Myosin Va plays a key role in nitrergic neurotransmission by transporting nNOSα to enteric varicosity membrane

Nitrergic neurotransmission at the smooth muscle neuromuscular junctions requires nitric oxide (NO) release that is dependent on the transport and docking of neuronal NO synthase (nNOS) α to the membrane of nerve terminals. However, the mechanism of translocation of nNOSα in actin-rich varicosities is unknown. We report here that the processive motor protein myosin Va is necessary for nitrergic neurotransmission. In wild-type mice, nNOSα-stained enteric varicosities colocalized with myosin Va and its tail constituent light chain 8 (LC8). In situ proximity ligation assay showed close association among nNOSα, myosin Va, and LC8. nNOSα was associated with varicosity membrane. Varicosities showed nitric oxide production upon stimulation with KCl. Intracellular microelectrode studies showed nitrergic IJP and smooth muscle hyperpolarizing responses to NO donor diethylenetriamine-NO (DNO). In contrast, enteric varicosities from myosin Va-deficient DBA (for dilute, brown, non-agouti) mice showed near absence of myosin Va but normal nNOSα and LC8. Membrane-bound nNOSα was not detectable, and the varicosities showed reduced NO production. Intracellular recordings in DBA mice showed reduced nitrergic IJPs but normal hyperpolarizing response to DNO. The nitrergic slow IJP was 9.1 ± 0.7 mV in the wild-type controls and 3.4 ± 0.3 mV in the DBA mice (P < 0.0001). Deficiency of myosin Va resulted in loss of nitrergic neuromuscular neurotransmission despite normal presence of nNOSα in the varicosities. These studies reveal the critical importance of myosin Va in nitrergic neurotransmission by facilitating transport of nNOSα to the varicosity membrane.     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

7, 8-diacetoxy-4-methylcoumarin induced cell death in human tumor cells is influenced by calreticulin

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity in presence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT and DAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells with different levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) to human glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neck carcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrial membrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells by application of small RNA interference enhanced the sensitivity by four folds while over expression of CRT in BMG-1 cells reduced their sensitivity to DAMC by ∼20% strongly suggesting the influence of CRT on DAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accompanied by changes in NFkB levels and over all protein acetylation status, besides increase in the NADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT is over-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRT transacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.     

Ammonia flux,physiological parameters,and <Emphasis Type="Italic">Symbiodinium</Emphasis> diversity in the anemonefish symbiosis on Red Sea coral reefs

Despite the ecological importance of anemonefish symbioses, little is known about how nutritional contributions from anemonefish interact with sea anemone physiology and Symbiodinium (endosymbiotic dinoflagellate) genetic identity under field conditions. On Red Sea coral reefs, we measured variation in ammonia concentrations near anemones, excretion rates of anemonefish, physiological parameters of anemones and Symbiodinium, and genetic identity of Symbiodinium within anemones. Ammonia concentrations among anemone tentacles were up to 49% above background levels, and anemonefish excreted ammonia significantly more rapidly after diurnal feeding than they did after nocturnal rest, similar to their excretion patterns under laboratory conditions. Levels of 4 physiological parameters (anemone protein content, and Symbiodinium abundance, chlorophyll a concentration, and division rate) were similar to those known for laboratory-cultured anemones, and in the field did not depend on the number of anemonefish per anemone or depth below sea surface. Symbiodinium abundance varied significantly with irradiance in the shaded reef microhabitats occupied by anemones. Most anemones at all depths harbored a novel Symbiodinium 18S rDNA variant within internal transcribed spacer region 2 (ITS2) type C1, while the rest hosted known ITS2 type C1. Association with Symbiodinium Clade C is consistent with the symbiotic pattern of these anemones on other Indo-Pacific reefs, but the C1 variant of Symbiodinium identified here has not been described previously. We conclude that in the field, anemonefish excrete ammonia at rapid rates that correlate with elevated concentrations among host anemone tentacles. Limited natural variation in anemonefish abundance may contribute to consistently high levels of physiological parameters in both anemones and Symbiodinium, in contrast to laboratory manipulations where removal of fish causes anemones to shrink and Symbiodinium to become less abundant.     

