Characterization of the mutT nucleoside triphosphatase of Escherichia coli.

The mutT protein, which prevents A:T----C:G transversions during DNA replication, has the following enzymatic properties. Although it prefers dGTP as a substrate, it hydrolyzes all of the canonical nucleoside triphosphates to some extent. It has no activity in the absence of divalent cations, is maximally activated by magnesium ions, and has a pH optimum of 9.0. Nucleoside triphosphates are hydrolyzed according to the following equation. dGTP----dGMP + PPi Studies with nucleotide analogues suggest that the enzyme may prefer the syn rather than the anti conformation of the nucleoside triphosphates, which might explain the role it plays in preventing mutations.     

The vomeronasal organ and associated structures of the fetal African elephant, Loxodonta africana (Proboscidea, Elephantidae)

The vomeronasal organ (VNO) is a chemosensory structure of the nasal septum found in most tetrapods. Although potential behavioural correlates of VNO function have been shown in two of the three elephant species, its morphology in Loxodonta africana has not been studied. The development of the VNO and its associated structures in the African elephant are described in detail using serially sectioned material from fetal stages. The results show that many components of the VNO complex (e.g. neuroepithelium, receptor‐free epithelium, vomeronasal nerve, paravomeronasal ganglia, blood vessels, vomeronasal cartilage) are well developed even in a 154‐day‐old fetus, in which the VNO opens directly into the oral cavity with only a minute duct present. However, the vomeronasal glands and their ducts associated with the VNO were developed only in the 210‐day‐old fetus. Notably, in this fetus, the vomeronasal–nasopalatine duct system had acquired a pathway similar to that described in the adult Asian elephant; the VNOs open into the oral cavity via the large palatal parts of the nasopalatine ducts, which are lined by a stratified squamous epithelium. The paired palatal ducts initially coursed anteriorly at an angle of 45° from the oral recess and/or the oral cavity mucosa, and merged into the vomeronasal duct. This study confirms the unique characteristics of the elephant VNO, such as its large size, the folded epithelium of the VNO tube, and the dorsomedial position of the neuroepithelium. The palatal position and exclusive communication of the VNO with the oral cavity, as well as the partial reduction of the nasopalatine duct, might be related to the unique elephantid craniofacial morphogenesis, especially the enormous growth of the tusk region, and can be seen as autapomorphies.     

Toxicological implications of a commercial formulation of deltamethrin (Decis®) in developing chick embryo

A deltamethrin containing insecticide formulation (Decis®) was evaluated for its toxic potential in developing chick embryos. For the present study, three water emulsified concentrations of Decis® (12.5 mg L^?1, 25 mg L^?1, and 50 mg L^?1) were used. Fertilized eggs of Gallus domesticus were immersed in these three concentrations of the insecticide for 60 min at 37°C on day 0 of incubation and kept for incubation till embryonic day 7. Recovered embryos were evaluated for teratogenic and biochemical changes. The results revealed that administration of Decis® at its lower concentrations (12.5 mg L^?1 and 25 mg L^?1) did not show any significant teratological changes but the significant number of abnormal survivors was observed at 50 mg L^?1 of dose concentration when compared with vehicle-treated control. Among biochemical changes, total glycogen and RNA contents of embryos was significantly decreased at 25 mg L^?1 and 50 mg L^?1 of Decis® concentrations. Similarly, significant alteration (p ≤ .05) was observed in alanine transaminase activity at 50 mg L^?1 concentration of Decis®. Thus, the present study concluded that the no-effect-level for developmental toxicity for Decis® is below the concentration of 25 mg L^?1 under standard laboratory conditions.     

NADPH binding to beta-subunit regulates inactivation of voltage-gated K(+) channels

Ancillary beta-subunits regulate the voltage-dependence and the kinetics of Kv currents. The Kvbeta proteins bind pyridine nucleotides with high affinity but the role of cofactor binding in regulating Kv currents remains unclear. We found that recombinant rat Kvbeta 1.3 binds NADPH (K(d)=1.8+/-0.02 microM) and NADP(+) (K(d)=5.5+/-0.9 microM). Site-specific modifications at Tyr-307 and Arg-316 decreased NADPH binding; whereas, K(d) NADPH was unaffected by the R241L mutation. COS-7 cells transfected with Kv1.5 cDNA displayed non-inactivating currents. Co-transfection with Kvbeta1.3 accelerated Kv activation and inactivation and induced a hyperpolarizing shift in voltage-dependence of activation. Kvbeta-mediated inactivation of Kv currents was prevented by the Y307F and R316E mutations but not by the R241L substitution. Additionally, the R316E mutation weakened Kvalpha-beta interaction. Inactivation of Kv currents by Kvbeta:R316E was restored when excess NADPH was included in the patch pipette. These observations suggest that NADPH binding is essential for optimal interaction between Kvalpha and beta subunits and for Kvbeta-induced inactivation of Kv currents.     

