Inducer effect of Tween 20 permeabilization treatment used for release of stored tropane alkaloids inDatura innoxia Mill. hairy root cultures

Summary The effects of Tween 20 as permeabilizing agent on tropane alkaloids fromDatura innoxia Mill. hairy root cultures have been studied. The kinetics of the alkaloid release is detailed and shows three different stages: an initial rapid increase of the alkaloid level within the roots and in the culture medium, followed by a slower but higher increase of the alkaloid concentration in the medium. During this phase, the alkaloid concentration within the roots returned to a lower value. Finally, after a longer time, the quantity of hyoscyamine in the medium decreased significantly with a variable rate. According to the total alkaloid content per flask determinations under different conditions, it is clearly demonstrated that Tween treatment permeabilized the roots, but also acted as an inducer.     

A case of hemoglobin D Punjab in Tunisia. Characterization and structural study

Hb D Punjab is a hemoglobin abnormality due to the substitution of a glutamic acid by a glutamic on the hemoglobin beta chain: beta 121 Glu----Gln. Authors report here the first case of this rare variant in a tunisian family.     

Haplotypes in tribal Indians bearing the sickle gene: evidence for the unicentric origin of the beta S mutation and the unicentric origin of the tribal populations of India

To determine the origin of sickle cell anemia (SS) in India, we analyzed haplotypes of the beta gene cluster in beta S-carrying individuals belonging to tribal populations living in the Nilgiris region of southern India and complemented the available data on tribes of east-central India. We found that in the Nilgiris tribes chromosomes bearing the beta S gene are linked in 91% of the cases to the "Asian" (Arab-Indian) haplotype (although 25% of the haplotypes had the epsilon polymorphic site negative, making the 5' portion of the haplotype identical with the African Senegal haplotype). These XmnI (+) chromosomes were associated with high G gamma expression (67.2 +/- 5.9%) and a high percentage of Hb F (15.5 +/- 7.9%; range, 6-25.3%). We have similar findings for tribal groups from west-central India (Gujarat). In east-central India we have confirmed the data of others, finding the same haplotype linked to beta S in tribes living in the east (Orissa, Andhra Pradesh). We conclude that the beta S gene in presently isolated and disperse tribal populations in India is associated with one predominant typical haplotype, suggesting a unicentric origin of the mutation in India. In addition, this finding implies a unicentric origin of the tribal populations themselves: The gene must have arisen and spread before tribal dispersion. Furthermore, we find extremely high frequencies of the (-alpha) haplotype in the Nilgiris (0.89) and in Gujarat (0.95). The beta S gene linkage to a high Hb F-expressing haplotype and the high incidence of alpha-thalassemia predict a mild phenotypical expression of sickle cell anemia in India.     

Nitric oxide,a biological effector

Nitric oxide has been used for more than 20 years as an electron paramagnetic resonance probe of oxygen binding sites in oxygen-carriers and oxygen-metabolizing metalloenzymes. The high reactivity of NO with oxygen and the superoxide anion and its high affinity for metalloproteins led biochemists to consider NO as a highly toxic compound for a living cell. This assertion has recently been reconsidered following a number of discoveries of great significance: the finding of the activation of guanylate cyclase by NO, the recognition that NO is the precursor of nitrite and nitrate ions released in the activation of macrophages by endotoxin and cytokities, evidence that NO is an Endothelium-Derived Relaxing Factor, and the discovery of NO-biosynthesis from l-arginine, a pathway common in various biological cell-to-cell signalling processes. It is now admitted that NO plays a key bioregulatory role within mammalian cells, between cells of different types and in the host defence response. In the present review we have attempted to give a general picture of what is known of the chemical, physical, biochemical and biophysical properties of NO among the various nitrogen oxides. We have focussed on the structural information that can be obtained by electron paramagnetic resonance spectroscopy of nitrosyl-metalloprotein complexes. Finally we have shown how molecular targets of nitric oxide can be characterized, within whole cells, by electron paramagnetic resonance spectroscopy.Abbreviations BCG Bacillus Calmette-Guérin - CcO cytochrome c oxidase - cGMP cyclic GMP - Cyt. cd ₁ cytochrome cd ₁ or nitrite reductase from Pseudomonas aeruginosa - DPG 2,3-diphosphoglycerate - EDRF endothelium-derived relaxing factor - EPR electron paramagnetic resonance - GC guanylate cyclase - GMN, GDN, GTN glyceryl mono-, di-, trinitrate - GSH, GSSG reduced and oxidized glutathione - GSH-ST glutathione S-transferase - Hb hemoglobin - Hb³⁺ ferrihemoglobin - IFN- interferon gamma - IHP inositol hexaphosphate - LPS lipopolysaccharide from E. coli - Mb myoglobin - NMMA N^G-monomethyl-l-arginine - P-450 cytochrome P-450 - P-420 cytochrome P-420 - P1, P2, P3, P7 isoperoxidases from turnip - SHF superhyperfine structure - TDO tryptophan 2,3-dioxygenase from Pseudomonas fuorescens - TNF tumor necrosis factor This review is based on a talk given by YH at the first European Meeting of Groupe d'Application de la Résonance Paramagnétique Electronique in Lyon, 10–11 January 1990. It has been up-dated to December 1990 Offprint requests to: Y. Henry     

