Netherlands Twin Register: a focus on longitudinal research.

In 1986 we began The Netherlands Twin Register (NTR) by recruiting young twins and multiples a few weeks or months after birth. Currently we register around 50% of all newborn multiples in The Netherlands. Their parents receive a questionnaire at registration and afterwards when the children are 2, 3, 5, 7, 10 and 12 years of age. Teachers are asked to rate the behavior of the children at ages 7, 10 and 12 years. Adolescent and young-adult twins were recruited through City Councils in the early 1990s. These twins, their parents and siblings participate in longitudinal survey studies that include items about health, fertility, lifestyle, addiction, personality and psychopathology, religion, socioeconomic status, and educational attainment. The total number of twins and multiples registered with the NTR is currently over 60,000. Subgroups of twins and siblings take part in studies of cognitive development, brain function and neuropsychological indices of attention processes, and molecular genetic studies of classical and behavioral cardiovascular risk factors. DNA samples are currently collected in selected twin families for two large linkage studies, which aim to find QTLs for anxious depression and for nicotine addiction. Sisters who are mothers of DZ twins contribute DNA samples for a linkage study of DZ twinning. Large cohorts of phenotyped family members from the general population are very valuable for genetic epidemiological studies and permit selection of informative families for gene finding studies.     

FKBP12 Modulation of the Binding of the Skeletal Ryanodine Receptor onto the II-III Loop of the Dihydropyridine Receptor

In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ of the II-III loop of the skeletal DHPR α₁-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu⁷²⁴-Pro⁷⁶⁰ antagonizes this effect. Two peptides, covering these sequences (peptide A_Sk and C_Sk, respectively) were immobilized on polystyrene beads. We demonstrate that peptide A_Sk binds to the skeletal isoform of RyR (RyR1) whereas peptide C_Sk does not. Using surface plasmon resonance detection, we show that 1) domain Thr⁶⁷¹-Leu⁶⁹⁰ is the only sequence of the II-III loop binding with RyR1 and 2) the interaction of peptide A_Sk with RyR1 is not modulated by Ca²⁺ (pCa 9-2) nor by Mg²⁺ (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC₅₀ = 10 nM) and inhibited by both rapamycin (IC₅₀ = 5 nM) and FK506. Peptide A_Sk induces a 300% increase of the opening probability of the RyR1 incorporated in lipid bilayer. Removal of FKBP12 from RyR1 completely abolishes this effect of domain A_Sk on RyR1 channel behavior. These results demonstrate a direct interaction of the RyR1 with the discrete domain of skeletal DHPR α₁-subunit corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ and show that the association of FKBP12 with RyR1 specifically modulates this interaction.     

A new translational repression element and unusual transcriptional control regulate expression of don juan during Drosophila spermatogenesis.

The Drosophila don juan (dj) gene encodes a basic protein that is expressed solely in the male germline and shows structural similarities to the linker histone H1. Don Juan is located in two different subcellular structures: in the nucleus during the phase of chromatin condensation and later in the mitochondrial derivatives starting with spermatid individualization. The don juan gene is transcribed in primary spermatocytes under the control of 23 bp upstream in combination with downstream sequences. During meiotic stages and in early spermatid stages don juan mRNA is translationally repressed for several days. Analysis of male sterile mutants which fail to undergo meiosis shows that release of dj mRNA from translational repression is independent of meiosis. In gel retardation assays 60 nucleotides at the end of the dj leader form four major complexes with proteins that were extracted from testes but not with protein extracts from ovaries. Transformation studies prove that in vivo 35 bp within that region of the dj mRNA is essential to confer translational repression. UV cross-linking studies show that a 62 kDa protein specifically binds to the same region within the 5′ untranslated region. The dj translational repression element, TRE, is distinct from the translational control element, TCE, described earlier for all members of the Mst(3)CGP gene family. Moreover, expression studies in several male sterile mutants reveal that don juan mRNA is translated in earlier developmental stages during sperm morphogenesis than the Mst(3)CGP mRNAs. This proves that translational activation of dormant mRNAs in spermatogenesis occurs at different time-points which are characteristic for each gene, an essential feature for coordinated sperm morphogenesis.     

