Response of Caligus rogercresseyi (Boxshall & Bravo, 2000) to treatment with Hydrogen Peroxide: Recovery of parasites, fish infestation and egg viability under experimental conditions.

Hydrogen peroxide (HP) is used to remove C. rogercresseyi from fish but little is known about its effect on this species. This study determined EC50 and concentration immobilizing 100% of specimens, capacity of parasites exposed to HP to recover and infest fish, and effect on survival into the copepodid stage. EC50 and concentration immobilizing 100% of specimens were estimated by exposing parasites for 20 min to 11 concentrations and evaluating effect at 1 and 24 h post-exposure. Capacity to recover and infest fish, and survival into copepodid were evaluated by exposing parasites and eggs to HP for 20 min. Recovery and fish infestation were evaluated at 25 and 24 h post-exposure, respectively. Eggs were grown until control reached the copepodid stage and survival calculated. EC50 was 709.8 ppm.100% immobilization was obtained at 825 ppm. Male and female recover 0.5 and 1 h post-exposure, respectively. Percentage of parasites exposed and not exposed to HP that were recovered on fish was not significantly different. Survival to copepodid was lower in those exposed to HP. HP effect is greater on copepodids, but 100% of the mobile stages are immobilized under 825 ppm causing detachment from fish and potentially driven away, reducing infestation risk.

The mutagenesis of a type IV secretion system locus of Piscirickettsia salmonis leads to the attenuation of the pathogen in Atlantic salmon, Salmo salar.

Piscirickettsiosis is a threatening infectious disease for the salmon industry, due to it being responsible for significant economic losses. The control of outbreaks also poses considerable environmental challenges. Despite Piscirickettsia salmonis having been discovered as the aetiological agent of the disease more than 25 years ago, its pathogenicity remains poorly understood. Among virulence factors identified so far, type four secretion systems (T4SS) seem to play a key role during the infection caused by the bacterium. We report here the genetic manipulation of P. salmonis by means of the transference of plasmid DNA in mating assays. An insertion cassette was engineered for targeting the icmB gene, which encodes a putative T4SS-ATPase and is carried by one of the chromosomal T4SS clusters found within the genome of P. salmonis PM15972A1, a virulent representative of the EM-90-like strain. The molecular characterization of the resulting mutant strain demonstrated that the insertion interrupted the target gene. Further in vitro testing of the icmB mutant showed a dramatic drop in infectivity as tested in CHSE-214 cells, which is in agreement with its attenuated behaviour observed in vivo. Altogether, our results demonstrate that, similar to other facultative intracellular pathogens, P. salmonis' virulence relies on an intact T4SS.

Prevalence, geographic distribution and phenotypic differences of Piscirickettsia salmonis EM-90-like isolates.

Early reports accounted for two main genotypes of Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis, placing the single isolate EM-90 apart from the prototypic LF-89 and related isolates. In this study, we provide evidence that, contrary to what has been supposed, the EM-90-like isolates are highly prevalent and disseminated across Chilean marine farms. Molecular analysis of 507 P. salmonis field isolates derived from main rearing areas, diverse hosts and collected over 6 years, revealed that nearly 50% of the entire collection were indeed typed as EM-90-like. Interestingly, these isolates showed a marked host preference, being recovered exclusively from Atlantic salmon (Salmo salar) samples. Although both strains produce undistinguishable pathological outcomes, differences regarding growth kinetics and susceptibility to the antibiotics and bactericidal action of serum could be identified. In sum, our results allow to conclude that the EM-90-like isolates represent an epidemiologically relevant group in the current situation of piscirickettsiosis. Based on the consistency between genotype and phenotype exhibited by this strain, we point out the need for genotypic studies that may be as important for the Chilean salmon industry as the continuous surveillance of antimicrobial susceptibility patterns.

Comprehensive antibiotic susceptibility profiling of Chilean Piscirickettsia salmonis field isolates.

