NMR studies of the variant-3 neurotoxin from Centruroides sculpturatus Ewing

We report a preliminary high-resolution proton nuclear magnetic resonance characterization of the variant-3 toxin from the scorpion Centruroides sculpturatus Ewing (range Southwestern USA). This toxin assumes a well defined folded conformation in aqueous solutions at room temperature and undergoes reversible thermal denaturation. A number of amide hydrogens exhibit exchange life times varying from several minutes to several hours. A few tentative assignments of the low field aromatic CH resonances has been made on the basis of 2D-COSY and NOE experiments. The upfield shifts exhibited by Trp-47 suggest a unique microenvironment for this residue. The NMR data suggest that there is some degree of correlation between the solution structure of the variant-3 toxin and its crystallographic structure. Our studies provide a basis for a detailed elucidation of the structure-function relationships of these interesting scorpion toxins which bind to the sodium channels of excitable membranes and delay sodium current inactivation.     

Epoxidation of aflatoxin B1 by Aspergillus flavus microsomes in vitro: interaction with DNA and formation of aflatoxin B1-glutathione conjugate

Metabolism of aflatoxin B1 (AFB1) by subcellular preparations of Aspergillus flavus is least understood. The results reported here have demonstrated for the first time the epoxidation of AFB1 and subsequent conjugation with glutathione (GSH). Microsomes prepared from toxigenic mycelia catalysed [3H]AFB1 to calf thymus DNA to a greater extent (approximately 2-fold) as compared to that of non-toxigenic. The binding of [3H]AFB1 to exogenous and A. flavus nuclear DNA catalyzed by A. flavus microsomes was found to be comparable with that of mammalian extrahepatic tissue such as lung. Addition of phenobarbitone to the growing cultures resulted in 1.5-fold increase in [3H]AFB1-DNA binding mediated by microsomes prepared from either of the two strains. Tolnaftate, an inhibitor of aflatoxin synthesis enhanced the epoxidation rate in a dose-related manner. The binding of [3H]AFB1 to DNA catalyzed by A. flavus microsomes was significantly reduced (50% of control) upon addition of hamster liver cytosol, thereby substantiating the formation of the carcinogen adduct with DNA as reported in mammalian tissues. The metabolite formed by subcellular preparation of A. flavus was found to be AFB1-GSH having Rf value (6.5) similar to that obtained for mammalian liver preparations.     

Synthesis of lysine-containing fragments of the Proteus mirabilis O27 O-specific polysaccharide and neoglycoconjugates therefrom.

Amide-linked lysine mono- and di-uronic acid fragments of the O-specific polysaccharide from P. mirabilis O27 have been synthesised. N epsilon-Boc-L-lysine tert-butyl ester was condensed with 2-azidoethyl glycosides of glucuronic acid and beta-D-GlcpNAc-(1----3)-beta-D-GlcpA. Transformation of the products into 2-acrylamidoethyl glycosides, followed by deprotection using trifluoroacetic acid, gave the target monomers that were converted into high-molecular-weight copolymer-type neoglycoconjugates.     

Resistance in certain maize cultivars to first and third-instar Chilo partellus larvae

The resistance/susceptibility levels of ten maize (Zea mays L.) cultivars to neonates and 3rd-instar larvae of Chilo partellus (Swinhoe) (Lepidoptera: Pyralidae) were measured in the field and screenhouse experiments. The maize cultivars tested were: Inbred A, Mp 704, V-37, Population 10, Hybrid 511, Katumani Composite B, ER-29 SVR, Poza Rica 7832, ICZ2-CM, and MMV 400. Most cultivars were found susceptible to 3rd-instar larvae based on the incidence of dead heart symptoms, foliar feeding and stem tunnelling caused by stem borers in both the field and screenhouse experiments. However, the maize cultivars V-37, MMV 400 and Poza Rica 7832 sustained significantly lesser damage than the other cultivars when infested with neonates or 3rd instar larvae.The screenhouse experiments conducted with five maize cultivars, i.e. Inbred A, Mp 704, V-37, ER-29 SVR, and Poza Rica 7832 showed that neonates fed less on the resistant than the susceptible maize cultivars but feeding by the 3rd instars was equally high on all the maize cultivars except Mp 704 on which the larvae fed less than the others. In the cultivars infested with neonates, the mean period for 50% moth emergence P₅₀ was longest on Mp 704 followed by Poza Rica 7832, ER-29 SVR, V-37 and Inbred A. In the cultivars infested with 3rd-instars, P₅₀ was longest for Poza Rica 7832 followed equally by the remaining cultivars.On the basis of the evidence presented we here conclude that neonates of C. partellus are sensitive to the growth inhibiting effects of the resistant maize cultivars Mp 704, V-37, and Poza Rica 7832 but the older larvae are insensitive to these effects of Mp 704 and V-37 but not of Poza Rica 7832.     

