Functional analysis of ligand-binding and signal transduction domains of CD69 and CD23 C-type lectin leukocyte receptors

CD69 and CD23 are leukocyte receptors with distinctive pattern of cell expression and functional features that belong to different C-type lectin receptor subfamilies. To assess the functional equivalence of different domains of these structurally related proteins, a series of CD69/CD23 chimeras exchanging the carbohydrate recognition domain, the neck region, and the transmembrane and cytoplasmic domains were generated. Biochemical analysis revealed the importance of the neck region (Cys68) in the dimerization of CD69. Functional analysis of these chimeras in RBL-2H3 mast cells and Jurkat T cell lines showed the interchangeability of structural domains of both proteins regarding Ca2+ fluxes, serotonin release, and TNF-alpha synthesis. The type of the signal transduced mainly relied on the cytoplasmic domain and was independent of receptor oligomerization. The cytoplasmic domain of CD69 transduced a Ca2+-mediated signaling that was dependent on the extracellular uptake of Ca2+. Furthermore, a significant production of TNF-alpha was induced through the cytoplasmic domain of CD69 in RBL-2H3 cells, which was additive to that promoted via FcepsilonRI, thus suggesting a role for CD69 in the late phase of reactions mediated by mast cells. Our results provide new important data on the functional equivalence of homologous domains of these two leukocyte receptors.     

Genomic organization of the human CD5 gene

CD5 is a member of the family of receptors which contain extracellular domains homologous to the type I macrophage scavenger receptor cysteine-rich (SRCR) domain. Here, we compare the exon/intron organization of the human CD5 gene with its mouse homologue, as well as with the human CD6 gene, the closest related member of the SRCR superfamily. The human CD5 gene spans about 24.5 kb and consists of at least 11 exons. These exons are conserved in size, number, and structure in the mouse CD5 homologue. No evidence for the biallelic polymorphism reported in the mouse could be found among a population of 100 individuals of different ethnic origins. The human CD5 gene maps to the Chromosome (Chr) 11q12.2 region, 82 kb downstream from the human CD6 gene, in a head-to-tail orientation, a situation which recalls that reported at mouse Chr 19. The exon/intron organization of the human CD5 and CD6 genes was very similar, differing in the size of intron 1 and the number of exons coding for their cytoplasmic regions. While several isoforms, resulting from alternative splicing of the cytoplasmic exons, have been reported for CD6, we only found evidence of a cytoplasmic tailless CD5 isoform. The conserved structure of the CD5 and CD6 loci, both in mouse and human genomes, supports the notion that the two genes may have evolved from duplication of a primordial gene. The existence of a gene complex for the SRCR superfamily on human Chr 11q (and mouse Chr 19) still remains to be disclosed.     

Monitoring sequence space as a test for the target of selection in viruses

An essential feature of viral quasispecies, predicted from quasispecies theory, is that the target of selection is the mutant distribution as a whole. To test molecularly the mutant composition selected from a viral quasispecies we reconstructed a mutant distribution using 19 antigenic variants of foot-and-mouth disease virus (FMDV). Each variant was marked by a specific amino acid replacement at a major antigenic site of the virus that conferred resistance to a monoclonal antibody (mAb). The variants were introduced in the mutant spectrum of a biological FMDV clone, at a frequency commonly found in FMDV quasispecies. The reconstructed quasispecies (and a number of control populations) were allowed to replicate in the presence or absence of the mAb. The mutant distribution that became dominant as a result of antibody selection included at least ten of the 19 mutants initially used to reconstruct the quasispecies. No such biased mutant repertoire was found in control populations. The results show that a mutant distribution was selected, and are incompatible with selection of an individual genome, which then generated multiple mutants upon further replication. An ample representation of variants immediately following a selection event should contribute to subsequent adaptability of the virus.     

