Supersensitive detection of T-2 toxin by the in situ synthesized π-conjugated molecularly imprinted nanopatterns. An in situ investigation by surface plasmon resonance combined with electrochemistry

A π-conjugated molecularly imprinted polymer (MIP) with nanopatterns for T-2 toxin (T-2) was prepared on SPR chip by in situ electropolymerization of 3-aminophenylboronicacid (3-APBA) with T-2. The complete removal of T-2 from polymer was confirmed in situ by SPR and EIS and also ex situ by SEM, EDAX, fluorescence microscopy and Raman spectroscopy. SEM image of T-2 MIP exhibited nanopatterns due to imprinting of T-2. The MIP of T-2 showed a linear response for T-2 from 2.1 fM to 33.6 fM with a detection limit of 0.1 fM (0.05 pg/mL). In this study, thermodynamic parameters such as change in Gibb's free energy (ΔG), change in enthalpy (ΔH) and change in entropy (ΔS) were determined and the values revealed that the interaction between T-2 and T-2 MIP as spontaneous, endothermic and entropy driven one. Moreover, interactions of very high concentration of interferents with T-2 MIP showed very less response due to the presence of nanopatterns of T-2 in the T-2 MIP. Equilibrium constant (12.7 fM) obtained in this study indicates the super binding affinity of T-2 with T-2 MIP. Moreover, the present methodology provides an outline to develop field-detection equipment capable of detecting T-2 toxin at or well below the guideline concentrations recommended by American subcommittee on military field drinking water.     

Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis

YagE is a 33 kDa prophage protein encoded by CP4-6 prophage element in Escherichia coli K12 genome. Here, we report the structures of YagE complexes with pyruvate (PDB Id 3N2X) and KDGal (2-keto-3-deoxy galactonate) (PDB Id 3NEV) at 2.2A resolution. Pyruvate depletion assay in presence of glyceraldehyde shows that YagE catalyses the aldol condensation of pyruvate and glyceraldehyde. Our results indicate that the biochemical function of YagE is that of a 2-keto-3-deoxy gluconate (KDG) aldolase. Interestingly, E. coli K12 genome lacks an intrinsic KDG aldolase. Moreover, the over-expression of YagE increases cell viability in the presence of certain bactericidal antibiotics, indicating a putative biological role of YagE as a prophage encoded virulence factor enabling the survival of bacteria in the presence of certain antibiotics. The analysis implies a possible mechanism of antibiotic resistance conferred by the over-expression of prophage encoded YagE to E. coli.     

New bichalcone analogs as NF-κB inhibitors and as cytotoxic agents inducing Fas/CD95-dependent apoptosis

A series of novel bichalcone analogs were synthesized and evaluated in lipopolysaccharide (LPS)-activated microglial cells as inhibitors of nitric oxide (NO) and for in vitro anticancer activity using a limited panel of four human cancer cell lines. All analogs inhibited NO production. Compounds 4 and 11 exhibited optimal activity with IC(50) values of 0.3 and 0.5 μM, respectively, and were at least 38-fold better than the positive control. A mechanism of action study showed that both compounds significantly blocked the nuclear translocation of NF-κB p65 and up-regulation of iNOS at 1.0 μM. Compound 4 and three other analogs (3, 20, and 23) exerted significant in vitro anticancer activity GI(50) values ranging from 0.70 to 13.10 μM. A mode of action study using HT-29 colon cancer cells showed that 23 acts by inducing apoptosis signaling.     

