Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors

Suspension cultures derived from gall calli which were obtained following infection with Agrobacterium tumefaciens (C58) were established in Coleus forskohlii. Cell line selection following single cell cloning or cell aggregate cloning was carried out to select cell lines capable of fast growth and for producing high level of forskolin. A fast growing cell line (GSO-5/7) thus selected was found to accumulate 0.021% forskolin in 42 days. The effect of cultural conditions on cell growth was studied to identify factors influencing biomass yield. Cell growth in suspension was found to be influenced significantly by carbon source, initial cell density and light or dark condition. Optimal cell growth (20 fold increase in biomass in a 42 day period) was obtained when the cells were grown in dark condition in B5O media containing 3% sucrose as sole carbon source with an initial cell density of 1.5 x 10(5) cells per ml. Forskolin accumulation was maximum (0.021%) in the stationary phase of cell growth. These suspension cultures showed continuous and stable production of forskolin.     

IL-4 Haplotype -590T, -34T and Intron-3 VNTR R2 Is Associated with Reduced Malaria Risk among Ancestral Indian Tribal Populations

Background

Interleukin 4 (IL-4) is an anti-inflammatory cytokine, which regulates balance between T_H1 and T_H2 immune response, immunoglobulin class switching and humoral immunity. Polymorphisms in this gene have been reported to affect the risk of infectious and autoimmune diseases.

Methods

We have analyzed three regulatory IL-4 polymorphisms; -590C>T, -34C>T and 70 bp intron-3 VNTR, in 4216 individuals; including: (1) 430 ethnically matched case-control groups (173 severe malaria, 101 mild malaria and 156 asymptomatic); (2) 3452 individuals from 76 linguistically and geographically distinct endogamous populations of India, and (3) 334 individuals with different ancestry from outside India (84 Brazilian, 104 Syrian, and 146 Vietnamese).

Results

The -590T, -34T and intron-3 VNTR R2 alleles were found to be associated with reduced malaria risk (P<0.001 for -590C>T and -34C>T, and P = 0.003 for VNTR). These three alleles were in strong LD (r²>0.75) and the TTR2 (-590T, -34T and intron-3 VNTR R2) haplotype appeared to be a susceptibility factor for malaria (P = 0.009, OR = 0.552, 95% CI = 0.356 –0.854). Allele and genotype frequencies differ significantly between caste, nomadic, tribe and ancestral tribal populations (ATP). The distribution of protective haplotype TTR2 was found to be significant (χ² ₃ = 182.95, p-value <0.001), which is highest in ATP (40.5%); intermediate in tribes (33%); and lowest in caste (17.8%) and nomadic (21.6%).

Conclusions

Our study suggests that the IL-4 polymorphisms regulate host susceptibility to malaria and disease progression. TTR2 haplotype, which gives protection against malaria, is high among ATPs. Since they inhabited in isolation and mainly practice hunter-gatherer lifestyles and exposed to various parasites, IL-4 TTR2 haplotype might be under positive selection.     

Interactive Effects of Abiotic Stress and Elevated CO2 on Physio-Chemical and Photosynthetic Responses in Suaeda Species

Suaeda fruticosa and S. monoica are important halophytes for ecological rehabilitation of saline lands. We report differential physio-chemical, photosynthetic, and chlorophyll fluorescence responses in these halophytes under 100 mM sodium chloride (NaCl), 50% strength (16.25 ppt) of seawater (SW)-imposed salinity, and 10% polyethylene glycol 6000 imposed osmotic stress at 380 (ambient) and 1200 (elevated) µmol mol^–1 CO₂ concentrations. SW salinity enhanced the growth in both species; however, compared with S. fruticosa, the S. monoica exhibited comparatively better growth and biomass accumulation under saline conditions at elevated CO₂. Results demonstrated better photosynthetic performances of S. monoica under stress conditions at both levels of CO₂, and this resulted in higher accumulation of carbon, nitrogen, sugar, and starch contents. S. monoica exhibited improved antenna size, electron transfer at PSII donor side, and efficient working of photosynthetic machinery at elevated CO₂, which might be due to efficient upstream utilization of reducing power to fix the CO₂. The δ¹³C results supported the operation of C₄ CO₂ fixation in S. monoica and C₃ or intermediate pathway of CO₂ fixation in S. fruticosa. Lower accumulation of reactive oxygen species, reduced membrane damage, lowered solute potential, and higher accumulation of proline and polyphenol contents indicated elevated CO₂-induced abiotic stress tolerance in Suaeda. Higher activity of antioxidant enzymes in both species at both levels of CO₂ help plants to combat the oxidative stress. Upregulation of NADP-dependent malic enzyme and NADP-dependent malate dehydrogenase genes indicated their role in abiotic stress tolerance as well as photosynthetic carbon (C) sequestration. Operation of C₄ type CO₂ fixation in S. monoica and an intermediate CO₂ fixation in S. fruticosa could be the possible reason for the superior photosynthetic efficiency of S. monoica under stress conditions at elevated CO₂.

