Mechanism of Nisin, Pediocin 34, and Enterocin FH99 Resistance in Listeria monocytogenes

Nisin-, pediocin 34-, and enterocin FH99-resistant variants of Listeria monocytogenes ATCC 53135 were developed. In an attempt to clarify the possible mechanisms underlying bacteriocin resistance in L. monocytogenes ATCC 53135, sensitivity of the resistant strains of L. monocytogenes ATCC 53135 to nisin, pediocin 34, and enterocin FH99 in the absence and presence of different divalent cations was assessed, and the results showed that the addition of divalent cations significantly reduced the inhibitory activity of nisin, pediocin 34, and enterocin FH99 against resistant variants of L. monocytogenes ATCC 53135. The addition of EDTA, however, restored this activity suggesting that the divalent cations seem to affect the initial electrostatic interaction between the positively charged bacteriocin and the negatively charged phospholipids of the membrane. Nisin-, pediocin 34-, and enterocin-resistant variants of L. monocytogenes ATCC 53135 were more resistant to lysozyme as compared to the wild-type strain both in the presence as well as absence of nisin, pediocin 34, and enterocin FH99. Ultra structural profiles of bacteriocin-sensitive L. monocytogenes and its bacteriocin-resistant counterparts revealed that the cells of wild-type strain of L. monocytogenes were maximally in pairs or short chains, whereas, its nisin-, pediocin 34-, and enterocin FH99-resistant variants tend to form aggregates. Results indicated that without a cell wall, the acquired nisin, pediocin 34, and enterocin FH99 resistance of the variants was lost. Although the bacteriocin-resistant variants appeared to lose their acquired resistance toward nisin, pediocin 34, and enterocin FH99, the protoplasts of the resistant variants appeared to be more resistant to bacteriocins than the protoplasts of their wild-type counterparts.     

ApoTome to visualize E-cadherin and p63 expression in oral pre-cancer

Precise histopathological localization of E-cadherin and p63 is of immense importance in understanding the integrity of oral mucosal stratified epithelium in normal and diseased conditions. Necessarily immunohistochemical imaging should have minimum bleaching impact on the dyes and ability to produce clear and crisp images. Here ApoTome provides an alternative with metal halide light source and structured illumination under the assistance of grids, along with integrated image processing modality to generate crisp images with digital interface. The current study demonstrates the applicability of such microscopic system in capturing fluorescence images of immunohistochemical sections of normal and precancerous biopsies in respect to the expression of p63 and E-cadherin in the epithelial cells. The ApoTome images localize the nuclear and membranous expressions of p63 and E-cadherin, respectively, with remarkable specificity. The findings on E-cadherin expression have enormous diagnostic significance as these images clearly differentiate the early and advanced stages of oral submucous fibrosis based on their cytoplasmic and membranous location. Thus, this study clearly depicts a remarkable performance of ApoTome with diagnostic significance.     

More than black and white: differences in predictors of obesity among Native Hawaiian/Pacific Islanders and European Americans

Although Native Hawaiians and Pacific Islanders exhibit the highest rates of obesity and associated chronic diseases of any racial/ethnic group, they remain vastly underrepresented in health research. In a cross-sectional survey of college students (N = 402) we examined BMI and health outcomes in an ethno-racially diverse rural sample of Native Hawaiian/Pacific Islanders (25.1%), Asian Americans (39.8%), and European Americans (35.1%). Measures assessed BMI, health status, health behaviors, frequency of exercise, and symptoms of psychiatric disorders (i.e., depression, anxiety, posttraumatic stress, and substance abuse and dependence). Regression analyses revealed that an overall model of five predictors (gender, race, regular exercise, difficulty sleeping, and anxiety) was significantly associated with obesity (P < 0.001) and correctly classified 84.2% of cases. A 30.7% of Native Hawaiians/Pacific Islanders were obese as compared with 9.2% of European Americans and 10.6% of Asian Americans. These findings suggest that Native Hawaiian/ Pacific Islanders are at high risk for obesity and associated medical comorbidities, but that regular physical activity may ameliorate this risk. Further, these results support the consideration of Native Hawaiians/Pacific Islanders as a distinct racial/ethnic subgroup separate from other Asian populations.     

