Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. Here we demonstrate that a multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. We also show that the phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. Here we define a novel ADF/cofilin phosphoregulatory complex and suggest a new mechanism for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton.     

In vivo interaction between serotonin and galanin receptors types 1 and 2 in the dorsal raphe: implication for limbic seizures

The neuropeptide galanin suppresses seizure activity in the hippocampus by inhibiting glutamatergic neurotransmission. Galanin may also modulate limbic seizures through interaction with other neurotransmitters in neuronal populations that project to the hippocampus. We examined the role of galanin receptors types 1 and 2 in the dorsal raphe (DR) in the regulation of serotonergic transmission and limbic seizures. Infusion of a mixed agonist of galanin receptors types 1 and 2 [galanin (1-29)] into the DR augmented the severity of limbic seizures in both rats and wild-type mice and concurrently reduced serotonin concentration in the DR and hippocampus as measured by immunofluorescence or HPLC. In contrast, injection of the galanin receptor type 2 agonist galanin (2-11) mitigated the severity of seizures in both species and increased serotonin concentration in both areas. Injection of both galanin fragments into the DR of galanin receptor type 1 knockout mice exerted anticonvulsant effects. Both the proconvulsant activity of galanin (1-29) and seizure suppression by galanin (2-11) were abolished in serotonin-depleted animals. Our data indicate that, in the DR, galanin receptors types 1 and 2 modulate serotonergic transmission in a negative and a positive fashion, respectively, and that these effects translate into either facilitation or inhibition of limbic seizures.     

Tissue tracking imaging for identifying the origin of idiopathic ventricular arrhythmias: a new role of cardiac ultrasound in electrophysiology

Several strategies for mapping ventricular outflow tract tachycardia have been reported as useful indices for differentiating between those originating from the right and the left side. Recently, tissue tracking imaging (TTI) has been demonstrated as a novel non-invasive modality for identifying the origin of outflow tract tachycardias. Tissue tracking imaging is an ultrasonographic technique that measures the myocardial motion amplitude towards the transducer in each region during systole, identifying regional myocardial displacement on the basis of myocardial velocities using color Doppler myocardial imaging principles. In this technique, the origin of the arrhythmia could be recognized as the site where the earliest color-coded signal (ECCS) appeared on the myocardium at the onset of the systole. In preliminary studies this modality was found to be useful in differentiating outflow tract ventricular tachycardias. ECCS was always found below or at the level of the pulmonary valve in all arrhythmias which could be ablated from the right ventricular outflow tract, while in those where the ECCS was above and close to the pulmonary valve it could be ablated from the left sinus of valsalva. These results indicate that TTI can provide detailed and accurate information on the arrhythmia origin of outflow tract tachycardia and may be useful for differentiating between an outflow tract tachycardia originating from the LV epicardium remote from the LSV and that from the LSV. Newer advances in echocardiographic technologies like high resolution, high frame rate real time three dimensional echocardiography with speckle tracking may further improve the precise localization of arrhythmias in the future.     

Gene expression intensity shapes evolutionary rates of the proteins encoded by the vertebrate genome

Natural selection leaves its footprints on protein-coding sequences by modulating their silent and replacement evolutionary rates. In highly expressed genes in invertebrates, these footprints are seen in the higher codon usage bias and lower synonymous divergence. In mammals, the highly expressed genes have a shorter gene length in the genome and the breadth of expression is known to constrain the rate of protein evolution. Here we have examined how the rates of evolution of proteins encoded by the vertebrate genomes are modulated by the amount (intensity) of gene expression. To understand how natural selection operates on proteins that appear to have arisen in earlier and later phases of animal evolution, we have contrasted patterns of mouse proteins that have homologs in invertebrate and protist genomes (Precambrian genes) with those that do not have such detectable homologs (vertebrate-specific genes). We find that the intensity of gene expression relates inversely to the rate of protein sequence evolution on a genomic scale. The most highly expressed genes actually show the lowest total number of substitutions per polypeptide, consistent with cumulative effects of purifying selection on individual amino acid replacements. Precambrian genes exhibit a more pronounced difference in protein evolutionary rates (up to three times) between the genes with high and low expression levels as compared to the vertebrate-specific genes, which appears to be due to the narrower breadth of expression of the vertebrate-specific genes. These results provide insights into the differential relationship and effect of the increasing complexity of animal body form on evolutionary rates of proteins.     