SeqHound: biological sequence and structure database as a platform for bioinformatics research

Background  

SeqHound has been developed as an integrated biological sequence, taxonomy, annotation and 3-D structure database system. It provides a high-performance server platform for bioinformatics research in a locally-hosted environment.     

Rab4b Is a Small GTPase Involved in the Control of the Glucose Transporter GLUT4 Localization in Adipocyte

Background

Endosomal small GTPases of the Rab family, among them Rab4a, play an essential role in the control of the glucose transporter GLUT4 trafficking, which is essential for insulin-mediated glucose uptake. We found that adipocytes also expressed Rab4b and we observed a consistent decrease in the expression of Rab4b mRNA in human and mice adipose tissue in obese diabetic states. These results led us to study this poorly characterized Rab member and its potential role in glucose transport.

Methodology/Principal Findings

We used 3T3-L1 adipocytes to study by imaging approaches the localization of Rab4b and to determine the consequence of its down regulation on glucose uptake and endogenous GLUT4 location. We found that Rab4b was localized in endosomal structures in preadipocytes whereas in adipocytes it was localized in GLUT4 and in VAMP2-positive compartments, and also in endosomal compartments containing the transferrin receptor (TfR). When Rab4b expression was decreased with specific siRNAs by two fold, an extent similar to its decrease in obese diabetic subjects, we observed a small increase (25%) in basal deoxyglucose uptake and a more sustained increase (40%) in presence of submaximal and maximal insulin concentrations. This increase occurred without any change in GLUT4 and GLUT1 expression levels and in the insulin signaling pathways. Concomitantly, GLUT4 but not TfR amounts were increased at the plasma membrane of basal and insulin-stimulated adipocytes. GLUT4 seemed to be targeted towards its non-endosomal sequestration compartment.

Conclusion/Significance

Taken our results together, we conclude that Rab4b is a new important player in the control of GLUT4 trafficking in adipocytes and speculate that difference in its expression in obese diabetic states could act as a compensatory effect to minimize the glucose transport defect in their adipocytes.     

Transfer of dysbiotic gut microbiota has beneficial effects on host liver metabolism

Gut microbiota dysbiosis has been implicated in a variety of systemic disorders, notably metabolic diseases including obesity and impaired liver function, but the underlying mechanisms are uncertain. To investigate this question, we transferred caecal microbiota from either obese or lean mice to antibiotic‐free, conventional wild‐type mice. We found that transferring obese‐mouse gut microbiota to mice on normal chow (NC) acutely reduces markers of hepatic gluconeogenesis with decreased hepatic PEPCK activity, compared to non‐inoculated mice, a phenotypic trait blunted in conventional NOD2 KO mice. Furthermore, transferring of obese‐mouse microbiota changes both the gut microbiota and the microbiome of recipient mice. We also found that transferring obese gut microbiota to NC‐fed mice then fed with a high‐fat diet (HFD) acutely impacts hepatic metabolism and prevents HFD‐increased hepatic gluconeogenesis compared to non‐inoculated mice. Moreover, the recipient mice exhibit reduced hepatic PEPCK and G6Pase activity, fed glycaemia and adiposity. Conversely, transfer of lean‐mouse microbiota does not affect markers of hepatic gluconeogenesis. Our findings provide a new perspective on gut microbiota dysbiosis, potentially useful to better understand the aetiology of metabolic diseases.     

