A hydrolytic ��-glutamyl transpeptidase from thermo-acidophilic archaeon Picrophilus torridus: binding pocket mutagenesis and transpeptidation

??-Glutamyl transpeptidase of a thermo-acidophilic archaeon Picrophilus torridus was cloned and expressed using E. coli Rosetta-pET 51b(+) expression system. The enzyme was expressed at 37 °C/200 rpm with ??-GT production of 1.99 U/mg protein after 3 h of IPTG induction. It was improved nearby 10-fold corresponding to 18.92 U/mg protein in the presence of 2 % hexadecane. The enzyme was purified by Ni²⁺-NTA with a purification fold of 3.6 and recovery of 61 %. It was synthesized as a precursor heterodimeric protein of 47 kDa with two subunits of 30 kDa and 17 kDa, respectively, as revealed by SDS-PAGE and western blot. The enzyme possesses hydrolase activity with optima at pH 7.0 and 55 °C. It was thermostable with a t _1/2 of 1 h at 50 °C and 30 min at 60 °C, and retained 100 % activity at 45 °C even after 24 h. It was inhibited by azaserine and DON and PMSF. Pt??-GT shared 37 % sequence identity and 53 % homology with an extremophile ??-GT from Thermoplasma acidophilum. Functional residues identified by in silico approaches were further validated by site-directed mutagenesis where Tyr327 mutated by Asn327 introduced significant transpeptidase activity.     

Inhibiting Cycloxygenase and Ornithine Decarboxylase by Diclofenac and Alpha-Difluoromethylornithine Blocks Cutaneous SCCs by Targeting Akt-ERK Axis

Non-melanoma skin cancer (NMSC) is the most common type of skin cancer in Caucasian populations. Its increasing incidence has been a major public health concern. Elevated expressions of ODC and COX-2 are associated with both murine and human NMSCs. Inhibition of these molecular targets singly employing their respective small molecule inhibitors showed limited success. Here, we show that combined blockade of ODC and COX-2 using their potent inhibitors, DFMO and diclofenac respectively abrogates growth of A431 epidermal xenograft tumors in nu/nu mice by more than 90%. The tumor growth inhibition was associated with a diminution in the proliferation and enhancement in apoptosis. The proliferation markers such as PCNA and cyclin D1 were reduced. TUNEL-positive apoptotic cells and cleaved caspase-3 were increased in the residual tumors. These agents also manifested direct target-unrelated effects. Reduced expression of phosphorylated MAPKAP-2, ERK, and Akt (ser⁴⁷³ & thr³⁰⁸) were noticed. The mechanism by which combined inhibition of ODC/COX attenuated tumor growth and invasion involved reduction in EMT. Akt activation by ODC+COX-2 over-expression was the key player in this regard as Akt inhibition manifested effects similar to those observed by the combined inhibition of ODC+COX-2 whereas forced over-expression of Akt resisted against DFMO+diclofenac treatment. These data suggest that ODC+COX-2 over-expression together leads to pathogenesis of aggressive and invasive cutaneous carcinomas by activating Akt signaling pathway, which through augmenting EMT contributes to tumor invasion.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.     

Target-mimicry based miRNA167-diminution ameliorates cotton somatic embryogenesis via transcriptional biases of auxin signaling associated miRNAs and genes

Plant Cell, Tissue and Organ Culture (PCTOC) - In vitro regeneration of cotton seems to be highly genotype dependent and typically ensues through the extraordinarily complex mechanism of somatic...     

Chloro and bromo-pyrazole curcumin Knoevenagel condensates augmented anticancer activity against human cervical cancer cells: design,synthesis, in silico docking and in vitro cytotoxicity analysis

Abstract

With an endeavor to develop novel curcumin analogs as potential anti-cancer agents, we designed and synthesized a series of Knoevenagel condensates by clubbing pyrazole carbaldehydes at the active methylene carbon atom of the curcumin backbone. Molecular docking studies were carried out to target the proposed derivatives on human kinase β (IKKβ), a potential anti-cancer target. The chloro derivative displayed five hydrogen bond interactions with a docking score of ?11.874?kcal/mol higher than curcumin (docking score =??7.434?kcal/mol). This was supported by the fact that the propellant shaped derivatives fitted aptly into the binding pocket. Molecular simulations studies were also conducted on the lead molecule and the results figured out that the stable complexes were developed as the minimal deviations per residue of protein within the range of 0.11–0.92 Å. The screened compounds were synthesized, characterized and evaluated in vitro for cytotoxicity against cervical cancer cell line, HeLa using standard cell proliferation assay. Chloro derivative and bromo analog demonstrated IC₅₀ (half maximal inhibitory concentration) value of 14.2 and 18.6 µg/ml, respectively, significantly lower than 42.4 µg/ml of curcumin and higher than 0.008 µg/ml of paclitaxel. Induction of apoptosis was evaluated in the terms of cleavage of caspase-3 enzyme and they also exhibited 69.6 and 65.4% of apoptosis significantly higher than 19.9% induced by curcumin. In conclusion, chloro and bromo derivatives must be evaluated under a set of stringent in vitro and in vivo parameters for translating in to a clinically viable product.

