In-silico screening of cancer associated mutation on PLK1 protein and its structural consequences

The Polo-like kinases (Plks) are a conserved subfamily of serine-threonine protein kinases that have significant roles in cell proliferation. The serine/threonine protein kinases or polo-like kinase 1 (PLK1) exist in centrosome during interphase and is an important regulatory enzyme in cell cycle progression during M phase. Mutations in mammalian PLK1 were found to be over expressed in various human cancers and it is disrupting the binding ability of polo box domain with target peptide. In this analysis we implemented a computational approach to filter the most deleterious and cancer associated mutation on PLK1 protein. We found W414F as the most deleterious and cancer associated by Polyphen 2.0, SIFT, I-mutant 3.0, PANTHER, PhD-SNP, SNP&GO, Mutpred and Dr Cancer tools. Molecular docking and molecular dynamics simulation (MDS) approach was used to investigate the structural and functional behavior of PLK1 protein upon mutation. MDS and docking results showed stability loss in mutant PLK1 protein. Due to mutation, PLK1 protein became more flexible and alters the dynamic property of protein which might affect the interaction with target peptide and leads to cell proliferation. Our study provided a well designed computational methodology to examine the cancer associated nsSNPs and their molecular mechanism. It further helps scientists to develop a drug therapy against PLK1 cancer-associated diseases.

Systemic pregabalin attenuates facial hypersensitivity and noxious stimulus-evoked release of glutamate in medullary dorsal horn in a rodent model of trigeminal neuropathic pain

Pregabalin is effective in treating many neuropathic pain conditions. However, the mechanisms of its analgesic effects remain poorly understood. The aim of the present study was to determine whether pregabalin suppresses facial mechanical hypersensitivity and evoked glutamate release in the medullary dorsal horn (MDH) in a rodent model of trigeminal neuropathic pain. Nociceptive mechanical sensitivity was assessed pre-operatively, and then post-operatively 1 h following pregabalin or vehicle (saline) treatment on post-operative days 2 and 5 following infraorbital nerve transection (IONX). In addition, an in vivo microdialysis probe was inserted into the exposed medulla post-operatively and dialysate samples were collected. Glutamate release was then evoked by mustard oil (MO) application to the tooth pulp, and the effects of pregabalin or vehicle were examined on the MDH glutamate release. Glutamate concentrations in the dialysated samples were determined by HPLC, and data analyzed by ANOVA. IONX animals (but not control animals) showed facial mechanical hypersensitivity for several days post-operatively. In addition, tooth pulp stimulation with MO evoked a transient release of glutamate in the MDH of IONX animals. Compared to vehicle, administration of pregabalin significantly attenuated the facial mechanical hypersensitivity as well as the MO-evoked glutamate release in MDH. This study provides evidence in support of recent findings pointing to the usefulness of pregabalin in the treatment of orofacial neuropathic pain.     

Anti-caspase-3 preconditioning increases proinsulin secretion and deteriorates posttransplant function of isolated human islets

Human islet isolation is associated with adverse conditions inducing apoptosis and necrosis. The aim of the present study was to assess whether antiapoptotic preconditioning can improve in vitro and posttransplant function of isolated human islets. A dose-finding study demonstrated that 200 μmol/L of the caspase-3 inhibitor Ac-DEVD-CMK was most efficient to reduce the expression of activated caspase-3 in isolated human islets exposed to severe heat shock. Ac-DEVD-CMK-pretreated or sham-treated islets were transplanted into immunocompetent or immunodeficient diabetic mice and subjected to static glucose incubation to measure insulin and proinsulin secretion. Antiapoptotic pretreatment significantly deteriorated graft function resulting in elevated nonfasting serum glucose when compared to sham-treated islets transplanted into diabetic nude mice (p < 0.01) and into immunocompetent mice (p < 0.05). Ac-DEVD-CMK pretreatment did not significantly change basal and glucose-stimulated insulin release compared to sham-treated human islets but increased the proinsulin release at high glucose concentrations (20 mM) thus reducing the insulin-to-proinsulin ratio in preconditioned islets (p < 0.05). This study demonstrates that the caspase-3 inhibitor Ac-DEVD-CMK interferes with proinsulin conversion in preconditioned islets reducing their potency to cure diabetic mice. The mechanism behind this phenomenon is unclear so far but may be related to the ketone CMK linked to the Ac-DEVD molecule. Further studies are required to identify biocompatible caspase inhibitors suitable for islet preconditioning.     