Mechanism of biochemical action of substituted 4-methylbenzopyran-2-ones. Part 9: comparison of acetoxy 4-methylcoumarins and other polyphenolic acetates reveal the specificity to acetoxy drug: protein transacetylase for pyran carbonyl group in proximity to the oxygen heteroatom

The evidences for the possible enzymatic transfer of acetyl groups (catalyzed by a transacetylase localized in microsomes) from an acetylated compound (acetoxy-4-methylcoumarins) to enzyme proteins leading to profound modulation of their catalytic activities was cited in our earlier publications in this series. The investigations on the specificity for transacetylase (TA) with respect to the number and positions of acetoxy groups on the benzenoid ring of coumarin molecule revealed that acetoxy groups in proximity to the oxygen heteroatom (at C-7 and C-8 positions) demonstrate a high degree of specificity to TA. These studies were extended to the action of TA on acetates of other polyphenols, such as flavonoids and catechin with a view to establish the importance of pyran carbonyl group for the catalytic activity. The absolute requirement of the carbonyl group in the pyran ring of the substrate for TA to function was established by the observation that TA activity was hardly discernible when catechin pentacetate and 7-acetoxy-3,4-dihydro-2,2-dimethylbenzopyran (both lacking pyran ring carbonyl group) were used as the substrates. Further, the TA activity with flavonoid acetates was remarkably lower than that with acetoxycoumarins, thus suggesting the specificity for pyran carbonyl group in proximity to the oxygen heteroatom. The biochemical properties of flavonoid acetates, such as irreversible activation of NADPH cytochrome C reductase and microsome-catalyzed aflatoxin B₁ binding to DNA in vitro were found to be in tune with their specificity to TA.     

Photophysical properties of 2-amino-9,10-anthraquinone: evidence for structural changes in the molecule with solvent polarity.

The photophysical properties of 2-amino-9,10-anthraquinone (2AAQ) have been investigated in different solvents and solvent mixtures and correlated with the Lippert-Mataga solvent polarity parameter, Deltaf. In the low solvent polarity region with Deltaf < ca. 0.1, the dye shows unusually high fluorescence quantum yields (Phif) and lifetimes (tauf) in comparison to those in other solvents of medium to high polarities. Similarly, the radiative rate constants (kf) are relatively lower and the non-radiative rate constants (knr) are relatively higher in the low polarity solvents in comparison to those in the medium to high polarity solvents. The current results have been rationalized assuming that the dye adopts different structural forms below and above the Deltaf value of approximately 0.1. It is inferred that in the low solvent polarity region the dye exists in a non-planar structure, with its 2-NH2 plane away from that of the 9,10-anthraquinone moiety in the ground state. In solvents of medium to high polarities, the dye exists in a polar intramolecular charge transfer (ICT) structure, where the amino lone pair of the 2-NH2 group is in strong resonance with the anthraquinone pi-cloud in the ground state. In all the solvents, however the dye is inferred to exist in the ICT structure in its excited (S1) state. Supportive evidence for the above hypothesis has been obtained from the solvent polarity effect on the Stokes' shifts for the dye. Quantum chemical studies on the structures of 2AAQ dye in the gas phase also give qualitative support for the inferences drawn from the photophysical properties of the dye in different solvents.     

Probing the steroid binding domain-like I (SBDLI) of the sigma-1 receptor binding site using N-substituted photoaffinity labels

Radioiodinated photoactivatable photoprobes can provide valuable insights regarding protein structure. Previous work in our laboratory showed that the cocaine derivative and photoprobe 3-[ (125)I]iodo-4-azidococaine ([ (125)I]IACoc) binds to the sigma-1 receptor with 2-3 orders of magnitude higher affinity than cocaine [Kahoun, J. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 1393-1397]. Using this photoprobe, we demonstrated the insertion site for [ (125)I]IACoc to be Asp188 [Chen, Y. (2007) Biochemistry 46, 3532-3542], which resides in the proposed steroid binding domain-like II (SBDLII) region (amino acids 176-194) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. An additional photoprobe based on the sigma-1 receptor ligand fenpropimorph, 1- N-(2-3-[ (125)I]iodophenyl)propane ([ (125)I]IAF), was found to label a peptide in both the SBDLII and steroid binding domain-like I (SBDLI) (amino acids 91-109) [Pal, A. (2007) Mol. Pharmacol. 72, 921-933]. In this report, we describe two novel strategically positioned carrier-free, radioiodinated photoaffinity labels specifically designed to probe the putative "nitrogen interacting region" of sigma-1 receptor ligands. These two novel photoprobes are (-)-methyl 3-(benzoyloxy)-8-2-(4-azido-3-[ (125)I]iodobenzene)-1-ethyl-8-azabicyclo[3.2.1]octane-2-carboxylate ([ (125)I]-N-IACoc) and N-propyl- N-(4-azido-3-iodophenylethyl)-3-(4-fluorophenyl)propylamine ([ (125)I]IAC44). In addition to reporting their binding affinities to the sigma-1 and sigma-2 receptors, we show that both photoaffinity labels specifically and covalently derivatized the pure guinea pig sigma-1 receptor (26.1 kDa) [Ramachandran, S. (2007) Protein Expression Purif. 51, 283-292]. Cleavage of the photolabeled sigma-1 receptor using Endo Lys C and cyanogen bromide (CNBr) revealed that the [ (125)I]-N-IACoc label was located primarily in the N-terminus and SBDLI-containing peptides of the sigma-1 receptor, while [ (125)I]IAC44 was found in peptide fragments consistent with labeling of both SBDLI and SBDLII.     

