Phage display selection can differentiate insecticidal activity of soybean cystatins

Plant cysteine proteinase inhibitors (phytocystatins) have been implicated as defensive molecules against Coleopteran and Hemipteran insect pests. Two soybean cystatins, soyacystatin N (scN) and soyacystatin L (scL), have 70% sequence identity but scN is a much more potent inhibitor of papain, vicilin peptidohydrolase and insect gut proteinases. When these cystatins were displayed on phage particles, papain-binding affinity and CPI activity of scN were substantially greater than those of scL, in direct correlation with their relative CPI activity as soluble recombinant proteins. Furthermore, scN substantially delayed cowpea weevil (Callosobruchus maculatus (F.)) growth and development in insect feeding bioassays, whereas scL was essentially inactive as an insecticide. Papain biopanning selection of phage-displayed soyacystatins resulted in a 200–1000-fold greater enrichment for scN relative to scL. These results establish that binding affinity of cystatins can be used in phage display biopanning procedures to select variants with greater insecticidal activity, illustrating the potential of phage display and biopanning selection for directed molecular evolution of biological activity of these plant defensive proteins.     

Identification and partial characterization of two species-specific repeat families in the great millet (Sorghum vulgare,Poaceae) genome

The 1.4 kbp Xba I and the 1.3 kbp EcoRI repeat families in great millet were partially characterized with respect to their genomic distribution and their homology with the EcoRI and Xba I families of five other millet DNAs. The digestions of great millet DNA using increasing amounts of the two enzymes show that these two families are disperse in nature. The hybridization of these two families to the genomic digests of great millet indicates that they are arranged in a clustered and scrambled manner. Similarly, the hybridization with the EcoRI and Xba I digests of five other millet DNAs reveals the speciesspecific nature of these two repeat families. The latter also hybridize to the total repetitive fraction of great millet isolated at a highly stringent temperature of 75°C suggesting that the members of these two families are probably largely homogeneous.     

Optimization of Culture Conditions for the Production of Extracellular Agarases from Newly Isolated Pseudomonas Aeruginosa AG LSL-11

Summary An agar-degrading bacterium capable of utilizing agar as sole source of carbon and energy was isolated from sea water by enrichment culture technique. The bacterium was identified as Pseudomonas aeruginosa and the culture conditions were standardized for the maximal production of extracellular agarases. The bacterium grew in the pH range 5.0–11.0, optimal between pH 7.0 and 8.0; temperature between 25 °C and 37 °C, optimal at 30 °C and sodium chloride concentration 0–8% and optimal at 2% respectively. The agarases secreted by Pseudomonas aeruginosa AG LSL-11 were inducible by agar and not by any other simple sugars tested. Maximal agarase production was observed at pH 8.0, and temperature 30 °C. The bacterium had no requirement for NaCl for both growth and production of agarases. The bacterium did not utilize other polysaccharides like ĸ-carrageenan, alginate, cellulose and CMC. The activity staining of partially purified agarase preparation after native-PAGE revealed the presence of three different agarases, agarase LSL-11a, LSL-11b and LSL-11c, whose molecular weights were estimated to be 76, 64 and 46 kDa respectively.     

Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture

An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain ofPseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F₄₂₀, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F₄₂₀ was three times that of FAD. Efficient utilization of alcohols in the presence of F₄₂₀ is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between thePseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance.     

Evidence for altered structure and impaired mitochondrial electron transport function in selenium deficiency

Selenium (Se) deficiency in the experimental models,Coturnix coturnix japonica andCorcyra cephalonica, resulted in impaired mitochondrial substrate oxidations and lowered thiol levels. Studies with respiratory inhibitors confirmed reduced mitochondrial electron transport enzyme activities, especially at cytochromec oxidase (COX), the terminal segment. Enhanced mitochondrial lipid peroxidation in Se deficiency was more pronounced in the heart tissue of the quail compared to other tissues. Glutathione peroxidase (GSH-Px) activity toward H₂O₂ and cumene hydroperoxide were generally low in the insect muscle tissue and activity toward H₂O₂ was maximal in the quail heart mitochondria that was not very sensitive to Se status. Lowered COX activity in Se deficiency was more directly correlated with the increased level of lipid peroxidation than with the GSH-Px activity measured, suggestive of Se mediated protective mechanisms independent of GSH-Px. Electron microscopic observations revealed structural changes such as loss of cristae with proliferative and degenerative changes of the mitochondria in Se deficiency. Involvement of Se in maintaining structure and functional efficiency of mitochondria is evident from the present study.     

