Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

Background: Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Methods: The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. Results: HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. Conclusion: The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.

Immune-derived opioids and peripheral antinociception

1. Recent findings have suggested a significant involvement of the immune system in the control of pain. Immune cells contain opioid peptides that are released within inflamed tissue and act at opioid receptors on peripheral sensory nerve endings. It is also apparent that different types of lymphocytes contain P-endorphin, memory T cells containing more beta -endorphin than naive cells. 2. These findings highlight an integral link between immune cell migration and inflammatory pain, The present review highlights immune system involvement in the site-directed control of inflammatory pain. 3. Full-length mRNA transcripts for opioid precursor proteins are expressed in immune cells. Increased expression of pro-opiomelanocortin mRNA and beta -endorphin has been demonstrated in stimulated lymphocytes and lymphocytes from animals with inflammation. 4. Cytokines and corticotropin-releasing factor (CRF) release opioids from immune cells, Potent peripheral analgesia due to direct injection of CRF can be blocked by antagonists to CRF, antibodies to opioid peptides, antisense to CRF and opioid receptor-specific antagonists. The release of opioid peptides from lymphocytes is calcium dependent and opioid receptor specific. Furthermore, endogenous sources of opioid peptides produce potent analgesia when implanted into the spinal cord. 5. Activated immune cells migrate directly to inflamed tissue using cell adhesion molecules to adhere to the epithelial surface of the vasculature in inflamed tissue. Lymphocytes that have been activated can express opioid peptides, Memory type T cells that contain opioid peptides are present within inflamed tissue; naive cells are not present in inflamed tissue and do not contain opioid peptides, Inhibiting the migration of memory type T cells into inflamed tissue by blocking selectins results in reduced numbers of beta -endorphin containing cells, a reduced quantity of beta -endorphin in inflamed paws and reduced stress- and CRF-induced peripheral analgesia. 6. Immunosuppression is associated with increased pain in patients. Moreover, immunosuppression results in decreased lymphocyte numbers as well as decreased analgesia in animal models.

Closed hybrid hierarchical reservoirs based on the deconstruction of the cellular native microenvironment

The stem cell niche organization and dynamics provide valuable cues for the development of mimetic environments that could have potential to stimulate the regenerative process. We propose the use of biodegradable biomaterials to produce closed miniaturised structures able to encapsulate different cell types or bioactive molecules. In particular, capsules are fabricated using the so-called layer-by-layer technology, where the consecutive (nano-sized) layers are well stabilized by electrostatic interactions or other weak forces. Using alginate-based spherical templates containing cells or other elements (e.g. proteins, magnetic nanoparticles, microparticles) it is possible to produce liquefied capsules that may entrap the entire cargo under mild conditions. The inclusion of liquefied micropcapsules may be used to produce hierarchical compartmentalised systems for the delivery of bioactive agents. The presence of solid microparticles inside such capsules offers adequate surface area for adherent cell attachment increasing the biological performance of these hierarchical systems, while maintain both permeability and injectability. We demonstrated that the encapsulation of distinct cell types (including mesenchymal stem cells and endothelial cells) enhances the osteogenic capability of this system, that could be useful in bone tissue engineering applications.

Contribution of the gap junction proteins Connexin40 and Connexin43 to the control of blood pressure

