In vivo humoral and cellular reactions, and fate of injected bacteria Aeromonas hydrophila in freshwater prawn Macrobrachium rosenbergii

Live non-opsonized and opsonized Aeromonas hydrophila were injected into juveniles of freshwater prawn Macrobrachium rosenbergii to study the cells involved in phagocytosis, distribution of bacteria, cellular reactions and clearance of both forms of bacteria from the system. The bacteria were rapidly distributed to various tissues viz., gills, heart, hepatopancreas within 1h, and the tissues revealed haemocytic nodule formation after 3 h of injection. There was rapid clearance of both the forms of bacteria from the circulation. However, clearance efficiency was significantly (P < 0.05) faster in the case of opsonized bacteria at 12 h after injection. Similarly, the nodule formation, that was prominent in cardiac musculature, was rapidly eliminated from the tissues of the group injected with opsonized bacteria as compared to non-opsonized bacteria injected group, thus confirming the existence of opsonic factors in haemolymph of this prawn. In another experiment, various dose levels of bacteria were injected intramuscularly into prawns and haemolymph was collected after 1, 6, 24, 72 h and 7 days of injection to study various immune parameters. Although, no major alterations in the total and differential haemocyte counts were observed in bacteria injected prawns compared to control, there was a significant decline in phenoloxidase activity in the highest dose bacteria injected group at the earlier phase and a rise in agglutinin levels at the later phase of the experimental period in the higher dose groups.     

Direct comparison of four bacterial source tracking methods and use of composite data sets

AIMS: Four bacterial source tracking (BST) methods, enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR), automated ribotyping using HindIII, Kirby-Bauer antibiotic resistance analysis (KB-ARA) and pulsed-field gel electrophoresis (PFGE) were directly compared using the same collection of Escherichia coli isolates. The data sets from each BST method and from composite methods were compared for library accuracy and their ability to identify water isolates. METHODS AND RESULTS: Potential sources of faecal pollution were identified by watershed sanitary surveys. Domestic sewage and faecal samples from pets, cattle, avian livestock, other nonavian livestock, avian wildlife and nonavian wildlife sources were collected for isolation of E. coli. A total of 2275 E. coli isolates from 813 source samples were screened using ERIC-PCR to exclude clones and to maximize library diversity, resulting in 883 isolates from 745 samples selected for the library. The selected isolates were further analysed using automated ribotyping with HindIII, KB-ARA and PFGE. A total of 555 E. coli isolates obtained from 412 water samples were analysed by the four BST methods. A composite data set of the four BST methods gave the highest rates of correct classification (RCCs) with the fewest unidentified isolates than any single method alone. RCCs for the four-method composite data set and a seven-way split of source classes ranged from 22% for avian livestock to 83% for domestic sewage. Two-method composite data sets were also found to be better than individual methods, having RCCs similar to the four-method composite and identification of the same major sources of faecal pollution. CONCLUSIONS: The use of BST composite data sets may be more beneficial than the use of single methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comprehensive comparisons using composite data from several BST methods. While the four-method approach provided the most desirable BST results, the use of two-method composite data sets may yield comparable BST results while providing for cost, labour and time savings.     

Influence of cholesterol on cancer progression and therapy

Cholesterol is a fundamental molecule necessary for the maintenance of cell structure and is vital to various normal biological functions. It is a key factor in lifestyle-related diseases including obesity, diabetes, cardiovascular disease, and cancer. Owing to its altered serum chemistry status under pathological states, it is now being investigated to unravel the mechanism by which it triggers various health complications. Numerous clinical studies in cancer patients indicate an alteration in blood cholesterol level (either decreased or increased) in comparison to normal healthy individuals. This article elaborates on our understanding as to how cholesterol is being hijacked in the malignancy for the development, survival, stemness, progression, and metastasis of cancerous cells. Also, it provides a glimpse of how cholesterol derived entities, alters the signaling pathway towards their advantage. Moreover, deregulation of the cholesterol metabolism pathway has been often reported to hamper various treatment strategies in different cancer. In this context, attempts have been made to bring forth its relevance in being targeted, in pre-clinical and clinical studies for various treatment modalities. Thus, understanding the role of cholesterol and deciphering associated molecular mechanisms in cancer progression and therapy are of relevance towards improvement in the management of various cancers.     

The use of coconut wastes for the production ofBacillus thuringiensis var.israelensis

Summary Good yields of crystal/spores and larvicidal activity were obtained whenBacillus thuringiensis var.israelensis was grown in coconut water and endosperm extracts. Coconut water contained 2.2 mg protein/ml, while the endosperm extracts contained 1–4 mg protein/ml extract. The carbohydrate content of the media ranged from 6–38.4 mg/ml.

