Effect of genotype and season on gynogenesis efficiency in Gerbera

The effects of season, genotype and their interaction on haploid production were evaluated on four genotypes responsive to gynogenesis. Naked mature unfertilised ovules, collected from April to October, were cultivated on modified MS basal medium plus 0.88 μM 6-benzyladenine and 0.57 μM indol-3-acetic acid. After 30–60 days, recovered calli were transferred to a MS basal medium with 8.8 μM BA and 0.57 μM IAA for regeneration. Genotype and season of ovule collection substantially interact in the recovery of haploid calli. Between the four genotypes analysed, two gave more calli in the spring, one in the autumn and the fourth showed only weak differences between seasons. Shoot recovery depended upon both the season of ovule collection and the genotype but no significant interaction was shown by our data. The ability to produce haploid callus is not predictive for efficient shoot regeneration. This revised version was published online in June 2006 with corrections to the Cover Date.     

Activation of smooth muscle and myenteric plexus cells of jejunum via Toll-like receptor 4

The cell types of the gut expressing Toll-like receptor 4, which recognizes specifically bacterial lipopolysaccharides, as well as the functionality of this receptor, have remained controversial. We aimed to clarify these issues. Mouse and human intestinal specimens were stained immunohistochemically to detect Toll-like receptor 4 expression. Smooth muscle and myenteric plexus cells but not enterocytes revealed receptor expression. Murine intestinal smooth muscle and myenteric plexus cells but not enterocytes showed nuclear translocation of nuclear factor-kappaB after in vivo stimulation with lipopolysaccharide. Moreover, lipopolysaccharide added to human jejunum biopsies free of epithelial cells induced release of interleukin-8 (IL-8). We can conclude that Toll-like receptor 4 is not expressed in epithelial layer, but rather on smooth muscle and myenteric plexus cells and that expression is functional. The expression of Toll-like receptor 4 on smooth muscle and myenteric plexus cells is consistent with the possibility that these cells are involved in intestinal immune defense; the low or absent expression of Toll-like receptor 4 on enterocytes might explain the intestinal epithelium hyporesponsiveness to the abundance of LPS in the intestinal lumen.     

Effects of analogues of inorganic phosphate and sodium ion on mineralization of matrix vesicles isolated from growth plate cartilage of normal rapidly growing chickens

The mechanism of matrix vesicle (MV) mineralization was studied using MVs isolated from normal growth plate tissue, as well as several putative intermediates in the MV mineralization pathway--amorphous calcium phosphate (ACP), calcium phosphate phosphatidylserine complex (CPLX) and hydroxyapatite (HAP). Radionuclide uptake and increase in turbidity were used to monitor mineral formation during incubation in synthetic cartilage lymph (SCL). Inhibitors of phosphate (Pi) metabolism, as well as replacing Na(+) with various cations, were used to study MV Pi transport, which had been thought to be Na(+)-dependent. MVs induced rapid mineralization approximately 3 h after addition to SCL; CPLX and HAP caused almost immediate induction; ACP required approximately 1 h. Phosphonoformate (PFA), a Pi analog, potently delayed the onset and reduced the rate of mineral formation of MV and the intermediates with IC(50)'s of 3-6 microM and approximately 10 microM, respectively. PFA:Pi molar ratios required to reduce the rate of rapid mineralization by 50% were approximately 1:30 for ACP, approximately 1:20 for HAP, approximately 1:3.3 for CPLX, and approximately 1:2.0 for MVs. MV mineralization was not found to be strictly Na(+)-dependent: substitution of Li(+) or K(+) for Na(+) had minimal effect; while N-methyl D-glucamine (NMG(+)) was totally inhibitory, choline(+) was clearly stimulatory. Na(+) substitutions had minimal effect on HAP- and CPLX-seeded mineral formation. However with ACP, NMG(+) totally blocked and choline(+) stimulated, just as they did MV mineralization. Thus, kinetic analyses indicate that ACP is a key intermediate, nevertheless, formation of CPLX appears to be the rate-limiting factor in MV mineralization.     

