Do pairing systems improve welfare of captive Red-Legged partridges (Alectoris rufa) in laying cages?

Although animal welfare has become an important premise in poultry, little attention has been paid to the effects of present-day rearing methods on the welfare of game birds, species released for hunting and re-establishment purposes. This work studied the effect of pairing methods on the welfare of the Red-Legged partridge kept in laying cages (4,500 cm2), a commonly hunted game species in Western Europe. Agonistic behavior and possible injuries caused by aggression were studied during the pairing and laying period in 2 types of couples: the forced type (n=24), 1 male and 1 female randomly chosen and placed in the same cage, and the free type (n=24), where the female had the opportunity to chose between 4 males, using the time spent by the female near each male as female choice parameter. Welfare of partridges was affected by pairing system, as aggressive behavior, divorces and injuries were observed in a higher rate in forced pairs (25% of pairs did not finish the productive cycle) than in free pairs (16.6%). In addition, more females were attacked in forced pairs, whereas in free pairs, the number of attacked males and mutual aggression was increased. Males tended to display more aggressive behavior than females, pecking mainly on the head and back of females. Although injuries were observed in a minor rate in free pairs, a higher mortality was reported in females compared with males from both free and forced pairs (6 females died in total). The poor welfare in a high percentage of laying pairs hampers the development of sustainable rearing methods for the species. Thus, farmers should consider avoiding forced pairing.

Does early antipredator training increase the suitability of captive red-legged partridges (Alectoris rufa) for releasing?

This study aimed to evaluate the postrelease survival and spatial distribution of farm-bred red-legged partridges (Alectoris rufa) that were subject to a prerelease training program based on exemplary behavior and alarm calls given by adults that acted as experienced demonstrators in simulated predator encounters (wooden raptor models and humans). Two groups of partridges were released in this study: trained (intensively reared birds accompanied by tutors) and control (chicks reared without tutors). Releases were conducted in the fall and winter-spring during 2 consecutive years using a total of 44 trained and 40 control radio-tagged partridges. Trained partridges showed statistically higher mean values of survival (72.7 d), home range (13.04 ha), and dispersion (549.58 m) compared with nontrained partridges, with most nontrained birds failing to survive more than 2 wk after release. Trained adult partridges showed the best survival results (105.2 d). Taking all birds into account, causes of death were attributed to terrestrial predators (45%), raptors (18.7%), hunting (11.3%), and unknown causes (25%). Although values of variables reported here were lower than those reported in wild counterparts, survival time and spatial behavior of trained birds were close to that of previous data of parent-reared partridges and higher than that of farm-bred birds. This study aimed to confirm the potential of prerelease training techniques in present-day rearing systems. Farm-bred game birds, which normally suffer from high predation rates after release, could highly benefit from the use of cost-effective training techniques based on learning from experienced adults.

Application of statistical experimental design to study the formulation variables influencing the coating process of lidocaine liposomes.

In this paper, we have used statistical experimental design to investigate the effect of several factors in coating process of lidocaine hydrochloride (LID) liposomes by a biodegradable polymer (chitosan, CH). These variables were the concentration of CH coating solution, the dripping rate of this solution on the liposome colloidal dispersion, the stirring rate, the time since the liposome production to the liposome coating and finally the amount of drug entrapped into liposomes. The selected response variables were drug encapsulation efficiency (EE, %), coating efficiency (CE, %) and zeta potential. Liposomes were obtained by thin-layer evaporation method. They were subsequently coated with CH according the experimental plan provided by a fractional factorial (2(5-1)) screening matrix. We have used spectroscopic methods to determine the zeta potential values. The EE (%) assay was carried out in dialysis bags and the brilliant red probe was used to determine CE (%) due to its property of forming molecular complexes with CH. The graphic analysis of the effects allowed the identification of the main formulation and technological factors by the analysis of the selected responses and permitted the determination of the proper level of these factors for the response improvement. Moreover, fractional design allowed quantifying the interactions between the factors, which will consider in next experiments. The results obtained pointed out that LID amount was the predominant factor that increased the drug entrapment capacity (EE). The CE (%) response was mainly affected by the concentration of the CH solution and the stirring rate, although all the interactions between the main factors have statistical significance.

