Characterization of maize chitinase-A, a tough allergenic molecule.

Food allergies are recognized as an increasing health concern. Proteins commonly identified as food allergens tend to have one of about 30 different biochemical activities. This leads to the assumption that food allergens must have specific structural features which causes their allergenicity. But these structural features are not completely understood. Uncovering the structural basis of allergenicity would allow improved diagnosis and therapy of allergies and would provide insights for safer food production. The availability of recombinant food allergens can accelerate their structural analysis and benefit specific studies in allergology. Plant chitinases are an example of food allergenic proteins for which structural analysis of allergenicity has only partially been reported. The recombinant maize chitinase, rChiA, was purified from Pichia pastoris extracellular medium by differential precipitation and cation exchange chromatography. Enzyme activity was evaluated by halo-assays and microcalorimetric procedures. rChiA modeling was performed by a two-step procedure, using the Swiss-Model server and Modeller software. Allergenicity of rChiA was verified by immunoblot assays with sera from allergic subjects. rChiA is active in the hydrolysis of glycol chitin and tetra-N-acetylchitotetraose and maintains its activity at high temperatures (70°C) and low pH (pH 3). The molecule is also reactive with IgE from sera of maize-allergic subjects. rChiA is a valuable molecule for further studies on structure-allergenicity relationships and as a tool for diagnosing allergies.

Fish fertilizer: a native north american practice?

The belief that the use of fish fertilizers originated among North American Indians, and was communicated as such by Squanto to the Plymouth settlers, has achieved the status of folklore and is therefore difficult to challenge. However, examination of the documentary evidence of Squanto's history and of native cultivation practices, and a cultural analysis of the implications of the use of fish fertilizer, have produced complementary lines of evidence. This evidence indicates that widely held beliefs about the "manner of the Indians" should be revised: Squanto's advice at Plymouth is probably best viewed as an interesting example of culture contact, one in which a native "culture-bearer" conveyed a technological idea from one group of Europeans to another.

Soluble proteome investigation of cobalt effect on the carotenoidless mutant of Rhodobacter sphaeroides.

AIMS: To investigate the surviving capability of Rhodobacter sphaeroides under phototrophic conditions in the presence of high cobalt concentration and its influence on the photosynthetic apparatus biosynthesis. METHODS AND RESULTS: Cells from R. sphaeroides strain R26.1 were grown anaerobically in a medium containing 5.0 mmol l(-1) cobalt ions and in a control medium. Metal toxicity was investigated comparing the soluble proteome of Co(2+)-exposed cells and cells grown in control medium by two-dimensional gel electrophoretic analysis. Significant changes in the expression level were detected for 43 proteins, the majority (35) being up-regulated. The enzyme porphobilinogen deaminase (PBGD) was found down-regulated and its activity was investigated. CONCLUSIONS: The up-regulated enzymes mainly belong to the general category of proteins and DNA degradation enzymes, suggesting that part of the catabolic reaction products can rescue bacterial growth in photosynthetically impaired cells. Furthermore, the down-regulation of PBGD strongly indicates that this key enzyme of the tetrapyrrole and bacteriochlorophyll synthesis is directly involved in the metabolic response. SIGNIFICANCE AND IMPACT OF THE STUDY: Data and experiments show that the cobalt detrimental effect on the photosynthetic growth of R. sphaeroides is associated with an impaired expression and functioning of PBGD.

Detection and quantification of Anaplasma marginale DNA in blood samples of cattle by real-time PCR.