Vibrio plantisponsor sp. nov., a diazotrophic bacterium isolated from a mangrove associated wild rice (Porteresia coarctata Tateoka)

Two Gram negative, facultatively anaerobic, halophilic, motile, slightly curved rod-shaped bacterial strains MSSRF60^T and MSSRF64 were isolated from the roots of a mangrove-associated wild rice collected in the Pichavaram mangroves, India. These strains possess the key functional nitrogenase gene nifH. Phylogenetic analysis based on the 16S rRNA, recA, gapA, mreB, gyrB and pyrH, gene sequences revealed that strains MSSRF60^T and MSSRF64 belong to the genus Vibrio, and had the highest sequence similarity with the type strains of Vibrio diazotrophicus LMG 7893^T (99.7, 94.8, 98.5, 97.9, 94.0 and 90.7%, respectively), Vibrio areninigrae J74^T (98.2, 87.5, 91.5, 88.9, 86.5 and 84.6% respectively) and Vibrio hispanicus LMG 13240^T (97.8, 87.1, 91.7, 89.8, 84.1 and 81.9%, respectively). The fatty acid composition too confirmed the affiliation of strains MSSRF60^T and MSSRF64 to the genus Vibrio. These strains can be differentiated from the most closely related Vibrio species by several phenotypic traits. The DNA G + C content of strain MSSRF60^T was 41.8 mol%. Based on phenotypic, chemotaxonomic, genotypic (multilocus sequence analysis using five genes and genomic fingerprinting using BOX-PCR) and DNA–DNA hybridization analyses, strains MSSRF60^T and MSSRF64 represent a novel species of the genus Vibrio, for which the name Vibrio plantipsonsor sp. nov. is proposed. The type strain is MSSRF60^T (=DSM 21026^T = LMG 24470^T = CAIM 1392^T).     

Protein-protein interactions: principles, techniques, and their potential role in new drug development

A vast network of genes is inter-linked through protein-protein interactions and is critical component of almost every biological process under physiological conditions. Any disruption of the biologically essential network leads to pathological conditions resulting into related diseases. Therefore, proper understanding of biological functions warrants a comprehensive knowledge of protein-protein interactions and the molecular mechanisms that govern such processes. The importance of protein-protein interaction process is highlighted by the fact that a number of powerful techniques/methods have been developed to understand how such interactions take place under various physiological and pathological conditions. Many of the key protein-protein interactions are known to participate in disease-associated signaling pathways, and represent novel targets for therapeutic intervention. Thus, controlling protein-protein interactions offers a rich dividend for the discovery of new drug targets. Availability of various tools to study and the knowledge of human genome have put us in a unique position to understand highly complex biological network, and the mechanisms involved therein. In this review article, we have summarized protein-protein interaction networks, techniques/methods of their binding/kinetic parameters, and the role of these interactions in the development of potential tools for drug designing.     

Using the Edmonton obesity staging system to predict mortality in a population-representative cohort of people with overweight and obesity

Background:

Anthropometric-based classification schemes for excess adiposity do not include direct assessment of obesity-related comorbidity and functional status and thus have limited clinical utility. We examined the ability of the Edmonton obesity staging system, a 5-point ordinal classification system that considers comorbidity and functional status, in predicting mortality in a nationally representative US sample.

Methods:

We analyzed data from the National Health and Human Nutrition Examination Surveys (NHANES) III (1988–1994) and the NHANES 1999–2004, with mortality follow-up through to the end of 2006. Adults (age ≥ 20 yr) with overweight or obesity who had been randomized to the morning session at the mobile examination centre were scored according to the Edmonton obesity staging system. We examined the relationship between staging system scores and mortality, and Cox proportional hazards models were adjusted for the presence of the metabolic syndrome or hypertriglyceridemic waist.

Results:

Over 75% of the cohort with overweight or obesity were given scores of 1 or 2. Scores of 4 could not be reliably assigned because specific data elements were lacking. Survival curves clearly diverged when stratified by scores of 0–3, but not when stratified by obesity class alone. Within the data from the NHANES 1988–1994, scores of 2 (hazard ratio [HR] 1.57; 95% confidence interval [CI] 1.16 to 2.13) and 3 (HR 2.69; 95% CI 1.98 to 3.67) were associated with increased mortality compared with scores of 0 or 1, even after adjustment for body mass index and the metabolic syndrome. We found similar results after adjusting for hypertriglyceridemic waist (i.e., waist circumference ≥ 90 cm and a triglyceride level ≥ 2 mmol/L for men; the corresponding values for women were ≥ 85 cm and ≥ 1.5 mmol/L), as well as in a cohort eligible for bariatric surgery.

Interpretation:

The Edmonton obesity staging system independently predicted increased mortality even after adjustment for contemporary methods of classifying adiposity. The Edmonton obesity staging system may offer improved clinical utility in assessing obesity-related risk and prioritizing treatment.Body mass index (BMI) is the most common measure used to classify excess adiposity. A BMI of 18.5–24.9 kg/m² is considered normal, a BMI of 25.0–29.9 kg/m² is considered overweight and a BMI of more than 30 kg/m² is considered obese.¹ Observational studies have consistently reported deleterious associations between elevated BMI and morbidity/mortality.² Each five-unit increment in BMI above 25 kg/m² is associated with increases of 29% for overall mortality, 41% for vascular mortality and 210% for diabetes-related mortality.³ Measures of central adiposity, including increased waist circumference, predict cardiometabolic risk independent of elevated BMI.⁴ Thus, measuring both BMI and central adiposity to classify and quantify obesity-related risk and assess the appropriateness of treatments such as starting the use of antiobesity drugs or bariatric surgery is recommended.^1,5BMI is a useful population-based tool to classify adiposity and estimate its prevalence.⁶ However, BMI possesses well-known limitations at the individual level,⁷ including the inability to directly distinguish between lean and fat tissue. Thus, at a given BMI, substantial variation in adiposity can occur.⁸ Furthermore, neither BMI nor waist circumference directly reflects the presence of underlying obesity-related comorbidity, reduced quality of life or diminished functional status — elements that are widely considered to be critically important to the clinical assessment of patients with excess body weight. For example, BMI thresholds are currently used to determine eligibility for bariatric surgery. This approach has been criticized, and recommendations for scoring systems that incorporate assessments of comorbidity have been proposed as alternatives.⁹Recently, we proposed a new clinical staging system that ranks people with excess adiposity on a 5-point ordinal scale, while incorporating obesity-related comorbidities and functional status into the assessment (Box 1).⁶ The Edmonton obesity staging system is intended to complement anthropometric measures, but it requires further validation. In this study, we examine the population distribution of Edmonton obesity staging system scores and assess the system’s ability to predict mortality independent of anthropometric indices in a large, nationally representative US sample.

Box 1:

The Edmonton obesity staging system

Isolation,Cloning and Characterization of Resistance Gene Analogues in Pearl Millet Based on Conserved Nucleotide‐binding Sites

Sequence analysis of plant disease resistance genes shows similarity among themselves, with the presence of conserved motifs common to the nucleotide‐binding site (NBS). Oligonucleotide degenerate primers designed from the conserved NBS motifs encoded by several plant disease resistance genes were used to amplify resistance gene analogues (RGAs) corresponding to the NBS sequences from the genomic DNA of various plant species. Using specific primers designed from the conserved NBS regions, 22 RGAs were cloned and sequenced from pearl millet (Pennisetum glaucum L. Br.). Phylogenetic analysis of the predicted amino acid sequences grouped the RGAs into nine distinct classes. GenBank database searches with the consensus protein sequences of each of the nine classes revealed their conserved NBS domains and similarity to other known R genes of various crop species. One RGA 213 was mapped onto LG1 and LG7 in the pearl millet linkage map. This is the first report of the isolation and characterization of RGAs from pearl millet, which will facilitate the improvement of marker‐assisted breeding strategies.