Corticotropin-releasing Factor (CRF) and Urocortin Promote the Survival of Cultured Cerebellar GABAergic Neurons Through the Type 1 CRF Receptor

Corticotropin releasing factor (CRF) is known to be involved in the stress response and in some degenerative brain disorders. In addition, CRF has a role as a neuromodulator in adult cerebellar circuits. Data from developmental studies suggest a putative role for CRF as a trophic factor during cerebellar development. In this study, we investigated the trophic role for CRF family of peptides by culturing cerebellar neurons in the presence of CRF, urocortin or urocortin II. Primary cell cultures of cerebella from embryonic day 18 mice were established, and cells were treated for either 1, 5 or 9 days with Basal Medium Eagles complete medium alone or complete medium with 1 µM CRF, urocortin, or urocortin II. The number of GABA-positive neurons in each treatment condition was counted at each culture age for monitoring the changes in neuronal survival. Treatment with 1 µM CRF or 1 µM urocortin increased the survival of GABAergic neurons at 6 days in vitro and 10 days in vitro, and this survival promoting effect was abolished by treatment with astressin in the presence of those peptides. Based on these data, we suggest that CRF or urocortin has a trophic role promoting the survival of cerebellar GABAergic neurons in cultures

Postnatal ontogeny of GABAB binding in rat brain

The postnatal development of GABAB binding sites in rat brain was studied by quantitative receptor autoradiography using [3H]GABA under selective conditions. Binding levels peak at regionally specific times during the first three weeks of life and then decline to adult levels. GABAB binding peaked in the globus pallidus, vestibular and spinal trigeminal nuclei, and the CA3 region of the hippocampus at postnatal day 3; in the striatum, nucleus accumbens, inferior olive, septum, dentate gyrus and CA1 region of the hippocampus at postnatal day 7; in the neocortex and thalamus at postnatal day 14; and in the medial geniculate at postnatal day 21. Following these regionally specific peaks, binding decreased to postnatal day 28 levels. Further significant decreases in binding were observed in all regions examined between postnatal day 28 and adulthood. Comparisons of binding site pharmacology reveal equipotent displacement of GABAB binding by several competitive agonists and antagonists in postnatal day 7 and adult rat brain, indicating that immature and adult binding sites have similar pharmacological properties with regard to these compounds. The GABAB receptor antagonist CGP 54626A, however, inhibited binding more potently in the postnatal day 7 thalamus and neocortex than in these areas in the adult brain. The guanyl nucleotide analogue guanosine 5'-O-(3-thiotriphasphate) inhibited GABAB binding extensively in both postnatal day 7 and adult brain. The non-competitive antagonist zinc also inhibited GABAB binding at both ages and was more potent in postnatal day 7 brain than in adult brain. Saturation analyses reveal two binding sites with similar affinities in both immature and adult rat brain, indicating that postnatal modulation of GABAB binding reflects changes in binding site density rather than modulation of binding site affinity. While immature GABAB binding sites share most pharmacological characteristics with adult binding sites and appear to be coupled to G-proteins at an early age, their interactions with zinc and CGP 54626A suggest that GABAB binding sites in immature brain may have a distinct pharmacological profile.Our data suggest significant regional and pharmacological changes in GABAB binding during development. The implications of these findings are discussed with regards to a possible role of GABAB receptors in the development of the central nervous system.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31372/1/0000285.pd

Overexpression of cathepsin K during silica-induced lung fibrosis and control by TGF-β

BACKGROUND: Lung fibrosis is characterized by tissue remodeling resulting from an imbalance between synthesis and degradation of extracellular organic matrices. To examine whether cathepsin(s) (Cat) are important in the development of pulmonary fibrosis, we assessed the expression of four Cat known for their collagenolytic activity in a model of silica-induced lung fibrosis. METHODS: Different strains of mice were transorally instilled with 2.5 mg crystalline silica or other particles. Cat expression (Cat K, S, L and B) was quantified in lung tissue and isolated pulmonary cells by quantitative RT-PCR. In vitro, we assessed the effect of different cytokines, involved in lung inflammatory and fibrotic responses, on the expression of Cat K by alveolar macrophages and fibroblasts. RESULTS: In lung tissue, Cat K transcript was the most strongly upregulated in response to silica, and this upregulation was intimately related to the fibrotic process. In mouse strains known for their differential response to silica, we showed that the level of Cat K expression following silica treatment was inversely related to the level of TGF-β expression and the susceptibility of these strains to develop fibrosis. Pulmonary macrophages and fibroblasts were identified as Cat K overproducing cells in the lung of silicotic mice. In vitro, Cat K was downregulated in mouse and human lung fibroblasts by the profibrotic growth factor TGF-β1. CONCLUSION: Altogether, these data suggest that while Cat K may contribute to control lung fibrosis, TGF-β appears to limit its overexpression in response to silica particles