Crystal structure of an enzyme displaying both inositol-polyphosphate-1-phosphatase and 3'-phosphoadenosine-5'-phosphate phosphatase activities: a novel target of lithium therapy.

Lithium cations exert profound and selective psychopharmacological effects on ameliorate manic-depressive psychosis. Although lithium is an effective drug for both treatment and prophylaxis of bipolar disorder, the precise mechanism of action is not well understood. Lithium acts as both an uncompetitive and non-competitive inhibitor of several lithium- sensitive phosphatases with regard to substrate and magnesium cofactor, respectively. In this work, we report the crystal structure and reaction mechanism of Rattus norvegicus 3'-phosphoadenosine 5'-phosphate and inositol 1,4-bisphosphate phosphatase (RnPIP), a recently identified target of lithium therapy. This Li(+)-sensitive enzyme plays a crucial role in several cellular processes, such as RNA processing, sulphation reactions and probably inositol recycling. RnPIP specifically removes the 3'-phosphate group of 3'-phosphoadenosine 5'-phosphate (PAP) and the 1'-phosphate group of inositol 1,4-bisphosphate (I(1),(4)P(2)) producing AMP and inositol 4'-phosphate, respectively. The crystal structure of RnPIP complexed with AMP, Pi and magnesium ions at 1.69 A resolution provides insight into the reaction mechanism of the hydrolysis of PAP. The core fold of the enzyme is equivalent to that found in other Li(+)-sensitive phosphatases, such as inositol monophosphatase, but molecular modelling of I(1),(4)P(2) in the RnPIP active site reveals important structural determinants that accommodate this additional substrate. RnPIP is potently inhibited by lithium and, as the accumulation of PAP inhibits a variety of proteins, including sulphotransferases and RNA processing enzymes, this dual specificity enzyme represents a potential target of lithium action, in addition to inositol monophosphatases.

X-ray structure of yeast Hal2p, a major target of lithium and sodium toxicity, and identification of framework interactions determining cation sensitivity.

The product of the yeast HAL2 gene (Hal2p) is an in vivo target of sodium and lithium toxicity and its overexpression improves salt tolerance in yeast and plants. Hal2p is a metabolic phosphatase which catalyses the hydrolysis of 3'-phosphoadenosine-5'-phosphate (PAP) to AMP. It is, the prototype of an evolutionarily conserved family of PAP phosphatases and the engineering of sodium insensitive enzymes of this group may contribute to the generation of salt-tolerant crops. We have solved the crystal structure of Hal2p in complex with magnesium, lithium and the two products of PAP hydrolysis, AMP and Pi, at 1.6 A resolution. A functional screening of random mutations of the HAL2 gene in growing yeast generated forms of the enzyme with reduced cation sensitivity. Analysis of these mutants defined a salt bridge (Glu238 ellipsis Arg152) and a hydrophobic bond (Va170 ellipsis Trp293) as important framework interactions determining cation sensitivity. Hal2p belongs to a larger superfamily of lithium-sensitive phosphatases which includes inositol monophosphatase. The hydrophobic interaction mutated in Hal2p is conserved in this superfamily and its disruption in human inositol monophosphatase also resulted in reduced cation sensitivity.

ABI2, a second protein phosphatase 2C involved in abscisic acid signal transduction in Arabidopsis.

The abi2-1 (abscisic acid insensitive) mutant of Arabidopsis thaliana shows abscisic acid (ABA) insensitivity with respect to seed germination and vegetative ABA responses. We identified the ABI2 gene by a combination of positional mapping and homology to ABI1. The ABI2 protein shows 80% amino acid sequence identity to ABI1, a protein phosphatase 2C (PP2C) involved in ABA signaling. The mutation that confers the abi2-1 phenotype is equivalent to the mutation previously identified in abi1-1 and the resulting Gly168Asp abi2 protein shows a reduced PP2C activity. Thus, a pair of highly homologous PP2Cs regulate ABA signaling.

