Prevalence of BRCA1 Mutations in Familial and Sporadic Greek Ovarian Cancer Cases

Germline mutations in the BRCA1 and BRCA2 genes contribute to approximately 18% of hereditary ovarian cancers conferring an estimated lifetime risk from 15% to 50%. A variable incidence of mutations has been reported for these genes in ovarian cancer cases from different populations. In Greece, six mutations in BRCA1 account for 63% of all mutations detected in both BRCA1 and BRCA2 genes. This study aimed to determine the prevalence of BRCA1 mutations in a Greek cohort of 106 familial ovarian cancer patients that had strong family history or metachronous breast cancer and 592 sporadic ovarian cancer cases. All 698 patients were screened for the six recurrent Greek mutations (including founder mutations c.5266dupC, p.G1738R and the three large deletions of exon 20, exons 23–24 and exon 24). In familial cases, the BRCA1 gene was consequently screened for exons 5, 11, 12, 20, 21, 22, 23, 24. A deleterious BRCA1 mutation was found in 43/106 (40.6%) of familial cancer cases and in 27/592 (4.6%) of sporadic cases. The variant of unknown clinical significance p.V1833M was identified in 9/698 patients (1.3%). The majority of BRCA1 carriers (71.2%) presented a high-grade serous phenotype. Identifying a mutation in the BRCA1 gene among breast and/or ovarian cancer families is important, as it enables carriers to take preventive measures. All ovarian cancer patients with a serous phenotype should be considered for genetic testing. Further studies are warranted to determine the prevalence of mutations in the rest of the BRCA1 gene, in the BRCA2 gene, and other novel predisposing genes for breast and ovarian cancer.     

Extra-adipocyte leptin release in human obesity and its relation to sympathoadrenal function

The link between the human sympathoadrenalmedullary system and the adipocyte hormone leptin is controversial. We measured total and regional norepinephrine spillover, epinephrine secretion rate, and extra-adipocyte leptin release in 22 lean [body mass index (BMI) < 26] and 20 obese (BMI > 28) normotensive men who underwent arterial and central venous catheterization. Because plasma clearance of leptin is primarily by renal removal, for men at steady state we could estimate whole body leptin release to plasma from renal plasma leptin extraction. Whole body leptin release was 1,950 +/- 643 (means +/- SE) ng/min in obese men and 382 +/- 124 ng/min in lean men (P < 0.05). Total and renal norepinephrine spillover rates correlated directly with whole body leptin secretion rate. Leptin is released from multiple nonadipocyte sites, which we tested by use of simultaneous arteriovenous blood sampling. We found a surprisingly large contribution of brain leptin release to the plasma leptin pool, 529 +/- 175 ng/min (> 40% whole body leptin release), with greater leptin release in obese than in lean men, 935 +/- 321 vs. 160 +/- 59 ng/min (P = 0.045). In parallel with leptin measurements, we also quantified brain serotonin turnover and jugular overflow of neuropeptide Y (NPY). Brain serotonin turnover was higher in obese than in lean men, 227 +/- 112 vs. 21 +/- 14 ng/min (P = 0.019), as was overflow of NPY from the brain, 12.9 +/- 1.4 vs. 5.3 +/- 2.2 ng/min (P = 0.042). These results suggest that leptin is released within the brain and at an increased rate in obese humans, in whom activation of brain serotonergic and NPY mechanisms also exists.     

Functional Interactions between BM88/Cend1, Ran-Binding Protein M and Dyrk1B Kinase Affect Cyclin D1 Levels and Cell Cycle Progression/Exit in Mouse Neuroblastoma Cells