     

Use of the random amplif|ed polymorphic DNA (RAPD) assay for the detection of DNA damage and mutations: possible implications of confounding factors

The aim of this study was to evaluate the potential of the random amplified polymorphic DNA (RAPD) assay to qualitatively detect the kinetics of benzo[a]pyrene (B[Ma]P)-induced DNA effects in the water flea Daphnia magna exposed to 25 and 50 µg l^-1 B[a]P for 7 and 6 days, respectively. Mortality was recorded on a daily basis in both experiments, and RAPD analysis was performed on samples collected every day following isolation of genomic DNA. The main changes occurring in RAPD profiles produced by the population of Daphnia magna exposed to 25 and 50µg l^-1 B[a]P was a decrease and increase in band intensity, respectively. Most of the changes occurring in the RAPD patterns were likely to be the result of B[a]P-induced DNA damage (B[a]P DNA adducts, oxidized bases, DNA breakages) and/or mutations (point mutations and large rearrangements). In addition, reproducible changes also occurred in the profiles generated by control Daphnia magna. The results lead us to suggest that, in addition to B[a]P-induced DNA damage and mutations, factors such as variation in gene expression, steady levels of genetic alterations and changes in metabolic processes could induce some changes in RAPD patterns. Nevertheless, our data suggest that DNA damage and mutations appear to be the main factors influencing RAPD patterns. This study also emphasizes that unexpected variation in control profiles is not always associated with artefacts.     

Contemporary human‐altered landscapes and oceanic barriers reduce bumble bee gene flow

Much of the world's terrestrial landscapes are being altered by humans in the form of agriculture, urbanization and pastoral systems, with major implications for biodiversity. Bumble bees are one of the most effective pollinators in both natural and cultivated landscapes, but are often the first to be extirpated in human‐altered habitats. Yet, little is known about the role of natural and human‐altered habitats in promoting or limiting bumble bee gene flow. In this study, I closely examine the genetic structure of the yellow‐faced bumble bee, Bombus vosnesenskii, across the southwestern US coast and find strong evidence that natural oceanic barriers, as well as contemporary human‐altered habitats, limit bee gene flow. Heterozygosity and allelic richness were lower in island populations, while private allelic richness was higher in island populations compared to mainland populations. Genetic differentiation, measured for three indices across the 1000 km study region, was significantly greater than the null expectation (F_ST = 0.041, F’_ST = 0.044 and D_est = 0.155) and correlated with geographic distance. Furthermore, genetic differentiation patterns were most strongly correlated with contemporary (2011) not past (2006, 2001) resistance maps calibrated for high dispersal limitation over oceans, impervious habitat and croplands. Despite the incorporation of dramatic elevation gradients, the analyses reveal that oceans and contemporary human land use, not mountains, are the primary dispersal barriers for B. vosnesenskii gene flow. These findings reinforce the importance of maintaining corridors of suitable habitat across the distribution range of native pollinators to promote their persistence and safeguard their ability to provide essential pollination services.     

Purification and partial characterization of an extracellular alginate lyase from <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> isolated from brown seaweed

The extracellular enzyme alginate lyase produced from marine fungus Aspergillus oryzae isolated from brown alga Dictyota dichotoma was purified, partially characterized, and evaluated for its sodium alginate depolymerization abilities. The enzyme characterization studies have revealed that alginate lyase consisted of two polypeptides with about 45 and 50 kDa each on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed 140-fold higher activity than crude enzyme under optimized pH (6.5) and temperature (35°C) conditions. Zn²⁺, Mn²⁺, Cu²⁺, Mg²⁺, Co²⁺ and NaCl were found to enhance the enzyme activity while (Ca²⁺, Cd²⁺, Fe²⁺, Hg²⁺, Sr²⁺, Ni²⁺), glutathione, and metal chelators (ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid) suppressed the activity. Fourier transform infrared and thin-layer chromatography analysis of depolymerized sodium alginate indicated the enzyme specificity for cleaving at the β-1,4 glycosidic bond between polyM and polyG blocks of sodium alginate and therefore resulted in estimation of relatively higher polyM content than polyG. Comparison of chemical shifts in ¹³C nuclear magnetic resonance spectra of both polyM and polyG from that of sodium alginate also showed further evidence for enzymatic depolymerization of sodium alginate.     

