Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions

Nucleolar organizer regions are nucleolar components that contain proteins that are stained selectively by silver methods; they can be identified as black dots throughout the nucleolus and are known as silver binding nucleolar organizer regions (AgNOR). The number of AgNOR is related to the cell cycle and the proliferative activity of the cells. We investigated AgNOR using exfoliative cytology smears of potentially malignant oral lesions. Eighty individuals were divided into four equal groups: healthy controls, oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. The mean number of AgNOR in each study group gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. The proliferative index was increased in the oral premalignant and malignant patients compared to normal subjects. The mean AgNOR size gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. Spherical shaped AgNOR were most common in controls, whereas large, clustered and kidney shapes were most common in oral squamous cell carcinoma. Multiparameter analysis of AgNOR in oral exfoliative smears is a simple, sensitive and cost-effective method for differentiating premalignant from malignant lesions and can be used in conjunction with routine cytomorphological evaluation.     

Multiple helical conformations of the helix‐turn‐helix region revealed by NOE‐restrained MD simulations of tryptophan aporepressor,TrpR

The nature of flexibility in the helix‐turn‐helix region of E. coli trp aporepressor has been unexplained for many years. The original ensemble of nuclear magnetic resonance (NMR structures showed apparent disorder, but chemical shift and relaxation measurements indicated a helical region. Nuclear Overhauser effect (NOE) data for a temperature‐sensitive mutant showed more helical character in its helix‐turn‐helix region, but nevertheless also led to an apparently disordered ensemble. However, conventional NMR structure determination methods require all structures in the ensemble to be consistent with every NOE simultaneously. This work uses an alternative approach in which some structures of the ensemble are allowed to violate some NOEs to permit modeling of multiple conformational states that are in dynamic equilibrium. Newly measured NOE data for wild‐type aporepressor are used as time‐averaged distance restraints in molecular dynamics simulations to generate an ensemble of helical conformations that is more consistent with the observed NMR data than the apparent disorder in the previously reported NMR structures. The results indicate the presence of alternating helical conformations that provide a better explanation for the flexibility of the helix‐turn‐helix region of trp aporepressor. Structures representing these conformations have been deposited with PDB ID: 5TM0. Proteins 2017; 85:731–740. © 2016 Wiley Periodicals, Inc.     

Screening of Chromium-Resistant Bacteria for Plant Growth-Promoting Activities

Chromium-resistant plant growth-promoting bacteria (PGPB) were isolated from the electroplating industry waste disposal site soils at Coimbatore, India, using LB medium. The strain tolerated chromium concentrations up to 500 mg Cr⁶⁺L^?1 on LB medium. The strain was identified as Pseudomonas sp. VRK3 based on its morphological, physiological, and biochemical characteristics following Bergey's manual of determinative bacteriology. Evaluation of plant growth-promoting parameters revealed the intrinsic ability of the strain for the production of indole-3-acetic acid (IAA), siderophore, and solubilization of insoluble phosphate. Pseudomonas sp. VRK3 utilized tryptophan as a precursor for the growth and production of IAA (105.77 μg mL^?1) and also exhibited the production of siderophore. The strain utilized tricalcium phosphate as the sole source of phosphate exhibiting a high rate of phosphate solubilization (0.49 μg mL^?1). Pseudomonas sp. VRK3 showed a significant extent of chromium uptake and accumulation in their cell walls. Furthermore, Pseudomonas sp. VRK3 was demonstrated to possess mobilization of chromium from the soil that would enhance chromium availability to the plant. Potential use of this Pseudomonas sp. VRK3 as PGPB needs further testing in enhancing the growth and chromium uptake by the plants in pots under nursery conditions.     