Modified peptides in anti-cancer vaccines: are we eventually improving anti-tumour immunity?

The discovery of tumour antigens recognized by T cells and the features of immune responses directed against them has paved the way to a multitude of clinical studies aimed at boosting anti-tumour T cell immunity as a therapeutic tool for cancer patients. One of the different strategies explored to ameliorate the immunogenicity of tumour antigens in vaccine protocols is represented by the use of optimized peptides or altered peptide ligands, whose amino acid sequence has been modified for improving HLA binding or TCR interaction with respect to native epitopes. However, despite the promising results achieved with preclinical studies, the clinical efficacy of this approach has not yet met the expectations. Although multiple reasons could explain the relative failure of altered peptide ligands as more effective cancer vaccines, the possibility that T cells primed by modified tumour peptides might may be unable to effectively cross-recognize tumour cells has not been sufficiently addressed. Indeed, the introduction of conservative amino acid substitutions may still produce diverse and unpredictable changes in the HLA/peptide interface, with consequent modifications of the TCR repertoire that can interact with the complex. This could lead to the expansion of a broad array of T cells whose TCRs may not necessarily react with equivalent affinity with the original antigenic epitope. Considering the results presently achieved with this vaccine approach, and the emerging availability of alternative strategies for boosting anti-tumour immunity, the use of modified tumour peptides could be reconsidered. This article is a symposium paper from the conference “Immunotherapy—From Basic Research to Clinical Applications”, Symposium of the Collaborative Research Center (SFB) 685, held in Tübingen, Germany, 6–7 March 2008.     

Characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains and selection of an isolate active against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis> and <Emphasis Type="Italic">Peridroma saucia</Emphasis>

Twelve Bacillus thuringiensis (Bt) strains, isolated from larvae and soil samples in Argentina, were molecularly and phenotypically characterized and their insecticidal activities against Spodoptera frugiperda and Peridroma saucia were determined. One isolate—Bt RT—produced more than 93% mortality on first instar larvae of both species, which was higher than that produced by the reference strain Bt 4D1. Bt RT carried a different cry gene profile than Bt 4D1. Scanning electron microscopy showed the presence of bipyramidal and cuboidal crystals. Phenotypic characterization revealed lytic enzymes that could contribute to Bt pathogenicity.     

Benzodiazepine receptor ligands. 8: synthesis and pharmacological evaluation of new pyrazolo[5,1-c] [1,2,4]benzotriazine 5-oxide 3- and 8-disubstituted: high affinity ligands endowed with inverse-agonist pharmacological efficacy

The synthesis and the binding study of new 3-arylesters and 3-heteroarylpyrazolo[5,1-c][1,2,4]benzotriazine 5-oxide 8-substituted are reported. The nature of these substituents (in terms of lipophilic and electronic features) seems to influence the binding affinity. High-affinity ligands were studied in mice in vivo for their pharmacological effects, considering six potential benzodiazepine actions: anxiolytic-like effects, muscle relaxant effects, motor coordination, anticonvulsant action, spontaneous motor activity, and ethanol-potentiating action. Compounds 4d and 6d showed an inverse-agonist profile. These compounds were evaluated also for their binding at benzodiazepine site on GABAA receptor complex (GABAA/BzR complex) subtype to evaluate their subtype selectivity.     

Microsatellite variability of Sardinian pine martens, Martes martes

The pine marten, Martes martes, is a medium-sized terrestrial carnivore associated with woodland habitats of the western Palearctic region. The present distribution area of the species also includes six islands of the western Mediterranean basin. The origin of these insular populations and their taxonomic status are still debated; their molecular characterization appears relevant for conservation purposes. To describe the genetic variability of the pine martens from Sardinia we characterized 40 insular and 14 Italian individuals at seven nuclear microsatellite loci. The identification of private alleles and the calculated F(ST) value of 0.074 revealed some genetic differentiation between the two populations, which accounts for the high percentages of correct allocation (96.39-98.80%) scored by the genotype assignment test. The presence of two distinct clusters corresponding to Sardinia and mainland Italy was further confirmed by the multivariate Factorial Correspondence Analysis of individual genotypes. Moreover, the genome of the Sardinian individuals bore signs of past demographic fluctuations, i.e. the presence of the monomorphic locus Ma-4, a lower allelic richness and a lower number of private alleles, which may derive from the combination of drift, founder effects, and human overexploitation. Anyway, if such events ever affected the Sardinian population, this is likely to have happened in the past since, according to our microsatellite data, the present-day population does not show evidence of recent bottlenecks or inbreeding, the Wilcoxon sign-rank test and the F(IS) index being not statistically significant (both P > 0.05). Based on this genetic evidence, we advance hypotheses about the distinctiveness of the Sardinian population and its significance for taxonomy and conservation.     