Production of highly active fungal milk-clotting enzyme by solid-state fermentation

Abstract

Cheese production is projected to reach 20 million metric tons by 2020, of which 33% is being produced using calf rennet (EC 3.4.23.4). There is shortage of calf rennet, and use of plant and microbial rennets, hydrolyze milk proteins non-specifically resulting in low curd yields. This study reports fungal enzymes obtained from cost effective medium, with minimal down streaming, whose activity is comparable with calf and Mucor rennet. Of the fifteen fungi that were screened, Mucor thermohyalospora (MTCC 1384) and Rhizopus azygosporus (MTCC 10195) exhibited the highest milk-clotting activity (MCA) of 18,383?±?486?U/ml and 16,373?± 558?U/ml, respectively. Optimization exhibited a 33% increase in enzyme production (30?g wheat bran containing 6% defatted soy meal at 30?°C, pH 7) for M. thermohyalospora. The enzyme was active from pH 5–10 and temperature 45–55?°C. Rhizopus azygosporus exhibited 31% increase in enzyme production (30?g wheat bran containing 4% defatted soy meal at 30?°C, pH 6) and the enzyme was active from pH 6–9 at 50?°C. Curd yields prepared from fungal enzyme extract decreased (5–9%), when compared with calf rennet and Mucor rennet. This study describes the potential of fungal enzymes, hitherto unreported, as a viable alternative to calf rennet     

Isolation and characterization of a novel <Emphasis Type="Italic">α</Emphasis>-amylase from a metagenomic library of Western Ghats of Kerala,India

In the present study, metagenomic library of Western Ghats soil sample was constructed in a fosmid vector (pCC1FOS) and screened for biocatalytic properties. The clones showed amylolytic activity on Luria-Bertani starch agar plates and one of them was studied in detail. The enzyme exhibited stability at elevated temperature with 60°C being the optimal temperature. The enzyme retained more than 30% activity after 60 min incubation at 80°C. It also showed more than 70% activity retention in 1.5 M NaCl solution. The pH optimum of the enzyme was at pH = 5.0. The enzyme possesses good activity in the presence of chelating and strong reducing agents with activity enhancements or retention being observed at 5 mM β-mercaptoethanol, dithiothreitol and N-bromosuccinimide. However, almost complete loss of activity was observed with 5 mM EDTA, while activity enhancement was observed upon incubation with Ca²⁺ suggesting it to be a Ca²⁺-dependent α-amylase, which was further confirmed by a thin-layer chromatography (TLC). The TLC run revealed that digestion pattern was similar to commercial α-amylase. The 16S rRNA gene sequence (GenBank accession number HQ680979) BLAST showed 95% similarities with Exiguobacterium sp. AFB-11 and AFB 18, with query sequence coverage of 99%.     

Endoplasmic reticulum-associated degradation (ERAD) and free oligosaccharide generation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, proteins with misfolded lumenal, membrane, and cytoplasmic domains are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L, -M, and -C, respectively. ERAD-L is N-glycan-dependent and is characterized by ER mannosidase (Mns1p) and ER mannosidase-like protein (Mnl1p), which generate Man(7)GlcNAc(2) (d1) N-glycans with non-reducing α1,6-mannosyl residues. Glycoproteins bearing this motif bind Yos9p and are dislocated into the cytoplasm and then deglycosylated by peptide N-glycanase (Png1p) to yield free oligosaccharides (fOS). Here, we examined yeast fOS metabolism as a function of cell growth in order to obtain quantitative and mechanistic insights into ERAD. We demonstrate that both Png1p-dependent generation of Man(7-10)GlcNAc(2) fOS and vacuolar α-mannosidase (Ams1p)-dependent fOS demannosylation to yield Man(1)GlcNAc(2) are strikingly up-regulated during post-diauxic growth which occurs when the culture medium is depleted of glucose. Gene deletions in the ams1Δ background revealed that, as anticipated, Mns1p and Mnl1p are required for efficient generation of the Man(7)GlcNAc(2) (d1) fOS, but for the first time, we demonstrate that small amounts of this fOS are generated in an Mnl1p-independent, Mns1p-dependent pathway and that a Man(8)GlcNAc(2) fOS that is known to bind Yos9p is generated in an Mnl1p-dependent, Mns1p-independent manner. This latter observation adds mechanistic insight into a recently described Mnl1p-dependent, Mns1p-independent ERAD pathway. Finally, we show that 50% of fOS generation is independent of ERAD-L, and because our data indicate that ERAD-M and ERAD-C contribute little to fOS levels, other important processes underlie fOS generation in S. cerevisiae.     