Summary Cells in tissues and organs coordinate their activities by communicating with each other through intercellular channels named gap junctions. These channels are conduits between the cytoplasmic compartments of adjacent cells, allowing the exchange of small molecules which may be crucial for hormone secretion. Renin is normally secreted in a regulated manner by specific cells of the juxtaglomerular apparatus located within the renal cortex. Gap junctional communication may be requisite to maintain an accurate functioning in coordination of renin-producing cells, more especially as renin is of paramount importance for the control of blood pressure. Connexin43 (Cx43) and Cx40 form gap junctions that link in vivo the cells of the juxtaglomerular apparatus. Cx43 links the endothelial cells, whereas gap junctions made of Cx40 connect the endothelial cells, the renin secreting cells, as well as the endothelial cells of to the renin-secreting cells of the afferent arteriole. The observation that loss of Cx40 results in chronic hypertension associated with altered vasomotion and signal conduction along arterioles, has lead us to suggest that connexins may contribute to control blood pressure by participating to the integration of various mechanical, osmotic and electrochemical stimuli involved in the control of renin secretion and by mediating the adaptive changes of the vascular wall induced by elevated blood pressure and mechanical stress. We therefore postulated that the absence of Cx40 could have deleterious effects on the coordinated functioning of the renin-containing cells, hence accounting for hypertension. In the first part of my thesis, we reported that Cx40-deficient mice (Cx40) are hypertensive due to increased plasma renin levels and numbers of renin-producing cells. Besides, we demonstrated that prostaglandins and nitric oxide, which are possible mediators in the regulation of renin secretion by the macula densa, exert a critical role in the mechanisms controlling blood pressure ín Cx40 knockout hypertensive mice. In view of previous studies that stated avessel-specifc increase in the expression of Cx43 during renin-dependent hypertension, we hypothesized that Cx43 channels are particularly well-matched to integrate the response of cells constituting the vascular wall to hypertensive conditions. Using transgenic mice in which Cx43 was replaced by Cx32, we revealed that the replacement of Cx43 by Cx32 is associated with decreased expression and secretion of renin and prevent the renin-dependent hypertension which is normally induced in the 2K1C model. To gain insights into the regulation of connexins in two separate tissues exposed to the same fluid pressure, the second part of my thesis work was dedicated to the study of the impact of chronic hypertension and related hypertrophy on the expression of the cardiovascular connexins (Cx40, Cx37, Cx43 and Cx45) in mouse aorta and heart. Our results documented that the expression of connexins is differentially regulated in mouse aorta. according to the models of hypertension. Thus, blood pressure induces mechanical forces that differentially alter the expression of vascular connexins in order to respond to an adaptation of the aortic wall observed under pathological conditions. Altogether these data provide the first evidences that intercellular communication mediated by gap junctions is required for a proper renin secretion from the juxtaglomerular apparatus in order to control blood pressure.

Characterization of the expression, regulation and function of Sorting Nexin 9 and Sorting Nexin 18

