Modeling and structural analysis of evolutionarily diverse S8 family serine proteases

Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.     

DNA damage response in adult stem cells: pathways and consequences

In contrast to postmitotic or short-lived somatic cells, tissue-specific stem cells must persist and function throughout life to ensure tissue homeostasis and repair. The enormous functional demands and longevity of stem cells raises the possibility that stem cells might be uniquely equipped to maintain genomic integrity in ways different than somatic cells. Indeed, evidence suggests that stem cell compartments possess unique properties that combine to either limit or, in some instances, accelerate DNA damage accrual.     

Fe(III)-salen and salphen complexes induce caspase activation and apoptosis in human cells

To explore the apoptotic and antitumor activities of metallo-salens, the authors have synthesized several Fe(III)-salen and salphen complexes and analyzed their effects on human cancer and noncancer cells. Their results demonstrated that Fe(III)-salen and salphen complexes affect cell viability and induce nuclear fragmentation and apoptosis in breast cancer (MCF7) cells. The IC(50) values for the active metallo-salen complexes ranged between 0.3 and 22 μM in MCF7 cells. Biochemically active Fe(III)-salen and salphen complexes induced caspase-3/7 activation and release of cytochrome c from the mitochondria to cytosol, suggesting the involvement of the mitochondrial pathway of apoptosis. Comparison of IC(50) values toward 3 different cell lines demonstrated that selected Fe(III)-salen complexes induce tumor cell-selective apoptosis in cultured cells. Overall, the studies demonstrated that Fe(III)-salen and salphen complexes induced efficient apoptosis in cultured human cells. The nature of the substituents and the bridging spacer between diamino groups play critical roles in determining the apoptotic activities of Fe(III)-salen and salphen complexes.     

Possible fasciocidal activity of methanol extract of Dregea volubilis leaves

In eastern and southern part of India Dregea volubilis (Family Asclepediaceae) is widely used as anthelmintic in traditional system of medicine. The present study was conducted to evaluate the fasciocidal activity of the methanol extract of D. volubilis leaves (MEDV) and to observe the drug’s effect on organisms through scanning electron microscopic (SEM) study. Live parasites (Trematode: Fasciola gigantica) were collected in 0.9% phosphate-buffered saline from the bile ducts of buffalo. Those were incubated in the said media at 37 ± 1 °C either as control, or with MEDV at 5, 10, 25, 50 and 100 mg/ml as test groups or with albendazole at 10 mg/ml as standard group. The efficacy of the extract was determined on the basis of paralysis (temporary loss of spontaneous movement of the organisms) and/or death of the liver flukes. Death was confirmed when the organisms lost their motility permanently and their motility could not be revived even when vigorously shaken or dipped in warm water. MEDV at all concentration effectively paralyzed first and then killed the liver flukes (p < 0.001). Maximum fasciocidal activity was found with concentration of 100 mg/ml at 38.83 ± 3.41 min. Through SEM study, severe damages were observed in both the suckers as well as on the tegumental surfaces of the treated liver flukes. The study confirmed the fasciocidal activity of the MEDV.     

Unexpected Histone H3 Tail-clipping Activity of Glutamate Dehydrogenase

Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro.     

Therapeutic potential of Lactobacillus ingluviei ADK10, a newly established probiotic organism against acetaminophen induced uremic rats

In the present study, Lactobacillus ingluviei ADK10 (Acc. No. JQ395039) from intestinal origin was tested for its probiotic characteristic as well as uremia ameliorating activity on acetaminophen induced uremic rats. The results revealed that L. ingluviei ADK10 was able to tolerate pH 3.0–9.0 and 0.5% bile salt along with good hydrophobicity (67%) and adherence index with Ht-29 cell line on 258/100 cells. It was susceptible to 20 antibiotics. The organism was able to degrade food ingredients, like starch and milk proteins. The strain showed significant growth inhibition of Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Shigella dysentery, Pseudomonas aeruginosa, and Klebsiella pneumoniae (average diameter of 10 mm). The therapeutic potentiality of this probiotic bacterium was tested against acetaminophen induced uremic rats. It was found that supplementation of L. ingluviei ADK10 for 14 days with food reduced severe increase of uremic profiles, such as blood urea (85%), creatinine (68%) and uric acid (41%) in comparison to the uremic rats. Moreover, during the feeding of rats with probiotic strain at a dose of 1×10⁹ bacteria, reduction of enterobacteria in faeces was observed. Our studies indicated that L. ingluviei ADK10 could be used as a health-promoting probiotic along with antiuremic efficacy.     

Molecular targeting of Akt by thymoquinone promotes G1 arrest through translation inhibition of cyclin D1 and induces apoptosis in breast cancer cells

Aim

Thymoquinone (TQ), the predominant bioactive constituent of black seed oil (Nigella Sativa), has been shown to possess antineoplastic activity against multifarious tumors. However, the meticulous mechanism of TQ on Akt mediated survival pathway is still unrevealed in breast cancer. Here, we investigated TQ's mechanism of action against PI3K/Akt signaling and its downstream targets by modulating proteins translational machinery, leading to apoptosis in cancer cells.

