Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.     

Development of acetylcholinesterase silica sol-gel immobilized biosensor--an application towards oxydemeton methyl detection

An amperometric silica sol-gel film immobilized biosensor doped with acetylcholinesterase was fabricated in the laboratory finding application in organophosphate detection based on enzyme inhibition. The substrate used was acetylthiocholine chloride and thiocholine released from the enzymatic hydrolysis was electrochemically oxidized giving larger anodic current at 0.5-0.6 V (versus Ag/AgCl reference). The dependencies of the current response on pH, enzyme loading and thermal stability of the developed biosensor were evaluated. The analytical performance of enzyme electrode towards substrate and inhibitor was investigated. Oxydemeton methyl was taken as a model compound for the inhibition studies. Linear calibration for oxydemeton methyl was obtained in the range of 2-200 ppb under the optimized conditions following an incubation time of 20 min. Treatment of the inhibited enzyme with pyridine-2-aldehyde restored 92% of its original activity. The sensor stored at -20 degrees C had a good storage and operational stability retaining 85% of its original activity for 60 successive measurements.     

Analysis of the germination of spores of Bacillus subtilis with temperature sensitive spo mutations in the spoVA operon

A Bacillus subtilis strain with a base substitution in the ribosome-binding site of spoVAC was temperature sensitive (ts) in sporulation and spores prepared at the permissive temperature were ts in L-alanine-triggered germination, but not in germination with Ca2+-dipicolinic acid (DPA) or dodecylamine. Spores of a ts spo mutant with a missense mutation in the spoVAC coding region were not ts for germination with l-alanine, dodecylamine or Ca2+-DPA. These findings are discussed in light of the proposal that SpoVA proteins are involved not only in DPA uptake during sporulation, but also in DPA release during nutrient-mediated spore germination.     

Geranyl chalcone derivatives with antifungal and radical scavenging properties from the leaves of Artocarpus nobilis

Antifungal activity guided fractionation of the n-butanol extract from the methanol extract of the leaves of Artocarpus nobilis furnished 2',4',4-trihydroxy-3'-geranylchalcone (1), 2 ',4',4-trihydroxy-3'-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]chalcone (2), 2',4',4-trihydroxy-3'-[2-hydroxy-7-methyl-3-methylene-6-octaenyl]chalcone (3), 2',3,4,4'-tetrahydroxy-3'-geranylchalcone (4), 2',3,4,4'-tetrahydroxy-3'-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]chalcone (5). The chalcones 3 and 5 are new natural products whereas 1 and 2 are reported first time from the family Moraceae. All these compounds showed good fungicidal activity against Cladosporium cladosporioides and high radical scavenging activity towards the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical in TLC bio-autography method.     

Flavonoids and andrographolides from Andrographis paniculata

Two flavonoids, identified as 5,7,2',3'-tetramethoxyflavanone and 5-hydroxy-7,2',3'-trimethoxyflavone, as well as several other flavonoids, andrographolide diterpenoids, and polyphenols, were obtained from the phytochemical investigation of the whole plant of Andrographis paniculata, a well known medicinal plant. The structures of these compounds were established with the aid of spectroscopic methods, including analysis by 2D NMR spectroscopy.     

Synthesis, characterization, EXAFS investigation and antibacterial activities of new ruthenium(III) complexes containing tetradentate Schiff base

A series of new hexa-coordinated ruthenium(III) complexes of the type [Ru(X)(2-atmp-ba)(EPh3)] (where H2-2-atmp-ba=N,N'-bis(2-aminothiophenol)benzoylacetone; X=Cl or Br; E=P or As) have been prepared by reacting [RuX3(EPh3)3] (where X=Cl or Br; E=P or As) with tetradentate Schiff base ligand (H2-2-atmp-ba) in 1:1 molar ratio. The complexes have been characterized by elemental analyses, Infra red, electronic, electron paramagnetic resonance spectroscopy and cyclic voltammetry. In order to confirm the coordination and structure of the complexes extended X-ray absorption fine structure spectroscopy (EXAFS) studies have been carried out. Based on the above data, an octahedral structure has been confirmed for the complexes. The new complexes were also screened for their antibacterial properties.     

Morphogenesis of bacillus spore surfaces

Spores produced by bacilli are encased in a proteinaceous multilayered coat and, in some species (including Bacillus anthracis), further surrounded by a glycoprotein-containing exosporium. To characterize bacillus spore surface morphology and to identify proteins that direct formation of coat surface features, we used atomic-force microscopy (AFM) to image the surfaces of wild-type and mutant spores of Bacillus subtilis, as well as the spore surfaces of Bacillus cereus 569 and the Sterne strain of Bacillus anthracis. This analysis revealed that the coat surfaces in these strains are populated by a series of bumps ranging between 7 and 40 nm in diameter, depending on the species. Furthermore, a series of ridges encircled the spore, most of which were oriented along the long axis of the spore. The structures of these ridges differ sufficiently between species to permit species-specific identification. We propose that ridges are formed early in spore formation, when the spore volume likely decreases, and that when the spore swells during germination the ridges unfold. AFM analysis of a set of B. subtilis coat protein gene mutants revealed three coat proteins with roles in coat surface morphology: CotA, CotB, and CotE. Our data indicate novel roles for CotA and CotB in ridge pattern formation. Taken together, these results are consistent with the view that the coat is not inert. Rather, the coat is a dynamic structure that accommodates changes in spore volume.     

Constitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial pathogens by induction of a hypersensitive response

Hypersensitive response-assisting protein (HRAP) has been previously reported as an amphipathic plant protein isolated from sweet pepper that intensifies the harpin(Pss)-mediated hypersensitive response (HR). The hrap gene has no appreciable similarity to any other known sequences, and its activity can be rapidly induced by incompatible pathogen infection. To assess the function of the hrap gene in plant disease resistance, the CaMV 35S promoter was used to express sweet pepper hrap in transgenic tobacco. Compared with wild-type tobacco, transgenic tobacco plants exhibit more sensitivity to harpin(Pss) and show resistance to virulent pathogens (Pseudomonas syringae pv. tabaci and Erwinia carotovora subsp. carotovora). This disease resistance of transgenic tobacco does not originate from a constitutive HR, because endogenous level of salicylic acid and hsr203J mRNA showed similarities in transgenic and wildtype tobacco under noninfected conditions. However, following a virulent pathogen infection in hrap transgenic tobacco, hsr203J was rapidly induced and a micro-HR necrosis was visualized by trypan blue staining in the infiltration area. Consequently, we suggest that the disease resistance of transgenic plants may result from the induction of a HR by a virulent pathogen infection.     

Total synthesis and evaluation of lamellarin alpha 20-Sulfate analogues

In order to explore the influence of sulfate groups on the bioactivity profiles of marine alkaloids of the lamellarin class, three such alkaloids, lamellarin alpha, lamellarin alpha 13,20-disulfate and lamellarin H, were synthesized and their activities against HIV-1 integrase and cancer cell lines were compared with those of lamellarin alpha 20-sulfate, which is a selective inhibitor of HIV-1 integrase. Lamellarin alpha does not inhibit HIV-1 integrase but shows moderate cytotoxicity with good cell line selectivity. Lamellarin alpha 13,20-disulfate is a moderate inhibitor of both HIV-1 integrase and cancer cell lines. Lamellarin H is a more potent inhibitor of HIV-1 integrase but lacked the specificity required to be medicinally useful.