Clobetasol and Halcinonide Act as Smoothened Agonists to Promote Myelin Gene Expression and RxRγ Receptor Activation

One of the causes of permanent disability in chronic multiple sclerosis patients is the inability of oligodendrocyte progenitor cells (OPCs) to terminate their maturation program at lesions. To identify key regulators of myelin gene expression acting at the last stages of OPC maturation we developed a drug repositioning strategy based on the mouse immortalized oligodendrocyte (OL) cell line Oli-neu brought to the premyelination stage by stably expressing a key factor regulating the last stages of OL maturation. The Prestwick Chemical Library^® of 1,200 FDA-approved compound(s) was repositioned at three dosages based on the induction of Myelin Basic Protein (MBP) expression. Drug hits were further validated using dosage-dependent reproducibility tests and biochemical assays. The glucocorticoid class of compounds was the most highly represented and we found that they can be divided in three groups according to their efficacy on MBP up-regulation. Since target identification is crucial before bringing compounds to the clinic, we searched for common targets of the primary screen hits based on their known chemical-target interactomes, and the pathways predicted by top ranking compounds were validated using specific inhibitors. Two of the top ranking compounds, Halcinonide and Clobetasol, act as Smoothened (Smo) agonists to up-regulate myelin gene expression in the Oli-neuM cell line. Further, RxRγ activation is required for MBP expression upon Halcinonide and Clobetasol treatment. These data indicate Clobetasol and Halcinonide as potential promyelinating drugs and also provide a mechanistic understanding of their mode of action in the pathway leading to myelination in OPCs. Furthermore, our classification of glucocorticoids with respect to MBP expression provides important novel insights into their effects in the CNS and a rational criteria for their choice in combinatorial therapies in de-myelinating diseases.     

AcMNPV ChiA protein disrupts the peritrophic membrane and alters midgut physiology of Bombyx mori larvae

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) chitinase A (ChiA) is a protein which promotes the final liquefaction of infected host larvae. The potential of this viral molecule as a new tool for insect control is explored here. The ChiA gene was isolated from the AcMNPV genome by PCR and expressed in E. coli. The recombinant protein, purified by affinity chromatography, showed both exo- and endo-chitinase activities and produced perforations on the peritrophic membrane (PM) of Bombyx mori larvae which increased in number and in size, in a dose-dependent manner. This structural alteration resulted into a significant increase of PM permeability to methylene blue and to the small neuropeptide proctolin. When the fifth instar larvae of B. mori were fed on a artificial diet supplemented with the recombinant ChiA, 100% mortality was observed at a dose of 1 microg/g of larval body weight (LW), while at sub-lethal doses of 0.56 microg/g LW, a reduced larval growth was recorded. These results indicate that AcMNPV-ChiA may offer interesting new opportunities for pest control.     

Identification in chromosome 8q11 of a region of homology with the g1 amplicon of the Y chromosome and functional analysis of the BEYLA gene

The male-specific region (MSY) of the Y chromosome contains genes involved mainly in male sex determination and in spermatogenesis. The majority of genes involved in male fertility are localized in multiple copies in the long arm of the Y chromosome, within specific regions defined as "ampliconic regions." It has been suggested that these genes derived from X-linked or autosomal ancestors during evolution, providing a benefit for male fertility when transposed onto the Y chromosome. So far, the autosomal origin has been demonstrated only for two MSY genes, DAZ and CDY. In the present study we report on the identification within chromosome 8q11.2 of a region homologous to the g amplicon, containing the VCY2 (approved gene symbol BPY2), TTTY4, and TTTY17 genes. A search for ancestor genes within the 8q11.2 region allowed us to identify a gene named BEYLA and to characterize the genomic organization and the expression patterns of this gene.     

