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.

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.

Factors associated with viral load suppression in HIV-infected pregnant women in Rio de Janeiro, Brazil.

Viral load (VL) near delivery is a determinant of mother-to-child transmission (MTCT) of HIV. To evaluate factors associated with an undetectable VL near delivery in HIV-infected pregnant women receiving highly active antiretroviral therapy (HAART) and non-HAART regimens, HIV-infected pregnant women with a detectable VL at entry and having used antiretrovirals for ≥4 weeks before delivery were selected. Multivariate analysis was employed using binary logistic unconditional models; the dependent variable was having a VL <400 copies/mL near delivery. VL suppression was achieved in 403/707 women (57%): 65.4% in the HAART group, but only 26% in the non-HAART group P = 0.001. Duration of HAART was correlated with VL suppression, with maximum benefit seen after ≥12 weeks of therapy (odds ratio [OR]: 2.51; 95% confidence interval [CI]: 1.72-3.65). CD4+ cell count near delivery (OR: 1.53; 95% CI: 1.06-2.20) and baseline VL (OR: 0.74; 95% CI: 0.58-0.94) were also independently associated with VL suppression. Overall MTCT rate was 1.6%. HAART for ≥12 weeks, baseline VL and CD4 cell count near delivery were independently associated with viral suppression near delivery.

Angiotensin converting enzyme (ACE) inhibitory peptides: production and implementation of functional food.

In recent decades, the most successful strategy for controlling blood pressure has been inhibition of the angiotensin-converting enzyme (ACE). ACE inhibitors of chemical synthesis (captopril, enalapril, ramipril and trandolapril) have been widely used clinically to reduce mortality in patients with heart failure, and in patients with recent myocardial infarction and heart failure or marked left ventricular dysfunction. In addition to preventive and therapeutic drugs, increased attention has been paid to identifying dietary compounds that may contribute to cardiovascular treatment and prevention. ACE inhibitory peptides, derived from a multitude of plant and animal proteins such as milk, soy or fish, represent sources of health-enhancing components. These ACE inhibitory peptides can be enzymatically released from precursor proteins in vitro and in vivo, respectively during food processing and gastrointestinal digestion. They have shown the ability to lower blood pressure by limiting the vasoconstrictory effects of Angiotensin II and potentiating the vasodilatory effects of Bradykinin. By using specific procedures they may be generated in or incorporated into functional foods for the development of 'natural' beneficial health products. Several products containing peptides with ACE inhibitory properties are currently on the market or in development. This review focuses on the use, application and future perspective of bioactive peptides with properties relevant to cardiovascular health.

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.

Haemorrhagic bowel syndrome in dairy cattle: possible role of Clostridium perfringens type A in the disease complex.

A survey based on clinical, pathological and microbiological investigations was performed on 11 Brown Swiss cattle affected with depression, anorexia, agalaxia, ruminal hypomotility, abdominal pain and melaena. In eight animals, macroscopical lesions consisted in haemorrhagic enteritis in the small intestine. Seven of eight isolates from tissue samples were identified as Clostridum perfringens type A, and four were identified as C. perfringens type A with the beta2 toxin gene. Based on these observations, animals were considered affected with haemorrhagic bowel syndrome.

Impact of oilseed rape expressing the insecticidal serine protease inhibitor, mustard trypsin inhibitor-2 on the beneficial predator Pterostichus madidus.

Abstract Insect-resistant transgenic plants have been suggested to have deleterious effects on beneficial predators feeding on crop pests, through transmission of the transgene product by the pest to the predator. To test this hypothesis, effects of oilseed rape expressing the serine protease inhibitor, mustard trypsin inhibitor -2 (MTI-2), on the predatory ground beetle Pterostichus madidus were investigated, using diamondback moth, Plutella xylostella as the intermediary pest species. As expected, oilseed rape expressing MTI-2 had a deleterious effect on the development and survival of the pest. However, incomplete pest mortality resulted in survivors being available to predators at the next trophic level, and inhibition studies confirmed the presence of biologically active transgene product in pest larvae. Characterization of proteolytic digestive enzymes of P. madidus demonstrated that adults utilize serine proteases with trypsin-like and chymotrypsin-like specificities; the former activity was completely inhibited by MTI-2 in vitro. When P. madidus consumed prey reared on MTI-2 expressing plants over the reproductive period in their life cycle, no significant effects upon survival were observed as a result of exposure to the inhibitor. However, there was a short-term significant inhibition of weight gain in female beetles fed unlimited prey containing MTI-2, with a concomitant reduction of prey consumption. Biochemical analyses showed that the inhibitory effects of MTI-2 delivered via prey on gut proteolysis in the carabid decreased with time of exposure, possibly resulting from up-regulation of inhibitor-insensitive proteases. Of ecological significance, consumption of MTI-2 dosed prey had no detrimental effects on reproductive fitness of adult P. madidus.

