In Silico Studies in Probing the Role of Kinetic and Structural Effects of Different Drugs for the Reactivation of Tabun-Inhibited AChE

We have examined the reactivation mechanism of the tabun-conjugated AChE with various drugs using density functional theory (DFT) and post-Hartree-Fock methods. The electronic environments and structural features of neutral oximes (deazapralidoxime and 3-hydroxy-2-pyridinealdoxime) and charged monopyridinium oxime (2-PAM) and bispyridinium oxime (Ortho-7) are different, hence their efficacy varies towards the reactivation process of tabun-conjugated AChE. The calculated potential energy surfaces suggest that a monopyridinium reactivator is less favorable for the reactivation of tabun-inhibited AChE compared to a bis-quaternary reactivator, which substantiates the experimental study. The rate determining barrier with neutral oximes was found to be ∼2.5 kcal/mol, which was ∼5.0 kcal/mol lower than charged oxime drugs such as Ortho-7. The structural analysis of the calculated geometries suggest that the charged oximes form strong O^…H and N^…H hydrogen bonding and C-H^…π non-bonding interaction with the tabun-inhibited enzyme to stabilize the reactant complex compared to separated reactants, which influences the activation barrier. The ability of neutral drugs to cross the blood-brain barrier was also found to be superior to charged antidotes, which corroborates the available experimental observations. The calculated activation barriers support the superiority of neutral oximes for the activation of tabun-inhibited AChE compared to charged oximes. However, they lack effective interactions with their peripheral sites. Docking studies revealed that the poor binding affinity of simple neutral oxime drugs such as 3-hydroxy-2-pyridinealdoxime inside the active-site gorge of AChE was significantly augmented with the addition of neutral peripheral units compared to conventional charged peripheral sites. The newly designed oxime drug 2 appears to be an attractive candidate as efficient antidote to kinetically and structurally reactivate the tabun-inhibited enzyme.     

Haptoglobin polymorphism among the tribal groups of southern Gujarat

BACKGROUND:

Gujarat is located at the western most point of the Indian subcontinent. Valsad and Surat districts are part of the ‘tribal belt’of Gujarat and constitute 29.1% of total tribal population of Gujarat. These tribal populations are a rich source of gaining insights in the patterns of genetic diversity and genetico-environmental disorders against the back drop of their ecological, historical and ethnographic aspects.

AIM:

The objectives were to find out a) the genetic diversity among the tribes of Gujarat with reference to haptoglobin (Hp) locus b) the relationship between Hp polymorphism and sickle cell anemia/trait.

MATERIALS AND METHODS:

431 individuals belonging to eight tribal groups were studied for Hp polymorphism using polyacrylamide disc gel electrophoresis (PAGE). Hb*S was screened by dithionate tube turbididy (DTT) test and confirmed using cellulose acetate membrane electrophoresis (CAME).

STATISTICAL ANALYSIS:

Allele frequency was calculated by direct gene counting method. Average heterozygosity and gene diversity were computed using software DISPAN. Analysis of molecular variance (AMOVA) was estimated using software ARLEQUIN version 3.1.

RESULTS AND CONCLUSIONS:

Pattern of allele frequency distribution showed preponderance of Hp² allele in all the eight tribal groups, which is in accordance with its frequency in different populations of Indian subcontinent. Total average heterozygosity (H_T) was found to be low (0.160) but the level of genetic differentiation (G_ST) was found to be moderately high (5.6%). AMOVA analysis indicated least among group variance between west and south Indian populations (-0.04%) indicating the affinities of the tribes of Gujarat with that of Dravidian speaking groups. Analysis of Hp phenotypes among sickle cell anemia/ trait individuals revealed a high frequency of Hp 0-0 phenotype (92.7%) among SS individuals as opposed to only 9.7% among AS individuals, reaffirming the selective advantage of HbAS state in relation to hemolytic disorders.     

