Karyotype of Brazilian Anopheles albitarsis sensu lato (Diptera:Culicidae)

Anopheles (Nyssorhynchus) albitarsis sensu lato is an important malaria vector in Brazil, especially in the Brazilian Amazon region. Chromosome preparations of fourth-instar larvae of A. albitarsis from Iranduba and Coari (AM) and Ilha Comprida (SP) were analyzed for karyotype determination and to improve cytogenetic identification of this species. Anopheles albitarsis possesses 2n = 6 chromosomes, with two pairs (submetacentric and metacentric) of autosomes and one pair of sex chromosomes, with X-Y dimorphism. The sex pair is homomorphic and acrocentric in females and heteromorphic in males, with a punctiform Y chromosome. Somatic pairing was detected in the prometaphase and metaphase chromosomes of the three A. albitarsis populations. Apparently, sex chromosome evolution in the Culicidae does not function as does evolution in the Culicidae, since it occurs in the subfamily Anophelinae, which possesses heteromorphic sex chromosomes and is regarded as primitive, based on several criteria. These karyotype data on the albitarsis complex reinforce the hypothesis that sex chromosome evolution in the subfamily Anophelinae is conserved, and the variation revealed in the mean size of chromosomes in three populations indicates that selective pressure in these populations is occurring only at a genetic level.     

The effects of deer exclusion on the development of a Mediterranean plant community affected by a wildfire

Despite the resilience of Mediterranean ecosystems to fire, the ecological restoration of burned plant communities can be hindered by ungulate herbivores, particularly in areas with high population densities. This study compares the postfire development of a shrub community with and without deer, after a wildfire occurred in 2003 in a protected area in Central Portugal. We monitored 12 fenced and 12 unfenced plots 2, 3, 4, and 8 years after fire. Within each plot, we established a linear transect and measured the monospecific canopy projections (plant patches). Five plant community indicators (patch number, average patch length, average patch height, patch cover, and patch phytovolume per square meter) were obtained. The diameter and height of individuals of the most abundant shrub species (Cistus salvifolius, Erica scoparia, Myrtus communis, Pistacia lentiscus, Rubus ulmifolius, and Ulex jussiaei) were also measured. These measurements were used as response variables in generalized linear mixed models in order to assess the effects of time‐after‐fire and fencing, on the development of the plant community. Patch height and phytovolume had a significantly higher growth in fenced plots. At the species level, C. salvifolius, M. communis, R. ulmifolius, and U. jussiaei showed a higher growth across time both in height and in diameter, in the absence of herbivory. This work shows that deer exclusion needs to be considered when aiming at the postfire restoration of Mediterranean shrub communities.     

Role of alpha adrenoceptors and nitric oxide on cardiovascular responses in acute and chronic hypertension

The contribution of α-adrenoceptors and nitric oxide (NO) on the alterations of sympathetically mediated cardiovascular responses after acute (AcH) and chronic (ChH) hypertension was evaluated in pithed aortic coarcted hypertensive rats. Pressor and tachycardia response produced by electrical stimulation of preganglionic sympathetic fibers or exogenous noradrenaline (NA) were recorded in the absence and presence of prazosin (α₁-antagonist), rauwolscine (α₂-antagonist), or N ^G-nitro-l-arginine methyl ester (l-NAME; an inhibitor of NO synthase). Compared with age-matched sham-operated rats (Nt), the pressor response produced by electrical stimulation or NA was smaller in AcH rats and larger in ChH rats. Prazosin caused a decrease of pressor response elicited by electrical stimulation or NA in all groups. However, this effect was higher in ChH. Rauwolscine produced a similar increase of sympathetically mediated pressor response in Nt and AcH rats. Nevertheless, this antagonist did not affect the sympathetically mediated pressor response in ChH rats. In addition, rauwolscine did not affect the NA-induced pressor response in all groups. The pressor response elicited by l-NAME was larger in all groups compared without l-NAME and in presence of l-arginine. Moreover, l-NAME in the presence of NA increased sympathetically mediated pressor response is in all groups, compared without it or in the presence of l-arginine. Compared with Nt, basally produced NO in aortic rings was increased in AcH but decreased in ChH. Collectively, our data suggest that decreased cardiovascular reactivity in AcH is due to an increase in basally produced NO. In ChH, enhanced cardiovascular response appears to be associated with a decrease in produced NO and an increase in released NA from sympathetic nerves.     

