Inhibition of catechol-O-methyltransferase increases estrogen-DNA adduct formation

The association found between breast cancer development and prolonged exposure to estrogens suggests that this hormone is of etiologic importance in the causation of the disease. Studies on estrogen metabolism, formation of DNA adducts, carcinogenicity, cell transformation, and mutagenicity have led to the hypothesis that reaction of certain estrogen metabolites, predominantly catechol estrogen-3,4-quinones, with DNA forms depurinating adducts [4-OHE1(E2)-1-N3Ade and 4-OHE(1)(E2)-1-N7Gua]. These adducts cause mutations leading to the initiation of breast cancer. Catechol-O-methyltransferase (COMT) is considered an important enzyme that protects cells from the genotoxicity and cytotoxicity of catechol estrogens, by preventing their conversion to quinones. The goal of the present study was to investigate the effect of COMT inhibition on the formation of depurinating estrogen-DNA adducts. Immortalized human breast epithelial MCF-10F cells were treated with 4-OHE2 (0.2 or 0.5 microM) for 24 h at 120, 168, 216, and 264 h postplating or one time at 1-30 microM 4-OHE2 with or without the presence of COMT inhibitor (Ro41-0960). The culture media were collected at each point, extracted by solid-phase extraction, and analyzed by HPLC connected with a multichannel electrochemical detector. The results demonstrate that MCF-10F cells oxidize 4-OHE2 to E1(E2)-3,4-Q, which react with DNA to form the depurinating N3Ade and N7Gua adducts. The COMT inhibitor Ro41-0960 blocked the methoxylation of catechol estrogens, with concomitant 3- to 4-fold increases in the levels of the depurinating adducts. Thus, low activity of COMT leads to higher levels of depurinating estrogen-DNA adducts that can induce mutations and initiate cancer.     

Strengths and weaknesses in the long-term sustainability of two sympatric seabreams (Argyrops spinifer and Rhabdosargus haffara,Sparidae)

Argyrops spinifer and Rhabdosargus haffara are two sympatric seabream species making important contributions to fisheries landings in the western Arabian/Persian Gulf. We identified the strengths and weaknesses in the long-term sustainability of A. spinifer and R. haffara stocks by integrating multiple sources of data, including fisheries catch and effort statistics, life history traits, scientific trawl surveys and historical length frequency distribution. Four strengths were identified in A. spinifer: wide distribution of juveniles, positive association to the network of de facto fishing exclusion areas created by hundreds of oil-gas facilities, early maturation and the existence of large and old individuals. A. spinifer suffers from two potential weaknesses: slow growth rate and higher exploitation pressure on the small-sized individuals. R. haffara, on the other hand, has a strength of having a short life span and a fast growth rate, characteristics that make it robust to unfavourable conditions. R. haffara suffers from two weaknesses: the lack of association to the oil and gas facilities, and the preference for nearshore shallow waters with stronger negative anthropogenic impacts. Identified strengths and weaknesses of these two sparids provided a preliminary assessment about their long-term sustainability, as well as a roadmap about how to develop different management strategies to meet specific objectives.     

Stability curves of laboratory evolved thermostable mutants of a Bacillus subtilis lipase

Shape of the protein stability curves changes to achieve higher melting temperature. Broadly, these changes have been classified as upward shift (increased ?G_s), rightward shift (increase in T_s) and flattening of the stability curves (decrease in ?C_p). Comparative studies on homologous mesophilic–thermophilic protein pairs highlighted the differential contribution of these three strategies amongst proteins. But unambiguous way of identification of the strategies, which will be preferred for a protein, is still not achieved. We have performed comparative thermodynamic studies using differential scanning calorimeter (DSC) on thermostable variants of a lipase from Bacillus subtilis. These variants are products of 1, 2, 3 and 4 rounds of directed evolution and harbor mutations having definite contribution in thermostability unlike natural thermophilic proteins. We have shown that upward and rightward shift in stability curves are prime strategies in this lipase. Our results along with that from the other study on laboratory evolved xylanase A suggest that optimization of suboptimal thermodynamic parameters is having a dominant influence in selection of thermodynamic strategies for higher thermostability.     

In-vitro antibacterial, antifungal and cytotoxic properties of metal-based furanyl derived sulfonamides

A new series of antibacterial and antifungal furanyl-derived sulfonamides and their cobalt (II), copper (II), nickel (II) and zinc (II) metal complexes have been synthesized, characterized and screened for their in-vitro antibacterial activity against four Gram-negative (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa and Salmonella typhi) and two Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacterial strains and, for in-vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glaberata. The results of these studies revealed that all compounds showed significant to moderate antibacterial activity. However, the zinc (II) complexes were found to be comparatively much more active as compared to the others. For antifungal activity generally, compounds (22) and (24) showed significant activity against Escherichia coli (a), (6) against Shigella flexeneri (b), (16) and (22) against Pseudomonas aeruginosa (c), (14) and (16) against Salmonella typhi (d), (9) against Staphylococcus aureus (e) and, (14) and (16) against Bacillus subtilis (f) fungal strains. The brine shrimp (Artemia salina) bioassay was also carried out to study their in-vitro cytotoxic properties. Only three compounds, (6), (10) and (23) displayed potent cytotoxic activity with LD50 = 1.8535 x 10(-4), 1.8173 x 10(-4) and 1.9291 x 10(-4) respectively.     

