Microwave-assisted synthesis of antimicrobial dihydropyridines and tetrahydropyrimidin-2-ones: novel compounds against aspergillosis

Ten 4-aryl-1,4-dihydropyridine and three 4-aryl-1,2,3,4-tetrahydropyrimidin-2-one derivatives have been synthesized and examined for their activity against pathogenic strains of Aspergillus fumigatus and Candida albicans. Although none of the three compounds belonging to pyrimidin-2-one series showed any activity against two pathogens, two of the compounds of the dihydropyridine series, that is, diethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate and dimethyl 4-(4-methoxyphenyl)-2,6-dimethyl-1,4-dihydropyridin-3,5-dicarboxylate, exhibited significant activity against A. fumigatus in disc diffusion, microbroth dilution and percent spore germination inhibition assays. The most active diethyl dihydropyridine derivative exhibited a MIC value of 2.92 microg/disc in disc diffusion and 15.62 microg/ml in microbroth dilution assays. The MIC(90) value of the most active compound by percent germination inhibition assay was found to be 15.62 microg/ml. The diethyl dicarboxylate derivative of dihydropyridine also exhibited appreciable activity against C. albicans. The in vitro toxicity of the most active diethyl dihydropyridine derivative was evaluated using haemolytic assay, in which the compound was found to be non-toxic to human erythrocytes even at a concentration of 625 microg/ml. The standard drug amphotericin B exhibited 100% lysis of erythrocytes at a concentration almost 16 times less than the safer concentration of the most active dihydropyridine derivative.     

