Cdc42 explores the cell periphery for mate selection in fission yeast.

How cells polarize in response to external cues is a fundamental biological problem. For mating, yeast cells orient growth toward the source of a pheromone gradient produced by cells of the opposite mating type. Polarized growth depends on the small GTPase Cdc42, a central eukaryotic polarity regulator that controls signaling, cytoskeleton polarization, and vesicle trafficking. However, the mechanisms of polarity establishment and mate selection in complex cellular environments are poorly understood. Here we show that, in fission yeast, low-level pheromone signaling promotes a novel polarization state, where active Cdc42, its GEF Scd1, and scaffold Scd2 form colocalizing dynamic zones that sample the periphery of the cell. Two direct Cdc42 effectors--actin cables marked by myosin V Myo52 and the exocyst complex labeled by Sec6 and Sec8--also dynamically colocalize with active Cdc42. However, these cells do not grow due to a block in the exocytosis of cell wall synthases Bgs1 and Bgs4. High-level pheromone stabilizes active Cdc42 zones and promotes cell wall synthase exocytosis and polarized growth. However, in the absence of prior low-level pheromone signaling, exploration fails, and cells polarize growth at cell poles by default. Consequently, these cells show altered partner choice, mating preferentially with sister rather than nonsister cells. Thus, Cdc42 exploration serves to orient growth for partner selection. This process may also promote genetic diversification.

Implication of Nerve Growth Factor in intestinal mucosal mast cell activity and colonic motor alterations in a model of ovalbumin-induced gut dysfunction in rats

We determined NGF involvement in MMCs and colonic motor alterations in an ovalbumin (OVA)-induced gut dysfunction model in rats. Animals received OVA (6 weeks), with/without simultaneous K252a (TrkA antagonist) treatment. MMCs, rat mast cell protease II (RMCPII) levels and colonic contractility in vitro were assessed. OVA increased MMC density and RMCPII concentration. Spontaneous contractility was similar in both groups and inhibited by K252a. Carbachol responses were increased by OVA in a K252a-independent manner. NO-synthase inhibition increased spontaneous activity in OVA-treated animals in a K252a-dependent manner. These observations support an involvement of NGF in the functional changes observed in this model.

Eokines: synthesis, storage and release from human eosinophils

Eosinophils are prominent inflammatory cells in asthma and other allergic disorders, as well as in helminthic parasite infections. Recently, eosinophils have been reported to synthesize and store a range of regulatory proteins within their secretory granules (eokines). Eokines comprise a group of cytokines, chemokines, and growth factors which are elaborated by eosinophils. These proteins, and the messages which encode them, appear to be identical to those produced by lymphocytes and other tissues. Interestingly, immunoreactivity to many of these eokines has been found to co-localize to the eosinophil´s secretory granules. In this review, we have discussed the repertoire of 18 eokines so far identified in eosinophils, and focused on four of these, namely, interleukin-2 (IL-2), IL-4, granulocyte/macrophage colony-stimulating factor (GM-CSF), and RANTES. These four eokines co-localize to the crystalloid granules in eosinophils, as shown in studies using subcellular fractionation and immunogold labeling in electron microscopy. During stimulation by physiological triggers, for example, with serum-coated particles, eosinophils release these mediators into the surrounding supernatant. In addition, eokines are likely to be synthesized within eosinophils rather than taken up by endocytosis, as show in detection of mRNA for each of these proteins using in situ hybridization, RT-PCR, and in the case of RANTES, in situ RT-PCR. Eokines synthesis and release from eosinophils challenges the commonly held notion that these cells act downstream of key elements in immune system, and indicate that they may instead belong to the afferent arm of immunity.

Perinatal nitric oxide therapy prevents adverse effects of perinatal hypoxia on the adult pulmonary circulation.

