Formation of Macromolecule Complex with Bacillus thuringiensis Cry1A Toxins and Chlorophyllide Binding 252-kDa Lipocalin-Like Protein Locating on Bombyx mori Midgut Membrane

P252, a 252-kDa Bombyx mori protein located on the larval midgut membrane, has been shown to bind strongly with Bacillus thuringiensis Cry1A toxins (Hossain et al. Appl Environ Microbiol 70:4604-4612, 2004). P252 was also shown to bind chlorophyllide (Chlide) to form red fluorescence-emitting complex Bm252RFP with significant antimicrobial activity (Pandian et al. Appl Environ Microbiol 74:1324-1331, 2008). In this article, we show that Cry1A toxin bound with Bm252RFP and Bm252RFP-Cry1A macrocomplex, with both antimicrobial and insecticidal activities, was formed. The insecticidal activity of Bm252RFP-Cry1Ab was reduced from an LD?? of 1.62 to 5.05 μg, but Bm252RFP-Cry1Aa and Bm252RFP-Cry1Ac did not show such reduction. On the other hand, the antimicrobial activity of Bm252RFP-Cry1Ab was shown to retain almost the same activity as Bm252RFP, while the other two complexes lost around 30% activity. The intensity of photo absorbance and fluorescence emission of Bm252RFP-Cry1Ab were significantly reduced compared to those of the other two complexes. Circular dichroism showed that the contents of Cry1Ab α-helix was significantly decreased in Bm252RFP-Cry1Ab but not in the other two toxins. These data suggested that the reduction of contents of α-helix in Cry1Ab affected the insecticidal activity of the macrocomplex but did not alter the antimicrobial moiety in the macrocomplex of Bm252RFP-Cry1Ab.     

Detection and imaging of superoxide in roots by an electron spin resonance spin-probe method

The detection, quantification, and imaging of short-lived reactive oxygen species, such as superoxide, in live biological specimens have always been challenging and controversial. Fluorescence-based methods are nonspecific, and electron spin resonance (ESR) spin-trapping methods require high probe concentrations and lack the capability for sufficient image resolution. In this work, a novel (to our knowledge), sensitive, small ESR imaging resonator was used together with a stable spin probe that specifically reacts with superoxide with a high reaction rate constant. This ESR spin-probe-based methodology was used to examine superoxide generated in a plant root as a result of an apical leaf injury. The results show that the spin probe rapidly permeated the plant's extracellular space. Upon injury of the plant tissue, superoxide was produced and the ESR signal decreased rapidly in the injured parts as well as in the distal part of the root. This is attributed to superoxide production and thus provides a means of quantifying the level of superoxide in the plant. The spin probe's narrow single-line ESR spectrum, together with the sensitive imaging resonator, facilitates the quantitative measurement of superoxide in small biological samples, such as the plant's root, as well as one-dimensional imaging along the length of the root. This type of methodology can be used to resolve many questions involving the production of apoplastic superoxide in plant biology.     

Production of biodiesel from mixed waste vegetable oil using an aluminium hydrogen sulphate as a heterogeneous acid catalyst

Al(HSO₄)₃ heterogeneous acid catalyst was prepared by the sulfonation of anhydrous AlCl₃. This catalyst was employed to catalyze transesterification reaction to synthesis methyl ester when a mixed waste vegetable oil was used as feedstock. The physical and chemical properties of aluminum hydrogen sulphate catalyst were characterized by scanning electron microscopy (SEM) measurements, energy dispersive X-ray (EDAX) analysis and titration method. The maximum conversion of triglyceride was achieved as 81 wt.% with 50 min reaction time at 220 °C, 16:1 molar ratio of methanol to oil and 0.5 wt.% of catalyst. The high catalytic activity and stability of this catalyst was related to its high acid site density (-OH, Brönsted acid sites), hydrophobicity that prevented the hydration of -OH group, hydrophilic functional groups (-SO₃H) that gave improved accessibility of methanol to the triglyceride. The fuel properties of methyl ester were analyzed. The fuel properties were found to be observed within the limits of ASTM D6751.     

AP-APSE dpol intein: A novel family A DNA polymerase intein domain

Inteins are "protein introns" that remove themselves from their host proteins through an autocatalytic protein-splicing. After their discovery, inteins have been quickly identified in organisms from all three kingdoms of life - eucarya, bacteria and archaea, but their distribution is sporadic. Here we report the identification and bioinformatics characterization of intein in DNA polymerase A gene of bacteriophage APSE (Acyrthosiphon pisum Secondary Endosymbiont bacteriophage) infecting the Aphid secondary endosymbionts of eukaryotic insects such as Acyrthosiphon pisum, Uroleucon rudbeckiae. The insertion site of intein within APSE family A DNA polymerase extein was identified to be dpola. Hence we propose this as a unique intein of family A DNA polymerase (dpola insertion site) and only reported intein in podoviridae family.     

