The exocyst complex and intracellular vesicles mediate soluble protein trafficking to the primary cilium.

The efficient transport of proteins into the primary cilium is a crucial step for many signaling pathways. Dysfunction of this process can lead to the disruption of signaling cascades or cilium assembly, resulting in developmental disorders and cancer. Previous studies on the protein delivery to the cilium were mostly focused on the membrane-embedded receptors. In contrast, how soluble proteins are delivered into the cilium is poorly understood. In our work, we identify the exocyst complex as a key player in the ciliary trafficking of soluble Gli transcription factors. In line with the known function of the exocyst in intracellular vesicle transport, we demonstrate that soluble proteins, including Gli2/3 and Lkb1, can use the endosome recycling machinery for their delivery to the primary cilium. Finally, we identify GTPases: Rab14, Rab18, Rab23, and Arf4 that are involved in vesicle-mediated Gli protein ciliary trafficking. Our data pave the way for a better understanding of ciliary transport and uncover transport mechanisms inside the cell.

Selective Serotonin Re-uptake Inhibitors (SSRIs) Induced Weight Changes: A Dose and Duration Dependent Study on Albino Rats.

INTRODUCTION: Selective Serotonin Re-uptake Inhibitors (SSRIs) are the most significant and safe drugs among the antidepressants. Fluoxetine is the prototype drug of SSRIs. Various clinical studies showed that SSRI causes change in body weight in patients. This study was conducted to know the extent of weight change with different doses for different durations. AIM: The aim of this study was to find out whether fluoxetine causes weight gain or weight loss, and to deduce the comparative weight change after intraperitoneal injection of fluoxetine for different duration and doses. MATERIALS AND METHODS: Present study was conducted on 72 adult (36 males and 36 females) albino rats, in 3 phases of 2 weeks, 4 weeks and 12 weeks duration. Each phase consisted of 24 (12 males and 12 females) albino rats. These 24 rats were further randomly subdivided into 4 Groups of 6 albino rats each (3 males & 3 females). Group 1(Control) received normal saline (vehicle). Rest 18 rats of each phase were experimental rats, of Group 2, Group 3 and Group 4 (6 rats each). Group 2, group 3 and group 4 experimental rats received 10mg/kg, 20 mg/kg and 40mg/kg of intraperitoneal injection of fluoxetine respectively. All rats were weighed on each day for growth monitoring. Data was subjected to statistical analysis (Mean, standard deviation and Student's t-Test). RESULTS: All experimental group rats which received fluoxetine showed decrease of body weight. Rats which received high doses of fluoxetine could not tolerate the drug for more than two weeks and died due to excessive body weight loss, loose stools and muscle twitching. CONCLUSION: Present study conclude that SSRIs can cause weight change in the form of decrease of body weight. This property of SSRIs can be used clinically by prescribing these drugs to obese psychiatric patient without any fear of withdrawal of drug.

Beclin 1 regulates growth factor receptor signaling in breast cancer.

Beclin 1 is a haploinsufficient tumor suppressor that is decreased in many human tumors. The function of beclin 1 in cancer has been attributed primarily to its role in the degradative process of macroautophagy. However, beclin 1 is a core component of the vacuolar protein sorting 34 (Vps34)/class III phosphatidylinositoI-3 kinase (PI3KC3) and Vps15/p150 complex that regulates multiple membrane-trafficking events. In the current study, we describe an alternative mechanism of action for beclin 1 in breast cancer involving its control of growth factor receptor signaling. We identify a specific stage of early endosome maturation that is regulated by beclin 1, the transition of APPL1-containing phosphatidyIinositol 3-phosphate-negative (PI3P(-)) endosomes to PI3P(+) endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P(-)/APPL(+)-signaling-competent compartment. As a result, suppression of BECN1 sustains growth factor-stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Our data identify a novel role for beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of beclin 1 expression would enhance breast cancer progression.

CR16 forms a complex with N-WASP in brain and is a novel member of a conserved proline-rich actin-binding protein family.

The Neuronal Wiskott-Aldrich syndrome protein (N-WASP) has emerged as a central regulator of the actin cytoskeleton with abilities to integrate multiple upstream signal inputs and transmit them to the Arp2/3 complex. Here, we demonstrate that native N-WASP is present in a tight complex with a proline-rich protein, CR16, which shares approximately 25% identity with WASP interacting protein. CR16 is encoded by a gene previously cloned as a glucocorticoid-regulated mRNA from a rat hippocampal cDNA library. Although N-WASP is expressed ubiquitously, full-length CR16 protein is found predominately in the brain. CR16 and N-WASP colocalize in primary hippocampal neurons and at the tips of their growth cone filopodia. In vitro, CR16 directly binds both monomeric and filamentous actin but does not affect the kinetics of actin polymerization mediated by N-WASP and the Arp2/3 complex. Sequence homologues of CR16 are found not only in other vertebrates but also in the invertebrate Caenorhabditis elegans and in yeast. Thus, CR16 and WASP interacting protein belong to a family of N-WASP-binding proteins.