A novel target of lithium therapy.

Phosphatases converting 3'-phosphoadenosine 5'-phosphate (PAP) into adenosine 5'-phosphate are of fundamental importance in living cells as the accumulation of PAP is toxic to several cellular systems. These enzymes are lithium-sensitive and we have characterized a human PAP phosphatase as a potential target of lithium therapy. A cDNA encoding a human enzyme was identified by data base screening, expressed in Escherichia coli and the 33 kDa protein purified to homogeneity. The enzyme exhibits high affinity for PAP (K(m)<1 microM) and is sensitive to subtherapeutic concentrations of lithium (IC(50)=0.3 mM). The human enzyme also hydrolyzes inositol-1, 4-bisphosphate with high affinity (K(m)=0.4 microM), therefore it can be considered as a dual specificity enzyme with high affinity (microM range) for both PAP and inositol-1,4-bisphosphate. Hydrolysis of inositol-1,4-bisphosphate was also inhibited by lithium (IC(50)=0.6 mM). Thus, we present experimental evidence for a novel target of lithium therapy, which could explain some of the side effects of this therapy.

Nonradioactive northwestern analysis using biotinylated riboprobes.

A nonradioactive modification of the Northwestern assay is described and applied to the detection of RNA-binding proteins. The nonradioactive assay is based on the use of biotinylated riboprobes, which are stable and easy to handle. Chemiluminescence is generated with streptavidin-conjugated peroxidase and provides even better sensitivity than the radioactive detection method to assay RNA binding to proteins bound to nitrocellulose.

Improved factor Xa cleavage of fusion proteins containing maltose binding protein.

The addition of five glycine residues at a position adjacent to the factor Xa cleavage site of a MBP-2C fusion protein, comprising maltose binding protein (MBP) and poliovirus 2C, allowed factor Xa to generate both of the component proteins. If, however, MBP-2C was without the above modification, it was cleaved only at a site located internally within poliovirus 2C, and this protein was not, therefore, generated by factor Xa cleavage. A simple procedure is described that uses PCR for the introduction of five glycines adjacent to the factor Xa recognition site.

Loss of the xeroderma pigmentosum group B protein binding site impairs p210 BCR/ABL1 leukemogenic activity.

Previous studies have demonstrated that p210 BCR/ABL1 interacts directly with the xeroderma pigmentosum group B (XPB) protein, and that XPB is phosphorylated on tyrosine in cells that express p210 BCR/ABL1. In the current study, we have constructed a p210 BCR/ABL1 mutant that can no longer bind to XPB. The mutant has normal kinase activity and interacts with GRB2, but can no longer phosphorylate XPB. Loss of XPB binding is associated with reduced expression of c-MYC and reduced transforming potential in ex-vivo clonogenicity assays, but does not affect nucleotide excision repair in lymphoid or myeloid cells. When examined in a bone marrow transplantation (BMT) model for chronic myelogenous leukemia, mice that express the mutant exhibit attenuated myeloproliferation and lymphoproliferation when compared with mice that express unmodified p210 BCR/ABL1. Thus, the mutant-transplanted mice show predominantly neutrophilic expansion and altered progenitor expansion, and have significantly extended lifespans. This was confirmed in a BMT model for B-cell acute lymphoblastic leukemia, wherein the majority of the mutant-transplanted mice remain disease free. These results suggest that the interaction between p210 BCR/ABL1 and XPB can contribute to disease progression by influencing the lineage commitment of lymphoid and myeloid progenitors.

The RhoGEF domain of p210 Bcr-Abl activates RhoA and is required for transformation.