Aniracetam Does Not Alter Cognitive and Affective Behavior in Adult C57BL/6J Mice

There is a growing community of individuals who self-administer the nootropic aniracetam for its purported cognitive enhancing effects. Aniracetam is believed to be therapeutically useful for enhancing cognition, alleviating anxiety, and treating various neurodegenerative conditions. Physiologically, aniracetam enhances both glutamatergic neurotransmission and long-term potentiation. Previous studies of aniracetam have demonstrated the cognition-restoring effects of acute administration in different models of disease. No previous studies have explored the effects of aniracetam in healthy subjects. We investigated whether daily 50 mg/kg oral administration improves cognitive performance in naïve C57BL/6J mice in a variety of aspects of cognitive behavior. We measured spatial learning in the Morris water maze test; associative learning in the fear conditioning test; motor learning in the accelerating rotarod test; and odor discrimination. We also measured locomotion in the open field test, anxiety through the elevated plus maze test and by measuring time in the center of the open field test. We measured repetitive behavior through the marble burying test. We detected no significant differences between the naive, placebo, and experimental groups across all measures. Despite several studies demonstrating efficacy in impaired subjects, our findings suggest that aniracetam does not alter behavior in normal healthy mice. This study is timely in light of the growing community of healthy humans self-administering nootropic drugs.     

Discovery of novel methanone derivatives acting as antimycobacterial agents

A series of pyrazoline derivatives were synthesized and in vitro activity against Mycobacterium tuberculosis H37Rv was carried out. Among the synthesized compounds, compounds (4d) and (4f) 4-aminophenyl-3-(3,4-dimethoxyphenyl)-6,7-dimethoxy-2,3,3a,4-tetrahydroindeno[1,2-c]pyrazol-2-ylmethanone and 4-aminophenyl-6,7-dimethoxy-3-phenyl-2,3,3a,4-tetrahydroindeno[1,2-c]pyrazol-2-ylmethanone were found to be the most active agent against M. tuberculosis H37Rv with a minimum inhibitory concentration of 10 μg/mL.     

Changes in Caenorhabditis elegans exposed to Vibrio parahaemolyticus

Vibrio parahaemolyticus, which owes its origin to the marine environment, is considered as one of the most common causes of infectious diarrhea worldwide. The present study investigated the pathogenicity of V. parahaemolyticus against the model organism, Caenorhabditis elegans. Infection in the host was localized with GFP-tagged V. parahaemolyticus using confocal laser scanning microscopy. The times required for causing infection, bacterial load in intestine, chemotactic response, and alteration in pharyngeal pumping were analyzed in the host system. In addition, the regulation of innate immune-related genes, lys-7, clec- 60, and clec-87, was analyzed using real-time PCR. The role of immune-responsible pmk-1 was studied using mutant strains. The pathogenicity of environmental strain CM2 isolated from the Gulf of Mannar, India was compared with that of a reference strain obtained from ATCC. The pathogen infected animals appeared to ward off infection by upregulating candidate antimicrobial genes for a few hours after the exposure, before succumbing to the pathogen. For the first time, the pathogenicity of V. parahaemolyticus at both the physiological and molecular levels has been studied in detail using the model organism C. elegans.     

Depletion of 14-3-3 protein exacerbates cardiac oxidative stress, inflammation and remodeling process via modulation of MAPK/NF-?B signaling pathways after streptozotocin-induced diabetes mellitus

Diabetic cardiomyopathy is associated with increased oxidative stress and inflammation. Mammalian 14-3-3 proteins are dimeric phosphoserine-binding proteins that participate in signal transduction and regulate several aspects of cellular biochemistry. The aim of the study presented here was to clarify the role of 14-3-3 protein in the mitogen activated protein kinase (MAPK) and nuclear factor-kB (NF-κB) signaling pathway after experimental diabetes by using transgenic mice with cardiac-specific expression of a dominant-negative 14-3-3 protein mutant (DN 14-3-3). Significant p-p38 MAPK activation in DN 14-3-3 mice compared to wild type mice (WT) after diabetes induction and with a corresponding up regulation of its downstream effectors, p-MAPK activated protein kinase 2 (MAPKAPK-2). Marked increases in cardiac hypertrophy, fibrosis and inflammation were observed with a corresponding up-regulation of atrial natriuretic peptide, osteopontin, connective tissue growth factor, tumor necrosis factor α, interleukin (IL)-1β, IL-6 and cellular adhesion molecules. Moreover, reactive oxygen species, left ventricular expression of NADPH oxidase subunits, p22 phox, p67 phox, and Nox4, and lipid peroxidation levels were significantly increased in diabetic DN 14-3-3mice compared to diabetic WT mice. Furthermore, myocardial NF-κB activation, inhibitor of kappa B-α degradation and mRNA expression of proinflammatory cytokines were significantly increased in DN 14-3-3 mice compared to WT mice after diabetes induction. In conclusion, our data suggests that depletion of 14-3-3 protein induces cardiac oxidative stress, inflammation and remodeling after experimental diabetes induction mediated through p38 MAPK, MAPKAPK-2 and NF-κB signaling.     

Purification of phosphofructokinase from rabbit skeletal muscle based on its ability to aggregate in absence of its effectors

Rabbit muscle phosphofructokinase was purified to homogeneity based on its property to form large aggregates with time at high concentration of its protein in absence of its effectors. The method involves no heat step or treatment with organic solvent or any ion-exchange columns. The enzyme thus prepared, however, exhibits the same kinetic properties as the enzyme purified by more drastic methods.