Symptom-based diagnosis of Huanglongbing (citrus greening) disease by PCR in sweet orange (Citrus sinensis Osbeck) and acid lime (Citrus aurantifolia Swingle)

Huanglongbing (HLB), previously known as citrus greening disease, is one of the most destructive diseases of citrus and responsible for the decline of citrus orchards in Andhra Pradesh (AP) and other citrus growing areas in the country. Polymerase chain reaction (PCR) amplification of 1160 bp fragment of 16S rDNA of HLB was observed in mottling symptoms, yellow vein symptoms, symptoms mixed with both citrus yellow mosaic virus and citrus greening and zinc deficiency-like symptoms; however, no amplification was observed in iron deficiency like symptoms. As the disease shows a variety of symptoms in infected trees, PCR detection could be useful for resolving confusion in identification of HLB between actual deficiency and deficiency like symptoms caused by HLB. In acid lime the midrib and veins of the leaves with both mottling and yellow vein symptoms were suitable for isolation of DNA and used as a template in the PCR test.     

Torrubiella pruinosa,a teliomorph of an entomopathogenic fungus Hirsutella versicolor of mango hopper (Idioscopus clypealis) from India

Torrubeilla pruinosa, a teliomorph of an anamorphic entomopathogenic fungus Hirsutella versicolor on mango hopper (Idioscopus clypealis) was observed. On the infected mango hopper, tiny pin head signs of ascomata were observed. Ascomata of T. pruinosa is pale brown or tawny brown, crowded, immersed in stroma, wall dark golden brown, 260–320?×?230–260?μm, asci clavate, hyaline and thin walled. The ascospores of T. pruinosa are fusiform, distoseptate with a faint tint of pigmentation, 17.5–25.0?×?5.0–7.5?μm. The anamorph stage of H. versicolor fungal hyphae is hyaline, septate and profusely branched, and conidiogenous (phialides) cells are hyaline, pear-shaped and smooth-walled with single or double sterigmata and rarely, multiple sterigmata. Each sterigmata bears single conidia which are hyaline, oval to pear-shaped.     

Synthesis,characterization and nucleic acid binding studies of mononuclear copper(II) complexes derived from azo containing O,O donor ligands

Abstract

Azo linked salicyldehyde and a new 2-hydroxy acetophenone based ligands (HL¹ and HL²) with their copper(II) complexes [Cu(L¹)₂] (1) and [Cu(L²)₂] (2) were synthesized and characterized by spectroscopic methods such as ¹H, 13C NMR, UV–Vis spectroscopy and elemental analyses. Calculation based on Density Functional Theory (DFT), have been performed to obtain optimized structures. Binding studies of these copper (II) complexes with calf thymus DNA (ct-DNA) and torula yeast RNA (t-RNA) were analyzed by absorption spectra, emission spectra and Viscosity studies and Molecular Docking techniques. The absorption spectral study indicated that the copper(II) complexes of 1 and 2 had intrinsic binding constants with DNA or RNA in the range of 7.6?±?0.2?×?10³?M^?1 or 6.5?±?0.3?×?10³M^?1 and 5.7?±?0.4?×?10⁴ M^?1 or 1.8?±?0.5?×?10³ M^?1 respectively. The synthesized compounds and nucleic acids were simulated by molecular docking to explore more details mode of interaction of the complexes and their orientations in the active site of the receptor.     

Strategies for the Production of Recombinant Protein in Escherichia coli

In the recent past years, a large number of proteins have been expressed in Escherichia coli with high productivity due to rapid development of genetic engineering technologies. There are many hosts used for the production of recombinant protein but the preferred choice is E. coli due to its easier culture, short life cycle, well-known genetics, and easy genetic manipulation. We often face a problem in the expression of foreign genes in E. coli. Soluble recombinant protein is a prerequisite for structural, functional and biochemical studies of a protein. Researchers often face problems producing soluble recombinant proteins for over-expression, mainly the expression and solubility of heterologous proteins. There is no universal strategy to solve these problems but there are a few methods that can improve the level of expression, non-expression, or less expression of the gene of interest in E. coli. This review addresses these issues properly. Five levels of strategies can be used to increase the expression and solubility of over-expressed protein; (1) changing the vector, (2) changing the host, (3) changing the culture parameters of the recombinant host strain, (4) co-expression of other genes and (5) changing the gene sequences, which may help increase expression and the proper folding of desired protein. Here we present the resources available for the expression of a gene in E. coli to get a substantial amount of good quality recombinant protein. The resources include different strains of E. coli, different E. coli expression vectors, different physical and chemical agents and the co expression of chaperone interacting proteins. Perhaps it would be the solutions to such problems that will finally lead to the maturity of the application of recombinant proteins. The proposed solutions to such problems will finally lead to the maturity of the application of recombinant proteins.     

