Construction of replicative and integrative plasmids for setting up the in vivo expression technology in Helicobacter pylori

Some pathogenic factors of Helicobacter pylori, a bacterium involved in peptic ulcer and gastric cancer, have already been identified using either global or particular approach, but there are still some orphan genes and unidentified pathogenic factors. One of the methods used successfully for the identification of virulence genes of many pathogens is the in vivo expression technology. We describe here the construction and sequences of three different plasmids, one integrative and two replicatives, for the identification of virulence genes by using in vivo expression technology in H. pylori, and of potential use in other bacteria such as Campylobacter spp. Moreover, the use of the green fluorescent protein could allow to classify the genes according to the strength of their expression and to identify those which are repressed upon interaction with gastric mucosa.     

Effects of high static magnetic field exposure on different DNAs

The effects of magnetic fields produced by permanent magnets on different DNA sources were investigated in vivo and in vitro. Escherichia coli DNA, plasmid, and amplification products of different lengths were used as the magnetic field target. The in vivo assays did not reveal any DNA alterations following exposure, demonstrating the presence of cell dependent mechanisms, such as the repair system and the buffering action of the heat shock proteins DNA K/J (Hsp 70/40). The in vitro assays displayed interactions between the magnetic field and DNA, revealing principally that magnetic field exposure induces DNA alterations in terms of point mutations. We speculate that the magnetic field can perturb DNA stability interacting with DNA directly or potentiating the activity of oxidant radicals. This genotoxic effect of the magnetic field, however, is minimized in living organisms due to the presence of protective cellular responses.     

Acetylcholine synthesis and possible functions during sea urchin development

Cholinergic neurotransmitter system molecules were found to play a role during fertilisation and early cell cycles of a large number of invertebrate and vertebrate organisms. In this study, we investigated the presence and possible function of choline acetyltransferase (ChAT, the biosynthetic enzyme of acetylcholine) in gametes of the sea urchin, Paracentrotus lividus, through localisation and functional studies. ChAT-like molecules were detected in oocytes, mature eggs and zygotes with indirect immunofluorescence methods. Positive immunoreactivity was found in the ovarian egg cytoplasm and surface as well as at the zygote surface. This suggests the eggs' capacity to autonomously synthesise acetylcholine (ACh), the signal molecule of the cholinergic system. Acetylcholinesterase (AChE, the lytic enzyme of acetylcholine) was also found in ovarian eggs, with a similar distribution; however, it disappeared after fertilisation. Ultrastructural ChAT localisation in sperms, which was carried out with the immuno-gold method, showed immunoreactivity in the acrosome of unreacted sperms and at the head surface of reacted sperms. In order to verify a functional role of ACh during fertilization and sea urchin development, in vivo experiments were performed. Exposure of the eggs before fertilisation to 1 mM ACh + 1 microM eserine caused an incomplete membrane depolarisation and consequently enhanced polyspermy, while lower concentrations of ACh caused developmental anomalies. The exposure of zygotes to 0,045 AChE Units/mL of sea water caused developmental anomalies as well, in 50% of the embryos. Altogether, these findings and other previously obtained results, suggest that the cholinergic system may subserve two different tasks during development, according to which particular type of ACh receptor is active during each temporal window. The first function, taking place in the course of fertilisation is a result of autonomously synthesised ACh in sperms, while the second function, taking place after fertilisation, is due to maternal ChAT molecules, assembled on the oolemma along with egg maturation and fertilisation processes.     