A biotinylated analog of the anti-proliferative prostaglandin A1 allows assessment of PPAR-independent effects and identification of novel cellular targets for covalent modification

The cyclopentenone prostaglandin (cyPG) PGA₁ displays potent anti-proliferative and anti-inflammatory effects. Therefore, PGA₁ derivatives are being studied as therapeutic agents. One major mechanism for cyPG action is the modification of protein cysteine residues, the nature of the modified proteins being highly dependent on the structure of the cyPG. Biotinylated cyPGs may aid in the proteomic identification of cyPG targets of therapeutic interest. However, for the identified targets to be relevant it is critical to assess whether biotinylated cyPGs retain the desired biological activity. Here we have explored the anti-inflammatory, anti-proliferative and cell stress-inducing effects of a biotinylated analog of PGA₁ (PGA₁-biotinamide, PGA₁-B), to establish its validity to identify cyPG–protein interactions of potential therapeutic interest. PGA₁ and PGA₁-B displayed similar effects on cell viability, Hsp70 and heme oxygenase-1 induction and pro-inflammatory gene inhibition. Remarkably, PGA₁-B did not activate PPAR. Therefore, this biotinylated analog can be useful to identify PPAR-independent effects of cyPGs. Protein modification and subcellular distribution of PGA₁-B targets were cell-type-dependent. Through proteomic and biochemical approaches we have identified a novel set of PGA₁-B targets including proteins involved in stress response, protein synthesis, cytoskeletal regulation and carbohydrate metabolism. Moreover, the modification of several of the targets identified could be reproduced in vitro. These results unveil novel interactions of PGA₁ that will contribute to delineate the mechanisms for the anti-proliferative and metabolic actions of this cyPG.     

Novel triazolopyridylbenzamides as potent and selective p38α inhibitors

A new class of p38α inhibitors based on a biaryl-triazolopyridine scaffold was investigated. X-ray crystallographic data of the initial lead compound cocrystallised with p38α was crucial in order to uncover a unique binding mode of the inhibitor to the hinge region via a pair of water molecules. Synthesis and SAR was directed towards the improvement of binding affinity, as well as ADME properties for this new class of p38α inhibitors and ultimately afforded compounds showing good in vivo efficacy.     

Complete Reversal of Coenzyme Specificity of Isocitrate Dehydrogenase from <Emphasis Type="Italic">Haloferax volcanii</Emphasis>

Haloferax volcanii Ds-threo-isocitrate dehydrogenase (ICDH) was highly expressed in bacteria as inclusion bodies. The recombinant enzyme was refolded, purified and characterized, and was found to be NADP-dependent like the wild-type protein. Sequence alignment of several isocitrate dehydrogenases from evolutionarily divergent organisms including H. volcanii revealed that the amino acid residues involved in coenzyme specificity are highly conserved. Our objective was to switch the coenzyme specificity of halophilic ICDH by altering these conserved amino acids. We were able to switch coenzyme specificity from NADP⁺ to NAD⁺ by changing five amino acids by site-directed mutagenesis (Arg291, Lys343, Tyr344, Val350 and Tyr390). The five mutants of ICDH were overexpressed in Escherichia coli as inclusion bodies and each recombinant ICDH protein was refolded and purified, and its kinetic parameters were determined. Coenzyme specificity did not switch until all five amino acids were substituted.     

Intrinsic pKas of ionizable residues in proteins: An explicit solvent calculation for lysozyme

Molecular dynamics simulations of triclinic hen egg white lysozyme in aqueous solution were performed to calculate the intrinsic pK_as of 14 ionizable residues. An all-atom model was used for both solvent and solute, and a single 180 ps simulation in conjunction with a Gaussian fluctuation analysis method was used. An advantage of the Gaussian fluctuation method is that it only requires a single simulation of the system in a reference state to calculate all the pK_as in the protein, in contrast to multiple simulations for the free energy perturbation method. pK_int shifts with respect to reference titratable residues were evaluated and compared to results obtained using a finite difference Poisson-Boltzmann (FDPB) method with a continuum solvent model; overall agreement with the direction of the shifts was generally observed, though the magnitude of the shifts was typically larger with the explicit solvent model. The contribution of the first solvation shell to the total charging free energies of the titratable groups was explicitly evaluated and found to be significant. Dielectric shielding between pairs of titratable groups was examined and found to be smaller than expected. The effect of the approximations used to treat the long-range interactions on the pK_int shifts is discussed. © 1994 Wiley-Liss, Inc.     

