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The Rhipicephalus sanguineus (Latreille) complex (Acari:Ixodidae) is composed of species with intra- and interspecific morphological variation that make their diagnosis difficult. In the present study, male specimens of the R. sanguineus complex were collected from dogs in six districts of three regions of Brazil and submitted to molecular and scanning electron microscopy (SEM) analyses. Analysis of COX1 gene, 12S rDNA, and D-loop rDNA shows that ticks classified as R. sanguineus form two different clades. Morphological comparisons using SEM found adult males to exhibit morphological differences in Haller’s organ, festoons, and adanal, spiracular, and genital plates, with the last having potential usefulness in distinguishing male specimens of the complex.

Amblyomma sculptum (Ixodida: Ixodidae) Berlese, 1888, a member of the Amblyomma cajennense complex, is the major vector of Brazilian spotted fever (BSF) in southeastern Brazil. In this study, the genetic diversity of A. sculptum populations in the state of Rio de Janeiro (RJ), Brazil, was investigated because genetic variability in tick populations may be related to vector competence. Samples of A. sculptum from 19 municipalities in 7 regions of RJ were subjected to DNA extraction, amplification and sequencing of D-loop, cytochrome oxidase II and 12S rDNA mitochondrial genes. These sequences were used to map the genetic diversity of this tick. Amblyomma sculptum populations are genetically diverse in RJ, especially in the South Centre and Highland regions. Few unique haplotypes were observed in all populations, and the majority of genetic variation found was among ticks within each population. Phylogenetic reconstruction reinforced the assumption that all the haplotypes identified in RJ belong to A. sculptum. However, some RJ haplotypes are closer to A. sculptum from Argentina than to A. sculptum from elsewhere in Brazil. In RJ, A. sculptum has high genetic diversity, although little genetic differentiation. Observations also indicated a high level of gene flow among the studied populations and no evidence of population structure according to region in RJ.

Amblyomma ovale (Ixodida: Ixodidae) Koch, 1844 is widely-reported in the neotropical region and is the main vector in the epidemic cycle of Rickettsia parkeri strain Atlantic rainforest, a bioagent of a milder variety of spotted fever (SF). Because species with wide geographical distributions are known to exhibit variations that influence their vectorial capacity, the present study aimed to analyze genetic diversity and rickettsia infection of A. ovale collected during the investigation and surveillance of SF cases in the Cerrado and Atlantic rainforest (ARF) Brazilian biomes. Samples had their DNA extracted, amplified and sequenced for 16S rDNA, 12S rDNA, cytochrome oxidase subunit II and D-loop markers for tick analyses, as well as the gltA, htrA, ompA and ompB genes for rickettsia detection. Between 11 and 33 A. ovale haplotypes were identified, all of them exclusive to areas within individual analyzed biome areas. The A. ovale populations appeared to be structured, with Cluster I restricted to Cerrado + ARF isolated in Caatinga and Cluster II to ARF continuous area. Rickettsia bellii, R. parkeri strain Atlantic rainforest (first report for Goias state, Cerrado), Rickettsia asemboensis (first record in A. ovale for Brazil) and Rickettsia felis (first detection in this ixodid) were identified. A. ovale clusters were not associated with rickettsia types.

The origin of New World anthropoids has received renewed attention since the advent of molecular dating methods that relax the assumption of a strict molecular clock. However, the studies conducted to date have estimated the time of the separation of New World and Old World anthropoids at values as different as 70 and 22 Ma. With the aim of investigating the source of the discrepancies in the inferred ages, we have compared the performance of mitochondrial and nuclear markers in two pairs of datasets. We show that in the larger genomic samples, the dates of the separation of New and Old World anthropoids estimated from nuclear and mitochondrial data are significantly different. The precision of the estimates demonstrated that both markers rendered significantly different estimates. However, parametric estimates from the large nuclear dataset were highly cross-correlated. Cross-correlation of absolute divergence times and evolutionary rates was as great as -96%. Consequently, the age estimates from the large nuclear data were not reproducible, because Markov chains were unable to reach the same parametric values independently, even with the adoption of additional information from calibration priors. Thus, because branch length decomposition was not achieved, a comparison of the genomic age estimates from nuclear and mitochondrial datasets was statistically impractical. We demonstrate the importance of examining the output of Markov chain Monte Carlo analyses for correlation between rate and time in studies that use phylogenomic datasets to examine the chronological scales of primate evolution.

Amblyomma sculptum (Ixodida: Ixodidae) Berlese, 1888 is the most important tick vector in Brazil, transmitting the bioagent of the most severe form of spotted fever (SF) in part of the Cerrado (in the states of Minas Gerais and São Paulo). In another part of the Cerrado (Central-West region of Brazil), a milder form of SF has been recorded. However, neither the rickettsia nor the vector involved have been characterized. The aim of the current study was to analyse genetic variation and the presence of rickettsia in A. sculptum in Cerrado, from silent areas and with the milder form of SF. Samples were subjected to DNA extraction, amplification and sequencing of 12S rDNA, cytochrome oxidase subunit II and D-loop mitochondrial genes (for tick population analyses), and gltA, htrA, ompA and gene D (sca4) genes for rickettsia researches. Exclusive haplotypes with low frequencies, high haplotype diversity and low nucleotide diversity, star-shaped networks and significant results in neutrality tests indicate A. sculptum population expansions in some areas. Rickettsia amblyommatis, Candidatus Rickettsia andeanae and Rickettsia felis were detected. The A. sculptum diversity is not geographically, or biome delimited, pointing to a different potential in vector capacity, possibly associated with differing tick genetic profiles.