Antibiotics have been extensively used against infections produced by Piscirickettsia salmonis, a fish pathogen and causative agent of piscirickettsiosis and one of the major concerns for the Chilean salmon industry. Therefore, the emergence of resistant phenotypes is to be expected. With the aim of obtaining a landscape of the antimicrobial resistance of P. salmonis in Chile, the susceptibility profiles for quinolones, florfenicol and oxytetracycline (OTC) of 292 field isolates derived from main rearing areas, different hosts and collected over 5 years were assessed. The results allowed for the determination of epidemiological cut-off values that were used to characterize the pathogen population. This work represents the first large-scale field study addressing the antimicrobial susceptibility of P. salmonis, providing evidence of the existence of resistant types with a high incidence of resistance to quinolones. Remarkably, despite the amounts and frequency of therapies, our results disclosed that the issue of resistance to florfenicol and OTC is still in the onset.

A closer look at specific therapeutic strategies in leukemia.

Leukemia-associated fusion genes are detected in a significant proportion of newly diagnosed cases, where genes encoding transcription factors are usually found at one of the breakpoints. Activated fusion proteins, such as PML-RARalpha and AML1-ETO, have been shown to inhibit cellular differentiation by recruitment of nuclear corepressor complexes, which maintain local histone deacetylase (HDAC) in a variety of hematologic lineage-specific gene promoters. This HDAC-dependent transcriptional repression appears as a common pathway in the development of leukemia and could represent an important target for new therapeutic agents. On the other hand, the Bcr-Abl oncoprotein shows high tyrosine kinase activity and deregulates signal transduction pathways involved normally in both apoptosis and proliferation. This aberrant activity is affected by signal transduction inhibitors (STIs), which block or prevent the oncogenic pathway. In this review, we present a closer look at our understanding of both the reversible transcriptional repression controlled by HDAC and the deregulated Bcr-Abl signal transduction. In addition, the application of low molecular weight drugs for human leukemia treatment based in this knowledge results in durable clinical remission and acceptable risk of toxic effects that should increase the cure rate. We hope that this review will provide timely information to the readers.

Estudio del polimorfismos de receptores Fcylla en pacientes chilenos con lupus erimatoso sistémico / Polymorphisms of Fcylla receptors in chilean patients with systemic lupus erythematosus

Background: Polymorphisms of Fc receptors for IgG (FcgR) have been proposed as a genetic factor that influences susceptibility for systemic lupus erythematosus (SLE). Human FcgRIIa has 2 codominantly expressed alleles, H131 and R131, which differ at amino acid position 131 in the second extracelular domain (histidine or arginine respectively) and differ substantially in their ability to bind human IgG2. The H131 allele binds IgG2 efficiently, whereas R131 binds it poorly. Because IgG2 is a poor activator of the classical complement pathway, the H131 is essential for the disposal of IgG2 immune complexes. Aim: To determine the distribution of FcgRIIA genes in a cohort of Chilean SLE patients, with or without a history of lupus nephritis. Patients and methods: We studied 52 Chilean SLE patients fulfilling the 1982 American College of Rheumatology (ACR) criteria, 20 of whom had a history of nephritis, and 44 ethnically matched disease-free controls. FcgRIIa allotypes were genotyped by PCR. Results: No significant association was observed between the low affinity FcgRII receptor (FcgRIIa-R131) and the presence of SLE or lupus nephritis. However, genotype frequencies in SLE patients but not in controls, departed from the proportions predicted by the Hardy-Weinberg equilibrium, suggesting this locus might be related to the disease. Conclusions: Our results suggest that in Chilean patients with SLE, as well as in many other populations, the R131 allotype is not a major factor predisposing to the development of SLE or lupus nephritis

Kinetic characteristics of nucleoside mono-, di- and triphosphatase activities of the periplasmic 5'-nucleotidase of Escherichia coli.