Galleria mellonella as a Model System for Studying Listeria Pathogenesis

Essential aspects of the innate immune response to microbial infection are conserved between insects and mammals. This has generated interest in using insects as model organisms to study host-microbe interactions. We used the greater wax moth Galleria mellonella, which can be reared at 37°C, as a model host for examining the virulence potential of Listeria spp. Here we report that Galleria is an excellent surrogate model of listerial septic infection, capable of clearly distinguishing between pathogenic and nonpathogenic Listeria strains and even between virulent and attenuated Listeria monocytogenes strains. Virulence required listerial genes hitherto implicated in the mouse infection model and was linked to strong antimicrobial activities in both hemolymph and hemocytes of infected larvae. Following Listeria infection, the expression of immune defense genes such as those for lysozyme, galiomycin, gallerimycin, and insect metalloproteinase inhibitor (IMPI) was sequentially induced. Preinduction of antimicrobial activity by treatment of larvae with lipopolysaccharide (LPS) significantly improved survival against subsequent L. monocytogenes challenge and strong antilisterial activity was detected in the hemolymph of LPS pretreated larvae. We conclude that the severity of septic infection with L. monocytogenes is modulated primarily by innate immune responses, and we suggest the use of Galleria as a relatively simple, nonmammalian model system that can be used to assess the virulence of strains of Listeria spp. isolated from a wide variety of settings from both the clinic and the environment.Listeriae are rod-shaped, motile, facultative, anaerobic Gram-positive bacteria that are ubiquitously distributed in the environment (28). Of the six species that comprise the genus Listeria, only L. monocytogenes and L. ivanovii are pathogenic and cause disease, while strains of the species L. innocua, L. welshimeri, L. seeligeri, and L. grayi are generally considered to be nonpathogenic (26). L. monocytogenes is a major food-borne pathogen, and listeriosis is an invasive disease that in its severest form can lead to meningitis, meningoencephalitis, septicemia, and abortions (38). Listeriosis occurs primarily in pregnant women, newborn infants, and the elderly as well as in immunocompromised patients, with a mortality rate of about 30% (22, 36). The virulence of L. monocytogenes has been linked to a 9.6-kb pathogenicity island designated vgc (virulence gene cluster) that comprises six genes encoding its major virulence determinants. These are (i) prfA, a master regulator of many known listerial virulence genes; (ii) hly, encoding listeriolysin, a hemolysin required for bacterial escape from the host primary vacuole to the host cytoplasm; (iii) two phospholipase genes denoted plcA and plcB, for facilitating lysis of host cell membranes; (iv) actA, encoding a surface bound protein that directs polymerization of host cell actin and is required for intracellular motility; and (v) mpl, encoding a metalloproteinase which is thought to work together with the plcB product to facilitate cell-to-cell spread (28). Presently, identification and characterization of novel virulence factors rely on assessing mutant bacteria for growth in the organs of infected mice. Nevertheless, the dependence on mouse infection models limits large-scale screening for additional mutants defective in their ability to grow in the host intracellularly or for those required to overcome host innate defenses (33).The possibility of addressing many aspects of mammalian innate immunity in invertebrates has opened a new arena for developing invertebrate models to study human infections. Recently the use of invertebrate models, in particular the fruit fly Drosophila melanogaster, has been introduced for the study of septic listerial infections (37). Listeria mutants attenuated for virulence in a mouse model exhibited lowered virulence in this model. The Drosophila model system has powerful genetic tools available and has thus provided deeper insights into molecular mechanisms of the interactions between Listeria and the insect innate immune system (1, 8-10, 18, 24). However, a recent study has shown that even nonpathogenic L. innocua strains cause lethal infections of Drosophila, limiting it use as a discerning model for the study of virulence potential among pathogenic L. monocytogenes isolates (32).We have a longstanding interest in host-pathogen interactions of the greater wax moth, Galleria mellonella, in particular with entomopathogenic microbes (55). Recently, Galleria has also emerged as a reliable model host to study the pathogenesis of many human pathogens (7, 11, 12, 17, 21, 30, 31, 39-42, 44, 46, 48-51). Among the advantages provided by the Galleria model host (e.g., low rearing costs, convenient injection feasibility, and status as an ethically acceptable animal model), it is of particular importance that Galleria has a growth optimum at 37°C, to which human pathogens are adapted and which is essential for synthesis of many virulence/pathogenicity factors. Significantly, a correlation between the virulence of a pathogen in G. mellonella and that in mammalian models has been established (16, 25).The innate immunity of Galleria is a complex, multicomponent response involving hemolymph coagulation, cellular phagocytosis, and phenol oxidase-based melanization. Importantly, killing of pathogens is achieved similarly to that in mammals, i.e., by enzymes (e.g., lysozymes), reactive oxygen species, and antimicrobial peptides (e.g., defensins). Galleria employs recognition of nonself microbe-associated molecular patterns by germ line-encoded receptors (e.g., Toll and peptidoglycan recognition proteins) (52). Recently, we have found that Galleria also senses pathogens by danger signaling, by detecting either nucleic acids released from damaged cells or peptides resulting from proteolytic cleavage of self proteins by matrix metalloproteinases (3-6).In this work we examined the Galleria model of septic infection for its ability to differentially distinguish between infections caused by strains with different virulence potentials in the mouse infection model, as well as in avirulent strains of Listeria. We found that the Galleria model is highly discriminatory in assessing the pathogenic potential of Listeria spp., and we observed a strong correlation with the virulence previously determined in the mouse model of infection. Here, we present data indicating that the Galleria model also replicates many aspects of innate immune function, such as the constitutive expressions of potential antimicrobial factors following infection. Also, prior induction of immunity in Galleria can protect larvae from septic infection with highly pathogenic L. monocytogenes.     

A fluorimetric semi-microplate format assay of protein carbonyls in blood plasma

Oxidative stress, originating from reactive oxygen species (ROS), has been implicated in aging and various human diseases. The ROS generated can oxidize proteins producing protein carbonyl derivatives. The level of protein carbonyls in blood plasma has been used as a measure of overall oxidative stress in the body. Classically, protein carbonyls have been quantitated spectrophotometrically by directly reacting them with 2,4-dinitrophenylhydrazine (DNPH). However, the applicability of this method to biological samples is limited by its low inherent sensitivity. This limitation has been overcome by the development of sensitive enzyme-linked immunosorbent assay (ELISA) methods to measure protein carbonyls. As part of the Healthy Aging in Neighborhoods of Diversity across the Lifespan (HANDL) study, oxidative stress in humans was quantified by measuring blood plasma protein carbonyls using the two commercially available ELISA kits and the spectrophotometric DNPH assay. Surprisingly, two ELISA methods gave very different values for protein carbonyls, both of which were different from the value of the spectrophotometric method. We have developed a fluorescent semi-microplate format assay of protein carbonyls involving direct reaction of protein carbonyls with fluorescein thiosemicarbazide that correlates (R = 0.992) with the direct spectrophotometric method. It has a coefficient of variation of 4.99% and is at least 100 times more sensitive than the spectrophotometric method.     

