DFT study of structure and vibrational spectra of ceramide 3: comparison to experimental data

The Fourier transform Raman and infrared (IR) spectra of the Ceramide 3 (CER3) have been recorded in the regions 200–3500 cm^? 1 and 680–4000 cm^? 1, respectively. We have calculated the equilibrium geometry, harmonic vibrational wavenumbers, electrostatic potential surfaces, absolute Raman scattering activities and IR absorption intensities by the density functional theory with B3LYP functionals having extended basis set 6-311G. This work is undertaken to study the vibrational spectra of CER3 completely and to identify the various normal modes with better wavenumber accuracy. Good consistency is found between the calculated results and experimental data for the IR and Raman spectra.     

172 Role of conserved water molecular triad in the recognition of IMP,NAD+ with Asp 274, Asn 303, Arg 322, and Asp 364 in both the isoform of hIMPDH

Inosine monophosphate dehydrogenase (IMPDH) plays an important role in the Guanosine monophosphate (GMP) biosynthesis pathway. As hIMPDH-II is involved in CML-Cancer, it is thought to be an active target for leukemic drug design. The importance of conserved water molecules in the salt-bridge-mediated interdomain recognition and loop-flap recognition of hIMPDH has already been indicated in some simulation studies (Bairagya et al., 2009, 2011a, 2011b, 2012; Mishra et al., 2012). In this work, the role of conserved water molecules in the recognition of Inosine monophosphate (IMP) and NAD⁺ (co-factor) to active site residues of both the isoforms has been investigated by all atoms MD-Simulation studies. During 25-ns dynamics of the solvated hIMPDH-II and I (1B3O and 1JCN PDB structures), the involvement of conserved water molecular triad (W _M, W _L and W _C) in the recognition of active site residues (Asp 274, Asn 303, Arg 322, and Asp 364), IMP and NAD⁺ has been observed (Figure 1). The H-bonding co-ordination of all three conserved water molecular centers is within 4–7 and their occupation frequency is 1.0. The H-bonding geometry and the electronic consequences of the water molecular interaction at the different residues (and also IMP and NAD⁺) may put forward some rational clues on antileukemic agent design.     

181 Integrative analysis of gene expression and protein–protein interaction networks in Glioblastoma

Glioblastoma multiforme (GBM) is the most malignant of all the brain tumors with very low median survival time of one year, as per Central Brain Tumor Registry of the USA, 2001. Efforts are ongoing to understand this disease pathogenesis in complete details. Global gene expression changes in GBM pathogenesis have been studied by several groups using microarray technology (e.g. Carro et al., 2010). One of the many approaches to ‘understand the control mechanisms underlying the observed changes in the activity of a biological process’ (Cline et al., 2007) is integration of gene expression and protein–protein interactions (PPI) datasets. Among several examples, aberrant activation of Wnt/β-catenin signaling pathway as well as sonic hedgehog (SHH) signaling pathway is reported in GBMs (Klaus & Birchmeier, 2008). Further, these two pathways are also involved in proliferation and clonogenicity of glioma cancer stem cells (Li et al., 2009), which are thought to play a role in glioma initiation, proliferation, and invasion, and are one of the important points of intervention. Hedgehog–Gli1 signaling is also found to regulate the expression of stemness genes. In this paper, analyses of the relationship between the significant differential expression of these and other genes and the connectivity as well as topological features of a PPI network would be discussed. This way, genes potentially overlooked when relying solely on expression profiles may be identified which can be biologically relevant as possible drug target/s or disease biomarker/s.     

Plant regeneration, genetic fidelity, and active ingredient content of encapsulated hairy roots of Picrorhiza kurrooa Royle ex Benth.

Among five hairy root lines of Picrorhiza kurrooa that were established through Agrobacterium rhizogenes, one (H7) was selected for encapsulation due to high accumulation of picrotin and picrotoxinin (8.3 and 47.6 μg/g DW, respectively). Re-grown encapsulated roots induced adventitious shoots with 73 % frequency on MS medium supplemented with 0.1 μM 6-benzylaminopurine, following 6 months of storage at 25 °C. Regenerated plantlets had 85 % survival after 2 months. Regenerants were of similar morphotype having increased leaf number and branched root system as compared to non-transformed plants. The transformed nature of the plants was confirmed through PCR and Southern blot analysis. Genetic fidelity analysis of transformed plants using RAPD and ISSR showed 5.2 and 3.6 % polymorphism, respectively. Phytochemical analysis also showed that picrotin and picrotoxinin content were similar in hairy root line and its regenerants.     

Comparative physico-kinetic properties of a homogenous purified β-glucosidase from Withania somnifera leaf

β-glucosidase from Withania somnifera (Solanaceae) leaf has been purified to homogeneity and characterized for its physico-kinetic properties. The enzyme purification was achieved through a sequence of gel filtration and ion-exchange column chromatography, and PAGE revealed the homogeneity purification status of the enzyme. The properties of the enzyme included an acidic pH optima (4.8), alkaline pI (8.7), meso-thermostabity, monomeric structure with subunit molecular weight of about 50 kDa, high affinity for substrate (K _m) for pNPG (0.19 mM) and high (105,263 M^?1 s^?1) catalytic efficiency (K _cat/K _m). The mesostable enzyme had a stringent substrate specificity restricted only to β-linked gluco-conjugate. The enzyme is optimally active at 40 °C with 12.4 kcal Mol^?1 activation energy, and was highly sensitive to d-gluconic acid lactone inhibition (94 % at 1 mM) with an apparent K _i 0.21 mM. The enzyme could catalyze transglucosylation of geraniol with pNPG as glucosyl donor, but not with cellobiose. Some of the physico-kinetic properties were noted to be novel when comprehensively compared with its counterparts from plant, animal and microbial counterparts. Nevertheless, the catalytic and other features of the enzyme were relatively closer to Oryza sativa among plants and Talaromyces thermophillus among fungi. Significance of building-up of a library of novel plant β-glucosidases for structural investigation to understand naturally evolved mechanistics of catalysis has been indicated.     

