Cardiac contractility modulation by electric currents applied during the refractory period

Inotropic effects of electric currents applied during the refractory period have been reported in cardiac muscle in vitro using voltage-clamp techniques. We investigated how electric currents modulate cardiac contractility in normal canine hearts in vivo. Six dogs were instrumented to measure regional segment length, ventricular volume (sonomicrometry), and ventricular pressure. Cardiac contractility modulating (CCM) electric currents (biphasic square pulses, amplitude +/-20 mA, total duration 30 ms) were delivered during the refractory period between pairs of electrodes placed on anterior and posterior walls. CCM significantly increased index of global contractility (E(es)) from 5.9 +/- 2.9 to 8.3 +/- 4.6 mmHg/ml with anterior CCM, from 5.3 +/- 1.8 to 8.9 +/- 4.0 mmHg/ml with posterior CCM, and from 6.1 +/- 2.6 to 11.0 +/- 7.0 mmHg/ml with combined CCM (P < 0.01, no significant change in volume axis intercept). End-systolic pressure-segment length relations showed contractility enhancement near CCM delivery sites, but not remotely. Relaxation was not influenced. CCM increased mean aortic pressure, but did not change peripheral resistance. Locally applied electrical currents enhanced global cardiac contractility via regional changes in myocardial contractility without impairing relaxation in situ.     

Slow molecular clocks in Old World monkeys,apes, and humans

Two longstanding issues on the molecular clock hypothesis are studied in this article. First, is there a global molecular clock in mammals? Although many authors have observed unequal rates of nucleotide substitution among mammalian lineages, some authors have proposed a global clock for all eutherians, i.e., a single global rate of 2.2 x 10(-9) substitutions per nucleotide site per year. We reexamine this issue using noncoding, nonrepetitive DNA from Old World monkeys (OWMs), chimpanzee, and human. First, using the minimal date of 6 MYA for the human-chimpanzee divergence and more than 2.5 million base pairs of genomic sequences from human and chimpanzee, we estimate a maximal rate of 0.99 x 10(-9) for noncoding, nonrepetitive genomic regions for these two species. This estimate is less than half of the proposed global rate and much smaller than the commonly used rate (3.5 x 10(-9)) for eutherians. Further, using a minimal date of 23 MYA for the human-OWM divergence, we estimate a maximal rate of 1.5 x 10(-9) for both introns and fourfold degenerate sites in humans and OWMs. In addition, with the New World monkey (NWM) lineage as an outgroup, we estimate that the rate of substitution in introns is 30% higher in the OWM lineage than in the human lineage. Clearly, there is no global molecular clock in eutherians. Second, although many studies have indicated considerable variation in the mutation rate among regions of the mammalian genome, a recent study proposed a uniform rate. Using new and existing intron sequence data from higher primates, we find significant rate variation among genomic regions and a positive correlation between the rate of substitution and the GC content, refuting the claim of a uniform rate.     

Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding

Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS. Glu143 is positioned near the P-loop and interacts with the Mg(2+) of Mg NTP-binding proteins. We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+). The structure showed a water molecule at the place where Mg(2+) bound to the wild type enzyme. Mutant proteins E143A, and even E143D and E143Q with conservative mutations, lacked enzyme activity and failed to complement the methionine auxotrophy of the E. coli folC mutant SF4, showing that Glu143 is an essential residue. Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally. The E. coli E146Q mutant FPGS bound ADP with the same affinity as the wild type enzyme but bound ATP with much lower affinity and had higher ATPase activity than the wild type enzyme. The mutant enzyme was defective in forming the acyl-phosphate reaction intermediate from ATP and dihydropteroate. The E. coli FPGS requires activation by dihydropteroate or tetrahydrofolate binding to allow full activity. In the absence of a pteroate substrate, only 30% of the total enzyme binds ATP. We suggest that dihydropteroate causes a conformational change to allow increased ATP binding. The mutant enzyme was similarly activated by dihydropteroate resulting in increased ADP binding.     

Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation

The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genus Leishmania. The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates of Leishmania donovani promastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification of Leishmania elongation factor-1alpha (EF-1alpha) as a SHP-1-binding protein. Purified Leishmania EF-1alpha, but not host cell EF-1alpha, bound directly to SHP-1 in vitro leading to its activation. Three independent lines of evidence indicated that Leishmania EF-1alpha may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [(35)S]methionine-labeled organisms contained Leishmania EF-1alpha. Second, confocal, fluorescence microscopy using Leishmania-specific antisera detected Leishmania EF-1alpha in the cytosol of infected cells. Third, co-immunoprecipitation showed that Leishmania EF-1alpha was associated with SHP-1 in vivo in infected cells. Finally, introduction of purified Leishmania EF-1alpha, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitric-oxide synthase expression in response to interferon-gamma. Thus, Leishmania EF-1alpha is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.     

Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(-/-) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(-/-) mice were 9-16% of that in Gclm(+/+) littermates. Cysteine levels in Gclm(-/-) mice were 9, 35, and 40% of that in Gclm(+/+) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an approximately 2-fold increase in K(m) for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(-/-) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H(2)O(2). We conclude that the Gclm(-/-) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.     

Proteinase suppression by E-cadherin-mediated cell-cell attachment in premalignant oral keratinocytes

The expression and activity of epithelial proteinases is under stringent control to prevent aberrant hydrolysis of structural proteins and disruption of tissue architecture. E-cadherin-dependent cell-cell adhesion is also important for maintenance of epithelial structural integrity, and loss of E-cadherin expression has been correlated with enhanced invasive potential in multiple tumor models. To address the hypothesis that there is a functional link between E-cadherin and proteinase expression, we have examined the role of E-cadherin in proteinase regulation. By using a calcium switch protocol to manipulate junction assembly, our data demonstrate that initiation of de novo E-cadherin-mediated adhesive contacts suppresses expression of both relative matrix metalloproteinase-9 levels and net urinary-type plasminogen activator activity. E-cadherin-mediated cell-cell adhesion increases both phosphatidylinositol 3'-kinase (PI3-kinase)-dependent AKT phosphorylation and epidermal growth factor receptor-dependent MAPK/ERK activation. Pharmacologic inhibition of the PI3-kinase pathway, but not the epidermal growth factor receptor/MAPK pathway, prevents E-cadherin-mediated suppression of proteinases and delays junction assembly. Moreover, inhibition of junction assembly with a function-blocking anti-E-cadherin antibody stimulates proteinase-dependent Matrigel invasion. As matrix metalloproteinase-9 and urinary-type plasminogen activator potentiate the invasive activity of oral squamous cell carcinoma, these data suggest E-cadherin-mediated signaling through PI3-kinase can regulate the invasive behavior of cells by modulating proteinase secretion.     

Hrs regulates endosome membrane invagination and tyrosine kinase receptor signaling in Drosophila

Signaling through tyrosine kinase receptors (TKRs) is thought to be modulated by receptor-mediated endocytosis and degradation of the receptor in the lysosome. However, factors that regulate endosomal sorting of TKRs are largely unknown. Here, we demonstrate that Hrs (Hepatocyte growth factor-regulated tyrosine kinase substrate) is one such factor. Electron microscopy studies of hrs mutant larvae reveal an impairment in endosome membrane invagination and formation of multivesicular bodies (MVBs). hrs mutant animals fail to degrade active epidermal growth factor (EGF) and Torso TKRs, leading to enhanced signaling and altered embryonic patterning. These data suggest that Hrs and MVB formation function to downregulate TKR signaling.     

Diapause development and acclimation regulating enzymes associated with glycerol synthesis in the Shonai ecotype of the rice stem borer larva,Chilo suppressalis walker

Overwintering larvae of the Shonai ecotype of the rice stem borer, Chilo suppressalis, enter diapause in early September and terminate diapause at the end of October. Cold acclimation at 0°C did not influence glycerol, trehalose or glycogen content in larvae collected on 22 September. Acclimation at 0°C increased the glycerol content and reduced the glycogen content significantly in larvae collected on 2 October and 22 November compared with acclimation at 15°C. These results indicate that overwintering larvae at different phases of diapause development respond differently to the low temperature stimulus for glycerol synthesis. Thus, we evaluated the metabolic rearrangements associated with glycerol synthesis during diapause development and after temperature acclimation. Larvae collected on 2 October were acclimated at 15°C for 15 and 60 days. Some of those acclimated at 15°C were then moved to 0°C for 15 days. The larvae acclimated at 15°C for 15 days were in deep diapause and accumulated little glycerol, while larvae acclimated at 15°C for 60 days were nearly ready to emerge from diapause and accumulated glycerol at 155.5 μmol/g. When larvae acclimated to 15°C for 15 days were transferred to 0°C, glycerol accumulation was stimulated to the same extent (ca 140 μmol/g) as it was in larvae that were acclimated to 15°C for 60 days and then transferred to 0°C. These results indicate that low temperature has a cumulative effect on glycerol production in larvae at different phases of diapause development. Glycerol accumulation was accomplished by activation of glycogen phosphorylase and inhibition of fructose-1,6-bisphosphatase, and activation of enzymes associated with glycerol synthesis, mainly glyceraldehyde-3-phosphatase and polyol dehydrogenase with glyceraldehyde activity.