Fine structure of the ventral nerve centre and interspecific identification of individual neurons in the enigmatic Chaetognatha

The enigmatic arrow worms (Chaetognatha) are marine carnivores and among the most abundant planktonic organisms. Their phylogenetic position has been heavily debated for a long time. Most recent molecular studies still provide a diverging picture and suggest arrow worms to be some kind of basal protostomes. In an effort to understand the organization of the nervous system in this clade for a broad comparison with other Metazoa we analysed the ultrastructure of the ventral nerve centre in Spadella cephaloptera by transmission electron microscopy. We were able to identify six different types of neurons in the bilateral somata clusters by means of the cytoplasmic composition (regarding the structure of the neurite and soma including the shape and eu-/heterochromatin ratio within the nucleus) as well as the size and position of these neurons. Furthermore, our study provides new insights into the neuropil composition of the ventral nerve centre and several other fine structural features. Our second goal was to examine if individually identifiable neurons are present in the ventral nerve centres of four chaetognath species, Sagitta setosa, Sagitta enflata, Pterosagitta draco, and Spadella cephaloptera. For that purpose, we processed whole mount specimens of these species for immunolocalization of RFamide-related neuropeptides and analysed them with confocal laser-scanning microscopy. Our experiments provide evidence for the interspecific homology of individual neurons in the ventral nerve centres of these four chaetognath species suggesting that the potential to generate serially arranged neurons with individual identities is part of their ground pattern.     

“In vitro” Antifungal activity of protease inhibitors

In the last five years, as HAART has become standard therapy in HIV seropositive or AIDS patients, changes have been noted in the numbers and types of opportunistic fungal infections in these cohorts of patients. Particularly, oropharyngeal candidiasis have become rare in HIV infected patients since the introduction of new anti-HIV drugs of the protease inhibitors type. At the Immunology Institute of the Universidad Central de Venezuela the most frequent protease inhibitors (PIs) used for the treatment of these patients have been: Nelfinavir (Viracept^TM, Roche),Indinavir (Crixivan^® Merck),Ritonavir (Norvir^®, Abbott),Saquinavir (Fortovase^®, Roche).Recently, we observed that recurrent candidiasis was less frequent and no Candidacould be isolated in our patients. A direct relation to the PIs was suspected. In order to assess the in vitro antifungal activity of the afore mentioned protease inhibitors on Candida sp., we used both the well diffusion test and the NCCLS broth microdilution test to assay 100 Candida sp. isolates from HIV seropositive or AIDS patients with syntomatic oropharyngeal Candida infection. In general, the data obtained with the well diffusion test were in agreement with those obtained by the broth microdilution test. All 100 isolates were susceptible to Saquinavir and 32 were susceptible to Indinavir using the NCCLS microdilution test,while 97 were susceptible to Saquinavir and 52 to Indinavir by the well diffusion test. From 17 C. albicans resistant to fluconazole, all were susceptible to Saquinavir by the NCCLS micro method and 16 by the well diffusion test. Our results showed anticandidal activity in vitro of PIs, mainly Saquinavir.This revised version was published online in October 2005 with corrections to the Cover Date.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

PHENOPSIS, an automated platform for reproducible phenotyping of plant responses to soil water deficit in Arabidopsis thaliana permitted the identification of an accession with low sensitivity to soil water deficit

The high-throughput phenotypic analysis of Arabidopsis thaliana collections requires methodological progress and automation. Methods to impose stable and reproducible soil water deficits are presented and were used to analyse plant responses to water stress. Several potential complications and methodological difficulties were identified, including the spatial and temporal variability of micrometeorological conditions within a growth chamber, the difference in soil water depletion rates between accessions and the differences in developmental stage of accessions the same time after sowing. Solutions were found. Nine accessions were grown in four experiments in a rigorously controlled growth-chamber equipped with an automated system to control soil water content and take pictures of individual plants. One accession, An1, was unaffected by water deficit in terms of leaf number, leaf area, root growth and transpiration rate per unit leaf area. Methods developed here will help identify quantitative trait loci and genes involved in plant tolerance to water deficit.     

The TWEAK–Fn14 system is a critical regulator of denervation-induced skeletal muscle atrophy in mice

Skeletal muscle atrophy occurs in a variety of clinical settings, including cachexia, disuse, and denervation. Inflammatory cytokines have been shown to be mediators of cancer cachexia; however, the role of cytokines in denervation- and immobilization-induced skeletal muscle loss remains unknown. In this study, we demonstrate that a single cytokine, TNF-like weak inducer of apoptosis (TWEAK), mediates skeletal muscle atrophy that occurs under denervation conditions. Transgenic expression of TWEAK induces atrophy, fibrosis, fiber-type switching, and the degradation of muscle proteins. Importantly, genetic ablation of TWEAK decreases the loss of muscle proteins and spared fiber cross-sectional area, muscle mass, and strength after denervation. Expression of the TWEAK receptor Fn14 (fibroblast growth factor–inducible receptor 14) and not the cytokine is significantly increased in muscle upon denervation, demonstrating an unexpected inside-out signaling pathway; the receptor up-regulation allows for TWEAK activation of nuclear factor κB, causing an increase in the expression of the E3 ubiquitin ligase MuRF1. This study reveals a novel mediator of skeletal muscle atrophy and indicates that the TWEAK–Fn14 system is an important target for preventing skeletal muscle wasting.     

