Genotypic background of the recipient plant is crucial for conferring RB gene mediated late blight resistance in potato.

BACKGROUND: Late blight, caused by oomycetes pathogen Phytophthora infestans (Mont.) de Bary, is the most devastating potato disease in the world. RB gene from Solanum bulbocastanum has been shown to impart broad spectrum resistance against P. infestans races. In this study Katahdin transgenic event SP951 was used as male parent to cross with the popular Indian potato cultivars viz., Kufri Bahar (KB) and Kufri Jyoti (KJ) to enhance the late blight resistance. RESULTS: Populations of 271 F1seedlings from the crosses KB × SP951 (87) and KJ × SP951 (184) were screened for inheritance of RB transgene through PCR and bioassay. Disease response based on AUDPC of different hybrid lines varied from immunity to complete susceptibility. High degree of resistance (<25% infection) was observed in KJ × SP951 derived seedlings (85.2%), whereas level of resistance in KB × SP951 (36.4% infection) derived seedlings was of low order. CONCLUSION: This study provides valuable genetic materials for development of potentially durable late blight resistant potato varieties. Besides, it also corroborates the fact that efficacy of R gene is not solely dependent on its presence in the variety but largely depends on the genetic background of the recipient genotype.

A 43 kDa recombinant plasmepsin elicits immune response in mice against Plasmodium berghei malaria.

Intraerythrocytic parasites degrade haemoglobin to make available nutrients for their growth and maturation. Plasmepsins, the aspartic proteases of Plasmodium play a significant role in haemoglobin degradation and are proposed as attractive drug targets. In the present study the gene which encodes plasmepsin in rodent malaria parasite, Plasmodium berghei, was cloned and expressed. The gene was sequenced and expressed in Escherichia coli BL21DE3 and a recombinant plasmepsin of molecular weight 43 kDa was obtained. The sequence obtained was analysed and compared with plasmepsins of other Plasmodium spp. Mice immunized with the recombinant plasmepsin induced a strong humoral immune response. ELISA and IFA performed on the serum of immunized mice showed high antibody titres. Along with this, in vivo study exhibited partial protection against P. berghei infection suggesting role of plasmepsin in malaria control.

Hypophosphatemic rickets associated with giant hairy nevus.

The association of multisystem pathologic conditions and epidermal nevi, known as the epidermal nevus syndrome, includes disorders of bone, central nervous system, eye, kidney, vasculature and skin. Rarely, congenital nevomelanocytic nevus also known as hairy nevus has also been reported in association with hypophosphatemic rickets. Studies suggest that phosphaturia, caused by circulating factors, called "phosphatonins" may be secreted by an epidermal or hairy nevus. We report here, a rare case of hypophosphatemic rickets associated with a giant hairy nevus in a 10-year-old boy.

Purification studies on a thermo-active amidase of Geobacillus pallidus BTP-5x MTCC 9225 isolated from thermal springs of Tatapani (Himachal Pradesh).

An intracellular aliphatic amide degrading inducible thermo-active amidase produced by Geobacillus pallidus BTP-5x MTCC 9225 was purified to apparent homogeneity using anion exchange and gel filtration chromatography, giving a yield of 6.7 % and a specific activity of 30.49 units mg(-1). The purified protein migrated as a single band of estimated molecular mass of 158 kDa (homo-tetramer) in 8 % polyacrylamide gel electrophoresis and ∼38.5 kDa in 12 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Optima of pH and temperature varied widely in broad pH range (pH 6-9) and temperature range (45-70 °C). The purified amidase was stable up to 6 h at 50 °C, with a t (1/2) of 7 h at 55 °C. The multimeric nature of the holozyme (tetramer) contributed to protection of the enzyme against thermal denaturation. The enzyme showed resistance to metal chelating agents (EDTA, 8-hydroxyquinoline, and sodium azide), explaining its non-metallic nature, and is strongly inhibited by thiol reagents that means cysteine is involved in catalysis. The amidase of G. pallidus BTP-5x preferentially hydrolyzed only small aliphatic amides and has a narrow substrate spectrum. The K (M) value for acrylamide is 10.54 mM, V (max) 45.19 µmol(-1) min(-1) mg(-1) protein, and k (cat) 4.29 min(-1). The sequence of amino acids of the purified enzyme MRHGDISSSHDTVGI appears similar to thermophilic amidases. Sequence analysis of the amidase gene showed that the enzyme is 347 amino-acid-long with a molecular weight of 38.4 kDa (as observed in SDS-PAGE), theoretical pI 5.38, and show strong similarity to thermostable amidases, possessing unique restriction sites.

