Granulocyte-macrophage colony-stimulating factor modulates tapasin expression in human neutrophils.
(41/9754)
Differential display-polymerase chain reaction (DD-PCR) was used to evaluate changes in mRNA expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) treated human neutrophils to better understand how this cytokine affects the functions of neutrophils at the molecular level. Although a variety of cDNA fragments were identified as modulated by GM-CSF with the use of DD-PCR, one fragment in particular, NGS-17 (neutrophil GM-CSF-stimulated fragment #17), was characterized. The NGS-17 fragment hybridized to a 3.8-kh mRNA that encodes for a protein of a predicted molecular mass of 47.6 kDa. After cloning and sequencing, this gene was found to code for the recently sequenced tapasin or TAP-A protein. Immunoprecipitation and immunoblotting studies using anti-tapasin antibodies showed that tapasin is expressed in neutrophils and is associated with the MHC class I-TAP complex. Moreover, tapasin expression was found to be induced by dimethyl sulfoxide and by retinoic acid in HL-60 cells. This is the first report on the expression of tapasin in human neutrophils. It provides novel information, at the molecular level, on how GM-CSF enhances the functions of these cells. (+info)
Screening of an intragenic second-site suppressor of purine-cytosine permease from Saccharomyces cerevisiae. Possible role of Ser272 in the base translocation process.
(42/9754)
The purine-cytosine permease from Saccharomyces cerevisiae mediates the active transport through the plasma membrane of adenine, hypoxanthine, guanine and cytosine using the proton electrochemical potential difference as an energy source. Analysis of the activity of strains mutated in a hydrophilic segment (371-377) of the polypeptidic chain has shown the involvement of this segment in the maintenance of the active three-dimensional structure of the carrier. In an attempt to identify permease domains that could interact functionally and/or physically with this segment, we looked for second-site mutations that could suppress the effects of amino acid changes in this region. This paper describes a positive screen that has allowed the isolation of one suppressor from a permease mutant displaying the N374I change (fcy2-20 allele), a substitution that induces a dramatic decrease in the affinity of the carrier for adenine, cytosine and hypoxanthine. The second-site mutation corresponds to the replacement of the Ser272 residue by Leu. Its suppressive effect is shown to be a partial restoration of the binding of cytosine and hypoxanthine to the permease. To test whether this second-site mutation is specific for the fcy2-20 allele, two double mutants were constructed (Fcy2pT213I, S272L and Fcy2pS272L, N377G). Results obtained with these two double mutants showed that the suppressive effect of S272 L replacement was not specific for the original N374I change. To understand the general effect of this amino acid replacement for the three distinct double mutants, a strain overexpressing Fcy2pS272I, was constructed. Kinetic analysis of this strain showed that, by itself, the S272 L change induced an improvement in the base-binding step that could account for its global suppressive effect. Moreover, S272 L induced a decrease in the turnover of the permease, thus showing the involvement of S272 in the translocation process. Taking into account the topological model of the permease proposed here, this Ser residue is probably located in a transmembrane amphipathic alpha-helix (TM5). The location and the observed decrease in the turnover of the carrier observed with the S272 L change lead us to propose that S272 could be part of a hydrophilic pore involved in the translocation of the base and/or the proton. (+info)
Studies on time-kill kinetics of different classes of antibiotics against veterinary pathogenic bacteria including Pasteurella, Actinobacillus and Escherichia coli.
(43/9754)
A systematic analysis of the bacteriostatic/bactericidal effect of several antibiotics used in veterinary medicine was carried out by time-kill kinetic analysis using P. haemolytica, P. multocida, A. pleuropneumoniae, and E. coli. The antibiotics tested were enrofloxacin, danofloxacin, erythromycin, tilmicosin, penicillin G, ceftiofur and tetracycline. Unexpectedly, the antibiotics well characterized as bacteriostatic agents against human pathogens such as tetracycline and macrolides, showed bactericidal activity against P. haemolytica and A. pleuropneumoniae. In contrast, tetracycline and erythromycin were bacteriostatic and tilmicosin was bactericidal against P. multocida. In addition, P. multocida was killed by fluoroquinolones at a slower rate than the other bacteria. Spectrum analysis revealed that ceftiofur and tilmicosin were good substrates of the universal efflux pump, AcrA/B, but penicillin and tetracycline were not. The fluoroquinolones were modest substrates for AcrA/B. (+info)
The choline-converting pathway in Staphylococcus xylosus C2A: genetic and physiological characterization.
