Highly-efficient, fluorescent, locus directed cre and FlpO deleter mice on a pure C57BL/6N genetic background

To facilitate the use of the new mutant resource developed in the mouse, we have generated Cre and FlpO deleter mice on a pure inbred C57BL/6N background. The new transgenic constructs were designed to drive either the Cre or FlpO recombinase, fused to a specific fluorescent marker, respectively the eGFP or the eYFP, and were inserted by homologous recombination in the neutral Rosa26 locus. They allow a rapid, cost-effective, and efficient identification of the carrier individuals through the coexpression of the fluorescent marker. The recombination efficiency of the two deleter lines, Gt(ROSA)26S or < tm1(ACTB-cre,-EGFP)Ics> and Gt(ROSA) 26S or < tm2(CAG-flpo, EYFP)Ics>, was carefully evaluated using five loxP-flanked or four FRT-flanked alleles located at different positions in the mouse genome. For each tested locus, we observed a 100% excision rate. The transgenic mice are easily distinguishable from wild type animals by their bright fluorescence that remains easily detectable until 10 days after birth. In the adult, fluorescence can still be detected in the unpigmented paws. Furthermore, they both display accumulation of the specific recombinase during oogenesis. These fluorescent 'Cre- and Flp- deleter' transgenic lines are valuable tools for the scientific community by their high and stable recombination efficiency, the simplicity of genotype identification and the maintenance of a pure genetic background when used to remove specific selection cassette or to induce complete loss-of-function allele.     

Abnormal electrophoretic mobility of spectrin tetramers in hereditary elliptocytosis

Summary Hereditary elliptocytosis (HE) is a genetically determined disorder of the red cell membrane. The main protein which composes the proteinaceous skeleton of the membrane is an elongated molecule named spectrin which is a heterodimer composed of two chains, and . In the membrane spectrin dimers are associated head-to-head to form tetrameric structures. We and other authors have reported that spectrin studied from many HE patients exhibited a dimer self-association defect (type I HE). A mutation in the head of the spectrin chain was mostly found in type I HE. We have previously described one of the three known spectrin pathological variants shown on mild tryptic digest pattern. This variant was characterized by the appearance of an abnormal 65,000-dalton peptide (Sp ^I/65). Using nondenaturating gel electrophoresis, we describe in this paper a triplicated pattern of the spectrin tetramer bands which is found in heterozygous HE cases displaying the 65,000-dalton variant. Study of a homozygous case allowed us to characterize the electrophoretic mobility of the abnormal symmetrical spectrin tetramer (₂ ^I/65-₂) and to study the correlation between the fraction of this abnormal symmetrical tetramer found in heterozygous patients and the amount of the 65,000-dalton peptide observed in spectrin tryptic digests.     

The essential role of lipids in Alzheimer's disease

In the absence of efficient diagnostic and therapeutic tools, Alzheimer's disease (AD) is a major public health concern due to longer life expectancy in the Western countries. Although the precise cause of AD is still unknown, soluble β-amyloid (Aβ) oligomers are considered the proximate effectors of the synaptic injury and neuronal death occurring in the early stages of AD. Aβ oligomers may directly interact with the synaptic membrane, leading to impairment of synaptic functions and subsequent signalling pathways triggering neurodegeneration. Therefore, membrane structure and lipid status should be considered determinant factors in Aβ-oligomer-induced synaptic and cell injuries, and therefore AD progression. Numerous epidemiological studies have highlighted close relationships between AD incidence and dietary patterns. Among the nutritional factors involved, lipids significantly influence AD pathogenesis. It is likely that maintenance of adequate membrane lipid content could prevent the production of Aβ peptide as well as its deleterious effects upon its interaction with synaptic membrane, thereby protecting neurons from Aβ-induced neurodegeneration. As major constituents of neuronal lipids, n-3 polyunsaturated fatty acids are of particular interest in the prevention of AD valuable diet ingredients whose neuroprotective properties could be essential for designing preventive nutrition-based strategies. In this review, we discuss the functional relevance of neuronal membrane features with respect to susceptibility to Aβ oligomers and AD pathogenesis, as well as the prospective capacities of lipids to prevent or to delay the disease.     

Characterization and localization of two forms of gonadotropin-releasing hormone (GnRH) in the spinal cord of the frog Rana ridibunda

