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).     

Techniques and high resolution DNA size markers for pulsed field gel electrophoresis

High resolution DNA size markers are described for pulsed field gel electrophoresis (PFGE). These markers provide resolution of 10–20 kbp over a size range from 10 kbp to more than 400 kbp and are produced by partial restriction digestion of lambda phage DNA concatemers (λ ladder). High resolution markers extending to over 400 kbp are made by partial restriction digestion of λ ladder embedded in agarose. Detailed methods are described for marker production and for DNA separation by contour-clamped homogeneous electric field (CHEF) electrophoresis. These markers and methods are useful for a variety of high resolution DNA mapping by PFGE.     

Alcohol dehydrogenase isoenzymes fromNicotiana tabacum include ADH of bothN. sylvestris andN. tomentosiformis

Electrophoretic separation of seed alcohol dehydrogenase (ADH) fromNicotiana tabacum on 12% starch gels at pH 7.8 produced only one band with an apparent R_f of 0.65, which confirmed earlier reports. The same was found with pollen ADH. However, in polyacrylamide gel isoelectric focusing, seed ADH separated into three distinct bands with apparent pI of 5.33, 5.42 and 5.50. The pI 5.33 isoenzyme was found to be the essential form inN. sylvestris seeds. The analysis of charge properties ofN. tomentosiformis seed ADH showed only one isoenzyme with pI of 5.56. These results present further evidence thatN. tabacum has arisen from a cross between aN. sylvestris predecessor and an ancestral type ofN. tomentosiformis. The presence of the pI 5.42 form inN. tabacum is consistent with the reported formation of heterodimeric ADH in tobacco hybrids.     

Yeast artificial chromosome cloning

Yeast artificial chromosome (YAC) cloning systems enable the cloning of DNA stretches of 50 to well over 2000 kb. This makes it possible to study large intact regions of DNA in detail, by restriction mapping the YAC to produce a physical map and by examining the YAC for coding sequences or genes. YACs are important for their ability to clone the complete sequences of large genes or gene complexes that exceed the size limit for cloning in conventional bacterial cloning vectors like plasmids (up to 10 kb), bacteriophage (15 kb), and cosmids (50 kb). A major advantage of cloning in yeast, a eukaryotc. is that many sequences that are unstable, underrepresented, or absent when cloned into prokaryotic systems, remain stable and intact in YAC clones. It is possible to reinlroduce YACs intact into mammalian cells where the introduced mammalian genes are expressed and used to study the functions of genes in the context of flanking sequences. The correct prolein processing mechanisms are present in the mammalian cells to ensure that a viable protein product is produced.     

Diversity in two candidate malaria vaccine antigens in different Indian strains ofPlasmodium falciparum

Polymorphism in two malarial antigens, merozoite surface antigen-1 (MSA-1) and ring erythrocyte surface antigen (RESA), has been characterized in four different Indian strains ofPlasmodium falciparum. The Indian strains were obtained from two malaria endemic regions of India, viz. Surat (Gujarat) and Delhi, and established in culture. Monoclonal and polyclonal antibodies raised against different domains of these antigens were used in the study. In MSA-1 a novel intragenic crossover was detected in the central conserved domain in two of the Indian strains. The repeat domain of RESA was found to be absent in the two strains ofP. falciparum isolated from Surat. These differences in immunoreactivity have been extended to the DNA level by appropriate PCR studies. MSA-1 and RESA are candidate vaccine antigens and these diversities will have an important bearing on the design of a suitable malaria vaccine.     

Techniques de détection du VIH dans les milieux biologiques: Application à la recherche du virus dans le sperme

Growing numbers of HIV seronegative women want to have children with HIV infected partners by artificial insemination. The most sensitive assays for showing the presence of the virus are the coculture method and the DNA-PCR that are able to detect proviral load. HIV is detected from non spermatozoal mononuclear cells, seminal fluid but it was not found in spermatozoa fraction. But we know, by in vitro studies, that HIV can bound to and enter spermatozoa. Thus artificial insemination between HIV seropositive man and seronegative woman lead to a risk of contamination even with purified spermatozoa. Today, any virological assay is able to affirm that specimen is not infectious.     

Mitochondrial DNA restriction maps ofMus booduga,Mus terricolor andMus musculus tytleri

Restriction maps of milochondrial DNA of the Indian pygmy field miceMus booduga andM. terricolor, and the house mouseM. musculus tytleri were determined with seven six-cutter restriction enzymes. The restriction map of the mitochondrial DNA of the laboratory mouseM. m. domesticus was used as a reference. Pairwise comparison was made of the mitochondrial DNAs for the presence or absence of the restriction sites, and per cent sequence divergence was calculated. The results show that the sequence divergence betweenbooduga andterricolor is 8-7% while thedomesticus-tytleri andbooduga-terricolor groups are divergent by 16-3%. The mtDNA sequence divergences we have obtained suggest that thebooduga-terricolor lineage might not have diverged before the Southeast Asiancervicolor-cookii-caroli lineage during evolution of these lineages of the subgenusMus as inferred by others earlier. On the other hand, it seems likely that these lineages evolved in parallel.     

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.     