A new species of Acanthobothrium (Cestoda: Tetraphyllidea: Onchobothriidae) from the ocellate river stingray, Potamotrygon motoro (Chondrichthyes: Potamotrygonidae), in Argentina

Examination of the spiral intestines of 44 freshwater stingrays, Potamotrygon motoro, from tributary rivers of the Parana River in Argentina, allowed for the collection of specimens of an undescribed species of Acanthobothrium. Acanthobothrium ramiroi n. sp. can be distinguished from all other congeners by the combination of the following characters: asymmetrical hooks (medial and lateral hooks conspicuously different in size and form, with axial prong of medial hooks stouter than abaxial prong), hook size (total length of medial hooks up to 242 microm, total length of lateral hooks up to 239 microm), bothridia not fused to the scolex proper at posterior ends, worm size (51-84 mm long), and the presence of a conspicuous vaginal sphincter. The new species is different from all other species of Acanthobothrium in freshwater potamotrygonids, except Acanthobothrium terezae, in having conspicuous asymmetrical hooks. The main differences that allow for the distinction between A. ramiroi and A. terezae include hook size, the way the bothridia are attached to the scolex proper, and the shape of the older gravid segments. The discovery of a new species of Acanthobothrium from a potamotrygonid extends our understanding of the diversity of the genus in freshwater stingrays in South America.     

Synthesis, characterization, and antibacterial activity of three palladium(II) complexes of tetracyclines

Pd(II) complexes with three antibiotics of the tetracycline family (tetracycline, doxycycline and chlortetracycline) were synthesized and characterized by elemental, thermogravimetric, and conductivity analyses, and infrared spectroscopy. The interactions between Pd(II) ions and tetracycline were investigated in aqueous solution by (1)H NMR. All the tetracyclines studied form 1:1 complexes with Pd(II) via the oxygen of the hydroxyl group at ring A and that of the amide group. The effect of the three complexes on the growth of bacterial strains sensitive and resistant to tetracycline was studied. The Pd(II) complex of tetracycline is practically as efficient as tetracycline in inhibiting the growth of two Escherichia coli (E. coli) sensitive bacterial strains and 16 times more potent against E. coli HB101/pBR322, a bacterial strain resistant to tetracycline. Pd(II) coordination to doxycycline also increased its activity in the resistant strain by a factor of 2.     

Alterations of the renal function and oxidative stress in renal tissue from rats chronically treated with aluminium during the initial phase of hepatic regeneration

Various indices of renal functions during the early stage of hepatic injury were studied in rats chronically treated with aluminum (Al) lactate. Tubular and hemodynamic parameters were analyzed four days after producing a 65% partial hepatectomy (PH). Water and sodium balances were also studied. Oxidative stress and the activity of Na-K-ATPase were determined in renal tissue. The rats were distributed in four groups: control, Al, PH, Al+PH. Al did not modify the hemodynamic renal functions and the PH-group reduced the glomerular filtrate rate (GFR). The Al + PH group presented a decrease in the renal blood flow and accentuated the GFR fall as compared with PH. The fractional excretion (FE) of water and sodium increased in the PH group. The rats chronically treated with Al and then submitted to the PH protocol developed a further increase in FE of water but a reduction in FE of sodium. Both PH and Al promoted an increase in the aldosterone. PH and Al induced a similar increase of the lipoperoxidation status with reduction of glutathione (GSH) and the activity of glutathione peroxidase (GSH-Px). The data indicated that Al is an inhibitor of catalase. The GSH and GSH-Px activity in the Al + PH group demonstrated a synergic effect of Al and PH. This work demonstrates that rats treated chronically with Al and submitted to another injury (such as hepatic damage) can aggravate renal functions, probably by increasing the oxidative state, at least in kidneys.     

Plant traits, environmental factors, and pollinator visitation in winter-flowering Helleborus foetidus (Ranunculaceae)

BACKGROUND AND AIMS: This study examined the effect of plant traits and environmental factors on pollinator visitation in the winter-flowering Helleborus foetidus (Ranunculaceae) in three distant regions in the Iberian Peninsula. METHODS: Geographical variation in floral visitor assemblage, plant traits and environmental factors were analysed during the flowering season. KEY RESULTS: Differences were found in all plant traits measured (number of open flowers, flower size, number of stamens per flower, and number of nectaries) both within and among regions, and differences among regions in all the environmental factors considered (air temperature, exposure to sunlight, canopy cover, and distance to the nearest neighbour). Differences were also found among regions in the probability that plants would be visited by pollinators. CONCLUSIONS: The results show that, although floral display (i.e. number of open flowers on a plant on a given day) consistently explained among-plant differences in visitation rate in all regions, visitation rate was not significantly affected by any other biological or environmental variable. In Helleborus foetidus, then, 'how' the plant is would seem to be more important than 'where' is it.     