A TaqMan-based real-time PCR assay was developed for the diagnosis of Anaplasma marginale infection of cattle. The established assay was proven to be highly specific, since no cross-reactions were observed with other Anaplasma species of ruminants, including the closely related Anaplasma centrale, or other haemoparasites of ruminants (Anaplasma bovis, Anaplasma ovis, Anaplasma phagocytophilum, Babesia bovis, Babesia bigemina, Theileria annulata and Theileria buffeli). The detection limit was equal to that of nested (n)PCR (10(1) copies of standard DNA and 3 x 10(1) infected erythrocytes ml(-1) of blood). The assay was also reproducible, as shown by satisfactory low intra-assay and inter-assay coefficients of variation. Fifty-four blood samples of ruminants (cattle, n = 51; sheep, n = 2; goats, n = 1), that had been tested previously by reverse line blot (RLB) hybridisation, were subjected to an nPCR assay and the newly established real-time PCR assay. By using real-time PCR, A. marginale DNA was detected in 39/51 bovine samples, with DNA titres ranging from 3.60 x 10(3) to 5.70 x 10(8) copies ml(-1) of blood, whereas sheep and goat samples tested negative. The concordance with nPCR was 100%, whereas a unique sample that had tested negative by RLB gave positive results by nPCR and real-time PCR. The established assay could overcome the limitations of existing diagnostic methods, allowing for simultaneous detection and quantification of the A. marginale DNA in bovine blood, that is essential to support the clinical diagnosis, to assess the carrier status of the animals and to evaluate the efficacy of vaccines and antirickettsial drugs.

Transcriptomic analysis of nickel exposure in Sphingobium sp. ba1 cells using RNA-seq.

Nickel acts as cofactor for a number of enzymes of many bacteria species. Its homeostasis is ensured by proteins working as ion efflux or accumulation systems. These mechanisms are also generally adopted to counteract life-threatening high extra-cellular Ni2+ concentrations. Little is known regarding nickel tolerance in the genus Sphingobium. We studied the response of the novel Sphingobium sp. ba1 strain, able to adapt to high Ni2+ concentrations. Differential gene expression in cells cultured in 10 mM Ni2+, investigated by RNA-seq analysis, identified 118 differentially expressed genes. Among the 90 up-regulated genes, a cluster including genes coding for nickel and other metal ion efflux systems (similar to either cnrCBA, nccCBA or cznABC) and for a NreB-like permease was found. Comparative analyses among thirty genomes of Sphingobium species show that this cluster is conserved only in two cases, while in the other genomes it is partially present or even absent. The differential expression of genes encoding proteins which could also work as Ni2+-accumulators (HupE/UreJ-like protein, NreA and components of TonB-associated transport and copper-homeostasis systems) was also detected. The identification of Sphingobium sp. ba1 strain adaptive mechanisms to nickel ions, can foster its possible use for biodegradation of poly-aromatic compounds in metal-rich environments.

PLMItRNA, a database for mitochondrial tRNA genes and tRNAs in photosynthetic eukaryotes.

The PLMItRNA database for mitochondrial tRNA molecules and genes in VIRIDIPLANTAE: (green plants) [Volpetti,V., Gallerani,R., DeBenedetto,C., Liuni,S., Licciulli,F. and Ceci,L.R. (2000) Nucleic Acids Res., 28, 159-162] has been enlarged to include algae. The database now contains 436 genes and 16 tRNA entries relative to 25 higher plants, eight green algae, four red algae (RHODOPHYTAE:) and two STRAMENOPILES: The PLMItRNA database is accessible via the WWW at http://bio-www.ba.cnr.it:8000/PLMItRNA.

PLANT-PIs: a database for plant protease inhibitors and their genes.

PLANT-PIs is a database developed to facilitate retrieval of information on plant protease inhibitors (PIs) and related genes. For each PI, links to sequence databases are reported together with a summary of the functional properties of the molecule (and its mutants) as deduced from literature. PLANT-PIs contains information for 351 plant PIs, plus several isoinhibitors. The database is accessible at http://bighost.area.ba.cnr.it/PLANT-PIs.

The gene coding for the mustard trypsin inhibitor-2 is discontinuous and wound-inducible.

The gene coding for the mustard trypsin inhibitor-2 has been isolated from a genomic library and characterized. Comparison of genomic and cDNA sequences indicates that the gene is interrupted by an intron of 193 bp. The eukaryotic peculiar regulatory sequences have been detected in the 5' flanking region of the gene. In addition, a decanucleotide has been detected that is highly similar to the proposed G-box and to the ABRE motifs required for the gene expression induced by methyl jasmonate and abscissic acid. Northern blot analysis demonstrates that the gene is expressed in immature seeds as well as in wounded leaves.