Periplasmic 5'-nucleotidase from Escherichia coli, in addition to the monophosphoesterase activity has a diphosphohydrolase activity, acting on nucleoside di- and triphosphates. We proposed that the monophosphoesterase and diphosphohydrolase activities have their own active site. This proposal is based on the different types of bonds being broken. Chemical modification with selective group reagents did not show differences in the essentiality of some residues, like histidyl, carboxyl and arginyl groups, of these two hydrolytic activities. While kinetic approaches employing the competition plot and unidirectional substrate inhibition point to that diphosphohydrolase activity (ATPase-ADPase) do not share the same active site with monophosphoesterase activity. Western blotting developed with polyclonal anti-placental apyrase antibody revealed a single protein in the periplasmic fraction of 66.5 kDa similar to the Mr of the purified enzyme by isoelectrofocusing.

Partial purification and immunohistochemical localization of ATP diphosphohydrolase from Schistosoma mansoni. Immunological cross-reactivities with potato apyrase and Toxoplasma gondii nucleoside triphosphate hydrolase.

ATP diphosphohydrolase from tegumental membranes of Schistosoma mansoni was solubilized with Triton X-100 plus deoxycholate and separated by preparative nondenaturing polyacrylamide gel electrophoresis. Two isoforms with ATP-hydrolytic activity were identified and excised from nondenaturing gels. For each of the active bands, two protein bands (63 and 55 kDa) were detected with Coomassie Blue staining, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blots developed with polyclonal anti-potato apyrase antibody revealed a single protein of 63 kDa, either with samples excised from active bands or with total S. mansoni tegument. Anti-potato apyrase antibody immobilized on Sepharose-Protein A depleted over 95% of ATPase and ADPase activities from detergent-solubilized tegument. Confocal laser scanning microscopy showed anti-potato apyrase antibody on the outer surface of S. mansoni tegument. A different antibody against a fusion protein derived from recently cloned Toxoplasma gondii nucleoside triphosphate hydrolase (Bermudes, D., Peck, K. R., Afifi, M. A., Beckers, C. J. M., and Joiner, K. A. (1994) J. Biol. Chem. 269, 29252-29260) revealed the same 63-kDa band in Western blots of S. mansoni tegument. Since anti-potato apyrase antibodies exhibited cross-reactivity with S. mansoni ATP diphosphohydrolase, we decided to gain further information on the primary structure of potato apyrase by sequencing the protein. Three novel peptides were obtained: amino-terminal sequence and two internal sequences from tryptic fragments. Eight sequences recently deposited in the data bank, including that of T. gondii nucleoside triphosphate hydrolase, have considerable homologies to potato apyrase suggesting a new family of nucleoside triphosphatases which contains a conserved motif (I/V)(V/M/I)(I/L/F/C)DAGS(S/T) near the amino-terminal. Antibody cross-reactivities in the present work suggest that conserved epitopes from S. mansoni ATP diphosphohydrolase are present in this family of nucleotide-splitting enzymes.

ATP-diphosphophydrolase activity in rat heart tissue.

Extracellular nucleotides interact with specific receptors on the cell surface and are locally metabolized by ecto-nucleotidases. Biochemical characterization of the ATPase and ADPase activities detected in rat heart sarcolemma, under conditions where mitochondrial ATPase and adenylate kinase were blocked, supports our proposal that both activities correspond to a single enzyme, known as ATP-diphosphohydrolase or apyrase. The physiological function of this enzyme could be dephosphorylation of the nucleotides present in the interstitial heart compartment acting together with 5'-nucleotidase. Both hydrolytic activities have similarities in: sarcolemma localization, bivalent metal ion dependence, optimum pH, effect of several amino acid residue modifiers, competitive inhibition of nucleotide analogs, and broad nucleoside di-and triphosphate specificity. The ATPase activity could not be separated from the ADPase either through isoelectrofocusing or electrophoresis under acid conditions.

ATP-diphosphohydrolase activity in rat renal microvillar membranes and vascular tissue.