Suppression of cell proliferation, DNA protein cross-links, and induction of apoptosis by vanadium in chemical rat mammary carcinogenesis

Vanadium, a dietary micronutrient, has recently been considered as an important pharmacological agent. The present investigation was carried out to ascertain its anticarcinogenic potential against an experimental rat mammary carcinogenesis. Female Sprague-Dawley rats were treated with 7,12dimethylbenz(alpha)anthracene (DMBA) (0.5 mg/100 g body weight) by a single tail vein injection in an oil emulsion. Vanadium (ammonium monovanadate) at a concentration of 0.5 ppm (4.27 micromol/L) was supplemented in drinking water and given ad libitum to the experimental group. The present study was an attempt to assess the effect of vanadium (ammonium monovanadate) on cell proliferation, apoptosis and histopathology in the mammary tissue. We also have examined DNA fragmentation and DNA protein cross-links (DPC) in the liver of rats as well. Immunohistochemical analysis indicated that early neoplasia in mammary tissue proceeds by a decrease in apoptotic cell death (ACD), which was also examined with TUNEL assay, rather than an increase in cell proliferation (P<0.01). DPC in liver were reduced by vanadium treatment (ANOVA, F=13.7, P<0.01). Agarose gel electrophoresis revealed DNA fragmentation in the vanadium-treated group, confirming apoptosis further. Results of the study indicate that the mammary preneoplasia is sensitive to vanadium intervention whereas normal proliferating cells are not.     

GalR represses galP1 by inhibiting the rate-determining open complex formation through RNA polymerase contact: a GalR negative control mutant

GalR represses the galP1 promoter by a DNA looping-independent mechanism. Equilibrium binding of GalR and RNA polymerase to DNA, and real-time kinetics of base-pair distortion (isomerization) showed that the equilibrium dissociation constant of RNA polymerase-P1 closed complexes is largely unaffected in the presence of saturating GalR, indicating that mutual antagonism (steric hindrance) of the regulator and the RNA polymerase does not occur at this promoter. In fluorescence kinetics with 2-AP labeled P1 DNA, GalR inhibited the slower of the two-step base-pair distortion process. We isolated a negative control GalR mutant, S29R, which while bound to the operator DNA was incapable of repression of P1. Based on these results and previous demonstration that repression requires the C-terminal domain of the alpha subunit (alpha-CTD) of RNA polymerase, we propose that GalR establishes contact with alpha-CTD at the last resolved isomerization intermediate, forming a kinetic trap.     

Probing the active-site requirements of human intestinal N-terminal maltase-glucoamylase: Synthesis and enzyme inhibitory activities of a six-membered ring nitrogen analogue of kotalanol and its de-O-sulfonated derivative

In order to probe the active-site requirements of the human N-terminal subunit of maltase-glucoamylase (ntMGAM), one of the clinically relevant intestinal enzymes targeted for the treatment of type-2 diabetes, the syntheses of two new inhibitors are described. The target compounds are structural hybrids of kotalanol, a naturally occurring glucosidase inhibitor with a unique five-membered ring sulfonium-sulfate inner salt structure, and miglitol, a six-membered ring antidiabetic drug that is currently in clinical use. The compounds comprise the six-membered ring of miglitol and the side chain of kotalanol or its de-O-sulfonated derivative. Inhibition studies of these hybrid molecules with human ntMGAM indicated that they are inhibitors of this enzyme with comparable K(i) values to that of miglitol (kotalanol analogue: 2.3±0.6μM; corresponding de-O-sulfonated analogue: 1.4±0.5μM; miglitol: 1.0±0.1μM). However, they are less active compared to kotalanol (K(i)=0.19±0.03μM). These results suggest that the (3)T(2) enzyme-bound conformation of the five-membered thiocyclitol moiety of the kotalanol class of compounds more closely resembles the (4)H(3) conformation of the proposed transition state for the formation of an enzyme-substrate covalent intermediate in the glycosidase hydrolase family 31 (GH31)-catalyzed reaction.