Phospholamban decreases the energetic efficiency of the sarcoplasmic reticulum Ca pump

We tested the hypothesis that increased Sarcoplasmic reticulum (SR) Ca content ([Ca](SRT)) in phospholamban knockout mice (PLB-KO) is because of increased SR Ca pump efficiency defined by the steady-state SR [Ca] gradient. The time course of thapsigargin-sensitive ATP-dependent (45)Ca influx into and efflux out of cardiac SR vesicles from PLB-KO and wild-type (WT) mice was measured at 100 nm free [Ca]. We found that PLB decreased the initial SR Ca uptake rate (0.13 versus 0.31 nmol/mg/s) and decreased steady-state (45)Ca content (0.9 versus 4.1 nmol/mg protein). Furthermore, at similar total SR [Ca], the pump-mediated Ca efflux rate was higher in WT (0.065 versus 0.037 nmol/mg/s). The pump-independent leak rate constant (k(leak)) was also measured at 100 nm free [Ca]. The results indicate that k(leak) was < 1% of pump-mediated backflux and was not different among nonpentameric mutant PLB (PLB-C41F), WT pentameric PLB (same expression level), and PLB-KO. Therefore differences in passive SR Ca leak cannot be the cause of the higher thapsigargin-sensitive Ca efflux from the WT membranes. We conclude that the decreased total SR [Ca] in WT mice is caused by decreased SR Ca influx rate, an increased Ca-pump backflux, and unaltered leak. Based upon both thermodynamic and kinetic analysis, we conclude that PLB decreases the energetic efficiency of the SR Ca pump.     

Ascorbic acid increases the yield of dopaminergic neurons derived from basic fibroblast growth factor expanded mesencephalic precursors

CNS precursors derived from E12 rat mesencephalon proliferate in the presence of basic fibroblast growth factor and differentiate in vitro into functional dopaminergic neurons, which upon transplantation alleviate behavioral symptoms in a rat model of Parkinson's disease. Here we show that the efficiency of dopaminergic differentiation decreases in the mesencephalic precursors that were proliferated or passaged for extended periods in vitro. Ascorbic acid treatment restored dopaminergic differentiation in these precursors and led to a greater than 10-fold increase in dopamine neuron yield compared with untreated cultures. The effect of ascorbic acid was stereospecific and could not be mimicked by any other antioxidants. The expression of sodium-dependent vitamin C transporter, a recently identified stereospecific ascorbic acid transporter, was maintained in mesencephalic precursors for extended in vitro periods. Pre-treatment of in vitro expanded mesencephalic precursors with ascorbic acid might facilitate the large-scale generation of dopaminergic neurons for clinical transplantation.     

Potential role of muscarinic receptors in schizophrenia

The role of muscarinic receptors in schizophrenia was investigated using the muscarinic agonist PTAC. PTAC was highly selective for muscarinic receptors, was a partial agonist at muscarinic M2/M4 receptors and an antagonist at M1, M3 and M5 receptors. PTAC was highly active in animal models predictive of antipsychotic behavior including inhibition of conditioned avoidance responding in rats and blockade of apomorphine-induced climbing behavior in mice. d-Amphetamine-induced Fos expression in rat nucleus accumbens was inhibited by PTAC, thus directly demonstrating the ability of PTAC to modulate DA activity. In electrophysiological studies in rats, PTAC acutely inhibited the firing of A10 DA cells and after chronic administration decreased the number of spontaneously firing DA cells in the A10 brain area. However, PTAC did not appreciably alter the firing of A9 DA cells. Thus, PTAC appears to have novel antipsychotic-like activity and these data suggest that muscarinic compounds such as PTAC may represent a new class of antipsychotic agents.     

Development of definitive endoderm from embryonic stem cells in culture

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury(+) population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury(+) cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.     

Salicylic acid dependent signaling promotes basal thermotolerance but is not essential for acquired thermotolerance in Arabidopsis thaliana

Salicylic acid (SA) is reported to protect plants from heat shock (HS), but insufficient is known about its role in thermotolerance or how this relates to SA signaling in pathogen resistance. We tested thermotolerance and expression of pathogenesis-related (PR) and HS proteins (HSPs) in Arabidopsis thaliana genotypes with modified SA signaling: plants with the SA hydroxylase NahG transgene, the nonexpresser of PR proteins (npr1) mutant, and the constitutive expressers of PR proteins (cpr1 and cpr5) mutants. At all growth stages from seeds to 3-week-old plants, we found evidence for SA-dependent signaling in basal thermotolerance (i.e. tolerance of HS without prior heat acclimation). Endogenous SA correlated with basal thermotolerance, with the SA-deficient NahG and SA-accumulating cpr5 genotypes having lowest and highest thermotolerance, respectively. SA promoted thermotolerance during the HS itself and subsequent recovery. Recovery from HS apparently involved an NPR1-dependent pathway but thermotolerance during HS did not. SA reduced electrolyte leakage, indicating that it induced membrane thermoprotection. PR-1 and Hsp17.6 were induced by SA or HS, indicating common factors in pathogen and HS responses. SA-induced Hsp17.6 expression had a different dose-response to PR-1 expression. HS-induced Hsp17.6 protein appeared more slowly in NahG. However, SA only partially induced HSPs. Hsp17.6 induction by HS was more substantial than by SA, and we found no SA effect on Hsp101 expression. All genotypes, including NahG and npr1, were capable of expression of HSPs and acquisition of HS tolerance by prior heat acclimation. Although SA promotes basal thermotolerance, it is not essential for acquired thermotolerance.     

Genome-wide identification of Arabidopsis coiled-coil proteins and establishment of the ARABI-COIL database

Increasing evidence demonstrates the importance of long coiled-coil proteins for the spatial organization of cellular processes. Although several protein classes with long coiled-coil domains have been studied in animals and yeast, our knowledge about plant long coiled-coil proteins is very limited. The repeat nature of the coiled-coil sequence motif often prevents the simple identification of homologs of animal coiled-coil proteins by generic sequence similarity searches. As a consequence, counterparts of many animal proteins with long coiled-coil domains, like lamins, golgins, or microtubule organization center components, have not been identified yet in plants. Here, all Arabidopsis proteins predicted to contain long stretches of coiled-coil domains were identified by applying the algorithm MultiCoil to a genome-wide screen. A searchable protein database, ARABI-COIL (http://www.coiled-coil.org/arabidopsis), was established that integrates information on number, size, and position of predicted coiled-coil domains with subcellular localization signals, transmembrane domains, and available functional annotations. ARABI-COIL serves as a tool to sort and browse Arabidopsis long coiled-coil proteins to facilitate the identification and selection of candidate proteins of potential interest for specific research areas. Using the database, candidate proteins were identified for Arabidopsis membrane-bound, nuclear, and organellar long coiled-coil proteins.     

Identification of bacilysin, chlorotetaine, and iturin a produced by Bacillus sp. strain CS93 isolated from pozol, a Mexican fermented maize dough

Three antimicrobial compounds produced by Bacillus sp. strain CS93 isolated from pozol were identified by using high-performance liquid chromatography and mass spectrometry. The three compounds were iturin, bacilysin, and chlorotetaine. Production of these compounds by CS93 could account for the medicinal properties attributed to pozol.