First Microsatellite Loci of Red Mullet (<Emphasis Type="Italic">Mullus barbatus</Emphasis>) and Their Application to Genetic Structure Analysis of Adriatic Shared Stock

In order to study the genetic structure of the Adriatic shared stock of red mullet (Mullus barbatus), we developed a set of dinucleotide microsatellite markers. A dinucleotide-enriched genomic library was obtained, and 6 polymorphic dinucleotide loci were successfully optimized. The markers showed high expected heterozygosity (from 0.68 to 0.92) and allele number (from 12 to 33); thus they appear to be suitable for detecting genetic differences in the population of red mullet. Four Adriatic samples were subsequently analyzed for microsatellite variation, and the results showed subtle but statistically significant genetic differentiation, indicating that the Adriatic red mullet may group into local, genetically isolated populations. No correlation between geographic distance and genetic differentiation was observed. In addition, the evidence of recent bottlenecks in the Adriatic samples indicates that the observed population subdivision might reflect random local allelic variations, generated by reproductive success, survival rates, or fishing pressure.     

A calcium-dependent bacterial surface protein is involved in the attachment of rhizobia to peanut roots

As part of a project to characterize molecules involved in the crack-entry infection process leading to nodule development, a microscopic assay was used to visualize the attachment of cells of Bradyrhizobium sp. strains SEMIA 6144 and TAL 1000 (labelled by introducing a plasmid expressing constitutively the green fluorescent protein GFP-S65T) to Arachis hypogaea L. (peanut). Qualitative and quantitative results revealed that attachment was strongly dependent on the growth phase of the bacteria. Optimal attachment occurred when bacteria were at the late log or early stationary phase. Cell surface proteins from the Bradyrhizobium sp. strains inhibited the attachment when supplied prior to the attachment assay. Root incubation with a 14-kDa protein (eluted from sodium dodecyl sulphate - gel electrophoresis of the cell surface fraction) prior to the attachment assay resulted in a strong decrease of attachment. The adhesin appeared to be a calcium-binding protein, since cells treated with EDTA were found to be able to bind to adhesin-treated peanut roots. Since this protein has properties identical to those reported for rhicadhesin, we propose that this adhesin is also involved in the attachment process of rhizobia to root legumes that are infected by the crack-entry process.     

Inhibition of pig liver and Zea mays L. polyamine oxidase: a comparative study

Polyamine oxidase (PAO) is involved in polyamine metabolism and production of hydrogen peroxide in animal and plants, thus representing a key system in development and programmed cell death. In the present study, the inhibitory effect of amiloride, p-aminobenzamidine, clonidine, 4',6-diamidino-2-phenyl-indole (DAPI), gabexate mesylate, guazatine, and N,N'-bis(2,3-butadienyl)-1,4-butane-diamine (MDL72527) on the catalytic activity of pig liver and Zea mays L. PAO, Lens culinaris L. and Pisum sativum L. and swine kidney copper amine oxidase, bovine trypsin, as well as neuronal constitutive nitric oxide synthase (NOS-I) was investigated. Moreover, agmatine and N(3) -prenylagmatine (G3) were observed to inhibit pig liver and Zea mays L. PAO, bovine trypsin, and NOS-I action, but were substrates for Lens culinaris L., Pisum sativum L. and swine kidney copper amine oxidase. Guazatine and G3 inhibited selectively Zea mays L. PAO with K(i) values of 7.5 x 10(-9) M and 1.5 x 10(-8) M, respectively (at pH 6.5 and 25.0 degrees C). As a whole, the data reported here represent examples of enzyme cross-inhibition, and appear to be relevant in view of the use of cationic L-arginine-and imidazole-based compounds as drugs.     

Transendothelial migratory pathways of V delta 1+TCR gamma delta+ and V delta 2+TCR gamma delta+ T lymphocytes from healthy donors and multiple sclerosis patients: involvement of phosphatidylinositol 3 kinase and calcium calmodulin-dependent kinase II

