), are able to inhibit HSV infection in cultured cells by affecting key steps of virus replication. As these complexes are candidates for a novel class anti-HSV drugs, further studies on their mutagenicity are of particular interest. In the present paper we present data showing that Zn(pic)(2) and Zn(asp)(2) do not express mutagenic effect in both prokaryotic (Salmonella typhimurium) and eukaryotic (Saccharomices cerevisiae) test systems.

Long-term results of silicone expander for moderate and severe Brown syndrome (Brown syndrome "plus").

BACKGROUND: The treatment of Brown syndrome has been undergoing an evolution toward more effective procedures with fewer operative interventions. Dr Kenneth Wright has introduced a procedure of superior oblique muscle tenotomy with a silicone expander to reduce the incidence of overcorrection. METHODS: There was a retrospective study of 20 eyes of 19 consecutive patients with moderate or severe Brown syndrome (Brown syndrome "plus"). Follow-up ranged from 12 to 72 months. The expander, which varies 6 to 10 mm in length, was placed in all patients in the tenotomized superior oblique muscle tendon 5 mm nasal to the nasal border of the superior rectus muscle using 7-0 or 8-0 Prolene suture without violating the inner layer of the intermuscular septum. The intermuscular septum was closed over the silicone expander. RESULTS: One hundred percent of patients had resolution of the down shoot in adduction and some or full ability to elevate the eye in adduction. Twenty percent of patients required reoperation (12.5% using 5-8 mm expanders) for overcorrection. Restriction of downgaze was not seen postoperatively. Patients often show an undercorrection 1 to 6 months postoperatively and improve or occasionally overcorrect at 1 to 2 years postoperatively. One patient with a 10-mm expander extruded the implant. DISCUSSION: Placement of a 5- to 8-mm silicone expander in the tenotomized superior oblique muscle tendon is an effective means of correcting Brown syndrome with a low rate of reoperation. Initial undercorrection should not discourage the surgeon because improvement may continue for up to 3 years. The goal of treatment should be to convert a moderate or severe Brown syndrome (Brown syndrome plus) to a mild Brown syndrome ("true" Brown syndrome). CONCLUSION: This technique reduces the need for either simultaneous or subsequent inferior oblique muscle weakening and represents an advance in the treatment of Brown syndrome.

[Genotoxic effect of cadmium chloride in various test-systems]. / Genotoksicheskii éffekt khloristogo kadmiia v razlichnykh test-sistemakh.

Many industrial regions of Bulgaria are contaminated with cadmium. Induction of various genetic damages by four concentrations of cadmium chloride was studied in various test systems. None of the tested concentrations induced gene mutations in Salmonella typhimurium. An increase in frequency of gene mutations, mitochondrial mutations, and intragene recombination was detected in Saccharomyces cerevisiae treated with the highest cadmium chloride concentration. A clastogenic effect and a significant decrease in mitotic index (MI) were induced in radicle meristem cells of Pisum sativum L. by the two highest cadmium chloride concentrations. Cadmium chloride was also shown to increase the frequency of sex-linked recessive lethals (SLRLs) and dominant lethals (DLs) in Drosophila germ cells. The results obtained in different test systems allow cadmium chloride to be considered a weak mutagen inducing various genetic damages.