Genomic acute myeloid leukemia-associated inv(16)(p13q22) breakpoints are tightly clustered.

The inv(16) and related t(16;16) are found in 10% of all cases with de novo acute myeloid leukemia. In these rearrangements the core binding factor beta (CBFB) gene on 16q22 is fused to the smooth muscle myosin heavy chain gene (MYH11) on 16p13. To gain insight into the mechanisms causing the inv(16) we have analysed 24 genomic CBFB-MYH11 breakpoints. All breakpoints in CBFB are located in a 15-Kb intron. More than 50% of the sequenced 6.2 Kb of this intron consists of human repetitive elements. Twenty-one of the 24 breakpoints in MYH11 are located in a 370-bp intron. The remaining three breakpoints in MYH11 are located more upstream. The localization of three breakpoints adjacent to a V(D)J recombinase signal sequence in MYH11 suggests a V(D)J recombinase-mediated rearrangement in these cases. V(D)J recombinase-associated characteristics (small nucleotide deletions and insertions of random nucleotides) were detected in six other cases. CBFB and MYH11 duplications were detected in four of six cases tested.

Brain spectrin isoforms during development of chick dorsal root ganglion cells in vivo and in vitro

Brain spectrin is one of the major cytoskeletal proteins associated with the plasma membrane. In many tissues this protein occurs in a variety of isoforms, for which at least three have been described in the brain: i) brain spectrin 240/235 is localized in neurons most prominently in axons and is present early during brain development. ii) Brain spectrin 240/235E is immunologicaly related to erythrocyte spectrin and restricted to somato-dendritic regions in neurons and to glia. It appears late in brain development. iii) A third form, brain spectrin 240/ 235A, is found exclusively in astrocytes. In this study we have investigated the appearance and distribution of brain spectrins 240/235 and 240/235E during embryonic chick dorsal root ganglia development in vivo and in vitro. This system provides a unique model due to the lack of dendrites on developing sensory neurons. Both isoforms first appeared at embryonic day 6. Brain spectrin 240/235 increased transiently around embryonic day 10 and 14, and was first expressed in ventrolateral neurons. It was localized abundantly in perikarya and their axons. This somato-axonal distribution pattern found in situ was also observed in vitro. In contrast, brain spectrin 240/235E only slightly increased between E6 and E15 and remained unchanged thereafter. It was localized mainly in small neurons of the mediodorsal area, where it was found as punctate staining in the cytoplasm, forming first a nuclear cap and in subsequent stages becoming distributed evenly throughout cytoplasm. This brain spectrin isoform was absent from axons, both in situ and in vitro. In conclusion, this study suggests i) that brain spectrin 240/235 may contribute towards the outgrowth, elongation and possibly maintenance of axonal processes, ii) that brain spcctrin 240/235E could be involved in the stablization of the cytoarchilecture of cell bodies in a sclected population of ganglion cells, and iii) that isoform expression of brain spectrin 240/235E in DRG cells may depend on environmental factors.

CATMA, a comprehensive genome-scale resource for silencing and transcript profiling of Arabidopsis genes.

BACKGROUND: The Complete Arabidopsis Transcript MicroArray (CATMA) initiative combines the efforts of laboratories in eight European countries 1 to deliver gene-specific sequence tags (GSTs) for the Arabidopsis research community. The CATMA initiative offers the power and flexibility to regularly update the GST collection according to evolving knowledge about the gene repertoire. These GST amplicons can easily be reamplified and shared, subsets can be picked at will to print dedicated arrays, and the GSTs can be cloned and used for other functional studies. This ongoing initiative has already produced approximately 24,000 GSTs that have been made publicly available for spotted microarray printing and RNA interference. RESULTS: GSTs from the CATMA version 2 repertoire (CATMAv2, created in 2002) were mapped onto the gene models from two independent Arabidopsis nuclear genome annotation efforts, TIGR5 and PSB-EuGène, to consolidate a list of genes that were targeted by previously designed CATMA tags. A total of 9,027 gene models were not tagged by any amplified CATMAv2 GST, and 2,533 amplified GSTs were no longer predicted to tag an updated gene model. To validate the efficacy of GST mapping criteria and design rules, the predicted and experimentally observed hybridization characteristics associated to GST features were correlated in transcript profiling datasets obtained with the CATMAv2 microarray, confirming the reliability of this platform. To complete the CATMA repertoire, all 9,027 gene models for which no GST had yet been designed were processed with an adjusted version of the Specific Primer and Amplicon Design Software (SPADS). A total of 5,756 novel GSTs were designed and amplified by PCR from genomic DNA. Together with the pre-existing GST collection, this new addition constitutes the CATMAv3 repertoire. It comprises 30,343 unique amplified sequences that tag 24,202 and 23,009 protein-encoding nuclear gene models in the TAIR6 and EuGène genome annotations, respectively. To cover the remaining untagged genes, we identified 543 additional GSTs using less stringent design criteria and designed 990 sequence tags matching multiple members of gene families (Gene Family Tags or GFTs) to cover any remaining untagged genes. These latter 1,533 features constitute the CATMAv4 addition. CONCLUSION: To update the CATMA GST repertoire, we designed 7,289 additional sequence tags, bringing the total number of tagged TAIR6-annotated Arabidopsis nuclear protein-coding genes to 26,173. This resource is used both for the production of spotted microarrays and the large-scale cloning of hairpin RNA silencing vectors. All information about the resulting updated CATMA repertoire is available through the CATMA database http://www.catma.org.