Effects of some indoor environmental factors on respiratory symptoms and lung function in a sample of young non smokers in North Italy

Summary We evaluated the effects of some indoor environmental factors in a non smoking subsample (n=381, age 8–19 years) of the general population living in the Po River Delta. Each subject completed an interviewer-administered standardized questionnaire on respiratory symptoms and risk factors. Acceptable maneuvers of forced vital capacity and slope of alveolar plateau of nitrogen were obtained in 96% and 59% of the subjects, respectively. In the houses there were more frequently natural gas for cooking (86%) than bottled gas (14%) and central heating (82%) than stove (18%). As regards passive smoking exposure, 18% of subjects had both parents smoking, 50% had one parent smoking. Significantly higher prevalence rates of wheeze, dyspnea, diagnosis of asthma were found in subjects of both sexes using bottled gas for cooking in comparison to those using natural gas, when also exposed to passive smoking. An insignificant trend towards higher symptom rates was shown by those using stove, instead of central heating. Lung function was affected only in females: those with both parents smoking had reduced forced expirograms, those with bottled gas for cooking or stove for heating had a decreased peak expiratory flow. Interactions of stove and passive smoking on peak expiratory flow and on slope of alveolar plateau were statistically significant. These findings confirm the mild adverse respiratory effects of certain home environment factors shown by other epidemiologic surveys in North Europe and in the USA. They have been a basis for the implementation, under the auspices of National Research Council and Electric Energy Authority, of future specific studies in which continuous monitoring of indoor pollutants and repeated recording of symptoms and lung function in North and Central Italy will be performed.     

In vitro modeling of matrix vesicle nucleation: synergistic stimulation of mineral formation by annexin A5 and phosphatidylserine

Annexins A5, A2, and A6 (Anx-A5, -A2, and -A6) are quantitatively major proteins of the matrix vesicle nucleational core that is responsible for mineral formation. Anx-A5 significantly activated the induction and propagation of mineral formation when incorporated into synthetic nucleation complexes made of amorphous calcium phosphate (ACP) and Anx-A5 or of phosphatidylserine (PS) plus ACP (PS-CPLX) and Anx-A5. Incorporation of Anx-A5 markedly shortened the induction time, greatly increasing the rate and overall amount of mineral formed when incubated in synthetic cartilage lymph. Constructed by the addition of Ca(2+) to PS, emulsions prepared in an intracellular phosphate buffer matched in ionic composition to the intracellular fluid of growth plate chondrocytes, these biomimetic PS-CPLX nucleators had little nucleational activity. However, incorporation of Anx-A5 transformed them into potent nucleators, with significantly greater activity than those made from ACP without PS. The ability of Anx-A5 to enhance the nucleation and growth of mineral appears to stem from its ability to form two-dimensional crystalline arrays on PS-containing monolayers. However, some stimulatory effect also may result from its ability to exclude Mg(2+) and HCO(-)(3) from nucleation sites. Comparing the various annexins for their ability to activate PS-CPLX nucleation yields the following: avian cartilage Anx-A5 > human placental Anx-A5 > avian liver Anx-A5 > or = avian cartilage Anx-A6 > cartilage Anx-A2. The stimulatory effect of human placental Anx-A5 and avian cartilage Anx-A6 depended on the presence of PS, since in its absence they either had no effect or actually inhibited the nucleation activity of ACP. Anx-A2 did not significantly enhance mineralization.