Allergological implication of the quaternary hexameric structure of the cockroach allergen Per a 3.

BACKGROUND: Cockroach allergens play a very important role in allergic diseases, especially asthma. The major allergen of the American cockroach (Periplaneta americana), Per a 3, naturally occurs as isoforms of hexamers. OBJECTIVE: The aim of this study was to investigate whether the hexameric structures of Per a 3 influence their allergenicity and immunogenicity. METHODS: Therefore, we compared the different effects of native hexamers and dissociated monomers of cockroach haemolymph (HL), containing almost only Per a 3 proteins (HL-Per a 3), on proliferation and T-helper type 1 (Th1)/Th2 cytokine production of human CD4(+) T cells in co-culture with allergen-pulsed monocyte-derived autologous dendritic cells (DC) as well as the leukotriene release of basophils. RESULTS: In P. americana-sensitized and non-sensitized donors the HL-Per a 3 monomers were internalized faster by immature DC and induced higher proliferation and IFN-gamma production than the hexamers. While in non-sensitized donors IL-4 and IL-5 as well as IL-10 production were also increased after stimulation with monomeric HL-Per a 3-pulsed DC, Th2 cytokine and IL-10 production were only enhanced in P. americana-sensitized donors using hexameric HL-Per a 3-pulsed DC. Furthermore, in the leukotriene release assay the monomers were less effective than the hexamers. CONCLUSION: Our data indicate that the quaternary structure can influence both allergenicity and immunogenicity, also depending on the sensitization status. The monomeric variant of Per a 3 allergens could be a possible candidate for a specific immunotherapy because the IgE-mediated allergic reaction and the Th2-inducing capacity are diminished while the Th1-inducing capacity is retained.

High-resolution mapping and chromosome landing at the root-know nematode resistance locus Ma from Myrobalan plum using a large-insert BAC DNA library.

The Ma gene for root-knot nematode (RKN)resistance from Myrobalan plum (Prunus cerasifera L.)confers a complete-spectrum and a heat-stable resistance to Meloidogvne spp., conversely to Mi-I from tomato,which has a more restricted spectrum and a reduced efficiency at high temperature. This gene was identified from a perennial self-incompatible near-wild rootstock species and lies in cosegregation with the SCAR marker SCAFLP2 on the Prunus linkage group 7 in a 2.3 cM interval between the SCAR SCAL19 and SSR pchgms6 markers. We initiated a map-based cloning of Ma and report here the strategy that rapidly led to fine mapping and direct chromosome landing at the locus. Three pairs of bulks, totaling 90 individuals from half-sibling progenies derived from the Ma-heterozygous resistant accession P.2175, were constructed using mapping data, and saturation of the Ma region was performed by bulked segregant analysis (BSA) of 320 AFLP primer pair combinations. The closest three AFLP markers were transformed into codominant SCARs or CAPS designatedSCAFLP3, SCAFLP4 and SCAFLP5. By completing the mapping population up to 1,332 offspring from P.2175,Ma and SCAFLP2 were mapped in a 0.8 cM interval between SCAFLP3 and SCAFLP4. A large-insert bacterial artificial chromosome (BAC) DNA library of P.2175,totaling 30,720 clones with a mean insert size of 145 kb and a 14-15x Prunus haploid genome coverage was constructed and used to land on the Ma spanning interval with few BAC clones. As P.2175 is heterozygous for the gene, we constructed the resistant and susceptible physical contigs by PCR screening of the library with codominant markers. Additional microsatellite markers were then designed from BAC subcloning or BAC end sequencing. In the resistant contig, a single 280 kb BAC clone was shown to carry the Ma gene; this BAC contains two flanking markers on each side of the gene as well as two cosegregating markers. These results should allow future cloning of the Ma gene in this perennial species.

Microsatellite genetic linkage maps of myrobalan plum and an almond-peach hybrid--location of root-knot nematode resistance genes.

