Palindromic unit-independent transposition of IS1397 in Yersinia pestis

Palindromic units (PUs) are intergenic repeated sequences scattered over the chromosomes of Escherichia coli and several other enterobacteria. In the latter, IS1397, an E. coli insertion sequence specific to PUs, transposes into PUs with sequences close to the E. coli consensus. Reasons for this insertion specificity can relate to either a direct recognition of the target (by its sequence or its structure) by the transposase or an interaction between a specific host protein and the PU target DNA sequence. In this study, we show that for Yersinia pestis, a species deprived of PUs, IS1397 can transpose onto its chromosome, with transpositional hot spots. Our results are in favor of a direct recognition of target DNA by IS1397 transposase.     

Experimental hyperphenylalaninemia provokes oxidative stress in rat brain

Tissue accumulation of L-phenylalanine (Phe) is the biochemical hallmark of human phenylketonuria (PKU), an inherited metabolic disorder clinically characterized by mental retardation and other neurological features. The mechanisms of brain damage observed in this disorder are poorly understood. In the present study we investigated some oxidative stress parameters in the brain of rats with experimental hyperphenylalaninemia. Chemiluminescence, total radical-trapping antioxidant potential (TRAP), superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activities were measured in the brain of the animals. We observed that chemiluminescence is increased and TRAP is reduced in the brain of hyperphenylalaninemic rats. Similar data were obtained in the in vitro experiments using Phe at various concentrations. CAT activity was significantly inhibited by Phe in vitro and in vivo, whereas GSH-Px activity was reduced in vivo but not in vitro and SOD activity was not altered by any treatment. The results indicate that oxidative stress may be involved in the neuropathology of PKU. However, further studies are necessary to confirm and extend our findings to the human condition and also to determine whether an antioxidant therapy may be of benefit to these patients.     

Cryopreservation of peripheral blood mononuclear cells does not significantly affect the levels of spontaneous apoptosis after 24-h culture

Studies performed with malaria patients living in endemic areas are frequently conducted in laboratories located hundreds of kilometer away from research centers, due to the difficulties in performing the assays in field conditions. Thus, we considered the potential indication of cryopreservation of peripheral blood mononuclear cells (PBMC), in most fieldwork, and decided to evaluate the effect of cryopreservation of PBMC on spontaneous apoptosis. The membrane integrity of PBMC was tested using three previously described protocols of cryopreservation. Cell samples were obtained from 19 healthy volunteers. Percentage of apoptotic nuclei in short-term PBMC cultures was determined by a sensitive method using 7-aminoactinomycin D followed by flow cytometry. Our results indicate that although cryopreservation can to some extent affect lymphocyte membrane integrity rates, flow cytometry analysis showed that frequencies of spontaneous apoptosis in cryopreserved cells were not significantly modified after 24-h culture. It is concluded that cryopreserved PBMC could be used for measuring spontaneous apoptosis and therefore, could be employed for the study of populations living in areas distant from research centers, allowing the comparative evaluation of samples obtained at different time.     

Anti-angiogenic effects of homocysteine on cultured endothelial cells

High levels of homocysteine induce a sustained injury on arterial endothelial cells which accelerates the development of thrombosis and atherosclerosis. Some of the described effects of homocysteine on endothelial cells are features shared with an anti-angiogenic response. Therefore, we studied the effects of homocysteine on key steps of angiogenesis using bovine aorta endothelial cells as a model. Homocysteine decreased proliferation and induced differentiation. Furthermore, 5 mM homocysteine produced strong inhibitions of matrix metalloproteinase-2 and urokinase, two proteolytic activities that play a key role in extracellular matrix re-modeling, and decreased migration and invasion, other two key steps of angiogenesis. This study demonstrates that homocysteine can inhibit several steps of the angiogenic process.     

Contribution of a mathematical modelling approach to the understanding of the ovarian function

The biological meaning of folliculogenesis is to free fertilisable oocytes at the time of ovulation. We approached the study of the control of follicular development at the level of follicular granulosa cells, on the experimental as well as mathematical modelling grounds. We built a mathematical model allowing for the processes of proliferation, differentiation and apoptosis. State variables correspond to the numbers of cells undergoing these different processes, while control variables correspond to the cellular transition rates. The model results raised the notion of proliferative resources, which leads to consider the optimal management of these resources and has motivated the settling of an experiment investigating the changes in the growth fraction within the granulosa throughout terminal development. We are now investigating the way gonadotrophins, and especially FSH, operate on granulosa cells, in order to account for the hormonal control of the divergent commitment of granulosa cells towards either proliferation, differentiation or apoptosis. We are thus focusing on the dynamics of cAMP production, which appears to be a keypoint in FSH signal transduction.     

