Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes in vitro

Effects of inorganic arsenicals on DNA synthesis in unsensitized human blood lymphocytes were biphasic: The chemicals at very low concentrations enhanced DNA synthesis, whereas higher concentrations inhibited DNA synthesis. The concentrations of arsenicals at which the maximum stimulating effect was found were 1×10^?5 M, 1×10^?6 or 2×10^?6 M, and 0.8×10^?6 or 1×10^?6 M for sodium arsenite exposure of 1 h, 3 d, and 6 d, respectively; for sodium arsenate, 1× 10^?5 M, 1×10^?5 M, and 2×10^?6 or 5×10^?6 M, respectively. Arsenicals must be present for the entire 6-d culture period to produce maximum stimulation of DNA synthesis in human lymphocytes. The longer exposure of the lymphocytes to arsenicals, the lower the concentrations of arsenicals at which the maximum stimulating effect on DNA synthesis was found. Stimulating effect of trivalent arsenic (sodium arsenite) on DNA synthesis was stronger than pentavalent arsenic (sodium arsenate), and the stronger the effect of trivalent arsenic than pentavalent, the longer exposure of the cells to the chemicals. Both sodium arsenite and sodium arsenate stimulated DNA synthesis in human lymphocytes to a lower degree than phytohemagglutinin (PHA).     

Hormone-like product of vascular tissue stimulating starch accumulation in pith explants of kale and endogenous cytokinins

When stem explants of kale (Brassica oleracea L. var.medullosa), containing pith parenchyma and a strip of vascular tissue, were cultured on simple sucrose medium, a hormone-like factor was transported from the vascular tissue to the adjacent pith, where it stimulated accumulation of starch. Similarly, up to a sevenfold increase of starch content in explants could be induced by cytokinins added to the culture medium. The relative stimulatory effect of several cytokinins (5×10^?6 M) and hormone-like product of vascular tissue (HPVT) in a typical experiment were: control (1.0), trans-zeatin (6.7), HPVT (6.2), N⁶-[2-isopentenyl]adenine (5.4), transzeatin riboside (5.2), N⁶-[2-isopentenyl]adenosine (5.4), kinetin (3.6), 6-benzylaminopurine (3.5), and adenine (2.1). Concentration of endogenous cytokinins was determined using ELISA (trans-zeatin, N⁶-[2-isopentenyl]adenine and their ribosides) andAmaranthus bioassay (total cytokinins). No effect of vascular tissue on the level of endogenous cytokinins in explants was found. The results support the conclusions of previous experiments that the HPVT stimulating starch accumulation is not a cytokinin.     

Superoxide dismutase level in response to paraquat and high temperature in the cyanobacteriumGloeocapsa sp

Paraquat (methylviologen) at concentrations above 0.05 mM inhibited the growth of photoautotrophic cyanobacteriumGleeocapsa sp. in axenic cultures. The growth rate was not affected by concentrations of 0.01 mM or less. This concentration resulted after a lag period in a moderate increase in superoxide dismutase level. After removal of paraquat, the cyanobacterium continued to generate higher levels of superoxide dismutase. There was a lag period of one hour before resumption of normal enzyme activity. Addition of puromycin at concentration of 0.5 mg cm^?3 had no effect on cell survival, but greatly enhanced the sensitivity of the culture to the toxicity of paraquat. The data showed an increase of SOD activity by temperatures above the normal growth temperature level. However, this increase was suppressed by chloramphenicol which revealed that the induction of superoxide dismutase by high temperatures was associated withde novo protein synthesis.     