Homology modeling and structural comparison of leucine rich repeats of toll like receptors 1-10 of ruminants

Toll-like receptors (TLRs) are transmembrane receptors composed of extra cellular leucine rich repeats (LRRs) that identify specific pathogen associated molecular patterns triggering a innate immune cascade. The LRR regions of TLR 1–10 proteins of goat (Capra hircus), sheep (Ovis aries), buffalo (Bubalus bubalis) and bovine (Bos taurus) were modeled using MODELLER 9v7 tool and validated. The similarities and variations of these 10 TLRs extracellular regions of each species were compared using online servers like FATCAT, SSM and SSAP. It was evident that the LRRs of TLRs like 1, 2, 3 and 6 showed structural convergence with <1 % RMSD deviation while TLRs like 5, 7, 8 and 9 had high divergence. Docking analysis showed that TLR 2, 3 and 7 of all the selected four ruminant species were able to bind with their corresponding ligands like Peptidoglycan (PGN), Poly I:C, Resiquimod (R-848) and Imiquimod. However, there were variations in the active site regions, interacting residues and the number of bonded interactions. Variations seen among TLR structures and their ligand binding characteristics is likely to be responsible for species and breed specific genetic resistance observed among species or breeds.     

Solid phase synthesis and biological evaluation of probestin as an angiogenesis inhibitor

Probestin is a potent aminopeptidase N (APN) inhibitor originally isolated from the bacterial culture broth. Here, we report probestin synthesis by solid phase peptide synthesis (SPPS) method and evaluated its activity to inhibit angiogenesis using a chicken embryo chorioallantoic membrane (CAM) assay and a CAM tumor xenograft model. Results from these studies demonstrate that probestin inhibits the angiogenic activity and tumor growth.     

Development of cell-active non-peptidyl inhibitors of cysteine cathepsins

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.     

NMK-TD-100, a Novel Microtubule Modulating Agent,Blocks Mitosis and Induces Apoptosis in HeLa Cells by Binding to Tubulin

Thiadiazoles are one of the most widely utilized agents in medicinal chemistry, having a wide range of pharmacologic activity. Microtubules (MTs) have always remained a sought-after target in rapidly proliferating cancer cells. We screened for the growth inhibitory effect of synthetic 5-(3-indolyl)-2-substituted-1,3,4-thiadiazoles on cancer cells and identified NMK-TD-100, as the most potent agent. Cell viability experiments using human cervical carcinoma cell line (HeLa cells) indicated that the IC₅₀ value was 1.42±0.11 µM for NMK-TD-100 for 48 h treatment. In further study, we examined the mode of interaction of NMK-TD-100 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. NMK-TD-100 induced arrest in mitotic phase of cell cycle, caused decline in mitochondrial membrane potential and induced apoptosis in HeLa cells. Immunofluorescence studies using an anti-α-tubulin antibody showed a significant depolymerization of the interphase microtubule network and spindle microtubule in HeLa cells in a concentration-dependent manner. However, the cytotoxicity of NMK-TD-100 towards human peripheral blood mononuclear cells (PBMC) was lower compared to that in cancer cells. Polymerization of tissue purified tubulin into microtubules was inhibited by NMK-TD-100 with an IC₅₀ value of 17.5±0.35 µM. The binding of NMK-TD-100 with tubulin was studied using NMK-TD-100 fluorescence enhancement and intrinsic tryptophan fluorescence of tubulin. The stoichiometry of NMK-TD-100 binding to tubulin is 1:1 (molar ratio) with a dissociation constant of ~1 µM. Fluorescence spectroscopic and molecular modeling data showed that NMK-TD-100 binds to tubulin at a site which is very near to the colchicine binding site. The binding of NMK-TD-100 to tubulin was estimated to be ~10 times faster than that of colchicine. The results indicated that NMK-TD-100 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided insights into its potential of being a chemotherapeutic agent.