Effect of culture conditions on Agrobacterium-mediated transformation in datura

A two step selection procedure is described for high frequency transformation and regeneration of transgenic plants by coculture of leaf discs of Datura innoxia with Agrobacterium tumefaciens carrying binary vectors. Leaf discs were cocultured with disarmed A. tumefaciens vectors pGS Glucl, pGSTRN943, pGV2260 and pBI121, and subcultured on regeneration media containing kanamycin. Kanamycinresistant, putatively transformed callus and vegetative buds were isolated, and subcultured on media containing reduced amounts of growth regulators and kanamycin to induce shooting. Rooted shoots produced normal fertile plants. Transformation frequency was related to duration of preculture, co-culture, and the bacterial strain used. With pGS Glue 1, a 3 day co-culture resulted in 70% of leaf discs being transformed. Transformation was confirmed by histochemical test for GUS activity, by the ability of leaf discs to initiate callus and from NPTII test, and Southern blot analysis. Progeny of the transgenic plants showed Mendelian segregation for kanamycin resistance.     

Abnormal Fossil Upper Molar of Pongo from Thailand: Quaternary Climatic Changes in Southeast Asia as a Possible Cause

We interpret an unusual left M ³ attributed to Pongo from the Late Middle Pleistocene of Thailand to be an abnormal tooth rather than a supernumerary molar. Its peculiar morphology cannot be related to classical known causes that affect the dental germ: Gemination, schizodontia, synodontia. The ontogenic mechanism that might have led to the morphology of the tooth perhaps reflects stress related to a phase of marked climatic changes during the Late Middle Pleistocene in Southeast Asia.     

Error rate for imputation from the Illumina BovineSNP50 chip to the Illumina BovineHD chip

Background

Imputation of genotypes from low-density to higher density chips is a cost-effective method to obtain high-density genotypes for many animals, based on genotypes of only a relatively small subset of animals (reference population) on the high-density chip. Several factors influence the accuracy of imputation and our objective was to investigate the effects of the size of the reference population used for imputation and of the imputation method used and its parameters. Imputation of genotypes was carried out from 50 000 (moderate-density) to 777 000 (high-density) SNPs (single nucleotide polymorphisms).

Methods

The effect of reference population size was studied in two datasets: one with 548 and one with 1289 Holstein animals, genotyped with the Illumina BovineHD chip (777 k SNPs). A third dataset included the 548 animals genotyped with the 777 k SNP chip and 2200 animals genotyped with the Illumina BovineSNP50 chip. In each dataset, 60 animals were chosen as validation animals, for which all high-density genotypes were masked, except for the Illumina BovineSNP50 markers. Imputation was studied in a subset of six chromosomes, using the imputation software programs Beagle and DAGPHASE.

Results

Imputation with DAGPHASE and Beagle resulted in 1.91% and 0.87% allelic imputation error rates in the dataset with 548 high-density genotypes, when scale and shift parameters were 2.0 and 0.1, and 1.0 and 0.0, respectively. When Beagle was used alone, the imputation error rate was 0.67%. If the information obtained by Beagle was subsequently used in DAGPHASE, imputation error rates were slightly higher (0.71%). When 2200 moderate-density genotypes were added and Beagle was used alone, imputation error rates were slightly lower (0.64%). The least imputation errors were obtained with Beagle in the reference set with 1289 high-density genotypes (0.41%).

Conclusions

For imputation of genotypes from the 50 k to the 777 k SNP chip, Beagle gave the lowest allelic imputation error rates. Imputation error rates decreased with increasing size of the reference population. For applications for which computing time is limiting, DAGPHASE using information from Beagle can be considered as an alternative, since it reduces computation time and increases imputation error rates only slightly.     