Synthesis and evaluation of antitubercular activity of glycosyl thio- and sulfonyl acetamide derivatives

A series of glycosyl thioacetamide and glycosyl sulfonyl acetamide derivatives have been prepared following a convenient reaction protocol and evaluated for their antitubercular activity against Mycobacterium tuberculosis H₃₇Rv. Amongst 32 compounds evaluated 3 compounds were effective in inhibiting mycobacterial growth at MIC of 6.25 μg/mL, 6 compounds at MIC of 3.125 μg/mL and 1 compound at MIC of 1.56 μg/mL. All active compounds were found nontoxic in Vero cell lines and mice bone marrow macrophages.     

Potent growth suppressive activity of curcumin in human breast cancer cells: Modulation of Wnt/β-catenin signaling

Abnormal activation of the Wnt/β-catenin signaling pathway and subsequent upregulation of β-catenin driven downstream targets—c-Myc and cyclin D1 is associated with development of breast cancer. The objective of our study was to determine if curcumin could modulate the key elements of Wnt pathway in breast cancer cells; an effect that might underscore its usefulness for chemoprevention/treatment of this malignancy. Curcumin showed a cytotoxic effect on MCF-7 cells with 50% inhibitory concentration (IC₅₀) of 35 μM; while IC₅₀ for MDA-MB-231 cells was 30 μM. Treatment with low cytostatic dose of 20 μM curcumin showed G₂/M arrest in both breast cancer cells. The effect of curcumin (20 μM) treatment on expression of Wnt/β-catenin pathway components in breast cancer cells (MCF-7 and MDA-MB-231) was analyzed by immunofluorescence and Western blotting. Curcumin was found to effectively inhibit the expression of several Wnt/β-catenin pathway components—disheveled, β-catenin, cyclin D1 and slug in both MCF-7 and MDA-MB-231. Immunofluorescence analysis showed that curcumin markedly reduced the nuclear expression of disheveled and β-catenin proteins. Further, the protein levels of the positively regulated β-catenin targets—cyclin D1 and slug, were downregulated by curcumin treatment. The expression levels of two integral proteins of Wnt signaling, GSK3β and E-cadherin were also altered by curcumin treatment. In conclusion, our data demonstrated that the efficacy of curcumin in inhibition of cell proliferation and induction of apoptosis might occur through modulation of β-catenin pathway in human breast cancer cells.     

A new approach to monitor expression of aldo–keto reductase proteins in mouse tissues

The aldo–keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan‐AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan‐AKR antibodies exhibited the immune specificities and immune sensitivities, Pan‐AKR‐P1 for AKR1B and AKR1C, Pan‐AKR‐P3 for AKR1C and Pan‐AKR‐P4 for all the AKR proteins. Pan‐AKR‐P4 antibody was employed to 2‐DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune‐reactive spots from each tissue. Protein identification with MS revealed that most immune‐positive spots were the members of AKR superfamily. Furthermore, Pan‐AKR‐P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances.     

Development and Characterization of 99mTc-timolol Maleate for Evaluating Efficacy of In Situ Ocular Drug Delivery System

In situ gel-forming systems have drawn much attention of current researchers to overcome the poor bioavailability from the conventional eye drops. The present work described formulation and pharmacoscintigraphic evaluation of timolol-maleate-loaded chitosan/hydroxy propyl methyl cellulose (HPMC)-based polymer matrix for enhanced ocular retention. Chitosan and HPMC ratio was optimized and formulation was characterized for various in vitro parameters. The ocular retention was studied on New Zealand rabbits by gamma scintigraphy, which is a very simple and noninvasive technique. For scintigraphy study, the drug timolol maleate was radiolabeled ^99mTc by direct labeling method using SnCl₂·2H₂O as reducing agent. The labeling procedure was optimized to get maximum labeling efficiency (>98%). In vitro stability of the radiolabeled drug (^99mTc-timolol maleate complex) was checked and it was found to be stable for up to 24 h. Plain drug eliminates rapidly as significant activity was recorded in kidney and bladder after 2 h of ocular administration. It was evident from the scintigraphic images and the time–activity curve plotted from the data that the plain drug solution cleared very rapidly from the corneal region and reached into systemic circulation via nasolachrymal drainage system, as significant activity was recorded in kidney and bladder after 2 h of ocular administration. Developed formulation cleared at a slow rate and remained at corneal surface for longer time duration. No radioactivity was observed in systemic circulation after 2 h. Ocular irritation of the developed formulation was also checked by hen’s egg chorioallantoic membrane test and formulation was found to be practically nonirritant. The study signified the potential of gamma scintigraphy in evaluation of novel drug delivery systems in a noninvasive manner.