Kinetic and chemical mechanisms of homocitrate synthase from Thermus thermophilus

The homocitrate synthase from Thermus thermophilus (TtHCS) is a metal-activated enzyme with either Mg(2+) or Mn(2+) capable of serving as the divalent cation. The enzyme exhibits a sequential kinetic mechanism. The mechanism is steady state ordered with α-ketoglutarate (α-Kg) binding prior to acetyl-CoA (AcCoA) with Mn(2+), whereas it is steady state random with Mg(2+), suggesting a difference in the competence of the E·Mn·α-Kg·AcCoA and E·Mg·α-Kg·AcCoA complexes. The mechanism is supported by product and dead-end inhibition studies. The primary isotope effect obtained with deuterioacetylCoA (AcCoA-d(3)) in the presence of Mg(2+) is unity (value 1.0) at low concentrations of AcCoA, whereas it is 2 at high concentrations of AcCoA. Data suggest the presence of a slow conformational change induced by binding of AcCoA that accompanies deprotonation of the methyl group of AcCoA. The solvent kinetic deuterium isotope effect is also unity at low AcCoA, but is 1.7 at high AcCoA, consistent with the proposed slow conformational change. The maximum rate is pH independent with either Mg(2+) or Mn(2+) as the divalent metal ion, whereas V/K(α-Kg) (with Mn(2+)) decreases at low and high pH giving pK values of about 6.5 and 8.0. Lysine is a competitive inhibitor that binds to the active site of TtHCS, and shares some of the same binding determinants as α-Kg. Lysine binding exhibits negative cooperativity, indicating cross-talk between the two monomers of the TtHCS dimer. Data are discussed in terms of the overall mechanism of TtHCS.     

The complement system

The complement system consists of a tightly regulated network of proteins that play an important role in host defense and inflammation. Complement activation results in opsonization of pathogens and their removal by phagocytes, as well as cell lysis. Inappropriate complement activation and complement deficiencies are the underlying cause of the pathophysiology of many diseases such as systemic lupus erythematosus and asthma. This review represents an overview of the complement system in an effort to understand the beneficial as well as harmful roles it plays during inflammatory responses.     

Common Myna Roosts Are Not Recruitment Centres

We studied communal roosting in the Common Myna (Acridotheres tristis) in the light of the recruitment centre hypothesis and predation at the roost. The number and sizes of flocks departing from and arriving at focal roosts were recorded over a two year period. We also recorded the sizes and behaviour of foraging flocks. We found that flock sizes of birds departing from roosts at sunrise were larger than those at the feeding site, suggesting that there was no recruitment from the roosts. Flocks entering the roosts during sunset were larger on average than those leaving the following sunrise, suggesting no consolidation of flocks in the morning. Flocks entering the roosts at sunset were also larger on average than those that had left that sunrise, although there was no recruitment at the feeding site. There was no effect of group size on the proportion of time spent feeding. Contrary to expectation, single birds showed lower apparent vigilance than birds that foraged in pairs or groups, possibly due to scrounging tactics being used in the presence of feeding companions. Thus, the recruitment centre hypothesis did not hold in our study population of mynas. Predation at dawn and dusk were also not important to communal roosting: predators near the roosts did not result in larger flocks, and resulted in larger durations of arrival/departure contrary to expectation. Since flock sizes were smallest at the feeding site and larger in the evening than in the morning, but did not coincide with predator activity, information transfer unrelated to food (such as breeding opportunities) may possibly give rise to the evening aggregations.