Cytokine/Chemokine HLDA8 Workshop panel report: analysis of receptors on lymphocytes from cord blood, normal and asthmatic subjects, and HIV positive patients

Chemokines and cytokines are involved in many processes, both physiological and pathological, particularly the recruitment, differentiation, activation, and proliferation of immune cells taking part in ontogenesis, inflammation, and cancer. It was assumed that chemokines and cytokines receptors are expressed in a regulated manner by human lymphocytes during ontogeny and later on, under the environmental stimulation of antigens they contribute to organogenesis, angiogenesis, and tissue remodeling, as well as modulating leukocyte effector functions. Using monoclonal antibodies classified by the Cytokine/Chemokine section of the 8th International Workshop on Human Leukocyte Differentiation Antigens, we analyzed human lymphocytes in blood samples drawn from the umbilical cord, normal adults, allergic and non-allergic asthma patients, HIV infected, and AIDS positive subjects. The main differences noted between adult and cord blood lymphocytes were related to CCR7 and CXCR4 receptors, which were more strongly expressed on cord blood lymphocytes, confirming the important role of these chemokines during development of the immune system. As with the HIV, CXCR4, and CCR5 co-receptors, we found no differences in CXCR4 expression between HIV and AIDS patients. However CCR5 was more strongly expressed in AIDS patients, which is likely to be associated with the evolution of disease. Further studies are needed to gain a better understanding of the functions of these molecules in the underlying pathogenesis of many diseases and to probe the use of the chemokine receptors as targets for therapeutic intervention.     

Evidence for a new species of <Emphasis Type="Italic">Anisakis</Emphasis> Dujardin, 1845: morphological description and genetic relationships between congeners (Nematoda: Anisakidae)

In the present study, a new biological species of Anisakis Dujardin, 1845, was detected in Kogia breviceps and K. sima from West Atlantic waters (coast of Florida) on the basis of 19 (nuclear) structural genes studied by multilocus allozyme electrophoresis. Fixed allele differences at 11 enzyme loci were found between specimens of both adults and larvae of the new species and the other Anisakis spp. tested. Reproductive isolation from A. brevispiculata Dollfus, 1968 was demonstrated by the lack of hybrid or recombinant genotypes in mixed infections in K. breviceps. Genetic distance of the new species from its closest relative, A. brevispiculata, was D(Nei)=0.79. The new species is morphologically different from the other species which have been genetically characterised and from the other Anisakis retained by Davey (1971) as valid or as species inquirendae: the name of Anisakis paggiae n. sp. is proposed for the new taxon. Anisakis Type II larvae (sensu Berland, 1961) from the European hake Merluccius merluccius in the northeastern Atlantic Ocean (Galician coast) and from the scabbard fish Aphanopus carbo in Central Atlantic waters (off Madeira), were identified as A. paggiae n. sp. Its genetic relationships with respect to the seven species previously characterised (A. simplex (Rudolphi, 1809) sensu stricto), A. pegreffii Campana-Rouget & Biocca, 1955, A. simplex, (A. typica (Diesing, 1860), A. ziphidarum Paggi et al., 1998, A. physeteris Baylis, 1923 and A. brevispiculata) were also inferred. Overall, a low genetic identity was detected at allozyme level between the eight Anisakis species. Interspecific genetic identity ranged from I(Nei)=0.68, between the sibling species of the A. simplex complex, to I(Nei)=0.00 (no alleles shared at the considered loci) when A. physeteris, A. brevispiculata and the new species were compared with the other species of the genus. Concordant topologies were obtained using both UPGMA and NJ tree analyses for the considered species. In both analyses, A. paggiae n. sp. clustered with A. brevispiculata. They also indicated two main clades, the first including A. physeteris, A. brevispiculata and A. paggiae n. sp., the second containing all of the remaining species (i.e. A. simplex (s.s.), A. pegreffii, A. simplex, A. typica and A. ziphidarum). A deep separation between these two main Anisakis clades, also supported by high bootstrap values at the major nodes, was apparent. This is also supported by differences in adult and larval morphology, as well as with respect to their main definitive hosts. A morphological key for distinguishing adult A. paggiae n. sp., A. physeteris and A. brevispiculata is presented. Allozyme markers for the identification of any life-history stage of the Anisakis spp. so far studied, as well as ecological data on their definitive host preferences and geographical distribution, are updated.     