Effects of Longevinex (modified resveratrol) on cardioprotection and its mechanisms of action

Although resveratrol has been proven to possess diverse health benefits, several recent reports have demonstrated conflicting results on some aspects of its effects, including its anti-aging properties. Considerable debate appears to exist on the dose and bioavailability of resveratrol, leading to the controversies on its effectiveness. To resolve the problem, we designed a study with a resveratrol formulation that contained resveratrol supplemented with 5% quercetin and 5% rice bran phytate (commercially known as Longevinex). These ingredients were micronized to increase the bioavailability. Sprague-Dawley rats were gavaged with either Longevinex or vehicle (5% quercetin plus 5% rice bran phytate), and rats were sacrificed after 1 or 3 months, when isolated working hearts were subjected to 30 min ischemia followed by 2 h of reperfusion. Longevinex-treated hearts, irrespective of the duration of treatments, revealed superior cardiac performance, reduced infarct size, and induction of survival signals as evidenced by increased Bcl2/Bax ratio and enhanced Akt phosphorylation. In contrast, LC3-II and Beclin were enhanced significantly after 3 months of Longevinex treatment, suggesting that autophagy occurred only after feeding Longevinex to rats for a prolonged period of time. Corroborating with the results of autophagy, Sirt1 and Sirt3 increased significantly only after 3 months of Longevinex treatment, suggesting that enhanced expression of Sirts correlated with induction of autophagy. In concert, Longevinex caused phosphorylation and nuclear translocation of FoxO1, FoxO3a, and FoxO4, indicating involvement of FoxOs with autophagy. Since Sirts and FoxOs are reliable markers of longevity, the results appear to suggest that Longevinex induces longevity after prolonged feeding via induction of autophagy, while it converts death signals into survival signals and provides cardioprotection within a relatively shorter period of time.     

Biochemical characterisation of Murray Valley encephalitis virus proteinase

Murray Valley encephalitis virus (MVEV) is a member of the flavivirus group, a large family of single stranded RNA viruses, which cause serious disease in all regions of the world. Its genome encodes a large polyprotein which is processed by both host proteinases and a virally encoded serine proteinase, non-structural protein 3 (NS3). NS3, an essential viral enzyme, requires another virally encoded protein cofactor, NS2B, for proteolytic activity. The cloning, expression and biochemical characterisation of a stable MVEV NS2B-NS3 fusion protein is described.     

Isolation of an unusual metabolite 2-allyloxyphenol from a marine actinobacterium, its biological activities and applications

A marine actinobacterium isolated from the Bay of Bengal, India and previously found to be producing an antimicrobial and cytotoxic terpenoid was further investigated for antimicrobial metabolites. The bacterium was preliminarily identified as a new species of the genus Streptomyces (strain MS1/7). The cell-free culture broth was extracted with n-butanol and purified using silica gel column chromatography and high-performance liquid chromatography. Molecular characterization was done using ESI mass, IR and ¹H and ¹³C NMR spectrometry. 2-Allyloxyphenol (MW 150; C₉H₁₀O₂), a synthetic drug and chemical intermediate, was obtained as a natural product for the first time. Serendipitous natural occurrence provided new insights into the synthetic molecule. 2-Allyloxyphenol was found to be inhibitory to 21 bacteria and three fungi in the minimum range 0.2–1.75 mg mL⁻¹ determined by agar dilution method. 2-Allyoxyphenol possesses strong antioxidant property (IC₅₀ 22 μg mL⁻¹, measured by 1, 1-diphenyl-2-picryl hydrazyl scavenging activity). Hydroxyl and allyloxy groups in 2-allyloxyphenol were responsible for antimicrobial and antioxidant activities. 2-Allyloxyphenol has marked resemblance to smoky aroma and is two to three times more active as an antimicrobial than some commercial smoke-flavour compounds. Absence of hemolytic toxicity, potential carcinogenicity, cytotoxicity and reports of toxic reactions in literature suggest possible application of 2-allyloxyphenol as a food preservative and an oral disinfectant.     

