Cannabinoid CB<Subscript>2</Subscript> Receptor-Mediated Anti-nociception in Models of Acute and Chronic Pain

The endocannabinoid system consists of cannabinoid CB₁ and CB₂ receptors, endogenous ligands and their synthesising/metabolising enzymes. Cannabinoid receptors are present at key sites involved in the relay and modulation of nociceptive information. The analgesic effects of cannabinoids have been well documented. The usefulness of nonselective cannabinoid agonists can, however, be limited by psychoactive side effects associated with activation of CB₁ receptors. Following the recent evidence for CB₂ receptors existing in the nervous system and reports of their up-regulation in chronic pain states and neurodegenerative diseases, much research is now aimed at shedding light on the role of the CB₂ receptor in human disease. Recent studies have demonstrated anti-nociceptive effects of selective CB₂ receptor agonists in animal models of pain in the absence of CNS side effects. This review focuses on the analgesic potential of CB₂ receptor agonists for inflammatory, post-operative and neuropathic pain states and discusses their possible sites and mechanisms of action. Jhaveri and Sagar joint first author.     

Decolorisation and detoxification of Direct Blue-15 by a bacterial consortium

Studies were carried out on decolorisation and biotransformation of the dye Direct Blue-15 into 3,3'-dimethoxybenzidine (O'-dianisidine) and a sulphonated derivative by a five-member bacterial consortium. Chromatographic studies revealed further complete biodegradation of 3,3'-dimethoxybenzidine coupled with release of ammonia, but the recalcitrant sulphonated derivative persisted. The microorganisms identified in the mixed consortium by 16S rDNA sequence analysis were Alcaligenes faecalis, Sphingomonas sp. EBD, Bacillus subtilis, Bacillus thuringiensis and Enterobacter cancerogenus. The cytotoxicity data showed a significant reduction in the toxicity (P<0.001) of the degraded dye as evidenced from the number of viable human polymorphonuclear leukocyte cells present.     

Role of Pterocarpus santalinus against mitochondrial dysfunction and membrane lipid changes induced by ulcerogens in rat gastric mucosa

Free radicals produced by ulcerogenic agents affect the TCA cycle enzymes located in the outer membrane of the mitochondria. Upon induction with ulcerogens, peroxidation of membrane lipids bring about alterations in the mitochondrial enzyme activity. This indicates an increase in the permeability levels of the mitochondrial membrane. The ability of PSE to scavenge the reactive oxygen species results in restoration of activities of TCA cycle enzymes. NSAIDs interfere with the mitochondrial beta-oxidation of fatty acids in vitro and in vivo, resulting in uncoupling of mitochondrial oxidative phosphorylation process. This usually results in diminished cellular ATP production. The recovery of gastric mucosal barrier function through maintenance of energy metabolism results in maintenance of ATP levels, as observed in this study upon treatment with PSE. Membrane integrity altered by peroxidation is known to have a modified fatty acid composition, a disruption of permeability, a decrease in electrical resistance, and increase in flip-flopping between monolayers and inactivated cross-linked proteins. The severe depletion of arachidonic acid in ulcer induced groups was prevented upon treatment with PSE. The acid inhibitory property of the herbal extract enables the maintenance of GL activity upon treatment with PSE. The ability to prevent membrane peroxidation has been traced to the presence of active constituents in the PSE. In essence, PSE has been found to prevent mitochondrial dysfunction, provide mitochondrial cell integrity, through the maintenance of lipid bilayer by its ability to provide a hydrophobic character to the gastric mucosa, further indicating its ability to reverse the action of NSAIDs and mast cell degranulators in gastric mucosa.     

Is low-frequency distribution of TGF-beta genotype associated with increased risk for end-stage renal disease?