Diversity and polymorphism in AHL-lactonase gene (aiiA) of Bacillus

To explore bacterial diversity for elucidating genetic variability in acylhomoserine lactone (AHL) lactonase structure, we screened 800 bacterial strains. It revealed the presence of a quorum quenching (QQ) AHL-lactonase gene (aiiA) in 42 strains. These 42 strains were identified using rrs (16S rDNA) sequencing as Bacillus strains, predominantly B. cereus. An in silico restriction endonuclease (RE) digestion of 22 AHL lactonase gene (aiiA) sequences (from NCBI database) belonging to 9 different genera, along with 42 aiiA gene sequences from different Bacillus spp. (isolated here) with 14 type II REs, revealed distinct patterns of fragments (nucleotide length and order) with four REs; AluI, DpnII, RsaI, and Tru9I. Our study reflects on the biodiversity of aiiA among Bacillus species. Bacillus sp. strain MBG11 with polymorphism (115Alanine > Valine) may confer increased stability to AHL lactonase, and can be a potential candidate for heterologous expression and mass production. Microbes with ability to produce AHL-lactonases degrade quorum sensing signals such as AHL by opening of the lactone ring. The naturally occurring diversity of QQ molecules provides opportunities to use them for preventing bacterial infections, spoilage of food, and bioremediation.     

Pancreatic β cell identity is maintained by DNA methylation-mediated repression of Arx

Adult pancreatic β cells can replicate during growth and after injury to maintain glucose homeostasis. Here, we report that β cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to α cells. We identified the lineage determination gene aristaless-related homeobox (Arx), as methylated and repressed in β cells, and hypomethylated and expressed in α cells and Dnmt1-deficient β cells. We show that the methylated region of the Arx locus in β cells was bound by methyl-binding protein MeCP2, which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of α cell lineage determination genes to maintain pancreatic β cell identity.     

Beneficial effects of banana (<Emphasis Type="Italic">Musa</Emphasis> sp. var. elakki bale) flower and pseudostem on hyperglycemia and advanced glycation end-products (AGEs) in streptozotocin-induced diabetic rats

Diabetes is a chronic health problem and major cause of death in most of the countries. Diet management plays an important role in controlling diabetes and its complications along with insulin and drugs. We have examined the effect of banana (Musa sp. var. elakki bale) flower and pseudostem on hyperglycemia and advanced glycation end-products (AGEs) in streptozotocin-induced diabetic rats. Our results indicated that banana flower and pseudostem have low glycemic index and have a high content of dietary fiber and antioxidants. Diabetic symptoms like hyperglycemia, polyuria, polyphagia, polydipsia, urine sugar, and body weight were ameliorated in banana flower- and pseudostem-treated rats. Increased glomerular filtration rate in the diabetic group (5.1 ± 0.22 ml/min) was decreased in banana flower-fed (2.5 ± 0.37 ml/min) and pseudostem-fed (3.0 ± 0.45 ml/min) groups and were significant at P < 0.001 and P < 0.01, respectively. Fructosamine and AGEs formed during diabetes were inhibited in treated groups when compared with the diabetic group. The diabetic group showed 11.5 ± 0.64 μg of AGEs/mg protein in kidney, whereas, in banana flower- and pseudostem-fed groups, it was reduced to 9.21 ± 0.32 and 9.29 ± 0.24 μg/mg protein, respectively, and were significant at P < 0.01. These findings suggest that banana flower and pseudostem have anti-diabetic and anti-AGEs properties and are beneficial as food supplements for diabetics.     

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via,mitochondrial mediated caspase-dependent mechanism

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.     

Structural characterization of angiotensin I-converting enzyme in complex with a selenium analogue of captopril

Human somatic angiotensin I-converting enzyme (ACE), a zinc-dependent dipeptidyl carboxypeptidase, is central to the regulation of the renin-angiotensin aldosterone system. It is a well-known target for combating hypertension and related cardiovascular diseases. In a recent study by Bhuyan and Mugesh [Org. Biomol. Chem. (2011) 9, 1356-1365], it was shown that the selenium analogues of captopril (a well-known clinical inhibitor of ACE) not only inhibit ACE, but also protect against peroxynitrite-mediated nitration of peptides and proteins. Here, we report the crystal structures of human testis ACE (tACE) and a homologue of ACE, known as AnCE, from Drosophila melanogaster in complex with the most promising selenium analogue of captopril (SeCap) determined at 2.4 and 2.35 ? resolution, respectively. The inhibitor binds at the active site of tACE and AnCE in an analogous fashion to that observed for captopril and provide the first examples of a protein-selenolate interaction. These new structures of tACE-SeCap and AnCE-SeCap inhibitor complexes presented here provide important information for further exploration of zinc coordinating selenium-based ACE inhibitor pharmacophores with significant antioxidant activity.