Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina

Summary In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron ) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron , was detected in five senescent subclones which all showed strong amplification of the intronic sequence (Sen DNA ).     

"In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules

The TSH effect on slice and the incubation medium cyclic AMP levels and T3 and T4 released from 8 autonomously functioning thyroid nodules (AFTN) and their respective perinodular (PN) tissues were examined. The thyroid slices were incubated in Eagle's Medium containing TSH (5 to 100 mU/ml) for 60 min and 300 min for tissue cyclic AMP generation and for cyclic AMP, T3 and T4 release, respectively. Basal cyclic AMP levels were not different either in AFTN and in PN slices or into the incubation medium. In both tissues TSH produced a similar cyclic AMP generation. In contrast, cyclic AMP released into the incubation medium was significantly higher in AFTN than in PN tissues, after TSH stimulation. Basal T3 values and TSH-stimulated T3 release in AFTN were not different from PN tissue. However, basal T4 levels were significantly higher in AFTN than in PN tissue as well as T4 released in response to TSH. In addition, T3/T4 ratio was lower in AFTN than in PN tissues. The cyclic AMP released into the incubation medium correlated with both T3 and T4 release in PN tissue but in the AFTN tissue no correlations were found. These findings suggest that the adenylate cyclase-cyclic AMP system is more sensitive to TSH-stimulation in AFTN when compared with PN tissue and that AFTN tissue has a preferential T4 secretion.     

Phylogenetic diversity and genome sizes of Astragalus (Fabaceae) in the Lebanon biogeographical crossroad

The Lebanese mountain range is an important zone of plant species richness and endemism where the genus Astragalus constitutes a principal component of plant biodiversity. Most of endemic Astragalus taxa, living in mountains and arid zones of Mounts Lebanon and anti-Lebanon, are characterized by a cushion, spiny vegetative form, named “tragacanthic”, which is a remarkable example of vegetative convergence evolution. Because of determination difficulties, taxonomic uncertainties, and discrepancy in the number of taxa listed according to authors, new data are hardly needed to improve systematics of Astragalus and to investigate the role of the Lebanese mountain range as refugia of biodiversity. Before this study only two values on the genome size of Astragalus were reported in the literature and no previous molecular studies had been carried out on Astragalus genus in Lebanon. We examined the utility of rDNA ITS molecular markers to distinguish Astragalus species of Lebanese mountain range and the variation range of their genome size. The main results revealed a striking diversity in Lebanese Astragalus species with the emphasis of a huge variation of genome sizes, an important inter-specific chromosome polymorphism and the existence of a high phylogenetic diversity. The strict endemic species of the Lebanese mountains are positioned throughout the phylogeny. These results confirm that the Lebanon and anti-Lebanon mounts constitute a third diversity center for Astragalus and that high altitude areas are important refugia of plant biodiversity despite centuries of exploitation by humans.     

Osteopontin mediates macrophage chemotaxis via α4 and α9 integrins and survival via the α4 integrin

Osteopontin (OPN) is highly expressed by macrophages and plays a key role in the pathology of several chronic inflammatory diseases including atherosclerosis and the foreign body reaction. However, the molecular mechanism behind OPN regulation of macrophage functions is not well understood. OPN is a secreted molecule and interacts with several integrins via two domains: the RGD sequence binding to α_v‐containing integrins, and the SLAYGLR sequence binding to α₄β₁, α₄β₇, and α₉β₁ integrins. Here we determined the role of OPN in macrophage survival, chemotaxis, and activation state. For survival studies, OPN treated‐bone marrow derived macrophages (BMDMs) were challenged with growth factor withdrawal and neutralizing integrin antibodies. We found that survival in BMDMs is mediated primarily through the α₄ integrin. In chemotaxis studies, we observed that migration to OPN was blocked by neutralizing α₄ and α₉ integrin antibodies. Further, OPN did not affect macrophage activation as measured by IL‐12 production. Finally, the relative contributions of the RGD and the SLAYGLR functional domains of OPN to leukocyte recruitment were evaluated in an in vivo model. We generated chimeric mice expressing mutated forms of OPN in myeloid‐derived leukocytes, and found that the SLAYGLR functional domain of OPN, but not the RGD, mediates macrophage accumulation in response to thioglycollate‐elicited peritonitis. Collectively, these data indicate that α₄ and α₉ integrins interacting with OPN via the SLAYGLR domain play a key role in macrophage biology by regulating migration, survival, and accumulation. J. Cell. Biochem. 114: 1194–1202, 2013. © 2012 Wiley Periodicals, Inc.     

Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses.     

Sphingosine kinases, sphingosine 1-phosphate, apoptosis and diseases

Sphingolipids are ubiquitous components of cell membranes and their metabolites ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) have important physiological functions, including regulation of cell growth and survival. Cer and Sph are associated with growth arrest and apoptosis. Many stress stimuli increase levels of Cer and Sph, whereas suppression of apoptosis is associated with increased intracellular levels of S1P. In addition, extracellular/secreted S1P regulates cellular processes by binding to five specific G protein coupled-receptors (GPCRs). S1P is generated by phosphorylation of Sph catalyzed by two isoforms of sphingosine kinases (SphK), type 1 and type 2, which are critical regulators of the "sphingolipid rheostat", producing pro-survival S1P and decreasing levels of pro-apoptotic Sph. Since sphingolipid metabolism is often dysregulated in many diseases, targeting SphKs is potentially clinically relevant. Here we review the growing recent literature on the regulation and the roles of SphKs and S1P in apoptosis and diseases.     

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

Infliximab therapy in rheumatoid arthritis and ankylosing spondylitis-induced specific antinuclear and antiphospholipid autoantibodies without autoimmune clinical manifestations: a two-year prospective study

Treatment of rheumatoid arthritis (RA) with infliximab (Remicade) has been associated with the induction of antinuclear autoantibodies (ANA) and anti-double-stranded DNA (anti-dsDNA) autoantibodies. In the present study we investigated the humoral immune response induced by infliximab against organ-specific or non-organ-specific antigens not only in RA patients but also in patients with ankylosing spondylitis (AS) during a two-year followup. The association between the presence of autoantibodies and clinical manifestations was then examined. The occurrence of the various autoantibodies was analyzed in 24 RA and 15 AS patients all treated with infliximab and in 30 RA patients receiving methotrexate but not infliximab, using the appropriate methods of detection. Infliximab led to a significant induction of ANA and anti-dsDNA autoantibodies in 86.7% and 57% of RA patients and in 85% and 31% of AS patients, respectively. The incidence of antiphospholipid (aPL) autoantibodies was significantly higher in both RA patients (21%) and AS patients (27%) than in the control group. Most anti-dsDNA and aPL autoantibodies were of IgM isotype and were not associated with infusion side effects, lupus-like manifestations or infectious disease. No other autoantibodies were shown to be induced by the treatment. Our results confirmed the occurrence of ANA and anti-dsDNA autoantibodies and demonstrated that the induction of ANA, anti-dsDNA and aPL autoantibodies is related to infliximab treatment in both RA and AS, with no significant relationship to clinical manifestations.