Genetic mapping and annotation of genomic microsatellites isolated from globe artichoke

Cynara cardunculus includes three taxa, the globe artichoke (subsp. scolymus L. Hegi), the cultivated cardoon (var. altilis) and their progenitor, the wild cardoon (var. sylvestris). Globe artichoke is an important component of the Mediterranean rural economy, but its improvement through breeding has been rather limited and its genome organization remains largely unexplored. Here, we report the isolation of 61 new microsatellite loci which amplified a total of 208 alleles in a panel of 22 C. cardunculus genotypes. Of these, 51 were informative for linkage analysis and 39 were used to increase marker density in the available globe artichoke genetic maps. Sequence analysis of the 22 loci associated with genes showed that 9 are located within coding sequence, with the repetitive domain probably being involved in DNA binding or in protein–protein interactions. The expression of the genes associated with 9 of the 22 microsatellite loci was demonstrated by RT-PCR.     

Organization and evolution of the cotG and cotH genes of Bacillus subtilis

The cotG and cotH genes of Bacillus subtilis encode two previously characterized spore coat proteins. The two genes are adjacent on the chromosome and divergently transcribed by σ(K), a sporulation-specific σ factor of the RNA polymerase. We report evidence that the cotH promoter maps 812 bp upstream of the beginning of its coding region and that the divergent cotG gene is entirely contained between the promoter and the coding part of cotH. A bioinformatic analysis of all entirely sequenced prokaryotic genomes showed that such chromosomal organization is not common in spore-forming bacilli. Indeed, CotG is present only in B. subtilis, B. amyloliquefaciens, and B. atrophaeus and in two Geobacillus strains. When present, cotG always encodes a modular protein composed of tandem repeats and is always close to but divergently transcribed with respect to cotH. Bioinformatic and phylogenic data suggest that such genomic organizations have a common evolutionary origin and that the modular structure of the extant cotG genes is the outcome of multiple rounds of gene elongation events of an ancestral minigene.     

Analysis of <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated from Patients Affected by Crohn’s Disease

The etiopathogenesis of Crohn’s disease (CD) is still controversial: several genetic, immunologic, and environmental factors, including some bacteria, have been implicated. This study has been devised to assess the involvement of Escherichia coli in CD. Seven E. coli strains were isolated from 14 biopsies obtained from ileocolic ulcers of patients affected by inflammatory bowel disease (IBD), including six with ulcerative colitis and eight with CD. Five strains, exclusively isolated from CD patients, were found inside mucosal cells. Different PCR techniques (for chuA, yjaA, TspE4.C2, escV, and bfpB genes) were performed and PFGE was carried out to characterize these bacteria in comparison with other E. coli strains isolated from non-IBD specimens. The correlation of these characters with bacterial invasiveness on intestinal (Caco-2) and phagocytic (U937) cells was assessed. Overall our pilot data suggest that five among eight strains isolated from CD patients belonged to the adherent-invasive E. coli (AIEC) group, and were invasive on Caco-2 cells and resistant to phagocytosis. These findings suggest that these bacteria could be considered target organisms whose elimination could reduce the intestinal inflammatory process and CD progression.     

High-resolution SNP scan of chromosome 6p21 in pooled samples from patients with complex diseases

We apply a high-throughput protocol of chip-based mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight; MALDI-TOF) as a method of screening for differences in single-nucleotide polymorphism (SNP) allele frequencies. Using pooled DNA from individuals with asthma, Crohn's disease (CD), schizophrenia, type 1 diabetes (T1D), and controls, we selected 534 SNPs from an initial set of 1435 SNPs spanning a 25-Mb region on chromosome 6p21. The standard deviations of measurements of time of flight at different dots, from different PCRs, and from different pools indicate reliable results on each analysis step. In 90% of the disease-control comparisons we found allelic differences of <10%. Of the T1D samples, which served as a positive control, 10 SNPs with significant differences were observed after taking into account multiple testing. Of these 10 SNPs, 5 are located between DQB1 and DRB1, confirming the known association with the DR3 and DR4 haplotypes whereas two additional SNPs also reproduced known associations of T1D with DOB and LTA. In the CD pool also, two earlier described associations were found with SNPs close to DRB1 and MICA. Additional associations were found in the schizophrenia and asthma pools. They should be confirmed in individual samples or can be used to develop further quality criteria for accepting true differences between pools. The determination of SNP allele frequencies in pooled DNA appears to be of value in assigning further genotyping priorities also in large linkage regions.     

Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice

The function of the clathrin coat in synaptic vesicle endocytosis is assisted by a variety of accessory factors, among which amphiphysin (amphiphysin 1 and 2) is one of the best characterized. A putative endocytic function of amphiphysin was supported by dominant-negative interference studies. We have now generated amphiphysin 1 knockout mice and found that lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain. Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts. Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions.     

Chemo-orientation responses in hymenopteran parasitoids induced by substrate-borne semiochemicals

Hymenopteran parasitoids can utilize substrate-borne semiochemicals released by conspecifics or by their hosts, increasing the likelihood of successful mating and host location. According to the literature, two substrate-borne chemo-orientation patterns can occur: (1) biased random searching, a non-directional reaction toward the chemicals (kinesis), and (2) trail-following searching, a directional response toward the source emitting the chemical compounds (taxis). These two different strategies can be adopted by parasitoids to locate hosts and mates. In host location, random searching is induced by allelochemicals indirectly associated with the host, whereas trail-following behavior is induced by allelochemicals directly emitted by the target organism. In mate finding, sex pheromones emitted by conspecifics can induce either the random searching or the trail-following behavior, although the spatial distribution of virgin conspecifics could be an important factor driving the evolution of substrate-borne chemo-orientation patterns. The chemical nature of substrate-borne semiochemicals has not yet been fully elucidated. Most studies have shown that crude extracts are biologically active for eliciting parasitoid arrestment response, but few studies have clearly characterized their chemical nature. However, experimental evidence indicates that cuticular lipids located in the external layer of insects’ bodies play a role in parasitoid–parasitoid and host–parasitoid communication. The ecological role of parasitoid chemo-orientation in host and mate location is discussed from a biological control perspective.     

Mucosal Adjuvant Activity of IL-2 Presenting Spores of Bacillus subtilis in a Murine Model of Helicobacter pylori Vaccination

The endospores of Bacillus subtilis are now widely used as a platform for presentation of heterologous proteins and due to their safety record and high resistance to harsh environmental conditions can be considered as potential vehicles for oral vaccination. In this research we show that recombinant B. subtilis spores presenting a fragment of the Helicobacter acinonychis UreB protein and expressing the ureB gene under vegetative promoter elicit a strong cellular immune response in orally immunized mice when co-administered with spores presenting IL-2. We show for the first time the successful application of two types of recombinant spores, one carrying an antigen and the other an adjuvant, in a single oral immunization.     

Flow‐mediated plasticity in the expression of stickleback nesting glue genes

Nest construction is an essential component of the reproductive behavior of many species, and attributes of nests – including their location and structure – have implications for both their functional capacity as incubators for developing offspring, and their attractiveness to potential mates. To maximize reproductive success, nests must therefore be suited to local environmental conditions. Male three‐spined sticklebacks (Gasterosteus aculeatus) build nests from collected materials and use an endogenous, glue‐like multimeric protein – “spiggin” – as an adhesive. Spiggin is encoded by a multigene family, and differential expression of spiggin genes potentially allows plasticity in nest construction in response to variable environments. Here, we show that the expression of spiggin genes is affected significantly by both the flow regime experienced by a fish and its nesting status. Further, we show the effects of flow on expression patterns are gene‐specific. Nest‐building fish exhibited consistently higher expression levels of the three genes under investigation (Spg‐a, Spg‐1, and Spg‐2) than non‐nesting controls, irrespective of rearing flow treatment. Fish reared under flowing‐water conditions showed significantly increased levels of spiggin gene expression compared to those reared in still water, but this effect was far stronger for Spg‐a than for Spg‐1 or Spg‐2. The strong effect of flowing water on Spg‐a expression, even among non‐nesters, suggests that the increased production of spiggin – or of spiggin rich in the component contributed by Spg‐a – may allow more rapid and/or effective nest construction under challenging high flow conditions.     