Rabies diagnosis for developing countries

Background

Canine rabies is a neglected disease causing 55,000 human deaths worldwide per year, and 99% of all cases are transmitted by dog bites. In N''Djaména, the capital of Chad, rabies is endemic with an incidence of 1.71/1,000 dogs (95% C.I. 1.45–1.98). The gold standard of rabies diagnosis is the direct immunofluorescent antibody (DFA) test, requiring a fluorescent microscope. The Centers for Disease Control and Prevention (CDC, Atlanta, United States of America) developed a histochemical test using low-cost light microscopy, the direct rapid immunohistochemical test (dRIT).

Methodology/Principal Findings

We evaluated the dRIT in the Chadian National Veterinary Laboratory in N''Djaména by testing 35 fresh samples parallel with both the DFA and dRIT. Additional retests (n = 68 in Chad, n = 74 at CDC) by DFA and dRIT of stored samples enhanced the power of the evaluation. All samples were from dogs, cats, and in one case from a bat. The dRIT performed very well compared to DFA. We found a 100% agreement of the dRIT and DFA in fresh samples (n = 35). Results of retesting at CDC and in Chad depended on the condition of samples. When the sample was in good condition (fresh brain tissue), we found simple Cohen''s kappa coefficient related to the DFA diagnostic results in fresh tissue of 0.87 (95% C.I. 0.63–1) up to 1. For poor quality samples, the kappa values were between 0.13 (95% C.I. −0.15–0.40) and 0.48 (95% C.I. 0.14–0.82). For samples stored in glycerol, dRIT results were more likely to agree with DFA testing in fresh samples than the DFA retesting.

Conclusion/Significance

The dRIT is as reliable a diagnostic method as the gold standard (DFA) for fresh samples. It has an advantage of requiring only light microscopy, which is 10 times less expensive than a fluorescence microscope. Reduced cost suggests high potential for making rabies diagnosis available in other cities and rural areas of Africa for large populations for which a capacity for diagnosis will contribute to rabies control.     

Intervention by nitric oxide, NO, and its oxide derivatives particularly in mammals

Nitric oxide (NO) is a natural and stable free radical produced in soil and water by the bacteriological reduction of nitrites and nitrates and in animals by the enzyme oxidation of L-arginine. NO is biosynthesised by finely regulated enzymatic systems called NO-synthases and readily diffuses through tissues. It reacts rapidly with hemoproteins and iron-sulphur centers to form nitrosylated compounds. It oxidises more slowly to form nitrogen oxides that nitrosate thiols into thionitrite. NO is transported in these various forms and released spontaneously or through yet unclear mechanisms into most cells; it also regulates oxygen consumption at the mitochondrial respiratory chain level through interaction with cytochrome oxidase. In the cardiovascular system, NO lowers blood pressure by activating a hemoprotein, the guanylate cyclase present in muscle cells; through such interaction it acts also as a neuromediator and neuromodulator in the nervous system. However, many of NO's roles result from rapid coupling to other radicals; for example, it reacts with the superoxide anion (O2-) to form oxoperoxinitrate (ONOO-, also known as peroxynitrite). This strong oxidant of metallic centers, thiols, and antioxidants is also able to convert tyrosine to 3-nitrotyrosine and to act upon tyrosine residues contained in proteins. The biological aspects of the roles of NO are presented with particular respect to the rapid interactions of NO with hemoproteins' iron and other radicals. Concurrently, NO oxidation enables nitrosation reactions primarily of thiols but ultimately of nucleic bases. The thionitrite function (R-S-NO) thus formed and the dimerisation and nitration of tyrosine residues are protein post-translational modifications that are being investigated in animals.     

The endogenous neurotransmitter, serotonin, modifies neuronal nitric oxide synthase activities

Serotonin, an important neurotransmitter, is colocalized with neuronal nitric oxide synthase (nNOS), a homodimeric enzyme which catalyzes the production of nitric oxide (NO(.-)) and/or oxygen species. As many interactions have been reported between the nitrergic and serotoninergic systems, we studied the effect of serotonin on nNOS activities. Our results reveal that nNOS is activated by serotonin as both NADPH consumption and oxyhemoglobin (OxyHb) oxidation were enhanced. The generation of L-citrulline from L-arginine (L-Arg) was not affected by serotonin in the range of 0-200 microM, suggesting an additional production of oxygen-derived species. But 5-hydroxytryptamine (5HT) induced the formation of both O and H(2)O(2) by nNOS, as evidenced by electron paramagnetic resonance (EPR) and by using specific spin traps. Overall, these results demonstrate that serotonin is able to activate nNOS, leading to the generation of reactive oxygen species (ROS) in addition to the NO(.-) production. Such a property must be considered in vivo as various nNOS-derived products mediate different signaling pathways.