Basal body-associated nucleation center for the centrin-based cortical cytoskeletal network in Paramecium

The infraciliary lattice, a contractile cortical cytoskeletal network of Paramecium, is composed of a small number of polypeptides including centrins. Its overall pattern reflects a hierarchy of structural complexity, from assembly and bundling of microfilaments to formation of polygonal meshes arranged in a continuous network subtending the whole cell surface, with local differentiations in the shape and size of the meshes. To analyse how the geometry of this complex network is generated and maintained, we have taken two approaches. Firstly, using monoclonal antibodies raised against the purified network, we have shown that all the component polypeptides colocalize, in agreement with previous biochemical data indicating that the infraciliary lattice is formed of large complexes comprising all the component polypeptides. Secondly, by taking advantage of different experimental conditions leading to disassembly of the network, we have followed its reassembly. Cytological analysis of the process revealed 1) that the network regrows exclusively from specific infraciliary lattice organizing centers (ICLOC), precisely localized near each basal body and, 2) that the global organization is not precisely controlled by genetic information but by the basal body pattern. Finally, slight ultrastuctural differences between reassembled and control lattices suggest that the organization of the filament bundles is partly templated by that of the preexisting ones.     

A New Type-II NADH Dehydrogenase from the Archaeon Acidianus ambivalens: Characterization and in vitro Reconstitution of the Respiratory Chain

A new type-II NADH dehydrogenase (NDH-II) was isolated from the hyperthermoacidophilic archaeon Acidianus ambivalens. This enzyme is a monomer with an apparent molecular mass of 47 kDa, containing a covalently bound flavin, and no iron–sulfur clusters. Upon isolation, NDH-II loses activity, which can, nevertheless, be restored by incubation with phospholipids. Catalytically, it is a proficient NADH:caldariella quinone oxidoreductase (130 mmol NADH oxidized/mg protein^-1/min^-1) but it can also donate electrons to synthetic quinones, strongly suggesting its involvement in the respiratory chain. The apparent K_m for NADH was found to be 6 M, both for the purified and membrane-integrated enzyme, thus showing that detergent solubilization and purification did not affect the substrate binding site. Further, it is the first example of a type-II NADH dehydrogenase that contains the flavin covalently attached, which may be related to the need to stabilize the otherwise labile cofactor in a thermophilic environment. A fully operative minimal version of Acidianus ambivalens respiratory system was successfully reconstituted into artificial liposomes, using three basic components isolated from the organism: the type-II NADH dehydrogenase, caldariella quinone, the organism-specific quinone, and the aa₃ type quinol oxidase. This system, which mimics the in vivo chain, is efficiently energized by NADH, driving oxygen consumption by means of the terminal oxidase.     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000     

microRNA-induced translational control of antiviral immunity by the cap-binding protein 4EHP

1. Download : Download high-res image (150KB)
2. Download : Download full-size image

     

Molecular epidemiology of isolates of the Cryptococcus neoformans species complex from Spain

To study genetic diversity of Cryptococcus neoformans species complex in Spain, 97 isolates of the yeast recovered from human, animal and environmental samples have been analysed using three molecular epidemiological techniques. One of these, URA5 gene fragment length polymorphism (RFLP) analysis, has been previously described as a molecular epidemiology tool. Thus, standard profiles and reference strains have been defined for it. In addition, 5S rDNA/IGS RFLP and [GACA]₄ microsatellite PCR fingerprinting were also used. Our results show five of the previously defined URA5 genotypes with a high frequency (33%) of the VNI type, which is in concordance with other studies. The high presence of VNIII pattern (28.9%) among our strains is remarkable and could be a specific feature of the isolates from our country. 5S rDNA/IGS RFLP showed a low intra-species discriminative power. Three different molecular profiles (S1-3), which showed a good correlation with the different species, varieties and genotypes, were obtained. [GACA]₄ microsatellite PCR-fingerprinting analysis showed a high variability of patterns among the studied strains. Molecular profiles represented in a dendrogram clustered strains in four main groups related with the source of the yeast and also in concordance with some of the described genotypes (VNI-IV and VGI).