Isolation and genetic characterization of mother-of-snow-white, a maternal effect allele affecting laterality and lateralized behaviors in zebrafish

In the present work we report evidence compatible with a maternal effect allele affecting left-right development and functional lateralization in vertebrates. Our study demonstrates that the increased frequency of reversed brain asymmetries in a zebrafish line isolated through a behavioral assay is due to selection of mother-of-snow-white (msw), a maternal effect allele involved in early stages of left-right development in zebrafish. msw homozygous females could be identified by screening of their progeny for the position of the parapineal organ because in about 50% of their offspring we found an altered, either bilateral or right-sided, expression of lefty1 and spaw. Deeper investigations at earlier stages of development revealed that msw is involved in the specification and differentiation of precursors of the Kupffer's vesicle, a structure homologous to the mammalian node. To test the hypothesis that msw, by controlling Kupffer's vesicle morphogenesis, controls lateralized behaviors related to diencephalic asymmetries, we analyzed left- and right-parapineal offspring in a "viewing test". As a result, left- and right-parapineal individuals showed opposite and complementary eye preference when scrutinizing a model predator, and a different degree of lateralization when scrutinizing a virtual companion. As maternal effect genes are expected to evolve more rapidly when compared to zygotic ones, our results highlight the driving force of maternal effect alleles in the evolution of vertebrates behaviors.     

P2X7-mediated Increased Intracellular Calcium Causes Functional Derangement in Schwann Cells from Rats with CMT1A Neuropathy

Charcot-Marie-Tooth (CMT) is the most frequent inherited neuromuscular disorder, affecting 1 person in 2500. CMT1A, the most common form of CMT, is usually caused by a duplication of chromosome 17p11.2, containing the PMP22 (peripheral myelin protein-22) gene; overexpression of PMP22 in Schwann cells (SC) is believed to cause demyelination, although the underlying pathogenetic mechanisms remain unclear. Here we report an abnormally high basal concentration of intracellular calcium ([Ca²⁺]_i) in SC from CMT1A rats. By the use of specific pharmacological inhibitors and through down-regulation of expression by small interfering RNA, we demonstrate that the high [Ca²⁺]_i is caused by a PMP22-related overexpression of the P2X7 purinoceptor/channel leading to influx of extracellular Ca²⁺ into CMT1A SC. Correction of the altered [Ca²⁺]_i in CMT1A SC by small interfering RNA or with pharmacological inhibitors of P2X7 restores functional parameters of SC (migration and release of ciliary neurotrophic factor), which are typically defective in CMT1A SC. More significantly, stable down-regulation of the expression of P2X7 restores myelination in co-cultures of CMT1A SC with dorsal root ganglion sensory neurons. These results establish a pathogenetic link between high [Ca²⁺]_i and impaired SC function in CMT1A and identify overexpression of P2X7 as the molecular mechanism underlying both abnormalities. The development of P2X7 inhibitors is expected to provide a new therapeutic strategy for treatment of CMT1A neuropathy.Charcot-Marie-Tooth disease type 1 (CMT1)³ is a progressive hereditary motor and sensory neuropathy, characterized by distal muscle wasting and weakness, foot deformities, and severe slowing of nerve conduction, because of progressive demyelination (1). With a prevalence of 1 case in 2500, CMT1 is the most common hereditary neurologic disorder, and in the majority of cases (CMT1A) the disease is associated with a duplication on chromosome 17p11.2 of the gene for PMP22 (peripheral myelin protein 22) (2). PMP22 is a 22-kDa glycoprotein mainly expressed by myelinating Schwann cells (SC) and localized in compact myelin (3). The transgenic rat model of CMT1A, obtained by overexpression of PMP22 (4), confirms a role of PMP22 in the pathogenesis of CMT1A. Both PMP22 overexpression because of gene duplication and point mutations of PMP22 are associated with a CMT1A phenotype.The biochemical mechanisms correlating PMP22 dysfunction with demyelination are still unclear. Some reports indicate that a perturbed homeostasis of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) might be causally involved in the demyelination process. Conditions inducing an increased [Ca²⁺]_i in SC impair cell differentiation and myelination (5, 6), similarly to what occurs in CMT1A. Incubation of intact rat nerves with Ca²⁺ and ionophores causes a progressive demyelination, spreading from the paranodes and invading regions of formerly compact myelin, which is dependent upon a rise in the [Ca²⁺]_i of SC (5).Additional evidence for the detrimental effect of a [Ca²⁺]_i elevation on myelin production by SC comes from application of ATP to murine SC monocultures, inducing an immediate and large increase in the [Ca²⁺]_i. As a result of ATP treatment, maturation and differentiation of SC, as well as expression of the myelin basic protein and production of compact myelin, are completely prevented (6). Taken together, the above observations indicate that abnormally elevated Ca²⁺ levels are causally related to impairment of myelin production by SC.In this study, we addressed the possible correlation between PMP22 overexpression and alteration of the [Ca²⁺]_i homeostasis in SC from a rat model of CMT1A. We recorded higher levels of basal [Ca²⁺]_i in affected than in control cells, and we identified the mechanisms responsible for the perturbation of the [Ca²⁺]_i levels in CMT1A SC. Experiments with pharmacological inhibitors and with small interfering RNA (siRNA) unequivocally demonstrated a correlation in CMT1A SC between overexpression of the purinergic receptor P2X7 and influx of extracellular [Ca²⁺]_i across this plasma membrane receptor/channel. In addition, correction of the abnormally elevated [Ca²⁺]_i levels by the use of a P2X7 antagonist or through down-regulation of the expression of P2X7 by transfection with siRNA or with short hairpin RNA-expressing plasmid (shRNA) restored the normal phenotype in CMT1A SC. These findings suggest that CMT1A should be considered as a “calcium disease.” Identification of P2X7 activation as the pathogenetic mechanism underlying demyelination may provide the rationale for a new therapeutic strategy for CMT1A, a disease with no currently available treatment.     

