Using reanalysis in crop monitoring and forecasting systems.

Weather observations are essential for crop monitoring and forecasting but they are not always available and in some cases they have limited spatial representativeness. Thus, reanalyses represent an alternative source of information to be explored. In this study, we assess the feasibility of reanalysis-based crop monitoring and forecasting by using the system developed and maintained by the European Commission- Joint Research Centre, its gridded daily meteorological observations, the biased-corrected reanalysis AgMERRA and the ERA-Interim reanalysis. We focus on Europe and on two crops, wheat and maize, in the period 1980-2010 under potential and water-limited conditions. In terms of inter-annual yield correlation at the country scale, the reanalysis-driven systems show a very good performance for both wheat and maize (with correlation values higher than 0.6 in almost all EU28 countries) when compared to the observations-driven system. However, significant yield biases affect both crops. All simulations show similar correlations with respect to the FAO reported yield time series. These findings support the integration of reanalyses in current crop monitoring and forecasting systems and point to the emerging opportunities linked to the coming availability of higher-resolution reanalysis updated at near real time.

Snapshot on drug-resistance rate and profiles in patients with chronic hepatitis B receiving nucleos(t)ide analogues in clinical practice.

While the selection of complex HBV drug-resistance patterns on therapeutic failure can compromise the efficacy of anti-HBV therapies, recent data show that patients failing treatment without drug-resistance have a rate of virological success close to drug-naive patients. The goal of this study is defining, in clinical practice, the burden of drug-resistance mutations in a cohort of patients treated with anti-HBV drugs. Prevalence and patterns of drug-resistance were analyzed by RT-sequencing in 204 patients infected chronically: 148 experiencing virological rebound (defined as an increase in serum HBV-DNA > 20 IU/ml after achieving virological success [HBV-DNA < 20 IU/ml]), and 56 null/partial responders (always detectable serum HBV-DNA [>20 IU/ml] within 48 weeks of therapy). The highest rate of drug-resistance was observed in patients experiencing virological rebound (prevalence, 79.1%). Conversely, almost half (46.4%) null/partial responders have no evidence of drug-resistance. The rate of drug-resistance was higher in patients treated with lamivudine (76.8% [109/142]) and telbivudine (83.3% [5/6]), followed by adefovir (62.5% [15/24]), and entecavir (52.2% [12/23]). Complex mutational patterns characterized by the co-presence of rtM204V/I-rtA181T/V (impairing the efficacy of all anti-HBV drugs) were detected in four patients (2.7%) with virological rebound. Drug-resistance is the main cause of failure to therapy in patients experiencing virological rebound, supporting the need of rapid switch to anti-HBV drugs with higher genetic barrier and potency (entecavir/tenofovir). Conversely, nearly half of null/partial responders shows no evidence of drug-resistance mutations, maintaining high chance of achieving therapeutic success with the same class of drug. In this setting, genotypic resistance may help in selecting patients still carrying wild-type viruses, that may take major benefits from antiviral treatment.

Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice.

Airway hyperresponsiveness is a key characteristic of human asthma and a marker for asthma-like conditions in animals. F1 mice derived from A/J and C57BL/6J display a phenotype which resembles the asthma-like phenotype of the A/J mice. Since airway responsiveness failed to segregate as a mendelian trait, we show significant linkage at two loci, Bhr1 (lod = 3.0) and Bhr2 (lod = 3.7) on chromosomes 2 and 15. A third locus, Bhr3 (lod = 2.83), maps to chromosome 17. Each of these loci maps near candidate loci implicated in the pathobiology of asthma. Our study represents the first linkages established through a genome-wide survey of airway hyperresponsiveness in any mammal.

T-lymphocytes regulate genetically determined airway hyperresponsiveness in mice.

Airway hyperresponsiveness (AHR) is a hallmark of asthma and a heritable polygenic trait in the mouse. In the mouse, candidate gene products of hematopoietic origin implicated in asthma mapped to the regions of the previously defined quantitative trait loci. Since hematopoietic cells have been implicated in the pathogenesis of asthma, we evaluated the role of hematopoietic cells in general and T cells specifically in the genetic modulation of native airway responsiveness in mice. Here, with the use of bone marrow transplantation, anti-T-cell monoclonal antibody treatment and T-cell transfer, we demonstrate that intrinsic non-atopic AHR is mediated by T lymphocytes. Our data support the novel concept that, in the absence of identified environmental influences, T cells enhance genetically determined airway responsiveness.

Treatment of allergic airway inflammation and hyperresponsiveness by antisense-induced local blockade of GATA-3 expression.