Ticks are blood-feeding arthropods and can harbor several bacteria, including the worldwide zoonotic disease Q-fever agent Coxiella burnetii. Recent studies have reported a distinct group of Coxiella mostly associated with Ixodidae ticks, including the primary endosymbionts of Amblyomma americanum. In the present work, a screening for Coxiella infection was performed by 16S ribosomal DNA (rDNA) gene analyses in 293 tick samples of 15 different species sampled worldwide, including Brazil, Colombia, Kenya, and China. Different Coxiella phylotypes were identified, and these putative symbiotic bacteria were detected in ten different Amblyomma tick species. Approximately 61 % of Rhipicephalus sanguineus and ∼37 % of Rhipicephalus microplus DNA samples were positive for Coxiella. Sequence analysis and phylogenetic reconstruction grouped all the detected Coxiella with Coxiella-like symbionts from different Ixodidae ticks. This well-defined clade clearly excludes known phylotypes of C. burnetii pathogens and other Coxiella spp. detected in different environmental samples and other invertebrate hosts.

A soluble fructose-1,6-bisphosphate aldolase enzyme has been purified 50.2-fold (2.36%) at the homogeneity from the electric organ of Electrophorus electricus by one step of DEAE- 52 anion exchange chromatography followed by Superose-12 gel filtration-FPLC. Like other aldolase enzymes the E. electricus protein is a dimer with two identical subunits of 45 kDa. The N-terminal (20 residues) revealed a high homology with S. aurata (75%, goldfish), R. ratus and M. musculus (mouse, 80%) enzymes.

Effects of the nitric oxide donors S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on Na+,K+-ATPase-rich membrane fragments purified from pig kidney outer medulla were studied using intrinsic fluorescence and ESR of spin-labeled membranes. These S-nitrosothiols differently affected the intrinsic fluorescence of Na+,K+-ATPase: GSNO induced a partial quenching, whereas SNAP produced no alteration. Quenching can be due to a direct modification of exposed tryptophan residues or to an indirect effect caused by reactions of nitrogen oxide reactive species with other residues or even with the membrane lipids. Pre-incubation of Na+,K+-ATPase with 0.4 mM GSNO resulted in a modest inhibition of ATPase activity (about 24%) measured under optimal conditions. Stearic acid spin-labeled at the 14th carbon atom (14-SASL) was used to investigate membrane fluidity and the protein–lipid interface. SNAP slightly increased the mobility of bulk lipids from Na+,K+-ATPase-rich membranes, but did not change the fraction of bulk to protein-interacting lipids. Conversely, treatment with GSNO extinguished the ESR signals from 14-SASL, indicating generation of free radicals with high affinity for the lipid moiety. Our results demonstrated that membranes influence bioavailability of reactive nitrogen species and bias the activity of different S-nitrosothiols.

Intravascular administration of recombinant adenovirus (rAd) in cancer patients has been well tolerated. However, dose-limiting hemodynamic responses associated with suppression of cardiac output have been observed at doses of 7.5 x 10(13) particles. While analysis of hemodynamic responses induced by small-molecule pharmaceuticals is well established, little is known about the cardiovascular effects of rAd. Telemetric cardiovascular (CV) monitoring in mice was utilized to measure hemodynamic events following intravascular rAd administration. Electrocardiogram analysis revealed a block in the SA node 3-4 min postinfusion, resulting in secondary pacemaking initiated at the AV node. This was associated with acute bradycardia, reduced blood pressure, and hypothermia followed by gradual recovery. Adenovirus-primed murine sera with high neutralizing antibody (nAb) titers could inhibit CV responses, whereas human sera with equivalent nAb titers induced by natural infection were, surprisingly, not inhibitory. Interestingly, repeat dosing within 2-4 h of the primary injection resulted in desensitization, resembling tachyphylaxis, for subsequent CV responses. Last, depletion of Kupffer cells prior to rAd infusion precluded induction of CV responses. These inhibitory effects suggest that rAd interactions with certain cells of the reticular endothelial system are associated with induction of CV responses. Significantly, these studies may provide insight into management of acute adverse effects following rAd systemic delivery, enabling a broadening of therapeutic index.

The Anopheles genus is a member of the Culicidae family and consists of approximately 460 recognized species. The genus is composed of 7 subgenera with diverse geographical distributions. Despite its huge medical importance, a consensus has not been reached on the phylogenetic relationships among Anopheles subgenera. We assembled a comprehensive dataset comprising the COI, COII and 5.8S rRNA genes and used maximum likelihood and Bayesian inference to estimate the phylogeny and divergence times of six out of the seven Anopheles subgenera. Our analysis reveals a monophyletic group composed of the three exclusively Neotropical subgenera, Stethomyia, Kerteszia and Nyssorhynchus, which began to diversify in the Late Cretaceous, at approximately 90 Ma. The inferred age of the last common ancestor of the Anopheles genus was ca. 110 Ma. The monophyly of all Anopheles subgenera was supported, although we failed to recover a significant level of statistical support for the monophyly of the Anopheles genus. The ages of the last common ancestors of the Neotropical clade and the Anopheles and Cellia subgenera were inferred to be at the Late Cretaceous (ca. 90 Ma). Our analysis failed to statistically support the monophyly of the Anopheles genus because of an unresolved polytomy between Bironella and A. squamifemur.

The effects of tricyclic antidepressants drugs (TCA) amitriptyline, imipramine and nortriptyline, on purified Electrophorus electricus (L.) acetylcholinesterase (AChE; acetylcholine hydrolase, EC 3.1.1.7) were studied using kinetic methods and specific fluorescent probe propidium. The antidepressants inhibited AChE activity by a non-competitive mechanism. Inhibition constants range from 200 to 400 microM. Dimethylated amitriptyline and imipramine were more potent inhibitors than the monomethylated nortriptyline. Fluorescence measurements using bis-quaternary ligand propidium were used to monitor ligand-binding properties of these cationic antidepressants to the AChE peripheral anionic site (PAS). This ligand exhibited an eight-fold fluorescence enhancement upon binding to the peripheral anionic site of AChE from E. electricus (L.) with K(D)=7 x 10(-7)M. It was observed that TCA drugs displaced propidium from the enzyme. On the basis of the displacement experiments antidepressant dissociation constants were determined. Similar values for the inhibition constants suggest that these drugs have similar affinity to the peripheral anionic site. The results also indicate that the catalytic active center of AChE does not participate in the interaction of enzyme with tricyclic antidepressants. These studies suggest that the binding site for tricyclic antidepressants is located at the peripheral anionic site of E. electricus (L.) acetylcholinesterase.

Acetylcholine Biosynthesis, Choline Acetyltransferase, Electrophorus electricus (L.), Electrocytes It is well known that the regulation of choline acetyltransferase (ChAT) activity, under physiological conditions, is important for the development and neuronal activities of cholinergic systems. The purification of ChAT has been obtained from many sources such as electric organs of fishes, Drosophila melonogaster, and mammals. We have prepared choline acetyl-transferase from a pool of supernatants obtained by differential centrifugation of electric organ homogenates from Electrophorus electricus (L.) in Tris-phosphate buffer, 0.05 m , pH 7.6. The first step of the enzyme purification was performed by ammonium sulfate precipitation at 40% and 80% . The precipitate at 80% was solubilized with sodium-phosphate buffer 0.05м , pH 7.6, dialyzed, chromatographed on DEAE-52 column and the active fraction submitted to FPLC system columns (Mono-Q: ion exchange -Superose-12: gel filtration). ChAT activity from the eluates was estimated by Fonnun’s method [Fonnun, 1975], with Acetyl-Coenzyme A tritium labelled ([3H ]AcCoA) as substrate, and the synthesis of 3HACh formed was measured. The peak from gel filtration showed a relative molecular mass of 80 kDa with highest activity in the order of 77,42 nmoles ACh/min/mg protein. This fraction was analyzed by SDS-PAGE and a band of 42 kDa was detected with Coomassie blue stain, indicating that the enzyme is formed by two subunits. Employing an antibody, the presence of ChAT was confirmed with the Western blotting technique. Isoelectrofocusing analysis demonstrated two isoforms with pI of 6,49 and 6,56, respectively.

The glyceraldehyde-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified to homogeneity from electric organ of Electrophorus electricus (L.) by a hydrophobic chroma­ tography method on deacetylcolchicine-Sepharose. The purification resulted in a 162 fold increase in specific activity of the GAPDH and final yield was approximately 37%. The purified enzyme showed a single band in SDS-PAGE, with an apparent molecular mass of 36 kDa. The purity of the colchicine-Sepharose isolated material was analysed by isoelectro-phocusing and immunoblotting using a heterologous rabbit serum anti-GAPDH. Sequence analysis of the 40-N-terminal amino acids, determined by Edman degradation, revealed its identity to other GAPDHs proteins being the largest number of identical amino acids to lobster (92.5%), rabbit muscle (85%) and human liver (80%) GAPDH.

The effect of denervation on the lipid metabolism and on the activity of (Na+-K+)ATPase isoforms from the membrane fraction P3, which corresponds to the innervated electrocyte membrane, was evaluated. On a discontinuous sucrose gradient, normal P3 membranes exhibit a bimodal (“a” and “b ” bands) distribution of the (Na+-K+)ATPase activity, which upon denervation changes to an unimodal (“c” band) distribution. Using these fractions, which have a higher (Na+-K+)ATPase activity, we characterized the lipids at the hydrophobic protein surface boundary, (i.e., the bulk lipids that surround the protein). The results confirm that these lipids consist of phospholipids and cholesterol. The quantitative composition of the phospholipids is similar for both isoform fractions obtained from the discontinuous gradient of normal membranes, with phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine representing about 90% of the total phospholipids. Sphingomyelin, phosphatidylinositol, diphosphatidylglycerol and phosphatidic acid were in the minority. However, in the single band obtained after denervation, the three major phospholipid components decreased to 70% of the total, and a significant increase in the other phospholipids and in cholesterol was observed. The high cholesterol content of the denervated fraction may confer membrane stabilization, as it is likely to cause a decrease in the membrane fluidity and consequently in the enzyme activity.

The electrogenic tissue of the electric eel Electrophorus electricus (L.) is distributed in three well-defined electric organs, the Main electric organ, Sach's organ and Hunter's organ. Sulfated glycosaminoglycan (GAG) composition was characterized in the three electric organs of the electric eel. Sulfated GAGs were analyzed in the electric organs using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed in the three electric organs that CS was the main sulfated GAG species detected, accompanied by small and diminutive amounts of CS/dermatan sulfate hybrid chains and heparan sulfate (HS), respectively. However, HS was not detected in the Sach's organ. CS was predominantly detected in the innervated membrane face of the electroplaques in the three electric organs. Our findings extend previous observations on the GAG composition in the electric organs of E. electricus and provide new information regarding the tissue distribution and location of CS.

Penaeid shrimps are an important resource in crustacean fisheries, representing more than the half of the gross production of shrimp worldwide. In the present study, we used a sample of wide-ranging diversity (41 shrimp species) and two mitochondrial markers (758 bp) to clarify the evolutionary relationships among Penaeidae genera. Three different methodologies of tree reconstruction were employed in the study: maximum likelihood, neighbor joining and Bayesian analysis. Our results suggest that the old Penaeus genus is monophyletic and that the inclusion of the Solenocera genus within the Penaeidae family remains uncertain. With respect to Metapenaeopsis monophyly, species of this genus appeared clustered, but with a nonsignificant bootstrap value. These results elucidate some features of the unclear evolution of Penaeidae and may contribute to the taxonomic characterization of this family.

The effect of mercury (Hg(2+)) on the activity of choline acetyltransferase (ChAT) from electrocytes of Electrophorus electricus (L.) was studied due to the importance of this enzyme and acetylcholine in many neurochemical functions such as arousal, learning, and memory.Mercury, which has affinity to thiol groups, acted as a potent inhibitor of ChAT, which was obtained by differential centrifugation and ammonium sulfate precipitation, at 80%, from the main electric organ homogenate.Mercury inhibition presents different kinetic behaviors for both enzyme substrates: noncompetitive to choline and of mixed type to AcCoA, with inhibition constants on the order of 0.5 to 1.0 microM. The enzyme activity was recovered using 2,3 dimercapto-propanol (BAL), a well-known chelate for sulphydryl groups and metals, which acted as a protecting agent and was able to revert the Hg(2+) inhibition at a concentration of 10 (-6) M. After treatment with this metal and in the presence of 2,3 dimercapto-propanol, 70% of the enzyme activity was recovered for AcCoA and 80% for choline.The observed inhibition is likely due to direct protein interaction, because the addition of BAL reversed the effects of HgCl(2) on ChAT activity. The results cast new light on the mechanisms of mercurial neurotoxicity.

The Mg(2+)-dependent (Na(+),K(+))ATPase maintains several cellular processes and is essential for cell excitability. In view of the importance of the enzyme activity, the interaction and binding affinities to substrates and metal ions have been studied. We determined the effect of Zinc ion (Zn(2+)) on the (Na(+),K(+))ATPase activity present in both conducting (non-innervated) and post-synaptic (innervated) membranes of electrocyte from Electrophorus electricus (L.). Zn(2+) is involved in many biological functions and is present in pre-synaptic nerve terminals. This metal, which has affinity for thiol groups, acted as a potent competitive inhibitor of (Na(+),K(+))ATPase of both membrane fractions, which were obtained by differential centrifugation of the E. electricus main electric organ homogenate. We tried to recover the enzyme activity using dithiothreitol, a reducing agent. Kinetic analysis showed that dithiothreitol acted as a non-essential non-competitive activator of (Na(+),K(+))ATPase from both membrane fractions and was able to revert the Zn(2+) inhibition at mM concentrations. In the presence of dithiothreitol, this metal behaved as a competitive inhibitor of (Na(+),K(+))ATPase in the non-innervated membrane fractions and presented a non-competitive inhibition of (Na(+),K(+))ATPase in innervated membrane fractions. This difference may be attributed to formation of a Zn-dithiothreitol complex, as well as the involvement of other binding sites for both agents. The consequences of the enzyme inhibition by Zn(2+) may be considered in regard to its neurotoxic effects.

Protein–lipid interactions are studied in normal and denervated electrocytes from Electrophorus electricus (L.) . Structural modifications of the lipid micro-environment encircling integral membrane proteins in membrane fractions presenting Na + ,K + -ATPase activity are investigated using ESR spectroscopy of stearic acid spin labeled at the 14th carbon (14-SASL). The microsomal fraction derived from the innervated electric organ exhibits, on a discontinuous sucrose gradient, a bimodal distribution of the Na + ,K + -ATPase activity, bands a and b . Band b is almost absent in microsomes from the denervated organ, and band a ′, with the same density as band a has lower Na + ,K + -ATPase activity. Band a ′ presents a larger ratio of protein-interacting lipids than band a . Analysis of the lipid stoichiometry at the protein interface indicates that denervation causes at least a twofold average decrease on protein oligomerization. Physical inactivity and denervation have similar effects on protein–lipid interactions. Denervation also influences the selectivity of proteins for fatty acids. Experiments in decreasing pH conditions performed to verify the influence of stearic acid negative charge on protein interaction revealed that denervation produces loss of charge selectivity. The observed modifications on molecular interactions induced by denervation may have importance to explain modulation of enzyme activity.

Acetylcholine is the neurotransmitter responsible for the transmission of impulses from cholinergic neurons to cells of innervated tissues. Its biosynthesis is catalyzed by the enzyme Choline acetyltransferase that is considered to be a phenotypically specific marker for cholinergic system. It is well known that the regulation of Choline acetyltransferase activity under physiological and pathological conditions is important for development and neuronal activities of cholinergic functions. We observed the distribution of Choline acetyltransferase in sections from the normal and denervated main electric organ sections of Electrophorus electricus (L.) by immunofluorescence using a anti-Choline acetyltransferase antibody. The animals were submitted to a surgical procedure to remove about 20 nerves and after 30 and 60 days, they were sacrificed. After 30 days, the results from immunohistochemistry demonstrated an increase on the Choline acetyltransferase distribution at denervated tissue sections when compared with the sections from the normal contralateral organ. A very similar labeling was observed between normal and denervated tissue sections of the animals after 60 days. However, Choline acetyltransferase activity (nmolesACh/ min/ mg of protein) in extracts obtained from electrocyte microsomal preparation, estimated by Fonnun's method (Fonnun 1975), was 70% lower in the denervated extracts.

The present investigation deals with the purification and the partial characterization of the soluble creatine kinase (CK) isoenzyme, isolated from the electric organ electrocyte of Electrophorus electricus (L.). Purification was performed by precipitation of the enzyme in the crude extract with ammonium sulfate (80%). The precipitate obtained was analyzed on an ion exchange column of diethylaminoethyl cellulose-52 (DEAE) followed by gel filtration on Superose 12 in a Fast Protein Liquid Chromatography (FPLC) system. Electrophoretic mobility of the active peak confirmed previous results identifying the hybrid isoenzyme MB in the eletrocyte cytoplasm. Electrocyte CK is a dimeric enzyme with two identical subunits of approximately 40 kDa as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The sequence analysis of the N-terminal peptide (14 amino acids) of the 40 kDa subunit showed homology with other CK enzymes from electric fish (Torpedo) and human muscle type CK. # 2000 Elsevier Science Ltd. All rights reserved.

We have determined the effects of mercury and cadmium on the creatine kinase activity of the electric organ of Electrophorus electricus (L.) which catalyses the transphosphorylation reaction between phosphocreatine and magnesium adenosine-5'-di-phosphate and has essential sulfhydryl groups. The kinetic effects of these heavy metals, which have high affinity for sulfhydryl groups, on the creatine kinase activity were analysed with the three reaction components: phosphocreatine, adenosine-5'-di-phosphate and magnesium. The kinetic data were analysed with a non-linear regression program (Sigmaplot for Windows). Both metals inhibit creatine kinase activity in the micromolar range, mercury being a more potent inhibitor than cadmium. With phosphocreatine as substrate, mercury behaved as a mixed partial hyperbolic inhibitor, non-competitive inhibitor with adenosine-5'-di-phosphate, and with magnesium mercury behaved as a competitive inhibitor. Cadmium inhibition was shown to be of a classical competitive nature with respect to both substrates, phosphocreatine or adenosine-5'-di-phosphate, and non-competitive when magnesium was the variable in the reaction mixture. The results suggest that the binding site of mercury is at or near the phosphocreatine site, but it is not the same as adenosine-5'-di-phosphate, whereas cadmium competes with these substrates to bind at the same sulphydryl site.

The effect of Mg(2+)-ATP on purified acetylcholinesterase (AChE) from electric tissue of Electrophorus electricus (L.) was studied. The enzymatic activities were measured with acetylcholine and acetylthiocholine as substrates. The kinetic parameters Vmax, Km and Hill coefficient (nH), for acetylcholine and acetylthiocholine were modified with Mg(2+)-ATP. It was shown that acetylcholinesterase presents an apparent activation at high concentration of substrates and an inhibition in the presence of Mg(2+)-ATP at low concentration of acetylcholine and acetylthiocholine. In addition, the data suggest that Mg(2+)-ATP induced an allosteric modulation of the acetylcholinesterase obtained from Electrophorous electricus (L.), and indicate an active adenosine triphosphate participation during cholinergic activity.

Study of the labeling of Schistosoma mansoni cercaria with technetium-99m (99mTc) at room temperature (25 degrees C) and 37 degrees C shows that the incorporation of radioactivity in this cercaria increases with the increase in stannous chloride concentration, reaching a constant value threshold at 130.00 microM. Strong binding of the 99mTc was obtained since the radioactivity was not washed out. The characteristic motion of the cercaria, labeled at 25 degrees C and 37 degrees C, was only modified at the concentration of 1300 microM for both temperatures, showing that the methodology here described can be applied to living structures. With this technique it is possible to obtain a cercariae suspension labeled with a radionuclide that is very inexpensive and readily available. Furthermore, it is a gamma emitter with a photon energy of 140 keV that would permit one to make scannings of the infected animals and causes less of an environmental impact and present fewer radioactive disposal problems than the long lived radionuclides.

1. 1. The behavior of lactate was studied during electrical Stimulation and influx was measured under resting conditions of rat soleus muscle. 2. 2. Lactate utilization was measured with (U-14C) lactate and results from electrical stimulation of the soleus muscle present evidence that this substance is mainly oxidized. 3. 3. Under resting conditions, lactate influx showed a saturable transport system with an apparent Km of 11 mM. This low affinity for lactate suggests that lactate transport has a limiting factor for the muscle. 4. 4. The increased lactate utilization under electrical stimulation (1,114 ± 344 μmol/g/hr, at 20 mM lactate) corresponds to increased lactate permeability as compared to the influx rate (20.81 ± 1.65 μmol/g/hr at 20 mM lactate) in resting conditions. 5. 5. Alanine, epinephrine or S.I.T.S. (4-amino-4'isothiocyanostilbene-2-2'-disulphonate) do not affect lactate permeability in the soleus muscle.

Curare, a drug which blocks nerve impulses at the myoneural junction, has been used in investigations aimed at the identification of the receptor substance of acetylcholine. This article reviews the progress so far.

We have previously demonstrated that Na+, K(+)-ATPase activity is present in both differentiated plasma membranes from Electrophorus electricus (L.) electrocyte. Considering that the alpha subunit is responsible for the catalytic properties of the enzyme, the aim of this work was to study the presence and localization of alpha isoforms (alpha1 and alpha2) in the electrocyte. Dose-response curves showed that non-innervated membranes present a Na+, K(+)-ATPase activity 2.6-fold more sensitive to ouabain (I50=1.0+/-0.1 microM) than the activity of innervated membranes (I50=2.6+/-0.2 microM). As depicted in [3H]ouabain binding experiments, when the [3H]ouabain-enzyme complex was incubated in a medium containing unlabeled ouabain, reversal of binding occurred differently: the bound inhibitor dissociated 32% from Na+, K(+)-ATPase in non-innervated membrane fractions within 1 h, while about 50% of the ouabain bound to the enzyme in innervated membrane fractions was released in the same time. These data are consistent with the distribution of alpha1 and alpha2 isoforms, restricted to the innervated and non-innervated membrane faces, respectively, as demonstrated by Western blotting from membrane fractions and immunohistochemical analysis of the main electric organ. The results provide direct evidence for a distinct distribution of Na+, K(+)-ATPase alpha-subunit isoforms in the differentiated membrane faces of the electrocyte, a characteristic not yet described for any polarized cell.

This report deals with on optical rotatory dispersion and viscosity study of hyaluronic acid with the electric organ of Electrophorus electricus (L.). This acidic polysaccharide exhibits an intrinsic Cotton effect (negative) around 212 nm in aqueous solutions. Extrinsic Cotton effects are developed by its interaction with cationic dyes which is an indication of ordered conformation in solution involving the polymer anionic sites. Viscosity measurements showed that hyaluronic acid behaves, in solution, as a polyelectrolyte with a decrease in intrinsic viscosity in media of high ionic strength and of low dielectric constant. With the quaternary ammonium compounds acetylcholine and gallamine triethyliodide, this polyelectrolyte also displays an interaction which affects both its optical rotatory dispersion and viscosity properties which is indicative of a conformational change.

The flagellar membrane of trypanosomatids has certain ultrastructural and antigenic characteristics that make its biochemical analysis very interesting. We have obtained a highly purified flagellar membrane fraction from epimastigotes of Trypanosoma cruzi and promastigotes of Herpetomonas samuelpessoai. The fractions consisted of regular spherical vesicles. Membrane fractions fixed in a glutaraldehyde solution containing filipin and freeze-fractured showed few intramembranous particles, and many protuberances indicative of filipin-sterol complexes. The protein composition of all the subflagellar fractions was analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The flagellar membrane fraction showed more than twenty bands. We wish particularly to point out the presence of six bands corresponding to proteins of 160, 135, 41, 31, 26 and 18 kDa which were not present in the axoneme-paraxial structure fraction. The blot of flagellar membrane proteins successively incubated with concanavalin A and horseradish peroxidase showed intense binding of the lectin to proteins of 117 and 87 kDa, and less strong binding to several other minor bands. Our results showed that although the membrane of the flagellum of trypanosomatids had few proteins, it seemed rich in glycosylated elements.

It was observed in thin sections that the flagellum from Herpetomonas megaseliae, a protozoan of the Trypanosomatidae family, has, besides the axoneme, a filamentous lattice-like structure, the paraxial rod. Crithidia deanei, a protozoan of the same family, does not possess a paraxial rod in its flagellum. The flagella of these two trypanosomatids were isolated. Cultures were sonified and fractionated thus resulting the following fractions: total homogenates, deflagellated cell bodies, crude flagellar fraction and purified flagellar fraction from both H. megaseliae and C. deanei. The flagellar fractions are very pure and the flagella have their structures well preserved: axonemes and paraxial structures, where they exist, are intact, associated with one another and surrounded by the flagellar membrane. A comparative analysis of the purified flagellar fractions from both trypanosomatids was made using polyacrylamide gel electrophoresis. Two protein bands, with molecular weights of 78,000 and 73,000 daltons, which were enriched along the process of purification of the flagella from H. megaseliae, were found to correspond to the main components of the paraxial structure. These two bands were not observed in the purified flagellar fraction from C. deanei.

L(+) lactate dehydrogenase (LDH) activity from the electric organ of Electrophorus electricus was measured in the presence of ATP in the forward (substrate lactate) and reverse (substrate pyruvate) enzymatic reactions. The I50 for ATP was first determined and then the kinetics of the reactions were investigated with either constant coenzyme (NAD or NADH) concentration and varying substrate (lactate or pyruvate) concentration, or, constant substrate and varying coenzyme concentration. The kinetic data showed that ATP inhibits LDH uncompetitively with respect to the reduced and the oxidized coenzyme. As for the substrates, ATP gives a mixed type inhibition for lactate and a noncompetitive inhibition for pyruvate.

Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freeze-fracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.

1. 1. Analysis of LDH isozymes from the electric organ of Electrophorus electricus (L.) was performed with Cellogel and disc electrophoresis. 2. 2. Several extracting media were employed for the enzyme analysis and only a single component with LDH activity was obtained, which had the same mobility as the muscle LDH of Electrophorus. 3. 3. Sucrose gradient ultracentrifugation analysis confirmed the presence of only one form of LDH in electric organ extracts. 4. 4. The identity of this form with muscle LDH confirms that the electric organ results from the differentiation of muscular tissue.

Establishment of the antiviral state by interferon does not impair differentiation of Friend cells. Interferon actually produces an increase in dimethylsulfoxide-induced hemoglobin synthesis. However, both the constitutive production and the induction of leukemia virus in these cells are inhibited by interferon.

1. 1. Creatine kinase (CPK) isozymes of extracts from the electric organ, dorsal muscle and brain of Electrophorus electricus (L.) were analysed with Cellogel electrophoresis. A single component corresponding to the MB-form was obtained for both electric organ and the dorsal muscle. The BB-form was present in the brain extract. 2. 2. Upon acetone fractionation of the aqueous extract of electric organ, the final fraction was submitted to gel filtration and presented a single peak of CPK activity. 3. 3. Characterization of this fraction by thin-layer gel filtration indicated an apparent molecular weight of 80,000 which corresponds to the enzyme dimeric structure. 4. 4. The implications of this finding with the muscular origin of the electric organ are discussed.

Membrane-bound (Na+ + K+)-ATPase activity from the non-innervated and innervated faces of Electrophorus electricus (L.) electric organ, obtained by differential centrifugation, was measured using AChE as an enzyme marker for membranes derived from the post-synaptic area (fraction P3) of the electrocyte. The effect of Li+ and Ba2+ on (Na+ + K+)-ATPase activity of the two membrane fractions (P2 and P3) was analysed with respect to K+ and Mg2+ ions, after the I50 estimation. The kinetics of the reactions with these cations were investigated showing that Li+ inhibits P2 uncompetitively and for P3 presented a mixed type inhibition. Ba2+ behaved as an hyperbolic mixed type inhibitor for P2 and a linear mixed type inhibitor for P3 fraction.

The study of the labelling of planaria with 99mTc shows that the incorporation of radioactivity in this platyhelminth increases with an increase in SnCl2 concentration from 0.13 to 1.3 microM, reaching a plateau in the range of 1.3-130 microM then decreasing with 1300 microM. At concentrations of 1.3 and 13 microM SnCl2, a stronger binding of 99mTc was obtained. The biological viability of the labelled planaria was not altered when the described methodology was used. The advantage of this new labelling technique is that it is possible to obtain a platyhelminth preparation labelled with a radionuclide that is very cheap, is easily available and is a gamma emitter with a photon energy of 140 keV.

L(+)lactate dehydrogenase (LDH) from the electric organ of Electrophorus electricus (L.) was purified by ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. Purified LDH was homogeneous when examined by polyacrylamide gel electrophoresis under nondenaturing conditions. Both LDH activity and protein were demonstrable in the same band. The mobility of the LDH-5 isozyme is characteristic of the muscle type enzyme. Isoelectric focusing showed a single molecular species of pIO 6.5 +/- 0.4. The apparent molecular weight was 140,000 (+/- 10%) on the basis of gel filtration of Sephadex G-200. The effect of organic acids on the enzyme activity towards pyruvate (NADH) and lactate (NAD) was determined spectrophotometrically at 340 nm. Sodium oxamate behaved as a mixed inhibitor when lactate (NAD) was the substrate, whereas ethyl oxamate was an uncompetitive inhibitor. Both the sodium salt and the ester of oxamic acid were competitive inhibitors when pyruvate (NADH) was the substrate.

L(+) lactate dehydrogenase (LDH) activity from cultured cells of Aedes albopictus was studied as a kinetic model of carbohydrate metabolism. Enzyme kinetics were studied in the forward (lactate as substrate) and reverse (pyruvate as substrate) reactions and the apparent Km values were obtained showing LDH higher affinity for pyruvate. The Hill coefficient values for each substrate were similar and indicate the existence of only one binding site on the enzyme. Isozyme analysis on cellulose-acetate electrophoresis presented a single band of LDH which presumably is of the LDH-5 type. The results obtained contribute to the assumption that Aedes albopictus cells have a predominance of anaerobic metabolism.

The localization of (Na+-K+) ATPase in the intact electrocyte of the electric organ of Electrophorus electricus (L.) and its subcellular fractions was investigated by biochemical and cytochemical methods. The distribution of AChE activity in the subcellular fractions was also comparatively analysed with this enzyme serving as a marker of the innervated membranes of the electrocyte. After application of cytochemical method of Farquhar and Palade to glutaraldehyde-fixed tissue, reaction was observed only at the membranes of vesicles localized at the periphery of the electrocyte. Previously fixed electrocytes, incubated in Ernst's medium showed reaction only at the vesicles whereas in unfixed tissue reaction also appeared at other membranes (surface and invaginations) of the anterior and posterior faces. This reaction was significantly inhibited in the presence of ouabain or in the absence of K+. Inhibition of Na+-K+-ATPase by glutaraldehyde fixation was also confirmed by biochemical analysis.

Detection of creatine kinase, which catalyzes the conversion of ADP and phosphocreatine to ATP and creatine, was performed on the electrocyte of Electrophorus electricus (L.) using a histoenzymological method based on the formation of blue colored formazan. The results indicate that the enzyme is mainly located within the cytoplasm of the electrolyte.

The concentration dependence of the self-association of hen egg-white lysozyme was studied spectrophotometrically at pH 6, 25 degrees C, and low ionic strength within a concentration range of 2.5–50 micrograms/ml. Of several possible mathematical models, an ideal or nearly ideal two-stage model representing an equilibrium between monomers and dimers and between dimers and trimers best describes the data. The dimerization and trimerization constants were found to be 2.5 x 10(-2) and 38 x 10(-2). Dialysis experiments confirmed that the mechanism involves three associating species. A "head-to-tail" contact between the associating sites was inferred from dialysis studies of the effect of indole and imidazole derivatives on lysozyme self-association.

1. We determined the effect of denervation on the lipid composition of the main electric organ and electrocyte postsynaptic membrane vesicles of Electrophorus electricus. 2. Lipid extracts of whole electric organ contain mainly cholesterol, triglycerides, phosphatidylcholine and phosphatidylethanolamine. After 30 days of denervation, the lipid composition of whole electric organ did not change appreciably. 3. Lipid extracts of the membrane vesicles were similar, except that they contained mainly free fatty acids rather than triglycerides. After 30 days of denervation, cholesterol concentration was increased and phospholipids were decreased in the membrane fraction, with higher relative concentrations of phosphatidylethanolamine and cerebrosides. 4. These results suggest that electrocyte membrane fluidity and permeability will change after 30 days of denervation.

Properties of L(+) lactate dehydrogenase (LDH) of Electrophorus electricus (L.) electric organ were studied, comparing the substrates pyruvate and lactate. Electric organ LDH is a soluble enzyme with a pH optimum of 7.4 for pyruvate and 9.0 for lactate. The apparent Km was lower for pyruvate (Km = 2.5 X 10(-4) M) than for lactate (Km = 1.5 X 10(-2) M). With lactate as a substrate at pH 7.4, malonate, oxalate and pyruvate inhibited competitively. For pyruvate as substrate at pH 9.0 malonate inhibited non-competitively and oxalate shiwed uncompetitive inhibition. The different effects of the carboxylic acids on LDH activity suggest different stereospecificities of the two enzyme-coenzyme complexes in the forward and reserve reactions. The reactions of electric organ LDH with substrates and inhibitors are consistent with electrophoretic analysis suggesting that the enzyme is of the M-type.

BackgroundThe hystricognath rodents of the New World, the Caviomorpha, are a diverse lineage with a long evolutionary history, and their representation in South American fossil record begins with their occurrence in Eocene deposits from Peru. Debates regarding the origin and diversification of this group represent longstanding issues in mammalian evolution because early hystricognaths, as well as Platyrrhini primates, appeared when South American was an isolated landmass, which raised the possibility of a synchronous arrival of these mammalian groups. Thus, an immediate biogeographic problem is posed by the study of caviomorph origins. This problem has motivated the analysis of hystricognath evolution with molecular dating techniques that relied essentially on nuclear data. However, questions remain about the phylogeny and chronology of the major caviomorph lineages. To enhance the understanding of the evolution of the Hystricognathi in the New World, we sequenced new mitochondrial genomes of caviomorphs and performed a combined analysis with nuclear genes.ResultsOur analysis supports the existence of two major caviomorph lineages: the (Chinchilloidea + Octodontoidea) and the (Cavioidea + Erethizontoidea), which diverged in the late Eocene. The Caviomorpha/phiomorph divergence also occurred at approximately 43 Ma. We inferred that all family-level divergences of New World hystricognaths occurred in the early Miocene.ConclusionThe molecular estimates presented in this study, inferred from the combined analysis of mitochondrial genomes and nuclear data, are in complete agreement with the recently proposed paleontological scenario of Caviomorpha evolution. A comparison with recent studies on New World primate diversification indicate that although the hypothesis that both lineages arrived synchronously in the Neotropics cannot be discarded, the times elapsed since the most recent common ancestor of the extant representatives of both groups are different.