Nocardia globerula NHB-2 nitrilase catalysed biotransformation of 4-cyanopyridine to isonicotinic acid.

Isonicotinic acid (INA) is an important pyridine derivative used in the manufacture of isoniazid (antituberculosatic drug) and other pharmaceutically important drugs. Nitrilase catalysed processes for the synthesis of pharmaceutically important acids from their corresponding nitriles are promising alternative over the cumbersome, hazardous, and energy demanding chemical processes. Nitrilase of Nocardia globerula NHB-2 (NitNHB2) is expressed in presence of isobutyronitrile in the growth medium (1.0% glucose, 0.5% peptone, 0.3% beef extract, and 0.1 % yeast extract, pH 7.5). NitNHB2 hydrolyses 4-cyanopyridine (4-CP) to INA without accumulation of isonicotinamide, which is common in the reaction catalysed via fungal nitrilases. The NitNHB2 suffers from substrate inhibition effect and hydrolysing activity up to 250 mM 4-CP was recorded. Complete conversion of 200 mM 4-CP to INA was achieved in 40 min using resting cell concentration corresponding to 10 U mL-1 nitrilase activity in the reaction. Substrate inhibition effect in the fed batch reaction (200 mM substrate feed/40min) led to formation of only 729 mM INA. In a fed batch reaction (100 mM 4-CP/20min), substrate inhibition effect was encountered after 7th feed and a total of 958 mM INA was produced in 400 min. The fed batch reaction scaled up to 1 L and 100% hydrolysis of 700 mM of 4-CP to INA at 35°C achieved in 140 min. The rate of INA production was 21.1 g h-1 mgDCW-1. This is the fastest biotransformation process ever reported for INA production with time and space productivity of 36 g L-1 h-1 using a bacterial nitrilase.

Biotransformation of Acetamide to Acetohydroxamic Acid at Bench Scale Using Acyl Transferase Activity of Amidase of Geobacillus pallidus BTP-5x MTCC 9225.

The bioprocess employing acyl transferase activity of intracellular amidase of Geobacillus pallidus BTP-5x MTCC 9225 was harnessed for the synthesis of pharmaceutically important acetohydroxamic acid. G. pallidus BTP-5x exhibited highest acyl transferase activity with acetamide: hydroxylamine in ratio of 1:5 in 0.1 M NaH2PO4/Na2HPO4 buffer (pH 7.5) at 65°C. In one liter fed-batch reaction containing 1:5 ratio of two substrates total of eight feedings of 0.05 M/20 min of acetamide were made and it was found that maximum acetohydroxamic production was achieved at 3:5 ratios of substrate and cosubstrate. In 1 l bench scale batch reaction containing 0.3 M acetamide, 0.5 M hydroxylamine in 0.1 M NaH2PO4/Na2HPO4 buffer (pH 7.5, 50°C, 400 rpm) and 0.5 mg/ml (dry cell weight) of whole cells of G. pallidus BTP-5x (as biocatalyst) resulted in an yield of 0.28 M of acetohydroxamic acid after 20 min reaction time at 50°C. The acetamide bioconversion rate was 90-95% (mol mol(-1)) and 51 g powder containing 40% (w/w) acetohydroxamic acid was recovered after lyophilization.

An improved nitrilase-mediated bioprocess for synthesis of nicotinic acid from 3-cyanopyridine with hyperinduced Nocardia globerula NHB-2.

Nitrilase of Nocardia globerula NHB-2 was induced by short-chain aliphatic nitriles (valeronitrile > isobutyronitrile > butyronitrile > propionitrile) and exhibited activity towards aromatic nitriles (benzonitrile > 3-cyanopyridine > 4-cyanopyridine > m-tolunitrile > p-tolunitrile). Hyperinduction of nitrilase (6.67 U mg (DCW) (-1), 18.7 U mL(-1)) was achieved in short incubation time (30 h, 30°C) through multiple feeding of isobutyronitrile in the growth medium. The nitrilase of this organism exhibits both substrate and product inhibition effects. In a fed batch reaction at 1 L scale using hyperinduced resting cells corresponding to 10 U mL(-1) nitrilase activity (1.5 mg(DCW) mL(-1)), a total of 123.11 g nicotinic acid was produced at a rate of 24 g h(-1) g (DCW) (-1).

Acrylamide synthesis using agar entrapped cells of Rhodococcus rhodochrous PA-34 in a partitioned fed batch reactor.

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10 degrees C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10 degrees C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4 degrees C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.