Fate of pathological prion (PrPsc92–138) in soil and water: prion-clay nanoparticle molecular dynamics

Pathogenic prion protein scrapie (PrP^sc) may contaminate soils for decades and remain in water in colloidal suspension, providing infection pathways for animals through the inhalation of ingested dust and soil particles, and drinking water. We used molecular dynamics simulations to understand the strong binding mechanism of this pathogenic peptide with clay mineral surfaces and compared our results to experimental works. We restricted our model to the moiety PrP(92–138), which is a portion of the whole PrP^sc molecule responsible for infectivity and modeled it using explicit solvating water molecules in contact with a pyrophyllite cleavage plane. Pyrophyllite is taken as a model for common soil clay, but it has no permanent structural charge. However, partial residual negative charges occur on the cleavage plane slab surface due to a slab charge unbalance. The charge is isotropic in 2D and it was balanced with K⁺ ions. After partially removing potassium ions, the peptide anchors to the clay surface via up to 10 hydrogen bonds, between protonated lysine or histidine residues and the oxygen atoms of the siloxane cavities. Our results provide insight to the mechanism responsible for the strong association between the PrP^sc peptide and clay nanoparticles and the associations present in contaminated soil and water which may lead to the infection of animals.     

ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo

Tumor cells invading three-dimensional matrices need to remodel the extracellular matrix (ECM) in their path. Many studies have focused on the role of extracellular proteases; however, cells with amoeboid or rounded morphologies are able to invade even when these enzymes are inhibited. Here, we describe the mechanism by which cells move through a dense ECM without proteolysis. Amoeboid tumor cells generate sufficient actomyosin force to deform collagen fibers and are able to push through the ECM. Force generation is elevated in metastatic MTLn3E cells, and this correlates with increased invasion and altered myosin light chain (MLC) organization. In metastatic cells, MLC is organized perpendicularly to the direction of movement behind the invading edge. Both the organization of MLC and force generation are dependent upon ROCK function. We demonstrate that ROCK regulates the phosphorylation of MLC just behind the invading margin of the cell. Imaging of live tumors shows that MLC is organized in a similar ROCK-dependent fashion in vivo and that inhibition of ROCK but not matrix-metalloproteases reduces cancer cell motility in vivo.     

Sds22, a PP1 phosphatase regulatory subunit, regulates epithelial cell polarity and shape [Sds22 in epithelial morphology]

Background  

How epithelial cells adopt their particular polarised forms is poorly understood. In a screen for genes regulating epithelial morphology in Drosophila, we identified sds22, a conserved gene previously characterised in yeast.     

Development of mutants of <Emphasis Type="Italic">Melanocarpus albomyces</Emphasis> for hyperproduction of xylanase

The wild type filamentous fungus, Melanocarpus albomyces, produces many commercially valuable enzymes, including Xylanases and Xylan-debranching enzymes with low activity. In this paper, we report for the first time the development of M. albomyces mutants from vegetative spores. Profuse sporulation of M. albomyces was induced on Potato Carrot Agar medium. These spores, when subjected to chemical mutation, led to the isolation of the hyper-xylanase producing mutant, viz, M. albomyces IITD3A. Various parameters including number of spores, nitrogen source and C/N ratio of the medium were optimized for production of xylanase by the mutant in a shake flask culture. Under controlled pH at 7.8, the mutant produced highly active xylanase with 415 IU/mL after 36 h of growth on soluble alkaline lignocellulosic extract in a 14-L fermentor. The overall productivity of xylanase was 8-fold higher than the wild type culture with11, 530 IU/L/h. The enzyme can be easily stored at 37°C for 50 days by addition of a small amount of the preservative — thiomersal. Also, for long term storage, a lyophilized powder form of the enzyme can be used which retained 100% of its activity for > 50 days. When assayed at pH 7.5 and temperature 55°C, the xylanase retained 100% of its original activity, and also at pH 9.0, it retained > 50% of its activity for 2 h, which is promising for its application in the pulp and paper industry.     