Recent studies in transgenic mice have revealed that expression of a dominant negative form of the transcription factor GATA-3 in T cells can prevent T helper cell type 2 (Th2)-mediated allergic airway inflammation in mice. However, it remains unclear whether GATA-3 plays a role in the effector phase of allergic airway inflammation and whether antagonizing the expression and/or function of GATA-3 can be used for the therapy of allergic airway inflammation and hyperresponsiveness. Here, we analyzed the effects of locally antagonizing GATA-3 function in a murine model of asthma. We could suppress GATA-3 expression in interleukin (IL)-4-producing T cells in vitro and in vivo by an antisense phosphorothioate oligonucleotide overlapping the translation start site of GATA-3, whereas nonsense control oligonucleotides were virtually inactive. In a murine model of asthma associated with allergic pulmonary inflammation and hyperresponsiveness in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate-labeled GATA-3 antisense oligonucleotides led to DNA uptake in lung cells associated with a reduction of intracellular GATA-3 expression. Such intrapulmonary blockade of GATA-3 expression caused an abrogation of signs of lung inflammation including infiltration of eosinophils and Th2 cytokine production. Furthermore, treatment with antisense but not nonsense oligonucleotides induced a significant reduction of airway hyperresponsiveness in OVA-sensitized mice to levels comparable to saline-treated control mice, as assessed by both enhanced pause (PenH) responses and pulmonary resistance determined by body plethysmography. These data indicate a critical role for GATA-3 in the effector phase of a murine asthma model and suggest that local delivery of GATA-3 antisense oligonucleotides may be a novel approach for the treatment of airway hyperresponsiveness such as in asthma. This approach has the potential advantage of suppressing the expression of various proinflammatory Th2 cytokines simultaneously rather than suppressing the activity of a single cytokine.

Contribution of nitric oxide synthases 1, 2, and 3 to airway hyperresponsiveness and inflammation in a murine model of asthma.

Asthma is a chronic disease characterized by increased airway responsiveness and airway inflammation. The functional role of nitric oxide (NO) and the various nitric oxide synthase (NOS) isoforms in human asthma is controversial. To investigate the role of NO in an established model of allergic asthma, mice with targeted deletions of the three known isoforms of NOS (NOS1, 2, and 3) were studied. Although the inducible (NOS2) isoform was significantly upregulated in the lungs of ovalbumin (OVA)-sensitized and -challenged (OVA/OVA) wild-type (WT) mice and was undetectable in similarly treated NOS2-deficient mice, airway responsiveness was not significantly different between these groups. OVA/OVA endothelial (NOS3)-deficient mice were significantly more responsive to methacholine challenge compared with similarly treated NOS1 and NOS1&3-deficient mice. Airway responsiveness in OVA/OVA neuronal (NOS1)-deficient and neuronal/endothelial (NOS1&3) double-deficient mice was significantly less than that observed in similarly treated NOS2 and WT groups. These findings demonstrate an important function for the nNOS isoform in controlling the inducibility of airway hyperresponsiveness in this model of allergic asthma.

Modulation of human cytidine deaminase by specific aminoacids involved in the intersubunit interactions.

An investigation was made of the role exerted by some residues supposed to be involved in the intersubunit interaction and also in the catalytic site of homotetrameric human cytidine deaminase (T-CDA). Attention was focused on Y33, Y60, R68, and F137 residues that are a part of a conserved region in most T-CDAs. Hence, a series of site-directed mutagenesis experiments was set up obtaining seven mutants: Y60G, Y33G, Y33F Y33S, F137A, R68G, and R68Q. Each active purified mutant protein was characterized kinetically, with a series of substrates and inhibitors, and the effect of temperature on enzyme activity and stability was also investigated. Circular dichroism (CD) experiments at different temperatures and in presence of small amounts of sodium dodecyl sulphate (SDS) were performed in all the soluble mutant CDAs. The results obtained by site-directed mutagenesis studies were compared to the crystallographic data of B. subtilis CDA and E. coli CDA and to molecular modeling studies previously performed on human CDA. The mutation of Y60 to glycine produced an enzyme with a more compact quaternary structure with respect to the wild-type; this mutation did not have a dramatic effect on cytidine deamination, but it slightly affected the binding with the substrate. None of the mutant CDAs in Y33 showed enzymatic activity; they existed only as monomers, indicating that this residue, located at the intersubunit interface, may be responsible for the correct folding of human CDA. The insertion of an alanine instead of phenylalanine at position 137 led to a soluble but completely inactive enzyme unable to form a tetramer, suggesting that F137 residue may be important for the assembling of the tetramer and also for the arrangement of the CDA active site. Finally, R68G and R68Q mutations revealed that the presence of the amino group seems to be important for the catalytic process but not for substrate binding, as already shown in B. subtilis CDA. The quaternary structure of R68Q was not affected by the mutation, as shown by the SDS-induced dissociation experiments and CD studies, whereas R68G dissociated very easily in presence of small amounts of SDS. These experiments indicated that in the human CDA, the side chain of arginine 68 involved in the catalytic process in one subunit active site might come from another subunit. The data obtained from these studies confirmed the presence of a complicated set of intersubunit interactions in the active site of human CDA, as shown in other T-CDAs.

