Functional Inactivation of Drosophila GCK Orthologs Causes Genomic Instability and Oxidative Stress in a Fly Model of MODY-2

Maturity-onset diabetes of the young (MODY) type 2 is caused by heterozygous inactivating mutations in the gene encoding glucokinase (GCK), a pivotal enzyme for glucose homeostasis. In the pancreas GCK regulates insulin secretion, while in the liver it promotes glucose utilization and storage. We showed that silencing the Drosophila GCK orthologs Hex-A and Hex-C results in a MODY-2-like hyperglycemia. Targeted knock-down revealed that Hex-A is expressed in insulin producing cells (IPCs) whereas Hex-C is specifically expressed in the fat body. We showed that Hex-A is essential for insulin secretion and it is required for Hex-C expression. Reduced levels of either Hex-A or Hex-C resulted in chromosome aberrations (CABs), together with an increased production of advanced glycation end-products (AGEs) and reactive oxygen species (ROS). This result suggests that CABs, in GCK depleted cells, are likely due to hyperglycemia, which produces oxidative stress through AGE metabolism. In agreement with this hypothesis, treating GCK-depleted larvae with the antioxidant vitamin B6 rescued CABs, whereas the treatment with a B6 inhibitor enhanced genomic instability. Although MODY-2 rarely produces complications, our data revealed the possibility that MODY-2 impacts genome integrity.     

Physical and functional association of cortactin with Syk in human leukemic cell line K562

Human leukemic cell line K562 is induced to differentiate into the megakaryocytic lineage by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA). We demonstrate here that TPA stimulation increases tyrosine phosphorylation of an 80-kDa protein at an early stage of megakaryocytic differentiation and that this 80-kDa protein is identical with cortactin. Since tyrosine kinase Syk was activated by TPA stimulation, we examined the possibility that cortactin is a potential substrate of Syk in K562 cells. TPA-induced tyrosine phosphorylation of cortactin was decreased profoundly by overexpression of dominant-negative Syk. Furthermore, cortactin was associated with Syk even before TPA stimulation. Since cortactin was previously referred as an 80/85-kilodalton pp60src substrate, we examined the association between Src and cortactin, whereas its association could not be detected. These data suggest that Syk phosphorylates cortactin in K562 cells upon TPA treatment.     

The effects of R-75,317 on antiglomerular basement membrane glomerulonephritis in rats

Platelet-activating factor (PAF) is a potent inflammatory mediator which is released by various inflammatory cells and produced by certain tissues, including the kidney. PAF has been shown to increase glomerular permeability to protein and to decrease glomerular filtration rate (GFR) by contracting mesangium. On the basis of these observations, it has been suspected that PAF may play a role as mediator of glomerular damage in glomerular nephritis. To examine this possibility, we studied the effects of a specific PAF antagonist, R-75,317, on the development of an experimental model of anti-glomerular basement membrane (anti-GBM) glomerulonephritis. Glomerulonephritis was initiated by injecting rabbit anti-rat GBM serum into rats. Proteinuria gradually developed after serum injection, plateaued at week 2, and remained at the high level of week 2 throughout the experimental period (6 wk). Chronic treatment with R-75,317 (10 mg/kg/day i.p.) tended to delay the onset of proteinuria and significantly accelerated the recovery phase. Creatinine clearance (Ccr) fell to 40% at week 3. R-75,317 treatment completely prevented this decline of Ccr. Histological changes in this model (glomerular hypertrophy, proliferation of mesangial matrix and interstitial fibrosis) were also ameliorated by the R-75,317 treatment. The results suggest that PAF may play a role in the development of glomerulonephritis and that PAF antagonists could be used in the treatment of human renal disease. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, May 1989.     

A Model of Weekly Labor Participation for a Belgian Synthetic Population

This paper presents a model of labor participation calibrated on a weekly basis; part-time and full-time employments are also considered. By applying the theory of random utility maximization we model households’ choices. In order to have a good temporal and spatial coverage the model is calibrated on three datasets, extracted from both national and regional travel surveys. The results are applied to synthetic households that reproduce the Belgian population. The proposed innovative methodology simulates synthetic agents by accounting both for households’ and individuals’ characteristics, while not suffering from the problem of the “zero cell value”. The results indicate that there is major day-to-day variability in working activity participation; in particular, on Wednesdays and Fridays the number of households working part-time or not working is particularly high. This is consistent with what was expected by the analysts. Working participation is a fundamental component in activity based models where work is considered a skeletal activity. The tools developed here can be useful to study how changes in population characteristics (i.e. increases of flexible working arrangements and of the number of women in the work force) affect activity participation and travel patterns.     