The BCR-ABL oncogene encodes an in-frame fusion protein containing N-terminal sequences derived from Bcr and C-terminal sequences derived from Abl. Bcr contains a centrally located Rho-specific guanine nucleotide exchange factor (RhoGEF) domain that is retained within p210 Bcr-Abl. Although this domain is subject to autoinhibition in the context of Bcr, here we show that it is constitutively activated in p210 Bcr-Abl. p210 Bcr-Abl can stimulate RhoA activation independently of its tyrosine kinase activity, and mutations within the RhoGEF domain that are predicted to eliminate RhoGEF activity inhibit RhoA activation. The RhoGEF mutant of p210 Bcr-Abl does not affect the tyrosine kinase activity of the molecule, nor the ability of p210 Bcr-Abl to interact with XPB through the RhoGEF domain. Despite retaining normal levels of tyrosine kinase activity, the RhoGEF mutant of p210 Bcr-Abl is impaired in transforming activity as measured by anchorage-independent growth. However, the mutant is still able to confer the phenotype of growth factor independence in myeloid cells, suggesting that some, but not all parameters of p210 Bcr-Abl transformation, are dependent upon a catalytically active RhoGEF domain. Collectively, these observations identify a gain-of-function activity attributable to the RhoGEF domain of p210 Bcr-Abl that is required to support the transformed phenotype.

Development of a citrus genome-wide EST collection and cDNA microarray as resources for genomic studies.

A functional genomics project has been initiated to approach the molecular characterization of the main biological and agronomical traits of citrus. As a key part of this project, a citrus EST collection has been generated from 25 cDNA libraries covering different tissues, developmental stages and stress conditions. The collection includes a total of 22,635 high-quality ESTs, grouped in 11,836 putative unigenes, which represent at least one third of the estimated number of genes in the citrus genome. Functional annotation of unigenes which have Arabidopsis orthologues (68% of all unigenes) revealed gene representation in every major functional category, suggesting that a genome-wide EST collection was obtained. A Citrus clementina Hort. ex Tan. cv. Clemenules genomic library, that will contribute to further characterization of relevant genes, has also been constructed. To initiate the analysis of citrus transcriptome, we have developed a cDNA microarray containing 12,672 probes corresponding to 6875 putative unigenes of the collection. Technical characterization of the microarray showed high intra- and inter-array reproducibility, as well as a good range of sensitivity. We have also validated gene expression data achieved with this microarray through an independent technique such as RNA gel blot analysis.

Protein phosphatase 2C (PP2C) function in higher plants.

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.

Poliovirus protein 2C has ATPase and GTPase activities.

Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids.

Poliovirus protein 2C contains two regions involved in RNA binding activity.

Poliovirus protein 2C is involved in poliovirus RNA replication, although the exact function of 2C is still unknown. Recently, it was shown that 2C can be purified to high levels when expressed as a fusion protein with maltose-binding protein (MBP). Evidence was presented that 2C has ATPase and GTPase activities; preliminary results also indicated that 2C interacts with RNA (Rodríguez, P.L., and Carrasco, L. (1993) J. Biol. Chem. 268, 8105-8110). In the present study, 20 variants of 2C have been generated, and their NTPase and RNA binding activities were analyzed. Moreover, an easy procedure to obtain genuine 2C after factor Xa cleavage of an MBP2-2C fusion protein is described. This work has determined that 2C has two regions involved in RNA binding: a NH2-terminal region located between amino acids 21 and 45 and a COOH-terminal region involving an Arg-rich region located between amino acids 312 and 319. Deletion of either the NH2- or COOH-terminal RNA-binding region abolishes RNA binding. Deletion of an internal region of protein 2C that includes the nucleotide-binding motif does not affect RNA binding, whereas this deletion destroys ATPase and GTPase activities. Therefore, the NTPase activity and the RNA binding capacity of protein 2C are located in different regions of the molecule.

Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol.

The mode of action of gliotoxin against poliovirus has been analyzed in detail. This fungal metabolite inhibits the appearance of poliovirus proteins when present from the beginning of infection but has no effect on viral translation when added at late times. In agreement with previous findings, this toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium. Analysis of the synthesis of poliovirus plus- or minus-stranded RNA in the presence of gliotoxin suggests that this compound effectively hampered both processes. This result contrasts with the mode of action of other inhibitors of poliovirus RNA synthesis, such as guanidine or flavones, that selectively block plus-stranded RNA synthesis and suggests that the target of gliotoxin differs from the target of guanidine, i.e., poliovirus protein 2C. Indeed, gliotoxin was found to be a potent inhibitor of poliovirus RNA synthesis in cell-free systems, using membranous crude replication complexes, a reaction that is not blocked by guanidine or Ro 09-0179. Moreover, in vitro activity of the purified poliovirus polymerase 3Dpol was efficiently inhibited by gliotoxin. These results indicate that this toxin acts on the poliovirus polymerase 3Dpol, providing the first description of an inhibitor of this viral enzyme.