Summary : Sorting nexin (SNX) family members play important roles in intracellular protein and membrane trafficking, The membrane-tubulating SNX9 protein has been shown to interact with multiple components of the endocytic machinery and to participate in clathrin-mediated endocytosis of cell surface receptors. It has not been investigated if SNX9 may also participate in other protein sorting pathways that involve vesicular transport, specifically the biogenesis of lysosome-related organelles (LROs). Closely related to SNX9 is SNXl8, whose function is largely unknown. In this work, we have characterized the expression of SNX9 and SNXl8 in LRO-containing cells and investigated their role in protein trafficking during the formation of LROs. Our results indicate that SNX9 and SNXl8 are not essential for the formation of LROs, nor for the sorting of melanosomal proteins. We investigated how the level of intracellular SNX9 protein is regulated and found that it is a substrate of the ubiquitin ligase Itch, a member of the NEDD4 family of E3 ubiquitin ligases. Itch ubiquitylates SNX9 and regulates SNX9 levels by enhancing its degradation. Using ? truncated proteins we found that the interaction with SNX9 is mediated by the proline-rich domain of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. We further showed that Itch binding is not affected by tyrosine phosphorylation of SNX9. Using lentivector-mediated siRNA techniques, we found that Itch regulates the level of melanosomal proteins, while knock-down of SNX9 does not alter their level. Interestingly, we revealed that silencing of SNXIS affects the amount of the melanosomal protein Melan-A, but also of SNX9, and that SNXl8 can interact with SNX9. Taken together, our results highlight that the pool of substrates of NEDD4 family E3 ligases extends to proteins containing SH3 domains and provide insight into the potential functions of SNXI8. Résumé : Les membres de la famille des Sorting Nexins (SNX) jouent des rôles importants dans le trafic intracellulaire de protéines et membranes. Il a été démontré que la protéine SNX9, qui génère les tubules membranaires, interagit avec plusieurs composants de la machinerie d'endocytose et participe à l'endocytose des récepteurs de surface mediée par la clathrine. Aucune étude n'a investigué si SNX9 pourrait aussi participer à d'autres voies de trafic de protéines tel que le transport vésiculaire, et plus particulièrement la biogenèse des organites lysosomaux ("lysosome-related organelles", LR©s). SNXl8 est similaire à SNX9, mais sa fonction est largement inconnue. Dans ce travail, nous avons caractérisé l'expression de SNX9 et SNX18 dans des cellules contenants des LROs et investigué leur rôle dans le trafic de protéines pendant la formation des LROS. Nos résultats indiquent que SNX9 et SNXI8 ne sont essentiels ni pour la formation des LR©s, ni pour le trafic de protéines mélanosomales. Nous avons examiné la régulation du niveau intracellulaire de la protéine SNX9 et avons trouvé qu'elle est un substrat de l'ubiquitine ligase Itch, un membre de la famille NEDD4 des ubiquitine ligases E3. Itch ubiquitine SNX9 et régule les niveaux de SNX9 en augmentant sa dégradation. En utilisant des protéines mutées nous avons découvert que l'interaction avec SNX9 est médiée par le domaine riche en proline de Itch, qui est différent du domaine conventionnel de reconnaissance WW, et par le domaine SH3 de SNX9. L'interaction avec le domaine riche en proline de Itch est essentielle pour l'ubiquitination et la dégradation de SNX9. De plus, nous avons montré que cette liaison n'est pas affectée par la phosphorylation des résidus tyrosine de SNX9. En utilisant des vecteurs lentiviraux exprimant des siARN, nous avons trouvé que Itch régule les niveaux de protéines mélanosomales, alors que l'extinction de l'expression de SNX9 ne change pas leurs niveaux. En autre, nous avons révélé que la diminution de SNXl8 affecte le niveau de la protéine mélanosomale Melan-A et de SNX9, et aussi que SNXl8 peut interagir avec SNX9. En résumé, nos résultats démontrent que l'ensemble des substrats de la famille NEDD4 des ubiquitine ligases E3 s'élargit aux protéines contenant des domaines SH3 et ouvrent des perspectives sur les fonctions potentielles de SNXl8.

Interrelationship between adipocytes and fibroblasts during acute damage to the subcutaneous adipose tissue of rats: an ultrastructural study

Ultrastructural phenotypic transitional features were noted between adult adipocytes and fibroblasts in the subcutaneous adipose tissue of the dorsal pad of normal adult Wistar rats of both sexes, weighing 180-260 g, after acute injury either by the implantation of small (1.8 x 1 x 0.4 cm) perforated plastic boxes or by local heat application. Soon after the inflicted damage, fat-containing cells presented variable shapes. Early after damage, some of these cells were round, adipocyte-like, with numerous and large cytoplasmic fat droplets. A few days later, fat-containing cells became elongated, with the fat droplets in their cytoplasm becoming smaller and less numerous. The cells also showed a prominent active rough endoplasmic reticulum and newly formed collagenous matrix accumulated in the interstices. Although current views consider adult adipocytes to be terminal cells, the present findings, in their time sequence, strongly suggest the transformation of adipocytes into fibroblasts after acute injury to adipose tissue.