Embryonic mortality in buffaloes synchronized and mated by AI during the seasonal decline in reproductive function

The aim was to determine the factors that contribute to embryonic mortality in buffaloes mated by AI during a period of increasing day length which corresponds to a natural decline in reproductive activity. Italian Mediterranean buffalo cows (n=243) showing regular estrous cycles were synchronized using the Ovsynch-TAI program and mated by AI at 16 and 40 h after the second injection of GnRH. Blood samples were collected on Days 10 and 20 after the first AI and assayed for progesterone (P4). Pregnancy diagnosis was undertaken on Days 26 and 40 after the first AI using rectal ultrasonography. Buffaloes with a conceptus on Day 26 but not on Day 40 were judged to have undergone embryonic mortality and for these animals uterine fluid was recovered by flushing and analysed for common infectious agents. Estrus synchronization was achieved in 86% of buffaloes and the pregnancy rate on Day 40 was 34%. Embryonic mortality between Days 26 and 40 occurred in 45% of buffaloes and was associated with the presence of significant infectious agents in only 10 buffaloes (8%). Concentrations of P4 on Day 10 after AI were higher (P<0.05) in buffaloes that established a pregnancy than in buffaloes that showed embryonic mortality that was not associated with infectious agents. Similarly, on Day 20 after AI P4 concentrations were higher (P<0.01) in pregnant buffaloes compared with non-pregnant buffaloes and buffaloes that had embryonic mortality. It is concluded that a reduced capacity for P4 secretion can explain around 50% of embryonic mortalities in buffaloes synchronised and mated by AI during a period of low reproductive activity and that other as yet unidentified factors also have a significant effect on embryonic survival.     

Determination of alpha-bisabolol in human blood by micro-HPLC-ion trap MS and head space-GC-MS methods

Alpha-bisabolol is a compound present in some essential oils, widely distributed in several plants, including camomile. Two different methods for analysing an essential oil, such as alpha-bisabolol in human blood are reported: the first uses micro-liquid chromatography-electrospray ionisation-mass spectrometry (muHPLC-ESI-MS), whereas the second is based on "head space" injection coupled to gas chromatography-mass spectrometry (HS-GC-MS). For LC-ESI-MS, human blood samples, spiked with alpha-bisabolol, were extracted with hexane and evaporated to dryness under air stream. The residue was then reconstituted with methanol and injected into a C18 column, connected to an ion trap mass spectrometer equipped with an ESI source. Spectra were recorded in the positive ion, selected ion monitoring mode. The detection limit of alpha-bisabolol in blood was 0.125 micromol/l. The preparation of samples for the analysis in HS-GC-MS was limited to blood dilution with water (0.5 ml blood + 1 ml water). Head space vials were heated at 125 degrees C for 1 h before automatic injection. The HS-GC-MS detection limit (0.13 micromol/l) was similar to that achieved with the muHPLC-ESI-MS method. Successful tests were performed to verify if alpha-bisabolol could be directly measured by the HS-GC-MS method in different biological samples (blood, urine, faeces, homogenate tissues) from rats treated with the camomile essential oil.     

Molten globule of bovine alpha-lactalbumin at neutral pH induced by heat,trifluoroethanol, and oleic acid: a comparative analysis by circular dichroism spectroscopy and limited proteolysis

The calcium-depleted form of alpha-lactalbumin (alpha-LA) at neutral pH can be induced to adopt a partly folded state or molten globule upon moderate heating, by dissolving the protein in aqueous TFE or by adding oleic acid. This last folding variant of the protein, named HAMLET, can induce apoptosis in tumor cells. The aim of the present work was to unravel from circular dichroism (CD) measurements and proteolysis experiments structural features of the molten globule of apo-alpha-LA at neutral pH. CD spectra revealed that the molten globule of apo-alpha-LA can be obtained upon mild heating at 45 degrees C, as well as at room temperature in the presence of 15% TFE or by adding to the protein solution 7.5 equivalents of oleic acid. Under these various conditions the far- and near-UV CD spectra of apo-alpha-LA are essentially identical to those of the most studied molten globule of alpha-LA at pH 2.0 (A-state). Proteolysis of the 123-residue chain of apo-alpha-LA by proteinase K at 4 degrees C occurs slowly as an all-or-none process leading to small peptides only. At 37 degrees C, proteinase K preferentially cleaves apo-alpha-LA at peptide bonds Ser34-Gly35, Gln39-Ala40, Gln43-Asn44, Phe53-Gln54, and Asn56-Asn57. All these peptide bonds are located at level of the beta-subdomain of the protein (chain region 34-57). Similar sites of preferential cleavage have been observed with the TFE- and oleic acid-induced molten globule of apo-alpha-LA. A protein species given by the N-terminal fragment 1-34 linked via the four disulfide bridges to the C-terminal fragment 54-123 or 57-123 can be isolated from the proteolytic mixture. The results of this study indicate that the same molten globule state of apo-alpha-LA can be obtained at neutral pH under mildly denaturing conditions, as indicated by using a classical spectroscopic technique such as CD and a simple biochemical approach as limited proteolysis. We conclude that the molten globule of alpha-LA maintains a native-like tertiary fold characterized by a rather well-structured alpha-domain and a disordered chain region encompassing the beta-subdomain 34-57 of the protein.     

