Amphibian Melanophore Technology as a Functional Screen for Antagonists of G-Protein Coupled 7-Transmembrane Receptors

Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-coupled receptors were established and evaluated, either as a primary screening utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors. Stably or transiently expressing melanophores responded selectively to respective effectors of the human calcium, cannabinoid, and neurokinin-1 receptors. Several selective cannabinoid receptor-binding inhibitors of known potency were characterized as agonists or antagonists of the human peripheral cannabinoid (CB(2)) receptor. The results were consistent with changes in cAMP content of hCB(2)-transfected human embryonic kidney (HEK) cells challenged with the same CB(2)-binding antagonists. A stable melanophore cell line expressing the human calcium receptor was used to screen a compound collection directly for functional antagonists, several of which were confirmed as antagonists in secondary screens by stimulating parathyroid hormone (PTH) secretion from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to toxic effects and validating melanophores as a primary screening modality. Also described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors. ()     

Cardiac troponin T forms a tetramer in vitro

Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction has led to the development of antibody-based assays for measuring cTnT serum concentration. We examined the behavior of cTnT in solution. Recombinant human cTnT3, the dominant isoform in the adult human heart, was used. The protein was pure and functional, as demonstrated by SDS-PAGE and surface plasmon resonance. cTnT3 was found to bind specifically and in a concentration-dependent manner to cTnC. Routine size exclusion chromatography suggested a higher-than-expected MW for cTnT. Using analytical ultracentrifugation, we found cTnT3 in solution to be mainly in the form of a tightly bound tetramer at concentrations as low as 4 micromol/L. Our sedimentation velocity and transmission electron microscopy results indicate that the tetramer's shape is elongated rather than globular. CTnT's self-association in solution is an important consideration in the design and interpretation of experiments with the aim of understanding the biochemical and biophysical properties of cTnT, its isoforms, and its mutants.     

High levels of Fis1, a pro-fission mitochondrial protein, trigger autophagy

Damaged mitochondria can be eliminated in a process of organelle autophagy, termed mitophagy. In most cells, the organization of mitochondria in a network could interfere with the selective elimination of damaged ones. In principle, fission of this network should precede mitophagy; but it is unclear whether it is per se a trigger of autophagy. The pro-fission mitochondrial protein Fis1 induced mitochondrial fragmentation and enhanced the formation of autophagosomes which could enclose mitochondria. These changes correlated with mitochondrial dysfunction rather than with fragmentation, as substantiated by Fis1 mutants with different effects on organelle shape and function. In conclusion, fission associated with mitochondrial dysfunction stimulates an increase in autophagy.     

Moderate physical training increases brain insulin concentrations in experimental diabetic rats

Insulin is an important modulator of growth and metabolic function in the central nervous system. The aim of this study was to investigate the influence of swimming physical training (at 32 degrees +/- 1 degree C, 1 hr/day, 5 days/week, with an overload equivalent to 5% of the body weight, for 4 weeks) on brain insulin concentrations in alloxan induced type 1 diabetic rats. Training attenuated hyperglycemia but had no effect on insulinemia in diabetic rats. Hematocrit and blood albumin values remained without changes. Brain insulin did not change in diabetic rats. However, physical training increased the concentration in both control and diabetic rats. It is concluded that in the present experimental conditions, diabetes had no influence on brain insulin, however moderate physical training increased the hormone in both control and diabetic animals.     

Lysophosphatidic acid receptor-dependent secondary effects via astrocytes promote neuronal differentiation

Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA(1)-LPA(5). In the nervous system, receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however, an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects, whereby LPA receptor-expressing cells exposed to, or "primed," by LPA may then act on other cells via distinct, yet LPA-initiated, mechanisms. In the present study, we examined cerebral cortical astrocytes as possible indirect mediators of the effects of LPA on developing cortical neurons. Cultured astrocytes express at least four LPA receptor subtypes, known as LPA(1)-LPA(4). Cerebral cortical astrocytes primed by LPA exposure were found to increase neuronal differentiation of cortical progenitor cells. Treatment of unprimed astrocyte-progenitor cocultures with conditioned medium derived from LPA-primed astrocytes yielded similar results, suggesting the involvement of an astrocyte-derived soluble factor induced by LPA. At least two LPA receptor subtypes are involved in LPA priming, since the priming effect was lost in astrocytes derived from LPA receptor double-null mice (LPA(1)((-/-))/LPA(2)((-/-))). Moreover, the loss of LPA-dependent differentiation in receptor double-null astrocytes could be rescued by retrovirally transduced expression of a single deleted receptor. These data demonstrate that receptor-mediated LPA signaling in astrocytes can induce LPA-dependent, indirect effects on neuronal differentiation.     

c-Met must translocate to the nucleus to initiate calcium signals

Hepatocyte growth factor (HGF) is important for cell proliferation, differentiation, and related activities. HGF acts through its receptor c-Met, which activates downstream signaling pathways. HGF binds to c-Met at the plasma membrane, where it is generally believed that c-Met signaling is initiated. Here we report that c-Met rapidly translocates to the nucleus upon stimulation with HGF. Ca(2+) signals that are induced by HGF result from phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate formation within the nucleus rather than within the cytoplasm. Translocation of c-Met to the nucleus depends upon the adaptor protein Gab1 and importin beta1, and formation of Ca(2+) signals in turn depends upon this translocation. HGF may exert its particular effects on cells because it bypasses signaling pathways in the cytoplasm to directly activate signaling pathways in the nucleus.     

"Mushroom cloud": a giant left ventricular pseudoaneurysm after a myocardial infarction due to myocardial bridging – a case report

Left ventricular pseudoaneurysm is an uncommon complication after transmural myocardial infarction, occurring when a free wall rupture is contained by adhesions of the overlying pericardium preventing acute tamponade. In this report, an unusual case of a 61 year-old male with a giant apical left ventricular pseudoaneurysm after an unnoticed myocardial infarction is presented. On coronary angiogram myocardial bridging of the distal left anterior descending artery was judged to be the infarct related lesion. The echocardiographic diagnosis allowed for a timely surgical intervention which resulted in the patient's full recovery.     

Defense Proteins from Vigna Unguiculata Seed Exudates: Characterization and Inhibitory Activity Against Fusarium Oxysporum

Plants exude a variety of substances through their roots, germinating seeds and aerial parts. Some of these released compounds seem to have an inhibitory effect against pathogens. The aim of this work was to investigate and identify antifungal proteins present in exudates from imbibed cowpea seeds (Vigna unguiculata (L.) Walp). The obtained exudation was analyzed in regard to specific protein activities by enzymatic or immunological assays for plant defense proteins, from 4 h to 48 h of seed imbibition. Our results show that cowpea seeds exudates present several defense related proteins characterized as β-1,3-glucanases, cystatins, vicilins and lipid transfer proteins (LTPs), as well as a storage vacuole membrane α-TIP protein, since the very first hours of imbibition. These exudates also have an “in vitro” inhibitory effect on the growth of the fungus Fusarium oxysporum f. sp. phaseoli. Our results suggest that seed exudates should promote seed protection from soil pathogens.