The F box protein S phase kinase-associated protein 2 regulates adipose mass and adipocyte number in vivo

Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin‐dependent kinase inhibitor p27 regulates activity of cyclin/cyclin‐dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1‐Cullin‐F‐box protein) complex targets proteins such as p27 for ubiquitin‐proteosome degradation; the F box protein S phase kinase‐associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2^?/?) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild‐type (WT), Skp2^?/?, and p27^?/?Skp2^?/? mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2^?/? fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2^?/? mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27^?/?Skp2^?/? double knockout mice. The Skp2^?/? decrements in adipocyte number and fat pad mass were totally reversed in p27^?/?Skp2^?/? mice. Adipogenesis was inhibited in MEFs from Skp2^?/? vs. WT mice, and this inhibition was absent in MEFs from p27^?/?Skp2^?/? mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia.     

Aplysia temptin - the 'glue' in the water-borne attractin pheromone complex

Temptin, a component of the complex of water-borne protein pheromones that stimulate attraction and mating behavior in the marine mollusk Aplysia, has sequence homology to the epidermal growth factor (EGF)-like domains of higher organisms that mediate protein-cell surface contact during fertilization and blood coagulation. In this work, recombinant temptin for structural and functional studies was produced in Escherichia coli using a cold shock promoter and purified by RP-HPLC. CD spectra confirmed a predominantly beta-sheet structure. Two disulfide bonds were determined via limited proteolysis and MS. One internal disulfide (Cys57-Cys77) was predicted from initial alignments with class I EGF-like domains; the second, between Cys18 and Cys103, could protect temptin against proteolysis in seawater and stabilize its interacting surface. A three-dimensional model of temptin was prepared with our MPACK suite, based on the Ca(2+)-binding, EGF-like domain of the extracellular matrix protein fibrillin. Two temptin residues, Trp52 and Trp79, which align with cysteine residues conserved in fibrillins, lie adjacent to and could stabilize the disulfide bonds and a proposed metal-binding loop. The water-borne pheromone attractin in egg cordon eluates is complexed with other proteins. Docking results with our model and the NMR structure of attractin suggest that one face of temptin interacts with the pheromone, perhaps controlling its access to the cellular receptors. Gel shifts confirmed that temptin complexes with wild-type attractin. These results indicate that temptin, analogous to the role of fibrillin in controlling transforming growth factor-beta concentration, modulates pheromone signaling by direct binding to attractin.     

Analysis of CCN2 promoter activity in PANC-1 cells: regulation by ras/MEK/ERK

Connective tissue growth factor (CTGF, CCN2) is overexpressed in pancreatic cancer. We mapped the minimal CCN2 promoter active in PANC-1 cells, a human pancreatic cancer cell line. Within this region, Sp1, BCE-1 and Ets elements were important for the activity of the CCN2 promoter. Constitutive hyperactivated ras is a hallmark of cancers, including that of the pancreas. Treatment of PANC-1 cells with the MEK inhibitor U0126 or the Sp1 inhibitor mithramycin reduced CCN2 mRNA and promoter activity. Mutation of the BCE-1, but not Sp1 or Ets, site abolished the responsiveness of the CCN2 promoter to U0126. Overexpressing constitutively active MEK1 or ras activated CCN2 promoter activity. Thus CCN2 is likely to act downstream of ras in PANC-1 cells. CCN2 is overexpressed in cancer cells. Activated ras/MEK/ERK is a hallmark of cancer, and we have shown that the elevated CCN2 expression in pancreatic cancer cells is dependent on this pathway.     

Phosphatidylinositol 4-kinase III-beta is required for Golgi maintenance and cytokinesis in Trypanosoma brucei

The parasitic protozoan Trypanosoma brucei contains two type III phosphatidylinositol 4-kinases (alpha and beta). We have cloned the gene encoding the T. brucei type III phosphatidylinositol 4-kinase beta (TbPI4KIII-beta), expressed the protein in COS-7 cells, and confirmed that the protein catalyzes the phosphorylation of phosphatidylinositol. Depletion of TbPI4KIII-beta in procyclic T. brucei by RNA interference (RNAi) resulted in inhibition of cell growth and a distorted cellular morphology. RNAi cells had a distorted Golgi apparatus, and lysosomal and flagellar pocket proteins were mislocalized. Ultrastructural analysis revealed the internal accumulation of a heterogeneous population of vesicles, abnormal positioning of organelles, and a loss of cell polarity. Scanning electron microcopy revealed a twisted phenotype, and dividing cells often exhibited a detached daughter flagellum and lacked a cleavage furrow. Cell cycle analysis confirmed that cells depleted of TbPI4KIII-beta have a postmitotic cytokinesis block that occurs after a single round of mitosis, suggestive of a specific cell cycle block. In summary, TbPI4KIII-beta is an essential protein in procyclic T. brucei, required for maintenance of Golgi structure, protein trafficking, normal cellular shape, and cytokinesis.     

