Overexpression of <Emphasis Type="Italic">MuHSP70</Emphasis> gene from <Emphasis Type="Italic">Macrotyloma uniflorum</Emphasis> confers multiple abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A 70-KD heat shock protein (HSP70) is one of the most conserved chaperones. It is involved in de novo protein folding and prevents the aggregation of unfolded proteins under lethal environmental factors. The purpose of this study is to characterise a MuHSP70 from horsegram (Macrotyloma uniflorum) and elucidating its role in stress tolerance of plants. A MuHSP70 was cloned and characterised from a natural drought stress tolerant HPK4 variety of horsegram (M. uniflorum). For functional characterization, MuHSP70 was overexpressed in transgenic Arabidopsis. Overexpression of MuHSP70 was found to provide tolerance to the transgenic Arabidopsis against various stresses such as heat, cold, drought, salinity and oxidative stress. MuHSP70 transgenics were observed to maintain the shoot biomass, root length, relative water content, and chlorophyll content during exposure to multi-stresses relative to non-transgenic control. Transgenic lines have further shown the reduced levels of MDA, H₂O₂, and proteolytic activity. Together, these findings suggest that overexpression of MuHSP70 plays an important role in improving abiotic stress tolerance and could be a crucial candidate gene for exploration in crop improvement program.     

Enhanced proteolysis leads to pre-mature cell death under the influence of elicitor like mycelial components from karnal bunt (tilletia indica) pathogen in wheat callus cultures

Calli raised from mature embryos of susceptible wheat cultivar WH 542 were used in the present study as in vitro bioassay system to study the influence of disease determinant(s) of Karnal bunt (Tilletia indica), a semi-biotrophic fungal pathogen of wheat. Influence of elicitor and conditioned medium (CM) prepared from fungal cultures of T. indica was investigated on induction of programmed cell death (PCD). Induction of PCD was observed as hypersensitive response (HR) in terms of browning at localized regions of callus cultures and induction of proteolytic enzyme(s). Elicitor treated calli showed higher induction of protease activity than untreated and CM-treated cultures, which showed not much change in the activity. It was further substantiated by gel protease assay and activation of caspase-3 like protein(s) in callus cultures that clearly suggested the presence of signaling molecule(s) in the fungal elicitor preparation rather than in conditioned medium. This study further demonstrated that only elicitor preparation possesses such molecule(s), which might be cell wall bound components, rather than secretory in nature as CM was unable to induce PCD in wheat callus cultivars.     

Experimental pathogenicity evaluation of Mycoplasma canadense from bovine mastitis in vitro and in vivo laboratory models

Mycoplasma canadense, a clinical isolate from milk of a mastitic buffalo, was experimentally tested for its pathogenic potential in hamster tracheal ring and rabbit fallopian tube explant organ cultures (in vitro) and rat and rabbit mammary gland (in vivo) models. The activity percentage reduction in M. canadense infected hamster tracheal rings was 99.1% in comparison to 16.4% in control rings. Mycoplasma canadense, also induced complete ciliostasis at 11-day post-infection in rabbit fallopian tube explants. Histopathological lesions in these infected organ cultures were loss of cilia, desquamation or denudation of epithelium, infiltration of inflammatory cells and proliferation of macrophages as well as oedema in lamina propria. At the end of the experiments, M. canadense organisms were reisolated in pure colonies from the infected but not the control organ cultures. In the rat and rabbit mammary glands, M. canadense organisms persisted upto 6-day and 7-day postinfection, respectively and caused histopathological changes suggestive of subacute to chronic mastitis during the experimental period. The results indicate that the tested M. canadense clinical isolate was virulent.     

Modified Nanoprecipitation Method for Preparation of Cytarabine-Loaded PLGA Nanoparticles

The present investigation was aimed at developing cytarabine-loaded poly(lactide-coglycolide) (PLGA)-based biodegradable nanoparticles by a modified nanoprecipitation which would have sustained release of the drug. Nine batches were prepared as per 3² factorial design to optimize volume of the co-solvent (0.22–0.37 ml) and volume of non-solvent (1.7–3.0 ml). A second 3² factorial design was used for optimization of drug: polymer ratio (1:5) and stirring time (30 min) based on the two responses, mean particle size (125 ± 2.5 nm), and percentage entrapment efficiency (21.8 ± 2.0%) of the Cyt-PLGA nanoparticles. Optimized formulation showed a zeta potential of −29.7 mV indicating good stability; 50% w/w of sucrose in Cyt-PLGA NP was added successfully as cryoprotectant during lyophilization for freeze-dried NPs and showed good dispersibility with minimum increase in their mean particle sizes. The DSC thermograms concluded that in the prepared PLGA NP, the drug was present in the amorphous phase and may have been homogeneously dispersed in the PLGA matrix. In vitro drug release from the pure drug was complete within 2 h, but was sustained up to 24 h from PLGA nanoparticles with Fickian diffusion. Stability studies showed that the developed PLGA NPs should be stored in the freeze-dried state at 2–8°C where they would remain stable in terms of both mean particle size and drug content for 2 months.     