Inheritance and linkage studies were carried out with microsatellite [or simple sequence repeat (SSR)] markers in a F(1) progeny including 101 individuals of a cross between Myrobalan plum ( Prunus cerasifera Ehrh) clone P.2175 and the almond (Prunus dulcis Mill.)-peach ( Prunus persica L. Batsch) hybrid clone GN22 ["Garfi" (G) almond x "Nemared" (N) peach]. This three-way interspecific Prunus progeny was produced in order to associate high root-knot nematode (RKN) resistances from Myrobalan and peach with other favorable traits for Prunus rootstocks from plum, peach and almond. The RKN resistance genes, Ma from the Myrobalan plum clone P.2175 and R(MiaNem) from the 'N' peach, are each heterozygous in the parents P.2175 and GN22, respectively. Two hundred and seventy seven Prunus SSRs were tested for their polymorphism. One genetic map was constructed for each parent according to the "double pseudo-testcross" analysis model. The Ma gene and 93 markers [two sequence characterized amplified regions (SCARs), 91 SSRs] were placed on the P.2175 Myrobalan map covering 524.8 cM. The R(MiaNem) gene, the Gr gene controlling the color of peach leaves, and 166 markers (one SCAR, 165 SSRs) were mapped to seven linkage groups instead of the expected eight in Prunus. Markers belonging to groups 6 and 8 in previous maps formed a single group in the GN22 map. A reciprocal translocation, already reported in a G x N F(2), was detected near the Gr gene. By separating markers from linkage groups 6 and 8 from the GN22 map, it was possible to compare the eight homologous linkage groups between the two maps using the 68 SSR markers heterozygous in both parents (anchor loci). All but one of these 68 anchor markers are in the same order in the Myrobalan plum map and in the almond-peach map, as expected from the high level of synteny within Prunus. The Ma and R(MiaNem)genes confirmed their previous location in the Myrobalan linkage group 7 and in the GN22 linkage group 2, respectively. Using a GN22 F(2) progeny of 78 individuals, a microsatellite map of linkage group 2 was also constructed and provided additional evidence for the telomeric position of R(MiaNem) in group 2 of the Prunus genome.

Location of independent root-knot nematode resistance genes in plum and peach.

Prunus species express different ranges and levels of resistance to the root-knot nematodes (RKN) Meloidogyne spp. In Myrobalan plum ( Prunus cerasifera), the dominant Ma gene confers a high-level and wide-spectrum resistance to the predominant RKN, Meloidogyne arenaria, Meloidogyne incognita, Meloidogyne javanica and the isolate Meloidogyne sp. Florida which overcomes the resistance of the Amygdalus sources. In Japanese plum ( Prunus salicina), a similar wide-spectrum dominant resistance gene, termed R(jap), has been hypothesized from an intraspecific segregating cross. In peach, two crosses segregating for resistance to both M. incognita and M. arenaria were used to identify single genes that each control both RKN species in the Shalil ( R(Mia557)) and Nemared ( R(MiaNem)) sources. Localisation of these genes was made possible using the RFLP and SSR- saturated reference Prunus map TxE, combined with a BSA approach applied to some of the genes. The Ma1 allele carried by the Myrobalan plum accession P.2175 was localised on the linkage group 7 at an approximate distance of 2 cM from the SSR marker pchgms6. In the Japanese plum accession J.222, the gene R(jap) was mapped at the same position in co-segregation with the SSR markers pchgms6 and CPPCT022. The peach genes R(Mia557) and R(MiaNem), carried by two a priori unrelated resistance sources, were co-localized in a subtelomeric position on linkage group 2. This location was different from the more centromeric position previously proposed by Lu et al. (1999) for the resistance gene Mij to M. incognita and M. javanica in Nemared, near the SSR pchgms1 and the STS EAA/MCAT10. By contrast, R(Mia557) and R(MiaNem) were flanked by STS markers obtained by Yamamoto and Hayashi (2002) for the resistance gene Mia to M. incognita in the Japanese peach source Juseitou. Concordant results for the three independent sources, Shalil, Nemared and Juseitou, suggest that these peach RKN sources share at least one major gene resistance to M. incognita located in this subtelomeric position. We showed that plum and peach genes are independent and, thus, can be pyramided into interspecific hybrid rootstocks based on the plum and peach species.

Age dependent increase of elastase type protease activity in mouse skin. Effect of UV-irradiation.

The effect of chronological aging and photoaging (UV-radiation) on elastase-type enzyme activity of hairless mouse skin was studied. Aging resulted in the increase of elastase type endopeptidase activity extractable from mouse skins. Both chronic UVA and UVB radiation resulted in a significant increase of elastase type activity. PBS extracted only small part of the elastase activity, UV-A produced an increase of about 90-120% according to the type of irradiation (xenon or UV-A SUN) and UV-B produced a 72% increase. Extraction by Triton X-100 suggested that most of the activity is bound to cells and fibrous structures. EDTA inhibited 80-90% of the elastase activity in chronologically aged skin extracts and also the activity induced by UVA radiation suggesting that metallo-elastase(s) are involved. About 30% of the UVB induced activity could only be inhibited by EDTA and about 50% by PMSF suggesting that irradiation by UVB increased more serine endopeptidase activity but also MMP-activity. Chronic UVA radiation produced an increase of skin elastase activity equivalent to that observed after 24 months of aging in non-irradiated animals (approximately 100 weeks) corresponding to approximately 90% of total life span of these mice. The total increase produced by UVB was less, but the strong increase of a serine elastase, presumably from PMN-s, appear to produce a much more pronounced biological activity as shown by the presence of fibronectin degradation products in skin extracts. Such degradation products were shown to exert harmful effects on tissues. These results may well have biological significance and distinguish chronological aging and photoaging.