Inhalation developmental neurotoxicity study of ethylbenzene in Crl-CD rats

BACKGROUND: This study was conducted to evaluate the potential adverse effects of whole-body inhalation exposure of F0 and F1 parental animals from a 2-generation reproduction study of ethylbenzene on nervous system functional and/or morphologic end points in the F2 offspring from four groups of male and female Crl:CD (SD)IGS BR rats. METHODS: Thirty rats/sex/group for F0 and 25/sex/group for F1 were exposed to 0, 25, 100, and 500 ppm ethylbenzene for six hours daily for at least 70 consecutive days prior to mating for the F0 and F1 generations. Inhalation exposure for the F0 and F1 females continued throughout mating and gestation through Gestation Day (GD) 20. On lactation days (LD) 1-4, the F0 and F1 females received no inhalation exposure, but instead were administered ethylbenzene in corn oil via oral gavage at dosages estimated to result in similar internal maternal exposure based upon PBPK modeling estimates (0, 26, 90, and 342 mg/kg/day, respectively, divided into three equal doses, approximately two hours apart). Inhalation exposure of the F0 and F1 females was reinitiated on LD 5 and continued through weaning on postnatal day (PND) 21. Survival, body weights, and physical landmarks were assessed in selected F2 offspring. Neurobehavioral development of one F2-generation treatment derived offspring/sex/litter was assessed in a functional observational battery (FOB; PND 4, 11, 22, 45, and 60), motor activity sessions (PND 13, 17, 21, and 61), acoustic startle testing (PND 20 and 60), a Biel water maze learning and memory task (initiated on PND 26 or 62), and in evaluations of whole-brain measurements and brain morphometric and histologic assessments (PND 21 and 72). RESULTS: There were no adverse effects on reproductive performance in either the F0 or F1 parental generations exposed to up to 500 ppm ethylbenzene [Faber et al. Birth Defects Res Part B 77:10-21, 2006]. In the current developmental neurotoxicity component, parental ethylbenzene exposure did not adversely affect offspring survival, clinical condition, body weight parameters, or acquisition of developmental landmarks of the F2-generation treatment derived offspring. There were no alterations in FOB parameters, motor activity counts, acoustic startle endpoints, or Biel water maze performance in offspring attributed to parental ethylbenzene exposure. A few isolated instances of statistically significant differences obtained in the treatment-derived groups occurred sporadically, and were attributed to unusual patterns of development and/or behavior in the concurrent control group. There were no exposure-related differences in any neuropathology parameters in the F2-generation treatment derived offspring. CONCLUSIONS: The no observed adverse effect level (NOAEL) for maternal reproductive toxicity, developmental toxicity, and developmental neurotoxicity in this study was considered to be 500 ppm/342 mg/kg/day ethylbenzene, the highest exposure level tested in the study.     

Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination

Nonhomologous end joining (NHEJ) eliminates DNA double-strand breaks (DSBs) in bacteria and eukaryotes. In Saccharomyces cerevisiae, there are pairwise physical interactions among the core complexes of the NHEJ pathway, namely Yku70-Yku80 (Ku), Dnl4-Lif1 and Mre11-Rad50-Xrs2 (MRX). However, MRX also has a key role in the repair of DSBs by homologous recombination (HR). Here we have examined the assembly of NHEJ complexes at DSBs biochemically and by chromatin immunoprecipitation. Ku first binds to the DNA end and then recruits Dnl4-Lif1. Notably, Dnl4-Lif1 stabilizes the binding of Ku to in vivo DSBs. Ku and Dnl4-Lif1 not only initiate formation of the nucleoprotein NHEJ complex but also attenuate HR by inhibiting DNA end resection. Therefore, Dnl4-Lif1 plays an important part in determining repair pathway choice by participating at an early stage of DSB engagement in addition to providing the DNA ligase activity that completes NHEJ.