A novel role of nectins in inhibition of the E-cadherin-induced activation of Rac and formation of cell-cell adherens junctions

Nectins are Ca(2+)-independent immunoglobulin (Ig)-like cell-cell adhesion molecules. The trans-interactions of nectins recruit cadherins to the nectin-based cell-cell adhesion, resulting in formation of cell-cell adherens junctions (AJs) in epithelial cells and fibroblasts. The trans-interaction of E-cadherin induces activation of Rac small G protein, whereas the trans-interactions of nectins induce activation of not only Rac but also Cdc42 small G protein. We showed by the fluorescent resonance energy transfer (FRET) imaging that the trans-interaction of E-cadherin induced dynamic activation and inactivation of Rac, which led to dynamic formation and retraction of lamellipodia. Moreover, we found here that the nectins, which did not trans-interact with other nectins (non-trans-interacting nectins), inhibited the E-cadherin-induced activation of Rac and reduced the velocity of the formation of the E-cadherin-based cell-cell AJs. The inhibitory effect of non-trans-interacting nectins was suppressed by the activation of Cdc42 induced by the trans-interactions of nectins. These results indicate a novel role of nectins in regulation of the E-cadherin-induced activation of Rac and formation of cell-cell AJs.     

Comparative analysis of gonadal morphology in six fish species of the Incertae Sedis genera in Characidae of occurrence in the São Francisco River Basin,Brazil

Martins, Y.S., Arantes, F.P., Sato, Y., dos Santos, J.E., Rizzo, E. and Bazzoli, N. 2012. Comparative analysis of gonadal morphology in six fish species of the Incertae Sedis genera in Characidae of occurrence in the São Francisco River Basin, Brazil. —Acta Zoologica (Stockholm) 93 : 48–56. This study presents a comparative analysis aiming to show the remarkable differences found in gonad morphology between six fish species from the genera Incertae Sedis in the Characidae family. Maturing ovaries of Astyanax fasciatus presented grayish color whilst the other species’ were yellowish in color. The cortical alveoli of the analyzed species are small (5.20 ± 1.44–8.28 ± 2.02 μm) and are formed by various discontinuous layers of vesicles except in the Bryconops affinis, where they are large (12.87 ± 2.41 μm) and form various continuous layers of vesicles. The pellucid zone is thin and varies from 2.44 ± 0.90 to 5.79 ± 1.90 μm and is thickest in B. affinis (10.57 ± 2.08 μm). Folds in the pellucid zone were observed in Astyanax bimaculatus, A. fasciatus, Hemigrammus marginatus, and Moenkhausia costae. The follicular cells of B. affinis are columnar (28.19 ± 13.71 μm), while they are squamous in other species (1.52 ± 0.64–2.12 ± 0.78 μm). This study showed that the morphologic and biometric characteristics of the B. affinis vitellogenic oocytes are statistically different than the analyzed other ones Incertae Sedis of the Characidae family. In advanced maturing testis of A. bimaculatus and A. fasciatus, the spermatozoa were embedded in acidophilic secretion which was gradually reabsorbed in the seminiferous tubules when males were partially spent.     

Involvement of stress kinase mitogen-activated protein kinase kinase 7 in regulation of mammalian circadian clock

The stress kinase mitogen-activated protein kinase kinase 7 (MKK7) is a specific activator of c-Jun N-terminal kinase (JNK), which controls various physiological processes, such as cell proliferation, apoptosis, differentiation, and migration. Here we show that genetic inactivation of MKK7 resulted in an extended period of oscillation in circadian gene expression in mouse embryonic fibroblasts. Exogenous expression in cultured mammalian cells of an MKK7-JNK fusion protein that functions as a constitutively active form of JNK induced phosphorylation of PER2, an essential circadian component. Furthermore, JNK interacted with PER2 at both the exogenous and endogenous levels, and MKK7-mediated JNK activation increased the half-life of PER2 protein by inhibiting its ubiquitination. Notably, the PER2 protein stabilization induced by MKK7-JNK fusion protein reduced the degradation of PER2 induced by casein kinase 1ε. Taken together, our results support a novel function for the stress kinase MKK7 as a regulator of the circadian clock in mammalian cells at steady state.     

