Chloride secretion by canine tracheal epithelium: I. Role of intracellular cAMP levels

Summary We measured the short-circuit current (I _sc) across canine tracheal epithelium and the intracellular cAMP levels of the surface epithelial cells in the same tissues to assess the role of cAMP as a mediator of electrogenic Cl secretion. Secretogogues fall into three classes: (i) epinephrine, prostaglandin (PG) E₁, and theophylline increase bothI _sc and cellular cAMP levels; (ii) PGF₂ and calcium ionophore A23187, increaseI _sc without affecting cell cAMP levels at the doses employed; and (iii) acetylcholine, histamine, and phenylephrine do not alter eitherI _sc or cAMP levels.These findings indicate that: (i) increases in cAMP or Ca activity stimulate electrogenic Cl secretion by the columnar cells of the surface epithelium; (ii) cAMP mediates the effects of PGE₁ and -adrenergic agonists; (iii) a strict correlation between cAMP levels and Cl secretion rate is not apparent from spontaneous variations in these parameters or from dose-response relations ofI _sc and cAMP to epinephrine concentration; and (iv) acetylcholine, histamine, and phenylephrine, agents that stimulate electrically-neutral NaCl secretion by submucosal glands, do not evoke cAMP-mediated, responses by the surface epithelium.Addition of 10^–6 m indomethacin (or other prostaglandin synthesis inhibitors) to the mucosal solution decreasesI _sc and cellular cAMP levels and reduces the release of PGE₂ into the bathing media by 80%. Indomethacin does not interfere with the subsequent secretory response to PGE₁. This suggests that endogenous prostaglandin production underlies the spontaneous secretion of Cl across canine tracheal epithelium under basal conditions.     

Oxygen cost of chloride transport in perfused rectal gland ofSqualus acanthias

Summary In the stimulated state, with glucose as substrate, oxygen uptake by the isolated perfused rectal gland is directly related to the rate of chloride secretion. Lactate production is negligible under aerobic conditions in the stimulated gland. A stoichiometric relationship exists between chloride transport and oxygen consumption, with a Cl/O₂ ratio of about 301, resembling that reported for sodium in mammalian kidneys. This ratio remains constant under varying degrees and modes of stimulation. The ratio does not change when the gland is induced to secrete chloride against varying electrochemical gradients by altering the concentration of urea in the perfusate.Established Investigator of the American Heart Association.     

Species-specific antigens ofMycoplasma hyopneumoniae and cross-reactions with other porcine mycoplasmas

Cell proteins from the porcine mycoplasmasMycoplasma hyorhinis, M. hyopneumoniae, andM. flocculare have been analyzed by SDS-gel electrophoresis and immunoblotting. The protein profiles ofM. hyopneumoniae andM. flocculare were similar, but the protein profile ofM. hyorhinis was quite different from the others. Antisera prepared against whole cells of the above three mycoplasmas were used in immunoblotting of electrophoretically separated antigens and in enzyme-linked immunosorbent assay. One major antigen, which had a molecular weight of 73 k, was found to be common to all three mycoplasmas. Another major antigen, with a molecular weight of 41 k, was common toM. hyopneumoniae andM. flocculare and may also be present inM. hyorhinis. Several antigens of comparatively high molecular weights (108 k, 102 k, 93 k, 89 k, and 87 k) seemed to be specific forM. hyopneumoniae. Three antisera prepared by immunization of rabbits with immunoprecipitates obtained by crossed immunoelectrophoresis ofM. hyopneumoniae were also used in blotting experiments. One of these antisera was found to be directed against the 73 k antigen common to the three porcine mycoplasmas investigated. The other two antisera were directed againstM. hyopneumoniae-specific antigens with molecular weights of 74 k, 58 k, 45 k, 44 k, and 38 k.     

Web servicing the biological office

Biologists routinely use Microsoft Office applications for standard analysis tasks. Despite ubiquitous internet resources, information needed for everyday work is often not directly and seamlessly available. Here we describe a very simple and easily extendable mechanism using Web Services to enrich standard MS Office applications with internet resources. We demonstrate its capabilities by providing a Web-based thesaurus for biological objects, which maps names to database identifiers and vice versa via an appropriate synonym list. The client application ProTag makes these features available in MS Office applications using Smart Tags and Add-Ins. AVAILABILITY: http://services.bio.ifi.lmu.de/prothesaurus/     

Unique mechanistic features of post-translational regulation of glutamine synthetase activity in Methanosarcina mazei strain Gö1 in response to nitrogen availability

