Chromosomal characterization of two species of genus <Emphasis Type="Italic">Steatogenys</Emphasis> (Gymnotiformes: Rhamphichthyoidea: Steatogenini) from the Amazon basin: sex chromosomes and correlations with Gymnotiformes phylogeny

Gymnotiformes are an important component of the Neotropical ichthyofauna and they are known for their ability to generate and detect electrical discharges. Phylogenetic relationships of Gymnotiformes are still not well understood. However, the monophyly of the superfamily Rhamphichthyoidea is well accepted, despite the position of tribe Steatogenini (Steatogenys, Hypopygus and Stegostenopos) within this superfamily is unclear. The genus Steatogenys includes three species that, together with Hypopygus and Stegostenopos, form tribe Steatogenini. Cytogenetic information is currently only available for Hypopygus lepturus. Here, we describe the karyotypes of Steatogenys elegans from four localities and S. duidae from two localities. S. elegans was found to have 2n = 50, ZZ/ZW (12m-sm/38st-a), while S. duidae had 2n = 50 (50m-sm). In S. elegans, constitutive heterochromatin (CH) was observed in the centromeric regions of all chromosomes, in the interstitial region of 1q, and in two blocks of Wq. In S. duidae, CH was observed in the centromeric and pericentromeric regions of all chromosomes, and in the interstitial regions of 2q, 3q, 5q, and 7q. Nucleolar organizer regions (NORs) were identified in the distal regions of one chromosome pair in each species. The CMA₃ fluorochrome (specific to G-C rich regions) coincided with the NORs in both species, and with the HC of S. elegans except on chromosome pair 5 and the W. The DAPI fluorochrome (specific to A-T rich regions) coincided with the CH of both species, and was very intense for chromosome pair 5 and the W of S. elegans. Our observations suggest that the ZZ/ZW system observed in S. elegans likely evolved through CH addition followed by a paracentric inversion. The chromosomal data described herein are consistent with the phylogenetic hypothesis that tribe Steatogenini should be positioned within family Ramphychthyidae.     

Fabric dependence of wave propagation in anisotropic porous media

Current diagnosis of bone loss and osteoporosis is based on the measurement of the bone mineral density (BMD) or the apparent mass density. Unfortunately, in most clinical ultrasound densitometers: 1) measurements are often performed in a single anatomical direction, 2) only the first wave arriving to the ultrasound probe is characterized, and 3) the analysis of bone status is based on empirical relationships between measurable quantities such as speed of sound (SOS) and broadband ultrasound attenuation (BUA) and the density of the porous medium. However, the existence of a second wave in cancellous bone has been reported, which is an unequivocal signature of poroelastic media, as predicted by Biot’s poroelastic wave propagation theory. In this paper, the governing equations for wave motion in the linear theory of anisotropic poroelastic materials are developed and extended to include the dependence of the constitutive relations upon fabric—a quantitative stereological measure of the degree of structural anisotropy in the pore architecture of a porous medium. This fabric-dependent anisotropic poroelastic approach is a theoretical framework to describe the microarchitectural-dependent relationship between measurable wave properties and the elastic constants of trabecular bone, and thus represents an alternative for bone quality assessment beyond BMD alone.     

BALB/c mice infected with antimony treatment refractory isolate of Leishmania braziliensis present severe lesions due to IL-4 production

Background

Leishmania braziliensis is the main causative agent of cutaneous leishmaniasis in Brazil. Protection against infection is related to development of Th1 responses, but the mechanisms that mediate susceptibility are still poorly understood. Murine models have been the most important tools in understanding the immunopathogenesis of L. major infection and have shown that Th2 responses favor parasite survival. In contrast, L. braziliensis–infected mice develop strong Th1 responses and easily resolve the infection, thus making the study of factors affecting susceptibility to this parasite difficult.

Methodology/Principal Findings

Here, we describe an experimental model for the evaluation of the mechanisms mediating susceptibility to L. braziliensis infection. BALB/c mice were inoculated with stationary phase promastigotes of L. braziliensis, isolates LTCP393(R) and LTCP15171(S), which are resistant and susceptible to antimony and nitric oxide (NO), respectively. Mice inoculated with LTCP393(R) presented larger lesions that healed more slowly and contained higher parasite loads than lesions caused by LTCP15171(S). Inflammatory infiltrates in the lesions and production of IFN-γ, TNF-α, IL-10 and TGF-β were similar in mice inoculated with either isolate, indicating that these factors did not contribute to the different disease manifestations observed. In contrast, IL-4 production was strongly increased in LTCP393(R)-inoculated animals and also arginase I (Arg I) expression. Moreover, anti-IL-4 monoclonal antibody (mAb) treatment resulted in decreased lesion thickness and parasite burden in animals inoculated with LTCP393(R), but not in those inoculated with LTCP15171(S).