Phytoplankton dynamics and structure: a comparative analysis in natural and man-made water bodies of different trophic state

Previous investigations on Sicilian man made lakes suggested that physical factors, along with the specific morphology and hydrology of the water body, are important in selecting phytoplankton species. In particular, the variations of the z _mix/z _eu ratio due to the operational procedure to which reservoirs are generally subject were recognised as a trigger allowing the assemblage shift. To investigate if these variations may be considered analogous to those occurring in natural lakes as trophic state and phytoplankton biomass increase, causing a transparency decrease and a contraction of the euphotic depth, phytoplankton were collected in two natural water bodies, one mesotrophic (Lake Biviere di Cesarò) the other eutrophic (Lake Soprano), and compared with those collected in two reservoirs with analogous trophic characteristics (Lake Rosamarina, mesotrophic and Lake Arancio, eutrophic). Particular attention was paid to the dynamics of two key groups: Cyanophytes and chlorophytes. In all four water bodies, transparency mainly depended on chlorophyll level. Annual average value of phytoplankton biomass in the mesotrophic environments was below 2.0 mg l^–1, whereas in the eutrophic systems it was well above 10 mg l^–1. All water bodies showed the presence of cyanophytes (e.g. Anabaena spp., Anabaenopsis spp., Microcystis spp., Planktothrix spp.) and chlorophytes (e.g. Chlamydomonas spp., Botryococcus spp., Oocystis spp., Scenedesmus spp., Pediastrum spp.), but their relative proportions and body size dimensions were different. In particular, small colonial chlorophytes and large-colony forming cyanophytes were most common in the most eutrophic water bodies, whereas larger colonies of green algae in those with a lower trophic state. The results showed that, under the same climatic conditions, autogenic (increase of biomass, decrease in light penetration and euphotic depth) and allogenic (use of the stored waters, anticipated breaking of the thermocline, increase of the mixing depth) processes may shift the structure of phytoplankton assemblage in the same direction even though the quantity of biomass remains linked to nutrient availability.     

Genetic analysis of Soil-Borne Cereal Mosaic Virus response in durum wheat: evidence for the role of the major quantitative trait locus QSbm.ubo-2BS and of minor quantitative trait loci

Genetic analysis of Soil-Borne Cereal Mosaic Virus (SBCMV) resistance in durum wheat was carried out using a population of 180 recombinant inbred lines (RILs) obtained from Simeto (susceptible) × Levante (resistant). The RILs were characterized for SBCMV response in the field under severe and uniform SBCMV infection in two growing seasons and genotyped with simple sequence repeat (SSR) and Diversity Arrays Technology^? markers. Transgressive segregation was observed for disease reaction as estimated by symptom severity scores and virus concentration in leaves. Heritability of the disease response was high, with h ² values consistently above 80%. A major quantitative trait locus (QTL) (QSbm.ubo-2BS) in the distal telomeric region of chromosome 2BS accounted for 60–70% of the phenotypic variation for symptom severity, 40–55% for virus concentration and 15–30% for grain yield. The favorable allele was contributed by Levante. Seven additional QTL influenced SBCMV resistance, with the low-susceptibility allele contributed by Levante at five QTL and by Simeto at the remaining two. The meta-QTL analysis carried out using the data from two mapping populations (Simeto × Levante and Meridiano × Claudio) suggests that in both populations SBCMV resistance is likely controlled by QSbm.ubo-2BS. Our results confine QSbm.ubo-2BS to a c. 2-cM-wide interval flanked by SSR markers that are already being used for marker-assisted selection.     