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex.

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane alpha-helix that may be involved in intramembrane protein-protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix alpha(0), formed by NS3 residues 12-23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.

Essential role for Pbx1 in corneal morphogenesis.

PURPOSE: The Pbx TALE (three-amino-acid loop extension) homeodomain proteins interact with class 1 Hox proteins, which are master regulators of cell fate decisions. This study was performed to elucidate the role of the Pbx1 TALE protein in the corneal epithelium of mice. METHODS: Pbx1(f/f) mice were crossed with mice containing Cre recombinase under the control of the K14 promoter. Subsequently, the eyes of these mice were dissected and prepared for histologic or molecular analysis. RESULTS: Tissue-specific deletion of Pbx1 in the corneal epithelium of mice resulted in corneal dystrophy and clouding that was apparent in newborns and progressively worsened with age. Thickening of the cornea epithelium was accompanied by stromal infiltration with atypical basal cells, severe disorganization of stromal collagen matrix, and loss of corneal barrier function. High epithelial cell turnover was associated with perturbed expression of developmental regulators and aberrant differentiation, suggesting an important function for Pbx1 in determining corneal identity. CONCLUSIONS: These studies establish an essential role of the Pbx1 proto-oncogene in corneal morphogenesis.

Serum sCD163 levels are associated with type 2 diabetes mellitus and are influenced by coffee and wine consumption: results of the Di@bet.es study.

OBJECTIVE Serum levels of soluble TNF-like weak inducer of apoptosis (sTWEAK) and its scavenger receptor CD163 (sCD163) have been linked to insulin resistance. We analysed the usefulness of these cytokines as biomarkers of type 2 diabetes in a Spanish cohort, together with their relationship to food consumption in the setting of the Di@bet.es study. RESEARCH DESIGN AND METHODS This is a cross-sectional, matched case-control study of 514 type 2 diabetes subjects and 517 controls with a Normal Oral Glucose Tolerance Test (NOGTT), using data from the Di@bet.es study. Study variables included clinical and demographic structured survey, food frequency questionnaire and physical examination. Serum concentrations of sTWEAK and sCD163 were measured by ELISA. Linear regression analysis determined which variables were related to sTWEAK and sCD163 levels. Logistic regression analysis was used to estimate odd ratios of presenting type 2 diabetes. RESULTS sCD163 concentrations and sCD163/sTWEAK ratio were 11.0% and 15.0% higher, respectively, (P<0.001) in type 2 diabetes than in controls. Following adjustment for various confounders, the OR for presenting type 2 diabetes in subjects in the highest vs the lowest tertile of sCD163 was [(OR), 2,01 (95%CI, 1,46-2,97); P for trend <0.001]. Coffee and red wine consumption was negatively associated with serum levels of sCD163 (P = 0.0001 and; P = 0.002 for coffee and red wine intake, respectively). CONCLUSIONS High circulating levels of sCD163 are associated with type 2 diabetes in the Spanish population. The association between coffee and red wine intake and these biomarkers deserves further study to confirm its potential role in type 2 diabetes.

Complex genetics in idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) is an important human disease model. Investigations of the genetics of IHH have facilitated insights into critical pathways regulating sexual maturation and fertility. IHH has been traditionally considered a monogenic disorder. This model holds that a single gene defect is responsible for the disease in each patient. In the case of IHH, 30% of cases are explained by mutations in one of eleven genes. In recent years, several lines of evidence have challenged the monogenic paradigm in IHH. First, disease-associated mutations display striking incomplete penetrance and variable expressivity within and across IHH families. Second, each locus is responsible for only a small percentage of cases. Third, more than one disease-associated mutation seems to be segregating in some families with IHH, and their combined or separate presence in individuals accounts for the variability in disease severity. Finally, IHH is not strictly a congenital and life-long disorder; occasionally it manifests itself during adulthood (adult-onset IHH); in other cases, the disease is not permanent, as evidenced by normal activity of the hypothalamic-pituitary-gonadal axis after discontinuation of treatment in adulthood (IHH reversal). Together, these observations suggest that IHH is not strictly a monogenic mendelian disease, as previously thought. Rather, it is emerging as a digenic, and potentially oligogenic disease, in which hormonal and/or environmental factors may critically influence genetic predisposition and clinical course. Future investigations of IHH should characterize the extent of the involvement of multiple genes in disease pathogenesis, and elucidate the contributions of epigenetic factors.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene.

