In vivo and in vitro analysis of cardiac troponin I phosphorylation

Adrenergic stimulation induces positive changes in cardiac contractility and relaxation. Cardiac troponin I is phosphorylated at different sites by protein kinase A and protein kinase C, but the effects of these post-translational modifications on the rate and extent of contractility and relaxation during beta-adrenergic stimulation in the intact animal remain obscure. To investigate the effect(s) of complete and chronic cTnI phosphorylation on cardiac function, we generated transgenic animals in which the five possible phosphorylation sites were replaced with aspartic acid, mimicking a constant state of complete phosphorylation (cTnI-AllP). We hypothesized that chronic and complete phosphorylation of cTnI might result in increased morbidity or mortality, but complete replacement with the transgenic protein was benign with no detectable pathology. To differentiate the effects of the different phosphorylation sites, we generated another mouse model, cTnI-PP, in which only the protein kinase A phosphorylation sites (Ser(23)/Ser(24)) were mutated to aspartic acid. In contrast to the cTnIAllP, the cTnI-PP mice showed enhanced diastolic function under basal conditions. The cTnI-PP animals also showed augmented relaxation and contraction at higher heart rates compared with the nontransgenic controls. Nuclear magnetic resonance amide proton/nitrogen chemical shift analysis of cardiac troponin C showed that, in the presence of cTnI-AllP and cTnI-PP, the N terminus exhibits a more closed conformation, respectively. The data show that protein kinase C phosphorylation of cTnI plays a dominant role in depressing contractility and exerts an antithetic role on the ability of protein kinase A to increase relaxation.     

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.     

Tetraspanin CD82 attenuates cellular morphogenesis through down-regulating integrin alpha6-mediated cell adhesion

Tetraspanin CD82 has been implicated in integrin-mediated functions such as cell motility and invasiveness. Although tetraspanins associate with integrins, it is unknown if and how CD82 regulates the functionality of integrins. In this study, we found that Du145 prostate cancer cells underwent morphogenesis on the reconstituted basement membrane Matrigel to form an anastomosing network of multicellular structures. This process entirely depends on integrin alpha6, a receptor for laminin. After CD82 is expressed in Du145 cells, this cellular morphogenesis was abolished, indicating a functional cross-talk between CD82 and alpha6 integrins. Interestingly, antibodies against other tetraspanins expressed in Du145 cells such as CD9, CD81, and CD151 did not block this integrin alpha6-dependent morphogenesis. We further found that CD82 significantly inhibited cell adhesion on laminin 1. Notably, the level of alpha6 integrins on the cell surface was down-regulated upon CD82 expression, although total cellular alpha6 protein levels remained unchanged in CD82-expressing cells. This down-regulation indicates that the diminished cell adhesiveness of CD82-expressing Du145 cells on laminin likely resulted from less cell surface expression of alpha6 integrins. As expected, CD82 physically associated with the integrin alpha6 in Du145-CD82 transfectant cells, suggesting that the formation of the CD82-integrin alpha6 complex reduces alpha6 integrin cell surface expression. Finally, the internalization of cell surface integrin alpha6 is significantly enhanced upon CD82 expression. In conclusion, our results indicate that 1) CD82 attenuates integrin alpha6 signaling during a cellular morphogenic process; 2) the decreased surface expression of alpha6 integrins in CD82-expressing cells is likely responsible for the diminished adhesiveness on laminin and, subsequently, results in the attenuation of alpha6 integrin-mediated cellular morphogenesis; and 3) the accelerated internalization of integrin alpha6 upon CD82 expression correlates with the down-regulation of cell surface integrin alpha6.     

Mechanistic differences in promoter DNA melting by Thermus aquaticus and Escherichia coli RNA polymerases

Formation of strand-separated, functional complexes at promoters was compared for RNA polymerases from the mesophile Escherichia coli and the thermophile Thermus aquaticus. The RNA polymerases contained sigma factors that were wild type or bearing homologous alanine substitutions for two aromatic amino acids involved in DNA melting. Substitutions in the sigmaA subunit of T. aquaticus RNA polymerase impair promoter DNA melting equally at temperatures from 25 to 75 degrees C. However, homologous substitutions in sigma70 render E. coli RNA polymerase progressively more melting-defective as the temperature is reduced below 37 degrees C. The effects of the mutations on the mechanism of promoter DNA melting were investigated by studying the interaction of wild type and mutant RNA polymerases with "partial promoters" mimicking promoter DNA where the nucleation of DNA melting had taken place. Because T. aquaticus and E. coli RNA polymerases bound these templates similarly, it was concluded that the different effects of the mutations on the two polymerases are exerted at a step preceding nucleation of DNA melting. A model is presented for how this mechanistic difference between the two RNA polymerase could explain our observations.     