Studies on lipid oxidation in fish phospholipid liposomes

Fish phospholipid liposomes were prepared and used as an artificial membrane system to study factors influencing-lipid oxidation. The extent of lipid oxidation was indexed by measuring the amount of thiobarbituric acid reactive substances (TBARS) produced. Fe²⁺, Fe³⁺, and Cu²⁺ were potent prooxidants in catalysing lipid oxidation. These metal ions induced lipid oxidation in a dose dependent manner. However, Zn²⁺, Ni²⁺, and Mn²⁺ did not significantly (p>0.05) affect lipid oxidation at all the concentrations (1, 10, or 100 μM) studied. Morin, luteolin (flavonoids), butein (chalcone), tannic acid, ellagic acid (polyphenols), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) (synthetic antioxidants) were potent antioxidants (producing <50% TBARS compared to control) of Fe²⁺-catalyzed lipid oxidation. Morin, luteolin, and butein possess two hydroxyl substituents, a C₄ ketone structure and a 2–3 double bond, all of which contributed to their antioxidative potential. Fe²⁺ caused some losses of polyunsaturated fatty acids (PUFA), whereas tannic acid protected the oxidation of several of the PUFA including C 16∶1 (Palmitoleic acid), C 18∶3 (Linolenic acid), C 20∶4 (Arachidonic acid), C 20∶5 (Eicosapentaenoic acid), and C 22∶6 (Docosahexaenoic acid).     

l-phenylalanine production by double auxotrophic multianalogue-resistant mutant ofArthrobacter globiformis

Tryptophan-plus-tyrosine double auxotrophic mutants resistant to fluorophenylalanine (PFP) and β-2-thienylalanine (TA) were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in mineral salts medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to 5-methyltryptophan (MT) and 3-nitrotyrosine (NT) from a double auxotrophic PFP^r and TA^r mutant. Under optimal cultural condition one mutant yielded 9.6g phenylalanine per L medium in flask culture. Enzymic activity of regulatory enzymes (deoxy-d-arabino-heptulosonate-7-phosphate synthase, chorismate mutase and prephenate dehydratase) were observed in the wild type, double auxotroph and double-auxotrophic multianalogue-resistant mutant.     

In vitro propagation ofWrightia tinctoria

In vitro method has been developed for propagation ofWrightia tinctoria R.Br. using cotyledonary node segments. Murashige and Skoog's (MS) medium supplemented with 5.0 mg dm^?3 of 6-benzylaminopurine (BAP) and 0.01 mg dm^?3 of naphthaleneacetic acid (NAA) induced up to eight shoots per explant with an average shoot length of 1.4 cm in 21 d. Three fold multiplication rate was achieved during every subculture of regenerated shoots on the same medium producing an average of 230 shoots per node within 84 d. Reduction in BAP concentration from 5.0 to 1.0 mg dm^?3 during subculture promoted shoot length without affecting the rate of multiplication. The differentiated shoots could be rooted by a dip treatment into preautoclaved indole-3-butyric acid (IBA-500 mg dm^?3 for 5 min) followed by their implantation onto MS medium containing 1/4 salts. Rooting was observed within 8–10 d in approximately 80% of shoots inoculated after IBA treatment. 15 d after rooting, the plantlets were transferred to culture bottles containing soil-Soilrite^TM (1∶1) and liquid nutrient solution comprising 1/4 MS salts. After their partial hardening in these bottles for 10 d they were transferred to pots containing soil-Soilrite (1∶1) mixture with 60% transplantation success. Methods are being standardized to improve the rate of survival and large scale field transfer.     

In vitro selection and characterization of a callus line ofVigna radiata resistant to NaCl,KCl and Na2SO4

A salt mixture resistant (SMR) cell line ofVigna radiata (L.) Wilczek was isolated by selection on agar solidified PC-L2 medium supplemented with NaCl, KCl and Na₂SO₄ (8∶1∶1) equimolar to 300 mol m^?3 NaCl, a concentration inhibitory to the wild-type non-selected cells (salt mixture sensitive, SMS). This line retained its resistance after subculture for 3 passages (3 months) on normal medium. The SMR line grew significantly better than SMS line at all the levels of salts, though less in saline medium than the SMR on normal medium. The growth of SMR line was significantly higher than that of SMS line under KCl stress. However, both the lines responded similarly to Na₂SO₄ at a concentration higher than 100 mol m^?3. The SMR line was found to be more sensitive to NaCl than SMS line. The SMR line under salt mixture stress maintained lower levels of Na⁺ and higher levels of K⁺ than SMS line. The SMR line failed to regenerate shoots, although rhizogenesis was observed on PC-L2 medium containing salt mixture (300 mol m^?3).     

