Growth inhibition and chromosomal instability of cultured marsupial (opossum) cells after treatment with DNA polymerase α inhibitor

The DNA replication mechanism has been well established for eutherian mammals (placental mammals such as humans, mice, and cattle), but not, to date, for metatherian mammals (marsupials such as kangaroos, koalas, and opossums). In this study, we found that dehydroaltenusin, a selective inhibitor of mammalian (eutherian) DNA polymerase α, clearly suppressed the growth of metatherian (opossum and rat kangaroo) cultured cells. In cultured opossum (OK) cells, dehydroaltenusin also suppressed the progression of DNA replication. These results suggest that dehydroaltenusin inhibits metatherian as well as eutherian DNA replication. Dehydroaltenusin treatment of OK cells engendered fluctuations in the numbers of chromosomes in the OK cells as well as inhibition of cell growth and DNA replication. This suggests that partial inhibition of DNA replication by dehydroaltenusin causes chromosomal instability in cultured cells.     

Conjugation of a peptide to mannan and its confirmation by tricine sodium dodecyl sulfate–polyacrylamide gel electrophoresis

The conjugation of polysaccharides to peptides is essential for antigen delivery and vaccine development. Herein, we show that tricine SDS-PAGE in combination with Coomassie Blue staining was adequate to determine the conjugation efficacy of a peptide (epitope 35–55 of myelin oligodendrocyte glycoprotein) to mannan. In addition, tricine SDS-PAGE and periodic acid–Schiff stains were able to monitor the redox state of mannan. Using the described protocol, more than 99.9% of a peptide containing five lysines at its N-terminus was confirmed conjugated to mannan.     

Sterilization of tendon allografts: a method to improve strength and stability after exposure to 50 kGy gamma radiation

Terminal sterilization of tendon allografts with high dose gamma irradiation has deleterious effects on tendon mechanical properties and stability after implantation. Our goal is to minimize these effects with radio protective methods. We previously showed that radio protection via combined crosslinking and free radical scavenging maintained initial mechanical properties of tendon allografts after irradiation at 50 kGy. This study further evaluates the tissue response and simulated mechanical degradation of tendons processed with radio protective treatment, which involves crosslinking in 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide followed by soaking in an ascorbate/riboflavin-5-phosphate solution. Control untreated and treated tendons were irradiated at 50 kGy and implanted in New Zealand White rabbit knees within the joint capsule for four and 8 weeks. Tendons were also exposed to cyclic loading to 20 N at one cycle per 12 s in a collagenase solution for 150 cycles, followed by tension to failure. Control irradiated tendons displayed increased degradation in vivo, and failed prematurely during cyclic processing at an average of 25 cycles. In contrast, radio protected irradiated tendons displayed greater stability following implantation over 8 weeks, and possessed strength at 59 % of native tendons and modulus equivalent to that of native tendons after cyclic loading in collagenase. These results suggest that radio protective treatment improves the strength and the stability of tendon allografts.     

DNA damage in thymocytes of mice after exposure of the whole body to combined action of cadmium ions and γ radiation

The effect of combined action of cadmium chloride at a dose of 0.5 mg/kg of body weight and γ-radiation of 1 Gy on induction of DNA damage in thymocytes and the total number of cells in the thymus of mice was studied. We found that injection of CdCl₂ 0.5 h prior to irradiation decreased the number of single-strand DNA breaks and the number of alkali-labile sites in thymocytes 48 h after irradiation as compared to the γ-radiation effect only. This effect was associated with a strong decrease in the total number of thymocytes in this organ as compared to the action of cadmium ions and γ radiation separately. This masked the general genotoxic effect of combined treatment and created an illusion of a radioprotective effect of cadmium ions. Injection of cadmium chloride into mice 24 h prior to irradiation was followed by an additive increase in the number of the single-strand DNA breaks and the number of alkali-labile sites in thymocytes as compared to the respective controls such as the separate effects of cadmium ions and irradiation. We revealed a simultaneous decrease in the part of DNA tightly bound to proteins, i.e., DNA-protein cross-links as compared to the effect of γ-radiation only. We did not observe any statistically significant changes in the total number of thymocytes as compared to the separate effects of cadmium ions and irradiation. Thus, our data show that exposure of murine thymocytes to combined action of cadmium ions and γ-radiation at the doses and with the methods of treatment used induced additive effects but not antagonistic effects or protection against radiation.     