Formulation of a strategy for the application of Duddingtonia flagrans to control caprine parasitic gastroenteritis

Experiments on the influence of egg density and varying quantities of chlamydospores on the nematode-trapping ability of Duddingtonia flagrans, influence of D. flagrans on the larval translation of gastrointestinal nematodes, doses of chlamydospores required for the effective control of gastrointestinal nematodosis and the epidemiology of nematode parasites were conducted in goats, which generated baseline data required for strategic application of the biocontrol agent. The nematode-trapping ability of D. flagrans, measured by numerical enumeration of infective third-stage larvae developed in the faecal culture, revealed that the efficacy is dependent on both nematode egg and chlamydospore density. Pasture plot studies revealed that D. flagrans, if deposited at the same time as nematode eggs, prevents translation of third-stage larvae of caprine nematodes from the faecal pats onto the grass blades. Feeding of 1 x 106 chlamydospores per kg body weight and above to goats virtually eliminated larvae from the pasture. Application of as few as 1 x 104 and 1 x 105 chlamydospores per kg body weight had a profound impact on larval recovery. The effect persisted as long as the chlamydospores were fed. Monthly faecal worm egg counts of adult goats maintained under a semi-intensive management system on the Chhattisgarh plain and pasture larval burden revealed that June to August were the months of high risk for nematodosis. Haemonchus was the dominant species recorded throughout the year. The present data can best be utilized by formulating a strategic control measure when the larval challenge to the animal is maximum (June to August), so as to prevent establishment of patent infection. The observations reinforced the strategy to be adopted for nematode parasite control in goats by applying the biocontrol option at the onset of the monsoon.     

An insight into the mechanism of inhibition of unusual bi-subunit topoisomerase I from Leishmania donovani by 3,3'-di-indolylmethane, a novel DNA topoisomerase I poison with a strong binding affinity to the enzyme

DIM (3,3'-di-indolylmethane), an abundant dietary component of cruciferous vegetables, exhibits a wide spectrum of pharmacological properties. In the present study, we show that DIM is a potent inhibitor of Leishmania donovani topoisomerase I with an IC50 of 1.2 microM. Equilibrium dialysis shows that DIM binds strongly to the free enzyme with a binding constant of 9.73x10(-9) M. The binding affinity of DIM to the small subunit is 8.6-fold more than that of the large subunit of unusual LdTOP1LS (bi-subunit L. donovani topoisomerase I). DIM stabilizes topoisomerase I-DNA cleavage complexes in vitro and also in vivo. Like CPT (camptothecin), DIM inhibits the religation step when the drug was added to preformed topoisomerase I-DNA binary complex. Hence, DIM is similar to CPT with respect to its ability to form the topoisomerase I-mediated 'cleavable complexes' in vitro and in vivo. But unlike CPT, DIM interacts with both free enzyme and substrate DNA. Therefore DIM is a non-competitive class I inhibitor of topoisomerase I. DIM also inhibits the relaxation activity of the CPT-resistant mutant enzyme LdTOP1Delta39LS (N-terminal deletion of amino acids 1-39 of LdTOP1LS). The IC50 values of DIM in simultaneous and enzyme pre-incubation relaxation assays were 3.6 and 2.9 muM respectively, which are higher than that of wild-type topoisomerase I (LdTOP1LS), indicating that the affinity of DIM to LdTOP1Delta39LS is less than that for LdTOP1LS. This is the first report on DIM as an L. donovani topoisomerase I poison. Our study illuminates a new mode of action of enzyme inhibition by DIM that might be exploited for rational drug design in human leishmaniasis.     

Oxalate contributes to the resistance of Gaillardia grandiflora and Lupinus sericeus to a phytotoxin produced by Centaurea maculosa

Centaurea maculosa Lam. is a noxious weed in western North America that produces a phytotoxin, (±)-catechin, which is thought to contribute to its invasiveness. Areas invaded by C. maculosa often result in monocultures of the weed, however; in some areas, North American natives stand their ground against C. maculosa and show varying degrees of resistance to its phytotoxin. Two of these resistant native species, Lupinus sericeus Pursh and Gaillardia grandiflora Van Houtte, were found to secrete increased amounts of oxalate in response to catechin exposure. Mechanistically, we found that oxalate works exogenously by blocking generation of reactive oxygen species in susceptible plants and reducing oxidative damage generated in response to catechin. Furthermore, field experiments show that L. sericeus indirectly facilitates native grasses in grasslands invaded by C. maculosa, and this facilitation can be correlated with the presence of oxalate in soil. Addition of exogenous oxalate to native grasses and Arabidopsis thaliana (L.) Heynh grown in vitro alleviated the phytotoxic effects of catechin, supporting the field experiments and suggesting that root-secreted oxalate may also act as a chemical facilitator for plant species that do not secrete the compound.