The GAUGAA Motif Is Responsible for the Binding between circSMARCA5 and SRSF1 and Related Downstream Effects on Glioblastoma Multiforme Cell Migration and Angiogenic Potential

Circular RNAs (circRNAs) are a large class of RNAs with regulatory functions within cells. We recently showed that circSMARCA5 is a tumor suppressor in glioblastoma multiforme (GBM) and acts as a decoy for Serine and Arginine Rich Splicing Factor 1 (SRSF1) through six predicted binding sites (BSs). Here we characterized RNA motifs functionally involved in the interaction between circSMARCA5 and SRSF1. Three different circSMARCA5 molecules (Mut1, Mut2, Mut3), each mutated in two predicted SRSF1 BSs at once, were obtained through PCR-based replacement of wild-type (WT) BS sequences and cloned in three independent pcDNA3 vectors. Mut1 significantly decreased its capability to interact with SRSF1 as compared to WT, based on the RNA immunoprecipitation assay. In silico analysis through the “Find Individual Motif Occurrences” (FIMO) algorithm showed GAUGAA as an experimentally validated SRSF1 binding motif significantly overrepresented within both predicted SRSF1 BSs mutated in Mut1 (q-value = 0.0011). U87MG and CAS-1, transfected with Mut1, significantly increased their migration with respect to controls transfected with WT, as revealed by the cell exclusion zone assay. Immortalized human brain microvascular endothelial cells (IM-HBMEC) exposed to conditioned medium (CM) harvested from U87MG and CAS-1 transfected with Mut1 significantly sprouted more than those treated with CM harvested from U87MG and CAS-1 transfected with WT, as shown by the tube formation assay. qRT-PCR showed that the intracellular pro- to anti-angiogenic Vascular Endothelial Growth Factor A (VEGFA) mRNA isoform ratio and the amount of total VEGFA mRNA secreted in CM significantly increased in Mut1-transfected CAS-1 as compared to controls transfected with WT. Our data suggest that GAUGAA is the RNA motif responsible for the interaction between circSMARCA5 and SRSF1 as well as for the circSMARCA5-mediated control of GBM cell migration and angiogenic potential.     

BRAF Gene and Melanoma: Back to the Future

As widely acknowledged, 40–50% of all melanoma patients harbour an activating BRAF mutation (mostly BRAF V600E). The identification of the RAS–RAF–MEK–ERK (MAP kinase) signalling pathway and its targeting has represented a valuable milestone for the advanced and, more recently, for the completely resected stage III and IV melanoma therapy management. However, despite progress in BRAF-mutant melanoma treatment, the two different approaches approved so far for metastatic disease, immunotherapy and BRAF+MEK inhibitors, allow a 5-year survival of no more than 60%, and most patients relapse during treatment due to acquired mechanisms of resistance. Deep insight into BRAF gene biology is fundamental to describe the acquired resistance mechanisms (primary and secondary) and to understand the molecular pathways that are now being investigated in preclinical and clinical studies with the aim of improving outcomes in BRAF-mutant patients.     

Role of anionic and nonionic surfactants on the control of particle size and latex colloidal stability in the seeded emulsion polymerization of butyl methacrylate

The evolution of the main colloidal parameters in the seeded starved‐feed semi‐continuous emulsion polymerization of butyl methacrylate (BMA) was investigated, with the main purpose of assessing the effectiveness of the semi‐empirical relationship S = K · S_S · ΔA/A_S as a tool to define the surfactant/monomer feed ratio (∝ K) best suited to achieve a target particle size. In particular, the effect of the type and amount of surfactant [i.e., anionic, sodium dodecyl sulfate (SDS), or nonionic, Brij 58P] added during the semi‐continuous stage was considered. Coagulum formation was never observed under the adopted experimental conditions. To detect the occurrence of secondary nucleation or particle aggregation, or both, the particle size and number of particles, the surface tension and the particle surface coverage ratio were correlated. The best results were obtained with SDS and 0.8 ≤ K ≤ 3. In fact, under the selected experimental conditions, only with SDS did the number of particles remain nearly constant throughout the polymerization at the value defined by the seed latex; the particle size distribution was highly monodisperse, and the final particle diameter closely matched the calculated one (～ 120 nm). The above semi‐empirical relationship based on the adjustable parameter K was validated by running test polymerizations aimed at lattices with well‐defined particle size. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 3083–3094, 2006