Somatic embryogenesis inCicer arietinum L: Influence of genotype and auxins

Plant regeneration through somatic embryogenesis was obtained in chickpea (Cicer arietinum L.) using immature cotyledons and immature embryonal axes as explants. 2,4-dichlorophenoxyacetic acid (2,4-D), 4-amino-3.5-6-trichloropicolinic acid (picloram) and 3.6-dichloro-0-anisc, acid (dicamba) in concentrations 1, 2, 5, 10 mg dm^?3 were found better than naphthaleneacetic acid (NAA) (10–20 mg dm^?3) for the induction of globular and heart-shaped somatic embryos. The embryos developed upto the dicotyledonary stage on medium supplemented with saccharose, mannitol and silver nitrate (AgNO₃) and developed further into plantlets on medium containing gibberellic acid (GA₃) and abscisic acid (ABA). The frequency of somatic embryogenesis was dependent on the genotype and auxins used.     

Plant radioresistance and DNA repair efficiency inChlamydomonas reinhardtii andPisum sativum

This paper compares the repair of DNA single strand breaks (ssb) induced by γ-radiation in two strains ofChlamydomonas reinhardtii (137C/+/ and UVS-I) and three lines ofPisum sativum (NN 131, 198, 140) differing in the degree of radioresistance. DNA ssb in cells exposed to γ-rays (50, 100, 200, 500 Gy) were measured by electrophoresis and alkaline unwinding method with subsequent chromatography on hydroxyapatite immediately after irradiation and after 30 min of post-irradiation incubation at 25°C. An increase of double-strand DNA (in%) was found in cells after 30 min post-irradiation incubation.C. reinhardtii strains displayed an equal level of DNA degradation and repair efficiency in the DNA single strand breaks. The radioresistant line N 198 ofP. sativum is characterized by a lower level of induced DNA ssb and higher efficiency of repair of these breaks as compared with less radioresistant lines NN 131 and 140.     

Production of ethylene,its precursors and implied enzyme activities in isolated chickpea embryonic axes during the onset of growth

The embryonic axes of chickpea (Cicer arietinum) seeds were used to quantify 1-(malonyl)aminocyclopropane-1-carboxylic acid (MACC), 1-aminocyclopropane-1-carboxylic acid (ACC), ethylene and some related enzymes during the initial 18 h imbibition period (anaerobic growth phase). Longer cold storage (stratification) of seeds produced higher levels of MACC and ACC. Maximum accumulation of MACC and malonyl-transferase activity occurred after 5 h of growth but MACC levels later became insignificant. ACC-synthase activity and endogenous ACC seem to reach a maximum 2 h after MACC accumulation. MACC-hydrolase activity was measured“in-vitro” and reached a maximum after 5.5 h of growth. These results suggest that endogenous MACC did not seem to be an end product; it may be involved in ACC production and the regulation of ethylene production before the emergence of the radicle. Ethylene-forming enzyme (EFE) activity reached a maximum after 12 h and ethylene production after 18 h of growth. The physiological implications of this temporal separation of MACC, ACC, ethylene and related enzymes is discussed.     

Characterization of mitochondrial DNA topoisomerase I fromNeurospora crassa

DNA topoisomeraseI isolated from the lower eukaryoteNeurospora crassa mitochondria was characterized. Molar mass of the enzyme in the native state is 120 kDa and 60–65 kDa when denatured. The pH optimum of the enzyme is 7.8 and the KCl optimum concentration is 40 mmol/L. This topoisomerase is independent of ATP and Mg²⁺. N-Ethylmaleimide, 4-chloromercuribenzoate, SDS, guanidinium chloride, polyethylene glycol, heparin and ethidium bromide inhibit its activity, while novobiocin, nalidixic acid, Triton X-100 and chloroquine do not. Polyamines, and histone, H1 stimulate the topoisomerase activity. We classify this DNA topoisomerase as typeI and eukaryotic. Conversion of the topoisomerase to a nonspecific endonuclease at increased temperature is proposed.     