BM88/Cend1 is a neuronal-lineage specific modulator with a pivotal role in coordination of cell cycle exit and differentiation of neuronal precursors. In the current study we identified the signal transduction scaffolding protein Ran-binding protein M (RanBPM) as a BM88/Cend1 binding partner and showed that BM88/Cend1, RanBPM and the dual specificity tyrosine-phosphorylation regulated kinase 1B (Dyrk1B) are expressed in mouse brain as well as in cultured embryonic cortical neurons while RanBPM can form complexes with either of the two other proteins. To elucidate a potential mechanism involving BM88/Cend1, RanBPM and Dyrk1B in cell cycle progression/exit, we transiently co-expressed these proteins in mouse neuroblastoma Neuro 2a cells. We found that the BM88/Cend1-dependent or Dyrk1B-dependent down-regulation of cyclin D1 is reversed following their functional interaction with RanBPM. More specifically, functional interaction of RanBPM with either BM88/Cend1 or Dyrk1B stabilizes cyclin D1 in the nucleus and promotes 5-bromo-2''-deoxyuridine (BrdU) incorporation as a measure of enhanced cell proliferation. However, the RanBPM-dependent Dyrk1B cytosolic retention and degradation is reverted in the presence of Cend1 resulting in cyclin D1 destabilization. Co-expression of RanBPM with either BM88/Cend1 or Dyrk1B also had a negative effect on Neuro 2a cell differentiation. Our results suggest that functional interactions between BM88/Cend1, RanBPM and Dyrk1B affect the balance between cellular proliferation and differentiation in Neuro 2a cells and indicate that a potentially similar mechanism may influence cell cycle progression/exit and differentiation of neuronal precursors.     

Nonyloxytryptamine mimics polysialic acid and modulates neuronal and glial functions in cell culture

Polysialic acid (PSA) is a major regulator of cell–cell interactions in the developing nervous system and in neural plasticity in the adult. As a polyanionic molecule with high water‐binding capacity, PSA increases the intercellular space generating permissive conditions for cell motility. PSA enhances stem cell migration and axon path finding and promotes repair in the lesioned peripheral and central nervous systems, thus contributing to regeneration. As a next step in developing an improved PSA‐based approach to treat nervous system injuries, we searched for small organic compounds that mimic PSA and identified as a PSA mimetic 5‐nonyloxytryptamine oxalate, described as a selective 5‐hydroxytryptamine receptor 1B (5‐HT_1B) agonist. Similar to PSA, 5‐nonyloxytryptamine binds to the PSA‐specific monoclonal antibody 735, enhances neurite outgrowth of cultured primary neurons and process formation of Schwann cells, protects neurons from oxidative stress, reduces migration of astrocytes and enhances myelination in vitro. Furthermore, nonyloxytryptamine treatment enhances expression of the neural cell adhesion molecule (NCAM) and its polysialylated form PSA‐NCAM and reduces expression of the microtubule‐associated protein MAP2 in cultured neuroblastoma cells. These results demonstrate that 5‐nonyloxytryptamine mimics PSA and triggers PSA‐mediated functions, thus contributing to the repertoire of molecules with the potential to improve recovery in acute and chronic injuries of the mammalian peripheral and central nervous systems.

     

RecB-dependent mutator phenotype in Neisseria meningitidis strains naturally defective in mismatch repair

Several invasive serogroup B meningococcal strains phylogenetically related to the lineage III (ET-24) exhibited a mutator phenotype as shown by mutagenicity assay using rifampicin-resistance as a selection marker. Hypermutation was associated to the presence of defective mutL alleles that were genetically characterized. Interestingly, the mutator phenotype was suppressed when a non-functional recB(ET-37) allele, derived from ET-37 meningococcal strains, replaced the functional recB allele in a lineage III strain. In contrast, the same gene replacement did not affect mutation frequencies in a mismatch repair-proficient strain. These results suggested that in MutL-deficient strains spontaneous mutations mostly arise from post-replicative DNA synthesis associated to the activity of the RecBCD recombination pathway.     

Plasmacytoid dendritic cells migrate in afferent skin lymph

Conventional dendritic cells enter lymph nodes by migrating from peripheral tissues via the lymphatic route, whereas plasmacytoid dendritic cells (pDC), also called IFN-producing cells (IPC), are described to gain nodes from blood via the high endothelial venules. We demonstrate here that IPC/pDC migrate in the afferent lymph of two large mammals. In sheep, injection of type A CpG oligodinucleotide (ODN) induced lymph cells to produce type I IFN. Furthermore, low-density lymph cells collected at steady state produced type I IFN after stimulation with type A CpG ODN and enveloped viruses. Sheep lymph IPC were found within a minor B(neg)CD11c(neg) subset expressing CD45RB. They presented a plasmacytoid morphology, expressed high levels of TLR-7, TLR-9, and IFN regulatory factor 7 mRNA, induced IFN-gamma production in allogeneic CD4(pos) T cells, and differentiated into dendritic cell-like cells under viral stimulation, thus fulfilling criteria of bona fide pDC. In mini-pig, a CD4(pos)SIRP(pos) subset in afferent lymph cells, corresponding to pDC homologs, produced type I IFN after type A CpG-ODN triggering. Thus, pDC can link innate and acquired immunity by migrating from tissue to draining node via lymph, similarly to conventional dendritic cells.     

