Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14

Statistical optimization of culture conditions for production of a widely suited detergent protease from Bacillus sp. RGR-14 was carried out using a two-step approach. A quick identification of the important factors with simple screening experiment was followed by application of complex response surface design for further optimization. The production of extracellular alkaline protease by Bacillus sp. was favored in the presence of complex carbon and nitrogen sources, viz. starch, casamino acid and soybean meal. A reduced quadratic model was found to fit the alkaline protease production. Response surface analysis revealed the significant role of phosphate ions in determining alkaline protease production. A steep, stretched out response surface showed direct relation between the level of protease production and casamino acid and starch concentration in the medium. A 12.85 fold increase in protease production could be obtained within the design space. Protease production was found to be repressed in the presence of high concentrations of casamino acid. The model could be validated in up to 2 l shake flasks (3914 U ml⁻¹). The same statistical design could explain economic protease production in cost-effective medium as well.     

Maternal footprints of Southeast Asians in North India

We have analyzed 7,137 samples from 125 different caste, tribal and religious groups of India and 99 samples from three populations of Nepal for the length variation in the COII/tRNA(Lys) region of mtDNA. Samples showing length variation were subjected to detailed phylogenetic analysis based on HVS-I and informative coding region sequence variation. The overall frequencies of the 9-bp deletion and insertion variants in South Asia were 1.9 and 0.6%, respectively. We have also defined a novel deep-rooting haplogroup M43 and identified the rare haplogroup H14 in Indian populations carrying the 9-bp deletion by complete mtDNA sequencing. Moreover, we redefined haplogroup M6 and dissected it into two well-defined subclades. The presence of haplogroups F1 and B5a in Uttar Pradesh suggests minor maternal contribution from Southeast Asia to Northern India. The occurrence of haplogroup F1 in the Nepalese sample implies that Nepal might have served as a bridge for the flow of eastern lineages to India. The presence of R6 in the Nepalese, on the other hand, suggests that the gene flow between India and Nepal has been reciprocal.     

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.     

Sequential liquid-liquid extraction of d-amino acid oxidase from Trigonopsis variabilis

A method for isolation of d-amino acid oxidase (DAAO) from disrupted Trigonopsis variabilis cells has been developed. In an aqueous two-phase system consisting of PEG6000 (220 g l^–1), potassium phosphate (110 g l^–1, K₂HPO₄ + KH₂PO₄ = 10.1:1, mol mol^–1) and dl-methionine (11 g l^–1), the major portion of cellular proteins (87%) was partitioned into the salt phase. By sequential extraction, 48% of DAAO was recovered in PEG phase, giving a yield of 211 U mg protein^–1.     

D-Aspartate oxidase and D-amino acid oxidase are localised in the peroxisomes of terrestrial gastropods

D-Aspartate oxidase and D-amino acid oxidase were found in high activity in the tissues of representative species of terrestrial gastropods. Analytical subcellular fractionation demonstrated that both of these oxidases co-localised with the peroxisome markers, acyl-CoA oxidase and catalase, in the digestive gland homogenate. Electron microscopy of peak peroxisome fractions showed particles of uniform size with generally well preserved variably electron-dense matrices bounded by an apparently single limiting membrane. Many of the particles exhibited a core region of enhanced electron density. Catalase cytochemistry of peak fractions confirmed the peroxisome identity of the organelles. Peroxisome-enriched subcellular fractions were used to investigate the properties of gastropod D-aspartate oxidase and D-amino acid oxidase activities. The substrate and inhibitor specificities of the two activities demonstrated that two distinct enzymes were present analogous to, but not identical to, the equivalent mammalian peroxisomal enzymes.     

Estrogen increases hepatic lipase levels in inbred strains of mice: A possible mechanism for estrogen-dependent lowering of high density lipoprotein

We have shown mouse to be an useful animal model for studies on the estrogen-mediated synthesis and secretion of lipoproteins since, unlike in rats, low density lipoprotein receptors are not upregulated in mice [3]. This results into the elevation of plasma levels of apolipoprotein (apo) B and apoE, and lowering of apoA-I-containing particles. The mechanisms of apoB and apoE elevation by estrogen have been elucidated [6], but the mechanism of lowering of plasma levels of HDL is still not known. Among other factors, apoA-I, cholesterol ester transfer protein (CETP), scavenger receptor B1 (SR-B1), and hepatic lipase are potential candidates that modulate plasma levels of HDL. Since estrogen treatment increased hepatic apoA-I mRNA and apoA-I synthesis, and mouse express undetectable levels of CETP, we tested the hypothesis that estradiol-mediated lowering of HDL in mice may occur through modulation of hepatic lipase (HL). Four mouse strains (C57L, C57BL, BALB, C3H) were administered supraphysiological doses of estradiol, and plasma levels of HDL as well as HL mRNA were quantitated. In all 4 strains estradiol decreased plasma levels of HDL by 30%, and increased HL mRNA 2–3 fold. In a separate experiment groups of male C57BL mouse were castrated or sham-operated, and low and high doses of estradiol administered. We found 1.4–2.5 fold elevation of HL mRNA with concomitant lowering of HDL levels. Ten other mouse strains examined also showed estradiol-induced elevation of HL mRNA, but the extent of elevation was found to be strain-specific. Based on these studies, we conclude that hepatic lipase is an important determinant of plasma levels of HDL and that HL mRNA is modulated by estrogen which in turn may participate in the lowering of plasma levels of HDL.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

THE STOMATA OF NELUMBO NUCIFERA: FORMATION,DISTRIBUTION AND DEGENERATION

Contrary to earlier reports, well-organized but fewer stomata develop on the lower surface of the leaves of Nelumbo nucifera Willd. during aerial growth. The stomata, however, become obliterated by the readjustment of neighboring epidermal cells. During initial stages of degeneration the guard cells show irregularly thickened walls, disintegrated nuclei, and highly vacuolated cytoplasm. Such abnormal features finally lead to the disappearance of stomata from the lower surface of leaves. The ontogeny, structure and distribution of stomata on leaves, perianth lobes, stamens, receptacles and carpels are described. The stomata are haplocheilic in development and are anomocytic (ranunculaceous) at maturity. The concept of a meristemoid and the significance of this study in taxonomy and phylogeny are discussed.