Cathepsin S activity regulates antigen presentation and immunity.

MHC class II molecules display antigenic peptides on cell surfaces for recognition by CD4(+) T cells. Proteolysis is required in this process both for degradation of invariant chain (Ii) from class II-Ii complexes to allow subsequent binding of peptides, and for generation of the antigenic peptides. The cysteine endoprotease, cathepsin S, mediates Ii degradation in human and mouse antigen-presenting cells. Studies described here examine the functional significance of cathepsin S inhibition on antigen presentation and immunity. Specific inhibition of cathepsin S in A20 cells markedly impaired presentation of an ovalbumin epitope by interfering with class II-peptide binding, not by obstructing generation of the antigen. Administration of a cathepsin S inhibitor to mice in vivo selectively inhibited activity of cathepsin S in splenocytes, resulting in accumulation of a class II-associated Ii breakdown product, attenuation of class II-peptide complex formation, and inhibition of antigen presentation. Mice treated with inhibitor had an attenuated antibody response when immunized with ovalbumin but not the T cell-independent antigen TNP-Ficoll. In a mouse model of pulmonary hypersensitivity, treatment with the inhibitor also abrogated a rise in IgE titers and profoundly blocked eosinophilic infiltration in the lung. Thus, inhibition of cathepsin S in vivo alters Ii processing, antigen presentation, and immunity. These data identify selective inhibition of cysteine proteases as a potential therapeutic strategy for asthma and autoimmune disease processes.

Inhibition of T cell costimulation abrogates airway hyperresponsiveness in a murine model.

Activation of naive T cells requires at least two signals. In addition to the well characterized interaction of the T cell antigen receptor with the antigen/MHC expressed on an antigen-presenting cell, T cell activation also requires costimulation by a second set of signals. The best characterized costimulatory receptor is CD28, which binds to a family of B7 ligands expressed on antigen-presenting cells. In asthma, although activated T cells play a role in the initiation and maintenance of airway inflammation, the importance of T cell costimulation in bronchial hyperresponsiveness had not been characterized. Therefore, we tested the hypothesis that inhibition of the CD28:B7 costimulatory pathway would abrogate airway hyperresponsiveness. Our results show that blockade of costimulation with CTLA4-Ig, a fusion protein known to prevent costimulation by blocking CD28:B7 interactions, inhibits airway hyperresponsiveness, inflammatory infiltration, expansion of thoracic lymphocytes, and allergen-specific responsiveness of thoracic T cells in this murine model of allergic asthma.

Interleukin-8 receptor modulates IgE production and B-cell expansion and trafficking in allergen-induced pulmonary inflammation.

We examined the role of the interleukin-8 (IL-8) receptor in a murine model of allergen-induced pulmonary inflammation using mice with a targeted deletion of the murine IL-8 receptor homologue (IL-8r-/-). Wild-type (Wt) and IL-8r-/- mice were systemically immunized to ovalbumin (OVA) and were exposed with either single or multiple challenge of aerosolized phosphate-buffered saline (OVA/PBS) or OVA (OVA/OVA). Analysis of cells recovered from bronchoalveolar lavage (BAL) revealed a diminished recruitment of neutrophils to the airway lumen after single challenge in IL-8r-/- mice compared with Wt mice, whereas multiply challenged IL-8r-/- mice had increased B cells and fewer neutrophils compared with Wt mice. Both Wt and IL-8r-/- OVA/OVA mice recruited similar numbers of eosinophils to the BAL fluid and exhibited comparable degrees of pulmonary inflammation histologically. Both total and OVA-specific IgE levels were greater in multiply challenged IL-8r-/- OVA/OVA mice than in Wt mice. Both the IL-8r-/- OVA/OVA and OVA/PBS mice were significantly less responsive to methacholine than their respective Wt groups, but both Wt and IL-8r mice showed similar degrees of enhancement after multiple allergen challenge. The data demonstrate that the IL-8r modulates IgE production, airway responsiveness, and the composition of the cells (B cells and neutrophils) recruited to the airway lumen in response to antigen.

