Cell-specific and concentration-dependent actions of interleukin-1 in acute brain inflammation

Interleukin (IL)-1 is a pivotal pro-inflammatory cytokine and an important mediator of both acute and chronic central nervous system (CNS) injuries. Despite intense research in CNS IL-1 biology over the past two decades, its precise mechanism of action in inflammatory responses to acute brain disorders remains largely unknown. In particular, much effort has been focussed on using in vitro approaches to better understand the cellular and signalling mechanisms of actions of IL-1, yet some discrepancies in the literature regarding the effects produced by IL-1β in in vitro paradigms of injury still exist, particularly as to whether IL-1 exerts neurotoxic or neuroprotective effects. Here we aim to review the cell-specific and concentration-dependent actions of IL-1 in brain cells, to depict the mechanism by which this cytokine induces neurotoxicity or neuroprotection in acute brain injury.     

Rapid Screening for Temperature-Sensitive Alleles in Plants

We developed a simple and fast method to identify temperature-sensitive alleles of essential plant genes. We used primary and tertiary structure information to identify residues in the core of the protein of interest. These residues were mutated and tested for temperature sensitivity, taking advantage of the exceptionally rapid 1-week complementation assay in the moss Physcomitrella patens. As test molecules, we selected the actin-binding proteins profilin and actin-depolymerizing factor, because they are essential and their loss-of-function phenotype can be fully rescued. Screening a small number of candidate mutants, we successfully identified temperature-sensitive alleles of both profilin and actin-depolymerizing factor. Plants harboring these alleles grew well at the permissive temperature of 20°C to 25°C but showed a complete loss of function at the restrictive temperature of 32°C. Notably, the profilin mutation identified in the moss gene can be transferred to profilins from other plant species, also rendering them temperature sensitive. The ability to routinely generate temperature-sensitive alleles of essential plant proteins provides a powerful tool for the study of gene function in plants.Conditional mutants are powerful genetic tools. In yeast, temperature-sensitive mutations have yielded a wealth of information regarding gene function and have aided immensely in the discovery and elucidation of many molecular pathways (Hartwell, 1967; Bonatti et al., 1972; Pringle, 1975; Novick and Botstein, 1985; Johnston et al., 1991; Balasubramanian et al., 1994; Chang et al., 1996, 1997; Iida and Yahara, 1999). In plants, a number of studies have generated temperature-sensitive alleles to study processes ranging from plant morphology to signal transduction (Lane et al., 2001; Whittington et al., 2001; Wiedemeier et al., 2002; Quint et al., 2005; Bannigan et al., 2006, 2007).In addition to temperature-dependent function, conditional expression can be generated in a variety of ways. A common strategy in mouse cells is to incorporate lox-p sites flanking the gene of interest (Sauer and Henderson, 1988; Orban et al., 1992; Vidali et al., 2006). Gene function is conditionally lost by the expression of cre recombinase that fuses the lox-p sites, deleting the intervening sequences. This method and others, such as inducible RNA interference (RNAi; Ketelaar et al., 2004), require long incubation times needed for gene expression and protein depletion. Due to the long time course for these studies, loss-of-function effects can be complicated with the development of the organism. In contrast, temperature-sensitive mutants are potentially fast acting, losing their function in some cases within minutes of exposure to the restrictive conditions (Novick and Botstein, 1985; Pruyne et al., 1998).In most cases, temperature-sensitive mutants are generated randomly and the elucidation of the gene harboring the mutation is uncovered by cloning the mutagenized gene. In plants, this is done by performing a chromosome walk to the mutagenized allele. In yeast, due to the ease of performing complementation, it is also possible to start with a gene of interest, mutagenize that gene, and screen for temperature-sensitive alleles (Shortle et al., 1984; Budd and Campbell, 1987; Mann et al., 1987). In plants, however, this process has not been widely used, presumably due to the time-consuming nature of performing complementation studies in planta.Here, we show that the moss Physcomitrella patens is an ideal plant suited for screening potential temperature-sensitive alleles of a gene of interest. To screen for a temperature-sensitive mutation, loss of the gene of interest must produce a measurable phenotype that can be rescued by reintroduction of the wild-type allele of the gene. We chose two proteins, profilin and actin-depolymerizing factor (ADF)/cofilin, as test molecules. Profilin and ADF are well-characterized actin-binding proteins that are important for cellular growth in plants (Staiger et al., 1994; Ramachandran et al., 2000; Dong et al., 2001; Vidali et al., 2001, 2007; Chen et al., 2002, 2003; McKenna et al., 2004; Augustine et al., 2008). In the moss P. patens, both profilin and ADF are essential for protonemal filament growth. Loss of profilin or ADF results in severely stunted plants, composed of morphologically abnormal cells (Vidali et al., 2007; Augustine et al., 2008). These phenotypes are fully rescued by expression of wild-type profilin or ADF, respectively.Moss has emerged as a facile plant system due to its ability to integrate exogenous DNA molecules by homologous recombination at frequencies enabling gene-targeting studies (Cove et al., 2006). In addition, moss is amenable to transient RNAi (Bezanilla et al., 2003, 2005), which enables the study of terminal phenotypes due to loss of essential genes, something that would not be possible if performing only gene knockout experiments. We have previously demonstrated the ability to knock down essential gene families and obtain quantitative rescue of the knockdown phenotypes (Vidali et al., 2007, 2009; Augustine et al., 2008). We have performed these studies using a rapid transient assay, which enables knock down and complementation studies to be performed within 1 week of transformation (Vidali et al., 2007). This is an extremely rapid assay that is unparalleled in other plant systems. Here, we use this complementation assay to screen for temperature-sensitive alleles of both profilin and ADF. Importantly, we show that the residue that confers temperature sensitivity in moss profilin can also render both Arabidopsis (Arabidopsis thaliana) and lily (Lilium longiflorum) profilins temperature sensitive, demonstrating a wider applicability to this rapid in planta complementation system.     