Developing Transgenic Jatropha Using the SbNHX1 Gene from an Extreme Halophyte for Cultivation in Saline Wasteland

Jatropha is an important second-generation biofuel plant. Salinity is a major factor adversely impacting the growth and yield of several plants including Jatropha. SbNHX1 is a vacuolar Na⁺/H⁺ antiporter gene that compartmentalises excess Na⁺ ions into the vacuole and maintains ion homeostasis. We have previously cloned and characterised the SbNHX1 gene from an extreme halophyte, Salicornia brachiata. Transgenic plants of Jatropha curcas with the SbNHX1 gene were developed using microprojectile bombardment mediated transformation. Integration of the transgene was confirmed by PCR and Rt-PCR and the copy number was determined by real time qPCR. The present study of engineering salt tolerance in Jatropha is the first report to date. Salt tolerance of the transgenic lines JL2, JL8 and JL19 was confirmed by leaf senescence assay, chlorophyll estimation, plant growth, ion content, electrolyte leakage and malondialdehyde (MDA) content analysis. Transgenic lines showed better salt tolerance than WT up to 200 mM NaCl. Imparting salt tolerance to Jatropha using the SbNHX1 gene may open up the possibility of cultivating it in marginal salty land, releasing arable land presently under Jatropha cultivation for agriculture purposes. Apart from this, transgenic Jatropha can be cultivated with brackish water, opening up the possibility of sustainable cultivation of this biofuel plant in salty coastal areas.     

In vivo and in vitro measurement of cell mediated immunity in Marek's disease: role of reticuloendothelial system

Dinitrofluorobenzene (DNFB) contact-sensitivity test and leucocyte adherence inhibition (LAI) test were performed in this study as in vivo and in vitro tests to measure the cell-mediated immunity (CMI) in chickens subjected to stimulation of reticuloendothelial (RE) system, depletion of RE system and other experimental groups after being challenged with Marek's disease (MD) virus. It was found that CMI was lower in the birds with depleted RE system and infected control birds, whereas CMI was higher in the birds with activated RE system and vaccinated birds as revealed by DNFB contact-sensitivity test. In cases of LAI test, the number of LAI-positive birds were highest in the chicks with depleted RE system particularly in 3rd and 4th month of age, when the incidence of MD was also maximum.     

Differential Expression of Antioxidant Enzymes During Degradation of Azo Dye Reactive black 8 in Hairy roots of Physalis minima L.

The enzymes involved in the protection of plant metabolism in presence of azo dye was characterized by studying activities of the role of antioxidant enzymes in the hairy roots (HRs) of Physalis minima L. during degradation of an azo dye, Reactive Black 8 (RB8). When the HRs were exposed to RB8 (30 mg L^?1), a nine fold increase in SOD activity was observed after 24 h, while 22 and 50 fold increase in activity was observed for POX and APX respectively after 72 h, whereas there was no significant change in activity of CAT. The activation of different antioxidant enzymes at different time intervals under dye stress suggests the synchronized functioning of antioxidant machinery to protect the HRs from oxidative damage. FTIR analysis confirmed the degradation of dye and the non-toxic nature of metabolites formed after dye degradation was confirmed by phytotoxicity study.     

Centella asiatica (L.) urban from Nepal: Quali-quantitative analysis of samples from several sites,and selection of high terpene containing populations for cultivation

Centella asiatica (L.) Urban is widely used in traditional medicine in many countries and in the formulation of drugs and cosmetics, and is therefore suitable as a trade item for the development of medicinal plants for the population of Nepal. The aim of this work was to select plant populations of C. asiatica with high contents of secondary metabolites growing in various localities in Nepal, and to enhance knowledge of the cultivation of this plant. Quali-quantitative analysis of bioactive triterpenes (asiaticoside and asiatic acid) and phenol derivatives (flavonoids and caffeoyl esters) was performed by HPLC-DAD-ELSD. The highest quantities of triterpenes and phenols were found in samples from the Gorkha and Chitwan districts. Regarding cultivated plants, soil fertilisation is critical, since over-rich soils affect secondary metabolite content. Plants growing in sand-rich soils produce more terpenes. This work provides indications on how to select high-terpene producing germplasm and recommendations for plant cultivation.