Two molecular variants of gonadotropin-releasing hormone (GnRH) have been previously characterized in the brain of amphibians, i.e., mammalian GnRH (mGnRH) and chicken GnRH-II (cGnRH-II). The aim of the present study was to identify the molecular forms of gonadotropin-releasing hormone and to localize gonadotropin-releasing hormone-containing elements in the spinal cord of the frog Rana ridibunda using highly specific antisera against mGnRH and cGnRH-II. High-performance liquid chromatography (HPLC) analysis combined with radioimmunoassay (RIA) detection revealed that frog spinal cord extracts contained both mGnRH and cGnRH-II. Immunohistochemical labeling revealed that the frog spinal cord was devoid of GnRH-containing cell bodies. In contrast, numerous GnRH-immunoreactive fibers were observed throughout the entire length of the cord. mGnRH immunoreactivity was only detected in the rostral region of the cord and consisted of varicose processes located in the vicinity of the central canal. cGnRH-II-positive fibers were found throughout the spinal cord, the density of immunoreactive processes decreasing gradually toward the caudal region. Two main cGnRH-II-positive fiber tracts with a rostrocaudal orientation were observed: a relatively dense fiber bundle surrounding the central canal, and a more diffuse plexus in the white matter. In addition, short, varicose cGnRH-II-positive processes with a radial orientation were present throughout the gray matter. These fibers were particularly abundant ventromedially and formed a diffuse network that ramified laterally to end in the vicinity of motoneurons. Taken together, these data indicate that the frog spinal cord, like the frog brain, contains two forms of GnRH. The presence of numerous cGnRH-II-immunoreactive fibers in the ventral horn suggests that cGnRH-II may influence the activity of a subpopulation of motoneurons.     

2′-and/or 3Y-Deoxy-β-L-pentofuranosyl Nucleoside Derivatives: Stereospecific Synthesis and Antiviral Activities

Abstract

Several L-enantiomers of nucleoside analogues were stereospecifically synthesized by a multi-step reaction from L-xylose and their antiviral properties were examined in vitro. Two of them, namely β-L-2′,3,′-dideoxycytidine (β-L-ddC) and its 5-fluoro derivative (β-L-FddC) were found to have potent anti-human immunodeficiency virus (HIV) and significant anti-hepatitis B virus (HBV) activities in cell cultures.     

Increased myocardial expression of RAMP1 and RAMP3 in rats with chronic heart failure

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) are potent vasodilators in humans and improved myocardial ischemia is observed after CGRP administration. Receptors for CGRP and ADM were already identified in heart. Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of the calcitonin receptor-like receptor (CRLR); co-expression of RAMP1 and CRLR results in a CGRP receptor, whereas the association of RAMP2 or RAMP3 with CRLR gives an ADM receptor. As CGRP and ADM may play a beneficial role in heart failure, we investigated whether the CGRP and ADM receptors are upregulated in chronic heart failure. We have used semi-quantitative RT-PCR and Western-blot analysis to detect and quantify the mRNA and the protein of RAMP1 and RAMP3 in both atria and ventricles of failing hearts 6 months after aortic banding in rats. Our results showed for the first time an up-regulation of RAMP1 and RAMP3 mRNAs and proteins in this model of cardiac failure. No change was observed in mRNAs coding for CRLR, RAMP2, RDC1 (canine orphan receptor), and ADM. The present results suggested after congestive heart failure in adult rats, an up-regulation of the CGRP receptor (by an increase in RAMP1 that is associated with CRLR) in atria and ventricles and of ADM receptor (by increased RAMP3 expression that is associated with CRLR) in atria. These findings support a functional role for CGRP and ADM receptors to compensate the chronic heart failure in rats.     

Pandemic influenza due to pH1N1/2009 virus: estimation of infection burden in Reunion Island through a prospective serosurvey, austral winter 2009

Background

To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season.

Methodology/Principal Findings

A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers ≥1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20–59 years) and 73.3% in the elderly (≥60 years) (P<0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P<0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P<0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive.

Conclusions

Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers ≥1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses.     

Functional Expression of Sinorhizobium meliloti BetS, a High-Affinity Betaine Transporter, in Bradyrhizobium japonicum USDA110

Among the Rhizobiaceae, Bradyrhizobium japonicum strain USDA110 appears to be extremely salt sensitive, and the presence of glycine betaine cannot restore its growth in medium with an increased osmolarity (E. Boncompagni, M. Østerås, M. C. Poggi, and D. Le Rudulier, Appl. Environ. Microbiol. 65:2072-2077, 1999). In order to improve the salt tolerance of B. japonicum, cells were transformed with the betS gene of Sinorhizobium meliloti. This gene encodes a major glycine betaine/proline betaine transporter from the betaine choline carnitine transporter family and is required for early osmotic adjustment. Whereas betaine transport was absent in the USDA110 strain, such transformation induced glycine betaine and proline betaine uptake in an osmotically dependent manner. Salt-treated transformed cells accumulated large amounts of glycine betaine, which was not catabolized. However, the accumulation was reversed through rapid efflux during osmotic downshock. An increased tolerance of transformant cells to a moderate NaCl concentration (80 mM) was also observed in the presence of glycine betaine or proline betaine, whereas the growth of the wild-type strain was totally abolished at 80 mM NaCl. Surprisingly, the deleterious effect due to a higher salt concentration (100 mM) could not be overcome by glycine betaine, despite a significant accumulation of this compound. Cell viability was not significantly affected in the presence of 100 mM NaCl, whereas 75% cell death occurred at 150 mM NaCl. The absence of a potential gene encoding Na⁺/H⁺ antiporters in B. japonicum could explain its very high Na⁺ sensitivity.