A Kunitz trypsin inhibitor of Entada scandens seeds: another member with single disulfide bridge

Sword bean (Entada scandens) is a tree climber that belongs to Mimosoideae, a subfamily of Leguminosae. A potent Kunitz type trypsin inhibitor (ESTI) was purified to homogeneity from Entada scandens seeds by sequential ammonium sulfate precipitation, affinity chromatography on trypsin-Sepharose and DEAE-Sephacel ion-exchange chromatography. ESTI is a single polypeptide chain of 19,766 Da. Both native PAGE as well as isoelectric focusing showed a single inhibitor species with a pI of 7.43. MALDI-TOF analysis also confirmed the monomeric nature. The amino-terminal sequence of ESTI reveals significant homology to the Kunitz-type protease inhibitors of legume plants. ESTI is unique in that it contains a single disulfide bridge, and unlike other inhibitors from Mimosoideae species is a single chain polypeptide. ESTI inhibited bovine trypsin with a stoichiometry of 1:1 and the apparent K(i) was 4.9 x 10(-9) M. In vitro assay showed that ESTI inhibited the midgut proteinase of the fifth instar larvae of Rice moth (Corcyra cephalonica) with an IC(50) of 26.4+/-0.01 nM. ESTI exhibits a mixed type competitive inhibition at lower concentration and pure competitive at higher inhibitor concentrations. Phylogenetic analyses depicted a clear divergence of single disulfide containing inhibitors from other tree legume Kunitz inhibitors. The homology of ESTI to Kunitz inhibitors together with the absence of Bowman-Birk type inhibitors in sword bean further supports the theory that there exists an evolutionary relationship between the families of inhibitors found in Leguminosae.     

Isolation of a novel rod shaped methanogen growing on methanol and 2-propanol

A methanogen utilizing methanol was isolated from a reactor anaerobically degrading leaves of Leucaena leucocephala. The cells are rods of length 2.2 m with diameter 0.46 m also occurring in short chains. The cells stain gram-positive and the G+C content is 52.8 mol%. Growth occurred optimally with methanol (0.1 M) at 37°C and pH 7.4 with a doubling time of 7.5 h. The preferred sulfur source was glutathione. In addition to methanol, the isolate utilized 2-propanol as carbon and hydrogen source and also grew effectively on H₂+CO₂. Electron microscopy revealed the cell enveloped in triple layers consisting of a cell membrane and a cell wall separated by an electron transparent space with the nuclear zone at the centre of the cytoplasm. Presence of cytochromes was evident in the membrane fractions. The isolate possesses novel features.     

Carcinogenic potential of Indian alcoholic beverage (country liquor).

Epidemiological studies reveal that alcohol consumption is a risk factor for the cancer of the mouth, larynx, esophagus and various other organs. Of the various alcoholic beverages consumed in India, country liquors are widely consumed and that too by the economically weak section of the society. The present paper describes the experiments designed to investigate the effect of one brand of country liquor from Maharashtra State, India (which was found to be more potent in our earlier mutagenicity studies) for its carcinogenicity in two strains of mice and Syrian golden hamsters. The experimental animals received 10% liquor in drinking water from 2 months of age for 16 months. One percent ethanol treated animals served as positive controls. Together with long term bioassays, the transplacental carcinogenic effect of country liquor in the offspring of treated mothers, as well as in the breeders themselves was also investigated. Pregnant mothers were fed 10% liquor through drinking water from 12th day of gestation till weaning of the progeny. Then offspring were allowed to live without further treatment and mothers continued to get liquor treatment. In long term bioassays, liquor caused 22% total tumor incidence in male BALB/c mice and 28% in male Swiss mice. In female Swiss mice and in hamsters, liquor did not show any pronounced effect on tumor incidence. Similar negative results were obtained in case of offspring of treated mothers, but the offspring of liquor treated mothers had higher mortality prior to weaning as compared to those of untreated mothers.