Characterization of recombinant mustard trypsin inhibitor 2 (MTI2) expressed in Pichia pastoris.

The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C-terminal amino acids of the precursor form on the inhibitor activity, the C-terminal precursor and the mature protein were both expressed. A third His-tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 40-160 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine beta-trypsin compared to the precursor and His-tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut proteases.

Effects of a mustard trypsin inhibitor expressed in different plants on three lepidopteran pests.

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco, arabidopsis and oilseed rape lines have been evaluated against three different lepidopteran insect pests. 1. Plutella xylostella (L.) larvae were the most sensitive to the ingestion of MTI-2. The inhibitor expressed at high levels in arabidopsis plants caused rapid and complete mortality. High mortality and significantly delayed larval development were also detectable in oilseed rape expressing MTI-2 at lower levels. 2. Mamestra brassicae (L.) larvae were sensitive only at high MTI-2 expression level, as obtained in transgenic tobacco and arabidopsis, whereas no effects were observed for larvae fed on plants showing relatively low expression levels such as those of oilseed rape lines. 3. Feeding bioassays with Spodoptera littoralis (Boisduval) larvae were carried out using the same oilseed rape lines, showing that at these low expression levels no mortality was observed although a delay in larval development did occur. The levels of insect gut proteolytic activities of the larvae still alive at the end of a 7 day feeding bioassay were usually higher than in the controls, but no new proteinases were expressed in any case. The combined results described in this paper demonstrate altogether the relevance of a case-by-case analysis [target insects and proteinase inhibitor (PI) level of expression in planta] in a PI-based strategy for plant protection.

Tick-borne diseases (TBDs) of dairy cows in a Mediterranean environment: a clinical, serological, and hematological study.

A longitudinal study of tick-borne diseases (TBDs) in Southern Italy was carried out by monitoring two dairy farms (A and B) located in the Apulia Region. On each farm ten calves and ten heifers were observed monthly from May 1999 to February 2001 for clinical signs and blood parameters; antibodies against Babesia bigemina and Anaplasma marginale using an ELISA test were also monitored for the first eight months of the study. Totals of 28 and 14 cases of TBDs were observed in the complete herds of Farms A and B, respectively. Timing of disease appearance, categories of animals affected and changes in blood parameters are discussed.

Evaluation of polymorphonuclear cell and monocyte functions in Leishmania infantum-infected dogs.

Leishmania infantum is the causative agent of canine leishmaniasis in the Mediterranean area. The aspects of the canine immune response which may explain the chronic severe disease in these animals have not been well investigated. Therefore, in this work we have evaluated the parasite killing ability by circulating polymorphonuclear (PMN) cells and monocytes in dogs with spontaneous leishmaniasis and in healthy dogs. Moreover, in order to analyse the mechanisms responsible for killing activity, we evaluated the oxidative burst of circulating PMN cells, in terms of O2- and H2O2 production. The killing ability of infected dog PMN cells and monocytes was significantly lower than in a group of healthy dogs and a group of infected dogs treated with meglumine antimoniate and without symptoms of leishmaniasis. Superoxide anion and hydrogen peroxide production by PMN cells was significantly lower in a group of Leishmania-infected dogs than in healthy dogs, suggesting that these mediators may be involved in the reduced killing activity of infected dogs.

Pharmacokinetics of N-methylglucamine antimoniate after intravenous, intramuscular and subcutaneous administration in the dog.

The pharmacokinetic profile of antimony in dogs was defined by administering it intravenously, intramuscularly and subcutaneously as N-methylglucamine antimoniate at a dose of about 25.65 mg of antimony kg-1 bodyweight. The results showed a different half-life for the three routes of administration: 20.5, 42.1 and 121.6 minutes for the intravenous, intramuscular and subcutaneous routes, respectively; peak time values (Tmax) were also different for the intramuscular (90 to 120 minutes) and subcutaneous (210 to 240 minutes) injection. The apparent bioavailability of antimony was > 100 per cent for the intramuscular and 100 per cent for the subcutaneous routes. The data obtained showed a relevant difference in the behaviour of the drug in the dog in comparison to that in humans.

