Association between the ACCN1 gene and multiple sclerosis in Central East Sardinia

Multiple genome screens have been performed to identify regions in linkage or association with Multiple Sclerosis (MS, OMIM 126200), but little overlap has been found among them. This may be, in part, due to a low statistical power to detect small genetic effects and to genetic heterogeneity within and among the studied populations. Motivated by these considerations, we studied a very special population, namely that of Nuoro, Sardinia, Italy. This is an isolated, old, and genetically homogeneous population with high prevalence of MS. Our study sample includes both nuclear families and unrelated cases and controls. A multi-stage study design was adopted. In the first stage, microsatellites were typed in the 17q11.2 region, previously independently found to be in linkage with MS. One significant association was found at microsatellite D17S798. Next, a bioinformatic screening of the region surrounding this marker highlighted an interesting candidate MS susceptibility gene: the Amiloride-sensitive Cation Channel Neuronal 1 (ACCN1) gene. In the second stage of the study, we resequenced the exons and the 3' untranslated (UTR) region of ACCN1, and investigated the MS association of Single Nucleotide Polymorphisms (SNPs) identified in that region. For this purpose, we developed a method of analysis where complete, phase-solved, posterior-weighted haplotype assignments are imputed for each study individual from incomplete, multi-locus, genotyping data. The imputed assignments provide an input to a number of proposed procedures for testing association at a microsatellite level or of a sequence of SNPs. These include a Mantel-Haenszel type test based on expected frequencies of pseudocase/pseudocontrol haplotypes, as well as permutation based tests, including a combination of permutation and weighted logistic regression analysis. Application of these methods allowed us to find a significant association between MS and the SNP rs28936 located in the 3' UTR segment of ACCN1 with p = 0.0004 (p = 0.002, after adjusting for multiple testing). This result is in tune with several recent experimental findings which suggest that ACCN1 may play an important role in the pathogenesis of MS.     

Heterologous protein production and delivery systems for Lactococcus lactis

Lactic acid bacteria (LAB), widely used in the food industry, are present in the intestine of most animals, including humans. The potential use of these bacteria as live vehicles for the production and delivery of heterologous proteins of vaccinal, medical or technological interest has therefore been extensively investigated. Lactococcus lactis, a LAB species, is a potential candidate for the production of biologically useful proteins. Several delivery systems have been developed to target heterologous proteins to a specific cell location (i.e., cytoplasm, cell wall or extracellular medium). A promising application of L. lactis is its use as an antigen delivery vehicle, for the development of live mucosal vaccines. The expression of heterologous proteins and antigens as well as the various delivery systems developed in L. lactis, and its use as an oral vaccine carrier are discussed.     

Red light imaging for programmed cell death visualization and quantification in plant–pathogen interactions

Studies on plant–pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant–pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.     

Coralloid root regeneration on Macrozamia megagametophites

Abstract

Megagametophytes of Macrozamia communis were incubated in White's Basal Medium and in White's Basal Medium modified with 2,4-D and kinetin. On the medium enriched with growth substances, regeneration of coralloid roots was induced. These are morphologically identical to sporophytic coralloid roots, without any endosymbiont and displaying negative geotropism. These results confirm the fact that coralloid roots represent an inherent feature of the root system of the Cycadales rather than being the result of induction by microbial factors. Therefore it is possible to suggest that coralloid roots represent vestigial pneumatophores.     

Chemotactic responsiveness toward ligands for CXCR3 and CXCR4 is regulated on plasma blasts during the time course of a memory immune response

Plasma blasts formed during memory immune responses emigrate from the spleen to migrate into the bone marrow and into chronically inflamed tissues where they differentiate into long-lived plasma cells. In this study, we analyze the chemokine responsiveness of plasma blasts formed after secondary immunization with OVA. Starting from day 4 and within approximately 48 h, OVA-specific plasma blasts emigrate from spleen and appear in the bone marrow. Although these migratory cells have lost their responsiveness to many B cell attracting chemokines, e.g., CXC chemokine ligand (CXCL)13 (B lymphocyte chemoattractant), they migrate toward CXCL12 (stromal cell-derived factor 1 alpha), and toward the inflammatory chemokines CXCL9 (monokine induced by IFN-gamma), CXCL10 (IFN-gamma-inducible protein 10), and CXCL11 (IFN-inducible T cell alpha chemoattractant). However, the responsiveness of plasma blasts to these chemokines is restricted to a few days after their emigration from the spleen, indicating a role for these molecules and their cognate receptors, i.e., CXCR3 and CXCR4, in the regulation of plasma blast migration into the bone marrow and/or inflamed tissues.     

Role of phospholipase C and D signalling pathways in vasopressin-dependent myogenic differentiation

Arg⁸-vasopressin (AVP) is a potent inducer of myogenic differentiation stimulating the expression of myogenic regulatory factors. To understand the mechanism of its effect on myogenesis, we investigated the early signals induced by AVP in myogenic target cells. In the rat skeletal muscle cell line L6, AVP selectively stimulates phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho) breakdown, through the activation of phospholipases C and D (PLC, PLD), as shown by the generation of Ins(1,4,5)P₃ and phosphatidylethanol (PtdEtOH), respectively. AVP induces the biphasic increase of sn-1,2-diacylglycerol (DAG) consisting in a rapid peak followed by a sustained phase, and the monophasic generation of phosphatidic acid (PA). Propranolol (a PA phosphatase inhibitor) and Zn²⁺ (a PLD inhibitor), abolish the sustained phase of DAG generation. Our data indicate that PtdIns-PLC activity is mainly responsible for the rapid phase of AVP-dependent DAG generation, whereas the sustained phase is dependent upon PtdCho-PLD activity and PA dephosphorylation, ruling out any significant role of DAG kinase. Modifications of PA level correlate with parallel changes of PLC activity, indicating a possible cross-talk between the two signal transduction pathways in the intact cell. PLD activation is elicited at AVP concentrations two orders of magnitude lower than those required for PLC activation. The differentiation of L6 myoblasts into multinucleated fibers is stimulated significantly by AVP at concentrations at which PLD, but not PLC, is activated. These data provide the first evidence for an important role of PLD in the mechanism of AVP-induced muscle differentiation. J. Cell. Physiol. 171:34–42, 1997. © 1997 Wiley-Liss, Inc.