(44/9754)
A Staphylococcus xylosus C2A gene cluster, which encodes enzymes in the pathway for choline uptake and dehydrogenation (cud), to form the osmoprotectant glycine betaine, was identified. The cud locus comprises four genes, three of which encode proteins with significant similarities to those known to be involved in choline transport and conversion in other organisms. The physiological role of the gene products was confirmed by analysis of cud deletion mutants. The fourth gene possibly codes for a regulator protein. Part of the gene cluster was shown to be transcriptionally regulated by choline and elevated NaCl concentrations as inducers. (+info)
Deduction of consensus binding sequences on proteins that bind IIAGlc of the phosphoenolpyruvate:sugar phosphotransferase system by cysteine scanning mutagenesis of Escherichia coli lactose permease.
(45/9754)
Mediated by the protein IIAGlc, the phosphoenolpyruvate:sugar phosphotransferase system plays a role in the regulation of activity of other sugar transport systems in Escherichia coli. By using a direct binding assay, a collection of single-Cys replacement mutants in cytoplasmic loops of lactose permease were evaluated for their capacity to bind IIAGlc. Selected Cys replacements in loops IV/V or VI/VII result in loss of binding activity. Analysis of the mutagenesis results together with multiple sequence alignments of a family of proteins that interacts with IIAGlc provides the basis for developing two regions of consensus sequence in those partner proteins necessary for binding to IIAGlc. The requirement for two interaction regions is interpreted in the regulatory framework of a substrate-dependent conformational change that brings those two regions into an orientation optimal for binding IIAGlc. (+info)
Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane.
(46/9754)
Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0 degrees C) and stage B (accumulated at 30 degrees C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled. (+info)
A nerve growth factor mimetic TrkA antagonist causes withdrawal of cortical cholinergic boutons in the adult rat.
(47/9754)
Cholinergic neurons respond to the administration of nerve growth factor (NGF) in vivo with a prominent and selective increase of choline acetyl transferase activity. This suggests the possible involvement of endogenous NGF, acting through its receptor TrkA, in the maintenance of central nervous system cholinergic synapses in the adult rat brain. To test this hypothesis, a small peptide, C(92-96), that blocks NGF-TrkA interactions was delivered stereotactically into the rat cortex over a 2-week period, and its effect and potency were compared with those of an anti-NGF monoclonal antibody (mAb NGF30). Two presynaptic antigenic sites were studied by immunoreactivity, and the number of presynaptic sites was counted by using an image analysis system. Synaptophysin was used as a marker for overall cortical synapses, and the vesicular acetylcholine transporter was used as a marker for cortical cholinergic presynaptic sites. No significant variations in the number of synaptophysin-immunoreactive sites were observed. However, both mAb NGF30 and the TrkA antagonist C(92-96) provoked a significant decrease in the number and size of vesicular acetylcholine transporter-IR sites, with the losses being more marked in the C(92-96) treated rats. These observations support the notion that endogenously produced NGF acting through TrkA receptors is involved in the maintenance of the cholinergic phenotype in the normal, adult rat brain and supports the idea that NGF normally plays a role in the continual remodeling of neural circuits during adulthood. The development of neurotrophin mimetics with antagonistic and eventually agonist action may contribute to therapeutic strategies for central nervous system degeneration and trauma. (+info)
Mechanisms of methotrexate resistance in osteosarcoma.
(48/9754)
High-dose methotrexate is a major component of current protocols for the treatment of osteosarcoma, but some tumors seem to be resistant. Potential mechanisms of resistance include decreased transport through the reduced folate carrier (RFC) and increased expression of dihydrofolate reductase (DHFR). To investigate methotrexate resistance, tumors were obtained from 42 patients with high-grade osteosarcoma. RFC and DHFR mRNA expression were studied by semiquantitative reverse transcription-PCR. The RFC and DHFR genes were studied for deletions and amplification by Southern blot. Thirteen of 20 (65%) osteosarcoma samples were found to have decreased RFC expression at the time of initial biopsy. At definitive surgery and relapse, 10 of 22 (45%) were found to have decreased RFC expression. Seventeen of 26 (65%) samples with a poor response to chemotherapy had decreased RFC expression, whereas 5 of 14 (36%) samples with a good response had a decrease (P = 0.03). None of the samples had an RFC gene deletion. Two of 20 samples (10%) showed increased DHFR expression at initial biopsy. The frequency of increased DHFR expression was significantly higher in metastatic or recurrent tumors (62%, P = 0.014). None of the samples showed evidence of DHFR gene amplification. The high frequency of decreased RFC expression in the biopsy material suggests that impaired transport of methotrexate is a common mechanism of intrinsic resistance in osteosarcoma. Increased DHFR expression in the pulmonary metastases may be a mechanism of acquired methotrexate resistance or a difference between primary and metastatic lesions. (+info)