Ultrahistopathology of enchytraeid oligochaetes (annelida) after exposure to pesticides—A means of identification of sublethal effects?

• 1.1. The effects of the commonly used insecticides E605f (parathion), Ripcord 10 (cypermethrin) and the fungicide DuPont Benomyl on chloragocytes of an enchytraeid species were studied by transmission electron microscopy.
• 2.2. Animals were exposed to concentrations varying between 0.1 × and 100 × the minimum concentration recommended for agricultural application.
• 3.3. Ultrastructural changes regarding nuclei, mitochondria and other cytoplasmic compartments were obvious and were correlated to concentration and exposition time of the pesticides.
• 4.4. The importance of ultrahistopathology for terrestrial bioassays and monitoring is discussed.

     

The role of nonhomologous end joining and homologous recombination in the clonogenic bystander effects of mammalian cells after exposure to counted 10 MeV protons and 4.5 MeV α-particles of the PTB microbeam

We have studied the dependence of clonogenic bystander effects on defects in the pathways of DNA double-strand break (DSB) repair and on linear energy transfer (LET). The single-ion microbeam of the Physikalisch-Technische Bundesanstalt (PTB) was used to irradiate parental Chinese hamster ovary cells or derivatives deficient in nonhomologous end joining (NHEJ) or homologous recombination (HR) in the G1-phase of the cell cycle. Cell nuclei were targeted with 10 MeV protons (LET = 4.7 keV/μm) or 4.5 MeV α-particles (LET = 100 keV/μm). During exposure, the cells were confluent, allowing signal transfer through both gap junctions and diffusion. When all cell nuclei were targeted with 10 MeV protons, approximately exponential survival curves were obtained for all three cell lines. When only 10% of all cell nuclei were targeted, a significant bystander effect was observed for parental and HR-deficient cells, but not for NHEJ-deficient cells. For all three cell lines, the survival data after exposure of all cell nuclei to 4.5 MeV α-particles could be fitted by exponential curves. When only 10% of all cell nuclei were targeted, significant bystander effects were obtained for parental and HR-deficient cells, whereas for NHEJ-deficient cells a small, but significant, bystander effect was observed only at higher doses. The data suggest that bystander cell killing is a consequence of un- or misrejoined DSB which occur in bystander cells during the S-phase as a result of the processing of oxidative bistranded DNA lesions. The relative contributions of NHEJ and HR to the repairing of DSB in the late S/G2-phase may affect clonogenic bystander effects.     

Long-term (39–51 hr) exposure to estradiol in silastic capsules and lordosis behavior of guinea pigs

Three dosage levels of estradiol (E₂) in Silastic capsules were administered to ovariectomized guinea pigs for periods of 39 or 51 hr. At 39 hr., a systemic injection of progesterone was given, and hourly testing began. Dose-dependency of various aspects of lordosis behavior was established at both time intervals, and the serum E₂ levels produced by the medium and high dosage levels bracketed those found at the proestrus peak in intact guinea pigs. Although there were no significant behavioral differences between the 39- and 51-hr groups at the individual dosage levels, when the data from all three dosage groups were collapsed, exposure for 51 rather than 39 hr was seen to produce increases in maximum lordosis and heat-duration measures. The results suggest that subtle facilitative effects of estrogen occur very late in the estrogen-conditioning process (even after progesterone is administered).     