Genetic Determinants of Antibiotic Resistance in Staphylococcus aureus Isolates from Milk of Mastitic Crossbred Cattle

Staphylococcus aureus is one of the major causes of mastitis in dairy animals and its resistance against multiple antimicrobials always remains crucial concern. Present investigation was carried out to detect the distribution of antibiotic-resistant genes of S. aureus isolates. Isolates (128) of S. aureus from mastitic milk were collected, tested for antibiotics with disc-diffusion method, and resistant genes mecA, linA, msrA msrB, vatA, vatB, vatC ermA, ermC tetK, tetM and aacA-D were detected by PCR. The phenotypic antibiotics resistance percent in S. aureus isolates was classified as tetracycline (36.7), gentamycin (30.5), streptomycin (26.6), kanamycin (25.8) and penicillin G (22.7). All the isolates were susceptible to vancomycin. Among isolates, 10.2% were observed as methicillin-resistant. The distribution of antibiotic-resistant genes was linA (51.6) followed by msrB (46.1), tetK + M (34.4), msrA and aacA-D (26.6%). Different antibiotic-resistant genes combinations (mecA/linA-2; mecA/aacA-D/tetK/linA/msrB-3; mecA/linA/msrA/msrB-3; aacA-D/linA/msrA/msrB-4; aacA-D/linA/msrB-7; linA/msrA/msrB-10; tetK/linA/msrA/msrB-11; aacA/tetK/linA/msrB-12 isolates) were observed. All the isolates lacked amplification of vatA, vatB, ermA and ermC genes. Molecular typing resulted genetic variation in protein A (6–12 repeats) and coagulase genes (A–E patterns) were observed. Coagulase A and D genotypes were more prevalent in antibiotic-resistant isolates, while E, B and C in susceptible ones. The significant observation was the prevalence of methicillin-resistant S. aureus, which were resistant to multiple antibiotics. Findings revealed the status of resistant isolates in herd that might be helpful in treatment, controlling of resistant strains and culling of cows for mastitis reduction.     

Temperature-dependent development and life table parameters of Thrips palmi (Thysanoptera: Thripidae) on eggplant

The life history of Thrips palmi Karny on eggplant (Solanum melongena L.) leaves was studied based on the age stage and two sex-life tables at 16, 19, 22, 25, and 31?°C. The intrinsic rate of increase (r) at these temperatures was 0.0427, 0.0566, 0.0979, 0.1738, and 0.2237?day^?1, respectively. The relationship among the gross reproductive rate (GRR), the net reproductive rate (R ₀), and the pre-adult survivorship (l _a) is consistent with R ₀?<?l _a?×?GRR?<?GRR for all results at different temperatures. The mean generation time was 47.52, 38.33, 29.52, 19.81, and 13.88?days, respectively. The developments of pre-adult and adult stages were faster in males than in females. The means of developmental periods for each developmental stage decreased with increases of temperature. The maximum life span of female adults was noted at 56.67?days, whereas that of males was 50.66?days at 16?°C. The maximum female fecundity (64.18?eggs/female) was recorded at 25?°C and the lowest (23.38 eggs/female) at 16?°C. Life table data could be used to project population growth, to design mass rearing programs, and to establish management tactics to control insect pests.     

Stress-Induced Changes in Peptidyl-Prolyl cis-trans Isomerase Activity of Sorghum bicolor Seedlings

Developmental changes and effects of various abiotic stresses on peptidyl prolyl cis-trans isomerase (PPIase) activity were studied in the seedlings of sorghum [Sorghum bicolor (L.) Moench cv. CSH-6]. The PPIase activity of sorghum seedlings markedly decreased after two days of germination. Up to 90 % of the PPIase activity was inhibited by cyclosporin-A. Maximal increase in specific PPIase activity in the 3-d-old seedlings was observed in response to osmotic stress and it was transient in nature. The stress-induced enhancement in PPIase activity, depending upon tissue and stress treatment, was due to induction of cyclophilins as well as other PPIases. Osmotic stress-induced enhancement in PPIase activity in the drought susceptible cv. SPRU-94008B was maximal in roots, as compared to shoots in the drought tolerant cv. ICSV-272. This revised version was published online in July 2006 with corrections to the Cover Date.     

Evaluation of efficacy of stabilizers on the thermostability of live attenuated thermo-adapted <Emphasis Type="Italic">Peste des petits ruminants</Emphasis> vaccines

In this study, thermo-adapted (Ta) PPR vaccines were assessed for their stability at 25, 37, 40, 42 and 45°C in lyophilized form using two extrinsic stabilizers {lactalbumin hydrolysate-sucrose (LS) and stabilizer E} and in reconstituted form with the diluents (1 mol/L MgSO₄ or 0.85% NaCl). The lyophilized vaccines showed an expiry period of 24–26 days at 25°C, 7–8 days at 37°C and 3–4 days at 40°C. LS stabilizer was superior at 42°C with a shelf-life of 44 h, whereas in stabilizer E, a 40 h shelf-life with a comparable half-life was observed. At 45°C, the half-life in stabilizer E was better than LS and lasted for 1 day. Furthermore, the reconstituted vaccine maintained the titre for 48 h both at 4°C and 25°C and for 24–30 h at 37°C. As both the stabilizers performed equally well with regard to shelf-life and half-life, the present study suggests LS as stabilizer as a choice for lyophilization with 0.85% NaCl diluent, because it has better performance at higher temperature. These Ta vaccines can be used as alternatives to existing vaccines for the control of the disease in tropical countries as they are effective in avoiding vaccination failure due to the breakdown in cold-chain maintenance, as this vaccine is considerably more stable at ambient temperatures.     

Iridoid glycosides from Gmelina arborea

Three iridoid glycosides 6-O-(3'-O-benzoyl)-alpha-l-rhamnopyranosylcatalpol (1a), 6-O-(3'-O-trans-cinnamoyl)-alpha-l-rhamnopyranosylcatalpol (2a) and 6-O-(3'-O-cis-cinnamoyl)-alpha-l-rhamnopyranosylcatalpol (3a) were isolated from aerial parts of Gmelina arborea and structures were elucidated by spectral analysis. Additionally a known iridoid 6-O-(3', 4'-O-dibenzoyl)-alpha-l-rhamnopyranosylcatalpol (4) was also isolated and identified.