Adverse events in utero are associated with the occurrence of chronic diseases in adulthood. We previously demonstrated in mice that perinatal hypoxia resulted in altered pulmonary circulation in adulthood, with a decreased endothelium-dependent relaxation of pulmonary arteries, associated with long-term alterations in the nitric oxide (NO)/cyclic GMP pathway. The present study investigated whether inhaled NO (iNO) administered simultaneously to perinatal hypoxia could have potential beneficial effects on the adult pulmonary circulation. Indeed, iNO is the therapy of choice in humans presenting neonatal pulmonary hypertension. Long-term effects of neonatal iNO therapy on adult pulmonary circulation have not yet been investigated. Pregnant mice were placed in hypoxia (13% O2) with simultaneous administration of iNO 5 days before delivery until 5 days after birth. Pups were then raised in normoxia until adulthood. Perinatal iNO administration completely restored acetylcholine-induced relaxation, as well as endothelial nitric oxide synthase protein content, in isolated pulmonary arteries of adult mice born in hypoxia. Right ventricular hypertrophy observed in old mice born in hypoxia compared to controls was also prevented by perinatal iNO treatment. Therefore, simultaneous administration of iNO during perinatal hypoxic exposure seems able to prevent adverse effects of perinatal hypoxia on the adult pulmonary circulation.

Nitric oxide mediates the microbicidal activity of eosinophils

There are several experimental evidences that nitric oxide (NO) is involved in the microbicidal activity of macrophages against a number of intracellular pathogens including Leishmania major, Trypanozoma cruzi, Toxoplasma gondii. It is also well known that eosinophils (EO) have microbicidal activity against many parasites such as Schistosoma mansoni, Trichinella spiralis, T. cruzi and L. amazonensis. The purpose of this study was to investigate if NO is involved in the microbicidal activity of EO against L. major. Eosinophils harvested from peritoneal cavity of rats released spontaneously after 24 and 48 hr a small amount of nitrite. This release was enhanced by the treatment of cells with IFN-gamma (200 IU/ml). This release was blocked by addition of the NO synthase inhibitor, L-NIO (100 mu M) into the culture. To determinate the leishmanicidal activity of eosinophils the parasites were incubated with activated eosinophils with IFN-gamma and the ability of surviving parasites to incorporate [³H]thymidine was evaluated. IFN-gamma-activated eosinophils were able to kill L. major and to release high levels of nitrite. The ability to destroy L. major and the release of NO were completely blocked by L-NIO. These results indicate that activated eosinophils release NO which is involved in the microbicidal activity of these cells against L. major.

The expression of nuclear 3,5,3' triiodothyronine receptors is induced in Schwann cells by nerve transection.

The effects of thyroid hormones on the nervous system are mediated by the presence of nuclear T3 receptors (NT3R). In this study, the expression of NT3R was investigated in spinal cord, dorsal root ganglia (DRG), or sciatic nerve of adult rats after immunostaining with a 2B3-NT3R monoclonal antibody which recognizes both alpha and beta types of NT3R. The specificity of this monoclonal antibody was confirmed by Western blots. The 2B3-NT3R monoclonal antibody recognized one band corresponding to a molecular weight of 57 kDa in extract of spinal cord or DRG. No staining was observed on immunoblot of intact sciatic nerve. In the spinal cord, the nuclei of the neurons and glial cells including both astrocytes and oligodendrocytes exhibited 2B3-NT3R immunoreactivity. While all the nuclei of the DRG sensory neurons expressed the NT3R, all the nuclei of the satellite and Schwann cells were devoid of any immunoreaction. In the sciatic nerve, the nuclei of the Schwann cells also lacked 2B3-NT3R-immunoreactivity. After sciatic nerve transection in vivo, Schwann cell nuclei, which never expressed NT3R in intact nerves of adult rats, displayed a clear 2B3-NT3R immunoreaction in proximal and distal stumps adjacent to the section. Double immunostaining with antibodies raised to 3-sulfogalactosylceramide or S100 confirmed that most of the NT3R containing nuclei belong to Schwann cells. In dissociated cell cultures grown in vitro from sciatic nerves, Schwann cells exhibited 2B3-NT3R immunoreactivity. These data suggest that the inhibition of NT3R expression in Schwann cells ensheathing axons in intact nerve is reversed when the axons are degenerating or lacking.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubility of cytoskeletal proteins in immunohistochemistry and the influence of fixation.

