Antibacterial and antidiarrhoeal effects of alkaloids of Holarrhena antidysenterica WALL

The alkaloids from the ethanolic extract of H. antidysenterica seeds were evaluated for their antibacterial activity against clinical isolates of enteropathogenic Escherichia coli (EPEC) in vitro, and their antidiarrhoeal activity on castor oil-induced diarrhoea in rats, in vivo. The plasmid DNA, whole cell lysate and outer membrane protein profile of a clinical isolate of EPEC was determined in presence of alkaloids of H. antidysenterica. The disc diffusion and agar well diffusion methods were used to evaluate the antibacterial efficacy. The alkaloids showed strong antibacterial activity against EPEC strains. In castor oil-induced diarrhoea, alkaloids reduced the diarrhoea with decrease in the number of wet faeces in pretreated rats at a dose of 200-800 mg/kg. The loss of plasmid DNA and suppression of high molecular weight proteins were observed on alkaloids treatment. Taking into account the multiple antibiotic resistance of EPEC, the results suggest usefulness of alkaloids of H. antidysenterica seeds as antibacterial and antidiarrhoeal agents.     

Purification and characterization of fibrinolytic protease from Bacillus amyloliquefaciens MCC2606 and analysis of fibrin degradation product by MS/MS

A serine protease with preference for fibrin protein was purified and characterized from Bacillus amyloliquefaciens MCC2606, isolated from dosa batter. The protease was purified using ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The degradation activity of the protease toward the fibrin was significantly higher compared with other protein substrates in the study. The molecular weight of the CFR15-protease was estimated to be 32?kDa based on SDS-PAGE. The purified enzyme exhibited both fibrinolytic and fibrinogenolytic activity. The optimum pH and temperature for the activity of the enzyme was found to be 10.5 and 45°C. A significant inhibition was seen with the protease inhibitors phenyl methyl sulphonyl fluoride and ethylene diamine tetra acetic acid and the activity of the enzyme was enhanced in presence of Mn²⁺. There was an observed increase in vitro activated partial thromboplastin time and prothrombin time of both time and dose dependent study. Among the four chains of fibrin, the β-chain of fibrin appears to be the primary component and site susceptible for CFR15-protease in early action as indicated by MS/MS analysis of initial degradation products. These results indicated that the CFR15-protease have the potential to be an effective fibrinolytic agent.     

Purification and Characterization of a Proteinase Inhibitor from Field Bean, Dolichos lablab perpureus L.

A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M _r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K _a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 10⁷ M^?1 and 3.1 × 10⁷ M^?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively.     

Indomethacin and juglone inhibit inflammatory molecules to induce apoptosis in colon cancer cells

Colorectal cancer (CRC) is the third most common fatal cancer. Indomethacin, a nonsteroidal anti‐inflammatory drug, is known to reduce the occurrence of CRC. This study evaluated the potential anticolon cancer effects of juglone (5‐hydroxy‐1,4‐naphthoquinone) in combination with indomethacin. Human colon adenocarcinoma cells (HT29) were subjected to treatment with indomethacin, juglone, and a combination of both. Morphological analysis, cell cycle regulation, and dual staining using acridine orange and ethidium bromide in control and treated cells revealed the apoptotic potential of these compounds. Bcl2 and inflammatory molecules (tumor necrosis factor‐α, nuclear factor kappa B, and Cox‐2) were found to be decreased with a concomitant increase in the expression of proapoptotic molecules (Bad, Bax, cytochrome c, and PUMA) as a result of the molecular regulation of Wnt, Notch, and peroxisome proliferator‐activated receptor‐γ signaling. Treatment with juglone was not as effective as with indomethacin; however, a combination of both was shown to be more effective, suggesting that juglone may be considered for therapeutic intervention of colon cancer.     

Rapid oligonucleotide-templated fluorogenic tetrazine ligations

Template driven chemical ligation of fluorogenic probes represents a powerful method for DNA and RNA detection and imaging. Unfortunately, previous techniques have been hampered by requiring chemistry with sluggish kinetics and background side reactions. We have developed fluorescent DNA probes containing quenched fluorophore-tetrazine and methyl-cyclopropene groups that rapidly react by bioorthogonal cycloaddition in the presence of complementary DNA or RNA templates. Ligation increases fluorescence with negligible background signal in the absence of hybridization template. Reaction kinetics depend heavily on template length and linker structure. Using this technique, we demonstrate rapid discrimination between single template mismatches both in buffer and cell media. Fluorogenic bioorthogonal ligations offer a promising route towards the fast and robust fluorescent detection of specific DNA or RNA sequences.     

