Cloning and expression of <Emphasis Type="Italic">β</Emphasis>-glucosidase gene from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> into <Emphasis Type="Italic">E. coli</Emphasis> BL 21 (DE3)

A 1.4 Kb fragment of Bacillus licheniformis ATCC 14580 encoding β-glucosidase was cloned and expressed in Escherichia coli. β-Glucosidase expressed by E. coli harboring cloned gene was located entirely in the intracellular fraction. Recombinant β-glucosidase protein was purified to homogeneity level and the molecular weight was found to be 53 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. It gave maximum activity at 50°C and pH 6. K _m and V _max were 0.206 mM and 1.26 U/mg, respectively, with p-nitrophenyl-β-D-glucopyranoside, while activation energy E_a, enthalpy of activation ?H and entropy of activation ΔS were found to be 66.31 kJ/mol, 64.04 kJ/mol and 48.28 J/mol/K, respectively. The pK_a1 and pK_a2 of the ionizable groups of active site residues involved in V_max were found to be 5.5 and 7.0, respectively. When the recombinant β-glucosidase protein was used as a member of consortium with endoglucanase and exoglucanase for the saccharification of wheat straw, 123% increase in saccharification was observed.     

Flower Senescence-Strategies and Some Associated Events

Different strategies of petal senescence and some important events associated with it have been discussed. On the basis of sensitivity to ethylene and associated symptoms of senescence, petal senescence has been classified into five different classes; besides changes in membrane permeability, autophagy and involvement of VPEs (Vacuolar processing enzymes), degradation of nucleic acids, protein turn over and remobilization of essential nutrients during petal senescence have been discussed. Nucleus appears to play a central role in administrating the execution of the events associated with petal senescence. Protein turn over appears to be an important factor governing petal senescence in both ethylene-sensitive and ethylene-insensitive flower systems and that the loss of membrane integrity, vacuolar autophagy and remobilization of essential nutrients being its important consequences. Autophagy seems to be a main process responsible for cell dismantling and remobilization of macromolecules besides final disintegration of nucleus. A large number of senescence-associated genes have been found to be differentially expressed during petal senescence. On the basis of the available literature, a schematic model representing some important events associated with petal senescence has been constructed. The review recommends that more elaborate work is required at cellular and organelle level to understand the ethylene-independent pathway and its execution in both ethylene-sensitive and ethylene-insensitive flower systems. It also recommends that ethylene sensitivity should not be generally assigned to plants at the family level on the basis of response of a few species in a family.     

Methyl jasmonate counteracts boron toxicity by preventing oxidative stress and regulating antioxidant enzyme activities and artemisinin biosynthesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> L.

Boron is an essential plant micronutrient, but it is phytotoxic if present in excessive amounts in soil for certain plants such as Artemisia annua L. that contains artemisinin (an important antimalarial drug) in its areal parts. Artemisinin is a sesquiterpene lactone with an endoperoxide bridge. It is quite expensive compound because the only commercial source available is A. annua and the compound present in the plant is in very low concentration. Since A. annua is a major source of the antimalarial drug and B stress is a deadly threat to its cultivation, the present research was conducted to determine whether the exogenous application of methyl jasmonate (MeJA) could combat the ill effects of excessive B present in the soil. According to the results obtained, the B toxicity induced oxidative stress and reduced the stem height as well as fresh and dry masses of the plant remarkably. The excessive amounts of soil B also lowered the net photosynthetic rate, stomatal conductance, internal CO₂ concentration and total chlorophyll content in the leaves. In contrast, the foliar application of MeJA enhanced the growth and photosynthetic efficiency both in the stressed and non-stressed plants. The excessive B levels also increased the activities of antioxidant enzymes, such as catalase, peroxidase and superoxide dismutase. Endogenous H₂O₂ and O₂⁻ levels were also high in the stressed plants. However, the MeJA application to the stressed plants reduced the amount of lipid peroxidation and stimulated the synthesis of antioxidant enzymes, enhancing the content and yield of artemisinin as well. Thus, it was concluded that MeJA might be utilized in mitigating the B toxicity and improving the content and yield of artemisinin in A. annua plant.     