Antibacterial, antifungal and cytotoxic properties of novel N-substituted sulfonamides from 4-hydroxycoumarin

A new series of 4-({[2, 4-dioxo-2H-chromen-3 (4H)-ylidene] methyl} amino) sulfonamides have been obtained by the condensation reaction of 4-hydroxycoumarin with various sulfonamides (sulfanilamide, sulfaguanidine, p-aminomethyl-sufanilamide, p-aminoethylsufanilamide, sulfathiazole, sulfamethoxazole, sulfamethazine and 4-[(2-amino-4-pyrimidinyl) amino] benzenesulfonamide) in the presence of an excess of ethylorthoformate. These compounds were screened for their in-vitro antibacterial activity against four Gram-negative (E. coli, S. flexneri, P. aeruginosa and S. typhi) and two Gram-positive (B. subtilis and S. aureus) bacterial strains and for in-vitro antifungal activity against T. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glaberata. Results revealed that a significant antibacterial activity was observed by compounds (4) and (5), (6) and (8) against two Gram-negative, (P. aeruginosa and S. typhi) and two Gram-positive (B. subtilis and S. aureus) species, respectively. Of these (4) was found to be the most active. Similarly, for antifungal activity compounds (3) and (8) showed significant activity against M. canis and, (6) and (8) against F. solani. The brine shrimp bioassay was also carried out to study their in-vitro cytotoxic properties and only two compounds, (4) and (8) possessing LD50 = 2.9072 x 10(-4) and 3.2844 x 10(-4) M, respectively, displayed potent cytotoxic activity against Artemia salina     

Virtual Electrophysiological Study of Atrial Fibrillation in Fibrotic Remodeling

Research has indicated that atrial fibrillation (AF) ablation failure is related to the presence of atrial fibrosis. However it remains unclear whether this information can be successfully used in predicting the optimal ablation targets for AF termination. We aimed to provide a proof-of-concept that patient-specific virtual electrophysiological study that combines i) atrial structure and fibrosis distribution from clinical MRI and ii) modeling of atrial electrophysiology, could be used to predict: (1) how fibrosis distribution determines the locations from which paced beats degrade into AF; (2) the dynamic behavior of persistent AF rotors; and (3) the optimal ablation targets in each patient. Four MRI-based patient-specific models of fibrotic left atria were generated, ranging in fibrosis amount. Virtual electrophysiological studies were performed in these models, and where AF was inducible, the dynamics of AF were used to determine the ablation locations that render AF non-inducible. In 2 of the 4 models patient-specific models AF was induced; in these models the distance between a given pacing location and the closest fibrotic region determined whether AF was inducible from that particular location, with only the mid-range distances resulting in arrhythmia. Phase singularities of persistent rotors were found to move within restricted regions of tissue, which were independent of the pacing location from which AF was induced. Electrophysiological sensitivity analysis demonstrated that these regions changed little with variations in electrophysiological parameters. Patient-specific distribution of fibrosis was thus found to be a critical component of AF initiation and maintenance. When the restricted regions encompassing the meander of the persistent phase singularities were modeled as ablation lesions, AF could no longer be induced. The study demonstrates that a patient-specific modeling approach to identify non-invasively AF ablation targets prior to the clinical procedure is feasible.     

Structural basis of TRAPPIII‐mediated Rab1 activation

The GTPase Rab1 is a master regulator of the early secretory pathway and is critical for autophagy. Rab1 activation is controlled by its guanine nucleotide exchange factor, the multisubunit TRAPPIII complex. Here, we report the 3.7 Å cryo‐EM structure of the Saccharomyces cerevisiae TRAPPIII complex bound to its substrate Rab1/Ypt1. The structure reveals the binding site for the Rab1/Ypt1 hypervariable domain, leading to a model for how the complex interacts with membranes during the activation reaction. We determined that stable membrane binding by the TRAPPIII complex is required for robust activation of Rab1/Ypt1 in vitro and in vivo, and is mediated by a conserved amphipathic α‐helix within the regulatory Trs85 subunit. Our results show that the Trs85 subunit serves as a membrane anchor, via its amphipathic helix, for the entire TRAPPIII complex. These findings provide a structural understanding of Rab activation on organelle and vesicle membranes.     

DF++ : an adaptive buffer-aware probabilistic delegation forwarding protocol for Delay Tolerant Network

The delegated forwarding (DF) curb transmissions by forwarding the message to a node that holds high quality value seen by the message. However, DF assumes infinite buffer space that is not possible in real time applications. In addition, quality value computation considers the encountering history and does not account for additional network parameters such as aging and transitive connectivity. In this paper, we have proposed a routing protocol called as DF++ that compute quality value based on probabilistic model used in PRoPHET protocol and forwards the message to current node by adaptive computation of available buffer space. We have compared performance of DF++ with DF, Epidemic and PRoPHET routing protocols. The proposed DF++ has higher delivery probability and fewer message drop and transmissions.     

The modification of the peptidyl transferase activity of 50-S ribosomal subunits, LiC1-split proteins and L16 ribosomal protein by ethoxyformic anhydride.

Ethoxyformic anhydride abolishes the peptidyl transferase activity of 50-S ribosomal subunits, LiC1 split proteins and L16. Hydroxylamine treatment results in reactivation. Erythromycin exhibits significant protection with 50-S ribosomal subunits. With LiC1 split proteins and L16 significant protection was exhibited only after reconstitution. The results indicate that the ethoxyformic anhydride is reacting with approximately six histidines in LiC1 split proteins and one in L16. Since L16 has been reported to contain a single histidine, the results presented indicate the involvement of this histidine in peptidyl transferase activity.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.