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.

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.

Analysis of mustard trypsin inhibitor-2 gene expression in response to developmental or environmental induction.

Transcription analysis of a mustard (Sinapis alba L.) serine proteinase inhibitor gene revealed identical 5' termini of mRNAs synthesized during seed maturation and chemical or wounding induction. Polyadenylation of mRNAs on multiple or single sites differentiated gene expression, increasing the availability of stable mRNAs during seed maturation compared with chemical and wounding induction. Expression of the beta-glucuronidase (GUS)-encoding region of the UidA reporter gene, detected under the control of deleted segments of the region flanking on the 5' side the mit-2 gene, identified a stretch of about 520 bp essential for gene expression. The presence in this region of two ABRE motifs is relevant for plant response to gene induction. Expression of GUS was detectable under different induction stimuli in several organs such as seedlings and leaves and was active to varying extents in the vascular tissues and meristem.

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.

Functional expression on bacteriophage of the mustard trypsin inhibitor MTI-2.

The mustard trypsin inhibitor MTI-2 is a potential tool in the study of interactions between pest insects and plants. It can be applied to study the adaptations of digestive proteases in pest insects. Phage display allows a rapid and exhaustive system for the selection of heterologous protein variants with novel specificities. Here we describe a bacteriophage expression system which permits functional expression of MTI-2 variants. Active and inactive mutants of MTI-2 are constructed and displayed on phage. These are used to demonstrate that an active variant can be selected from a background of 10,000 inactive mutants in four rounds of selection and amplification.

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.

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.

PLMItRNA, a database for tRNAs and tRNA genes in plant mitochondria: enlargement and updating.

The current version of PLMItRNA has been realized to constitute a database for tRNA molecules and genes identified in the mitochondria of all green plants ( Viridiplantae ). It is the enlargement of a previous database originally restricted to seed plants [Ceci,L.R., Volpicella,M., Liuni,S., Volpetti,V., Licciulli,F. and Gallerani,R. (1999) Nucleic Acids Res., 27, 156-157]. PLMItRNA reports information and multialignments on 254 genes and 16 tRNA molecules detected in 25 higher plants (one bryophyta and 24 vascular plants) and seven green algae. PLMItRNA is accessible via the WWW at http://bio-WWW.ba.cnr.it:8000/srs6/

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.

Identification of Theileria buffeli/orientalis and Babesia bigemina in Apulian cattle using molecular techniques and study of changes in blood parameters.

Recently several cases of theileriosis due to the haemoprotozoan Theileria buffeli/orientalis have been recorded in the Apulian region, Italy. In this area other tick-borne pathogens were usually identified such as Anaplasma marginale and Babesia bigemina. Outbreaks were recorded showing that these pathogens can be observed separately or in mixed infections. Sub-clinical cases and carrier animals were also previously identified. A lack of specific techniques could not rule out the presence of other haemoparasites such as T. annulata, B. divergens, B. bovis, Ehrlichia phagocytophila and E. bovis. Moreover little is known about the tick species involved in the dissemination of these diseases. Therefore more powerful techniques to specifically identify Theileria or Babesia species have been recently developed. A PCR technique and reverse line blotting (RLB) system to specifically identify six Theileria species and three Babesia species were used. T. buffeli/orientalis and B. bigemina were the only pathogens observed in the targeted animals. The authors also present some changes in blood parameters for the animals followed during this study.