Modelling the Stoichiometric Regulation of C-Rich Toxins in Marine Dinoflagellates

Toxin production in marine microalgae was previously shown to be tightly coupled with cellular stoichiometry. The highest values of cellular toxin are in fact mainly associated with a high carbon to nutrient cellular ratio. In particular, the cellular accumulation of C-rich toxins (i.e., with C:N > 6.6) can be stimulated by both N and P deficiency. Dinoflagellates are the main producers of C-rich toxins and may represent a serious threat for human health and the marine ecosystem. As such, the development of a numerical model able to predict how toxin production is stimulated by nutrient supply/deficiency is of primary utility for both scientific and management purposes. In this work we have developed a mechanistic model describing the stoichiometric regulation of C-rich toxins in marine dinoflagellates. To this purpose, a new formulation describing toxin production and fate was embedded in the European Regional Seas Ecosystem Model (ERSEM), here simplified to describe a monospecific batch culture. Toxin production was assumed to be composed by two distinct additive terms; the first is a constant fraction of algal production and is assumed to take place at any physiological conditions. The second term is assumed to be dependent on algal biomass and to be stimulated by internal nutrient deficiency. By using these assumptions, the model reproduced the concentrations and temporal evolution of toxins observed in cultures of Ostreopsis cf. ovata, a benthic/epiphytic dinoflagellate producing C-rich toxins named ovatoxins. The analysis of simulations and their comparison with experimental data provided a conceptual model linking toxin production and nutritional status in this species. The model was also qualitatively validated by using independent literature data, and the results indicate that our formulation can be also used to simulate toxin dynamics in other dinoflagellates. Our model represents an important step towards the simulation and prediction of marine algal toxicity.     

Karyology,pollen and seed morphology,and distribution of eight endangered species in the Veneto region (Northern Italy)

ABSTRACT

Eight endangered species of the Veneto region were examined from the karyological and micromorphological points of view. Their geographical distribution, the exsiccata of Herbarium Venetum (HV-PAD) and conservation problems were also considered. These species are: Cortusa matthioli L., a taxon sporadically distributed in Veneto with 2n=24 chromosomes; Calianthemum kernerianum Freyn ex Kerner, only known on Mt. Baldo (Verona) and with 2n=4x=32; Scrophularia vernalis L. with 2n=28; Kosteletzkya pentacarpos (L.) Ledeb., an entity with no regional conservation regulations and with 2n=34; Athamanta vestina A.Kerner, endemic to Italy and with 2n=22; Hottonia palustris L. with 2n=20 and in danger because of the destruction of its habitat; Sagittaria sagttifolia L. with 2n=22; Trapa natans L., a protected species in Veneto with 2n=36 and 2n=48.     

Medullary thyroid carcinoma (MTC) and RET proto-oncogene: Mutation spectrum in the familial cases and a meta-analysis of studies on the sporadic form

Medullary thyroid carcinoma (MTC) is an uncommon malignant tumor arising from the calcitonin-producing parafollicular cells (C cells) of thyroid. It accounts for 5–10% of all thyroid cancers, and it mostly occurs as a sporadic entity (sMTC), but a familial pattern (fMTC) is also possible. RET proto-oncogene germline mutations are crucial for the onset and the progression of fMTC, and the occurrence of single nucleotide polymorphisms could predispose to the sporadic form. In order to clarify the role of this gene in MTC, we carefully reviewed the PubMed database using appropriate terms. First, we summarized current knowledge of the germline RET mutations, mutation spectrum, and prevalence. We then performed a meta-analysis on the available case-control association studies for sMTC. Finally, we carried out in silico predictions of the best associated variants in the attempt to better define their role in the disease. To date, a total of 39 different RET germline mutations have been identified in fMTC families. The most affected codons are 609, 611, 618, 620 (exon 10) and 634 (exon 11), encoding for the extracellular cysteine-rich domain, and codons 768 (exon 13) and 804 (exon 14) of the intracellular tyrosine kinase domain. Six polymorphisms with at least three studies were included in the meta-analysis (A45A [rs1800858], G691S [rs1799939], L769L [rs1800861], S836S [rs1800862], S904S [rs1800863], and IVS1-126G>T [rs2565206]). The meta-analysis demonstrated a modest association of sMTC susceptibility with S836S and a strong association with the IVS1-126G>T polymorphism. Besides RET polymorphisms, we also investigated the role of a few other low-penetrance alleles of genes involved in the RET pathway or in xenobiotic metabolism, but none of these were confirmed. Thus, despite the well-known molecular basis of fMTC, the genetic variants of the sporadic form are still poorly understood, and functional analyses are needed to better understand the consequence of such RET variants and to improve our knowledge on the disease.