Identification and characterization of the enzymatic activity of zeta-crystallin from guinea pig lens. A novel NADPH:quinone oxidoreductase.

zeta-Crystallin is a major protein in the lens of certain mammals. In guinea pigs it comprises 10% of the total lens protein, and it has been shown that a mutation in the zeta-crystallin gene is associated with autosomal dominant congenital cataract. As with several other lens crystallins of limited phylogenetic distribution, zeta-crystallin has been characterized as an "enzyme/crystallin" based on its ability to reduce catalytically the electron acceptor 2,6-dichlorophenolindophenol. We report here that certain naturally occurring quinones are good substrates for the enzymatic activity of zeta-crystallin. Among the various quinones tested, the orthoquinones 1,2-naphthoquinone and 9,10-phenanthrenequinone were the best substrates whereas menadione, ubiquinone, 9,10-anthraquinone, vitamins K1 and K2 were inactive as substrates. This quinone reductase activity was NADPH specific and exhibited typical Michaelis-Menten kinetics. Activity was sensitive to heat and sulfhydryl reagents but was very stable on freezing. Dicumarol (Ki = 1.3 x 10(-5) M) and nitrofurantoin (Ki = 1.4 x 10(-5) M) inhibited the activity competitively with respect to the electron acceptor, quinone. NADPH protected the enzyme against inactivation caused by heat, N-ethylmaleimide, or H2O2. Electron paramagnetic resonance spectroscopy of the reaction products showed formation of a semiquinone radical. The enzyme activity was associated with O2 consumption, generation of O2- and H2O2, and reduction of ferricytochrome c. These properties indicate that the enzyme acts through a one-electron transfer process. The substrate specificity, reaction characteristics, and physicochemical properties of zeta-crystallin demonstrate that it is an active NADPH:quinone oxidoreductase distinct from quinone reductases described previously.     

Identification of nitroxide radioprotectors.

The nitroxide Tempol, a stable free radical, has recently been shown to protect mammalian cells against several forms of oxidative stress including radiation-induced cytotoxicity. To extend this observation, six additional water-soluble nitroxides with different structural features were evaluated for potential radioprotective properties using Chinese hamster V79 cells and clonogenic assays. Nitroxides (10 mM) were added 10 min prior to radiation exposure and full radiation dose-response curves were determined. In addition to Tempol, five of the six nitroxides afforded in vitro radioprotection. The best protectors were found to be the positively charged nitroxides, Tempamine and 3-aminomethyl-PROXYL, with protection factors of 2.3 and 2.4, respectively, compared with Tempol, which had a protection factor of 1.3. 3-Carboxy-PROXYL, a negatively charged nitroxide, provided minimal protection. DNA binding characteristics as studied by nonequilibrium dialysis of DNA with each of the nitroxides demonstrated that Tempamine and 3-amino-methyl-PROXYL bound more strongly to DNA than did Tempol. Since DNA is assumed to be the target of radiation-induced cytotoxicity, differences in protection may be explained by variabilities in affinity of the protector for the target. This study establishes nitroxides as a general class of new nonthiol radioprotectors and suggests other parameters that may be exploited to find even better nitroxide-induced radioprotection.     

Allozyme variation among biotypes of the brown planthopperNilaparvata lugens in the Philippines

Allozyme variation was studied in threeNilaparvata lugens biotypes infesting specific rice varieties and a biotype infesting a weed grass,Leersia hexandra. Of the 20 enzymes inN. lugens for which activity was noted, 9 were polymorphic. Eleven enzyme loci were monomorphic for the same allele in all biotype populations; the rest were polymorphic for two or more alleles. The mean number of alleles per polymorphic locus was 2.3, while the mean number of alleles per locus was 1.5; heterozygosity ranged from 0.02 to 0.06 (biotype 1 > biotype 3 >Leersia-infesting biotype > biotype 2). Allelic frequency differences were observed in five loci among the four biotypes. However, the coefficient of genetic identity (I) of 0.99+ showed that the four biotype populations were genetically close relatives or merely populations ofN. lugens undergoing genetic differentiation. This work was partly supported by a financial grant received from the Directorate for Technical Cooperation and Humanitarian Aid, Switzerland.     

Influence of phosphate and zinc on growth,nodulation and mineral composition of chickpea (Cicer arietinum L.) under salt stress

Zn²⁺ at 5 ppm and phosphate at 20 and 40 ppm improved the growth and nodulation of chickpea (Cicer arietinum L.) at two levels of salinity (4.34 and 8.3 dS m^–1). Augmentation with Zn²⁺ at 5 ppm provided protection to the plant under saline conditions by reducing the Na⁺:K⁺ ratio in the shoot. The shoot nitrogen content with 5 ppm Zn²⁺ and 20 ppm phosphate was equal to that of a non-saline control. No significant effect on nitrogenase activity was observed.     

In vitro growth of urinaryEscherichia coli related to siderophore production

Thirty seven strains ofEscherichia coli isolated from the urine of patients with acute symptomatic urinary tract infection were examined for siderophore production: hydroxamate (aerobactin) and phenolate (enterochelin). All the strains were found to produce varying amounts of enterochelin. With the chemical assay, 24.3% strains were aerobactin producers, while 43.2% were positive in the bio-assay. All the aerobactin producers carried the aerobactin receptor on their surface. Attempts to correlate siderophore production with growth in minimal and iron-depleted medium showed that there was a positive quantitative correlation between enterochelin production and growth of organisms under iron depletion. Aerobactin production failed to give an additional advantage of growth to strains producing enterochelin.