Picrorhiza kurrooa: current status and tissue culture mediated biotechnological interventions

Picrorhiza kurrooa, one of the important plant species among the various medicinal plants, is endemic to Himalaya. As the plant is useful in the treatment of various diseases, e.g., hepatic disorders, gastric troubles, anemia, asthma, etc., illegal collection from the wild is increasing and now this plant is banned for export in any form and listed as ‘endangered’. Ecological studies carried out on this species in last few decades suggested that the availability of this species in its specific habitats is comparatively lower than other associate species. Possible factors responsible for this depletion are increasing demand in the pharmaceutical industries, habitat specificity, heavy exploitation from the wild, unorganized cultivation practices etc. Biotechnology is playing a crucial role to conserve this important plant species. The past 23 years have witnessed a progressive biotechnological advances made in P. kurrooa. People have published various reports on establishments of in vitro culture techniques including micropropagation, synthetic seed production, plant regeneration via callus-mediated shoot organogenesis, adventitious shoot regeneration, genetic transformation through Agrobacterium rhizogenes, secondary metabolite analysis etc. This review attempts to focus on present ecological status and provide a comprehensive account on the tissue culture-mediated biotechnological interventions made in P. kurrooa for improvement and conservation of this medicinally important plant.     

Unravelling the beneficial role of microbial contributors in reducing the allelopathic effects of weeds

The field of allelopathy is one of the most fascinating but controversial processes in plant ecology that offers an exciting, interdisciplinary, complex, and challenging study. In spite of the established role of soil microbes in plant health, their role has also been consolidated in studies of allelopathy. Moreover, allelopathy can be better understood by incorporating soil microbial ecology that determines the relevance of allelopathy phenomenon. Therefore, while discussing the role of allelochemicals in plant–plant interactions, the dynamic nature of soil microbes should not be overlooked. The occurrence and toxicity of allelochemicals in soil depend on various factors, but the type of microflora in the surroundings plays a crucial role because it can interfere with its allelopathic nature. Such microbes could be of prime importance for biological control management of weeds reducing the cost and ill effects of chemical herbicides. Among microbes, our main focus is on bacteria—as they are dominant among other microbes and are being used for enhancing crop production for decades—and fungi. Hence, to refer to both bacteria and fungi, we have used the term microbes. This review discusses the beneficial role of microbes in reducing the allelopathic effects of weeds. The review is mainly focused on various functions of bacteria in (1) reducing allelopathic inhibition caused by weeds to reduce crop yield loss, (2) building inherent defense capacity in plants against allelopathic weed, and (3) deciphering beneficial rhizospheric process such as chemotaxis/biofilm, degradation of toxic allelochemicals, and induced gene expression.     

Structural–functional insights of single and multi-domain Capsicum annuum protease inhibitors

Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural–functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. β-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K_i ～ 57 μM) than -15 (～184 μM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity.     

Synthesis and antioxidant evaluation of (S,S)- and (R,R)-secoisolariciresinol diglucosides (SDGs)

Secoisolariciresinol diglucosides (SDGs) (S,S)-SDG-1 (major isomer in flaxseed) and (R,R)-SDG-2 (minor isomer in flaxseed) were synthesized from vanillin via secoisolariciresinol (6) and glucosyl donor 7 through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives (S,S)-8 and (R,R)-9. Synthetic (S,S)-SDG-1 and (R,R)-SDG-2 exhibited potent antioxidant properties (EC₅₀ = 292.17 ± 27.71 μM and 331.94 ± 21.21 μM, respectively), which compared well with that of natural (S,S)-SDG-1 (EC₅₀ = 275.24 ± 13.15 μM). These values are significantly lower than those of ascorbic acid (EC₅₀ = 1129.32 ± 88.79 μM) and α-tocopherol (EC₅₀ = 944.62 ± 148.00 μM). Compounds (S,S)-SDG-1 and (R,R)-SDG-2 also demonstrated powerful scavenging activities against hydroxyl [natural (S,S)-SDG-1: 3.68 ± 0.27; synthetic (S,S)-SDG-1: 2.09 ± 0.16; synthetic (R,R)-SDG-2: 1.96 ± 0.27], peroxyl [natural (S,S)-SDG-1: 2.55 ± 0.11; synthetic (S,S)-SDG-1: 2.20 ± 0.10; synthetic (R,R)-SDG-2: 3.03 ± 0.04] and DPPH [natural (S,S)-SDG-1: EC₅₀ = 83.94 ± 2.80 μM; synthetic (S,S)-SDG-1: EC₅₀ = 157.54 ± 21.30 μM; synthetic (R,R)-SDG-2: EC₅₀ = 123.63 ± 8.67 μM] radicals. These results confirm previous studies with naturally occurring (S,S)-SDG-1 and establish both (S,S)-SDG-1 and (R,R)-SDG-2 as potent antioxidants and free radical scavengers for potential in vivo use.