Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. (C) 2011 Elsevier B.V. All rights reserved.

Characterization of a Local Renin-Angiotensin System in Rat Gingival Tissue

Background: The systemic renin-angiotensin system (RAS) promotes the plasmatic production of angiotensin (Ang) II, which acts through interaction with specific receptors. There is growing evidence that local systems in various tissues and organs are capable of generating angiotensins independently of circulating RAS. The aims of this study were to investigate the expression and localization of RAS components in rat gingival tissue and evaluate the in vitro production of Ang II and other peptides catalyzed by rat gingival tissue homogenates incubated with different Ang II precursors. Methods: Reverse transcription - polymerase chain reaction assessed mRNA expression. Immunohistochemical analysis aimed to detect and localize renin. A standardized fluorimetric method with tripeptide hippuryl-histidyl-leucine was used to measure tissue angiotensin-converting enzyme (ACE) activity, whereas high performance liquid chromatography showed products formed after the incubation of tissue homogenates with Ang I or tetradecapeptide renin substrate (TDP). Results: mRNA for renin, angiotensinogen, ACE, and Ang II receptors (AT(1a), AT(1b), and AT(2)) was detected in gingival tissue; cultured gingival fibroblasts expressed renin, angiotensinogen, and AT(1a) receptor. Renin was present in the vascular endothelium and was intensely expressed in the epithelial basal layer of periodontally affected gingival tissue. ACE activity was detected (4.95 +/- 0.89 nmol histidyl-leucine/g/minute). When Ang I was used as substrate, Ang 1-9 (0.576 +/- 0.128 nmol/mg/minute), Ang II (0.066 +/- 0.008 nmol/mg/minute), and Ang 1-7 (0.111 +/- 0.017 nmol/mg/minute) were formed, whereas these same peptides (0.139 +/- 0.031, 0.206 +/- 0.046, and 0.039 +/- 0.007 nmol/mg/minute, respectively) and Ang 1 (0.973 +/- 0.139 nmol/mg/minute) were formed when TDP was the substrate. Conclusion: Local RAS exists in rat gingival tissue and is capable of generating Ang II and other vasoactive peptides in vitro. J Periodontol 2009;80:130-139.

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases in hypertensive vascular remodeling

Structural vascular changes in two-kidney, one-clip (2K-1C) hypertension may result from increased matrix metalloproteinase (MMP)-2 activity. MMP-2 activation is regulated by other MMPs, including transmembrane-MMPs, and by tissue inhibitors of MMPs (TIMPs). We have investigated the localization of MMP-2, -9, -14, and TIMPs 1-4 in hypertensive aortas and measured their levels by zymography/Western blotting and immunohistochemistry. Gelatinolytic activity was assayed in tissues by in situ zymography. Sham-operated and 2K-1C hypertensive rats were treated with doxycycline (or vehicle) for 8 weeks, and the systolic blood pressure was monitored weekly. Doxycycline attenuated 2K-1C hypertension (165 +/- 11.7 mmHg versus 213 +/- 7.9 mm Hg in hypertensive controls, P<0.01), and completely prevented increase in the thicknesses of the media and the intima in 2K-1C animals (P<0.01). Increased amounts of MMP-2, -9, and -14 were found in hypertensive aortas, as well as enhanced gelatinolytic activity. A gradient in the localization of MMP-2, -9, and -14 was found, with increased amounts detected in the intima, at sites with higher gelatinolytic activity. Doxycycline attenuated hypertension induced increases in all the 3 investigated MMPs in both the media and the intima (all P<0.05). but it did not change the amounts of TIMPs 1-4 (P>0.05). Therefore, an imbalance between increased amounts of MMPs at the tissue level without a corresponding increase in the quantities of TIMPs, particularly in the intima and inner media layers, appears to account for the increased proteolytic activity found in 2K-1C hypertension-induced maladaptive vascular remodeling. (C) 2009 Elsevier B.V. All rights reserved.

Bril: A novel bone-specific modulator of mineralization

In the course of attempting to define the bone ""secretome"" using a signal-trap screening approach, we identified a gene encoding a small membrane protein novel to osteoblasts. Although previously identified in silico as ifitm5, no localization or functional studies had been undertaken on this gene. We characterized the expression patterns and localization of this gene in vitro and in vivo and assessed its role in matrix mineralization in vitro. The bone specificity and shown role in mineralization led us to rename the gene bone restricted ifitm-like protein (Bril). Bril encodes a 14.8-kDa 1.34 arnino acid protein with two transmembrane domains. Northern blot analysis showed bone-specific expression with no expression in other embryonic or adult tissues. In situ hybridization and immunohistochemistry in mouse embryos showed expression localized on the developing bone. Screening of cell lines showed Bril expression to be highest in osteoblasts, associated with the onset of matrix maturation/mineralization, suggesting a role in bone formation. Functional evidence of a role in mineralization was shown by adenovirus-mediated Brit overexpression and lentivirus-mediated Bril shRNA knockdown in vitro. Elevated Bril resulted in dose-dependent increases in mineralization in UMR106 and rat primary osteoblasts. Conversely, knockdown of Bril in MC3T3 osteoblasts resulted in reduced mineralization. Thus, we identified Bril as a novel osteoblast protein and showed a role in mineralization, possibly identifying a new regulatory pathway in bone formation.