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2.

We report the cloning of both the cDNA and the corresponding genomic sequence of a new PP2C from Arabidopsis thaliana, named AtP2C-HA (for homology to ABI1/ABI2). The AtP2C-HA cDNA contains an open reading frame of 1536 bp and encodes a putative protein of 511 amino acids with a predicted molecular mass of 55.7 kDa. The AtP2C-HA protein is composed of two domains, a C-terminal PP2C catalytic domain and a N-terminal extension of ca. 180 amino acid residues. The deduced amino acid sequence is 55% and 54% identical to ABI1 and ABI2, respectively. Comparison of the genomic structure of the ABI1, ABI2 and AtP2C-HA genes suggests that they belong to a multigene family. The expression of the AtP2C-HA gene is up-regulated by abscisic acid (ABA) treatment.

A role for 3AB protein in poliovirus genome replication.

The poliovirus polypeptide 3AB, the precursor of the genome-bound VPg protein, stimulates in vitro the synthesis of poly(U) directed by the viral polymerase 3Dpol (Lama, J., Paul, A., Harris, K., and Wimmer, E. (1994) J. Biol. Chem. 269, 66-70), suggesting that 3AB could be modulating the activity of the viral polymerase in poliovirus-infected cells. To address the exact function of 3AB in the viral replication cycle, a biochemical and molecular genetic analysis of 3AB has been carried out. 3AB protein bound RNA probes in two different assays, and amino acid positions implicated in the RNA binding activity of 3AB were determined. Mutant proteins with reduced RNA binding activity were unable to stimulate 3Dpol polymerase activity. Purified protein 3A showed no RNA binding or 3Dpol stimulatory activity, but 3A and VPg mutations conferred a synergistic effect on the 3AB functions. Polioviruses encoding for these mutant 3ABs were constructed. These mutant viruses translated their RNA genomes in vitro and processed their polyproteins as wild type virus did. Cells infected with 3AB mutant viruses showed over 90% inhibition in the accumulation of plus and minus viral RNA strands and more than 100-fold reduction of virus yield at 4 h postinfection. Our results suggest that 3AB protein functions in vivo as a co-factor of the viral polymerase and that the activity of 3AB may be regulated by proteolytic processing.

CtCdc55p and CtHa13p: two putative regulatory proteins from Candida tropicalis with long acidic domains.

The salt-tolerance gene HAL3 from Saccharomyces cerevisiae encodes a novel regulatory protein (Hal3p) which modulates the expression of the ENA1 sodium-extrusion ATPase (Ferrando et al., Mol. Cell. Biol. vol. 15, 1995, pp. 5470-5481). Hal3p contains an essential acidic domain rich in aspartates at its carboxyl terminus. We have isolated two cross-hybridizing genes from a genomic library of Candida tropicalis. One of the genes (CtHAL3) is a true homolog of HAL3 and it partially complements the salt sensitivity of a S. cerevisiae hal3 mutant. The activity of CtHAL3 was equivalent to that of an open reading frame (YKL088w) identified by genome sequencing of S. cerevisiae and with homology to HAL3. The other cross-hybridizing gene (CtCDC55) is a CDC55 homolog, encoding a protein with an internal acidic domain not present in the S. cerevisiae CDC55 product. Cdc55p is a regulatory subunit of protein phosphatase 2A and CtCDC55 complements the cold sensitivity of a S. cerevisiae cdc55 mutant. The presence of acidic domains in different putative regulatory proteins may suggest a role for this type of domain in molecular interactions.