Salicylic acid mitigates salinity stress by improving antioxidant defence system and enhances vincristine and vinblastine alkaloids production in periwinkle [<Emphasis Type="Italic">Catharanthus roseus</Emphasis> (L.) G. Don]

A pot experiment was conducted to find out whether the foliar spray of salicylic acid (SA) could successfully ameliorate the adverse effects of salinity stress on periwinkle. Thirty-day-old plants were supplied with Control; 0 mM NaCl + 10⁻⁵ M SA (T₁); 50 mM NaCl + 0 SA (T₂); 100 mM NaCl + 0 SA (T₃); 150 mM NaCl + 0 SA (T₄); 50 mM NaCl + 10⁻⁵ M SA (T₅); 100 mM NaCl + 10⁻⁵ M SA (T₆); 150 mM NaCl + 10⁻⁵ M SA (T₇). The plants were sampled 90 days after sowing to assess the effect of SA on stressed and unstressed plants. Salt stress significantly reduced the growth attributes including plant height, leaf-area index, shoot and root fresh weights, shoot and root dry weights. Increasing NaCl concentrations led to a gradual decrease in photosynthetic parameters and activities of nitrate reductase and carbonic anhydrase. Ascorbic acid, total alkaloids and antioxidants enzymes superoxide dismutase, catalase and peroxidase also declined in NaCl-treated plants. The plants, undergoing NaCl stress, exhibited a significant increase in electrolyte leakage and proline content. Foliar application of SA (10⁻⁵ M) reduced the damaging effect of salinity on plant growth and accelerated the restoration of growth processes. It not only improved the growth parameters but also reversed the effects of salinity. Total alkaloid content was improved by SA application both in unstressed and stressed plants. The highest level of total alkaloid content recorded in leaves of SA-treated stressed plants was 11.1%. Foliar spray of SA overcame the adverse effect of salinity by improving the content of vincristine (14.0%) and vinblastine (14.6%) in plants treated with 100 M NaCl.     

Trichoderma harzianum SQR-T037 rapidly degrades allelochemicals in rhizospheres of continuously cropped cucumbers

To alleviate the stress of continuous cropping for cucumber continuous cropping (CCC) system, a beneficial fungus Trichoderma harzianum SQR-T037 (SQR-T037) was isolated and applied to soil to degrade allelochemicals exuded from cucumber plants in a Rhizobox experiment. The following phenolic acids (PAs), classified as allelochemicals, were isolated and identified from cucumber rhizospheres: 4-hydroxybenzoic acid, vanillic acid, ferulic acid, benzoic acid, 3-phenylpropionic acid, and cinnamic acid. Mixed PAs added in potato dextrose broth, each with 0.2 gram per liter, were completely degraded by SQR-T037 after 170 h of incubation. In Rhizobox experiments, inoculation of SQR-T037 in the CCC soil also degraded the PAs exuded from cucumber plant roots. This degradation was 88.8% for 4-hydroxybenzoic acid, 90% for vanillic acid, 95% for benzoic acid, and 100% for ferulic acid, 3-phenylpropionic acid, and cinnamic acid at 45 days after plantation. Simultaneously, a significant (p ≥ 0.05) decrease in the disease index of Fusarium wilt and an increase in dry weights of cucumber plants were obtained in pot experiments by application of SQR-T037. This was mostly attributed to degradation of PAs exuded from cucumber roots in CCC soil by SQR-T037 and alleviation of the allelopathic stress. Application of beneficial microorganisms, such as SQR-T037 that biodegrades allelochemicals, is a highly efficient way to resolve the problems associated with continuous cropping system.     