Nck and phosphatidylinositol 4,5-bisphosphate synergistically activate actin polymerization through the N-WASP-Arp2/3 pathway.

The Wiskott-Aldrich syndrome protein (WASP) and its relative neural WASP (N-WASP) regulate the nucleation of actin filaments through their interaction with the Arp2/3 complex and are regulated in turn by binding to GTP-bound Cdc42 and phosphatidylinositol 4,5-bisphosphate. The Nck Src homology (SH) 2/3 adaptor binds via its SH3 domains to a proline-rich region on WASP and N-WASP and has been implicated in recruitment of these proteins to sites of tyrosine phosphorylation. We show here that Nck SH3 domains dramatically stimulate the rate of nucleation of actin filaments by purified N-WASP in the presence of Arp2/3 in vitro. All three Nck SH3 domains are required for maximal activation. Nck-stimulated actin nucleation by N-WASP.Arp2/3 complexes is further stimulated by phosphatidylinositol 4,5-bisphosphate, but not by GTP-Cdc42, suggesting that Nck and Cdc42 activate N-WASP by redundant mechanisms. These results suggest the existence of an Nck-dependent, Cdc42-independent mechanism to induce actin polymerization at tyrosine-phosphorylated Nck binding sites.

WIP regulates N-WASP-mediated actin polymerization and filopodium formation.

Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.

Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4, 5-bisphosphate.

Neuronal Wiskott-Aldrich Syndrome protein (N-WASP) transmits signals from Cdc42 to the nucleation of actin filaments by Arp2/3 complex. Although full-length N-WASP is a weak activator of Arp2/3 complex, its activity can be enhanced by upstream regulators such as Cdc42 and PI(4,5)P(2). We dissected this activation reaction and found that the previously described physical interaction between the NH(2)-terminal domain and the COOH-terminal effector domain of N-WASP is a regulatory interaction because it can inhibit the actin nucleation activity of the effector domain by occluding the Arp2/3 binding site. This interaction between the NH(2)- and COOH termini must be intramolecular because in solution N-WASP is a monomer. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) influences the activity of N-WASP through a conserved basic sequence element located near the Cdc42 binding site rather than through the WASp homology domain 1. Like Cdc42, PI(4,5)P(2) reduces the affinity between the NH(2)- and COOH termini of the molecule. The use of a mutant N-WASP molecule lacking this basic stretch allowed us to delineate a signaling pathway in Xenopus extracts leading from PI(4, 5)P(2) to actin nucleation through Cdc42, N-WASP, and Arp2/3 complex. In this pathway, PI(4,5)P(2) serves two functions: first, as an activator of N-WASP; and second, as an indirect activator of Cdc42.

The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly.

Although small GTP-binding proteins of the Rho family have been implicated in signaling to the actin cytoskeleton, the exact nature of the linkage has remained obscure. We describe a novel mechanism that links one Rho family member, Cdc42, to actin polymerization. N-WASP, a ubiquitously expressed Cdc42-interacting protein, is required for Cdc42-stimulated actin polymerization in Xenopus egg extracts. The C terminus of N-WASP binds to the Arp2/3 complex and dramatically stimulates its ability to nucleate actin polymerization. Although full-length N-WASP is less effective, its activity can be greatly enhanced by Cdc42 and phosphatidylinositol (4,5) bisphosphate. Therefore, N-WASP and the Arp2/3 complex comprise a core mechanism that directly connects signal transduction pathways to the stimulation of actin polymerization.

Implication of the GABA(B) receptor in gamma vinyl-GABA's inhibition of cocaine-induced increases in nucleus accumbens dopamine.

Previously, we demonstrated that gamma vinyl-GABA (GVG, Vigabatrin) dose-dependently inhibits cocaine-induced increases in dopamine (DA) concentrations in both the rodent and primate brain. Furthermore, it abolishes cocaine self-administration and conditioned place preference, while having no effect on locomotor activity or drug delivery to the brain. In an effort to better understand the mechanisms underlying this inhibition, we examined the effect of the selective GABA(B) receptor antagonist SCH 50911 on the GVG-induced decrease in cocaine's elevation of extracellular DA concentrations in the nucleus accumbens (NACC). Cocaine administration alone (20 mg/kg i.p.) produced a 480% increase in extracellular NACC DA levels. GVG (300 mg/kg i.p.) significantly reduced this increase by 25% (P<0.01). In sharp contrast, extracellular DA levels increased to 550% after the sequential administration of SCH 50911 (3 mg/kg i.p.), GVG, and cocaine. This increase is significantly different than GVG and cocaine (P<0.05) but similar to cocaine alone. These results demonstrate that the GABA(B) antagonist SCH 50911 was able to completely abolish GVG's inhibition of cocaine-induced increases in DA in the NACC and implicates the GABA(B) receptor in the mechanism underlying this inhibition.

The Arp2/3 complex mediates actin polymerization induced by the small GTP-binding protein Cdc42.