Patterns of child fosterage in rural northern Thailand

Evolutionary theory guides an investigation of foster parent selection in two northern Thai villages with different biosocial environments: one village has high levels of labour migration and divorce, and growing numbers of parental death due to HIV/AIDS, while the other village has lower migration, divorce and parental mortality levels. Focus groups examine mothers' and fathers' motivations and ideals regarding foster caretaker selection, and quantitative family surveys examine real fostering outcomes: specifically, the laterality (matrilateral versus patrilateral) and genetic distance of the foster caretakers of all ever-fostered children in these two villages. As predicted, in environments of high marital stability and paternity certainty, parents seem to prefer close genetic kin from either side as foster parents for their children. In low marital stability and paternity certainty environments, parents trust their own lateral kin, regardless of genetic distance, over close genetic kin from the other side. The striking exception to this pattern, however, occurs in the case of parental death, in which case children are fostered to the deceased parent's kin, regardless of the child's sex or other factors. In general, the foster parents for girls are selected with more care, reflecting the daughter/female preference expected in traditionally matrilineal, matrilocal societies. An ordered decision-making pathway for foster parent selection is proposed, taking into consideration the key factors of (a) the circumstances driving the fostering decision, (b) the gender of the child, (c) the gender of the key decision-making parent, and (d) the degree of marital and population fluidity (and subsequent, paternity certainty) in the village.     

Factors affecting entomopathogenic nematodes (Steinernematidae) for control of overwintering codling moth (Lepidoptera: Tortricidae) in fruit bins

Fruit bins infested with diapausing codling moth larvae, Cydia pomonella (L.), are a potential source of reinfestation of orchards and may jeopardize the success of mating disruption programs and other control strategies. Entomopathogenic nematodes (EPNs) were tested as a potential means of control that could be applied at the time bins are submerged in dump tanks. Diapausing cocooned codling moth larvae in miniature fruit bins were highly susceptible to infective juveniles (IJs) of Steinernema carpocapsae (Weiser) and Steinernema feltiae (Filipjev) in a series of experiments. Cocooned larvae are significantly more susceptible to infection than are pupae. Experimental treatment of bins in suspensions of laboratory produced S. feltiae ranging from 10 to 100 IJs/ml of water with wetting agent (Silwet L77) resulted in 51-92% mortality. The use of adjuvants to increase penetration of hibernacula and retard desiccation of S. feltiae in fruit bins resulted in improved efficacy. The combination of a wetting agent (Silwet L77) and humectant (Stockosorb) with 10 S. feltiae IJs/ml in low and high humidity resulted in 92-95% mortality of cocooned codling moth larvae versus 46-57% mortality at the same IJ concentration without adjuvants. Immersion of infested bins in suspensions of commercially produced nematodes ranging from 10 to 50 IJs/ml water with wetting agent in an experimental packing line resulted in mortality in cocooned codling moth larvae of 45-87 and 56 - 85% for S. feltiae and S. carpocapsae, respectively. Our results indicate that EPNs provide an alternative nonchemical means of control that could be applied at the time bins are submerged in dump tanks at the packing house for flotation of fruit.     

Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current

We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin-sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor-mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.     

Supplementation with N-acetylcysteine and taurine failed to restore glutathione content in liver of streptozotocin-induced diabetics rats but protected from oxidative stress

Rats were rendered diabetic with streptozotocin and supplemented or not with N-acetylcysteine (NAC) and taurine (TAU). The liver was examined for the quantity of glutathione (GSH), both total and oxidised (GSSG), by HPLC assay. Moreover, the liver expression of gamma-glutamyl-cysteine synthetase, cysteine dioxygenase and heme oxygenase 1 was evaluated. Streptozotocin-diabetic rats showed decreased levels of liver glutathione (GSH); dietary supplementation with the antioxidants NAC and TAU failed to restore liver GSH to the level of control rats. Gamma-glutamyl-cysteine synthetase expression was not reduced in the diabetic rats, so the low hepatic GSH level in the supplemented diabetic rats cannot be ascribed to decreased expression of the biosynthetic key enzyme. Moreover, the diabetic rats showed no evidence of increased expression of cysteine dioxygenase, which could have indicated that NAC-derived cysteine was consumed in metabolic pathways different from GSH synthesis. However, NAC+TAU treatment provided partial protection from glutathione oxidation in the liver of diabetic rats; moreover, the antioxidant treatment reduced the hepatic overexpression of heme oxygenase 1 (HO-1) mRNA which was detected in the diabetic rats. In conclusion, although NAC was not able to restore liver GSH levels, the antioxidant treatment restrained GSH oxidation and HO-1 overexpression, which are markers of cellular oxidative stress: diabetic rats probably exploit NAC as an antioxidant itself rather than as a GSH precursor.