Main methods

MDA-MB-468 and T-47D cells were treated with TQ and evaluated for its anticancer activity through phase distribution and western blot. Modulatory effects of TQ on Akt were affirmed through kinase and drug potential studies.

Key findings

Studies revealed G₁ phase arrest till 24 h incubation with TQ while extended exposure showed phase shift to subG₁ indicating apoptosis, supported by suppression of cyclin D1, cyclin E and cyclin dependent kinase inhibitor p27 expression. Immunoblot and membrane potential studies revealed mitochondrial impairment behind apoptotic process with upregulation of Bax, cytoplasmic cytochrome c and procaspase-3, PARP cleavage along with Bcl-2, Bcl-xL and survivin downregulation. Moreover, we construed the rationale behind mitochondrial dysfunction by examining the phosphorylation status of PDK1, PTEN, Akt, c-raf, GSK-3β and Bad in TQ treated cells, thus ratifying the involvement of Akt in apoptosis. Further, the consequential effect of Akt inhibition by TQ is proven by translational repression through deregulated phosphorylation of 4E-BP1, eIF4E, S6R and p70S6K.

Significance

Our observations for the first time may provide a new insight for the development of novel therapies for Akt overexpressed breast cancer by TQ.     

A Realistic Host-Vector Transmission Model for Describing Malaria Prevalence Pattern

Malaria continues to be a major public health concern all over the world even after effective control policies have been employed, and considerable understanding of the disease biology have been attained, from both the experimental and modelling perspective. Interactions between different general and local processes, such as dependence on age and immunity of the human host, variations of temperature and rainfall in tropical and sub-tropical areas, and continued presence of asymptomatic infections, regulate the host-vector interactions, and are responsible for the continuing disease prevalence pattern. In this paper, a general mathematical model of malaria transmission is developed considering short and long-term age-dependent immunity of human host and its interaction with pathogen-infected mosquito vector. The model is studied analytically and numerically to understand the role of different parameters related to mosquitoes and humans. To validate the model with a disease prevalence pattern in a particular region, real epidemiological data from the north-eastern part of India was used, and the effect of seasonal variation in mosquito density was modelled based on local climactic data. The model developed based on general features of host-vector interactions, and modified simply incorporating local environmental factors with minimal changes, can successfully explain the disease transmission process in the region. This provides a general approach toward modelling malaria that can be adapted to control future outbreaks of malaria.     

Synthesis of interpenetrating network hydrogel from poly(acrylic acid-co-hydroxyethyl methacrylate) and sodium alginate: Modeling and kinetics study for removal of synthetic dyes from water

Several interpenetrating network (IPN) hydrogels were made by free radical in situ crosslink copolymerization of acrylic acid (AA) and hydroxy ethyl methacrylate in aqueous solution of sodium alginate. N,N′-methylenebisacrylamide (MBA) was used as comonomer crosslinker for making these crosslink hydrogels. All of these hydrogels were characterized by carboxylic content, FTIR, SEM, XRD, DTA–TGA and mechanical properties. Swelling, diffusion and network parameters of the hydrogels were studied. These hydrogels were used for adsorption of two important synthetic dyes, i.e. Congo red and methyl violet from water. Isotherms, kinetics and thermodynamics of dye adsorption by these hydrogels were also studied.     

Trifluoroethanol-induced conformational change of tetrameric and monomeric soybean agglutinin: Role of structural organization and implication for protein folding and stability

2,2,2-Trifuoroethanol (TFE)-induced conformational structure change of a β-sheet legume lectin, soybean agglutinin (SBA) has been investigated employing its exclusive structural forms in quaternary (tetramer) and tertiary (monomer) states, by far- and near-UV CD, FTIR, fluorescence, low temperature phosphorescence and chemical modification. Far-UV CD results show that, for SBA tetramer, native atypical β-conformation transforms to a highly α-helical structure, with the helical content reaching 57% in 95% TFE. For SBA monomer, atypical β-sheet first converts to typical β-sheet at low TFE concentration (10%), which then leads to a nonnative α-helix at higher TFE concentration. From temperature-dependent studies (5–60 °C) of TFE perturbation, typical β-sheet structure appears to be less stable than atypical β-sheet and the induced helix entails reduced thermal stability. The heat induced transitions are reversible except for atypical to typical β-sheet conversion. FTIR results reveal a partial α-helix conversion at high protein concentration but with quantitative yield. However, aggregation is detected with FTIR at lower TFE concentration, which disappears in more TFE. Near-UV CD, fluorescence and phosphorescence studies imply the existence of an intermediate with native-like secondary and tertiary structure, which could be related to the dissociation of tetramer to monomer. This has been further supported by concentration dependent far-UV CD studies. Chemical modification with N-bromosuccinimide (NBS) shows that all six tryptophans per monomer are solvent-exposed in the induced α-helical conformation. These results may provide novel and important insights into the perturbed folding problem of SBA in particular, and β-sheet oligomeric proteins in general.