Trapping of the thioacylglyceraldehyde-3-phosphate dehydrogenase intermediate from Bacillus stearothermophilus. Direct evidence for a flip-flop mechanism

The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8A resolution. Formation of the intermediate was obtained by diffusion of the natural substrate within the crystal of the holoenzyme in the absence of inorganic phosphate. To define the soaking conditions suitable for the isolation and accumulation of the intermediate, a microspectrophotometric characterization of the reaction of GAPDH in single crystals was carried out, following NADH formation at 340 nm. When compared with the structure of the Michaelis complex (Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., and Aubry, A. (2003) J. Biol. Chem. 278, 12968-12976) the 206-210 loop is shifted and now forms part of the so-called "new P(i)" site. The locations of both the O1 atom and the C3-phosphate group of the substrate are also changed. Altogether, the results provide evidence for the flipping of the C3-phosphate group occurring concomitantly or after the redox step.     

The isoform-specific region of the Na,K-ATPase catalytic subunit: role in enzyme kinetics and regulation by protein kinase C

Comparisons of the primary structures of the Na,K-ATPase alpha-isoforms reveal the existence of regions of structural divergence, suggesting that they are involved in unique functions. One of these regions is the isoform-specific region (ISR), located near the ATP binding site in the major cytoplasmic loop. To evaluate its importance, we constructed mutants of the rodent wild-type alpha1 and alpha3 isoforms in which the ISR was replaced with irrelevant sequences, i.e., the analogous region from the rat gastric H,K-ATPase catalytic subunit or a region from the human c-myc oncogene. Opossum kidney (OK) cells were transfected with wild-type rat alpha1, alpha3, or their corresponding chimeras and selected in ouabain. Introduction of either mutant produced ouabain-resistant colonies, consistent with functional expression of the chimeric protein and indicating that the ISR is not essential for overall Na,K-ATPase function. The introduced chimeras were then characterized enzymatically by measuring the relative rate of K(+) and Li(+) deocclusions. Results showed that exchanges of both alpha1 and alpha3 ISRs significantly modified the sensitivity for the enzyme to either K(+) or Li(+). Subsequent treatment of the cells with phorbol esters revealed an altered Na,K-ATPase transport in response to protein kinase C activation for the alpha1 chimeras. No changes were observed for the alpha3 isoform, suggesting that it is not sensitive to PKC regulation. These results demonstrated that the ISR plays an important role in ion deocclusion and in the response to PKC (only for the alpha1 isoform).     

Systematic screening of LNA/2'-O-methyl chimeric derivatives of a TAR RNA aptamer

We synthesized and evaluated by surface plasmon resonance 64 LNA/2'-O-methyl sequences corresponding to all possible combinations of such residues in a kissing aptamer loop complementary to the 6-nt loop of the TAR element of HIV-1. Three combinations of LNA/2'-O-methyl nucleoside analogues where one or two LNA units are located on the 3' side of the aptamer loop display an affinity for TAR below 1nM, i.e. one order of magnitude higher than the parent RNA aptamer. One of these combinations inhibits the TAR-dependent luciferase expression in a cell assay.     

DMD and IL1RAPL1: two large adjacent genes localized within a common fragile site (FRAXC) have reduced expression in cultured brain tumors

Common fragile sites (CFSs) are large regions of profound genomic instability found in all individuals. Spanning the center of the two most frequently expressed CFS regions, FRA3B (3p14.3) and FRA16D (16q23.2), are the 1.5 Mb FHIT gene and the 1.0 Mb WWOX gene. These genes are frequently deleted and/or altered in many different cancers. Both FHIT and WWOX have been demonstrated to function as tumor suppressors, both in vitro and in vivo. A number of other large CFS genes have been identified and are also frequently inactivated in multiple cancers. Based on these data, several additional very large genes were tested to determine if they were derived from within CFS regions, but DCC and RAD51L1 were not. However, the 2.0 Mb DMD gene and its immediately distal neighbor, the 1.8 Mb IL1RAPL1 gene are CFS genes contained within the FRAXC CFS region (Xp21.2-->p21.1). They are abundantly expressed in normal brain but were dramatically underexpressed in every brain tumor cell line and xenograft (derived from an intracranial model of glioblastoma multiforme) examined. We studied the expression of eleven other large CFS genes in the same panel of brain tumor cell lines and xenografts and found reduced expression of multiple large CFS genes in these samples. In this report we show that there is selective loss of specific large CFS genes in different cancers that does not appear to be mediated by the relative instability within different CFS regions. Further, the inactivation of multiple large CFS genes in xenografts and brain tumor cell lines may help to explain why this type of cancer is highly aggressive and associated with a poor clinical outcome.