The ubiquitin E3 ligase MARCH7 is differentially regulated by the deubiquitylating enzymes USP7 and USP9X

Protein modification by one or more ubiquitin chains serves a critical signalling function across a wide range of cellular processes. Specificity within this system is conferred by ubiquitin E3 ligases, which target the substrates. Their activity is balanced by deubiquitylating enzymes (DUBs), which remove ubiquitin from both substrates and ligases. The RING-CH ligases were initially identified as viral immunoevasins involved in the downregulation of immunoreceptors. Their cellular orthologues, the Membrane-Associated RING-CH (MARCH) family represent a subgroup of the classical RING genes. Unlike their viral counterparts, the cellular RING-CH proteins appear highly regulated, and one of these in particular, MARCH7, was of interest because of a potential role in neuronal development and lymphocyte proliferation. Difficulties in detection and expression of this orphan ligase lead us to search for cellular cofactors involved in MARCH7 stability. In this study, we show that MARCH7 readily undergoes autoubiquitylation and associates with two deubiquitylating enzymes – ubiquitin-specific protease (USP)9X in the cytosol and USP7 in the nucleus. Exogenous expression and short interfering RNA depletion experiments demonstrate that MARCH7 can be stabilized by both USP9X and USP7, which deubiquitylate MARCH7 in the cytosol and nucleus, respectively. We therefore demonstrate compartment-specific regulation of this E3 ligase through recruitment of site-specific DUBs.     

Image-based modeling of hemodynamics in coronary artery aneurysms caused by Kawasaki disease

Kawasaki Disease (KD) is the leading cause of acquired pediatric heart disease. A subset of KD patients develops aneurysms in the coronary arteries, leading to increased risk of thrombosis and myocardial infarction. Currently, there are limited clinical data to guide the management of these patients, and the hemodynamic effects of these aneurysms are unknown. We applied patient-specific modeling to systematically quantify hemodynamics and wall shear stress in coronary arteries with aneurysms caused by KD. We modeled the hemodynamics in the aneurysms using anatomic data obtained by multi-detector computed tomography (CT) in a 10-year-old male subject who suffered KD at age 3?years. The altered hemodynamics were compared to that of a reconstructed normal coronary anatomy using our subject as the model. Computer simulations using a robust finite element framework were used to quantify time-varying shear stresses and particle trajectories in the coronary arteries. We accounted for the cardiac contractility and the microcirculation using physiologic downstream boundary conditions. The presence of aneurysms in the proximal coronary artery leads to flow recirculation, reduced wall shear stress within the aneurysm, and high wall shear stress gradients at the neck of the aneurysm. The wall shear stress in the KD subject (2.95-3.81 dynes/sq cm) was an order of magnitude lower than the normal control model (17.10-27.15 dynes/sq cm). Particle residence times were significantly higher, taking 5 cardiac cycles to fully clear from the aneurysmal regions in the KD subject compared to only 1.3 cardiac cycles from the corresponding regions of the normal model. In this novel quantitative study of hemodynamics in coronary aneurysms caused by KD, we documented markedly abnormal flow patterns that are associated with increased risk of thrombosis. This methodology has the potential to provide further insights into the effects of aneurysms in KD and to help risk stratify patients for appropriate medical and surgical interventions.     

First evidence of pathogenicity of V234I mutation of hVAPB found in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis is a motor neurodegenerative disease which is characterized by progressive loss of motor neurons followed by paralysis and eventually death. In human, VAMP-associated protein B (VAPB) is the causative gene of the familial form of ALS8. Previous studies have shown that P56S and T46I point mutations of hVAPB are present in this form of ALS. Recently, another mutation, V234I of hVAPB was found in one familial case of ALS. This is the first study where we have shown that V234I-VAPB does not form aggregate like other two mutants of VAPB and localizes differently than the wild type VAPB. It induces Ubiquitin aggregation followed by cell death. We propose that V234I-VAPB exhibits the characteristics of ALS in spite of not having the typical aggregation property of different mutations in various neurodegenerative diseases.     

Comparative analysis of some biochemical responses of three indica rice varieties during polyethylene glycol-mediated water stress exhibits distinct varietal differences

Extensive investigation into plant response and adaptation to diverse osmotic stresses like high salt/dehydration/low temperature, involving a broad spectrum of cellular physiological and biochemical changes, is essential to unravel intrinsic mechanism to mitigate against such stresses. In our previous communications, we conducted biochemical analyses of indica rice varieties, subjected to exogenous salt/abscisic acid-mediated oxidative stress. The aim of this study was to compare differential biochemical responses of the salt-sensitive (IR-29), salt-tolerant (Pokkali) and aromatic (Pusa Basmati or PB) rice varieties during polyethylene glycol (PEG)-induced dehydration stress. The greater susceptibility of IR-29 and PB, to water scarcity, was reflected by the higher toxic Na⁺ and putrescine accumulation, considerable decrease in (reduced/oxidized) glutathione, maximal increment in protease activity and greater downregulation of nitrate reductase activity. On the other hand, Pokkali appeared to suffer lesser damages as evidenced from much lower endogenous Na⁺ but higher K⁺, Ca²⁺ and Mg²⁺ accumulation, registering the highest levels of osmolytes like glycinebetaine and higher polyamines (spermidine and spermine) accounting to improved relative water content, higher (reduced/oxidized) glutathione, maximal induction of the enzyme phenylalanine ammonia-lyase and practically unhindered nitrate reductase activity, following PEG treatment. The highest induction of sugars and proline in IR-29 and PB probably played the osmoprotective/antioxidative functions, enabling to a certain extent to heighten their lipoxygenase inhibition or H₂O₂ scavenging potential, more than Pokkali, to ward off oxidative damages and sustain survival under critical dehydrated situations. Thus, the salt-tolerant Pokkali also showed prominent dehydration-tolerance properties, whereas the aromatic rice PB, almost identical in their biochemical responses to IR-29, showed greater sensitivity to PEG-mediated water deficit.     

Steroid hormones and embryo development in ovariectomized hamsters

Preimplantation embryo development was arrested at the premorula stage in hamsters ovariectomized on Day 1 of pregnancy. This effect was reversed when 500 micrograms progesterone were administered daily. Oestradiol-17 beta given alone had no significant effect on the cleavage rate or blastocyst formation, but a synergistic response was evident when a suboptimal (30 micrograms) dose of progesterone was given with 50 ng oestradiol-17 beta. Cholesterol and hydrocortisol had no effect on embryo development.     

Efforts in diagnosing early leprosy using serological techniques

Skin scrapings obtained from the lesions of leprosy patients of all types showed 96 % positivity to the serum antibody competition test using monoclonal antibody (ML04)to 35 kDa antigen of Mycobacterium leprae. Further, in vitro culture of full thickness skin biopsies from lepromatous patients were noted to release IgG antibodies toM. leprae with a peak antibody response at 48 h. The significance of this local antibody response toM. leprae in skin has been discussed for its possible use in diagnosing early leprosy.