Tyrosine phosphorylation of alpha-actinin in activated platelets

The integrin alpha(IIb)beta(3) mediates tyrosine phosphorylation of a 105-kDa protein (pp105) in activated platelets. We have partially purified a 105-kDa tyrosine-phosphorylated protein from platelets stimulated with phorbol 12-myristate 13-acetate and obtained the sequence of an internal 12-mer peptide derived from this protein. The sequence was identical to human alpha-actinin sequences deposited in the Swiss Protein Database. alpha-Actinin, a 105-kDa protein in platelets, was subsequently purified from activated platelets by four sequential chromatographic steps. Fractions were analyzed by Western blotting and probed with alpha-actinin and anti-phosphotyrosine antibodies. The distribution of alpha-actinin and pp105 overlapped throughout the purification. Furthermore, in the course of this purification, a 105-kDa tyrosine-phosphorylated protein was only detected in fractions that contained alpha-actinin. The purified alpha-actinin protein was immunoprecipitated with antibodies to phosphotyrosine in the absence but not in the presence of phenyl phosphate. alpha-Actinin resolved by two-dimensional gel electrophoresis of activated platelet lysates was recognized by the antibodies to phosphotyrosine, whereas pretreatment of the platelets with bisindolylmaleimide, a protein kinase C inhibitor that prevents tyrosine phosphorylation of pp105, inhibited the reactivity of the antibodies to phosphotyrosine with alpha-actinin. Taken together, these data demonstrate that a fraction of alpha-actinin is tyrosine-phosphorylated in activated platelets.     

Recovery of Trichuris tenuis Chandler, 1930, from camelids (Lama glama and Vicugna vicugna) in Argentina

A survey of whipworms was conducted in llamas and vicu?as in northwestern Argentina. Fecal examinations of a group of 14 llamas (April 1995-March 1996) and 69 vicu?as (November 1996) indicated a high prevalence (usually >50%) of Trichuris sp. in these hosts. Prevalence was highest during July-November 1995 that also coincided with the highest mean fecal egg count. During postmortem examinations of 1 llama and 1 vicu?a, specimens of Trichuris tenuis were recovered from the cecum/large intestine of each camelid. This is the first report of T. tenuis in South America, and the first report in the vicu?a. It is suggested that T. tenuis is the typical whipworm of aboriginal camelids.     

The life cycle of Amblyomma neumanni Ribaga, 1902 (Acari: Ixodidae) in the laboratory

A colony of Amblyomma neumanni was started with engorged females collected from cattle in the province of Salta (24° 51S, 65° 33W), Argentina. The larvae and nymphs were fed on rabbits and the adults on calves. The non-parasitic stages were maintained in darkness at 27 ± 1°C and 83–86% RH. The life cycle (pre-feeding period not tested) had a mean duration of 205.7 days. The mean time (days) for the different phases of the cycle were as follows: feeding of females 8.8, pre-oviposition 23.8, oviposition 41.4, minimum egg incubation 76.1, feeding of larvae 8.5, pre-moult to nymphs 16.4, feeding of nymphs 7.9 and pre-moult to adults 22.8. The mean recovery rates of larvae, nymphs and females were 83.8, 85.6 and 89.3%, respectively. The nymphs moulting to females were heavier (8.1 ± 2.34 mg) than those moulting to males (6.0 ± 2.34 mg; p < 0.01), but their range of engorgement weight showed overlap (2.3–16.2 versus 2.2–12.8 mg, respectively). Two gynandromorphs were detected between the nymphs. A comparison of biological parameters of A. neumanni with other American Amblyomma species from mammals is presented. © Rapid Science Ltd. 1998     

Coralline algae indicate Pleistocene evolution from deep,open platform to outer barrier reef environments in the northern Great Barrier Reef margin