Anti-Tumor Action,Clinical Biochemistry Profile and Phytochemical Constituents of a Pharmacologically Active Fraction of S. crispus in NMU-Induced Rat Mammary Tumour Model

Cancer patients seek alternative remedies such as traditional medicinal plants for safe and effective treatment and help overcome the side effects of conventional therapy. Current knowledge indicates that extracts of Strobilanthes crispus of the Acanthaceae family exhibit potent anticancer properties in vitro and are non-toxic in vivo. S. crispus was also reported to be protective against chemical hepatocarcinogenesis. We previously showed that a bioactive fraction of S. crispus leaves also synergized with tamoxifen to cause apoptosis of human breast cancer cell lines without damaging non-malignant epithelial cells. The present study aimed to evaluate the antitumor effect of S. crispus dichloromethane fraction (F3) using N-methyl-N-Nitrosourea (NMU)-induced rat mammary tumor model. Tumor regression was observed in 75% of the rats following 8-week oral administration of F3 with no secondary tumour formation and no signs of anemia or infection. However, no improvement in the liver and renal function profiles was observed. Major constituents of F3 were identified as lutein, 13¹-hydroxy-13²-oxo-pheophytin a, campesterol, stigmasterol, β-sitosterol, pheophytin a and 13²-hydroxy-pheophytin a. These compounds however, may not significantly contribute to the antitumor effect of F3.     

Neutralization of radical toxicity by temperature-dependent modulation of extracellular SOD activity in coral bleaching pathogen Vibrio shiloi and its role as a virulence factor

Vibrio shiloi is the first and well-documented bacterium which causes coral bleaching, particularly, during summer, when seawater temperature is between 26 and 31°C. Coral bleaching is the disruption of the symbiotic association between coral hosts and their photosynthetic microalgae zooxanthellae. This is either due to lowered resistance in corals to infection or increased virulence of the bacterium at the higher sea surface temperature. The concentration of the oxygen and resulting oxygen radicals produced by the zooxanthellae during photosynthesis are highly toxic to bacteria, which also assist corals in resisting the infection. Hence, in this study we examined the effect of different temperatures on the activity of a novel extracellular SOD in V. shiloi. We also partially characterized the SOD and clearly confirmed that the extracellular SOD produced by V. shiloi is Mn–SOD type, as it was not inhibited by H₂O₂ or KCN. Performing chemical susceptibility killing assay, we confirmed that extracellular SOD may act as first line of defense for the bacteria against the reactive oxygen species. Since, increased activity of novel Mn–SOD at higher temperature, leads to the neutralization of radical toxicity and facilitates the survival of V. shiloi. Hence, the extracellular Mn–SOD may be considered as a virulence factor.     

Morin fosters apoptosis in experimental hepatocellular carcinogenesis model

Here we investigated the in vivo effect of morin (500 ppm in diet) in fostering apoptosis in diethylnitrosamine (DEN) (200 mg/kg bodyweight) mediated experimental hepatocellular carcinogenesis model. We analyzed the expression of cytosolic protein Akt and their important apoptotic downstream targets like caspase-9, Bcl-2, Bax, GSK-3βin vivo, by immunoblot analysis. In silico docking studies indicated that morin could serve as a better inhibitor than the classical PI3K inhibitor LY294002. The results obtained from in vivo studies confirm this. We also demonstrate here that morin's interaction with a defined set of amino acids of PI3K p110γ catalytic subunit resulted in the down-regulation of p-Akt^Ser473, p-Akt^Thr308 and total Akt causing the attenuation of its downstream targets in DEN-induced hepatocellular carcinoma. Further, morin caused the up-regulation of tumor suppressor PTEN, an important negative regulator of Akt, thus initiating apoptosis. Supplementation of morin to experimental animals modulated Bcl-2/Bax ratio causing the release of cyt C and up-regulation of caspase-3 and -9. Morin was also found to prevent the Akt-mediated suppression of GSK-3β possibly causing cell cycle arrest at the G1/S phase. These observations were supported by the DNA fragmentation and transmission electron microscopy results, which showed the occurrence of apoptosis. In conclusion, our findings demonstrate that morin begets apoptosis in DEN-induced hepatocellular carcinoma.