Testicule et sperme dans le Syndrome de Klinefelter chez l'adulte

Klinefelter's syndrome is characterised by azoospermia, gynecomastia and hypogonadotropic hypogonadism. The testes are reduced in volume. Microscopically, seminiferous tubules show a reduced diameter and a thickening of peritubular sheath. The seminiferous epithelium contains Sertoli cells with a clear typical nucleus and a heterogeneous nucleolus. Usually there are no germ cells. When present they degenerate at the spermatocyte stage. Very few achieve a complete maturation until the spermatozoon form. The interstitial gland appears relatively hypertrophied, due to the decrease in volume in the other components of the testis. Sometimes Leydig cells display an adenomatous morphology. Therefore the steroidogenic function is impaired. Azoospermia is a major event in Klinefelter's syndrome but sometimes restricted to severe oligozoospermia. In that cases, fertility may be somewhat preserved, and the caryotype usually reveals a mosaicism, 47,XY/47,XXY with a better prognosis than in pure syndrome.     

Effects of albumin and adenosine phosphates on iron transfer from ferric lactate

Ferric lactate is known to modify Ca²⁺ uptake by the cells. To enlighten the role of protein and ATP in this phenomenon, iron transfer from ferric lactate to albumin and adenosine polyphosphates was determined by electrophoresis. The order of iron affinity was ATP>ADP>AMP for the polyphosphates, and albumin does not compete for iron binding with the polyphosphates. The iron transfer to ATP was also observed in vivo by adsorption chromatography of the adenosine polyphosphates fraction from blood plasma of mice injected with ferric lactate plus ATP. In vitro iron and calcium uptake by Ehrlich ascites tumor cells showed that albumin and ATP decreased iron uptake, whereas calcium incorporation is diminished by albumin but augmented by ATP. This difference might be explained by albumin binding of ferric lactate that is inhibited from reaching cell structures, whereas ATP, known to be an inhibitor of iron polimerization, facilitates it.     

The interaction of the vanadyl (IV) cation with chondroitin sulfate A

The interaction of VO²⁺ with the muchopolysaccharide chondroitin sulfate A (CSA) has been investigated by electron absorption spectroscopy and infrared measurements in aqueous solutions at different pH-values and ligand to metal ratios up to 6:1. The generation of a VO(CSA)₂ species could be demonstrated. Coordination of the oxocation through the carboxylate group and the glycosidic oxygen of thed-glucuronate moieties is suggested. Infrared spectra of some poorly characterized solid VO/CSA complexes point to the same bonding characteristics. Preliminary results obtained at higher ligand to metal ratios suggest a different coordination behavior.     

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).     

The role of seed coats in seed viability

The seed coat is the seed’s primary defense against adverse environmental conditions. A hard seed coat protects the seed not only from mechanical stress but also from microorganism invasion and from temperature and humidity fluctuations during storage. Phenolic compounds in the seed coat contribute to seed hardness and inhibition of microorganism growth. During germination, the seed coat protects the seed from hydration stress and electrolyte leakage.     

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.     

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).     

CMVH,HSV et procréation

At least two members of the herpesviridae family, the human cytomegaloviruses (HCMV) and the herpes simplex viruses (HSV) can be found in human semen; but the role of the germ cells in the sexual transmission of these viruses is not clear. In teenagers and the adult population, sexual contact is considered to be a common mode of HCMV acquisition. HCMV was isolated from semen specimens of 33 % of HIV infected homosexual men, 20 % of HIV uninfected homosexual men and only of 2,4 % of healthy heterosexual men. Virus particles could be demonstrated by electron microscopy examination inside the sperm head as well as in the seminal liquid but at present, there is no direct evidence either for HCMV transmission via fertilization or for induction of fetal anomalies by vertical transmission. Transmission via donor semen is undoubtedly possible although not yet described and it may be safer to employ HCMV seronegative donor for all recipients, regardless of the recipient’s serologic status. The development of serologic assays that differentiate the two serotypes of HSV demonstrated the worldwide distribution of genital HSV-2 which has been increasing in many developed countries throughout the last two decades. In several studies, HSV-2 has been recovered from the male reproductive tract, specifically the prostate, seminal vesicles, vasa and testes, in the absence of active lesions. In contrast, tissue cultures of semen sampled during lesion-free periods had been uniformly negative for HSV. However recently, one report documents transmission of HSV-2 via therapeutic donor insemination and illustres the fact that semen might be a vehicle of transmission of HSV. At present, it is adequate to recommend that men, with a history of a recurrent genital herpes or who have a sexual partner with such a history, are excluded as potential semen donors. Further, in the near future, with the increase of asymptomatic viral shedding from the genital tract, the presence of HSV-2 antibody could be added as an exclusion criterion.     

Enzyme activities ofChlorophyllum molybditis andCortinarius melliolens at different sporophore stages

The intracellular enzyme activities ofChlorophyllum molybditis (Mayerex. Fr.) Massee andCortinarius melliolens Fries were determined at different stages of sporophore maturity. In both mushroom species, total amylase, α-amylase, proteinase, lipase, peroxidase, catalase and polyphenol oxidase activities were increased with sporophore maturity. In contrast, glucose-6-phosphatase activity was higher in the young sporophore than in very young and mature ones. All the enzymes assayed except cellulase and β-amylase showed greater activity in the pilei than in the stipes.C. molybditis showed greater total amylase, α-amylase, cellulase, proteinase, catalase and glucose-6-phosphatase activities thanC. melliolens.     

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.     

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.