Antixenosis,tolerance and genetic analysis of some rice landraces for resistance to Nilaparvata lugens (Stål.)

Twenty-six rice landraces from West Bengal, India were evaluated for antixenosis and tolerance against brown planthopper (BPH) biotype 4 at the Bidhan Chandra Krishi Viswavidyalaya (BCKV), West Bengal. High levels of resistance were observed in six landraces, namely Badshabhog, Gamra, Haldichuri, Janglijata, Kalabhat and Khara. These phenotypically resistant rice landraces including Ptb33 exhibited lowest feeding rate, fecundity, nymphal and adult preference, survival, plant dry weight loss per mg of BPH dry weight produced (PDWL), and higher functional plant loss index (FPLI), more days to wilt and unhatched eggs compared with the susceptible check Swarna. All the landraces were classified into four major clusters at 10 unit distance by the scale of similarity during genetic diversity analysis through 21 gene-linked SSR markers of BPH resistance. Some phenotypically resistant landraces were gathered under the major cluster I indicating their analogous genetic history, while some were grouped with susceptible landraces exhibiting their genetic variation. The resistant landraces can be used as potential donors in the breeding programme for the development of rice varieties with resistance to BPH.     

STATISTICAL APPROACH FOR THE ENHANCED PRODUCTION OF COLD-ACTIVE β-GALACTOSIDASE FROM Thalassospira frigidphilosprofundus: A NOVEL MARINE PSYCHROPHILE FROM DEEP WATERS OF BAY OF BENGAL

In the present investigation Thalassospira frigidphilosprofundus, a novel species from the deep waters of the Bay of Bengal, was explored for the production of cold-active β-galactosidase by submerged fermentation using marine broth medium as the basal medium. Effects of various medium constituents, namely, carbon, nitrogen source, pH, and temperature, were investigated using a conventional one-factor-at-a-time method. It was found that lactose, yeast extract, and bactopeptones are the most influential components for β-galactosidase production. Under optimal conditions, the production of β-galactosidase was found to be 3,864 U/mL at 20 ± 2°C, pH 6.5 ± 0.2, after 48 hr of incubation. β-Galactosidase production was further optimized by the Taguchi orthogonal array design of experiments and the central composite rotatable design (CCRD) of response surface methodology. Under optimal experimental conditions the cold-active β-galactosidase enzyme production from Thalassospira frigidphilosprofundus was enhanced from 3,864 U/mL to 10,657 U/mL, which is almost three times higher than the cold-active β-galactosidase production from the well-reported psychrophile Pseudoalteromonas haloplanktis.     

Differentiation of Isomeric Purine and Pyrimidine Mononucleotides by Fast Atom Bombardment Tandem Mass Spectrometry

Abstract

The use of positive ion fast atom bombardment mass-analysed ion kinetic energy (FAB/MIKE) spectroscopy to differentiate the 2′, 3′-and 5′-monophosphate isomers of adenosine, guanosine and cytidine is described.     

Analgesic and anti-inflammatory studies of cyclopeptide alkaloid fraction of leaves of Ziziyphus nummularia

Ziziyphus nummularia (family: Rhamnaceae) is a thorny small bush, grows in abundance in the grazing lands of the arid areas of Rajasthan, India. It is an important ethnomedicinal plant of the Thar Desert; local inhabitants use every part of the plant as medicine. Kernels are prescribed in pregnancy as soporific, antiemetic and for relieving abdominal pain. The insect gall is powered and given orally with water to cure bone fracture. Crushed root is applied on the paining shoulder of the bullock. The decoction of leaves is used for the treatment of cough and cold; leaves are also regarded as diaphoretic and prescribed in typhoid. Paste of leaves is used for healing of cuts, boils and cutaneous disease. It is widely used in pain and inflammatory conditions.Z. nummularia contains a unique group of alkaloids known as cyclopeptide alkaloids, in continuation of our work carried out on the leaves of Z. nummularia, present study was initiated to explore antiinflammatory and analgesic potential of cyclopeptide alkaloids isolated from the leaves of Z. nummularia (IFZN). Anti-inflammatory activity was tested against rat paw oedema, mouse peritonitis and cotton pellet granuloma. For screening of analgesic activity, acetic acid induced writhing, tail flick and hot plate test were performed.IFZN 30 mg/kg shows the anti-oedematogenic effect against paw oedema induced by carrageenan, dextran, serotonin and histamine; IFZN 20 and 30 mg/kg were found to have highly significant anti-nociceptive effects.Result of pharmacological studies indicated that IFZN is a potent and efficacious analgesic agent. The analgesic activity of IFZN is mediated by the peripheral as well as central pathways.     

S-Adenosyl-Homocysteine Is a Weakly Bound Inhibitor for a Flaviviral Methyltransferase

The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.     