Transport of pipecolic acid in adult and developing mouse brain

In an effort to develop an animal model of hyperpipecolatemia, the uptake of pipecolic acid (PA) in the brain and changes of PA levels in serum following administration ofd,l-PA were studied in the mouse using a new sensitive HPLC-EC method. Following i.p. injections (250 mg/kg) to adult male mice, the brain concentration peaks at 5–10 min (40 nmol/g). The level remains relatively stable up to 5 hrs and then declines slowly to 24 hrs. In serum, the level of PA increases rapidly to reach the maximum value at 10 min and then decreases rapidly in the first hour and continues to decline more slowly to 24 hrs. The net uptake of PA following administration of various amounts ofd,l-PA is saturable at low doses (3.9–15.6 mg/kg), and it increases linearly at higher doses in a dose-dependent manner up to the maximum dose (500 mg/kg) used in the present study. Kinetic analysis suggests the presence of two kinds of transport systems. These findings are in good agreement with the previous results usingd,l-[³H]PA in the mouse (7) andl-[¹⁴C]PA in the rat (13). There were no significant differences between uptake ofd-pipecolic acid andl-pipecolic acid (250 mg/kg, i. p., 10 min), suggesting the absence of stereospecificity for PA uptake in the mouse brain. Developmental changes in net brain uptake of PA following injections ofd,l-PA (250 mg/kg, s.c., 10 min) showed an age-dependent decrease which continues until adult levels are reached at four weeks after birth. The results suggest that the blood brain barrier (BBB) for PA is completed during the first month of life. Following administration ofd,l-PA (250 mg/kg, s.c.) to pregnant mice during the period 19–21 days of gestation, PA level increases in fetal brain to a maximum value at 2 hrs (420 nmol/g). This level is unchanged during 24 hrs. The maximum level of PA in fetal serum is reached at 30 min to 1 hr. The level gradually decreases after 1 hr over 24 hrs. These results indicate that PA taken up by the placenta and into the brain is transported from the fetal circulation. Our results also demonstrate that a higher amount of PA is taken up by the fetal than the adult brain. This finding is important in order to develop an animal model of hyperpipecolatemia in which high brain levels of PA should mimick those of human hyperpipecolatemic patients. Our results strongly support the hypothesis that high levels of PA present in brain during fetal life may exert a devastating effect on the development of the human CNS in hyperpipecolatemic children.     

Identification and characterization of pipecolic acid binding sites in mouse brain

Pipecolic acid (PA, piperidine-2-carboxylic acid) is the major product of lysine metabolism in the mammalian brain (Giacobini et al., 1980). In this study we have characterized the binding of [³H]PA to P₂ fraction membranes and its distribution in the mouse brain. The binding was found to be saturable (70 nM), temperature and Na⁺ and Cl^– dependent. A high affinity binding site with an apparentK _D of 33.2 nM and aB _max of 0.2 pmol/mg protein was demonstrated. The regional distribution of [³H]PA specific binding in mouse brain showed the highest concentration in cerebral cortex, thalamus and olfactory bulb. Unlabeled PA (10^–3–10^–11M) displaced specific binding of [³H]PA in a concentration dependent manner. Out of several substances tested, only proline showed a similar pattern of displacement. Pre-incubation of the membrane preparation with GABA (10^–3–10^–11M) resulted in either an increase or decrease of [³H]PA binding depending on the concentrations of GABA and PA. These results suggest a modulatory action of GABA on PA binding sites. The postnatal development of [³H]PA specific binding was studied in the whole brain of the mouse. [³H]Pipecolic acid binding increased progressively (8-fold) from one day after birth to 16 days. Following this developmental peak, the binding decreased gradually to 30 days at which age, adult values were attained.