AMF-26, a novel inhibitor of the Golgi system, targeting ADP-ribosylation factor 1 (Arf1) with potential for cancer therapy

ADP-ribosylation factor 1 (Arf1) plays a major role in mediating vesicular transport. Brefeldin A (BFA), a known inhibitor of the Arf1-guanine nucleotide exchange factor (GEF) interaction, is highly cytotoxic. Therefore, interaction of Arf1 with ArfGEF is an attractive target for cancer treatment. However, BFA and its derivatives have not progressed beyond the pre-clinical stage of drug development because of their poor bioavailability. Here, we aimed to identify novel inhibitors of the Arf1-ArfGEF interaction that display potent antitumor activity in vivo but with a chemical structure distinct from that of BFA. We exploited a panel of 39 cell lines (termed JFCR39) coupled with a drug sensitivity data base and COMPARE algorithm, resulting in the identification of a possible novel Arf1-ArfGEF inhibitor AMF-26, which differed structurally from BFA. By using a pulldown assay with GGA3-conjugated beads, we demonstrated that AMF-26 inhibited Arf1 activation. Subsequently, AMF-26 induced Golgi disruption, apoptosis, and cell growth inhibition. Computer modeling/molecular dynamics (MD) simulation suggested that AMF-26 bound to the contact surface of the Arf1-Sec7 domain where BFA bound. AMF-26 affected membrane traffic, including the cis-Golgi and trans-Golgi networks, and the endosomal systems. Furthermore, using AMF-26 and its derivatives, we demonstrated that there was a significant correlation between cell growth inhibition and Golgi disruption. In addition, orally administrated AMF-26 (83 mg/kg of body weight; 5 days) induced complete regression of human breast cancer BSY-1 xenografts in vivo, suggesting that AMF-26 is a novel anticancer drug candidate that inhibits the Golgi system, targeting Arf1 activation.     

Regulation of a proteinaceous elicitor-induced Ca2+ influx and production of phytoalexins by a putative voltage-gated cation channel, OsTPC1, in cultured rice cells

Pathogen/microbe- or plant-derived signaling molecules (PAMPs/MAMPs/DAMPs) or elicitors induce increases in the cytosolic concentration of free Ca(2+) followed by a series of defense responses including biosynthesis of antimicrobial secondary metabolites called phytoalexins; however, the molecular links and regulatory mechanisms of the phytoalexin biosynthesis remains largely unknown. A putative voltage-gated cation channel, OsTPC1 has been shown to play a critical role in hypersensitive cell death induced by a fungal xylanase protein (TvX) in suspension-cultured rice cells. Here we show that TvX induced a prolonged increase in cytosolic Ca(2+), mainly due to a Ca(2+) influx through the plasma membrane. Membrane fractionation by two-phase partitioning and immunoblot analyses revealed that OsTPC1 is localized predominantly at the plasma membrane. In retrotransposon-insertional Ostpc1 knock-out cell lines harboring a Ca(2+)-sensitive photoprotein, aequorin, TvX-induced Ca(2+) elevation was significantly impaired, which was restored by expression of OsTPC1. TvX-induced production of major diterpenoid phytoalexins and the expression of a series of diterpene cyclase genes involved in phytoalexin biosynthesis were also impaired in the Ostpc1 cells. Whole cell patch clamp analyses of OsTPC1 heterologously expressed in HEK293T cells showed its voltage-dependent Ca(2+)-permeability. These results suggest that OsTPC1 plays a crucial role in TvX-induced Ca(2+) influx as a plasma membrane Ca(2+)-permeable channel consequently required for the regulation of phytoalexin biosynthesis in cultured rice cells.     

