Differences in calmodulin and calmodulin-binding proteins in phasic and tonic smooth muscles

To determine whether densities ofcalmodulin (CaM) and CaM-binding proteins are related to phasic andtonic behavior of smooth muscles, we quantified these proteins in theopossum esophageal body (EB) and lower esophageal sphincter (LES),which represent phasic and tonic smooth muscles, respectively. Gelelectrophoresis, immunoprecipitation, Western blot, and hemagglutininepitope-tagged CaM (HA-CaM) overlay assay with quantitative scanningdensitometry and phosphorylation measurements were used. Total proteincontent in the two smooth muscles was similar (~30 mg protein/gfrozen tissue). Total tissue concentration of CaM was significantly(25%) higher in EB than in LES (P < 0.05).HA-CaM-binding proteins were qualitatively similar in LES and EBextracts. Myosin, myristoylated alanine-rich C kinase substrateprotein, Ca²⁺/CaM kinase II, and calponin contents werealso similar in the two muscles. However, content and total activity ofmyosin light chain kinase (MLCK) and content of caldesmon (CaD) werethree- to fourfold higher in EB than in LES. Increased CaM and MLCKcontent may allow for a wide range of contractile force varying fromcomplete relaxation in the basal state to a large-amplitude,high-velocity contraction in EB phasic muscle. Increased content ofCaD, which provides a braking mechanism on contraction, may furthercontribute to the phasic contractile behavior. In contrast, low CaM,MLCK, and CaD content may be responsible for a small range ofcontractile force seen in tonic muscle of LES.

     

Toxicity and disruption of midgut physiology in larvae of the European corn borer,Ostrinia nubilalis,by anion transporter blockers

In this study, four blockers of anion transporters (ATs) belonging to four different classes of organic acids, including DIDS (4, 4'‐diisothiocyanatostilbene‐2, 2'‐ disulfonic acid; a stilbene disulfonic acid), NPPB [(5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid; an anthranilic acid)], 9‐AC (anthracene‐9‐carboxylic acid; an aromatic carboxylic acid), and IAA‐94 (indanyloxy acetic acid; an indanyloxy alkanoic acid), were tested for their toxicity against the European corn borer (ECB), Ostrinia nubilalis. All the AT blockers inhibited the growth of larvae, increased the developmental time, and decreased survival compared to controls, when second‐instar ECB larvae were fed for seven days on treated diet. In general, DIDS and NPPB were the most active compounds, with the rank order of activity being DIDS>NPPB>IAA‐94>9‐AC. All the AT blockers decreased the midgut alkalinity in fifth‐instar larvae when fed for 3 h on treated diet. Effective concentrations required for 50% decrease in midgut alkalinity (EC₅₀) ranged between 29.1 and 41.2 ppm and the rank order of activity was NPPB>DIDS>IAA‐94>9‐AC. Similarly, all the tested AT blockers inhibited ³⁶Cl^? uptake from the midgut lumen in fifth‐instar larvae when fed for 3 h on treated diet. Concentrations required for 50% inhibition of ³⁶Cl^? uptake (IC₅₀) ranged between 7.4 and 11.0 ppm and the rank order of activity was DIDS>NPPB>9‐AC >IAA‐94. Modest to highly strong positive correlations observed among growth, midgut alkalinity, and midgut Cl^? ion transport in AT blocker–fed larvae suggested that these effects are causally related to each other. Finally, AT blockers have the potential to become good candidates for development of insecticides with a unique mode of action. © 2009 Wiley Periodicals, Inc.     

Performance of an anaerobic baffled reactor and hybrid constructed wetland treating high-strength wastewater in Nepal—A model for DEWATS

Centralized wastewater treatment systems require sophisticated technologies and skilled manpower for their operation and maintenance (O&M). These systems have huge construction as well as O&M costs. Therefore, a Decentralized Wastewater Treatment System (DEWATS) rather than a centralized system might be especially beneficial in developing countries. A model for DEWATS is developed in Nepal with Anaerobic Baffled Reactor (ABR) and hybrid Constructed Wetland (CW). The DEWATS treats high-strength wastewater from 80 households (400 PE). This paper summarizes the performance of the DEWATS from July 2006 to August 2007 in the removal efficiencies of TSS, BOD₅, COD, NH₄–N, TP and FC. The ABR is very effective in the removal of organic pollutants and could achieve TSS removal up to 91%, BOD₅ up to 78% and COD up to 77%. The average removal efficiencies of the DEWATS is 96% TSS, 90% BOD₅, 90% COD, 70% NH₄–N, 26% TP and 98% FC.     