For accurate and quantitative immunohistochemical localization of antigens it is crucial to know the solubility of tissue proteins and their degree of loss during processing. In this study we focused on the solubility of several cytoskeletal proteins in cat brain tissue at various ages and their loss during immunohistochemical procedures. We further examined whether fixation affected either solubility or immunocytochemical detectability of several cytoskeletal proteins. An assay was designed to measure the solubility of cytoskeletal proteins in cryostat sections. Quantity and quality of proteins lost or remaining in tissue were measured and analyzed by electrophoresis and immunoblots. Most microtubule proteins were found to be soluble in unfixed and alcohol fixed tissues. Furthermore, the microtubule proteins remaining in the tissue had a changed cellular distribution. In contrast, brain spectrin and all three neurofilament subunits were insoluble and remained in the tissue, allowing their immunocytochemical localization in alcohol-fixed tissue. Synapsin I, a protein associated with the spectrin cytoskeleton, was soluble, and aldehyde fixation is advised for its immunohistochemical localization. With aldehyde fixation, the immunoreactivity of some antibodies against neurofilament proteins was reduced in axons unveiling novel immunogenic sites in nuclei that may represent artifacts of fixation. In conclusion, protein solubility and the effects of fixation are influential factors in cytoskeletal immunohistochemistry, and should be considered before assessments for a quantitative distribution are made.

Portal veins of mice infected with Schistosoma mansoni exhibit an increased reactivity to 5-hydroxytryptamine

In chronic severe infection with Schistosoma mansoni, portal hypertension and related vascular alterations usually develop as a consequence of granulomatous response to eggs. In order to investigate a putative direct effect of worms on the reactivity of their host portal vein, mice infected only with male worms were used in the present study. An higher reactivity to 5-hydroxytryptamine (5-HT) characterized by an increase in the maximal contraction and sensitivity was observed in portal vein from infected mice compared to healthy mice. Blockade of NO-synthase with l-NAME induced a small increase in 5-HT potency in portal vein from non-infected mice without changing the amplitude of the contractions, whereas it did not alter the reactivity of veins from infected mice. The present results show that unisexual infection of mice with male S. mansoni increased the reactivity of the portal vein to 5-HT which seems to be partially related to an alteration in the nitric oxide release by endothelium.

Protective effect of intravitreal administration of tresperimus, an immunosuppressive drug, on experimental autoimmune uveoretinitis.

PURPOSE: To test the efficiency of locally administrated tresperimus in experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced in Lewis rats by S-antigen (S-Ag) immunization. Three intravitreal injections of tresperimus (prevention or prevention/treatment protocols) were performed at different time points after immunization. The pharmacokinetics of tresperimus was evaluated in the ocular tissues and plasma. The in vitro effect of tresperimus was evaluated on macrophages. EAU was graded clinically and histologically. Blood ocular barrier permeability was evaluated by protein concentration in ocular fluids. Immune response to S-Ag was examined by delayed type hypersensitivity, the expression of inflammatory cytokines in lymph nodes, ocular fluids and serum by multiplex ELISA, and in ocular cells by RT-PCR. RESULTS: In vitro, tresperimus significantly reduced the production of inflammatory cytokines by lipopolysaccharide-stimulated macrophages. In vivo, in the treatment protocol, efficient tresperimus levels were measured in the eye but not in the plasma up to 8 days after the last injection. Tresperimus efficiently reduced inflammation, retinal damage, and blood ocular barrier permeability breakdown. It inhibited nitric oxide synthase-2 and nuclear factor κBp65 expression in ocular macrophages. IL-2 and IL-17 were decreased in ocular media, while IL-18 was increased. By contrast, IL-2 and IL-17 levels were not modified in inguinal lymph nodes draining the immunization site. Moreover, cytokine levels in serum and delayed type hypersensitivity to S-Ag were not different in control and treated rats. In the prevention/treatment protocol, ocular immunosuppressive effects were also observed. CONCLUSIONS: Locally administered tresperimus appears to be a potential immunosuppressive agent in the management of intraocular inflammation.