The small GTP-binding protein Cdc42 is thought to induce filopodium formation by regulating actin polymerization at the cell cortex. Although several Cdc42-binding proteins have been identified and some of them have been implicated in filopodium formation, the precise role of Cdc42 in modulating actin polymerization has not been defined. To understand the biochemical pathways that link Cdc42 to the actin cytoskeleton, we have reconstituted Cdc42-induced actin polymerization in Xenopus egg extracts. Using this cell-free system, we have developed a rapid and specific assay that has allowed us to fractionate the extract and isolate factors involved in this activity. We report here that at least two biochemically distinct components are required, based on their chromatographic behavior and affinity for Cdc42. One component is purified to homogeneity and is identified as the Arp2/3 complex, a protein complex that has been shown to nucleate actin polymerization. However, the purified complex alone is not sufficient to mediate the activity; a second component that binds Cdc42 directly and mediates the interaction between Cdc42 and the complex also is required. These results establish an important link between a signaling molecule, Cdc42, and a complex that can directly modulate actin networks in vitro. We propose that activation of the Arp2/3 complex by Cdc42 and other signaling molecules plays a central role in stimulating actin polymerization at the cell surface.

Kinetic and mechanistic analysis of nonenzymatic, template-directed oligoribonucleotide ligation.

The role of divalent cations in the mechanism of pyrophosphate-activated, template-directed oligoribonucleotide ligation has been investigated. The dependence of the reaction rate on Mg2+ concentration suggests a kinetic scheme in which a Mg2+ ion must bind before ligation can proceed. Mn2+, Ca2+, Sr2+, and Ba2+ can also catalyze the reaction. Although Pb2+ and Zn2+ do not catalyze the reaction in the absence of other divalent ions, they significantly modulate the reaction rate when added in the presence of Mg2+, with Pb2+ stimulating the reaction (up to 65-fold) and Zn2+ inhibiting the reaction. The logarithm of the ligation rate increases linearly, with slope of 0.95, as a function of pH, indicating that the reaction involves a single critical deprotonation step. The ligation rates observed with the different divalent metal ion catalysts (Mn2+ > Mg2+ > Ca2+ > Sr2+ = Ba2+) vary inversely with the pKa values of their bound water molecules. The pH profile and these relative ligation rates suggest a mechanism in which a metal-bound hydroxide ion located near the ligation junction promotes catalysis, most likely by deprotonation of the hydroxl nucleophile. The effects of changing either the leaving group or the attacking hydroxyl, together with the large delta S(++) value for oligonucleotide ligation (about -20 eu), are consistent with an associative transition state.

Nonenzymatic, template-directed ligation of oligoribonucleotides is highly regioselective for the formation of 3'-5' phosphodiester bonds.

We have found that nonenzymatic, template-directed ligation reactions of oligoribonucleotides display high selectivity for the formation of 3'-5' rather than 2'-5' phosphodiester bonds. Formation of the 3'-5'-linked product is favored regardless of the metal ion catalyst or the leaving group, and for several different ligation junction sequences. The degree of selectivity depends on the leaving group: the ratio of 3'-5'- to 2'-5'-linked products was 10-15:1 when the 5'-phosphate was activated as the imidazolide, and 60-80:1 when the 5'-phosphate was activated by the formation of a 5'-triphosphate. Comparison of oligonucleotide ligation reactions with previously characterized single nucleotide primer extension reactions suggests that the strong preference for 3'-5'-linkages in oligonucleotide ligation is primarily due to occurence of ligation within the context of an extended Watston-Crick duplex. The ability of RNA to correctly self-assemble by template-directed ligation is an intrinsic consequence of its chemical structure and need not be imposed by an external catalyst (i.e., an enzyme polymerase); RNA therefore provides a reasonable structural basis for a self-replicating system in a prebiological world.

Electrocardiographic diagnosis of left ventricular hypertrophy in the presence of left bundle branch block: an echocardiographic correlation.

Surface electrocardiographic findings of 10 cases of left bundle branch block with echocardiographically determined left ventricular hypertrophy were compared with another 10 cases of left bundle branch block without left ventricular hypertrophy. Horizontal plane QRS- and T-wave angle > or = 150 degrees was highly sensitive (70%) and specific (90%) in diagnosing left ventricular hypertrophy in the presence of left bundle branch block. This diagnosis criterion was better than any other conventional criterion described before.

Isolated right ventricular infarction.

The literature on isolated right ventricular infarction is reviewed and local experience is reported. Chronic lung disease is an important risk factor. Chest pain and breathlessness are common. Syncope and sudden collapse can also occur. Rhythm disorders include sinus bradycardia, atrial fibrillation and ventricular tachycardia or fibrillation. Atrioventricular block is rare. Hypotension and a right-sided fourth heart sound are common. Cautious use of slow-release nitroglycerin is not hazardous in the absence of hypotension. High doses of steroids and anticoagulants can be helpful. The prognosis is usually good, although sudden collapse can occur due to ventricular fibrillation, rupture of the right ventricular free wall or massive pulmonary embolism.