Coralline algae from a drill core in Ribbon Reef 5 have been used to interpret changes in the depositional palaeoenvironments in the northern Australian Great Barrier Reef over the last 790 ka. Three main coralline algal assemblages, each dominated by members of a particular subfamily, have been distinguished by quantitative analysis: (1) mastophoroid assemblages, usually occurring as crusts on corals, are typical of the shallowest reef settings; (2) lithophylloid assemblages within algal nodules may represent shallow-water, cooler environments or deeper reef subenvironments; and (3) melobesioid assemblages are characteristic of deeper-water platform areas. The algal assemblages between 96 and 210 m b.s.f. (metre below sea floor) record a fluctuating but progressive shallowing-upwards from deep, outer-platform to shallower, non-reefal depositional environments. Two intervening episodes dominated by mastophoroids represent two phases of reef growth. The section above 96 m b.s.f. comprises several stacked reefs in which mastophoroid crusts similar to the present-day shallow-water assemblages predominate.Electronic Supplementary Material Supplementary material is available for this article at .Communicated by: Geological Editor P.K. Swart     

Validation of the Diagnostic Interview for Genetic Studies (DIGS) in Colombia

An interview tool, Diagnostic Interview for Genetic Studies (DIGS 3.0), was translated into Spanish for application in studies of psychiatric disorders in Colombia. Two Spanish translations of the original English version of DIGS were prepared and back-translated into English. A review committee verified the linguistic and cultural equivalence of the translations. The evaluator and test-retest reliability were assessed calculating Cohen's kappa for samples of 65 and 91 patients respectively. DIGS proved valid in both appearance and content. The confidence interval (C.I.) was excellent for schizophrenia (kappa = 0.81, C.I. 95% = 0.68-0.93), bipolar disorder (kappa = 0.87, C.I. 95% = 0.75-0.99), major depressive disorder (kappa = 0.86, C.I. 95% = 0.70-1.00), and for a normal diagnosis (kappa = 0.65, C.I. 95% = 0.41-0.89); it was good for other psychiatric diagnosis (kappa = 0.65, C.I. 95% = 0.41-0.89) and poor for schizoaffective disorder (kappa = 0.37, C.I. 95% = -0.02-0.76). Test-retest reliability was excellent for all diagnoses (kappa > 0.8), except for "other psychiatric diagnoses" (kappa = 0.64, C.I. 95% = 0.31-0.96). The Spanish translation of the DIGS was comprehensible, with face and content validity, and good test-retest and evaluator reliability. This translation will be a useful tool for genetic studies of psychiatric disorders in Latin America, particularly where schizophrenia and affective disorders are involved.     

The -308 polymorphism in the promoter region of the tumor necrosis factor-alpha (TNF-alpha) gene and ex vivo lipopolysaccharide-induced TNF-alpha expression in patients with aggressive periodontitis and/or type 1 diabetes mellitus

Several single-nucleotide polymorphisms (SNPs) have been identified in the TNF-alpha gene promoter. The transition G-->A at position -308 generates the TNF-alpha1 (G/G) and TNF-alpha2 (G/A or A/A) alleles, where the polymorphic TNF-alpha2 allele is associated with a high, in vitro TNF-alpha expression and an increased susceptibility to diverse illnesses. Here we study the association of the -308 TNF-alpha SNP with the susceptibility for developing aggressive periodontitis (AP), AP combined with type 1 diabetes mellitus (DM) and DM. We also explore the TNF-alpha capability expression and the presence of the -308 polymorphism. For this purpose we recruited 27 individuals with AP (AP+ group), 27 individuals with AP combined with DM (AP+/DM+ group), and 27 individuals with DM without signs of periodontitis upon clinical examination (DM+ group). The control group was comprised of 30 subjects. Genotyping for TNF-alpha promoter was performed by PCR-RFLP analysis. For TNF-alpha expression we used a blood culture system.     

Effect of ischemia on soluble and particulate guanylyl cyclase-mediated cGMP synthesis in cardiomyocytes

The effect of simulated ischemia [hypoxia, no glucose, extracellular pH (pH(o)) 6.4] on cGMP synthesis induced by stimulation of soluble (sGC) or particulate guanylyl cyclase (pGC) was investigated in adult rat cardiomyocytes. Intracellular cGMP content was measured after stimulation of sGC by S-nitroso-N-penicillamine (SNAP) or stimulation of pGC by natriuretic peptides [urodilatin (Uro), atrial natriuretic peptide (ANP), or C-type natriuretic peptide (CNP)] for 1 min in the presence of phosphodiesterase inhibitors. After 2 h of simulated ischemia, a decrease of >50% was observed in pGC-dependent cGMP synthesis, but no significant change was observed in sGC-dependent cGMP synthesis. The reduction in cGMP synthesis caused by simulated ischemia was mimicked by extracellular acidosis (pH(o) 6.4), which decreased pGC-mediated cGMP synthesis without altering sGC-mediated cGMP synthesis. An extreme sensitivity of pGC activity to low pH was also observed in membrane cell fractions. Hypoxia without acidosis (pH(o) 7.4) profoundly depressed cellular ATP content but did not change the response to SNAP, Uro, or ANP (selective agonists of pGC type A receptor). Only cGMP synthesis in response to CNP (a selective agonist of pGC type B receptor) was significantly reduced by ATP depletion. These data support the relevance of intracellular pH as a modulator of cGMP and suggest that, in ischemic cardiomyocytes, synthesis of cGMP would be mainly nitric oxide dependent.     

Iron porphyrin treatment extends survival in a transgenic animal model of amyotrophic lateral sclerosis

Oxidative damage, produced by mutant Cu/Zn superoxide dismutase (SOD1), may play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating motor neuron degenerative disease. A novel approach to antioxidant therapy is the use of metalloporphyrins that catalytically scavenge a wide range of reactive oxygen and reactive nitrogen species. In this study, we examined the therapeutic potential of iron porphyrin (FeTCPP) in the G93A mutant SOD1 transgenic mouse model of ALS. We found that intraperitoneal injection of FeTCPP significantly improved motor function and extended survival in G93A mice. Similar results were seen with a second group of mice wherein treatment with FeTCPP was initiated at the onset of hindlimb weakness-roughly equivalent to the time at which treatment would begin in human patients. FeTCPP-treated mice also showed a significant reduction in levels of malondialdehyde (a marker of lipid peroxidation), in total content of protein carbonyls (a marker of protein oxidation), and increased neuronal survival in the spinal cord. These results therefore provide further evidence of oxidative damage in a mouse model of ALS, and suggest that FeTCPP could be beneficial for the treatment of ALS patients.     

Ligand-induced caveolae-mediated internalization of A1 adenosine receptors: morphological evidence of endosomal sorting and receptor recycling

The involvement of caveolae in the internalization of A(1) adenosine receptors (A(1)R) and the receptor sorting and recycling was studied in the smooth muscle cell line DDT(1)MF-2, by binding assays, by confocal microscopy, and at the structural level. The use of cholera toxin-binding subunit adsorbed to gold as a specific probe for labeling the ganglioside GM(1) and immunoelectron microscopy techniques showed that agonist stimulation produced a clustering and sequestration of adenosine receptors in caveolae. Furthermore, pull-down experiments showed there to be a direct interaction between the C-terminal domain of A(1)R and caveolin-1. Addition of exogenous adenosine deaminase (ADA), a protein that binds to A(1)R and acts as a receptor activity modifying protein (RAMP) stimulated R-PIA-induced A(1) receptor internalization. Finally, the sorting and recycling of A(1)R/ADA complexes was analyzed. Detailed electron microscopy revealed that A(1)R/ADA complexes internalize together through caveolae, are differentially sorted in endosomes, and are recycled back to the cell surface by different groups of recycling endosomes. These results give insight into the spatiotemporal regulation and traffic of A(1)R and RAMPs.