Comparing detection and location performance of perpendicular and parallel broadside GPR antenna orientations

GPR (Ground Penetrating Radar) results are shown for perpendicular broadside and parallel broadside antenna orientations. Performance in detection and localization of concrete tubes and steel tanks is compared as a function of acquisition configuration. The comparison is done using 100 MHz and 200 MHz center frequency antennas. All tubes and tanks are buried at the geophysical test site of IAG/USP in Sao Paulo city, Brazil. The results show that the long steel pipe with a 38-mm diameter was well detected with the perpendicular broadside configuration. The concrete tubes were better detected with the parallel broadside configuration, clearly showing hyperbolic diffraction events from all targets up to 2-m depth. Steel tanks were detected with the two configurations. However, the parallel broadside configuration was generated to a much lesser extent an apparent hyperbolic reflection corresponding to constructive interference of diffraction hyperbolas of adjacent targets placed at the same depth. Vertical concrete tubes and steel tanks were better contained with parallel broadside antennas, where the apexes of the diffraction hyperbolas better corresponded to the horizontal location of the buried target disposition. The two configurations provide details about buried targets emphasizing how GPR multi-component configurations have the potential to improve the subsurface image quality as well as to discriminate different buried targets. It is judged that they hold some applicability in geotechnical and geoscientific studies. (C) 2009 Elsevier B.V. All rights reserved.

Regulation of Multidrug Resistance-Associated Protein 2 by Calcium Signaling in Mouse Liver

Mu hiding resistance associated protein 2 (Mrp2) is a canalicular transporter responsible for organic anion secretion into bile. Mrp2 activity is regulated by insertion into the plasma membrane; however, the factors that control this are not understood. Calcium (Ca(2+)) signaling regulates exocytosis of vesicles in most cell types, and the type II inositol 1,4,5-triphosphate receptor (InsP(3)R2) regulates Ca(2+) release in the canalicular region of hepatocytes. However, the role of InsP(3)R2 and of Ca(2+) signals in canalicular insertion and function of Mrp2 is not known. The aim of this study was to determine the role of InsP(3)R2-mediated Ca(2+) signals in targeting Mrp2 to the canalicular membrane. Livers, isolated hepatocytes, and hepatocytes in collagen sandwich culture from wild-type (WT) and InsP(3)R2 knockout (KO) mice were used for western blots, confocal immunofluorescence, and time-lapse imaging of Ca(2+) signals and of secretion of a fluorescent organic anion. Plasma membrane insertion of green fluorescent protein (GFP)-Mrp2 expressed in HepG2 cells was monitored by total internal reflection microscopy. InsP(3)R2 was concentrated in the canalicular region of WT mice but absent in InsP(3)R2 KO livers, whereas expression and localization of InsP(3)R1 was preserved, and InsP(3)R3 was absent from both WT and KO livers. Ca(2+) signals induced by either adenosine triphosphate (ATP) or vasopressin were impaired in hepatocytes lacking InsP(3)R2. Canalicular secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was reduced in KO hepatocytes, as well as in WT hepatocytes treated with 1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetra-acetic acid (BAPTA). Moreover, the choleretic effect of tauroursodeoxycholic acid (TUDCA) was impaired in InsP(3)R2 KO mice. Finally, ATP increased GFP-Mrp2 fluorescence in the plasma membrane of HepG2 cells, and this also was reduced by BAPTA. Conclusion: InsP(3)R2-mediated Ca(2+) signals enhance organic anion secretion into bile by targeting Mrp2 to the canalicular membrane. (HEPATOLOGY 2010;52:327-337)

Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed ""RaTART"" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.