Morphological, Biochemical and Molecular Characterization of Trichoderma harzianum Isolates for their Efficacy as Biocontrol Agents

The random amplified polymorphic DNA (RAPD) procedure was used to examine the genetic variability among 8 isolates of Trichoderma harzianum , and their ability to antagonize Sclerotium rolfsii using a dual culture assay was correlated with RAPD profiles. Eight oligodeoxynucleotide primers were selected for the RAPD assays, which resulted in 86 bands for 8 isolates of T . harzianum . The data were entered into a binary matrix and a similarity matrix was constructed using the DICE similarity (SD) index. An unweighted pair grouping mathematical averaging (UPGMA) cluster based on SD values was generated using the NTSYS computer program. A mean coefficient of similarity obtained for pairwise comparisons was c. 30% and it showed that the variability among the isolates of T. harzianum was very high. Using the dual culture method in antagonism experiments, the T. harzianum isolates were classified in to antagonism classes. Further, T. harzianum isolates were screened for chitinase and β-1,3-glucanase activity. RAPD was efficient in demonstrating the high intraspecific genetic variation among isolates. The dendrogram did not show the grouping of isolates by their level of antagonism. Relationship among polymorphism existent, the aggressiveness and the origin of isolates were not found.     

Select pyrimidinones inhibit the propagation of the malarial parasite,Plasmodium falciparum

Plasmodium falciparum, the Apicomplexan parasite that is responsible for the most lethal forms of human malaria, is exposed to radically different environments and stress factors during its complex lifecycle. In any organism, Hsp70 chaperones are typically associated with tolerance to stress. We therefore reasoned that inhibition of P. falciparum Hsp70 chaperones would adversely affect parasite homeostasis. To test this hypothesis, we measured whether pyrimidinone-amides, a new class of Hsp70 modulators, could inhibit the replication of the pathogenic P. falciparum stages in human red blood cells. Nine compounds with IC₅₀ values from 30 nM to 1.6 μM were identified. Each compound also altered the ATPase activity of purified P. falciparum Hsp70 in single-turnover assays, although higher concentrations of agents were required than was necessary to inhibit P. falciparum replication. Varying effects of these compounds on Hsp70s from other organisms were also observed. Together, our data indicate that pyrimidinone-amides constitute a novel class of anti-malarial agents.     

Specificities of Calreticulin Transacetylase to acetoxy derivatives of 3-alkyl-4-methylcoumarins: Effect on the activation of nitric oxide synthase

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and modulates their biological activities. CRTAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (DAMC). We have studied earlier, the influence of acetoxy groups on the benzenoid ring, the effect of reduction of double bond at C-3 and C-4 position, the effect of methyl/phenyl group at C-4, and the influence of position of carbonyl group with respect to oxygen heteroatom in the benzopyran nucleus, for the catalytic activity of CRTAase. In this communication, we have extended our previous work; wherein we studied the influence of an alkyl group (ethyl, hexyl and decyl) at the C-3 position of the acetoxy coumarins on the CRTAase activity. The substitution at C-3 position of coumarin nucleus resulted in the reduction of CRTAase activity and related effects. Accordingly the formation of NO in platelets by C-3 alkyl substituted acetoxy coumarins was found to be much less compared to the unsubstituted analogs. In addition the alkyl substitution at C-3 position exhibited the tendency to form radicals other than NO.     

A model for the evaluation of domain based classification of GPCR

G-Protein Coupled Receptors (GPCR) are the largest family of membrane bound receptor and plays a vital role in various biological processes with their amenability to drug intervention. They are the spotlight for the pharmaceutical industry. Experimental methods are both time consuming and expensive so there is need to develop a computational approach for classification to expedite the drug discovery process. In the present study domain based classification model has been developed by employing and evaluating various machine learning approaches like Bagging, J48, Bayes net, and Naive Bayes. Various softwares are available for predicting domains. The result and accuracy of output for the same input varies for these software''s. Thus, there is dilemma in choosing any one of it. To address this problem, a simulation model has been developed using well known five softwares for domain prediction to explore the best predicted result with maximum accuracy. The classifier is developed for classification up to 3 levels for class A. An accuracy of 98.59% by Naïve Bayes for level I, 92.07% by J48 for level II and 82.14% by Bagging for level III has been achieved.     

Functional and Probiotic Attributes of an Indigenous Isolate of Lactobacillus plantarum

Background

Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.

Methods and Findings

An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.

Conclusion

The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.