Attenuation of 4-nitroquinoline 1-oxide induced in vitro lipid peroxidation by green tea polyphenols

Lipid peroxidation is believed to play an important role in pathogenesis of diseases. 4-Nitroquiunoline 1-oxide (4-NQO) a potent oral carcinogen, widely used for induction of oral carcinogenesis, was found to induce lipid peroxidation in vivo and in vitro. Green tea contains high content of polyphenols, which are potent antioxidants. Thus green tea polyphenols (GP) can play a protective role in 4-NQO induced in vitro lipid peroxidation. 4-NQO at the concentration of 1.5 mM was found to induce lipid peroxidation in 5% liver homogenate in phosphate buffered saline and extent of lipid peroxidation at the different time intervals 0, 15, 30 and 45 min where studied by assessing parameters such as hydroxyl radical production (OH), thiobarbituric acid reactants (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT). It was found that addition of 4-NQO caused an increase in OH and TBARS level and a decrease in activity of SOD, CAT and the levels of GSH. Simultaneous addition of GP 10 mg/ml significantly decreased lipid peroxidation and increased in antioxidant status. Thus, we conclude that GP, a potent antioxidant, was found to nullify 4-NQO induced lipid peroxidation in vitro and 4-NQO acts initially by causing oxidative stress and leads to carcinogenesis.     

Extracellular enzymatic activity profiles in fungi isolated from oil-rich environments

About 34 wild fungal species associated with edible oil mill wastes were isolated by the serial dilution technique. Methods for rapid screening of fungal species against production of extracellular enzymes such as amylase, protease, cellulase, and lipase are reported. Among all the species, Aspergillus versicolor exhibited high amlylolytic and gelatinolytic activity, whereas Penicillium citrinum showed only high amylolytic activity. Maximum cellulolytic activity was recorded for Absidia corymbifera, As. niger, Cunninghamella echinulata, Curvularia lunata, Fusarium solani, Mucor racemosus, Paecilomyces variotii, and Syncephalastrum racemosum. The fungal species Ab. corymbifera, As. fumigatus, As. japonicus, As. nidulans, As. terreus, Cun. verticillata, Cur. pallescens, F. oxysporum, Geotrichum candidum, M. racemosus, Pe. citrinum, Pe. frequentans, Rhizopus stolonifer, and Trichoderma viride exhibited maximum lipase activity. This study confirms the isolated fungi present on a wide range of substrates in the ambient environment, and these results could provide basic data for further investigations on fungal extracellular enzymes.First two authors equally contributed to this work     

Benchmarking and analysis of protein docking performance in Rosetta v3.2

RosettaDock has been increasingly used in protein docking and design strategies in order to predict the structure of protein-protein interfaces. Here we test capabilities of RosettaDock 3.2, part of the newly developed Rosetta v3.2 modeling suite, against Docking Benchmark 3.0, and compare it with RosettaDock v2.3, the latest version of the previous Rosetta software package. The benchmark contains a diverse set of 116 docking targets including 22 antibody-antigen complexes, 33 enzyme-inhibitor complexes, and 60 'other' complexes. These targets were further classified by expected docking difficulty into 84 rigid-body targets, 17 medium targets, and 14 difficult targets. We carried out local docking perturbations for each target, using the unbound structures when available, in both RosettaDock v2.3 and v3.2. Overall the performances of RosettaDock v2.3 and v3.2 were similar. RosettaDock v3.2 achieved 56 docking funnels, compared to 49 in v2.3. A breakdown of docking performance by protein complex type shows that RosettaDock v3.2 achieved docking funnels for 63% of antibody-antigen targets, 62% of enzyme-inhibitor targets, and 35% of 'other' targets. In terms of docking difficulty, RosettaDock v3.2 achieved funnels for 58% of rigid-body targets, 30% of medium targets, and 14% of difficult targets. For targets that failed, we carry out additional analyses to identify the cause of failure, which showed that binding-induced backbone conformation changes account for a majority of failures. We also present a bootstrap statistical analysis that quantifies the reliability of the stochastic docking results. Finally, we demonstrate the additional functionality available in RosettaDock v3.2 by incorporating small-molecules and non-protein co-factors in docking of a smaller target set. This study marks the most extensive benchmarking of the RosettaDock module to date and establishes a baseline for future research in protein interface modeling and structure prediction.     

Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice

Sarcolipin (SLN) inhibits sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pumps. To evaluate the physiological significance of SLN in skeletal muscle, we compared muscle contractility and SERCA activity between Sln-null and wild-type mice. SLN protein expression in wild-type mice was abundant in soleus and red gastrocnemius (RG), low in extensor digitorum longus (EDL), and absent from white gastrocnemius (WG). SERCA activity rates were increased in soleus and RG, but not in EDL or WG, from Sln-null muscles, compared with wild type. No differences were seen between wild-type and Sln-null EDL muscles in force-frequency curves or maximum rates of force development (+dF/dt). Maximum relaxation rates (-dF/dt) of EDL were higher in Sln-null than wild type across a range of submaximal stimulation frequencies, but not during a twitch or peak tetanic contraction. For soleus, no differences were seen between wild type and Sln-null in peak tetanic force or +dF/dt; however, force-frequency curves showed that peak force during a twitch and 10-Hz contraction was lower in Sln-null. Changes in the soleus force-frequency curve corresponded with faster rates of force relaxation at nearly all stimulation frequencies in Sln-null compared with wild type. Repeated tetanic stimulation of soleus caused increased (-dF/dt) in wild type, but not in Sln-null. No compensatory responses were detected in analysis of other Ca(2+) regulatory proteins using Western blotting and immunohistochemistry or myosin heavy chain expression using immunofluorescence. These results show that 1) SLN regulates Ca(2+)-ATPase activity thereby regulating contractile kinetics in at least some skeletal muscles, 2) the functional significance of SLN is graded to the endogenous SLN expression level, and 3) SLN inhibitory effects on SERCA function are relieved in response to repeated contractions thus enhancing relaxation rates.     

Survival of bundleless hair cells and subsequent bundle replacement in the bullfrog's saccule

Our senses of hearing and balance depend upon hair cells, the sensory receptors of the inner ear. Millions of people suffer from hearing and balance deficits caused by damage to hair cells as a result of exposure to noise, aminoglycoside antibiotics, and antitumor drugs. In some species such damage can be reversed through the production of new cells. This proliferative response is limited in mammals but it has been hypothesized that damaged hair cells might survive and undergo intracellular repair. We examined the fate of bullfrog saccular hair cells after exposure to a low dose of the aminoglycoside antibiotic gentamicin to determine whether hair cells could survive such treatment and subsequently be repaired. In organ cultures of the bullfrog saccule a combination of time‐lapse video microscopy, two‐photon microscopy, electron microscopy, and immunocytochemistry showed that hair cells can lose their hair bundle and survive as bundleless cells for at least 1 week. Time‐lapse and electron microscopy revealed stages in the separation of the bundle from the cell body. Scanning electron microscopy (SEM) of cultures fixed 2, 4, and 7 days after antibiotic treatment showed that numerous new hair bundles were produced between 4 and 7 days of culture. Further examination revealed hair cells with small repaired hair bundles alongside damaged remnants of larger surviving bundles. The results indicate that sensory hair cells can undergo intracellular self‐repair in the absence of mitosis, offering new possibilities for functional hair cell recovery and an explanation for non‐proliferative recovery. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 81–92, 2002; DOI 10.1002/neu.10002     

Mutualistic Biofilm Communities Develop with Porphyromonas gingivalis and Initial,Early, and Late Colonizers of Enamel

