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.     

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.     

Th1-Biased Immunomodulation and Therapeutic Potential of Artemisia annua in Murine Visceral Leishmaniasis

Background

In the absence of vaccines and limitations of currently available chemotherapy, development of safe and efficacious drugs is urgently needed for visceral leishmaniasis (VL) that is fatal, if left untreated. Earlier we reported in vitro apoptotic antileishmanial activity of n-hexane fractions of Artemisia annua leaves (AAL) and seeds (AAS) against Leishmania donovani. In the present study, we investigated the immunostimulatory and therapeutic efficacy of AAL and AAS.

Methodology/Principal Findings

Ten-weeks post infection, BALB/c mice were orally administered AAL and AAS for ten consecutive days. Significant reduction in hepatic (86.67% and 89.12%) and splenic (95.45% and 95.84%) parasite burden with decrease in spleen weight was observed. AAL and AAS treated mice induced the strongest DTH response, as well as three-fold decrease in IgG1 and two-fold increase in IgG2a levels, as compared to infected controls. Cytometric bead array further affirmed the elicitation of Th1 immune response as indicated by increased levels of IFN-γ, and low levels of Th2 cytokines (IL-4 and IL-10) in serum as well as in culture supernatant of lymphocytes from treated mice. Lymphoproliferative response, IFN-γ producing CD4⁺ and CD8⁺ T lymphocytes and nitrite levels were significantly enhanced upon antigen recall in vitro. The co-expression of CD80 and CD86 on macrophages was significantly augmented. CD8⁺ T cells exhibited CD62L^low and CD44^hi phenotype, signifying induction of immunological memory in AAL and AAS treated groups. Serum enzyme markers were in the normal range indicating inertness against nephro- and hepato-toxicity.

Conclusions/Significance

Our results establish the two-prong antileishmanial efficacy of AAL and AAS for cure against L. donovani that is dependent on both the direct leishmanicidal action as well as switching-on of Th1-biased protective cell-mediated immunity with generation of memory. AAL and AAS could represent adjunct therapies for the treatment of leishmaniasis, either alone or in combination with other antileishmanial agents.     

A Statistical Thermodynamic Model for Investigating the Stability of DNA Sequences from Oligonucleotides to Genomes

We describe the development and testing of a simple statistical mechanics methodology for duplex DNA applicable to sequences of any composition and extensible to genomes. The microstates of a DNA sequence are modeled in terms of blocks of basepairs that are assumed to be fully closed (paired) or open. This approach generates an ensemble of bubblelike microstates that are used to calculate the corresponding partition function. The energies of the microstates are calculated as additive contributions from hydrogen bonding, basepair stacking, and solvation terms parameterized from a comprehensive series of molecular dynamics simulations including solvent and ions. Thermodynamic properties and nucleotide stability constants for DNA sequences follow directly from the partition function. The methodology was tested by comparing computed free energies per basepair with the experimental melting temperatures of 60 oligonucleotides, yielding a correlation coefficient of −0.96. The thermodynamic stability of genic/nongenic regions was tested in terms of nucleotide stability constants versus sequence for the Escherichia coli K-12 genome. It showed clear differentiation of the genes from promoters and captures genic regions with a sensitivity of 0.94. The statistical thermodynamic model presented here provides a seemingly new handle on the challenging problem of interpreting genomic sequences.     

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.     

Microprojectile bombardment mediated genetic transformation of embryo axes and plant regeneration in cumin (<Emphasis Type="Italic">Cuminum cyminum</Emphasis> L.)

Pre-cultured cumin embryos were bombarded under 27 inches Hg vacuum, 25 mm distance from rupture disc to macrocarrier, 10 mm macrocarrier flight distance using 1100 psi rupture disc and 9 cm microprojectile travel distance. An average of 110 embryos was used per shot and 91% embryos showed transient GUS expression after 24 h. Shoot tips and roots of T₀ plantlets exhibited GUS expression done after 3 months of bombardment. Transformation was confirmed with PCR amplification of 0.96 and 1.3 kb band of hptII and gus genes respectively from T₀ transgenics and southern blot analysis using PCR amplified DIG labeled hptII gene as probe. It is the first successful attempt of transformation of cumin plant through direct gene transfer using particle gene gun and adequately exhibiting the possibility of stable transformation in cumin.     

Professional and consumer insecticides for management of adult Japanese beetle on hybrid tea rose

In many states, Japanese beetle, Popilliajaponica Newman (Coleoptera: Scarabeidae), is no longer quarantined, and management is left to professional applicators and consumers. Adult management in hybrid tea rose, Rosa L., was compared among biorational insecticides, novel imidacloprid applications (tablet, gel, and root dip), and conventional insecticides. Efficacy of biorational insecticides used by consumers varied widely and may not offer predictable management: mortality was 3.0% with Garlic Barrier, 5.0% with Monterey Neem Oil, 15.1% with Pygenic (1.4% pyrethrins), and 27.3% with Orange Guard (D-limonene). Only JB Killer (0.02% pyrethrins plus 0.2% piperonyl butoxide) had mortality of 90.9%, probably due to piperonyl butoxide. Professional biorationals did not show significant mortality: 7.7% with Azatin XL (azadirachtin) and 3.7% Conserve (spinosad). In contrast, conventional insecticides demonstrated significant mortality; 88.4% with Decathlon 20 WP (cyfluthrin) and 83.3% with Discus SC (imidacloprid plus cyfluthrin). New imidacloprid applications (tablet, gel, and root dip) worked as well as standard drench and granular methods, but they showed 9.1-42.7% mortality. However, beetles were incapacitated as demonstrated by inability to walk (82-106-s flip time) compared with controls (30-s flip time). No phytotoxicity was observed in any treatments. However, some imidacloprid treatments produced growth enhancement: higher leaf chlorophyll (1X, 3X granular, and one tablet), and larger leaf area and higher nitrogen (3X granular, drench). The highest (active ingredient) imidacloprid was in 3X granular treatment, which in an unplanned infestation, showed highest numbers of twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). Effects of imidacloprid on leaf quality and mite outbreaks deserves research.     

Acetylcholine receptor gating at extracellular transmembrane domain interface: the cys-loop and M2-M3 linker

Acetylcholine receptor channel gating is a propagated conformational cascade that links changes in structure and function at the transmitter binding sites in the extracellular domain (ECD) with those at a "gate" in the transmembrane domain (TMD). We used Phi-value analysis to probe the relative timing of the gating motions of alpha-subunit residues located near the ECD-TMD interface. Mutation of four of the seven amino acids in the M2-M3 linker (which connects the pore-lining M2 helix with the M3 helix), including three of the four residues in the core of the linker, changed the diliganded gating equilibrium constant (K(eq)) by up to 10,000-fold (P272 > I274 > A270 > G275). The average Phi-value for the whole linker was approximately 0.64. One interpretation of this result is that the gating motions of the M2-M3 linker are approximately synchronous with those of much of M2 (approximately 0.64), but occur after those of the transmitter binding site region (approximately 0.93) and loops 2 and 7 (approximately 0.77). We also examined mutants of six cys-loop residues (V132, T133, H134, F135, P136, and F137). Mutation of V132, H134, and F135 changed K(eq) by 2800-, 10-, and 18-fold, respectively, and with an average Phi-value of 0.74, similar to those of other cys-loop residues. Even though V132 and I274 are close, the energetic coupling between I and V mutants of these positions was small (< or =0.51 kcal mol(-1)). The M2-M3 linker appears to be the key moving part that couples gating motions at the base of the ECD with those in TMD. These interactions are distributed along an approximately 16-A border and involve about a dozen residues.