PMP70 knock-down generates oxidative stress and pro-inflammatory cytokine production in C6 glial cells

By using RNA interference (RNAi) in rat C6 glial cells, we previously generated the cell line abcd3kd in which the peroxisomal half-transporter PMP70 was stably knocked-down. The observations that abcd3kd cells had peroxisomal beta-oxidation impairment and an increase of hexacosenoic acid in cholesterol ester fraction, indicated an overlapping function of PMP70 with adrenoleukodystrophy protein (ALDP), the peroxisomal half-transporters involved in X-linked adrenoleukodystrophy (X-ALD). The objective of the present study was to investigate whether PMP70 could affect some oxidative and inflammatory parameters, since many findings indicate oxidative damage in the brain of ALD patients and inflammation is a hallmark of the cerebral forms of X-ALD. We thus measured parameters indicative of oxidative stress, the expression or activity of antioxidant enzymes, and the production of some pro-inflammatory cytokines. Our results show that, due to inducible nitric oxide synthase up-regulation, abcd3kd cell line produces higher levels of nitrites than native C6 cells. The enhanced production of superoxide and thiobarbituric acid-reactive substances, the increased expression of mitochondrial superoxide dismutase, and the reduction of catalase and glutathione peroxidase activities confirm the presence of an oxidative process. We then measured the concentrations of TNFalpha, IFNgamma, and IL-12 and we observed that abcd3kd cells produce higher amounts of pro-inflammatory cytokines compared to native C6 cells. By using neutralizing antibodies against IL-12, not only inflammatory parameters significantly decrease, but nitrite and superoxide production is also affected. This demonstrates that oxidative status of abcd3kd cells is not a direct PMP70 knock-down consequence, but depends on IL-12 release. The scenery induced by the knock-down of PMP70 in C6 cells recall the oxidative and inflammatory status observed in human X-ALD and thus reinforce the idea that PMP70 could affect the clinical course of the disease.     

Influence of specific fermentation conditions on natural microflora of pomace in “Grappa” production

As reported in the European Community regulation, grappa is a spirit beverage made in Italy from marc that has been steam distilled or distilled after the addition of water. Grape marc from red grapes has already undergone alcoholic fermentation with the must and can be distilled immediately. Grape marc from white grapes does not contain ethanol but contains sugars that are fermented by spontaneous anaerobic fermentation during a storage period. The characteristic aroma of grappa consists of a large number of volatile compounds, which arise from various sources, the most important of which is yeast. Very few studies have been undertaken to characterize the natural populations of yeast during the fermentation of grape marc. The goal of this study was to understand how different pHs, temperatures and yeast starter cultures affect the growth and dynamics of yeast species involved in pomace fermentation, which could be the basis for improving the final quality of grappa production. We found that a temperature of 15°C has the greatest effect on improving the quality of the product. Unfortunately, due to the solid state of the grape marc and the impossibility of its mixing, it appears that acidification and the addition of yeast starter cultures during the silage period are not effective.     