Porphyromonas gingivalis is present in dental plaque as early as 4 h after tooth cleaning, but it is also associated with periodontal disease, a late-developing event in the microbial successions that characterize daily plaque development. We report here that P. gingivalis ATCC 33277 is remarkable in its ability to interact with a variety of initial, early, middle, and late colonizers growing solely on saliva. Integration of P. gingivalis into multispecies communities was investigated by using two in vitro biofilm models. In flow cells, bacterial growth was quantified using fluorescently conjugated antibodies against each species, and static biofilm growth on saliva-submerged polystyrene pegs was analyzed by quantitative real-time PCR using species-specific primers. P. gingivalis could not grow as a single species or together with initial colonizer Streptococcus oralis but showed mutualistic growth when paired with two other initial colonizers, Streptococcus gordonii and Actinomyces oris, as well as with Veillonella sp. (early colonizer), Fusobacterium nucleatum (middle colonizer), and Aggregatibacter actinomycetemcomitans (late colonizer). In three-species flow cells, P. gingivalis grew with Veillonella sp. and A. actinomycetemcomitans but not with S. oralis and A. actinomycetemcomitans. Also, it grew with Veillonella sp. and F. nucleatum but not with S. oralis and F. nucleatum, indicating that P. gingivalis and S. oralis are not compatible. However, P. gingivalis grew in combination with S. gordonii and S. oralis, demonstrating its ability to overcome the incompatibility when cultured with a second initially colonizing species. Collectively, these data help explain the observed presence of P. gingivalis at all stages of dental plaque development.Removal of dental plaque by routine oral hygiene procedures is followed by a repetition of a species succession that starts with initially colonizing streptococci and actinomyces (5, 16). Other species follow as early, middle, and late colonizers, which establishes the following developmental process: successive attachment of saliva-suspended species to already attached bacteria and formation of multispecies communities.Attachment is a critical event essential to preventing the bacteria from being swallowed by salivary flow. Initial colonizers bind to host-derived receptors in the salivary pellicle coating of the tooth enamel. The remainder of typical plaque development occurs by accretion of saliva-suspended species and growth of attached bacteria, thereby increasing the microbial diversity. Adherence of suspended single cells to attached cells is called coadhesion (1). Some suspended cells are already coaggregated and adhere to attached cells as coaggregates; coaggregation is defined as the specific cell-to-cell recognition and adherence of genetically distinct cell types (8). All human oral bacterial species exhibit coaggregation. For example, Streptococcus oralis coaggregates with Streptococcus gordonii (intrageneric coaggregation). Both species pair with Actinomyces oris (intergeneric coaggregation), and all of them coaggregate with Fusobacterium nucleatum (multigeneric coaggregation). Multispecies communities composed of coaggregating species characterize dental plaque biofilms in vivo (3, 17, 18).To increase our understanding of interactions among species, we have employed two in vitro model systems and are testing numerous combinations of seven species for their ability to grow on saliva as their sole nutritional source (20, 21). First, we reported that F. nucleatum (middle colonizer) failed to grow when paired with S. oralis but grew well when A. oris was included in the three-species biofilm (20), indicating specificity by F. nucleatum for the presence of a particular initial colonizer. Recently, we showed that Aggregatibacter actinomycetemcomitans (late colonizer and periodontopathogen) exhibited mutualistic relationships with F. nucleatum and Veillonella sp. (early colonizer and commensal organism), illustrating the ability of commensals and pathogens to grow together (21).Porphyromonas gingivalis, another periodontopathogen, forms three-species communities with F. nucleatum and S. gordonii (11). Proteomics of P. gingivalis in this three-species community revealed a broad increase in proteins involved in protein synthesis, suggesting that a multispecies relationship is advantageous for the porphyromonad (11). This research group had previously reported the presence of differentially regulated porphyromonad genes when P. gingivalis and S. gordonii were together in biofilms (22). Thus, P. gingivalis responds to the presence of other oral species.P. gingivalis is detected in dental plaque samples within 6 h after professional tooth cleaning (5, 13), and its numbers increase in periodontally diseased sites (15). It forms biofilms with S. gordonii but not with Streptococcus mutans (12) or Streptococcus cristatus (23). P. gingivalis required a preformed streptococcal substratum for its incorporation into a biofilm (12). Partner specificity was also noted among four fresh isolates of P. gingivalis, which showed no coaggregation with a variety of oral actinomyces, aggregatibacteria, capnocytophagae, and streptococci (9) but coaggregated with F. nucleatum (7, 10). We show here that P. gingivalis exhibits widespread mutualism with initial, early, middle, and late colonizers but also shows specificity with initially colonizing streptococci, which could help explain its early appearance in the development of dental plaque biofilms. The relationship of porphyromonads with initial, early, middle, and later colonizers during biofilm growth on saliva as a sole nutritional source has not been explored previously. We hypothesize that the ability of P. gingivalis to coaggregate with S. gordonii and A. oris (initial colonizers), Veillonella sp. (early colonizer), F. nucleatum (middle colonizer), and A. actinomycetemcomitans (late colonizer) allows these bacteria to form multispecies biofilm communities.     

Toxicity of Buprofezin on the Survival of Embryo and Larvae of African Catfish,Clarias gariepinus (Bloch)

Buprofezin is an insect growth regulator and widely used insecticide in Malaysia. The present study evaluated the toxic effects of buprofezin on the embryo and larvae of African catfish (Clarias gariepinus) as a model organism. The embryos and larvae were exposed to 7 different concentrations (0, 0.05, 0.5, 5, 25, 50 and 100 mg/L) of buprofezin. Each concentration was assessed in five replicates. Eggs were artificially fertilized and 200 eggs and larvae were subjected to a static bath treatment for all the concentrations. The mortality of embryos was significantly increased with increasing buprofezin concentrations from 5 to 100 mg/L (p< 0.05). However, the mortality was not significantly different (p<0.05) among the following concentrations: 0 (control), 0.05, 0.5 and 5 mg/L. Data obtained from the buprofezin acute toxicity tests were evaluated using probit analysis. The 24 h LC₅₀ value (with 95% confidence limits) of buprofezin for embryos was estimated to be 6.725 (3.167-15.017) mg/L. The hatching of fish embryos was recorded as 68.8, 68.9, 66.9, 66.4, 26.9, 25.1 and 0.12% in response to 7 different concentrations of buprofezin, respectively. The mortality rate of larvae significantly (p<0.05) increased with increasing buprofezin concentrations exposed to 24-48 h. The 24 and 48 h LC₅₀ values (with 95% confidence limits) of buprofezin for the larvae was estimated to be 5.702 (3.198-8.898) and 4.642 (3.264-6.287) mg/L respectively. There were no significant differences (p>0.05) in the LC₅₀ values obtained at 24 and 48 h exposure times. Malformations were observed when the embryos and larvae exposed to more than 5 mg/L. The results emerged from the study suggest that even the low concentration (5 mg/L) of buprofezin in the aquatic environment may have adverse effect on the early embryonic and larval development of African catfish.