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Resumen Utilización de desechos de coco para la producción de Bacillus thuringiensisvar. israelensis Agua de coco y extractos de endosperma se han utilizado para el cultivo deBacillus thuringiensis var.israelensis obteniendo un buen rendimiento en cristales/esporas y una buena actividad larvicida. El agua de coco contenía 2.2 mg de proteína/ml y los extractos de endosperma 1–4 mg/protei'na/ml de extracto. El contenido en hidratos de carbono de los medios utilizados varió entre 6–38.4 mg/ml.

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Résumé Utilisation des déchets de noix de coco pour la production de Bacillus thurigiensisvar. israelensisDe bons rendements en cristaux, spores et activité larvicide ont été obtenus en cultivantBacillus thurigiensis var.israelensis dans l'eau et les extraits endospermiques de noix de coco. L'eau des noix contient 2,2 mg de protéines/ml et les extraits d'endosperme 1–4 mg. La teneur des milieux en carbohydrates était comprise entre 6–38,4 mg/ml.

     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

A catalytically inactive form of protein kinase C-associated kinase/receptor interacting protein 4, a protein kinase C beta-associated kinase that mediates NF-kappa B activation,interferes with early B cell development

Protein kinase C-associated kinase (PKK)/receptor interacting protein 4 (RIP4) is a protein kinase C (PKC) beta-associated kinase that links PKC to NF-kappaB activation. The kinase domain of PKK is similar to that of RIP, RIP2, and RIP3. We show in this study that PKK is expressed early during lymphocyte development and can be detected in common lymphoid progenitor cells. Targeting of a catalytically inactive version of PKK to lymphoid cells resulted in a marked impairment in pro-B cell generation in the bone marrow. Although peripheral B cell numbers were markedly reduced, differentiation into follicular and marginal zone B cells was not defective in these mice. B-1a and B-1b B cells could not be detected in these mice, but this might be a reflection of the overall defect in B cell production observed in these animals. In keeping with a possible link to PKCbeta, peripheral B cells in these mice exhibit a defect in anti-IgM-mediated proliferation. These studies suggest that PKK may be required early in B cell development and for BCR-mediated B cell proliferation.     

Novel drug designing approach for dual inhibitors as anti-inflammatory agents: implication of pyridine template

Compounds incorporating thiophene moiety, a pi excess five membered heterocycle, have attracted a great deal of research interest, owing to the therapeutic utility of the template as useful drug molecular scaffolding. We report the synthesis and pharmacological evaluation of thiophenes substituted with 4-methanesulfonyl benzoyl moiety at the fifth position of the ring, as possible anti-inflammatory lead candidates. The aryl sulfonyl methyl thiophene analogs AP29, AP82, and AP37, when screened for anti-inflammatory activity in carrageenin induced rat paw edema, an acute in vivo model, exhibited moderate to good activity at a dose level of 100 mg/kg body weight P.o compared to Ibuprofen. In a five day formalin induced rat paw edema, a chronic in vivo anti-inflammatory model, candidates AP29, AP82, and AP37 inhibited the disease progression by 53%, 34%, and 65%, respectively on the fifth day, at a dose level of 100 mg/kg body weight P.o compared to Rofecoxib, Ibuprofen, and Dexamethasone at therapeutic doses which gave a protection of 53.8%, 81.5%, and 81.5%, respectively. The replacement of the 4-methanesulfonyl benzoyl moiety in AP82 with the pyridine template, 3,5-dimethyl-4-methoxy-2-pyridyl function, gave rise to AP84, which was less active in the acute model, but gave 54% and 75% protection both during the first day and fifth day, respectively, in the chronic model. A dual mechanism of action is proposed for AP84, a non-steroidal drug which has exhibited remarkable activity when compared to the steroid dexamethasone. These results open up new avenues in designing novel anti-inflammatory drugs as dual inhibitors with the incorporation of a pyridine template as part of the pharmacophore.     

Enzymatic digestion--a safe and rapid technique for individual separation of Macrobrachium rosenbergii embryos for cryopreservation studies

A new, safe, and rapid technique for the individual separation of the embryos of giant freshwater prawn Macrobrachium rosenbergii de Man is described. Two protease enzymes, e.g., trypsin and collagenase were used. Embryos in the advanced stage of development (gray embryos with eyespot and heart beat) were selected for the study. Treatment with collagenase and trypsin at respective concentrations of 0.05 and 0.25% for 30 min resulted in 100% separation of 35-40 mg of embryonic mass (approximately 180 embryos). A chelating agent, EDTA (ethylenediaminetetraacetic acid disodium salt: dihydrate) at 400 mg l(-1) enhanced the activity of trypsin. Trypsin and collagenase, when used together, were found to act synergistically. The separated embryos revealed no morphological injury when observed under the microscope. Further, in vitro hatching of the separated embryos was successful indicating that the present technique is safe and effective in achieving individual separation of prawn embryos.