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

Studies on thermal stability of human cytidine deaminase.

The thermal stability of human cytidine deaminase (CDA), an enzyme involved in pyrimidine metabolism was investigated. With this in view, the residues R68 and Y60, supposed to be involved in the intersubunit interactions and in the catalytic site of CDA, were mutated to glutamine and glycine, respectively. Thermal stability experiments were performed on the purified mutants by means of circular dichroism and enzymatic assays. The results obtained should be useful for designing more efficient cytidine based drugs for chemotherapy.

Mini-myoglobin: native-like folding of the NO-derivative.

Mini-myoglobin is a polypeptide fragment (residues 32-139) obtained by limited proteolysis of horse heart apomyoglobin and reconstituted with the natural heme. Its functional properties are very similar to those of native myoglobin and therefore it may represent a model for testing the functional role of the protein fragment encoded by the central exon of myoglobin gene (residues 31-105). Here we have investigated some properties of the nitric oxide derivative of mini-myoglobin in comparison with those of NO-myoglobin, to provide more structural information on the heme pocket residues in addition to that already acquired by electron paramagnetic resonance of the cobalt-substituted mini-myoglobin. At pH 7.0, optical and circular dichroism Soret spectra, as well as electron paramagnetic resonance spectra reveal that the heme orientation in the pocket and the coordination state of the ferrous iron in NO-mini-myoglobin are similar to those of the whole protein. The spectra of the NO-mini-myoglobin complex are very sensitive to pH changes at variance to what is observed for the NO-myoglobin derivative in the same pH range (5.5-9.5). In particular, increasing or decreasing pH from 7.0, results in a decrease of the extinction coefficient and of the ellipticity in the Soret region and in a change of the shape of the electron paramagnetic resonance signal. The spectral changes are diagnostic for a transition from a hexa-coordinated (at pH 7.0) to a penta-coordinated heme (at pH 5.5 or 9.5), with the proximal histidine-iron bond either broken or stretched dramatically. Thus, although mini-myoglobin is able to bind NO in a geometry similar to that of the native protein, the resulting NO derivative shows a much higher pH dependence, suggesting that the two lacking side domains are mainly involved in enhancing the stability of the hemoprotein core.

Mini-myoglobin: preparation and reaction with oxygen and carbon monoxide.

A domain of 108 amino acid residues (32 to 139), obtained by digestion of horse heart apomyoglobin with clostripain, was found to bind protoheme in a 1 to 1 molar ratio. This domain is 33 amino acid residues larger than the protein segment encoded by the central exon in seal myoglobin. Flash photolysis experiments have shown that reconstituted "mini-myoglobin" is very similar to myoglobin in the combination reaction with carbon monoxide and with oxygen, and in the oxygen replacement reaction by carbon monoxide. These experiments provide for the first time direct evidence for the presence of a structural and functional domain, closely corresponding to the segment encoded by the central exon of the myoglobin gene, which contains the information for binding the natural heme and for maintaining the native folding typical of a respiratory protein.

Mini-myoglobin. The structural significance of haem-ligand interactions.

The properties of purified mini-myoglobin, the fragment 32-139 of horse heart myoglobin reconstituted with protohaem, have been investigated from a structural and functional view point. The recovery of secondary structure observed in the carbon monoxide derivative of mini-myoglobin, as shown by circular dichroism, and the overall similarity of the haem pocket to that of myoglobin, as deduced from the fluorescence properties of the complex with 1-anilino-8-naphthalene sulphonate, indicate that, in the presence of the constraints imposed by the haem and its ligands, the miniprotein reacquires a conformation close to that of native myoglobin. These spectroscopic data parallel the conclusions drawn from the results of ligand combination and dissociation kinetics; stopped-flow experiments indicate that carbon monoxide and oxygen bind to mini-myoglobin with rates almost identical with those of myoglobin itself. The significance of mini-myoglobin as a model of an oxygen-carrying protein, with some of the expected functional characteristics of an ancestor haemoprotein, is discussed, with reference to the mosaic structure of the myoglobin gene and the role of different exons in the evolution of proteins.

Mini-myoglobin. Electron paramagnetic resonance and reversible oxygenation of the cobalt derivative.