Physiological responses to Cd and Zn in two Cd/Zn hyperaccumulating Thlaspi species

In a model hyperaccumulation study a Cd/Zn hyperaccumulator Thlaspi caerulescens accession Ganges and a recently reported Cd/Zn hyperaccumulator Thlaspi praecox grown in increasing Cd and Zn concentrations in the substrate and in field collected polluted soil were compared. Plant biomass, concentrations of Cd and Zn, total chlorophylls and anthocyanins, antioxidative stress parameters and activities of selected antioxidative enzymes were compared. Increasing Cd, but not Zn in the substrate resulted in the increase of biomass of roots and shoots of T. praecox and T. caerulescens. The two species hyperaccumulated Cd in the shoots to a similar extent, whereas T. caerulescens accumulated more Zn in the shoots than T. praecox. Cadmium amendment decreased total chlorophyll concentration and glutathione reductase activity, and increased non-protein thiols concentration only in T. praecox, suggesting that it is less tolerant to Cd than T. caerulescens. In the field-contaminated soil, T. caerulescens accumulated higher Cd concentrations; but as T. praecox produced higher biomass, both species have similar ability to extract Cd.     

Cilostazol and pentoxifylline decrease angiogenesis,inflammation, and fibrosis in sponge-induced intraperitoneal adhesion in mice

AimsAdhesion formation following abdominal intervention is an abnormal peritoneal healing process. Our aim was to investigate the effects of controlling adhesion development by inhibiting its key components (angiogenesis, inflammation and fibrosis) using phosphodiesterase (PDE) inhibitors.Main methodsTwo PDE inhibitors including cilostazol a PDE3 inhibitor (40 and 400 mg/kg), and pentoxifylline (PTX), a PDE 1–5 inhibitor (50 and 500 mg/kg) were used for a period of 7 days to inhibit angiogenesis, inflammation, and fibrosis in a murine model of sponge-induced peritoneal adhesion. Angiogenesis was assessed by hemoglobin content, vascular endothelial growth factor (VEGF) levels, and morphometric analysis. Accumulation of neutrophils and macrophages was determined by measuring myeloperoxidase (MPO) and N-acetylglucosaminidase (NAG) activities, respectively. Levels of TNF-α were also determined. Fibrosis was assessed by determining the amount of collagen in the implant; TGF-β1 levels in the implant were also measured.Key findingsOur results show that the treatments attenuated the main components of the adhesion tissue by reducing the amount of fibrovascular tissue that infiltrated the sponge matrix (wet weight). Hemoglobin content and VEGF levels were also decreased by approximately 40%. Neutrophil accumulation was unaffected by the compounds. However, NAG activity was reduced by pentoxifylline, but not by cilostazol. These compounds also decreased the levels of the pro-inflammatory and pro-fibrogenic cytokines TNF-α and TGF-β1, respectively, and collagen synthesis.SignificanceOur results suggest that cilostazol and PTX decreased the development of peritoneal adhesions in the model, which might be associated with cyclic nucleotide modulation. Therapies to intervene in these pathways may be beneficial for the prevention of these lesions.     

Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital microscopic studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression of l-selectin and β2-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to fMLP, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation.     