Identification of Key Contributory Factors Responsible for Vascular Dysfunction in Idiopathic Recurrent Spontaneous Miscarriage

Poor endometrial perfusion during implantation window is reported to be one of the possible causes of idiopathic recurrent spontaneous miscarriage (IRSM). We have tested the hypothesis that certain angiogenic and vasoactive factors are associated with vascular dysfunction during implantation window in IRSM and, therefore, could play a contributory role in making the endometrium unreceptive in these women. This is a prospective case-controlled study carried out on 66 women with IRSM and age and BMI matched 50 fertile women serving as controls. Endometrial expression of pro-inflammatory (IL-1β, TNF-α, IFN-γ, TGF-β1), anti-inflammatory (IL-4, -10), angiogenesis-associated cytokines (IL-2, -6, -8), angiogenic and vasoactive factors including prostaglandin E2 (PGE2), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), nitric oxide (NO) and adrenomedullin (ADM) were measured during implantation window by ELISA. Subendometrial blood flow (SEBF) was assessed by color Doppler ultrasonography. Multivariate analysis was used to identify the significant factor(s) responsible for vascular dysfunction in IRSM women during window of implantation and further correlated with vascular dysfunction. Endometrial expression of pro-inflammatory cytokines and PGE2 were up-regulated and anti-inflammatory and angiogenesis-associated cytokines down-regulated in IRSM women as compared with controls. Further, the angiogenic and vasoactive factors including VEGF, eNOS, NO and ADM were found to be down-regulated and SEBF grossly affected in these women. Multivariate analysis identified IL-10, followed by VEGF and eNOS as the major factors contributing towards vascular dysfunction in IRSM women. Moreover, these factors strongly correlated with blood flow impairment. This study provides an understanding that IL-10, VEGF and eNOS are the principal key components having a contributory role in endometrial vascular dysfunction in women with IRSM. Down-regulation of these factors is also associated with impaired endometrial perfusion which possibly makes the endometrium unreceptive that may eventually cause early pregnancy loss.     

The RECQL4 protein,deficient in Rothmund–Thomson syndrome is active on telomeric D-loops containing DNA metabolism blocking lesions

Telomeres are critical for cell survival and functional integrity. Oxidative DNA damage induces telomeric instability and cellular senescence that are associated with normal aging and segmental premature aging disorders such as Werner Syndrome and Rothmund–Thomson Syndrome, caused by mutations in WRN and RECQL4 helicases respectively. Characterizing the metabolic roles of RECQL4 and WRN in telomere maintenance is crucial in understanding the pathogenesis of their associated disorders. We have previously shown that WRN and RECQL4 display a preference in vitro to unwind telomeric DNA substrates containing the oxidative lesion 8-oxoguanine. Here, we show that RECQL4 helicase has a preferential activity in vitro on telomeric substrates containing thymine glycol, a critical lesion that blocks DNA metabolism, and can be modestly stimulated further on a D-loop structure by TRF2, a telomeric shelterin protein. Unlike that reported for telomeric D-loops containing 8-oxoguanine, RECQL4 does not cooperate with WRN to unwind telomeric D-loops with thymine glycol, suggesting RECQL4 helicase is selective for the type of oxidative lesion. RECQL4's function at the telomere is not yet understood, and our findings suggest a novel role for RECQL4 in the repair of thymine glycol lesions to promote efficient telomeric maintenance.     

Improved Glucose Control and Reduced Body Weight in Rodents with Dual Mechanism of Action Peptide Hybrids

Combination therapy is being increasingly used as a treatment paradigm for metabolic diseases such as diabetes and obesity. In the peptide therapeutics realm, recent work has highlighted the therapeutic potential of chimeric peptides that act on two distinct receptors, thereby harnessing parallel complementary mechanisms to induce additive or synergistic benefit compared to monotherapy. Here, we extend this hypothesis by linking a known anti-diabetic peptide with an anti-obesity peptide into a novel peptide hybrid, which we termed a phybrid. We report on the synthesis and biological activity of two such phybrids (AC164204 and AC164209), comprised of a glucagon-like peptide-1 receptor (GLP1-R) agonist, and exenatide analog, AC3082, covalently linked to a second generation amylin analog, davalintide. Both molecules acted as full agonists at their cognate receptors in vitro, albeit with reduced potency at the calcitonin receptor indicating slightly perturbed amylin agonism. In obese diabetic Lep^ob/Lep ^ob mice sustained infusion of AC164204 and AC164209 reduced glucose and glycated haemoglobin (Hb_A1c) equivalently but induced greater weight loss relative to exenatide administration alone. Weight loss was similar to that induced by combined administration of exenatide and davalintide. In diet-induced obese rats, both phybrids dose-dependently reduced food intake and body weight to a greater extent than exenatide or davalintide alone, and equal to co-infusion of exenatide and davalintide. Phybrid-mediated and exenatide + davalintide-mediated weight loss was associated with reduced adiposity and preservation of lean mass. These data are the first to provide in vivo proof-of-concept for multi-pathway targeting in metabolic disease via a peptide hybrid, demonstrating that this approach is as effective as co-administration of individual peptides.     

Improved nearest-neighbor parameters for the stability of RNA/DNA hybrids under a physiological condition

The stability of Watson–Crick paired RNA/DNA hybrids is important for designing optimal oligonucleotides for ASO (Antisense Oligonucleotide) and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 techniques. Previous nearest-neighbour (NN) parameters for predicting hybrid stability in a 1 M NaCl solution, however, may not be applicable for predicting stability at salt concentrations closer to physiological condition (e.g. ∼100 mM Na⁺ or K⁺ in the presence or absence of Mg²⁺). Herein, we report measured thermodynamic parameters of 38 RNA/DNA hybrids at 100 mM NaCl and derive new NN parameters to predict duplex stability. Predicted ΔG°₃₇ and T_m values based on the established NN parameters agreed well with the measured values with 2.9% and 1.1°C deviations, respectively. The new results can also be used to make precise predictions for duplexes formed in 100 mM KCl or 100 mM NaCl in the presence of 1 mM Mg²⁺, which can mimic an intracellular and extracellular salt condition, respectively. Comparisons of the predicted thermodynamic parameters with published data using ASO and CRISPR–Cas9 may allow designing shorter oligonucleotides for these techniques that will diminish the probability of non-specific binding and also improve the efficiency of target gene regulation.