Mini-myoglobin, obtained by limited proteolysis of horse heart myoglobin (residues 32 to 139), represents a good model for testing the correlation between an exon and a protein domain. We have shown that ligand binding kinetics, spectral and folding features of mini-myoglobin are very similar to those of native myoglobin. In order to develop further the analysis of the structure-function relationship in this mini-protein, mini-globin was reconstituted with the heme moiety in which iron is replaced by cobalt. The Soret absorption spectra of oxy and deoxy cobaltous mini-myoglobin are very similar to those of cobaltous myoglobin derivatives; in addition. Co-mini-myoglobin binds oxygen reversibly with an n value approximately 1 and a p50 value of 45 to 50 mm Hg (the same as Co-myoglobin). Oxy Co-mini-myoglobin shows a well-resolved electron paramagnetic resonance (e.p.r.) spectrum typical of an oxygenated hemoprotein, while the spectrum of the deoxy derivative, although similar to that of deoxy Co-myoglobin, displays a lower resolution of the complex hyperfine structure. Moreover, photodissociation experiments on oxy Co-mini-myoglobin allow e.p.r. detection of an intermediate state, already observed in most hemoproteins and diagnostic for the interaction of bound oxygen with the distal histidine residue. Thus, reconstitution of mini-globin with cobalt protoprophyrin IX has provided, for the first time, a stable oxygenated complex that reflects a correct folding of the protein surrounding the heme pocket and possesses the functional behaviour typical of a hemoprotein.

Reptile heme protein structure: X-ray crystallographic study of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin at 2.0 A resolution.

The X-ray crystal structures of the aquo-met and cyano-met derivatives of the loggerhead sea turtle (Caretta caretta) myoglobin have been determined at 2.0 A resolution (R = 0.182, and 0.178, respectively). The results here reported, representing the first reptile globin solved by X-ray crystallography, have been analyzed in parallel with data for related monomeric hemoproteins, and indicate a strong overall structural similarity between the loggerhead sea turtle and mammalian myoglobins, reflected by the 63% amino acid identity of their primary structures. The root-mean-square deviation between the entire polypeptide backbones of loggerhead sea turtle and sperm whale myoglobins, after structure superposition, is 0.83 A. Upon cyanide binding to the protein distal site, the iron-bound water molecule present in the aquo-met form is displaced by the incoming ligand. Cyanide is oriented towards the inner part of the heme distal site forming a Fe-C-N angle of 133 degrees.

Human semen inhibits T rosette formation through an opiate mediated mechanism.

Seminal plasma contains high levels of opioid peptides and both seminal plasma and endogenous opioids can influence the immune system. In order to investigate whether these two findings can be related, semen was collected from 7 normal subjects, and assayed for beta-endorphin content and for its in vitro ability to inhibit the total T rosette formation of human lymphocytes in the presence or in the absence of 10(-6) M naloxone, an universal opiate antagonist. The results were as follows: 1) immunoreactive beta-endorphin content in seminal plasma was 4 to 12 times higher than the peripheral plasma levels detected in the same subjects (76.1 +/- 42.1 SD vs 10.5 +/- 2.0 SD pg/ml); 2) increasing concentrations of seminal plasma (1%, 5%, and 10%) in RPMI 1640 significantly depressed the T rosette formation ability of lymphocytes; and 3) the simultaneous addition to the incubation mixture of 10(-6) M naloxone prevented the phenomenon, while naloxone per se was ineffective. The possibility that endogenous opioids may play a role in the immunomodulatory action of human semen is suggested.

PHA-ICC, ADCC and NK in patients with ANLL in CR: human fibroblastic interferon fails to increase NK-active cell frequency.

PHA-ICC, ADCC and NK activity of PBL were studied in ten patients with ANLL in CR and in eighteen normal controls in the presence and absence of HFIF. No statistically significant differences were recorded among the two groups with regard to basic lymphocyte functions. Although the parameters of lymphocyte function remained analogous for those tested, the analysis at the single cell level revealed that HFIF stimulation increases the number of NK active cells and target binding cells among normals, but not in leukemic patients.

Deficiency of natural killer activity, but not of natural killer binding, in patients with lymphoadenopathy syndrome positive for antibodies to HTLV-III.

Blood lymphocytes (BL) of eleven patients with lymphoadenopathy syndrome (LAS) were studied for natural killer (NK) activity against the K562 cell line (using both the standard 51Cr release assay and the single-cell cytotoxicity assay on poly-L-lysine-coated coverslips) and for surface phenotype (employing OKT4, OKT8 and Leu7 monoclonal antibodies). A significant reduction in NK activity and in NK active cells was detected, while the percentage of target binding cells was not affected. Furthermore, the OKT4/OKT8 ratio was found to be inverted, and the Leu7+ subpopulation expanded. The patients had high titers of anti-HTLV-III antibodies. This study indicates that defective NK activity in LAS is secondary to an abnormality in the lytic event itself and not in target binding.