Conclusion/Significance

We conclude that the ability of L. braziliensis isolates to induce Th2 responses affects the susceptibility to infection with these isolates and contributes to the increased virulence and severity of disease associated with them. Since these data reflect what happens in human infection, this model could be useful to study the pathogenesis of the L. braziliensis infection, as well as to design new strategies of therapeutic intervention.     

Multicenter evaluation of mycobacteria identification by PCR restriction enzyme analysis in laboratories from Latin America and the Caribbean

The identification of mycobacterial species in clinical isolates is essential for making patient care decisions. Polymerase chain reaction (PCR) restriction enzyme analysis (PRA) is a simple and rapid identification method, based on amplification of 441 bp of the hsp65 gene and restriction with BstEII and HaeIII. As a contribution to the validation of PRA, a multicenter study was performed in eight laboratories located in Argentina, Brazil, Colombia, Chile, and Guadeloupe. Each laboratory received 18 coded isolates from the collection of the Institute of Tropical Medicine (Antwerp, Belgium), representing duplicates of nine laboratory strains: Mycobacterium terrae CIPT 140320001, Mycobacterium scrofulaceum CIPT 140220031, Mycobacterium flavescens ATCC 14474, Mycobacterium triviale ATCC 23292, Mycobacterium nonchromogenicum ATCC 19530, Mycobacterium chitae ATCC 19627, Mycobacterium abscessus ATCC 19977, Mycobacterium kansasii ATCC 12478, and Mycobacterium peregrinum ATCC 14467. A detailed protocol including amplification, enzymatic digestion, and gel preparation was provided to each laboratory. Two laboratories identified correctly all 18 (100%) isolates, one identified correctly 17 (94.5%), two identified 14 (77.7%), one identified 11 (61%), and two identified 8 (44.4%) isolates. Errors detected in laboratories with more than 77% accuracy were associated with electrophoresis running conditions and an unspecific amplicon produced by a single strain. Lower accuracy was mainly related to inappropriate use of DNA markers and insufficient training in interpretation of patterns. In conclusion, the PRA method was readily implemented in some Latin American and Caribbean laboratories of mycobacteria, but improvements in critical points, as gel running conditions and training in interpretiation of patterns, are needed in order to improve accuracy. In others, improvement in critical points is still necessary.     

XNP-1/ATR-X acts with RB, HP1 and the NuRD complex during larval development in C. elegans

Mutations in the XNP/ATR-X gene cause several X-linked mental retardation syndromes in humans. The XNP/ATR-X gene encodes a DNA-helicase belonging to the SNF2 family. It has been proposed that XNP/ATR-X might be involved in chromatin remodelling. The lack of a mouse model for the ATR-X syndrome has, however, hampered functional studies of XNP/ATR-X. C. elegans possesses one homolog of the XNP/ATR-X gene, named xnp-1. By analysing a deletion mutant, we show that xnp-1 is required for the development of the embryo and the somatic gonad. Moreover, we show that abrogation of xnp-1 function in combination with inactivation of genes of the NuRD complex, as well as lin-35/Rb and hpl-2/HP1 leads to a stereotyped block of larval development with a cessation of growth but not of cell division. We also demonstrate a specific function for xnp-1 together with lin-35 or hpl-2 in the control of transgene expression, a process known to be dependent on chromatin remodelling. This study thus demonstrates that in vivo XNP-1 acts in association with RB, HP1 and the NuRD complex during development.     

Frequencies of the four major Amerindian mtDNA haplogroups in the population of Montevideo, Uruguay

mtDNA Amerindian polymorphisms were studied in 108 inhabitants of Montevideo, Uruguay, using PCR RFLP analysis. Amerindian haplogroups were found in 20.4% of the sample. The frequency of Amerindian polymorphisms in Montevideo differed significantly from that observed in Tacuarembó, a city about 400 km away, indicating the high level of variation within Uruguay. Results for mitochondrial markers indicate that admixture occurred primarily as a result of Amerindian females mating with European males.     