Down-regulation of NOSII gene expression by iontophoresis of anti-sense oligonucleotide in endotoxin-induced uveitis.

Transcorneoscleral iontophoresis was used to enhance ocular penetration of a 21-bp NH(2) protected anti-NOSII oligonucleotides (ODNs) (fluorescein or infrared-41 labeled) in Lewis rats. Both histochemical localization and acrylamide sequencing gels were used. To evaluate the potential to down-regulate NOSII expression in the rat model of endotoxin-induced uveitis (EIU), anti-sense NOSII ODN, scrambled ODN or saline were iontophorezed into these animals' eyes. Iontophoresis facilitated the penetration of intact ODNs into the intraocular tissues of the rat eye and only the eyes receiving ODNs and electrical current demonstrated intact ODNs within the ocular tissues of both segments of the eye. Iontophoresis of anti-NOSII ODN significantly down-regulated the expression of NOSII expression in iris/ciliary body compared to the saline or scrambled ODN treated eyes. Nitrite production was also significantly reduced in the anti-NOSII applied eyes compared to those treated with saline. Using this system, intraocular delivery of ODNs can be significantly enhanced increasing the potential for successful gene therapy for human eye diseases.

Transcriptional control of the hydrogen cyanide biosynthetic genes hcnABC by the anaerobic regulator ANR and the quorum-sensing regulators LasR and RhlR in Pseudomonas aeruginosa.

Virulence factors of Pseudomonas aeruginosa include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The hcnABC genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of P. aeruginosa PAO1. The hcnA promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional lacZ fusions. The promoter sequence displayed an FNR/ANR box at -42.5 bp upstream of T2 and a lux box centered around -42.5 bp upstream of T1. Expression of the hcn genes was completely abolished when this lux box was deleted or inactivated by two point mutations in conserved nucleotides. The lux box was recognized by both LasR [activated by N-(oxododecanoyl)-homoserine lactone] and RhlR (activated by N-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective P. aeruginosa mutants and in the N-acyl-homoserine lactone-negative heterologous host P. fluorescens CHA0. A second, less conserved lux box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the hcn promoter. Together, these data indicate that expression of the hcn promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.

Functional analysis of pyochelin-/enantiopyochelin-related genes from a pathogenicity island of Pseudomonas aeruginosa strain PA14.

Genomic islands are foreign DNA blocks inserted in so-called regions of genomic plasticity (RGP). Depending on their gene content, they are classified as pathogenicity, symbiosis, metabolic, fitness or resistance islands, although a detailed functional analysis is often lacking. Here we focused on a 34-kb pathogenicity island of Pseudomonas aeruginosa PA14 (PA14GI-6), which is inserted at RGP5 and carries genes related to those for pyochelin/enantiopyochelin biosynthesis. These enantiomeric siderophores of P. aeruginosa and certain strains of Pseudomonas protegens are assembled by a thiotemplate mechanism from salicylate and two molecules of cysteine. The biochemical function of several proteins encoded by PA14GI-6 was investigated by a series of complementation analyses using mutants affected in potential homologs. We found that PA14_54940 codes for a bifunctional salicylate synthase/salicyl-AMP ligase (for generation and activation of salicylate), that PA14_54930 specifies a dihydroaeruginoic acid (Dha) synthetase (for coupling salicylate with a cysteine-derived thiazoline ring), that PA14_54910 produces a type II thioesterase (for quality control), and that PA14_54880 encodes a serine O-acetyltransferase (for increased cysteine availability). The structure of the PA14GI-6-specified metabolite was determined by mass spectrometry, thin-layer chromatography, and HPLC as (R)-Dha, an iron chelator with antibacterial, antifungal and antitumor activity. The conservation of this genomic island in many clinical and environmental P. aeruginosa isolates of different geographical origin suggests that the ability for Dha production may confer a selective advantage to its host.