The role of phosphoinositide 3-kinase C2alpha in insulin signaling

The members of the class II phosphoinositide 3-kinase (PI3K) family can be activated by several stimuli, indicating that these enzymes can regulate many intracellular processes. Nevertheless, to date, there has been no definitive identification of their in vivo product, their mechanism(s) of activation, or their precise intracellular roles. By metabolic labeling, we here identify phosphatidylinositol 3-phosphate as the sole in vivo product of the insulin-dependent activation of PI3K-C2alpha, confirming the emerging role of such a phosphoinositide in signaling. We demonstrate that activation of PI3K-C2alpha involves its recruitment to the plasma membrane and that activation is mediated by the GTPase TC10. This is the first report showing a membrane targeting-mediated mechanism of activation for PI3K-C2alpha and that a small GTP-binding protein can activate a class II PI3K isoform. We also demonstrate that PI3K-C2alpha contributes to maximal insulin-induced translocation of the glucose transporter GLUT4 to the plasma membrane and subsequent glucose uptake, definitely assessing the role of this enzyme in insulin signaling.     

Mice with a disruption of the imprinted Grb10 gene exhibit altered body composition, glucose homeostasis, and insulin signaling during postnatal life

The Grb10 adapter protein is capable of interacting with a variety of receptor tyrosine kinases, including, notably, the insulin receptor. Biochemical and cell culture experiments have indicated that Grb10 might act as an inhibitor of insulin signaling. We have used mice with a disruption of the Grb10 gene (Grb10Delta2-4 mice) to assess whether Grb10 might influence insulin signaling and glucose homeostasis in vivo. Adult Grb10Delta2-4 mice were found to have improved whole-body glucose tolerance and insulin sensitivity, as well as increased muscle mass and reduced adiposity. Tissue-specific changes in insulin receptor tyrosine phosphorylation were consistent with a model in which Grb10, like the closely related Grb14 adapter protein, prevents specific protein tyrosine phosphatases from accessing phosphorylated tyrosines within the kinase activation loop. Furthermore, insulin-induced IRS-1 tyrosine phosphorylation was enhanced in Grb10Delta2-4 mutant animals, supporting a role for Grb10 in attenuation of signal transmission from the insulin receptor to IRS-1. We have previously shown that Grb10 strongly influences growth of the fetus and placenta. Thus, Grb10 forms a link between fetal growth and glucose-regulated metabolism in postnatal life and is a candidate for involvement in the process of fetal programming of adult metabolic health.     

Maternally-inherited Grb10 reduces placental size and efficiency

The control of foetal growth is poorly understood and yet it is critically important that at birth the body has attained appropriate size and proportions. Growth and survival of the mammalian foetus is dependent upon a functional placenta throughout most of gestation. A few genes are known that influence both foetal and placental growth and might therefore coordinate growth of the conceptus, including the imprinted Igf2 and Grb10 genes. Grb10 encodes a signalling adapter protein, is expressed predominantly from the maternally-inherited allele and acts to restrict foetal and placental growth. Here, we show that following disruption of the maternal allele in mice, the labyrinthine volume was increased in a manner consistent with a cell-autonomous function of Grb10 and the enlarged placenta was more efficient in supporting foetal growth. Thus, Grb10 is the first example of a gene that acts to limit placental size and efficiency. In addition, we found that females inheriting a mutant Grb10 allele from their mother had larger litters and smaller offspring than those inheriting a mutant allele from their father. This grandparental effect suggests Grb10 can influence reproductive strategy through the allocation of maternal resources such that offspring number is offset against size.