PII-like signal transduction proteins are found in all three domains of life and have been shown to play key roles in the control of bacterial nitrogen assimilation. This communication reports the first target protein of an archaeal PII-like protein, representing a novel PII receptor. The GlnK(1) protein of the methanogenic archaeon Methanosarcina mazei strain Go1 interacts and forms stable complexes with glutamine synthetase (GlnA(1)). Complex formation with GlnK(1) in the absence of metabolites inhibits the activity of GlnA(1). On the other hand, the activity of this enzyme is directly stimulated by the effector molecule 2-oxoglutarate. Moreover, 2-oxoglutarate antagonized the inhibitory effects of GlnK(1) on GlnA(1) activity but did not prevent GlnK(1)/GlnA(1) complex formation. On the basis of these findings, we hypothesize that besides the dominant effector molecule 2-oxoglutarate, the nitrogen sensor GlnK(1) allows finetuning control of the glutamine synthetase activity under changing nitrogen availabilities and propose the following model. (i) Under nitrogen limitation, increasing concentrations of 2-oxoglutarate stimulate maximal GlnA(1) activity and transform GlnA(1) into an activated conformation, which prevents inhibition by GlnK(1). (ii) Upon a shift to nitrogen sufficiency after a period of nitrogen limitation, GlnA(1) activity is reduced by decreasing internal 2-oxoglutarate concentrations through diminished direct activation and by GlnK(1) inhibition.     

Phylogenetic mapping of intron positions: a case study of translation initiation factor eIF2gamma

Eukaryotic translation initiation factor 2 (eIF2) is a G protein that delivers the methionyl initiator tRNA to the small ribosomal subunit and releases it upon GTP hydrolysis after the recognition of the initiation codon. eIF2 is composed of three subunits, alpha, beta, and gamma. Subunit gamma shows the strongest conservation, and it confers both tRNA and GTP/GDP binding. Using intron positioning and protein sequence alignment, here we show that eIF2gamma is a suitable phylogenetic marker for eukaryotes. We determined or completed the sequences of 13 arthropod eIF2gamma genes. Analyzing the phylogenetic distribution of 52 different intron positions in 55 distantly related eIF2gamma genes, we identified ancient ones and shared derived introns in our data set. Obviously, intron positioning in eIF2gamma is evolutionarily conserved. However, there were episodes of complete and partial intron losses followed by intron gains. We identified 17 clusters of intron positions based on their distribution. The evolution of these clusters appears to be connected with preferred exon length and can be used to estimate the relative timing of intron gain because nearby precursor introns had to be erased from the gene before the new introns could be inserted. Moreover, we identified a putative case of intron sliding that constitutes a synapomorphic character state supporting monophyly of Coleoptera, Lepidoptera, and Diptera excluding Hymenoptera. We also performed tree reconstructions using the eIF2gamma protein sequences and intron positioning as phylogenetic information. Our results support the monophyly of Viridoplantae, Ascomycota, Homobasidiomyceta, and Apicomplexa.     

Copper related toxic effects on cellular protein metabolism in human astrocytes

INTRODUCTION: Copper overload due to a defect in the ATPase 7B mediated copper excretion within hepatocytes produces the phenotype of Wilson disease. The overload of hepatocytes with copper results in necrotic liver cells and is accompanied by a high concentration of blood copper levels. That occurs to be the reason for increasing neurological copper concentration. Although copper is linked to oxidation, there are no data on the direct copper related effects in human brain cells. AIM: To test the copper induced changes in protein oxidation in human astrocyte like cells. METHODS: We used U87 cells as model for human astrocytes. Cells were treated with increasing concentrations of copper(II)-chloride in Dulbeccos minimal essential medium. Subsequently, at different time points we investigated: cellular growth, cellular survival under copper treatment, the concentration of oxidized tryptophane in GADPH in vitro as well as the carbonyl concentration and the concentration of oxidized proteins in vivo in U87 glial cells. RESULTS: The viability of cells decreased with both increasing copper concentration and duration of treatment. The concentration of oxidized proteins was directly correlated to the increase of copper concentration and duration of exposure. CONCLUSION: These observations demonstrate the similarities between copper treatment and treatment with other commonly used oxidants, including hydrogen peroxide. Furthermore, the vulnerability of astrocytes towards copper exposure could be demonstrated. Therefore, these data give further insights into understanding of copper metabolism, which in turn is important to reveal the exact pathological mechanism in copper related diseases such as Wilson disease.     

Wound-healing defect of CD18(-/-) mice due to a decrease in TGF-beta1 and myofibroblast differentiation

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(-/-) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers alpha-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(-/-) mice, suggesting an impaired myofibroblast differentiation. TGF-beta signalling was clearly involved since TGF-beta1 and TGF-beta receptor type-II protein levels were decreased, while TGF-beta(1) injections into wound margins fully re-established wound closure. Since, in CD18(-/-) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(-/-) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-beta1. Indeed, in neutrophil-macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-beta1 release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing.