Taxonomic and Phylogenetic Relationships Between Species of the Genus Culex (Diptera: Culicidae) From Brazil Inferred From the Cytochrome c Oxidase I Mitochondrial Gene

Species of the genus Culex Linnaeus have been incriminated as the main vectors of lymphatic filariases and are important vectors of arboviruses, including West Nile virus. Sequences corresponding to a fragment of 478 bp of the cytochrome c oxidase subunit I gene, which includes part of the barcode region, of 37 individuals of 17 species of genus Culex were generated to establish relationships among five subgenera, Culex, Phenacomyia, Melanoconion, Microculex, and Carrollia, and one species of the genus Lutzia that occurs in Brazil. Bayesian methods were employed for the phylogenetic analyses. Results of sequence comparisons showed that individuals identified as Culex dolosus, Culex mollis, and Culex imitator possess high intraspecific divergence (3.1, 2.3, and 3.5%, respectively) when using the Kimura two parameters model. These differences were associated either with distinct morphological characteristics of the male genitalia or larval and pupal stages, suggesting that these may represent species complexes. The Bayesian topology suggested that the genus and subgenus Culex are paraphyletic relative to Lutzia and Phenacomyia, respectively. The cytochrome c oxidase subunit I sequences may be a useful tool to both estimate phylogenetic relationships and identify morphologically similar species of the genus Culex.

Resurrection of Two Species From Synonymy of Anopheles (Nyssorhynchus) strodei Root, and Characterization of a Distinct Morphological Form From the Strodei Complex (Diptera: Culicidae)

Anopheles albertoi Unti and Anopheles arthuri Unti are revived from the synonymy with Anopheles strodei Root, and a distinct morphological form (designated in this study as Anopheles CP Form) from the Strodei Complex of Anopheles (Nyssorhynchus) is characterized. The male genitalia of An. arthuri and An. albertoi are described and illustrated for the first time. An. strodei, An. arthuri, and An. albertoi were first distinguished based on scanning electron microphotos of the eggs, and then each egg type was associated with diagnostic characters of the male genitalia. Identification of Anopheles CP Form was based on morphological characters of the male genitalia, characterized and illustrated in this study. Molecular phylogenetic analysis was most clear when an outgroup was not included, in which case using the nuclear white gene, or the white gene in combination with the mitochondrial cytochrome c oxidase subunit I (COI) gene, clearly separated these four taxa. When Anopheles quadrimaculatus Say and Anopheles stephensi Liston were included as an outgroup, combined white and COI data resolved An. strodei and An. albertoi, whereas An. arthuri was not well resolved. The single sequence of Anopheles CP Form was recovered well separated from other groups in all analyses.

Studies on Anopheles (Kerteszia) homunculus Komp (Diptera: Culicidae)

The present findings suggest that Anopheles (Kerteszia) homunculus may comprise more than one species. The rDNA ITS2 sequence data corroborate the presence of An. homunculus l.s. in Mata Atlantica, southern Brazil, and suggest that specimens from Trinidad may belong to an unnamed morphologically similar species. There is a need for additional studies to establish the geographical distribution of An. homunculus l.s. in continental South America and in Trinidad, especially in southern Mata Atlantica, Brazil.

Redescription of Anopheles (Nyssorhynchus) lutzii, and Resurrection of Anopheles guarani From Synonymy With An. lutzii (Diptera: Culicidae)

Anopheles lutzii Cruz (Diptera: Culicidae) is redescribed using specimens collected in Pariquera-Acu, Vale do Ribeira, state of Sao Paulo, southeastern Mata Atlantica, Brazil. Specimens of An. lutzii from Vale do Ribeira and two females from Nova Friburgo, Rio de Janeiro, are compared with three syntypes of An. lutzii, deposited in the Instituto Oswaldo Cruz, Rio de Janeiro. Comparisons of external morphology of specimens from the type locality of Anopheles guarani Shannon demonstrate it is a valid species, and that Anopheles niger Theobald is conspecific with Anopheles guarani stat. rev. The adult male, male terminalia, fourth-instar larva, and pupa of An. guarani stat. rev. are described for the first time. Diagnostic characters of the male and female, male terminalia, fourth-instar larva and pupa of An. lutzii and An. guarani stat. rev. are illustrated. An. guarani stat. rev. is herein resurrected from the synonymy with An. lutzii, and Anopheles niger comb. nov. is transferred from the synonymy with An. lutzii to the synonymy of An. guarani stat. rev.

A novel bradykinin potentiating peptide isolated from Bothrops jararacussu venom using catallytically inactive oligopeptidase EP24.15

Characterization of the peptide content of venoms has a number of potential benefits for basic research, clinical diagnosis, development of new therapeutic agents, and production of antiserum. Here, we use a substrate-capture assay that employs a catalytically inactive mutant of thimet oligopeptidase (EC 3.4.24.15; EP24.15) to identify novel bioactive peptides in Bothrops jararacussu venom. Of the peptides captured with inactive EP24.15 and identified by mass spectrometry, three were previously identified bradykinin-potentiating peptides (BPP), < ENWPHPQIPP (Xc), < EGGWPRPGPEIPP (XIIIa) and < EARPPHPPIPP (XIe) (where < E is a pyroglutamyl residue). In addition, we identified a novel BPP peptide containing additional AP amino acids in the C-terminus (< EARPPHPPIPPAP); this novel peptide was named BPP-AP. Next, dermal and muscle microcirculations were visualized using intravital microscopy to establish the roles of peptides BPP-XIe and BPP-AP in this process. After local administration of peptide BPP-XIe (0.5 mu g.mu L-1), leukocyte rolling flux and adhesion were increased by fivefold in post-capillary venules, without any increments in vasodilatation of arterioles compared to control experiments. In contrast, local administration of BPP-AP (0.5 mu g.mu L-1) potently induced vasodilatation of arterioles (nearly 100% increase compared with the vehicle saline control), with only a small increase in leukocyte rolling flux. Therefore, the novel BPP-AP described herein has pharmacological advantages compared to the BPP-XIe. The present study further suggests that inactive oligopeptidase EP24.15 is a useful tool for the isolation of bioactive peptides from crude biological samples.

Argininosuccinate Synthetase Is a Functional Target for a Snake Venom Anti-hypertensive Peptide ROLE IN ARGININE AND NITRIC OXIDE PRODUCTION

Bj-BPP-10c is a bioactive proline-rich decapeptide, part of the C-type natriuretic peptide precursor, expressed in the brain and in the venom gland of Bothrops jararaca. We recently showed that Bj-BPP-10c displays a strong, sustained anti-hypertensive effect in spontaneous hypertensive rats (SHR), without causing any effect in normotensive rats, by a pharmacological effect independent of angiotensin-converting enzyme inhibition. Therefore, we hypothesized that another mechanism should be involved in the peptide activity. Here we used affinity chromatography to search for kidney cytosolic proteins with affinity for Bj-BPP-10c and demonstrate that argininosuccinate synthetase (AsS) is the major protein binding to the peptide. More importantly, this interaction activates the catalytic activity of AsS in a dose-dependent manner. AsS is recognized as an important player of the citrulline-NO cycle that represents a potential limiting step in NO synthesis. Accordingly, the functional interaction of Bj-BPP-10c and AsS was evidenced by the following effects promoted by the peptide: (i) increase of NO metabolite production in human umbilical vein endothelial cell culture and of arginine in human embryonic kidney cells and (ii) increase of arginine plasma concentration in SHR. Moreover, alpha-methyl-DL-aspartic acid, a specific AsS inhibitor, significantly reduced the anti-hypertensive activity of Bj-BPP-10c in SHR. Taken together, these results suggest that AsS plays a role in the anti-hypertensive action of Bj-BPP-10c. Therefore, we propose the activation of AsS as a new mechanism for the anti-hypertensive effect of Bj-BPP-10c in SHR and AsS as a novel target for the therapy of hypertension-related diseases.

Triangular ruthenium acetate clusters containing the bis(pyridyl)propane ligand and their inclusion chemistry with beta-cyclodextrin

The triruthenium carboxylate cluster [Ru(3)O(OAc)(6)(py)(2)(bpp)](+) (OAc = acetate) containing the bridging 1,3-bis(4-pyridyl)propane (bpp) ligand, and its dimeric species [{Ru(3)O(OAc)(6)(py(2))}(2)(mu-bpp)](2+) were synthesized in order to investigate their inclusion compounds with beta-cyclodextrin (beta-CD). Characterization of the complexes was carried out based on spectroscopic, electrochemical and spectroelectrochemical techniques, while the formation of inclusion complexes was evaluated using (1)H NMR/NOESY spectroscopy. Since bpp is a flexible ligand, a DFT study was carried out in order to characterize its conformational isomers and their possible role in the host-guest chemistry with beta-CD. Instead of observing the formation of inclusion compounds with different stoichiometries, we observed the formation of 1:1 bpp/beta-CD compounds in which the bpp ligand assumes different conformations. The assembly of polymetallic rotaxane species was successfully demonstrated by monitoring the (1)H NMR spectra of the monomeric cluster species in the presence of aquapentacyanoferrate(II) ions and beta-CD.

A novel physiological property of snake bradykinin-potentiating peptides-Reversion of MK-801 inhibition of nicotinic acetylcholine receptors

The first naturally occurring angiotensin-converting enzyme (ACE) inhibitors described are pyroglutamyl proline-rich oligopeptides, found in the venom of the viper Bothrops jararaca, and named as bradykinin-potentiating peptides (BPPs). Biochemical and pharmacological properties of these peptides were essential for the development of Captopril, the first active site-directed inhibitor of ACE, currently used for the treatment of human hypertension. However, a number of data have suggested that the pharmacological activity of BPPs could not only be explained by their inhibitory action on enzymatic activity of somatic ACE. In fact, we showed recently that the strong and long-lasting anti-hypertensive effect of BPP-10c [BPP-10c on acetylcholine receptor function in the PC12 pheochromocytoma cell line, which following induction to neuronal differentiation expresses most of the nicotinic receptor subtypes. BPP-10c did not induce receptor-mediated ion flux, nor potentiated carbamoylcholine-provoked receptor activity as determined by whole-cell recording. This peptide, however, alleviated MK-801-induced inhibition of nicotinic acetylcholine receptor activity. Although more data are needed for understanding the mechanism of the BPP-10c effect on nicotinic receptor activity and its relationship with the anti-hypertensive activity, this work reveals possible therapeutic applications for BPP-10c in establishing normal acetylcholine receptor activity. (C) 2008 Elsevier Inc. All rights reserved.