Hypoxic contraction of small pulmonary arteries from normal and endotoxemic rats: fundamental role of NO.

The present study was aimed at examining the role of nitric oxide (NO) in the hypoxic contraction of isolated small pulmonary arteries (SPA) in the rat. Animals were treated with either saline (sham experiments) or Escherichia coli lipolysaccharide [LPS, to obtain expression of the inducible NO synthase (iNOS) in the lung] and killed 4 h later. SPA (300- to 600-micrometer outer diameter) were mounted as rings in organ chambers for the recording of isometric tension, precontracted with PGF2alpha, and exposed to either severe (bath PO2 8 +/- 3 mmHg) or milder (21 +/- 3 mmHg) hypoxia. In SPA from sham-treated rats, contractions elicited by severe hypoxia were completely suppressed by either endothelium removal or preincubation with an NOS inhibitor [NG-nitro-L-arginine methyl ester (L-NAME), 10(-3) M]. In SPA from LPS-treated rats, contractions elicited by severe hypoxia occurred irrespective of the presence or absence of endothelium and were largely suppressed by L-NAME. The milder hypoxia elicited no increase in vascular tone. These results indicate an essential role of NO in the hypoxic contractions of precontracted rat SPA. The endothelium independence of HPV in arteries from LPS-treated animals appears related to the extraendothelial expression of iNOS. The severe degree of hypoxia required to elicit any contraction is consistent with a mechanism of reduced NO production caused by a limited availability of O2 as a substrate for NOS.

Intestinal bacteria condition dendritic cells to promote IgA production.

Immunoglobulin (Ig) A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role in limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. We show in mice that Peyer's Patch-derived dendritic cells (PP-DC) exhibit a specialized phenotype allowing the promotion of IgA production by B2 cells. This phenotype included increased expression of the retinaldehyde dehydrogenase 1 (RALDH1), inducible nitric oxide synthase (iNOS), B cell activating factor of the tumor necrosis family (BAFF), a proliferation-inducing ligand (APRIL), and receptors for the neuropeptide vasoactive intestinal peptide (VIP). The ability of PP-DC to promote anti-CD40 dependent IgA was partially dependent on retinoic acid (RA) and transforming growth factor (TGF)-beta, whilst BAFF and APRIL signaling were not required. Signals delivered by BAFF and APRIL were crucial for CD40 independent IgA production, although the contribution of B2 cells to this pathway was minimal. The unique ability of PP-DC to instruct naïve B cells to differentiate into IgA producing plasma cells was mainly imparted by the presence of intestinal commensal bacteria, and could be mimicked by the addition of LPS to the culture. These data indicate that exposure to pathogen-associated molecular patterns present on intestinal commensal bacteria condition DC to express a unique molecular footprint that in turn allows them to promote IgA production.

Maturation-dependent neurotoxicity of lead acetate in vitro: implication of glial reactions.

Despite a wealth of data on the neurotoxic effects of lead at the cellular and molecular levels, the reasons for its development-dependent neurotoxicity are still unclear. Here, the maturation-dependent effects of lead acetate were analyzed in immature and differentiated brain cells cultured in aggregates. Markers of general cytotoxicity as well as cell-type-specific markers of glial and neuronal cells showed that immature brain cells were more sensitive to lead than the differentiated counterparts, demonstrating that the development-dependent neurotoxicity of lead can be reproduced in aggregating brain cell cultures. After 10 days of treatment, astrocytes were found to be more affected by lead acetate than neurons in immature cultures, and microglial cells were strongly activated. Eleven days after cessation of the treatment, lead acetate caused a partial loss of astrocytes and an intense reactivity of the remaining ones. Furthermore, microglial cells expressed a macrophagic phenotype, and the loss of activity of neuron-specific enzymes was aggravated. In differentiated cultures, no reactive gliosis was found. It is hypothetized that the intense glial reactions (microgliosis and astrogliosis) observed in immature cultures contribute to the development-dependent neurotoxicity of lead.