Differential regulation of FGF-2 in neurons and reactive astrocytes of axotomized rat hypoglossal nucleus. A possible therapeutic target for neuroprotection in peripheral nerve pathology

Despite the favorable treatment of cranial nerve neuropathology in adulthood, some cases are resistant to therapy leading to permanent functional impairments In many cases, suitable treatment is problematic as the therapeutic target remains unknown Basic fibroblast growth factor (bFGF, FGF 2) is involved in neuronal maintenance and wound repair following nervous system lesions It is one of few neurotrophic molecules acting in autocrine, paracrine and intracrine fashions depending upon specific circumstances Peripheral cranial somatic motor neurons, i e hypoglossal (XII) neurons, may offer a unique opportunity to study cellular FGF 2 mechanisms as the molecule is present in the cytoplasm of neurons and in the nuclei of astrocytes of the central nervous system FGF-2 may trigger differential actions during development, maintenance and lesion of XII neurons because axotomy of those cells leads to cell death during neonatal ages, but not in adult life Moreover, the modulatory effects of astroglial FGF 2 and the Ca+2 binding protein S100 beta have been postulated in paracrine mechanisms after neuronal lesions In our study, adult Wistar rats received a unilateral crush or transection (with amputation of stumps) of XII nerve, and were sacrificed after 72 h or 11 days Brains were processed for immunohistochemical localization of neurofilaments (NF), with or without counterstaining for Nissl substance, ghat fibrillary acidic protein (GFAP, as a marker of astrocytes), S100 beta and FGF-2 The number of Nissl positive neurons of axotomized XII nucleus did not differ from controls The NF immunoreactivity increased in the perikarya and decreased in the neuropil of axotomized XII neurons 11 days after nerve crush or transection An astrocytic reaction was seen in the ipsilateral XII nucleus of the crushed or transected animals 72 h and 11 days after the surgery The nerve lesions did not change the number of FGF-2 neurons in the ipsilateral XII nucleus, however, the nerve transection increased the number of FGF-2 ghat profiles by 72 h and 11 days Microdensitometric image analysis revealed a short lasting decrease in the intensity of FGF 2 immunoreactivity in axotomized XII neurons by 72 h after nerve crush or transection and also an elevation of FGF-2 in the ipsilateral of ghat nuclei by 72h and 11 days after the two lesions S100 beta decreased in astrocytes of 11-day transected XII nucleus The two-color immunoperoxidase for the simultaneous detection of the GFAP/FGF-2 indicated FGF-2 upregulation in the nuclei of reactive astrocytes of the lesioned XII nucleus Astroglial FGF-2 may exert paracrine trophic actions in mature axotomized XII neurons and might represent a therapeutic target for neuroprotection in peripheral nerve pathology (C) 2009 Elsevier GmbH All rights reserved

Molecular characterization of a putative heat shock protein cognate gene in Rhynchosciara americana

An hsc70 homologue gene (Rahsc70) of the diptera Rhynchosciara americana was isolated and characterized. We were able to determine the mRNA sequence from an EST of salivary gland cDNA library, and a Rahsc70 cDNA cassette was used as a probe to isolate the genomic region from a genomic library. The mRNA expression of this gene parallels the 2B puff expansion, suggesting its involvement in protein processing, since this larval period corresponds to a high synthetic activity period. During heat shock stress conditions, hsc70 expression decreased. In situ hybridization of polytene chromosomes showed that the Rahsc70 gene is located near the C3 DNA puff. The cellular localization of Hsc70 protein showed this protein in the cytoplasm and in the nucleus.

Neurodegeneration and Increased Production of Nitrotyrosine, Nitric Oxide Synthase, IFN-gamma and S100 beta Protein in the Spinal Cord of IL-12p40-Deficient Mice Infected with Trypanosoma cruzi

Background/Aim: Chagas` disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100 beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100 beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. Methods: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100 beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. Results: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p<0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p<0.01) and NOS (544%, p<0.001). Moreover, the intensity of nitrotyrosine (16%, p<0.01), NOS (38%, p<0.05) and S100 beta (21%, p<0.001) immunoreactivities were also augmented. No IFN-gamma labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled.Conclusion: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100 beta may contribute to NOS activation in the absence of IL-12. Copyright (C) 2009 S. Karger AG, Basel

Slower rescue of ER homeostasis by the unfolded protein response pathway associated with common variable immunodeficiency

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of unfolded protein response (UPR) in the pathogenesis of the disease. Augmented unspliced X-box binding protein 1 (XBP-1) mRNA concurrent with co-localization of IgM and BiP/GRP78 were found in one CVID patient. At confocal microscopy analysis this patient`s cells were enlarged and failed to present the typical surface distribution of IgM, which accumulated within an abnormally expanded endoplasmic reticulum. Sequencing did not reveal any mutation on XBP-1, neither on IRE-1 alpha that could potentially prevent the splicing to occur. Analysis of spliced XBP-1, IRE-1 alpha and BiP messages after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these endoplasmic reticulum (ER) stressors by presenting waves of transcription of these three genes, this patient`s cells presented lower rates of transcription, not reaching the same level of response of healthy subjects even after 48 h of ER stress. Treatment with DMSO rescued IgM and IgG secretion as well as the expression of spliced XBP-1. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia. (C) 2008 Elsevier Ltd. All rights reserved.