An investigation into the functional role of the D1:1 and D1:2 polypeptides in photosystem II in cyanobacteria : the effect of changing PSI/PSII ratio on photoinhibition in Synechococcus sp. PCC7942 /

The cyanobacterium Synechococcus sp. PCC 7942 (Anacystis nidulans R2) adjusts its photosynthetic function by changing one of the polypeptides of photosystem II. This polypeptide, called Dl, is found in two forms in Synechococcus sp. PCC 7942. Changing the growth light conditions by increasing the light intensity to higher levels results in replacement of the original form of D 1 polypeptide, D 1: 1, with another form, D 1 :2. We investigated the role of these two polypeptides in two mutant strains, R2S2C3 (only Dl:l present) and R2Kl (only Dl:2 present) In cells with either high or low PSI/PSII. R2S2C3 cells had a lower amplitude for 77 K fluorescence emission at 695 nm than R2Kl cells. Picosecond fluorescence decay kinetics showed that R2S2C3 cells had shorter lifetimes than R2Kl cells. The lower yields and shorter lifetimes observed in the D 1 and Dl:2 containing cells. containing cells suggest that the presence of D 1: 1 results in more photochemical or non-photochemical quenching of excitation energy In PSII. One of the most likely mechanisms for the increased quenching in R2S2C3 cells could be an increased efficiency in the transfer of excitation energy from PSII to PSI. However, photophysical studies including 77 K fluorescence measurements and picosecond time resolved decay kinetics comparing low and high PSI/PSII cells did not support the hypothesis that D 1: 1 facilitates the dissipation of excess energy by energy transfer from PSII to PSI. In addition physiological studies of oxygen evolution measurements after photoinhibition treatments showed that the two mutant cells had no difference in their susceptibility to photoinhibition with either high PSI/PSII ratio or low PSI/PSII ratio. Again suggesting that, the energy transfer efficiency from PSII to PSI is likely not a factor in the differences between Dl:l and Dl:2 containing cells.

Anatomic and molecular characterization of the 1 endocrine pancreas of a teleostean fish: Atlantic wolffish (Anarhichas lupus)

Background: The biologic attributes of the endocrine pancreas and the comparative endocrinology of islet amyloid polypeptide (IAPP) of fish are not well described in the literature. This study describes the endocrine pancreas of one teleostean fish. Ten captive Atlantic wolffish (Anarhichas lupus) from the Montreal Biodome were submitted for necropsy and their pancreata were collected. Results: Grossly, all the fish pancreata examined contained 1-3 nodules of variable diameter (1-8 mm). Microscopically, the nodules were uniform, highly cellular, and composed of polygonal to elongated cells. Immunofluorescence for pancreatic hormones was performed. The nodules were immunoreactive for insulin most prominent centrally, but with IAPP and glucagon only in the periphery of the nodules. Exocrine pancreas was positive for chromogranin A. Not previously recognized in fish, IAPP immunoreactivity occurred in α, glucagon-containing, cells and did not co-localize with insulin in β cells. The islet tissues were devoid of amyloid deposits. IAPP DNA sequencing was performed to compare the sequence among teleost fish and the potency to form amyloid fibrils. In silico analysis of the amino acid sequences 19-34 revealed that it was not amyloidogenic. Conclusions: Amyloidosis of pancreatic islets would not be expected as a spontaneous disease in the Atlantic wolffish. Our study underlines that this teleost fish is a potential candidate for pancreatic xenograft research.

TRPV1 receptors are involved in protein nitration and Muller cell reaction in the acutely axotomized rat retina

