A trs20 Mutation That Mimics an SEDT‐Causing Mutation Blocks Selective and Non‐Selective Autophagy: A Model for TRAPP III Organization

TRAPP is a multisubunit complex that functions in membrane traffic. Mutations in the mammalian TRAPP protein C2 are linked to the skeletal disorder spondyloepiphyseal dysplasia tarda (SEDT) that is thought to arise from an inability to secrete procollagen from the endoplasmic reticulum. Here, we show that C2 binds to the SNARE protein Syntaxin 5 and this interaction is weakened by an SEDT‐causing missense mutation (D47Y). Interestingly, the equivalent mutation (D46Y) in the yeast C2 homolog Trs20p does not block anterograde traffic but did affect endocytosis. The trs20D46Y mutation interfered with the interaction between Trs20p and Trs85p (TRAPP III‐specific subunit), Trs120p and Trs130p (TRAPP II‐specific subunits). Size exclusion chromatography suggested that this yeast mutation destabilized the TRAPP III complex that is involved in autophagy. We further show that this mutation blocks both the selective cytosol‐to‐vacuole (cvt) pathway as well as non‐selective autophagy. We demonstrate that the apparent molecular size of the TRAPP III complex is dependent upon membranes, and that the presence of TRAPP III is dependent upon Atg9p. Finally, we demonstrate that lipidated Bet3p is enriched in TRAPP III and that lipidation increases the efficiency of autophagy. Our study suggests that Trs20p acts as an adaptor for Trs85p and Trs120p and reveals complexities in TRAPP III assembly and function. The implications of C2D47Y in SEDT are discussed .     

Chemical synthesis of S-ribosyl-L-homocysteine and activity assay as a LuxS substrate

Bacterial quorum sensing is mediated by autoinducers, small signaling molecules generated by bacteria. It has been proposed that the LuxS enzyme converts S-ribosyl-L-homocysteine to 4,5-dihydroxy-2,3-pentanedione, the precursor of autoinducer 2 (AI-2). We report here a chemical synthesis of S-ribosyl-L-homocysteine and its analogue using Mitsunobu coupling. Chemically synthesized ribosylhomocysteine has been confirmed as a substrate for LuxS in both an enzyme assay and a whole cell quorum sensing assay. The chemical entities of products from the LuxS reaction were also established. Several ribosylhomocysteine analogues have been tested as LuxS inhibitors.     

Nanosized paclitaxel-loaded niosomes: formulation,in vitro cytotoxicity,and apoptosis gene expression in breast cancer cell lines

Molecular Biology Reports - In this study, the optimized niosomal formulation containing paclitaxel using non-ionic surfactants and cholesterol was designed and its cytotoxic effects against...     

The Effects of Selenium Supplementation on Clinical Symptoms and Gene Expression Related to Inflammation and Vascular Endothelial Growth Factor in Infertile Women Candidate for In Vitro Fertilization

Biological Trace Element Research - This study was performed to determine the effects of selenium supplementation on clinical symptoms and gene expression related to inflammatory markers in...     

Increased release of serotonin from rat ileum due to dexfenfluramine

Plasma levels of serotonin are elevated in primary pulmonary hypertension even after bilateral lung transplantation, suggesting a possible etiologic role. Serotonin is released primarily from the small intestine. Anorectic agents, such as dexfenfluramine, which can cause pulmonary hypertension, are known to inhibit potassium channels in vascular smooth muscle cells. We examined the hypothesis that dexfenfluramine may stimulate release of serotonin from the ileum by inhibition of K+ channels. In an isolated loop of rat ileum perfused with a physiological salt solution, the administration of dexfenfluramine, its major metabolite D-norfenfluramine, the potassium channel blocker 4-aminopyridine (5 mM), and caffeine (30 mM) increased serotonin levels in the venous effluent. Potassium chloride (60 mM) tended to increase serotonin levels. In genetically susceptible individuals, dexfenfluramine may induce pulmonary hypertension by increasing cytosolic calcium in enterochromaffin cells of the small intestine, thus releasing serotonin and causing vasoconstriction. This work indicates that dexfenfluramine and its major metabolite d-norfenfluramine can increase serotonin release from the small intestine.     

Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1

Calcineurin is a serine/threonine protein phosphatase that plays a critical role in many physiologic processes such as T-cell activation, skeletal myocyte differentiation, and cardiac hypertrophy. We previously showed that active MEKK3 is capable of stimulating calcineurin/nuclear factor of activated T-cells (NFAT) signaling in cardiac myocytes through phosphorylation of modulatory calcineurin-interacting protein 1 (MCIP1). However, the protein kinases that function downstream of MEKK3 to mediate MCIP1 phosphorylation and the mechanism of MCIP1-mediated calcineurin regulation have not been defined. Here, we show that MEK5 and big MAP kinase 1 (BMK1) function downstream of MEKK3 in a signaling cascade that induces calcineurin activity through phosphorylation of MCIP1. Genetic studies showed that BMK1-deficient mouse lung fibroblasts failed to mediate MCIP1 phosphorylation and activate calcineurin/NFAT in response to angiotensin II, a potent NFAT activator. Conversely, restoring BMK1 to the deficient cells restored angiotensin II-mediated calcineurin/NFAT activation. Thus, using BMK1-deficient mouse lung fibroblast cells, we provided the genetic evidence that BMK1 is required for angiotensin II-mediated calcineurin/NFAT activation through MICP1 phosphorylation. Finally, we discovered that phosphorylated MCIP1 dissociates from calcineurin and binds with 14-3-3, thereby relieving its inhibitory effect on calcineurin activity. In summary, our findings reveal a previously unrecognized essential regulatory role of mitogen-activated protein kinase signaling in calcineurin activation through the reversible phosphorylation of a calcineurin-interacting protein, MCIP1.     

Mast cells and inflammation

Mast cells are well known for their role in allergic and anaphylactic reactions, as well as their involvement in acquired and innate immunity. Increasing evidence now implicates mast cells in inflammatory diseases where they are activated by non-allergic triggers, such as neuropeptides and cytokines, often exerting synergistic effects as in the case of IL-33 and neurotensin. Mast cells can also release pro-inflammatory mediators selectively without degranulation. In particular, IL-1 induces selective release of IL-6, while corticotropin-releasing hormone secreted under stress induces the release of vascular endothelial growth factor. Many inflammatory diseases involve mast cells in cross-talk with T cells, such as atopic dermatitis, psoriasis and multiple sclerosis, which all worsen by stress. How mast cell differential responses are regulated is still unresolved. Preliminary evidence suggests that mitochondrial function and dynamics control mast cell degranulation, but not selective release. Recent findings also indicate that mast cells have immunomodulatory properties. Understanding selective release of mediators could explain how mast cells participate in numerous diverse biologic processes, and how they exert both immunostimulatory and immunosuppressive actions. Unraveling selective mast cell secretion could also help develop unique mast cell inhibitors with novel therapeutic applications. This article is part of a Special Issue entitled: Mast cells in inflammation.     

Calreticulin regulates insulin receptor expression and its downstream PI3 Kinase/Akt signalling pathway

Defects in insulin signalling and glucose metabolism are associated with the development of diabetes. Insulin signalling is initiated by the binding of insulin to its receptor and triggering cascades of events including activation of PI3kinase/Akt signalling pathway. Calreticulin (CRT) is a calcium binding chaperone molecule located in the endoplasmic reticulum. Targeted deletion of CRT in mice is embryonic lethal as a result of developmental and metabolic abnormalities. Rescued CRT null mice develop severe hypoglycemia the reason for which is not known. In addition, ventricular cardiomyocytes isolated from CRT null (crt-/-) mice have increased glycogen deposits. Therefore, the aim of this study was to investigate the changes in the glucose uptake and insulin signalling pathway (mainly PI3 kinase/Akt) in the absence of CRT. Here we show a significant increase in the glucose uptake by the crt-/- cells. This increase was accompanied by a significant increase in both insulin receptor beta expression, Insulin receptor substrate-1 phosphorylation, GLUT-1 expression and in insulin stimulated Akt phosphorylation and kinase activity in the crt-/- cells. Intriguingly, the increased expression of insulin receptor beta in the crt-/- was due to decreased levels of p53 protein. The current study is the first evidence for the up-regulation of insulin receptor density and activity in the absence of CRT function.