We have previously reported that the Vdelta2(+)TCRgammadelta(+) T lymphocyte subset, expressing the NK receptor protein 1a (NKRP1a; CD161), is expanded in patients with relapsing-remitting multiple sclerosis and uses this molecule to migrate through endothelium. In this work, we show that Vdelta1(+) and Vdelta2(+) gammadelta T lymphocytes use distinct signal transduction pathways to accomplish this function. Indeed, we have found that Vdelta1(+) cells lack NKRP1a and selectively express the platelet endothelial cell adhesion molecule 1 (PECAM1; CD31), which drives transendothelial migration of this cell subset, at variance with Vdelta2(+) T cells, which are PECAM1 negative and use NKRP1a for transmigration. Interestingly, when Vdelta2(+) T cells were pretreated with two specific inhibitors of the calcium calmodulin-dependent kinase II KN62 and KN93, but not with the inactive compound KN92, the number of migrating cells and the rate of transmigration were significantly decreased. In turn, the phosphatidylinositol 3 kinase blockers wortmannin and LY294002 exerted a dose-dependent inhibition of Vdelta1(+) cell migration. Finally, NKRP1a and PECAM1 engagement led to activation of different signal transduction pathways: indeed, oligomerization of NKRP1a on Vdelta2(+) T cells activates calcium calmodulin-dependent kinase II, while occupancy of PECAM1 on Vdelta1(+) cells triggers the phosphatidylinositol 3 kinase-dependent Akt/protein kinase Balpha activation. These findings suggest that subsets of gammadelta T lymphocytes may migrate to the site of lesion in multiple sclerosis using two different signaling pathways to extravasate.     

Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei invasion

Wounding chickpea (Cicer arietinum) internodes or cotyledons resulted in an increase in the steady-state level of copper amine oxidase (CuAO) expression both locally and systemically. Dissection of the molecular mechanisms controlling CuAO expression indicated that jasmonic acid worked as a potent inducer of the basal and wound-inducible CuAO expression, whereas salicylic acid and abscisic acid caused a strong reduction of the wound-induced CuAO expression, without having any effect on the basal levels. Epicotyl treatment with the CuAO mechanism-based inhibitor 2-bromoethylamine decreased hydrogen peroxide (H(2)O(2)) levels in all the internodes, as evidenced in vivo by 3,3'-diaminobenzidine oxidation. Moreover, inhibitor pretreatment of wounded epicotyls resulted in a lower accumulation of H(2)O(2) both at the wound site and in distal organs. In vivo CuAO inhibition by 2-bromoethylamine after inoculation of resistant chickpea cv Sultano with Ascochyta rabiei resulted in the development of extended necrotic lesions, with extensive cell damage occurring in sclerenchyma and cortical parenchyma tissues. These results, besides stressing the fine-tuning by key signaling molecules in wound-induced CuAO regulation, demonstrate that local and systemic CuAO induction is essential for H(2)O(2) production in response to wounding and indicate the relevance of these enzymes in protection against pathogens.     

Caspase-1 levels in biological fluids from patients with multiple sclerosis and from patients with other neurological and non-neurological diseases

In multiple sclerosis (MS), pathological white matter damage in the central nervous system is sustained by immune-inflammatory response. Caspase-1 plays a pivotal role in immune-mediated inflammation, as it regulates the cellular export of IL-1beta and IL-18. We carried out a preliminary in vitro study of the kinetics of extracellular caspase-1 release. We then measured caspase-1 levels in paired serum and cerebrospinal fluid (CSF) samples of 75 MS patients, 15 healthy subjects, and patients with other neurological diseases. Paired synovial fluid and serum samples of patients with juvenile idiopathic arthritis, and paired sputum and serum samples of asthma patients were also studied. Mean serum caspase-1 concentrations did not differ between groups. Caspase-1 was detected in the CSF of patients with acute, but not stable, MS [7.5 +/- (SEM) 0.9 pg/ml; test's sensitivity, 56% and specificity, 100%]. Its levels correlated with pleocytosis. The highest mean caspase-1 levels were found in the arthritic synovial fluids (945.5 +/- 126.6 pg/ml, which correlated with erythrocyte sedimentation rate), and in the sputum samples (370.1 +/- 71.0 pg/ml, which correlated with the number of macrophages in the sputum). On condition that caspase-1 is determined in the fluids pertaining to the disease-specific inflammatory sites, its level is a reliable marker of ongoing immune-inflammatory response. The enzyme measurement in CSF can also help define state-trait in MS.