Quantitative fluorescenct analysis of different conformational forms of DNA bound to the dye, 4′,6-diamidine-2-phenylindole,and sepasated by gel electrophoresis

A quantitative fluorescent method for estimating the amounts of different conformational forms of the same DNA on agarose gels is described. Supercoiled, open circular, and linear forms of PM2 DNA and fluorescent dye (4′,6-diamidine-2-phenylindole) were used. The results are compared with respective radiometric estimations and are shown to be highly reproducible.     

Corticosteroid,catecholamine and glucose plasma levels in rabbits after repeated exposure to a novel environment or administration of (1–24)ACTH or insulin

The responsiveness of the adrenal cortex and the sympathoadrenal-medullary system to stress factors and administration of (1–24) ACTH and insulin was studied in adult rabbits. In comparison to untreated animals, exposure to a novel environment for 10 min followed by artery puncture on 6 consecutive days elicited a moderate increase of corticosteroid (C), norepinephrine (NE) and epinephrine (E) plasma levels. Intramuscular injection of 50 μg/kg body weight (1–24) ACTH increased C, NE and E plasma levels. Saline injection resulted in elevated NE levels; C, E and glucose remained unchanged. After injection of 1.0 IU/kg body weight insulin C levels were higher than those found after exposure to a novel environment for 10mmin followed by artery puncture; similarly, NE and E were increased.In accordance with results obtained in the rat or mouse the sympathoadrenal-medullary system in the rabbit is stimulated by stress factors such as handling, artery puncture or injection of (1–24) ACTH or insulin. In contrast the adrenal cortex can be stimulated only to a certain extent by these manipulations. An increased activation of adrenal cortex cells occurs only after insulin, a maximum stimulation only after (1–24) ACTH administration.     

50 Years after the Death of D.A. Sabinin (1889–1951)

Russian Journal of Plant Physiology -     

Temperature gradient capillary electrophoresis (TGCE)–a tool for the high-throughput discovery and mapping of SNPs and IDPs

Temperature gradient capillary electrophoresis (TGCE) can be used to distinguish heteroduplex from homoduplex DNA molecules and can thus be applied to the detection of various types of DNA polymorphisms. Unlike most single nucleotide polymorphism (SNP) detection technologies, TGCE can be used even in the absence of prior knowledge of the sequences of the underlying polymorphisms. TGCE is both sensitive and reliable in detecting SNPs, small InDel (insertion/deletion) polymorphisms (IDPs) and simple sequence repeats, and using this technique it is possible to detect a single SNP in amplicons of over 800 bp and 1-bp IDPs in amplicons of approximately 500 bp. Genotyping data obtained via TGCE are consistent with data obtained via gel-based detection technologies. For genetic mapping experiments, TGCE has a number of advantages over alternative heteroduplex-detection technologies such as celery endonuclease (CELI) and denaturing high-performance liquid chromatography (dHPLC). Multiplexing can increase TGCEs throughput to 12 markers on 94 recombinant inbreds per day. Given its ability to efficiently and reliably detect a variety of subtle DNA polymorphisms that occur at high frequency in genes, TGCE shows great promise for discovering polymorphisms and conducting genetic mapping and genotyping experiments.Electronic Supplementary Material Supplementary material is available for this article at     

Response of rice to different forms of urea and phosphorus fertilization under intermediate deepwater conditions (15–50 cm)

Summary Under intermediate deepwater condition with water depths of 15–35 cm during most of the crop growth period, sulphur coated urea and urea supergranule placement was superior to prilled urea in increasing the relative uptake and grain yield (23–25%) of rice. A semi-tall variety (CR 1016) responded better to the nitrogen application than a tall variety (CR 1030).The crop fertilized with N produced more number of tillers and grain yield than the unfertilized crop under complete submergence for 6 days at seedling establishement stage (8 days after transplanting) followed by waterlogging of 25±5 cm throughout the crop growth period. Application of phosphorus together with nitrogen increased the grain yield (9–14%) over nitrogen alone. Crop fertilized with these two nutrients (NP) increased their uptake and yield components.     