Ecto-nucleotidases may have a role in the regulation of purinoceptor-mediated responses. ATP-diphosphohydrolase or apyrase has been described as an ecto-nucleotidase, which is characterized by a low specificity for its substrates and bivalent cations. The aim of this work was to demonstrate the presence of apyrase as an ecto-enzyme in the rat kidney. ATPase-ADPase activities of the renal microvillar membrane preparation, which correspond to "right side out' membranes, were characterized. The detection of ATP-diphosphohydrolase in the renal vasculature was done through perfusion of isolated rat kidney. ATPase-ADPase activities of the microvillar membrane preparation and apyrase share similar kinetic properties. These include: low substrate and bivalent metal specificities and insensitivity towards inhibitors like: oligomycin, ouabain, verapamil, levamisole and Ap5A. The M(r) or native ATPase and ADPase activities was determined by the 60Co irradiation-inactivation technique being around 65 kDa for both hydrolytic activities. Immunowestern blot analysis also supports the presence of apyrase in microvilli. Perfusion of isolated rat kidney with ATP and ADP, in the presence or absence of different inhibitors or apyrase antibodies indicated the existence of this enzyme in the vascular endothelium. The identification of ATP-diphosphohydrolase as an ecto-enzyme both in microvilli and vasculature support the proposal that the enzyme may have an important role in the extracellular metabolism of nucleotides.

Comparison of the biochemical properties, regulation and function of ATP-diphosphohydrolase from human placenta and rat kidney.

ATP-diphosphohydrolase (apyrase. EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metals, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similarities in Mr. bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specific ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.

Comparison of the biochemical properties, regulation and function of ATP-diphosphohydrolase from human placenta and rat kidney

ATP-diphosphohydrolase (apyrase, EC 3.6.1.5) has both ATPase and ADPase activity that are stimulated by bivalent metais, with Ca2+ being the most effective. The possible physiological function of this enzyme, associated with placental and renal microvilli, is related to the extracellular metabolism of nucleotides. A comparison of the biochemical properties of human placenta and rat kidney apyrase is presented, showing similaiities in Mr, bivalent metal stimulation, nucleotide nonspecificity, insensitivity towards specifjc ATPase inhibitors, and lack of essential sulfhydryl and aliphatic hydroxyl groups. We describe the treatment of membrane preparations from both tissues with different detergents and the isoelectric focusing of the solubilized proteins to partially purify apyrase. An ectoenzyme localization is assigned both in microvillus membranes and in the vasculature on the basis of organ perfusion experiments with nucleotides in the presence of antibodies. Placental and kidney microvillus membranes inhibited ADP-induced platelet aggregation, in agreement with an extracellular role. Initial studies on enzyme regulation suggested the existence of at least two types of modulatory proteins: an activating protein in the cytosol of both tissues, and an inhibitory protein associated with placental microsomes. Possible hormonal regulation was investigated in kidneys using in vivo estradiol treatment, but only slight changes in total apyrase activity were observed.

Characterization of ATP-diphosphohydrolase from rat mammary gland.

ATP-diphosphohydrolase (or apyrase) hydrolyses nucleoside di- and triphosphates in the presence of millimolar concentration of divalent cations. It is insensitive towards sulfhydryl and aliphatic hydroxyl-selective reagents and to specific inhibitors of ATPases. We present further evidence that ATPase and ADPase activities present in rat mammary gland correspond to apyrase. Two kinetic approaches have been employed, competition plot and chemical modification with group-selective reagents. The M(r) of these activities was determined by 60Co radiation-inactivation. The kinetic approaches employed, competition plot (which discriminate whether competitive reactions occur at the same site) and chemical modification, point to the presence of a single protein which hydrolyses ATP and ADP. The similar M(r) values of ATPase and ADPase activities also support this proposal. ATPase and ADPase activities of mammary gland show a similar sensitivity or insensitivity towards several chemical modifiers. These results suggest that this enzyme is ATP-diphosphohydrolase, also known as apyrase. The results obtained are compared with the ones obtained by us and other authors with the enzyme isolated from other sources.

Human placental ATP-diphosphohydrolase: biochemical characterization, regulation and function.

1. Kinetic and physico-chemical studies on human placental microsomal fraction confirmed that the ATPase and ADPase activities detected in this fraction correspond to the enzyme ATP-diphosphohydrolase or apyrase (EC 3.6.1.5). These include substrate specificity, and coincident M(r) and pI values of both ATPase-ADPase activities. 2. This enzyme hydrolyses both the free unprotonated and cation-nucleotide complex, the catalytic efficiency for the latter being considerably higher. 3. Microsomal apyrase is insensitive to ouabain and Ap5A. The highly purified enzyme was only inhibited by o-vanadate, DES and slightly by DCCD. 4. Apyrase seems to be a glycoprotein from its interaction with Concanavalin-A. 5. Preliminary studies on the essential amino acid residues suggest the participation of Arg, Lys and His residues, and discard the requirement of -SH, COO-, -OH, and probably also Tyr and Trp. 6. Two kinetic modulatory proteins of apyrase were detected in placental tissue. An activating protein was found in the soluble fraction and an inhibitory protein was loosely bound to the membranes. 7. The proposed in vivo function for apyrase is related to the inhibition of platelet aggregation due to its ADPase activity, which is supported by the direct effect on washed platelets and by its plasma membrane localization.

Changes in apyrase activity in uterus and mammary gland during the lactogenic cycle.

1. The purpose of this present research was to explore the possible roles of ATP-diphosphohydrolase (apyrase) in two tissues with high energetic demands during cell proliferation and differentiation. 2. Changes in apyrase activities during the pregnancy lactation cycle were examined in the rat uterus and mammary gland. 3. A significant decrease in apyrase activity (ATPase-ADPase) was observed in the pregnant uterus; this observation correlates with a minor inhibitory effect on platelet aggregation. 4. In mammary gland, the enzyme activity increases during lactation in parallel with an increase in blood supply, synthesis of glycoproteins and cell proliferation. 5. Apyrase activity did not change during the estrous cycle. Estradiol administration to rats slightly increased (20%) both ATPase-ADPase activities. 6. The probable function of apyrase is finally discussed, based on its substrate specificity and subcellular localization.

ATPase-ADPase activities of rat placental tissue.

1. Calcium-stimulated ATPase-ADPase activities were studied in a microsomal fraction of rat placental tissue. 2. The kinetic characteristics correspond to those of ATP-diphosphohydrolase, also known as apyrase (E.C. 3.6.1.5). 3. These characteristics include the lack of specificity towards nucleoside di- and triphosphates, activation by Ca2+, Mg2+ or Mn2+, insensitivity to specific inhibitors of some ATPase and absence of an effect of sulphydryl reagents. 4. Chemical modification of tyrosine, tryptophan, arginine and carboxylic residues decreases both ATPase and ADPase activities. 5. The substrate analogue, 5'-(beta, gamma-methylene)triphosphate, protected both enzyme activities against all the modifying amino acid reagents tested. 6. Placental fractions (homogenate and microsomes) inhibit ADP-dependent platelet aggregation. 7. The solubilized microsomal enzyme has a molecular mass of 67 kDa by size-exclusion chromatography; the pI is 9.36. 8. A differential effect is observed on the activation produced by Concanavalin A on microsomal and solubilized fractions when treated in the presence and absence of alpha-methylmannoside.

Comparative subcellular distribution of apyrase from animal and plant sources. Characterization of microsomal apyrase.

1. Apyrase (ATP: diphosphohydrolase) has been found in the microsomal fraction of rat salivary gland, mammary gland and uterus. 2. This enzyme, already described in plant tissue, is mainly present as a soluble polypeptide in tubers of Solanum tuberosum. 3. A fraction of this enzyme is associated with the microsomal fraction with a higher specific activity than the soluble one, for either ATP or ADP as substrate. 4. Apyrase bound to microsomes from rat and potato tissues was characterized in its substrate specificity and effect of inhibitors. 5. The Km values for ATP and ADP, optimum pH and metal ion requirement were determined. 6. A characteristic common to the microsomal and soluble apyrases is the stimulatory effect of a potato activator protein of soluble plant apyrase. 7. The microsomal-bound apyrase from rat and potato tissues were solubilized and subjected to size-exclusion chromatography. 8. The mammary gland and salivary gland apyrases eluted as molecular aggregates, in contrast to the uterus and potato enzyme.