Loss of ICAT gene function leads to arrest of ureteric bud branching and renal agenesis

ICAT, inhibitor of beta-catenin and T cell factor, or Ctnnbip1, is a negative regulator of the Wnt signaling pathway that interferes with the interaction between beta-catenin and T cell factor. Some ICAT-deficient (ICAT-/-) embryos exhibit unilateral or bilateral renal agenesis. In this study, we investigated developmental processes in the ICAT-/- kidney. ICAT was highly expressed in both the ureteric bud (UB) and the surrounding metanephric mesenchymal (MM) cells in the metanephros of embryonic day E11.5-E13.5 wild-type (ICAT+/+) mouse. In the E12.5-ICAT-/- metanephros, UB branching was delayed, and a T-shaped, bifurcated UB was frequently seen; this was never seen in the E12.5-ICAT+/+ metanephros. More apoptotic MM cells were detected in the ICAT-/- metanephros than in the ICAT+/+ metanephros. These results suggest that the loss of ICAT gene function causes the arrest of UB branching and the apoptotic death of MM cells, resulting in renal agenesis.     

Lyso-GM3, its dimer,and multimer: their synthesis,and their effect on epidermal growth factor-induced receptor tyrosine kinase

Glycosphingolipids, particularly gangliosides, are known to modulate growth factor receptor tyrosine kinase. A well-documented example is the inhibitory effect of GM3 on kinase associated with epidermal growth factor receptor (EGFR) in human epidermoid carcinoma A431 cells. Lyso-GM3 was detected as a minor component in A431 cells, and may function as an auxiliary factor in GM3-dependent inhibition of EGFR. We studied the inhibitory effect of chemically synthesized GM3, lyso-GM3, and its derivatives, on EGFR function, based on their interaction in membrane microdomain, with the following major findings: (1) GM3, EGFR, and caveolin coexist, but tetraspanins CD9 and CD82 are essentially absent, within the same low-density membrane fraction, separated by sucrose density gradient ultracentrifugation. (2) Strong interaction between EGFR and GM3 was indicated by increasing binding of EGFR to GM3-coated polystyrene beads, in a GM3 dose-dependent manner. Confocal microscopy results suggested that three components in the microdomain (GM3, EGFR, and caveolin) are closely associated. (3) Lyso-GM3 or lyso-GM3 dimer strongly inhibited EGFR kinase activity, in a dose-dependent manner, while lyso-GM3 trimer and tetramer did not. >50 μM lyso-GM3 was cytolytic, while >50 μM lyso-GM3 dimer was not cytolytic, yet inhibited EGFR kinase strongly. Thus, lyso-GM3 and its dimer exert an auxiliary effect on GM3-induced inhibition of EGFR kinase and cell growth, and lyso-GM3 dimer may be a good candidate for pharmacological inhibitor of epidermal tumor growth.     

Epistasis between hyperglycemic QTLs revealed in a double congenic of the OLETF rat

Glucose homeostasis is believed to be regulated by multiple genetic components, in addition to numerous external factors. It is therefore crucial to dissect and understand what roles each causative gene plays in maintaining proper glucose metabolism. In OLETF (Otsuka Long-Evans Tokushima Fatty) rat, a model of polygenic type 2 diabetes, at least 14 quantitative trait loci (QTLs) influencing plasma glucose levels were identified. In congenic strains some of the OLETF allelic variants were shown to increase glucose levels. In this study the focus was on two of the hyperglycemic loci, Nidd1/of and Nidd2/of. Congenic rats possessing OLETF genome fragment at either locus showed similar levels of mild hyperglycemia. A newly established double congenic rat showed a further aggravation of hyperglycemia. The Nidd1/of locus was also shown to function in the reduction of plasma leptin levels and fat weights, while the Nidd2/of locus led to increased plasma insulin and fat weights. Interestingly, both plasma leptin and fat weights reverted to the control levels in the double congenic rat. These results indicate that there is an epistatic interaction between the two loci. However, it is unlikely that the abnormal level of enhanced glucose homeostasis is mediated, at least not directly, by leptin or fat mass.