Disposition of antimony and aminosidine in dogs after administration separately and together: implications for therapy of leishmaniasis.

The pharmacokinetic behaviour of aminosidine (15 mg kg-1) and antimony (25.65 mg kg-1 as N-methylglucamine antimoniate), administered subcutaneously either separately or together was studied on four dogs. The results demonstrated that antimony (Sb) did not significantly modify the kinetics of aminosidine (AM) but that the kinetic behaviour of the metal was markedly influenced by the antibiotic, as shown by the differences in mean residence time (MRT), elimination rate constant (Kel) and area under the curve (AUC) with and without the antibiotic (MRT[Sb] = 243.8 +/- 29.5 minutes, MRT[Sb+AM] = 1067.9 +/- 199.2 minutes; Kel[Sb] = 0.008 +/- 0.001 min-1, Kel[Sb+AM] = 0.0015 +/- 0.0003 min-1; AUC[Sb] = 21,024.6 +/- 4448.5 micrograms min ml-1, AUC[Sb+AM] = 130,478.5 +/- 30,481.7 micrograms min ml-1). The persistence of high serum concentrations of antimony when it was administered with aminosidine suggests that the therapeutic doses commonly used should be reduced and that the interval between administration should be increased to avoid the metal reaching toxic concentrations.

Identification and mapping of tRNA genes on the Helianthus annuus mitochondrial genome.

The physical map for seventeen tRNA genes on the mitochondrial genome of the dicotyledonous plant Helianthus annuus has been established. Eleven are genuine mitochondrial genes, while the other six show a high degree of similarity with the chloroplast counterparts. The genes, with the exception of the genuine trnS(GCT) and of the chloroplast-like trnV and trnP, are expressed. The comparison of the organization of some tRNA genes in the H. annuus mitochondrial genome with that of similar genes detectable in other plants reveals that their association is common to several dicotyledons.

GTG as translation initiation codon in the apocytochrome b gene of sunflower mitochondria.

Sunflower mitochondrial DNA contains a single copy of the cob gene. The gene begins with the unusual GTG initiation codon and lies in a transcription unit having a different organization with respect to that common to the other six known sequences from plant species which include both monocotyledons and dicotyledons.

Identification and mapping of trnI, trnE and trnfM genes in the sunflower mitochondrial genome.

Three sunflower mitochondrial HindIII restriction fragments containing the tRNA genes trnI, trnE and trnfM have been sequenced. The genes are present in single copy on the whole genome and are transcribed. Hybridization experiments and sequence analysis of the HindIII fragments allowed the precise mapping and orientation of each gene on the sunflower mitochondrial genome.

Tick-borne diseases of livestock in Italy: general review and results of recent studies carried out in the Apulia region.

This paper reviews basic information on the knowledge of tick-borne diseases, babesiosis, anaplasmosis and theileriosis, in horses, cattle, sheep and goats in Italy with particular reference to the southern part of the country. It is stressed that there is limited knowledge about the parasite species present, their vectors, distribution, prevalence and impact on livestock production and there is the need to assess their epidemiology. A study carried out in the Apulia region to assess the prevalence of Anaplasma marginale infection in 1,648 cattle showed a seroprevalence of 17% using the Card Agglutination Test. In another study in the same region a microscopic prevalence of 78% for Theileria spp. was found in 60 cows. Afterwards using the IFAT test the Theileria sp. was identified as Theileria buffeli/orientalis.

Circulating immune complexes and autoantibodies in canine leishmaniasis.

In sera of 35 dogs with generalised leishmaniasis elevated levels of circulating immune complexes were detected by the 125I-C1q binding test. Furthermore, anti-heart muscle and anti-smooth muscle antibodies were detected by an indirect immunofluorescence technique in sera of infected dogs.