Mesenchymal cell survival in airway and interstitial pulmonary fibrosis

Fibrotic reactions in the airways of the lung or the pulmonary interstitium are a common pathologic outcome after exposure to a wide variety of toxic agents, including metals, particles or fibers. The survival of mesenchymal cells (fibroblasts and myofibroblasts) is a key factor in determining whether a fibroproliferative response that occurs after toxic injury to the lung will ultimately resolve or progress to a pathologic state. Several polypeptide growth factors, including members of the platelet-derived growth factor (PDGF) family and the epidermal growth factor (EGF) family, are prosurvival factors that stimulate a replicative and migratory mesenchymal cell phenotype during the early stages of lung fibrogenesis. This replicative phenotype can progress to a matrix synthetic phenotype in the presence of transforming growth factor-β1 (TGF-β1). The resolution of a fibrotic response requires growth arrest and apoptosis of mesenchymal cells, whereas progressive chronic fibrosis has been associated with mesenchymal cell resistance to apoptosis. Mesenchymal cell survival or apoptosis is further influenced by cytokines secreted during Th1 inflammation (e.g., IFN-γ) or Th2 inflammation (e.g., IL-13) that modulate the expression of growth factor activity through the STAT family of transcription factors. Understanding the mechanisms that regulate the survival or death of mesenchymal cells is central to ultimately developing therapeutic strategies for lung fibrosis

Free amino acids, nitrate, and nitrate reductase in nitrogen fixation by soybean nodules

Due to the character of the original source materials and the nature of batch digitization, quality control issues may be present in this document. Please report any quality issues you encounter to [email protected], referencing the URI of the item.Bibliography: leaves 33-36.Not availabl

Isolation and characterization of two peptides specific to soybean nodules

Typescript (photocopy).Two low molecular weight peptides, each with a molecular weight of about 5.000, were isolated from soybean nodules. The levels of these peptides were positively correlated with the level of nitrogenase activity. These peptides are nodule-specific and bind ammonia and ureides. Peptide R is composed of 16.57% glutamic acid, and 15.02% aspartic acid, 13.26% alanine, 11.45% proline, 10.45% serine, and low levels of other common amino acids. Peptide Y contains 54.26% aspartic acid and 22.03% glutamic acid, and low levels of other common amino acids. Observations suggest that each peptide contains a transitions metal such as cobalt, copper, molybdenum, or nickel. Spectroscopic analysis revealed undistinguished spectra. Spectra of each peptide show similarities between them and rule out any possibility of either being a nitrogenase fragment. Two chromophores are common to peptides R and Y; peptide Y contains one additional chromophore. ORD spectra indicate a low amount of ordered structure for both peptides. Incorporation of [^15]N from [^15]N2 into free amino acids and the two peptides was measured using plasma desorption mass spectrometry. A 2.1 atom % [^15]N excess was seen for aspartic acid and a 5.7 atom % [^15]N excess for glutamic acid. A 3.2 atom % [^15]N excess incorporation was found for ureides bound to combined peptide sample. A MW 147 peak remains unidentified. Cyanide reduction, an activity associated with nitrogenase, may be enhanced by peptide Y. both peptides act on cyanide but neither reduce acetylene. The non-specificity of substrates common t nitrogenases is not observed with the peptides. Cyanide reduction by both nitrogenase and a combine peptide sample was monitored by [^13]C-NMR. Three transient peaks were observed which were identified as oxygenated imine compounds. The peptides found are suspected to be involved in the transfer of ammonia from the site of reduction to the site of assimilation, acting as carriers... The appearance and disappearance of NMR signal due to transient compounds produced during cyanide reduction was the first observation of intermediates formed during a reduction catalyzed by nitrogenase. These intermediates are the first direct evidence supporting the hypothesis that reduction of dinitrogen occurs via diimide and hydrazine intermediates