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.     

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.     

Synthesis and topoisomerase I inhibitory properties of luotonin A analogues

Luotonin A, a naturally occurring pyrroloquinazolinoquinoline alkaloid, has been previously demonstrated to be a topoisomerase I poison. A number of luotonin A derivatives have now been prepared through the condensation of anthranilic acid derivatives and 1,2-dihydropyrrolo[3,4-b]quinoline-3-one in the presence of phosphorus oxychloride. When dichloromethane was used as solvent the reaction proceeded to a single product. In contrast when the reaction was carried out in tetrahydrofuran or in phosphorus oxychloride, an additional isomeric product was obtained. The luotonin A analogues were evaluated for their ability to effect stabilization of the covalent binary complex formed between human topoisomerase I and DNA, and for cytotoxicity toward a yeast strain expressing the human topoisomerase I.     

Rapid unfolding of a domain populates an aggregation-prone intermediate that can be recognized by GroEL

Some amino acid substitutions in phage P22 coat protein cause a temperature-sensitive folding (tsf) phenotype. In vivo, these tsf amino acid substitutions cause coat protein to aggregate and form intracellular inclusion bodies when folded at high temperatures, but at low temperatures the proteins fold properly. Here the effects of tsf amino acid substitutions on folding and unfolding kinetics and the stability of coat protein in vitro have been investigated to determine how the substitutions change the ability of coat protein to fold properly. The equilibrium unfolding transitions of the tsf variants were best fit to a three-state model, N if I if U, where all species concerned were monomeric, a result confirmed by velocity sedimentation analytical ultracentrifugation. The primary effect of the tsf amino acid substitutions on the equilibrium unfolding pathway was to decrease the stability (DeltaG) and the solvent accessibility (m-value) of the N if I transition. The kinetics of folding and unfolding of the tsf coat proteins were investigated using tryptophan fluorescence and circular dichroism (CD) at 222 nm. The tsf amino acid substitutions increased the rate of unfolding by 8-14-fold, with little effect on the rate of folding, when monitored by tryptophan fluorescence. In contrast, when folding or unfolding reactions were monitored by CD, the reactions were too fast to be observed. The tsf coat proteins are natural substrates for the molecular chaperones, GroEL/S. When native tsf coat protein monomers were incubated with GroEL, they bound efficiently, indicating that a folding intermediate was significantly populated even without denaturant. Thus, the tsf coat proteins aggregate in vivo because of an increased propensity to populate this unfolding intermediate.     

NMR and temperature-jump measurements of de novo designed proteins demonstrate rapid folding in the absence of explicit selection for kinetics

We address the importance of natural selection in the origin and maintenance of rapid protein folding by experimentally characterizing the folding kinetics of two de novo designed proteins, NC3-NCAP and ENH-FSM1. These 51 residue proteins, which adopt the helix-turn-helix homeodomain fold, share as few as 12 residues in common with their most closely related natural analog. Despite the replacement of up to 3/4 of their residues by a computer algorithm optimizing only thermodynamic properties, the designed proteins fold as fast or faster than the 35,000 s(-1) observed for the closest natural analog. Thus these de novo designed proteins, which were produced in the complete absence of selective pressures or design constraints explicitly aimed at ensuring rapid folding, are among the most rapidly folding proteins reported to date.     

In vitro RNA replication directed by replicase complexes isolated from the subgenomic replicon cells of hepatitis C virus

Replication of hepatitis C virus (HCV) RNA in virus-infected cells is believed to be catalyzed by viral replicase complexes (RCs), which may consist of various virally encoded nonstructural proteins and host factors. In this study, we characterized the RC activity of a crude membrane fraction isolated from HCV subgenomic replicon cells. The RC preparation was able to use endogenous replicon RNA as a template to synthesize both single-stranded (ss) and double-stranded (ds) RNA products. Divalent cations (Mg2+ and Mn2+) showed different effects on RNA synthesis. Mg2+ ions stimulated the synthesis of ss RNA but had little effect on the synthesis of ds RNA. In contrast, Mn2+ ions enhanced primarily the synthesis of ds RNA. Interestingly, ss RNA could be synthesized under certain conditions in the absence of ds RNA, and vice versa, suggesting that the ss and ds RNA were derived either from different forms of replicative intermediates or from different RCs. Pulse-chase analysis showed that radioactivity incorporated into the ss RNA was chased into the ds RNA and other larger RNA species. This observation indicated that the newly synthesized ss RNA could serve as a template for a further round of RNA synthesis. Finally, 3' deoxyribonucleoside triphosphates were able to inhibit RNA synthesis in this cell-free system, presumably through chain termination, with 3' dGTP having the highest potency. Establishment of the replicase assay will facilitate the identification and evaluation of potential inhibitors that would act against the entire RC of HCV.