Direct identification in food samples of Listeria spp. and Listeria monocytogenes by molecular methods

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.     

Microbiological characterisation of Burkholderia cepacia isolates from cystic fibrosis patients: investigation of the exopolysaccharides produced

Eleven strains of Burkholderia cepacia were isolated directly from clinical specimens: 10 from sputum of cystic fibrosis patients, and one from a vaginal swab. They were biochemically identified using API20NE and confirmed by a PCR-based assay. The genomovar characterisation obtained by specific PCR amplification revealed seven strains belonging to genomovar I, three belonging to genomovar IIIA and one belonging to genomovar IV. All isolates were also typed by ribotyping and random amplification of polymorphic DNA analysis. Some of the characterised strains were examined for the ability to produce exopolysaccharides, with the aim of correlating the genomovar with the exopolysaccharide structure. The polysaccharides were analysed by means of methylation analysis and 1H-NMR spectroscopy in order to determine structural similarities. It was shown that different strains are capable of producing chemically different polysaccharides.     

Antimicrobial activity of Bac7 fragments against drug-resistant clinical isolates

Ten peptides from 13 to 35 residues in length and covering the whole sequence of the Pro-rich peptide Bac7 were synthesized to identify the domain responsible for its antimicrobial activity. At least 16 residues of the highly cationic N-terminal sequence were required to maintain the activity against Gram-negative bacteria. The fragments Bac7(1–35) and, to a lesser extent, Bac7(1–16) proved active against a panel of antibiotic-resistant clinical isolates of Gram-negative bacteria, with the notable exception of Burkholderia cepacia. In addition, when tested against fungi, the longer fragment was also active against collection strains and clinical isolates of Cryptococcus neoformans, but not towards clinical isolates of Candida albicans.     

Effects of <Emphasis Type="SmallCaps">L</Emphasis>-mono Methyl-arginine, <Emphasis Type="Italic">N</Emphasis>-Acetyl-cysteine and Diphenyleniodonium on Free Radical Release in C6 Glial Cells Enriched in Hexacosenoic Acid

Previously, we have shown that C6 glial cells enriched in hexacosenoic acid (HA) incubated with oxidative stressors released higher amounts of nitric oxide (NO) products and superoxide (O^–₂), compared to native C6 cells. In the present study, we examined the effects of pre-treatment with some of free radical release inhibitors. The aim was to determine the origin of the enhanced generation of NO and superoxide, and to test the possibility of preventing it. Pre-treatment with L-mono-methyl-arginine and N-acetyl-cysteine in oxidized low-density lipoprotein (ox-LDL) exposed HA cells, inhibited not only nitrite but also superoxide production suggesting that (O^–₂) anion could partially derive from inducible NO synthase. We also observed that ox-LDL treatment of HA cells reduced the intracellular glutathione levels and activated extracellular signal-related kinases. Since this signalling is related to neurotoxic effect, our data substantiate the role of the free radicals in X-linked adrenoleukodystrophy pathogenesis, as HA cells have been used as an in vitro model for this disease.     

Linked foreign T-cell help activates self-reactive CTL and inhibits tumor growth

Transgenic mice expressing membrane-bound OVA under the rat insulin promoter, RIP-mOVA, has previously been suggested to display deletional tolerance toward the dominant CTL epitope, SIINFEKL, and provide an elegant model system to test the hypothesis that the lack of T cell help contributes to the tolerance. To understand how the CD8 tolerance is maintained in these mice, a set of neo-self-Ags, OVA, modified to contain a foreign Th peptide, were constructed and tested for their ability to induce CTL responses in RIP-mOVA mice. Immunization with these Th peptide-modified OVA molecules and not with the wild-type OVA induced self-reactive CTLs recognizing dominant CTL peptide, SIINFEKL. Importantly, immunization with the modified OVA constructs also prevented the growth of OVA-expressing tumors in transgenic mice. Since endogenous OVA Th peptides did not contribute toward breaking self CTL tolerance, these results also highlighted a very robust CD4 T cell tolerance toward OVA in RIP-mOVA mice that has not been previously described. These results therefore provide direct evidence that it is the tolerance in the CD4 Th cell compartment that helps maintain the CTL tolerance against self-Ag in these mice. Since the CTL tolerance can be broken or bypassed by foreign Th peptides inserted into a self Ag, potential of using this approach in generating effective therapeutic cancer vaccines is discussed.