We report here the protein expression of TRPV1 receptor in axotomized rat retinas and its possible participation in mechanisms involved in retinal ganglion cell (RGC) death. Adult rats were subjected to unilateral, intraorbital axotomy of the optic nerve, and the retinal tissue was removed for further processing. TRPV1 total protein expression decreased progressively after optic nerve transection, reaching 66.2% of control values 21 days after axotomy. The number of cells labeled for TRPV1 in the remnant GCL decreased after 21 days post-lesion (to 63%). Fluoro-jade B staining demonstrated that the activation of TRPV1 in acutely-lesioned eyes elicited more intense neuronal degeneration in the GCL and in the inner nuclear layer than in sham-operated retinas. A single intraocular injection of capsazepine (100 mu M), a TRPV1 antagonist, 5 days after optic nerve lesion, decreased the number of GFAP-expressing Muller cells (72.5% of control values) and also decreased protein nitration in the retinal vitreal margin (75.7% of control values), but did not affect lipid peroxidation. Furthermore, retinal explants were treated with capsaicin (100 mu M), and remarkable protein nitration was then present, which was reduced by blockers of the constitutive and inducible nitric oxide synthases (7-NI and aminoguanidine, respectively). TRPV1 activation also increased GFAP expression, which was reverted by both TRPV1 antagonism with capsazepine and by 7-NI and aminoguanidine. Given that Muller cells do not express TRPV1, we suppose that the increased GFAP expression in these cells might be elicited by TRPV1 activation and by its indirect effect upon nitric oxide overproduction and peroxynitrite formation. We incubated Fluorogold pre-labeled retinal explants in the presence of capsazepine (1 mu M) during 48 h. The numbers of surviving RGCs stained with fluorogold and the numbers of apoptotic cells in the GCL detected with TUNEL were similar in lesioned and control retinas. We conclude that TRPV1 receptor expression decreased after optic nerve injury due to death of TRPV1-containing cells. Furthermore, these data indicate that TRPV1 might be involved in intrinsic protein nitration and Muller cell reaction observed after optic nerve injury. (C) 2010 Elsevier Ltd. All rights reserved.

Increased glial-derived neurotrophic factor in the small intestine of rats infected with the tapeworm, Hymenolepis diminuta

The neurotrophin, glial-derived neurotrophic factor (GDNF), is essential for the development of the enteric nervous system (ENS) in both the embryo and neonate and may be important for maintenance and plasticity of ENS. The tapeworm, Hymenolepis diminuta, altered the number of cells containing GNDF in the host's jejunum and ileum. Numbers and locations of GDNF-containing cells were determined by applying monoclonal anti-GDNF antibody to intestinal segments collected from infected and uninfected age-matched rats during the initial 34 days post-infection (dpi). Most cells staining positive for GDNF were present in the lamina propria of the jejunum and ileum from both infected and uninfected rats. The co-localization of staining by the antibodies, anti-GDNF and anti-ED2 (a nuclear specific antibody for resident macrophages) indicated that at least 74% of the cells staining for GDNF were macrophages. Mast cells did not stain with the anti-GDNF antibody. The increased number of GDNF+ cells in the infected rat intestine suggests that this neurotrophin may play a role in the neural and mucosal responses to lumenal tapeworm infection.

How to Perform and Interpret Testicular Fine Needle Aspiration in Stallions

Although several methods of testicular biopsy have been proposed previously, testicular fine needle aspiration (FNA) has proved to be the simplest, the most rapid, inexpensive, and overall the least invasive technique for obtaining testicular biopsies Testicular FNA is indicated for fertility investigations in stallions with oligozoospermia or azoospermia It is also used for differential diagnosis of testicular enlargement After sedation the stallion's testis is punctured to obtain testicular parenchyma samples containing cells mainly from the seminiferous epithelium the material obtained is used to perform smears which are analyzed for identification and quantification of term cells and Sertoli cells The results are based on the presence of the cell types found in the smears and the proportions of Sertoli cells per germ cells In addition to being a very useful diagnostic tool, testicular FNA is also used for follow-up examinations, as it is minimally invasive

Visceral pigmentation in four Dendropsophus species (Anura: Hylidae): Occurrence and comparison

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Visceral Pigmentation in Three Species of the Genus Scinax (Anura: Hylidae): Distinct Morphological Pattern

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Histological characterization of cellular types during Scinax fuscovarius oogenesis (Lutz) (Anura, Hylidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)