An application of capsid-specific artificial ankyrin repeat proteinproduced in E. coli for immunochromatographic assay as a surrogate for antibody

Immunochromatographic strip test is a unique type of rapid test that has been developed for use as part of a diagnostic kit for the rapid detection of antibodies and/or other proteins of interest. For the detection of target proteins, most of the commercial tests are assembled based on the conjugation of colloidal gold particles to monoclonal antibodies embedded within the conjugate pad of a strip test. In this study, we tested the novel concept of using an artificial non-antibody structure for generating a colloidal gold conjugate (CGC). We exploited the property of an ankyrin repeat protein that specifically binds to the HIV-1 capsid protein termed Ank^GAG1D4. This construct was applied as a model structure to create Ank1D4-CGC and used as a new type of visible detector system and termed it ankyrin-based immunochromatographic strip (ABIS) test. The ABIS test was shown to be highly sensitive with a lower limit of detection of the target protein at 0.1 μg/ml. Moreover, the ABIS test was not only highly sensitive but also shared a level of specificity within the same range of the commercial test kit. The results of the studies presented herein therefore demonstrate the novel application of an artificial non-immunoglobulin structure (ankyrin repeat protein) as the new line of a visible detector using a rapid diagnostic test with characteristics that have the potential to be superior to those that utilize antibody-based tests.     

Synthesis of Silver Embedded Poly(o-Anisidine) Molybdophosphate Nano Hybrid Cation-Exchanger Applicable for Membrane Electrode

Poly(o-anisidine) molybdophosphate was expediently obtained by sol-gel mixing of Poly(o-anisidine) into the inorganic matrices of molybdophosphate, which was allowed to react with silver nitrate to the formation of poly(o-anisidine) molybdophosphate embedded silver nano composite. The composite was characterized by Fourier Transform Infrared Spectroscopy, X-ray powder diffraction, UV-Vis Spectrophotometry, Fluorescence Spectroscopy, Scanning Electron Microscopy/Energy-dispersive X-ray Spectroscopy and Thermogravimertic Analysis. Ion exchange capacity and distribution studies were carried out to understand the ion-exchange capabilities of the nano composite. On the basis of highest distribution studies, this nano composite cation exchanger was used as preparation of heavy metal ion selective membrane. Membrane was characterized for its performance as porosity and swelling later on was used for the preparation of membrane electrode for Hg(II), having better linear range, wide working pH range (2–4.5) with fast response in the real environment.     

Functional and Evolutionary Characterization of the CONSTANS Gene Family in Short-Day Photoperiodic Flowering in Soybean

CONSTANS (CO) plays a central role in photoperiodic flowering control of plants. However, much remains unknown about the function of the CO gene family in soybean and the molecular mechanisms underlying short-day photoperiodic flowering of soybean. We identified 26 CO homologs (GmCOLs) in the soybean genome, many of them previously unreported. Phylogenic analysis classified GmCOLs into three clades conserved among flowering plants. Two homeologous pairs in Clade I, GmCOL1a/GmCOL1b and GmCOL2a/GmCOL2b, showed the highest sequence similarity to Arabidopsis CO. The mRNA abundance of GmCOL1a and GmCOL1b exhibited a strong diurnal rhythm under flowering-inductive short days and peaked at dawn, which coincided with the rise of GmFT5a expression. In contrast, the mRNA abundance of GmCOL2a and GmCOL2b was extremely low. Our transgenic study demonstrated that GmCOL1a, GmCOL1b, GmCOL2a and GmCOL2b fully complemented the late flowering effect of the co-1 mutant in Arabidopsis. Together, these results indicate that GmCOL1a and GmCOL1b are potential inducers of flowering in soybean. Our data also indicate rapid regulatory divergence between GmCOL1a/GmCOL1b and GmCOL2a/GmCOL2b but conservation of their protein function. Dynamic evolution of GmCOL regulatory mechanisms may underlie the evolution of photoperiodic signaling in soybean.     

Synthetic cannabinoids: Analysis and metabolites

Cannabimimetics (commonly referred to as synthetic cannabinoids), a group of compounds encompassing a wide range of chemical structures, have been developed by scientists with the hope of achieving selectivity toward one or the other of the cannabinoid receptors CB1 and CB2. The goal was to have compounds that could possess high therapeutic activity without many side effects. However, underground laboratories have used the information generated by the scientific community to develop these compounds for illicit use as marijuana substitutes. This chapter reviews the different classes of these “synthetic cannabinoids” with particular emphasis on the methods used for their identification in the herbal products with which they are mixed and identification of their metabolites in biological specimens.