Gene optimization leads to robust expression of human respiratory syncytial virus nucleoprotein and phosphoprotein in human cells and induction of humoral immunity in mice

Human respiratory syncytial virus (HRSV) is the major pathogen leading to respiratory disease in infants and neonates worldwide. An effective vaccine has not yet been developed against this virus, despite considerable efforts in basic and clinical research. HRSV replication is independent of the nuclear RNA processing constraints, since the virus genes are adapted to the cytoplasmic transcription, a process performed by the viral RNA-dependent RNA polymerase. This study shows that meaningful nuclear RNA polymerase II dependent expression of the HRSV nucleoprotein (N) and phosphoprotein (F) proteins can only be achieved with the optimization of their genes, and that the intracellular localization of N and P proteins changes when they are expressed out of the virus replication context. Immunization tests performed in mice resulted in the induction of humoral immunity using the optimized genes. This result was not observed for the non-optimized genes. In conclusion, optimization is a valuable tool for improving expression of HRSV genes in DNA vaccines. (c) 2009 Elsevier B.V. All rights reserved.

Dynamics of a class of ODEs more general than almost periodic

A temporally global solution, if it exists, of a nonautonomous ordinary differential equation need not be periodic, almost periodic or almost automorphic when the forcing term is periodic, almost periodic or almost automorphic, respectively. An alternative class of functions extending periodic and almost periodic functions which has the property that a bounded temporally global solution solution of a nonautonomous ordinary differential equation belongs to this class when the forcing term does is introduced here. Specifically, the class of functions consists of uniformly continuous functions, defined on the real line and taking values in a Banach space, which have pre-compact ranges. Besides periodic and almost periodic functions, this class also includes many nonrecurrent functions. Assuming a hyperbolic structure for the unperturbed linear equation and certain properties for the linear and nonlinear parts, the existence of a special bounded entire solution, as well the existence of stable and unstable manifolds of this solution are established. Moreover, it is shown that this solution and these manifolds inherit the temporal behaviour of the vector field equation. In the stable case it is shown that this special solution is the pullback attractor of the system. A class of infinite dimensional examples involving a linear operator consisting of a time independent part which generates a C(0)-semigroup plus a small time dependent part is presented and applied to systems of coupled heat and beam equations. (C) 2010 Elsevier Ltd. All rights reserved.

First-order-reversal-curve analysis of Pr-Fe-B-based exchange spring magnets

Ribbons of nominal composition (Pr(9.5)Fe(84.5)B(6))(0.96)Cr(0.01)(TiC)(0.03) were produced by arc-melting and melt-spinning the alloys on a Cu wheel. X-ray diffraction (XRD) reveals two main phases, one based upon alpha-Fe and the other upon Pr(2)Fe(14)B. The ribbons show exchange spring behavior with H (c) = 12.5 kOe and (BH)(max) = 13.6 MGOe when these two phases are well coupled. Transmission electron microscopy revealed the coupled behavior is observed when the microstructure consists predominantly of alpha-Fe grains (diameter similar to 100 nm.) surrounded by hard material containing Pr(2)Fe(14)B. The microstructure is discussed in terms of a calculation by Skomski and Coey. A first-order-reversal-curve (FORC) analysis was performed for both a well-coupled sample and a poorly coupled sample. The FORC diagrams show two strong peaks for both the poorly coupled sample and for the well-coupled material. In both cases, the localization of the FORC probability suggests magnetizing interactions between particles. Switching field distributions were calculated and are consistent with the sample microstructure.

First-order-reversal-curve analysis of Pr-Fe-B-based nanocomposites

Ribbons of nominal composition (Pr(9.5)Fe(84.5)B(6))(0.96)Cr(0.01)(TiC)(0.03) were produced by arc-melting and melt-spinning the alloys on a Cu wheel. X-ray diffraction reveals two main phases, one based upon alpha-Fe and the other upon Pr(2)Fe(14)B. The ribbons show exchange spring behavior with H(c)=12.5 kOe and (BH)(max)= 13.6 MGOe when these two phases are well coupled. Transmission electron microscopy revealed that the coupled behavior is observed when the microstructure consists predominantly of alpha-Fe grains(diameter similar to 100 nm.) surrounded by hard material containing Pr(2)Fe(14)B. A first-order-reversal-curve (FORC) analysis was performed for both a well-coupled sample and a partially-coupled sample. The FORC diagrams show two strong peaks for both the partially-coupled sample and for the well coupled material. In both cases, the localization of the FORC probability suggests demagnetizing interactions between particles. Switching field distributions were calculated and are consistent with the sample microstructure. (C) 2009 Elsevier B.V. All rights reserved.