Fat absorptive processes in the intestine of the Antarctic fish Notothenia coriiceps (Richardson, 1844)

Absorption of lipids by the enterocytes of Notothenia coriiceps, an omnivorous Antarctic and subAntarctic fish, was studied by light and electron microscopy. The lipids are absorbed by the anterior and middle intestine segments. They appear as fat droplets that measure from 0.5 to 7 μm of diameter and which accumulate in the apical cytoplasm within the first 24 h and seem to be the main fat storage form in the enterocytes. Fat is also observed as lipid particles with 60–300 nm inside the rough endoplasmic reticulum and cytoplasmic smooth vesicles. The epithelial intercellular space and the mucosal inner lamina contain lipid particles, which probably are the fat transport form. Our observations show that an intense lipid absorptive process takes place in N. coriiceps digestive system, due to the great extension of the intestine involved and due to the great lipid accumulation found in the epithelial compartment.     

Age-dependent changes in diastolic Ca and Na concentrations in dystrophic cardiomyopathy: Role of Ca entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca²⁺ concentration ([Ca²⁺]_d) and diastolic Na⁺ concentration ([Na⁺]_d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd³⁺)-sensitive Ca²⁺ entry and inositol triphosphate (IP₃) signaling pathways in abnormal [Ca²⁺]_d and [Na⁺]_d were investigated. Our results showed an age-dependent increase in both [Ca²⁺]_d and [Na⁺]_d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd³⁺ treatment significantly reduced both [Ca²⁺]_d and [Na⁺]_d at all ages. In addition, blockade of the IP₃-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd³⁺ normalized both [Ca²⁺]_d and [Na⁺]_d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca²⁺ and Na⁺ overload mediated at least in part by enhanced Ca²⁺ entry through Gd³⁺ sensitive transient receptor potential channels (TRPC), and by IP₃ receptors.     

Interactions between Seagrass Complexity,Hydrodynamic Flow and Biomixing Alter Food Availability for Associated Filter-Feeding Organisms

Seagrass shoots interact with hydrodynamic forces and thereby a positively or negatively influence the survival of associated species. The modification of these forces indirectly alters the physical transport and flux of edible particles within seagrass meadows, which will influence the growth and survivorship of associated filter-feeding organisms. The present work contributes to gaining insight into the mechanisms controlling the availability of resources for filter feeders inhabiting seagrass canopies, both from physical (influenced by seagrass density and patchiness) and biological (regulated by filter feeder density) perspectives. A factorial experiment was conducted in a large racetrack flume, which combined changes in hydrodynamic conditions, chlorophyll a concentration in the water and food intake rate (FIR) in a model active filter-feeding organism (the cockle). Results showed that seagrass density and patchiness modified both hydrodynamic forces and availability of resources for filter feeders. Chlorophyll a water content decreased to 50% of the initial value when densities of both seagrass shoots and cockles were high. Also, filter feeder density controlled resource availability within seagrass patches, depending on its spatial position within the racetrack flume. Under high density of filter-feeding organisms, chlorophyll a levels were lower between patches. This suggests that the pumping activity of cockles (i.e. biomixing) is an emergent key factor affecting both resource availability and FIR for filter feeders in dense canopies. Applying our results to natural conditions, we suggest the existence of a direct correlation between habitat complexity (i.e. shoot density and degree of patchiness) and filter feeders density. Fragmented and low-density patches seem to offer both greater protection from hydrodynamic forces and higher resource availability. In denser patches, however, resources are allocated mostly within the canopy, which would benefit filter feeders if they occurred at low densities, but would be limiting when filter feeder were at high densities.