End-stage renal disease has been associated with an inflammatory state. TGF-beta plays a critical role in antiinflammation counteracting inflammatory cytokines, wound healing, and tissue repair. We, therefore, speculated the protective role of TGF-beta in renal inflammation rather than inducing fibrosis. Three polymorphisms of TGF-beta (713-8delC), i.e., C deletion in intron sequence 8 base prior to exon-5 by PCR-RFLP and codon-10, Leu/Pro, and codon-25, Arg/Pro by Amplification Refractory Mutation System (ARMS-PCR) techniques were genotyped in 228 end-stage renal disease (ESRD) patients and 180 controls. Linkage disequilibrium (LD) and haplotype analysis was performed by Arlequin software. Our data showed positive association between codon-10 polymorphism and ESRD risk (P < 0.001; OR 4.845, 95% CI 2.57-9.11 for Pro/Pro). However, genotype frequencies were comparable in patients and controls for 713-8delC, while in the case of codon-25, a trend of higher frequency of Pro/Pro genotype (16.2% versus 10.0%) was observed but the P-value did not reach significant (P = 0.187). Significant association of codon-10 Pro/Pro was observed in patients with glomerulonephritis (P = 0.001; OR 4.138, 95%CI 2.1-8.13). LD was found significant between codon-10 and 25 (P = 0.021). Haplotype "Pro-Pro" showed 1.8-fold higher risk for ESRD (p = 0.003; OR = 1.867, 95%CI = 1.229-2.838). A combined analysis of the effect of TGF-beta (codon-10) with C-deletion and codon-25 showed significant difference for TGF-beta(10)-TGF-beta(C-del) (P = 0.010). In conclusion, the present study suggests that low-producing genotype (Pro/Pro) of TGF-beta (codon-10) polymorphism is associated with ESRD. Haplotype analysis further suggested that "Pro-Pro" (low producer) is associated with higher risk for ESRD. Thus, high-producing genotype of TGF-beta may be beneficial and may play a potential role in the resolution of renal inflammation.     

Possible involvement of extracellular polymeric substrates of Antarctic cyanobacterium Nostoc sp. strain SO-36 in adaptation to harsh environments

Journal of Plant Research - Cyanobacteria are some of the primary producers in extremely cold biospheres such as the Arctic, Antarctic, and vast ice sheets. Many genera of cyanobacteria are...     

Abiotic Elicitors Influence Antioxidative Enzyme Activities and Shelf Life of Carrot During Storage Under Refrigerated Conditions

The objective of the present study was to investigate the effectiveness of the post-harvest treatments of abiotic elicitors, that is, calcium chloride (CaCl₂) and salicylic acid (SA) on physicochemical and biochemical parameters in relation to activities of antioxidative enzymes in carrot to enhance shelf life. Carrot of variety Punjab Carrot Red was harvested, washed, surface dried and treated with CaCl₂ (1, 1.5 and 2%) or SA (1, 1.5 and 2 mM) for 5 min, while distilled water was used as the control. Treated as well as untreated carrots were placed in open trays and stored under refrigerated (5 ± 1 °C, 90% RH) conditions for 63 days. Treatment of carrots with CaCl₂ and SA showed a reduction in changes in physiological weight, color, total soluble solids, ascorbic acid, titratable acidity, total phenolics, carotenoids, antioxidant activity and TBA reactive compound as compared to untreated samples. Higher activities of antioxidative enzymes, that is, catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), peroxidase (POD), dehydro-ascorbate-reductase (DHAR) and monodehydro-ascorbate-reductase (MDHAR), were found in treated carrots as compared to untreated carrots during the whole storage period. SA treatment exhibited more usefulness in maintaining the quality of carrot than CaCl₂ treatment. Among all the treatments, 1.5 mM SA exhibited the highest antioxidative enzyme activities and slowest changes in biochemical quality of carrot during storage. Thus, 1.5 mM SA can be used to extend the shelf life of carrot during refrigerated storage.