Characterisation of a detergent-stable alkaline protease from a novel thermophilic strain Paenibacillus tezpurensis sp. nov. AS-S24-II

An alkaline-protease-producing bacterial strain (AS-S24-II) isolated from a soil sample in Assam is a Gram-stain-positive, catalase-positive, endospore-forming rod and grows at temperatures ranging from 30 °C to 60 °C and salinity ranging from 0% to 7% (w/v) NaCl. Phenotypic characterisation, chemotaxonomic properties, presence of Paenibacillus-specific signature sequences, and ribotyping data suggested that the strain AS-S24-II represents a novel species of the genus Paenibacillus, for which the name Paenibacillus tezpurensis sp. nov. (MTCC 8959) is proposed. Phylogenetic analysis revealed that P. lentimorbus strain DNG-14 and P. lentimorbus strain DNG-16 represent the closest phylogenetic neighbour of this novel strain. Alkaline protease production (598 × 10³ U l⁻¹) by P. tezpurensis sp. nov. in SmF was optimised by response surface method. A laundry-detergent-stable, Ca²⁺-independent, 43-kDa molecular weight alkaline serine protease from this strain was purified with a 1.7-fold increase in specific activity. The purified protease displayed optimum activity at pH 9.5 and 45–50 °C temperature range and exhibited a significant stability and compatibility with surfactants and most of the tested commercial laundry detergents at room temperature. Further, the protease improved the wash performance of detergents, thus demonstrating its feasibility for inclusion in laundry detergent formulations.     

In vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD)

The objective of the present study was to evaluate the in vitro toxicity of bithionol and bithionol sulphoxide to Neoparamoeba spp., the causative agent of amoebic gill disease (AGD). The current treatment for AGD-affected Atlantic salmon involves bathing sea-caged fish in freshwater for a minimum of 3 h, a labour-intensive and costly exercise. Previous attempts to identify alternative treatments have suggested bithionol as an alternate therapeutic, but extensive in vitro efficacy testing has not yet been done. In vitro toxicity to Neoparamoeba spp. was examined using amoebae isolated from the gill of AGD-affected Atlantic salmon and exposing the parasites to freshwater, alumina (10 mg l(-1)), seawater, bithionol or bithionol sulphoxide at nominal concentrations of 0.1, 0.5, 1, 5 and 10 mg l(-1) in seawater. The numbers of viable amoebae were counted using the trypan blue exclusion method at 0, 24, 48 and 72 h. Both bithionol and bithionol sulphoxide demonstrated in vitro toxicity to Neoparamoeba spp. at all concentrations examined (0.1 to 10 mg l(-1) over 72 h), with a comparable toxicity to freshwater observed for both chemicals at concentrations > 5 mg l(-1) following a 72 h treatment. Freshwater remained the most effective treatment, with only 6% viable amoebae seen after 24 h and no viable amoebae observed after 48 h.     

Comparative inhibitory activity of 3'- and 5'-functionalized nucleosides on ribonuclease A

Modified nucleosides, molecules, functionalized with various polar groups at different positions have been synthesized to rationalize the impact of structural modification on their inhibitory activity. Agarose gel and precipitation assays indicate their improved inhibitory activity on ribonuclease A (RNase A). Kinetic experiments clearly categorize them as competitive inhibitors of RNase A with improved inhibition constant (K(i)) values (37±9, 67±6, and 193±7μM for compounds 10, 3, and 7, respectively). The preferential hydrogen bonding network formation between His-12 and His-119 of RNase A with the polar carboxylic and amino groups of these compounds has been evidenced from the docking studies. The relationship between structural modifications and inhibitory activity of these compounds is further justified in terms of energetics using PEARLS.