Responses of cattle to the combined exposure to diurnal temperature rhythm (−5 to 25°C) and to simulated high-altitude (4,000 m)

Eight 1/2-year old calves were exposed in a climatized altitude chamber to the following four conditions: 400 and 4,000 m at constant T_a (17°C), 400 and 4,000 m at alternating T_a (–5° to 25°C). Each exposure lasted for 24 h and for the rhythmic conditions included a cold night and warm midday hours, supplemented by infrared heaters. During exposure, hourly measurements were made of heart rate, respiratory rate, rectal and three skin temperatures. Every 3-h blood samples were collected for the determination of 10 blood variables. The following main results were obtained: (a) Altitude alone caused increases in respiratory rate, heart rate, erythrocyte number, haemoglobin, specific gravity of blood and plasma, LDH and all four body temperatures. (b) In the rhythmic exposures, high correlation coefficients were found between ambient temperature on the one hand and skin temperatures (0.88 to 0.94), rectal temperature (–0.43) and respiratory rate (0.49) on the other hand. A change in ambient temperature by 1°C lead, on average, to a change in ear temperature by 1.2°C. (c) in response to falling ambient temperature during the night, rectal temperature and heart rate increased. This was interpreted as indicating a compensatory elevation in meta bolic heat production. At the same time, there was haemoconcentration as shown by elevations in erythrocyte number, haematocrit and haemoglobin. This haemoconcentration might have reflected splenic discharge, possibly supplemented by some loss of water from the plasma. (d) The warm environmental conditions around midday produced mild heat responses in terms of elevated values for respiratory rate, heart rate and body temperatures. (e) It is concluded that the rhythmic temperature with alternating stress of cold and mild heat, especially in combination with high altitude, was a strain on the animals and that they were forced to expend extra energy for combatting altitude- and temperature stress, energy which no longer would be available for productive processes.Presented at the Eight International Congress of Biometeorology, 9–14 September 1979, Shefayim, Israel.     

Use of capillary (zone) electrophoresis for determining felinine and it’s application to investigate the stability of felinine

Summary. A rapid capillary electrophoresis method was established to quantify felinine (2-amino-7-hydroxy-5,5-dimethyl-4-thiaheptanoic acid) in cat urine and used to investigate felinine stability. Synthetic felinine was stable in the presence of oxygen while 11% of the natural felinine in urine disappeared after 4 days exposure to air. Both synthetic felinine and the natural felinine (in urine) were stable for up to 3 months when stored at –5°C and 20°C. Thirty percent of the synthetic felinine was lost after 5 hours at 100°C while 95% of the natural felinine disappeared after only 2 hours at the same temperature. The recovery of felinine under certain conditions was greater than 100%. It is possible that acetyl-felinine may be present in the urine and that it is deacetylated during incubation. Overall synthetic felinine was found to be stable but the felinine in cat urine much less so. Other compounds present in the urine may contribute to the decomposition of felinine.     

Dynamics of bacterial communities during solid-state fermentation using agro-industrial wastes to produce poly-γ-glutamic acid,revealed by real-time PCR and denaturing gradient gel electrophoresis (DGGE)

The dynamics of bacterial communities play an important role in solid-state fermentation (SSF). Poly-γ-glutamic acid (γ-PGA) was produced by Bacillus amyloliquefaciens C1 in SSF using dairy manure compost and monosodium glutamate production residuals as basic substrates. The production of γ-PGA reached a maximum of 0.6% after 20 days fermentation. Real-time polymerase chain reaction showed the amount of total bacteria reached 3.95 × 10⁹ 16S rDNA copies/g sample after 30 days, which was in good accordance with the 4.80 × 10⁹ CFU/g obtained by plate counting. Denaturing gradient gel electrophoresis profile showed a reduction of microbial diversity during fermentation, while the inoculum, B. amyloliquefaciens C1, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbial, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce γ-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.