Safety of trivalent chromium complexes: no evidence for DNA damage in human HaCaT keratinocytes

Several studies have demonstrated beneficial effects of supplemental trivalent Cr in subjects with reduced insulin sensitivity with no documented signs of toxicity. However, recent studies have questioned the safety of supplemental trivalent Cr complexes. The objective of this study was to evaluate the cytotoxic and genotoxic potential of the Cr(III) complexes (histidinate, picolinate, and chloride) used as nutrient supplements compared with Cr(VI) dichromate. The cytotoxic and genotoxic effects of the Cr complexes were assessed in human HaCaT keratinocytes. The concentrations of Cr required to decrease cell viability were assessed by determining the ability of a keratinocyte cell line (HaCaT) to reduce tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. DNA damage using the Comet assay and the production of 8-hydroxy-2′-deoxyguanosine were also determined with and without hydrogen peroxide-induced stress. The LC50 for human cultured HaCaT keratinocytes was 50 μM for hexavalent sodium dichromate and more than 120-fold higher for Cr chloride (6 mM) and Cr histidinate (10 mM). For Cr picolinate at saturating concentration (120 μM) the LC50 was not attained. High Cr(III) concentrations, 250 μM Cr as Cr chloride and Cr histidinate and 120 μM Cr picolinate (highest amount soluble in the system), not only did not result in oxidative DNA damage but exhibited protective antioxidant effects when cells were exposed to hydrogen peroxide-induced oxidative stress. These data further support the low toxicity of trivalent Cr complexes used in nutrient supplements.     

Comparative efficiency of microbial enzyme preparations versus pancreatin for in vitro alimentary protein digestion

Utilisation of microbial enzymes may represent an alternative strategy to the use of conventional pancreatin obtained from pig pancreas for the treatment of severe pancreatic insufficiency. In this study, we focused on the capacity of two microbial preparations for their capacity to digest alimentary proteins (caseins and soya proteins) in comparison with pancreatin. These microbial enzymatic preparations were found to be able to generate small, medium-size and larger polypeptides from caseins and soya proteins but were inactivated at pH 3.0. As determined by Liquid Chromatography–Mass Spectrometry analysis, microbial enzymes generated very different peptides from caseins when compared with peptides generated through pancreatin action. These microbial preparations were characterised by relatively low trypsin- and low carboxypeptidase-like activities but high chymotrypsin-like activities and strong capacity for cleavage of caseins at the methionine sites. Although the efficiency of these microbial preparations to increase the rate of absorption of nitrogen-containing compounds in severe pancreatic insufficiency remains to be tested in vivo, our in vitro data indicate proteolytic capacities of such preparations for alimentary protein digestion.     

Receptor trafficking via the perinuclear recycling compartment accompanied by cell division is necessary for permanent neurotensin cell sensitization and leads to chronic mitogen-activated protein kinase activation

Most G protein-coupled receptors are internalized after interaction with their respective ligand, a process that subsequently contributes to cell desensitization, receptor endocytosis, trafficking, and finally cell resensitization. Although cellular mechanisms leading to cell desensitization have been widely studied, those responsible for cell resensitization are still poorly understood. We examined here the traffic of the high affinity neurotensin receptor (NT1 receptor) following prolonged exposure to high agonist concentration. Fluorescence and confocal microscopy of Chinese hamster ovary, human neuroblastoma (CHP 212), and murine neuroblastoma (N1E-115) cells expressing green fluorescent protein-tagged NT1 receptor revealed that under prolonged treatment with saturating concentrations of neurotensin (NT) agonist, NT1 receptor and NT transiently accumulated in the perinuclear recycling compartment (PNRC). During this cellular event, cell surface receptors remained markedly depleted as detected by both confocal microscopy and (125)I-NT binding assays. In dividing cells, we observed that following prolonged NT agonist stimulation, NT1 receptors were removed from the PNRC, accumulated in dispersed vesicles inside the cytoplasm, and subsequently reappeared at the cell surface. This NT binding recovery allowed for constant cell sensitization and led to a chronic activation of mitogen-activated protein kinases p42 and p44. Under these conditions, the constant activation of NT1 receptor generates an oncogenic regulation. These observations support the potent role for neuropeptides, such as NT, in cancer progression.