Fasting is associated with significant changes in nutrient metabolism, many of which are governed by transcription factors that regulate the expression of rate-limiting enzymes. One factor that plays an important role in the metabolic response to fasting is the peroxisome proliferator-activated receptor alpha (PPARalpha). To gain more insight into the role of PPARalpha during fasting, and into the regulation of metabolism during fasting in general, a search for unknown PPARalpha target genes was performed. Using subtractive hybridization (SABRE) comparing liver mRNA from wild-type and PPARalpha null mice, we isolated a novel PPARalpha target gene, encoding the secreted protein FIAF (for fasting induced adipose factor), that belongs to the family of fibrinogen/angiopoietin-like proteins. FIAF is predominantly expressed in adipose tissue and is strongly up-regulated by fasting in white adipose tissue and liver. Moreover, FIAF mRNA is decreased in white adipose tissue of PPARgamma +/- mice. FIAF protein can be detected in various tissues and in blood plasma, suggesting that FIAF has an endocrine function. Its plasma abundance is increased by fasting and decreased by chronic high fat feeding. The data suggest that FIAF represents a novel endocrine signal involved in the regulation of metabolism, especially under fasting conditions.

Localization of the cannabinoid CB1 receptor and the 2-AG synthesizing (DAGLα) and degrading (MAGL, FAAH) enzymes in cells expressing the Ca(2+)-binding proteins calbindin, calretinin, and parvalbumin in the adult rat hippocampus.

The retrograde suppression of the synaptic transmission by the endocannabinoid sn-2-arachidonoylglycerol (2-AG) is mediated by the cannabinoid CB1 receptors and requires the elevation of intracellular Ca(2+) and the activation of specific 2-AG synthesizing (i.e., DAGLα) enzymes. However, the anatomical organization of the neuronal substrates that express 2-AG/CB1 signaling system-related molecules associated with selective Ca(2+)-binding proteins (CaBPs) is still unknown. For this purpose, we used double-label immunofluorescence and confocal laser scanning microscopy for the characterization of the expression of the 2-AG/CB1 signaling system (CB1 receptor, DAGLα, MAGL, and FAAH) and the CaBPs calbindin D28k, calretinin, and parvalbumin in the rat hippocampus. CB1, DAGLα, and MAGL labeling was mainly localized in fibers and neuropil, which were differentially organized depending on the hippocampal CaBPs-expressing cells. CB(+) 1 fiber terminals localized in all hippocampal principal cell layers were tightly attached to calbindin(+) cells (granular and pyramidal neurons), and calretinin(+) and parvalbumin(+) interneurons. DAGLα neuropil labeling was selectively found surrounding calbindin(+) principal cells in the dentate gyrus and CA1, and in the calretinin(+) and parvalbumin(+) interneurons in the pyramidal cell layers of the CA1/3 fields. MAGL(+) terminals were only observed around CA1 calbindin(+) pyramidal cells, CA1/3 calretinin(+) interneurons and CA3 parvalbumin(+) interneurons localized in the pyramidal cell layers. Interestingly, calbindin(+) pyramidal cells expressed FAAH specifically in the CA1 field. The identification of anatomically related-neuronal substrates that expressed 2-AG/CB1 signaling system and selective CaBPs should be considered when analyzing the cannabinoid signaling associated with hippocampal functions.

Genome-wide association study identifies variants associated with progression of liver fibrosis from HCV infection.

Polymorphisms in IL28B were shown to affect clearance of hepatitis C virus (HCV) infection in genome-wide association (GWA) studies. Only a fraction of patients with chronic HCV infection develop liver fibrosis, a process that might also be affected by genetic factors. We performed a 2-stage GWA study of liver fibrosis progression related to HCV infection. We studied well-characterized HCV-infected patients of European descent who underwent liver biopsies before treatment. We defined various liver fibrosis phenotypes on the basis of METAVIR scores, with and without taking the duration of HCV infection into account. Our GWA analyses were conducted on a filtered primary cohort of 1161 patients using 780,650 single nucleotide polymorphisms (SNPs). We genotyped 96 SNPs with P values <5 × 10(-5) from an independent replication cohort of 962 patients. We then assessed the most interesting replicated SNPs using DNA samples collected from 219 patients who participated in separate GWA studies of HCV clearance. In the combined cohort of 2342 HCV-infected patients, the SNPs rs16851720 (in the total sample) and rs4374383 (in patients who received blood transfusions) were associated with fibrosis progression (P(combined) = 8.9 × 10(-9) and 1.1 × 10(-9), respectively). The SNP rs16851720 is located within RNF7, which encodes an antioxidant that protects against apoptosis. The SNP rs4374383, together with another replicated SNP, rs9380516 (P(combined) = 5.4 × 10(-7)), were linked to the functionally related genes MERTK and TULP1, which encode factors involved in phagocytosis of apoptotic cells by macrophages. Our GWA study identified several susceptibility loci for HCV-induced liver fibrosis; these were linked to genes that regulate apoptosis. Apoptotic control might therefore be involved in liver fibrosis.