Subcellular localization of ribonuclease isoenzymes in tobacco mesophyll protoplasts and their changes induced by infection of PVY

Changes in the content and subcellular localization of ribonuclease isoenzymes were determined in mesophyll protoplasts prepared fromNicotiana tabacum L. cv. Samsun from healthy and potato virus Y (PVY) infected plants. Intact chloroplasts, mitochondria and soluble cytosolic proteins were obtained after protoplast disintegration by means of differential centrifugation. The 1 000g pellet from healthy protoplasts contained 7.3 %, the 15 000g pellet 13.5 % and 15 000g supernatant 82.1 % of the total activity of ribonucleases. The 1 000g pellet from infected protoplasts contained 10.4%, the 15 000g pellet 10.0% and 15 000g supernatant 89.6 % of the total activity of ribonucleases. The activity of these enzymes in infected protoplasts was enhanced in crude homogenate to 137.0 % (P<0.001), in 1 000g pellet to 194.8 % (P<0.001), in 15 000g pellet to 101.3 % (NS), and in 15 000g supernatant to 149.4 % (P<0.001) of that in healthy noninoculated protoplasts.     

Metabolic relevance of selenium in the insectCorcyra cephalonica

Requirement, uptake, and subcellular distribution of Na₂ ⁷⁵SeO₃ in the larvae of the insectC. cephalonica was investigated. That Se is well tolerated byC. cephalonica upto an added level of 2 ppm in the diet is suggested by the observed increase in body weight, total protein, and succinate dehydrogenase levels. Significant increases in the State 3 respiration ensued with Se supplementation up to 2 ppm in the mitochondrial oxidation of D-glycerol 1-phosphate, succinate and NADH, along with concomitant unaltered State 4 respiration, leading to enhanced RCR values. Maximal uptake of⁷⁵Se was registered in the larvae maintained on basal diet when subjected to short-term exposure to 0.5 ppm⁷⁵Se level. When exposure level was further increased up to 20 ppm, the observed decrease in the uptake of⁷⁵Se suggested that Se status of larvae itself controlled the tissue uptake. Subcellular distribution pattern revealed maximal incorporation of⁷⁵Se (cpm/g tissue) in the supernatant fraction, whereas, maximal specific⁷⁵Se activity (cpm/mg protein) was associated with the mitochondrial fraction. Autoradiography of the soluble fractions indicated the presence of single selenoprotein in the larval group with short term 2 ppm⁷⁵Se exposure. Inherent Se controls both the extent and the nature of distribution of mitochondrial⁷⁵Se incorporation. Uptake of⁴⁵Ca by the insect mitochondria was enhanced by dietary Se up to 2 ppm but was unaffected by addition ofin vitro ⁷⁵Se in the medium. A more fundamental role for Se in the mitochondrial energy metabolism emerges from these studies.     

Cytological relationships of selected species ofPanicum L.

The cytological investigation of 12 taxa ofPanicum L. revealed that the vast majority of them have the basic number x=9 at different ploidy levels. The basic number x=8 was recorded only in the tetraploid speciesP. maximum with 2n=32. The diploid number 2n=18 was encountered inP. capillare, P. laevifolium, P. antidotale andP. coloratum (2) with 3B-chromosomes recorded in the latter species. The tetraploid chromosome number 2n=36 was found to exist inP. miliaceum, P. miliare, P. coloratum (1) andP. virgatum. The hexaploid number 2n=54 was recorded inP. bulbosum, P. dichotomiflorum andP. esculentum. The karyotypes of all accessions were mostly symmetrical and mainly comprised of meta- and submetacentric chromosomes with little variation in length among them within each karyotype. Investigation of chromosome association during metaphase I of meiosis revealed that the frequency of bivalents/cell was the highest among all investigated diploid, tetraploid and hexaploid accessions. Univalents were also frequently encountered in various accessions. These results may indicate that segmental alloploidy has been the major process by which polyploid species have originated.     