Effect of dietary conjugated linoleic acid isomers on water and glycerol permeability of kidney membranes

Conjugated linoleic acid (CLA) refers to a group of positional and geometrical isomers of linoleic acid in which the double bonds are conjugated. Dietary CLA has been associated with various health benefits although details of its molecular mode of action remain elusive. The effect of CLA supplemented to palm oil-based diets in Wistar rats, as a mixture of both or isolated c9,t11 and t10,c12 isomers, was examined on water and glycerol membrane permeability of kidney proximal tubule. Although water permeability was unaltered, an increase in glycerol permeability was obtained for the group supplemented with CLA mixture, even though the activation energy for glycerol permeation remained high. This effect was correlated with an increased CLA isomeric membrane incorporation for the same dietary group. These results suggest that diet supplementation with CLA mixture, in contrast to its individual isomers, may enhance membrane fluidity subsequently raising kidney glycerol reabsorption.     

Is communal burrowing or burrow sharing a benefit of group living in the lesser cavy <Emphasis Type="Italic">Microcavia australis</Emphasis>?

Burrow systems play an important role in the life of rodents in arid environments. The objectives of this study were to examine the hypothesis that group living is beneficial to the semifossorial rodent, and determine whether Microcavia australis (Geoffroy and d’Orbigny, 1833) burrows communally and/or shares burrow systems. I related the structure of burrow systems to the number of cavies inhabiting them, in two habitats with different soil hardness and different plant cover (El Leoncito and Ñacuñán). El Leoncito has a harsh climate, with lower plant density and softer soil than Ñacñuán. A total of 18 burrow systems were characterized at Ñacuñán, and 12 at El Leoncito. Social groups at El Leoncito have a higher number of individuals than at Ñacuñán, but the structure of burrow systems in softer soil is narrower (small area size), with fewer holes, less slope and depth of galleries, and with no relationship between the number of holes and burrow area. Therefore, considering the development of the burrow system as an indicator of the cost of burrowing, I conclude that communal burrowing to reduce the energetic cost of burrowing per capita is not the primary cause of cavy sociality. M. australis were not active diggers, because digging behaviour was rarely recorded at either site. Burrow systems of cavies persisted over the years of study, occupied by the same cavies and new offspring, and digging new burrow systems and tunnels was a relatively rare event at both sites. Under the burrow-sharing hypothesis, sociality could prevail in M. australis that regularly dig to build and maintain a burrow system which they use for a long time.     

Nonflowers near the base of extant angiosperms? Spatiotemporal arrangement of organs in reproductive units of Hydatellaceae and its bearing on the origin of the flower

Reproductive units (RUs) of Trithuria, the sole genus of the early-divergent angiosperm family Hydatellaceae, are compared with flowers of their close relatives in Cabombaceae (Nymphaeales). Trithuria RUs combine features of flowers and inflorescences. They differ from typical flowers in possessing an "inside-out" morphology, with carpels surrounding stamens; furthermore, carpels develop centrifugally, in contrast to centripetal or simultaneous development in typical flowers. Trithuria RUs could be interpreted as pseudanthia of two or more cymose partial inflorescences enclosed within an involucre, but the bractlike involucral phyllomes do not subtend partial inflorescences and hence collectively resemble a typical perianth. Teratological forms of T. submersa indicate a tendency to fasciation and demonstrate that the inside-out structure-the primary feature that separates RUs of Hydatellaceae from more orthodox angiosperm flowers-can be at least partially modified, thus producing a morphology that is closer to an orthodox flower. The Trithuria RU could be described as a "nonflower", i.e., a structure that contains typical angiosperm carpels and stamens but does not allow recognition of a typical angiosperm flower. The term nonflower could combine cases of secondary loss of flower identity and cases of a prefloral condition, similar to those that gave rise to the angiosperm flower. Nonhomology among some angiosperm flowers could be due to iterative shifts between nonfloral construction and flower/inflorescence organization of reproductive organs. Potential testing of these hypotheses using evolutionary-developmental genetics is explored using preliminary data from immunolocalization of the floral meristem identity gene LEAFY in T. submersa, which indicated protein expression at different hierarchical levels.     

γ-Tubulin ring complexes regulate microtubule plus end dynamics

γ-Tubulin is critical for the initiation and regulation of microtubule (MT) assembly. In Drosophila melanogaster, it acts within two main complexes: the γ-tubulin small complex (γ-TuSC) and the γ-tubulin ring complex (γ-TuRC). Proteins specific of the γ-TuRC, although nonessential for viability, are required for efficient mitotic progression. Until now, their role during interphase remained poorly understood. Using RNA interference in Drosophila S2 cells, we show that the γ-TuRC is not critical for overall MT organization. However, depletion of any component of this complex results in an increase of MT dynamics. Combined immunofluorescence and live imaging analysis allows us to reveal that the γ-TuRC localizes along interphase MTs and that distal γ-tubulin spots match with sites of pause or rescue events. We propose that, in addition to its role in nucleation, the γ-TuRC associated to MTs may regulate their dynamics by limiting catastrophes.