alpha1-Adrenoceptors stimulate a Galphas protein and reduce the transient outward K+ current via a cAMP/PKA-mediated pathway in the rat heart

alpha(1)-Adrenoceptor stimulation prolongs the duration of the cardiac action potentials and leads to positive inotropic effects by inhibiting the transient outward K(+) current (I(to)). In the present study, we have examined the role of several protein kinases and the G protein involved in I(to) inhibition in response to alpha(1)-adrenoceptor stimulation in isolated adult rat ventricular myocytes. Our findings exclude the classic alpha(1)-adrenergic pathway: activation of the G protein G(alphaq), phospholipase C (PLC), and protein kinase C (PKC), because neither PLC, nor PKC, nor G(alphaq) blockade prevents the alpha(1)-induced I(to) reduction. To the contrary, the alpha(1)-adrenoceptor does not inhibit I(to) in the presence of protein kinase A (PKA), adenylyl cyclase, or G(alphas) inhibitors. In addition, PKA and adenylyl cyclase activation inhibit I(to) to the same extent as phenylephrine. Finally, we have shown a functional coupling between the alpha(1)-adrenoceptor and G(alphas) in a physiological system. Moreover, this coupling seems to be compartmentalized, because the alpha(1)-adrenoceptor increases cAMP levels only in intact cells, but not in isolated membranes, and the effect on I(to) disappears when the cytoskeleton is disrupted. We conclude that alpha(1)-adrenoceptor stimulation reduces the amplitude of the I(to) by activating a G(alphas) protein and the cAMP/PKA signaling cascade, which in turn leads to I(to) channel phosphorylation.     

Chytrid fungus parasitizing the wild amphibian Leptodactylus ocellatus (Anura: Leptodactylidae) in Argentina

The present contribution is the first report of parasitosis by a chytrid fungus in wild anuran amphibians in Argentina, as well as the first case of amphibian mortality documented to date in Argentina. We report the presence of the chytrid fungus in dead adult Leptodactylus ocellatus. It has been suggested that chytridiomycosis is the main cause of death in several amphibian populations worldwide. Our study demonstrates that chytridiomycosis afflicts L. ocellatus, a common widespread amphibian species, and is the first report of chytridiomycosis in the Argentinian lowlands. The occurrence at this latitude would indicate an extended distribution of this fungus in wildlife populations. It is also the first report of amphibian mortality due to chytrid fungus in our country. It is noteworthy that the site of collection is situated very close to sea level in a temperate climate zone and that this represents the southern most record for South American wild amphibians.     

Effect of GLP-1 on glucose transport and its cell signalling in human myocytes

Glucagon-like peptide-1 (GLP-1) controls glucose metabolism in extrapancreatic tissues participating in glucose homeostasis, through receptors not associated to cAMP. In rat hepatocytes, activation of PI3K/PKB, PKC and PP-1 mediates the GLP-1-induced stimulation of glycogen synthase. We have investigated the effect of GLP-1 in normal human myocytes, and that of its structurally related peptides exendin-4 (Ex-4) and its truncated form 9-39 (Ex-9) upon glucose uptake, and the participation of cellular enzymes proposed to mediate insulin actions. GLP-1 and both exendins activated, like insulin, PI3K/PKB and p42/44 MAPK enzymes, but p70s6k was activated only by GLP-1 and insulin. GLP-1, Ex-4 and Ex-9, like insulin, stimulated glucose uptake; wortmannin blocked the action of GLP-1, insulin and Ex-9, and reduced that of Ex-4; PD98059 abolished the effect of all peptides/hormones, while rapamycin blocked that of insulin and partially prevented that of GLP-1. H-7 abolished the action of GLP-1, insulin and Ex-4, while Ro 31-8220 prevented only the Ex-4 and Ex-9 effect. In conclusion, GLP-1, like insulin, stimulates glucose uptake, and this involves activation of PI3K/PKB, p44/42 MAPKs, partially p70s6k, and possibly PKC; Ex-4 and Ex-9 both have GLP-1-like effect upon glucose transport, in which both share with GLP-1 an activation of PI3K/PKB--partially in the case of Ex-4--and p44/42 MAPKs but not p70s6k.