Dose-finding study with nicotine as a proconvulsant agent in PTZ-induced seizure model in mice

The present study was conducted to investigate the possible interaction between low doses of nicotine and pentylenetetrazole (PTZ) in vivo and also to evaluate the influence of nicotine on the antiseizure efficacy of topiramate and sodium valproate in the PTZ-induced seizure model in mice. Graded dose–response study with nicotine showed the CD50 value for nicotine at 6.76 mg/kg. i.p. Subtheshold dose of nicotine (4 mg/kg, i.p.) pretreatment significantly decreased the CD50 value for PTZ from 47.86 mg/kg, i.p. (of PTZ per se) to 31.62 mg/kg, i.p. Sodium valproate but not topiramate, significantly inhibited PTZ-induced seizures in mice with an ED50 value of 177.83 mg/kg, i.p. Nonconvulsive dose of nicotine (1 mg/kg, i.p.) significantly antagonized the protective efficacy of sodium valproate against PTZ-induced seizures and increased the ED50 value to 338.84 mg/kg, i.p. PTZ-induced seizures significantly increased the mouse brain levels of MDA and reduced the level of GSH while sodium valproate reversed such changes. Nicotine pretreatment reversed the anti-lipid peroxidative action of sodium valproate in the PTZ-induced seizure model in mice. The study highlighted the convulsant as well as proconvulsant role of nicotine and established dose discrimination for nicotine as a proconvulsant agent and an anti-antiseizure agent. The study bears significant clinical relevance particularly amongst epileptic smokers who may show failure of efficacy of antiepileptic agents and present with breakthrough seizure attacks on exposure to nicotine.     

Fine needle aspiration cytology of minor salivary gland tumours of the palate

Fine needle aspiration cytology of minor salivary gland tumours of the palate This retrospective study was carried out to review aspirates from minor salivary gland tumours of the palate and to assess the problems encountered in their diagnosis, especially the cytological diagnosis of newer entities such as polymorphous low grade adenocarcinoma (PLGA). Fifty-five cases of palatal salivary gland tumours aspirated over a period of 16 years were reviewed. Histology was available in 26 cases. Pleomorphic adenoma (27 cases) was the most common benign cytodiagnosis. Eleven aspirates were malignant tumours of which eight cases were adenoid cystic carcinoma and three cases were mucoepidermoid carcinoma. Seven cases were diagnosed on fine needle aspiration as suggestive of PLGA. However histological confirmation was available in only one of these cases. Concordance between the initial and revised typings of the tumours was seen in only 28 cases (54%) in the present study. Initially 18 of the 51 tumours (35.3%) could not be typed; and after review, only three could not be typed. Three cases of oncocytoma could be diagnosed on review only. Palatal salivary gland tumours, although relatively uncommon, are difficult to diagnose cytologically. This is more so in cases of newer entities such as PLGA, as their cytological diagnosis is still not well characterized.     

PDK1 regulates cancer cell motility by antagonising inhibition of ROCK1 by RhoE

In three-dimensional matrices cancer cells move with a rounded, amoeboid morphology that is controlled by ROCK-dependent contraction of acto-myosin. In this study, we show that PDK1 is required for phosphorylation of myosin light chain and cell motility, both on deformable gels and in vivo. Depletion of PDK1 alters the localization of ROCK1 and reduces its ability to drive cortical acto-myosin contraction. This form of ROCK1 regulation does not require PDK1 kinase activity, but instead involves direct binding of PDK1 to ROCK1 at the plasma membrane; PDK1 competes directly with RhoE for binding to ROCK1. In the absence of PDK1, negative regulation by RhoE predominates, causing reduced acto-myosin contractility and motility. This work uncovers a novel non-catalytic role for PDK1 in regulating cortical acto-myosin and cell motility.