Effect of 2,4-dichlorophenoxyacetic acid on nuclear division in stem segments ofPisum sativum L. cultivatedin vitro

We have investigated the influence of 2,4-dichlorophenoxyacetic acid (2,4-D) on the appearance of nuclear fragments, caused by direct nuclear division, as well as on mitotic activity in cultivated internodial stem segments ofPisum sativum L., cv. Bördi, during 180 d of cultivation. Direct nuclear fragmentation (dNF) was indicated by the shape and structure of the nucleus as well as by the occurrence of 1C- and 3C-values of DNA, investigated cytophotometrically. The dNF occurred during the whole cultivation period in segments treated by 2,4-D in concentrations from 4 to 32 mg 1^?1. In the presence of 2 mg 1^?1 of 2,4-D the dNF existed in the explants only up to 90 d. Mitotic activity was not observed in the 2,4-D-free control but occurred during the whole cultivation period when 2,4-D was added in concentrations from 2 to 16 mg l^?1. In the presence of 32 mg l^?1 of 2,4-D the level of mitotic activity was very low at the beginning and ceased after 60 d in culture.     

Flow cytometric estimation of nuclear DNA amount in diploid bananas (Musa acuminata andM. balbisiana)

Cell nuclei were isolated from leaf tissues of wild banana (Musa balbisiana, M. acuminata ssp.banksii andM. acuminata ssp.errans) and of the two vegetative clones of diploid cultivar “Pisang Mas”. Relative fluorescence intensity was measured on propidium iodide-stained nuclei by flow cytometry. Nuclei isolated fromGlycine max with known nuclear genome size were used as internal standard to determine nuclear DNA content ofMusa in absolute units. The results of the study showed that the size of nuclear genome ofMusa is smaller than previously estimated. In general, it is smaller in comparison with many other angiosperms. Furthermore, it was found that nuclear DNA content ofM. balbisiana (genome BB) is significantly lower than that ofM. acuminata subspecies and cultivars (genome AA). This finding should permit estimation of genome composition in triploidMusa clones with expected hybrid composition. Flow cytometry is proposed as a useful technique with potential applications in taxonomy, breeding and biotechnology ofMusa.     

The distribution of BrKα/RbKα peak intensity ratio in human whole blood samples

A preliminary study was conducted to compare the BrKα/RbKα peak intensity ratio in colorectal cancer (CRC) patients and healthy subjects (controls) in Isreal. Blood samples were obtained from 61 CRC patients and 124 controls. The controls represent a normal population from different areas in Israel. Three hundred microliters of wet whole blood samples were analyzed by the EDXRF method. A weighted mean of 2.45±0.38 Br/Rb ratio was obtained for CRC patients, as compared with 3.28±0.40 Br/Rb ratio for controls. The mean value for controls represents only 112 subjects, since 12 individuals of the control group suffered from some other diseases and therefore were not included for the mean value calculation in this group. The results indicate that the Br/Rb ratio in CRC patients is significantly lower (p<0.05) than in controls. No significant difference was found in Br/Rb ratio correlated to age and sex. It was not possible to draw final conclusion concerning the relationship between the Br/Rb ratio and the malignancy stage, since there was a relatively small number of cases in each stage under investigation.     

Restriction endonucleases R.EcoKI and R.EcoR124I are probably located in different environments within the bacterial cell

We describe the phenomenon of a transient state of R124I restriction deficiency after long-term storage of theE. coli[pCP1005] strain at 4°C, or after growth of the culture in synthetic M9 medium with the nonmutagenic solvent dimethyl sulfoxide. The unusual high reversion from the R⁺ ₁₂₄ to the R^? ₁₂₄ phenotype was observed only inE. coli strain transformed with the high-copy number plasmid pCP1005 carryingECoR124IhsdR, M and S genes cloned, but not with strains carrying the natural conjugative plasmid R124. The effect of both treatments on the expression ofEcoR124I phenotype in relation to the possible location of R.EcoR124I restriction endonuclease inE. coli is discussed.