A novel combinatorial biocatalytic approach for producing antibacterial compounds effective against Mycobacterium tuberculosis (TB)

Two bacterial hosts expressing cloned aromatic oxygenases were used to catalyze the oxidation and polymerization of indole and related substrates, creating mixtures of indigoid compounds comprised of novel dimers and trimers. Crude extracts and purified compounds were tested for their ability to inhibit the growth of Gram-positive organisms, in general, and Mycobacterium tuberculosis (TB), in particular. Of the 74 compounds tested against M. tuberculosis, ~66 % had minimum inhibitory concentrations (MIC) of 5 μg/ml or less. The most effective antibiotic found was designated SAB-P1, a heterodimer of indole and anthranil, which had a MIC of 0.16 μg/ml, and did not inhibit kidney cells (IC⁵⁰) at concentrations of >8 μg/ml. Combinatorial biocatalysis was used to create a series of halogenated derivatives of SAB-P1 with a wider therapeutic window. None of the derivatives had MIC values that were superior to SAB-P1, but some had a wider therapeutic window because of decreased kidney cell toxicity. Generally, the indigoid dimers that were effective against TB appeared to be specific for TB. Some of the trimers generated, however, had a broader spectrum of activity inhibiting not only TB (MIC?=?1.1 μg/ml) but also the growth of Mycobacterium smegmatis MC2 155, Bacillus cereus, Enterococcus faecalis, Staphylococcus epidermidis, Bacillus subtilis 168, and Clostridium acetobutylicum. The structure of two of the novel dimers (SAB-C4 and SAB-P1) and a trimer (SAB-R1) were solved using X-ray crystallography.     

alpha beta Spectrin coiled coil association at the tetramerization site

On the basis of sequence homology studies, it has been suggested that the association of human erythrocytes alpha and beta spectrin at the tetramerization site involves interactions between helices. However, no empirical details are available, presumably due to the experimental difficulties in studying spectrin molecules because of its size and/or its structural flexibility. It has been speculated that erythrocyte tetramerization involves helical bundling rather than coiled coil association. We have used recombinant spectrin peptides to model alpha and beta spectrin to study their association at the tetramerization site. Two alpha peptides, Sp alpha 1-156 and Sp alpha 1-368, and one beta peptide, Sp beta 1898-2083, were used as model peptides to demonstrate the formation of the alpha beta complex. We also found that the replacement of R28 in Sp alpha 1-368 to give Sp alpha 1-368R28C abolished complex formation with the beta peptide. Circular dichroism techniques were used to monitor the secondary structures of the individual peptides and of the complex, and the results showed that both Sp alpha 1-156 and Sp beta 1898-2083 peptides in solution, separately, included helices that were not paired with other helices in the absence of their binding partners. However, in a mixture of Sp alpha 1-156 and Sp beta 1898-2083 and formation of the alpha beta complex, the unpaired helices associated to form coiled coils. Since the sequences of these two peptides that are involved in the coiled coil association are derived from a native protein, the information obtained from this study also provides insight toward a better understanding of naturally occurring coiled coil subunit-subunit association.     

Association of non-heterocystous cyanobacteria with crop plants

Cyanobacteria have the ability to form associations with organisms from all domains of life, notably with plants, which they provide with fixed nitrogen, among other substances. This study was aimed at developing artificial associations between non-heterocystous cyanobacteria and selected crop plants. We isolated several non-heterocystous cyanobacteria from various rice fields. The cultures were tested for their capacity to produce the plant hormone indole-3-acetic acid (IAA), and the possible role of IAA in the association of cyanobacteria with seedling roots was evaluated. Axenic cultures were co-inoculated with 10-day-old plant seedlings of Triticum aestivum, Vigna radiata and Pisum sativum and incubated for 1 week. Cyanobacterial association with the roots of these seedlings was quantified by measuring chlorophyll-a. Cyanobacterial association with the roots was observed by light microscopy as well as by confocal laser scanning microscopy (CLSM). Based on sequence analysis of the 16S rRNA gene, the isolates were identified as Synechocystis sp., Chroococcidiopsis sp., Leptolyngbya sp., and Phormidium sp. CLSM observations revealed the intimate association of cyanobacteria with the seedling roots as well as invasion of the roots and root cells. Strains producing IAA were more efficient in the colonization of the roots than those that lacked this ability. IAA-producing cyanobacteria possess a tryptophan-dependent pathway, and these cyanobacteria showed IAA synthesis activity in the presence of roots in media lacking tryptophan. Based on the results of this study, we conclude that non-heterocystous cyanobacteria also have the potential for use in agriculture to improve the growth and yield of crop plants that do not naturally form associations with cyanobacteria.     

In vitro antioxidant activity of Juglans regia L. bark extract and its protective effect on cyclophosphamide-induced urotoxicity in mice

Walnut (Juglans regia L.) bark has been claimed to possess anti-inflammatory, blood purifying, anticancer, depurative, diuretic and laxative activities. It contains several therapeutically active constituents, especially polyphenols. We studied the antioxidant potential of aqueous extract of walnut bark and its modulatory effect on cyclophosphamide (CP)-induced urotoxicity in Swiss albino male mice. Free radical-scavenging activity of extract was assessed in four in vitro assays. The phenolic and flavonolic contents of the extract were also measured. Walnut bark extract treatment (150 mg/kg p.o. x 10 days) resulted in protective restoration of decreased antioxidants in CP-treated (18 mg/kg i.p. x 10 days) animals. CP treatment caused decreases in the activities of catalase (CAT), glutathione peroxidase (GP), glutathione reductase (GR) and glutathione S-transferase (GST) and in the glutathione (GSH) content in urinary bladder and a significant concomitant increase in lipid peroxidation (LPO). Administration of extract restored all the antioxidants significantly and lowered the elevated LPO in the bladder. A correlation between radical scavenging capacities of the extract with phenolic content was observed thus justifying its antioxidant potential against oxidative stress-mediated urotoxicity in mice. Walnut is reported to possess antiproliferative activity. Its protective effect on CP-induced toxicity in bladder is a promising activity, which warrants possible clinical investigations on this medicinal plant.     