Fusant Trichoderma HF9 with enhanced extracellular chitinase and protein content

Strain improvement was carried out to obtain higher chitinase and protein by inter-specific protoplast fusion between Trichoderma harzianum and Trichoderma viride. Fusant HF9 and parental strains of Trichoderma were compared for chitinase and protein production. 1% of glucose, sucrose and fungal cell wall (Rhizoctonia solani), were used as carbon source for cultivation of Trichoderma and fungal cell wall was the best to induce chitinase and protein. Usage of 0.5% colloidal chitin for the fungal growth under aerated conditions at pH 6.5 and 28°C led to higher chitinase and protein production. In these conditions fusant Trichoderma HF9 in comparison with parent strains had 3-, 2.5- and 1.5-fold increase of total chitinase, specific chitinase and protein, respectively. SDS-PAGE analysis revealed that it had 9 major protein bands with up-regulation compared to parent strains. Amino acid analysis showed that protein of culture filtrate of T. harzianum, T. viride and fusant Trichoderma HF9 had 8, 6 and 10 amino acids, respectively. The results obtained suggested that fusant HF9 could be an integration of T. harzianum and T. viride through protoplast fusion.     

Salmonella detection in foods and feeds in 27 hours by an enzyme immunoassay

A heterogeneous enzyme immunoassay (EIA), which could be completed within 27 h, was developed for the detection of salmonellae in foods. Samples were subjected to the usual non-selective enrichment for 16–18 at 35°C and to a short (6 h) post-enrichment in a moderately selective broth. The EIA was carried out on polystyrene microtitration plates. A pooled polyvalent Salmonella flagellar antiserum and a protein A-alkaline phosphatase conjugate were used. The sensitivity of the enzyme immunoassay compared favorably with that of the conventional cultural technique for detection of Salmonella in 40 naturally contaminated food and feed samples. No sample was positive only by the cultural technique; samples positive only by the enzyme immunoassay were observed for feeds. Some specimens yielded high background values indicating possible interference from food proteins.     

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.     

Targeting the single-strand G-rich overhang of telomeres with PNA inhibits cell growth and induces apoptosis of human immortal cells

Telomeres are believed to stabilize chromosomes through several mechanisms that are dependent upon specific DNA-DNA and protein-DNA interactions. Telomeres are maintained by the enzyme telomerase. Telomerase activity, which is below detectable level in almost all types of diploid cells, is re-activated in most immortal and cancer cells. For this study, we designed peptide nucleic acid (PNA) oligonucleotides targeted to the telomeric G-rich strand, and tested their efficacy to reverse the immortality of transformed human fibroblasts. Anti-telomere PNAs, transfected into human fibroblasts along with a selectable marker, resulted in a significant reduction in colony size and elicited cell death by apoptosis. This PNA inhibitor does not inhibit telomerase activity in vitro, suggesting a distinct cellular mechanism from known PNA inhibitors. A combination of this class of PNA inhibitor with a PNA that does block telomerase activity resulted in nearly complete inhibition of colony growth, induction of apoptosis, and an apparent reduction in telomere length. Each effect was greater than that evoked by either agent alone, indicating enhanced efficacy for therapeutic approaches that target multiple, distinct mechanism of telomere maintenance.     

The minor anionic form of arylsulphatase B (arylsulphatase Bm) of monkey brain. Purification and phosphoprotein nature

The anionic form of arylsulphatase B (arylsulphatase Bm) was purified to apparent homogeneity from monkey brain through steps involving chromatography on diethylaminoethyl-cellulose, Blue-Sepharose, Biogel HTP and finally Biogel P-300 gel filtration. The molecular weight of the purified enzyme as deduced by gel filtration on Biogel P-300 and by sodium dodecylsulphate gel electrophoresis was ∼ 30,000.Escherichia coli alkaline phosphatase treatment of arylsulphatase Bm resulted in the conversion of upto 84% of the enzyme into a less charged form of enzyme, that could not bind to diethylaminoethyl cellulose. Potassium phosphate an inhibitor of alkaline phosphatase prevented this conversion. Upon acid hydrolysis the purified enzyme yielded approximately 7.0 mol of inorganic phosphate per mol of protein.Vibrio cholerae neuraminidase treatment did not alter the charge on arylsulphatase Bm.