     

Capture and display of antibodies secreted by hybridoma cells enables fluorescent on-cell screening

Hybridoma methods for monoclonal antibody (mAb) cloning are a mainstay of biomedical research, but they are hindered by the need to maintain hybridomas in oligoclonal pools during antibody screening. Here, we describe a system in which hybridomas specifically capture and display the mAbs they secrete: On-Cell mAb Screening (OCMS?). In OCMS?, mAbs displayed on the cell surface can be rapidly assayed for expression level and binding specificity using fluorescent antigens with high-content (image-based) methods or flow cytometry. OCMS? demonstrated specific mAb binding to poliovirus and rabies virus by forming a cell surface IgG “cap”, as a universal assay for anti-viral mAbs. We produced and characterized OCMS?-enabled hybridomas secreting mAbs that neutralize poliovirus and used fluorescence microscopy to identify and clone a human mAb specific for the human N-methyl-D-aspartate receptor. Lastly, we used OCMS? to assess expression and antigen binding of a recombinant mAb produced in 293T cells. As a novel method to physically associate mAbs with the hybridomas that secrete them, OCMS? overcomes a central challenge to hybridoma mAb screening and offers new paradigms for mAb discovery and production.     

Analysis of inter-/intra-E-plate repeatability in the real-time cell analyzer

Over the past decade, the real-time cell analyzer (RTCA) has provided a good tool to the cell-based in vitro assay. Unlike the traditional systems that label the target cells with luminescence, fluorescence, or light absorption, RTCA monitors cell properties using noninvasive and label-free impedance measuring. However, realization of the maximum value of RTCA for applications will require assurance of within-experiment repeatability, day-to-day repeatability, and robustness to variations in conditions that might occur from different experiments. In this article, the performance and variability of RTCA is evaluated and a novel repeatability index (RI) is proposed to analyze the intra-/inter-E-plate repeatability of RTCA. The repeatability assay involves six cell lines and two media (water [H₂O] and dimethyl sulfoxide [DMSO]). First, six cell lines are exposed to the media individually, and time-dependent cellular response curves characterized as a cell index (CI) are recorded by RTCA. Then, the variations along sampling time and among repeated tests are calculated and RI values are obtained. Finally, a discriminating standard is set up to evaluate the degree of repeatability. As opposed to the standardized methodologies, it is shown that the presented index can give the quantitative evaluation for repeatability of RTCA within E-plate and variation on different days.     

Opioid Receptor Function Is Regulated by Post-endocytic Peptide Processing

Most neuroendocrine peptides are generated in the secretory compartment by proteolysis of the precursors at classical cleavage sites consisting of basic residues by well studied endopeptidases belonging to the subtilisin superfamily. In contrast, a subset of bioactive peptides is generated by processing at non-classical cleavage sites that do not contain basic residues. Neither the peptidases responsible for non-classical cleavages nor the compartment involved in such processing has been well established. Members of the endothelin-converting enzyme (ECE) family are considered good candidate enzymes because they exhibit functional properties that are consistent with such a role. In this study we have explored a role for ECE2 in endocytic processing of δ opioid peptides and its effect on modulating δ opioid receptor function by using selective inhibitors of ECE2 that we had identified previously by homology modeling and virtual screening of a library of small molecules. We found that agonist treatment led to intracellular co-localization of ECE2 with δ opioid receptors. Furthermore, selective inhibitors of ECE2 and reagents that increase the pH of the acidic compartment impaired receptor recycling by protecting the endocytosed peptide from degradation. This, in turn, led to a substantial decrease in surface receptor signaling. Finally, we showed that treatment of primary neurons with the ECE2 inhibitor during recycling led to increased intracellular co-localization of the receptors and ECE2, which in turn led to decreased receptor recycling and signaling by the surface receptors. Together, these results support a role for differential modulation of opioid receptor signaling by post-endocytic processing of peptide agonists by ECE2.     

Dendrobium tamenglongense sp. nov. (Orchidaceae) from Manipur,India

Dendrobium tamenglongense R. Kishor, Y. N. Devi, H. B. Sharma, J. Tongbram & S. P. Vij sp. nov. from Manipur, India is described and illustrated as a new species. The new species differs from D. jaintianum in having larger and glabrous leaves with acute apices, green sepal apices, a 1–3‐flowered inflorescence, smaller and non‐fragrant flowers and a 3‐lobed labellum with fimbriate midlobe.