Nitrate and (per)chlorate reduction pathways in (per)chlorate-reducing bacteria

The reduction of (per)chlorate and nitrate in (per)chlorate-reducing bacteria shows similarities and differences. (Per)chlorate reductase and nitrate reductase both belong to the type?II DMSO family of enzymes and have a common bis(molybdopterin guanine dinucleotide)molybdenum cofactor. There are two types of dissimilatory nitrate reductases. With respect to their localization, (per)chlorate reductase is more similar to the dissimilatory periplasmic nitrate reductase. However, the periplasmic, unlike the membrane-bound, respiratory nitrate reductase, is not able to use chlorate. Structurally, (per)chlorate reductase is more similar to respiratory nitrate reductase, since these reductases have analogous subunits encoded by analogous genes. Both periplasmic (per)chlorate reductase and membrane-bound nitrate reductase activities are induced under anoxic conditions in the presence of (per)chlorate and nitrate respectively. During microbial (per)chlorate reduction, molecular oxygen is generated. This is not the case for nitrate reduction, although an atypical reaction in nitrite reduction linked to oxygen formation has been described recently. Microbial oxygen production during reduction of oxyanions may enhance biodegradation of pollutants under anoxic conditions.     

Development of ARMS-PCR assay for genotyping of Pro12Ala SNP of PPARG gene: a cost effective way for case–control studies of type 2 diabetes in developing countries

Type 2 diabetes (T2D) is a prevalent metabolic disorder across the globe. Research is underway on various aspects including genetics to understand and control the global epidemic of diabetes. Recently, several SNPs in various genes have been associated with T2D. These association studies are mainly carried out in the developed countries through Genome Wide Association Scans, with follow-up replication/validation studies by high-throughput genotyping techniques (e.g. Taqman Technology). Although, similar studies could be conducted in developing countries, however, the limiting factors are the associated cost and expertise. These factors hamper research into the genetic association and replication studies from low-income countries to figure out the role of putatively associated SNPs in diabetes. Although, there are several SNP detection methods (e.g. Taqman assay, Dot-blot, PCR-RFLP, DGGE, SSCP) but these are either expensive or labor intensive or less sensitive. Hence, our aim was to develop a low-cost method for the validation of PPARG (Pro12Ala, CCA>GCA) SNP (rs1801282) for its association with T2D. Here, we developed a cost-effective and rapid amplification refractory mutation specific-PCR (ARMS-PCR) method for this SNP detection. We successfully genotyped PPARG SNPs (Pro12Ala) in human samples and the validity of this method was confirmed by DNA sequencing of a few representative samples for the three different genotypes. Furthermore, ARMS-PCR was applied to T2D patients and control samples for the screening of this SNP.     

Determination of absolute configuration and binding efficacy of benzimidazole-based FabI inhibitors through the support of electronic circular dichroism and MM-GBSA techniques

We have previously reported benzimidazole-based compounds to be potent inhibitors of FabI for Francisella tularensis (FtFabI), making them promising antimicrobial hits. Optically active enantiomers exhibit markedly differing affinities toward FtFabI. The IC₅₀ of benzimidazole (?)-1 is ～100× lower than the (+)-enantiomer, with similar results for the 2 enantiomers. Determining the absolute configuration for these optical compounds and elucidating their binding modes is important for further design. Electronic circular dichroism (ECD) quantum calculations have become important in determining absolute configurations of optical compounds. We determined the absolute configuration of (?)/(+)-1 and (?)/(+)-2 by comparing experimental spectra and theoretical density functional theory (DFT) simulations of ECD spectra at the B3LYP/6-311+G(2d, p) level using Gaussian09. Comparison of experimental and calculated ECD spectra indicates that the S configuration corresponds to the (?)-rotation for both compounds 1 and 2, while the R configuration corresponds to the (+)-rotation. Further, molecular dynamics simulations and MM-GBSA binding energy calculations for these two pairs of enantiomers with FtFabI show much tighter binding MM-GBSA free energies for S-1 and S-2 than for their enantiomers, R-1 and R-2, consistent with the S configuration being the more active one, and with the ECD determination of the S configuration corresponding to (?) and the R configuration corresponding to (+). Thus, our computational studies allow us to assign (?) to (S)- and (+) to (R)- for compounds 1 and 2, and to further evaluate structural changes to improve efficacy.     

Pcal_0768, a hyperactive 4-α-glucanotransferase from <Emphasis Type="Italic">Pyrobacculum calidifontis</Emphasis>

Genome sequence of hyperthermophilic archaeon Pyrobaculum calidifontis revealed the presence of an open reading frame, Pcal_0768, corresponding to a putative 4-α-glucanotranferase belonging to glycoside hydrolases (GH) family 77. We have produced, in Escherichia coli, and purified recombinant Pcal_0768 which exhibited high disproportionation (690 U mg^?1) activity. To the best of our knowledge, this is the highest ever reported activity for any member of family GH77. Maltooligosaccharides, when used as sole substrates, were disproportionated into linear maltooligohomologues. The analysis of the reaction end products revealed no evidence for the production of cycloamyloses. Catalytic activity of the enzyme remained unchanged in the presence or the absence of ionic and nonionic detergents. γ-cyclodextrin, an inhibitor of 4-α-glucanotransferases, did not show any inhibitory effect on Pcal_0768 activity. These properties make Pcal_0768 a potential candidate for starch processing industry.