Hepatitis C virus drives the unconstrained monoclonal expansion of VH1-69-expressing memory B cells in type II cryoglobulinemia: a model of infection-driven lymphomagenesis

Chronic hepatitis C virus infection causes B cell lymphoproliferative disorders that include type II mixed cryoglobulinemia and lymphoma. This virus drives the monoclonal expansion and, occasionally, the malignant transformation of B cells producing a polyreactive natural Ab commonly encoded by the V(H)1-69 variable gene. Owing to their property of producing natural Ab, these cells are reminiscent of murine B-1 and marginal zone B cells. We used anti-Id Abs to track the stages of differentiation and clonal expansion of V(H)1-69(+) cells in patients with type II mixed cryoglobulinemia. By immunophenotyping and cell size analysis, we could define three discrete stages of differentiation of V(H)1-69(+) B cells: naive (small, IgM(high)IgD(high)CD38(+)CD27(-)CD21(high)CD95(-)CD5(-)), "early memory" (medium-sized, IgM(high)IgD(low)CD38(-)CD27(+)CD21(low)CD95(+)CD5(+)), and "late memory" (large-sized, IgM(low)IgD(low-neg)CD38(-)CD27(low)CD21(low-neg)CD5(-)CD95(-)). The B cells expanded in cryoglobulinemia patients have a "memory" phenotype; this fact, together with the evidence for intraclonal variation, suggests that antigenic stimulation by hepatitis C virus causes the unconstrained expansion of activated V(H)1-69(+) B cells. In some cases, these cells replace the entire pool of circulating B cells, although the absolute B cell number remains within normal limits. Absolute monoclonal V(H)1-69(+) B lymphocytosis was seen in three patients with cryoglobulinemia and splenic lymphoma; in two of these patients, expanded cells carried trisomy 3q. The data presented here indicate that the hepatitis C virus-driven clonal expansion of memory B cells producing a V(H)1-69(+) natural Ab escapes control mechanisms and subverts B cell homeostasis. Genetic alterations may provide a further growth advantage leading to an overt lymphoproliferative disorder.     

Segregation of the Qb‐SNAREs GS27 and GS28 into Golgi Vesicles Regulates Intra‐Golgi Transport

The Golgi apparatus is the main glycosylation and sorting station along the secretory pathway. Its structure includes the Golgi vesicles, which are depleted of anterograde cargo, and also of at least some Golgi‐resident proteins. The role of Golgi vesicles remains unclear. Here, we show that Golgi vesicles are enriched in the Qb‐SNAREs GS27 (membrin) and GS28 (GOS‐28), and depleted of nucleotide sugar transporters. A block of intra‐Golgi transport leads to accumulation of Golgi vesicles and partitioning of GS27 and GS28 into these vesicles. Conversely, active intra‐Golgi transport induces fusion of these vesicles with the Golgi cisternae, delivering GS27 and GS28 to these cisternae. In an in vitro assay based on a donor compartment that lacks UDP‐galactose translocase (a sugar transporter), the segregation of Golgi vesicles from isolated Golgi membranes inhibits intra‐Golgi transport; re‐addition of isolated Golgi vesicles devoid of UDP‐galactose translocase obtained from normal cells restores intra‐Golgi transport. We conclude that this activity is due to the presence of GS27 and GS28 in the Golgi vesicles, rather than the sugar transporter. Furthermore, there is an inverse correlation between the number of Golgi vesicles and the number of inter‐cisternal connections under different experimental conditions. Finally, a rapid block of the formation of vesicles via COPI through degradation of ϵCOP accelerates the cis‐to‐trans delivery of VSVG. These data suggest that Golgi vesicles, presumably with COPI, serve to inhibit intra‐Golgi transport by the extraction of GS27 and GS28 from the Golgi cisternae, which blocks the formation of inter‐cisternal connections .     

A PNA microarray for tomato genotyping

The design and development of a PNA microarray designed for the simultaneous identification of several SNPs characteristic of seven different tomato varieties is described. Highly selective arginine-based monomer containing PNAs (Arg-PNAs) have been used in order to obtain very selective probes. Seven modified PNA probes were synthesised and their binding properties in solution were studied. PNA-microarrays based on these probes were prepared and applied to SNP discrimination in model experiments using oligonucleotide mixtures simulating the different sequences of the seven tomato varieties. The strength and the limitations of such a system for SNP recognition are thoroughly discussed.