Communicated by Ramaswamy H. Sarma     

Detection of begomovirus associated with okra leaf curl disease

Leaf curl and yellow vein mosaic viral disease is the major constraint on okra (Abelmoschus esculentus L.) production in India. Amplified fragment sequence of DNA-β showed highest similarity of 91.7% with Bhendi yellow vein mosaic virus-Tamil Nadu (AJ308425, NC_003405) and lowest similarity of 48.5% with OKLCV (NC_004093), whereas coat protein specific amplified sequence showed highest homology with isolate of Madurai, Haryana, Ludhiana and lowest homology of 92% with Mesta yellow vein mosaic Bahraich virus (MYVMBV) (EU360303). The results obtained in the present study confirm that both the viral diseases of okra reported in southern India are caused by a begomovirus associated with DNA-β in which the plants show leaf curl symptoms and never develops yellow vein mosaic and those plants which show yellow vein mosaic, never develops leaf curl symptoms even in the same rows and field. The okra leaf curl is an emerging virus disease in India.     

Establishment and characterization of an epithelial cell line from thymus of Catla catla (Hamilton, 1822)

A cell line, CTE, derived from catla (Catla catla) thymus has been established by explant method and subcultured for more than 70 passages over a period of 400 days. The cell line has been maintained in L-15 (Leibovitz) medium supplemented with 10% fetal bovine serum. CTE cell line consists of homogeneous population of epithelial-like cells and grows optimally at 28 °C. Karyotype analysis revealed that the modal chromosome number of CTE cells was 50. Partial amplification, sequencing and alignment of fragments of two mitochondrial genes 16S rRNA and COI confirmed that CTE cell line originated from catla. Significant green fluorescent signals were observed when the cell line was transfected with phrGFP II-N mammalian expression vector, indicating its potential utility for transgenic and genetic manipulation studies. The CTE cells showed strong positivity for cytokeratin, indicating that cell line was epithelial in nature. The flow cytometric analysis of cell line revealed a higher number of cells in S-phase at 48 h, suggesting a high growth rate. The extracellular products of Vibrio cholerae MTCC 3904 were toxic to the CTE cells. This cell line was not susceptible to fish betanodavirus, the causative agent of viral nervous necrosis in a large variety of marine fish.     

A non-surgical method for induction of lung cancer in Wistar rats using a combination of NNK and high dietary fats

Lung cancer is one of the most common malignant neoplasms all over the world. Smoking and a number of constituents of tobacco are responsible for development of lung tumours; however, the deleterious effects of tobacco-derived carcinogen, nitrosamine 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (nicotine-derived nitrosamine ketone (NNK)) remain unmatched. We report the development of a novel rodent model by administering multiple doses of NNK to male Wistar rats and feeding them with high-fat and low-protein diet. Tumour cells in lungs were observed in approximately 98 % rats after 8 months of NNK treatment, as evident by histopathological analysis. This rodent model showed slow progression of lung tumours which has helped us to assess early indicators of oxidative damage in lungs by studying the levels of lipid peroxidation and antioxidant parameters. LPO was elevated by 46.94 %, SOD, CAT, GSH and GR activity was decreased by 48.67 %, 22.04 %, 21.46 % and 20.85 %, respectively in serum of NNK treated rats when compared with control. These findings suggest that increased oxidative stress can represent a risk factor for the development of chronic disease in early future. This new animal model is an attempt to greatly facilitate studies of the pathophysiology, biochemistry and therapy of lung cancer.     

Effect of montmorillonite on morphology,glass transition and crystallinity of the xylitol-plasticized bionanocomposites

High amylose based nanocomposites plasticized by xylitol were prepared via twin-screw extrusion. The synergistic interaction in the xylitol-plasticized nanocomposite was studied via various characterization methods and the unique behavior of the xylitol-plasticized nanocomposite had been discussed. As revealed in the XRD and TEM results, good intercalated/exfoliated morphology had been achieved in all the nanocomposites. Furthermore, the expansion of nanoclay basal spacing was related to the xylitol/nanoclay ratio. DSC analysis clearly proved the unique crystallization process of xylitol-plasticized samples. Moreover, in the crystallization domain results, two domains sized at approximately 93.7 Å and 346 Å were found. This observation points to a two-level complex effect from two aggregate domains; one, the re-aggregation of certain number of silicate layers into domains which trap some of the amylose polymer chains, and two, the bulk drying process which combines smaller amylose crystalline domains within a larger amorphous high amylose matrix.     

4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway

4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G₂/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested.