Ral-GTPase interacts with the beta1 subunit of Na+/K+-ATPase and is activated upon inhibition of the Na+/K+ pump

Na+/K+-ATPase functions as both an ion pump and a signal transducer. Cardiac glycosides partially inhibit Na+/K+-ATPase, causing activation of multiple interrelated growth pathways via the Na+/K+-ATPase/c-Src/epidermal growth factor receptor complex. Such pathways include Ras/MEK/ERK and Ral/RalGDS cascades, which can lead to cardiac hypertrophy. In search of novel Ral-GTPase binding proteins, we used RalB as the bait to screen a human testes cDNA expression library using the yeast 2-hybrid system. The results demonstrated that 1 of the RalB interacting clones represented the C-terminal region of the beta1 subunit of Na+/K+-ATPase. Further analysis using the yeast 2-hybrid system and full-length beta1 subunit of Na+/K+-ATPase confirmed the interaction with RalA and RalB. In vitro binding and pull-down assays demonstrated that the beta1 subunit of Na+/K+-ATPase interacts directly with RalA and RalB. Ral-GTP pull-down assays demonstrated that short-term ouabain treatment of A7r5 cells, a rat aorta smooth muscle cell line, caused activation of Ral GTPase. Maximal activation was observed 10 min after ouabain treatment. Ouabain-mediated Ral activation was inhibited upon the stimulation of Na+/K+-ATPase activity by Ang II. We propose that Ral GTPase is involved in the signal transducing function of Na+/K+-ATPase and provides a possible molecular mechanism connecting Ral to cardiac hypertrophy during diseased conditions.     

Cdk5 Modulation of mitogen-activated protein kinase signaling regulates neuronal survival

Cdk5, a cyclin-dependent kinase, is critical for neuronal development, neuronal migration, cortical lamination, and survival. Its survival role is based, in part, on "cross-talk" interactions with apoptotic and survival signaling pathways. Previously, we showed that Cdk5 phosphorylation of mitogen-activated protein kinase kinase (MEK)1 inhibits transient activation induced by nerve growth factor (NGF) in PC12 cells. To further explore the nature of this inhibition, we studied the kinetics of NGF activation of extracellular signal-regulated kinase (Erk)1/2 in cortical neurons with or without roscovitine, an inhibitor of Cdk5. NGF alone induced an Erk1/2-transient activation that peaked in 15 min and declined rapidly to baseline. Roscovitine, alone or with NGF, reached peak Erk1/2 activation in 30 min that was sustained for 48 h. Moreover, the sustained Erk1/2 activation induced apoptosis in cortical neurons. Significantly, pharmacological application of the MEK1 inhibitor PD98095 to roscovitine-treated cortical neurons prevented apoptosis. These results were also confirmed by knocking down Cdk5 activity in cortical neurons with Cdk5 small interference RNA. Apoptosis was correlated with a significant shift of phosphorylated tau and neurofilaments from axons to neuronal cell bodies. These results suggest that survival of cortical neurons is also dependent on tight Cdk5 modulation of the mitogen-activated protein kinase signaling pathway.     

Ajuba, a Novel LIM Protein, Interacts with Grb2, Augments Mitogen-Activated Protein Kinase Activity in Fibroblasts, and Promotes Meiotic Maturation of Xenopus Oocytes in a Grb2- and Ras-Dependent Manner

LIM domain-containing proteins contribute to cell fate determination, the regulation of cell proliferation and differentiation, and remodeling of the cell cytoskeleton. These proteins can be found in the cell nucleus, cytoplasm, or both. Whether and how cytoplasmic LIM proteins contribute to the cellular response to extracellular stimuli is an area of active investigation. We have identified and characterized a new LIM protein, Ajuba. Although predominantly a cytosolic protein, in contrast to other like proteins, it did not localize to sites of cellular adhesion to extracellular matrix or interact with the actin cytoskeleton. Removal of the pre-LIM domain of Ajuba, including a putative nuclear export signal, led to an accumulation of the LIM domains in the cell nucleus. The pre-LIM domain contains two putative proline-rich SH3 recognition motifs. Ajuba specifically associated with Grb2 in vitro and in vivo. The interaction between these proteins was mediated by either SH3 domain of Grb2 and the N-terminal proline-rich pre-LIM domain of Ajuba. In fibroblasts expressing Ajuba mitogen-activated protein kinase activity persisted despite serum starvation and upon serum stimulation generated levels fivefold higher than that seen in control cells. Finally, when Ajuba was expressed in fully developed Xenopus oocytes, it promoted meiotic maturation in a Grb2- and Ras-dependent manner.     

Comparative studies on myosin ATPase of a flying and nonflying bird.

Myosin was purified from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird. Ki (ADP) of myosin ATPase of pigeon is higher, but the Km (ATP) is lower than that of fowl. The specific activity (mumole of Pi liberated/min/mg protein) is higher for the fowl. A0.5 (CaCl2) of myosin of both pigeon and fowl is similar. However, the two proteins differ in their interactions with ADP, ATP and p-chloromercuribenzoate. The two proteins have the same tyrosine, tryptophan and sulfhydryl contents. The electrophoretic patterns of the two myosins on SDS-polyacrylamide gels are different. These studies show significant molecular differences in the myosin derived from the flight muscles of a flying (pigeon) and a nonflying (fowl) bird.     

Miniaturised hybrid immunoassay for high sensitivity analysis of aflatoxin M1 in milk

An ultra-sensitive sandwich ELISA was developed for detection of AFM1 in milk. The assay involved the immobilization of rat monoclonal antibody of AFM1 in 384 microtiter plate to capture AFM1 antigen. This was detected by tracer secondary rabbit poly-clonal antibody labelled with horseradish peroxidase upon addition of a luminol-based substrate. Milk samples with different fat percentage were analyzed after pre-treatment. Linear range of AFM1 detection 250-6.25 pg/mL was achieved in 3% fat milk. The miniaturised assay (10 μL) enabled ultra trace analysis of AFM1 in milk with much improved lower limit of detection at 0.005 pg/mL. A sensitive magnetic nanoparticles (MNPs) based ELISA was also developed and coupled with micro plate ELISA for analysis in milk. The hybrid-assay, by coupling the 1°Ab immobilized MNPs column with microwell plate assay enabled simultaneous measurement of low (0.5 pg/mL) and high AFM1 contamination (200 pg/mL). The most promising feature of this MNPs-ELISA is the small column size, high capture efficiency and lower cost over other reported materials. The proposed assay can be deployed for simultaneous analysis and monitoring of AFM1 in milk.     

Efficacy of a low-cost, inactivated whole-cell oral cholera vaccine: results from 3 years of follow-up of a randomized, controlled trial

Background

Killed oral cholera vaccines (OCVs) have been licensed for use in developing countries, but protection conferred by licensed OCVs beyond two years of follow-up has not been demonstrated in randomized, clinical trials.

Methods/Principal Findings

We conducted a cluster-randomized, placebo-controlled trial of a two-dose regimen of a low-cost killed whole cell OCV in residents 1 year of age and older living in 3,933 clusters in Kolkata, India. The primary endpoint was culture-proven Vibrio cholerae O1 diarrhea episodes severe enough to require treatment in a health care facility. Of the 66,900 fully dosed individuals (31,932 vaccinees and 34,968 placebo recipients), 38 vaccinees and 128 placebo-recipients developed cholera during three years of follow-up (protective efficacy 66%; one-sided 95%CI lower bound = 53%, p<0.001). Vaccine protection during the third year of follow-up was 65% (one-sided 95%CI lower bound = 44%, p<0.001). Significant protection was evident in the second year of follow-up in children vaccinated at ages 1–4 years and in the third year in older age groups.

Conclusions/Significance

The killed whole-cell OCV conferred significant protection that was evident in the second year of follow-up in young children and was sustained for at least three years in older age groups. Continued follow-up will be important to establish the vaccine''s duration of protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00289224","term_id":"NCT00289224"}}NCT00289224.     

Perceived Morbidity,Healthcare-Seeking Behavior and Their Determinants in a Poor-Resource Setting: Observation from India

BackgroundTo control the double burden of communicable and non-communicable diseases (NCDs), in the developing world, understanding the patterns of morbidity and healthcare-seeking is critical. The objective of this cross-sectional study was to determine the distribution, predictors and inter-relationship of perceived morbidity and related healthcare-seeking behavior in a poor-resource setting.MethodsBetween October 2013 and July 2014, 43999 consenting subjects were recruited from 10107 households in Malda district of West Bengal state in India, through multistage random sampling, using probability proportional-to-size. Information on socio-demographics, behaviors, recent ailments, perceived severity and healthcare-seeking were analyzed in SAS-9.3.2.ResultsRecent illnesses were reported by 55.91% (n=24600) participants. Among diagnosed ailments (n=23626), 50.92% (n=12031) were NCDs. Respiratory (17.28%,n=7605)), gastrointestinal (13.48%,n=5929) and musculoskeletal (6.25%,n=2749) problems were predominant. Non-qualified practitioners treated 53.16% (n=13074) episodes. Older children/adolescents [adjusted odds ratio for private healthcare providers(AOR_Pri)=0.76, 95% confidence interval=0.71-0.83) and for Govt. healthcare provider(AOR_Govt)=0.80(0.68-0.95)], females [AOR_Govt=0.80(0.73-0.88)], Muslims [AOR_Pri=0.85(0.69-0.76) and AOR_Govt=0.92(0.87-0.96)], backward castes [AOR_Govt=0.93(0.91-0.96)] and rural residents [AOR_Pri=0.82(0.75-0.89) and AOR_Govt=0.72(0.64-0.81)] had lower odds of visiting qualified practitioners. Apparently less severe NCDs [acid-peptic disorders: AOR_Pri=0.41(0.37-0.46) & AOR_Govt=0.41(0.37-0.46), osteoarthritis: AOR_Pri=0.72(0.59-0.68) & AOR_Govt=0.58(0.43-0.78)], gastrointestinal [AOR_Pri=0.28(0.24-0.33) & AOR_Govt=0.69(0.58-0.81)], respiratory [AOR_Pri=0.35(0.32-0.39) & AOR_Govt=0.46(0.41-0.52)] and skin infections [AOR_Pri=0.65(0.55-0.77)] were also less often treated by qualified practitioners. Better education [AOR_Pri=1.91(1.65-2.22) for ≥graduation], sanitation [AOR_Pri=1.58(1.42-1.75)] and access to safe water [AOR_Pri=1.33(1.05-1.67)] were associated with healthcare-seeking from qualified private practitioners. Longstanding NCDs [chronic obstructive pulmonary diseases: AOR_Pri=1.80(1.46-2.23), hypertension: AOR_Pri=1.94(1.60-2.36), diabetes: AOR_Pri=4.94(3.55-6.87)] and serious infections [typhoid: AOR_Pri=2.86(2.04-4.03)] were also more commonly treated by qualified private practitioners. Potential limitations included temporal ambiguity, reverse causation, generalizability issues and misclassification.ConclusionIn this poor-resource setting with high morbidity, ailments and their perceived severity were important predictors for healthcare-seeking. Interventions to improve awareness and healthcare-seeking among under-privileged and vulnerable population with efforts to improve the knowledge and practice of non-qualified practitioners probably required urgently.     

Caspase-3 deficiency reveals a physiologic role for Smac/DIABLO in regulating programmed cell death

Inhibitor of apoptosis protein (IAP)-binding proteins such as Grim, Reaper and HID have been shown to exert a critical role in regulating caspase activity in species such as D. Melanogaster. However, a comparable role for the mammalian homologue of second mitochondrial-derived activator of caspase/direct IAP-binding protein with low pI (Smac/DIABLO) has yet to be clearly established in vivo. Despite tremendous interest in recent years in the use of so-called Smac mimetics to enhance chemotherapeutic potency, our understanding of the true physiologic nature of Smac/DIABLO in regulating programmed cell death (PCD) remains elusive. In order to critically evaluate the role of Smac/DIABLO in regulating mammalian PCD, deficiency of caspase-3 was used as a sensitizing mutation in order to reduce aggregate levels of executioner caspase activity. We observe that combinatorial deletion of Diablo and Casp3, but neither alone, results in perinatal lethality in mice. Consistent with this, examination of both intrinsic and extrinsic forms of PCD in lines of murine embryonic fibroblasts demonstrate that loss of Smac/DIABLO alters both caspase-dependent and caspase-independent intrinsic PCD. Comparative small interfering RNA inhibition studies of X-linked inhibitor of apoptosis, cellular inhibitor of apoptosis (cIAP)-1, cIAP-2, caspase-6 and -7 in both wild-type and Casp3/Diablo DKO mouse embryonic fibroblast lineages, supports a model in which Smac/DIABLO acts to enhance the early phase executioner caspase activity through the modulation of inhibitory interactions between specific IAP family members and executioner caspases-3 and -7.