Androgen- and estrogen-dependent regulation of insulin-degrading enzyme in subcellular fractions of rat prostate and uterus

Innumerous data support the fact that insulin-degrading enzyme (IDE) is the primary enzymatic mechanism for initiating and controlling cellular insulin degradation. Nevertheless, insulin degradation is unlikely to be the only cellular function of IDE, because it appears that some cellular effects of insulin are mediated by IDE as a regulatory protein. Insulin-degrading enzyme shows a significant correlation with various cellular functions, such as cellular growth and differentiation, and the expression of IDE is developmentally regulated. Besides insulin, other substrates are also degraded by IDE, including various growth-promoting peptides. It has also been shown that IDE enhances the binding of androgen to DNA in the nuclear compartment. It is also known that the androgen hormones have a stimulatory effect on prostate growth, and that estradiol stimulates uterine growth. To establish whether IDE is regulated by a cellular prostate/uterine growth stimulus, the present study assessed whether IDE was modified in quantity and activity during proliferative conditions (castration + testosterone in the male rat, or castration + estradiol or the proestrus phase of the estrous cycle in the female rat) and autolysis (castration or the metestrus phase of the estrous cycle) using cytosolic and nuclear fractions of rat prostate and cytosolic fractions of rat uterus. The activity and amount of IDE decreased in the cytosolic fraction with castration and during metestrus, and increased with testosterone or estradiol treatment and during proestrus. In the nuclear fraction, the quantity of the IDE followed the same pattern observed in the cytosolic fraction, although without degradative activity. The data presented here suggest that IDE may participate in prostatic and uterine growth and that the testosterone or estradiol and/or prostate and uterus insulin-like growth factors may be important factors for the expression and regulation of IDE in the prostate and uterus.     

Seasonal modulation of free radical metabolism in estivating land snails Helix aspersa

We investigated the regulation of free radical metabolism in Helix aspersa snails during a cycle of 20-day estivation and 24-h arousal in summer in comparison with estivation/arousal in winter-snails. In winter-snails (J. Exp. Biol. 206, 675-685, 2003), we had already observed an increase in the selenium-dependent glutathione-peroxidase (Se-GPX) activity in foot muscle and hepatopancreas and in the contents of hepatopancreas GSH-equivalents (GSH-eq=GSH+2 GSSG) during estivation compared with 24-h aroused snails. Summer-estivation prompted a 3.6-fold increase in Se-GPX activity in hepatopancreas, though not in foot muscle. Total-superoxide dismutase and catalase activities in hepatopancreas decreased (by 30-40%) during summer-estivation; however, no changes occurred in the activities of glutathione reductase, glutathione S-transferase and glucose-6-phosphate dehydrogenase in the two organs. GSH-eq levels were increased (by 54%) in foot muscle during estivation, but were unchanged in hepatopancreas. In contrast with winter-snails, oxidative stress markers (lipid peroxidation, carbonyl protein, and the GSSG/GSH-eq ratio) were unaltered during estivation/arousal in summer. These results demonstrate that seasonality modulates not only the absolute activities/levels of antioxidants (enzymes and GSH-eq) in H. aspersa, but also the regulatory process that controls the snail's antioxidant capacity during estivation/arousal. These results suggest that H. aspersa has an "internal clock" controlling the regulation of free radical metabolism in the different seasons.     

The active site is the least stable structure in the unfolding pathway of a multidomain cold-adapted alpha-amylase

The cold-active alpha-amylase from the Antarctic bacterium Pseudoalteromonas haloplanktis (AHA) is the largest known multidomain enzyme that displays reversible thermal unfolding (around 30 degrees C) according to a two-state mechanism. Transverse urea gradient gel electrophoresis (TUG-GE) from 0 to 6.64 M was performed under various conditions of temperature (3 degrees C to 70 degrees C) and pH (7.5 to 10.4) in the absence or presence of Ca2+ and/or Tris (competitive inhibitor) to identify possible low-stability domains. Contrary to previous observations by strict thermal unfolding, two transitions were found at low temperature (12 degrees C). Within the duration of the TUG-GE, the structures undergoing the first transition showed slow interconversions between different conformations. By comparing the properties of the native enzyme and the N12R mutant, the active site was shown to be part of the least stable structure in the enzyme. The stability data supported a model of cooperative unfolding of structures forming the active site and independent unfolding of the other more stable protein domains. In light of these findings for AHA, it will be valuable to determine if active-site instability is a general feature of heat-labile enzymes from psychrophiles. Interestingly, the enzyme was also found to refold and rapidly regain activity after being heated at 70 degrees C for 1 h in 6.5 M urea. The study has identified fundamental new properties of AHA and extended our understanding of structure/stability relationships of cold-adapted enzymes.