The GeneQuiz web server: protein functional analysis through the Web.
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Amino Acid Sequence↗
Biomedical subjects
C Leroy
Publications and source records attributed to C Leroy.
Involvement of the PP2C-like phosphatase Ptc2p in the DNA checkpoint pathways of Saccharomyces cerevisiae.
RAD53 encodes a conserved protein kinase that acts as a central transducer in the DNA damage and the DNA replication checkpoint pathways in Saccharomyces cerevisiae. To identify new elements of these pathways acting with or downstream of RAD53, we searched for genes whose overexpression suppressed the toxicity of a dominant-lethal form of RAD53 and identified PTC2, which encodes a protein phosphatase of the PP2C family. PTC2 overexpression induces hypersensitivity to genotoxic agents in wild-type cells and is lethal to rad53, mec1, and dun1 mutants with low ribonucleotide reductase activity. Deleting PTC2 specifically suppresses the hydroxyurea hypersensitivity of mec1 mutants and the lethality of mec1Delta. PTC2 is thus implicated in one or several functions related to RAD53, MEC1, and the DNA checkpoint pathways.
Alleles↗
Molecular and functional characterization of an amphibian urea transporter.
We report the characterization of a frog (Rana esculenta) urea transporter (fUT). The cloned cDNA is 1.4 kb long and contains a putative open reading frame of 1203 bp. In frog urinary bladder, the gene is expressed as two mRNAs of 4.3 and 1.6 kb. The fUT protein is 63.1 and 56.3% identical to rat UT-A2 and UT-B1, respectively. The internal duplication of UT-A2 and UT-B, as well as the double LP box urea transporter signature sequence were found in this amphibian urea transporter. When expressed in Xenopus oocytes, fUT induced a 10-fold increase in urea permeability, which was blocked by both phloretin and mercurial reagents. The fUT protein did not transport thiourea, but the fUT-mediated urea transport was strongly inhibited by this compound. Thus, this amphibian urea transporter displays transport characteristics in between those of UT-A2 and UT-B.
Amino Acid Sequence↗
Automated genome sequence analysis and annotation.
MOTIVATION: Large-scale genome projects generate a rapidly increasing number of sequences, most of them biochemically uncharacterized. Research in bioinformatics contributes to the development of methods for the computational characterization of these sequences. However, the installation and application of these methods require experience and are time consuming. RESULTS: We present here an automatic system for preliminary functional annotation of protein sequences that has been applied to the analysis of sets of sequences from complete genomes, both to refine overall performance and to make new discoveries comparable to those made by human experts. The GeneQuiz system includes a Web-based browser that allows examination of the evidence leading to an automatic annotation and offers additional information, views of the results, and links to biological databases that complement the automatic analysis. System structure and operating principles concerning the use of multiple sequence databases, underlying sequence analysis tools, lexical analyses of database annotations and decision criteria for functional assignments are detailed. The system makes automatic quality assessments of results based on prior experience with the underlying sequence analysis tools; overall error rates in functional assignment are estimated at 2.5-5% for cases annotated with highest reliability ('clear' cases). Sources of over-interpretation of results are discussed with proposals for improvement. A conservative definition for reporting 'new findings' that takes account of database maturity is presented along with examples of possible kinds of discoveries (new function, family and superfamily) made by the system. System performance in relation to sequence database coverage, database dynamics and database search methods is analysed, demonstrating the inherent advantages of an integrated automatic approach using multiple databases and search methods applied in an objective and repeatable manner. AVAILABILITY: The GeneQuiz system is publicly available for analysis of protein sequences through a Web server at http://www.sander.ebi.ac. uk/gqsrv/submit
Amino Acid Sequence↗
Assessment of the expression of candidate human plasma membrane phospholipid scramblase in Scott syndrome cells.
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Adult↗
Pulmonary sarcoidosis with cutaneous leishmaniasis.
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Adrenal Cortex Hormones↗
MView: a web-compatible database search or multiple alignment viewer.
UNLABELLED: MView is a tool for converting the results of a sequence database search into the form of a coloured multiple alignment of hits stacked against the query. Alternatively, an existing multiple alignment can be processed. In either case, the output is simply HTML, so the result is platform independent and does not require a separate application or applet to be loaded. AVAILABILITY: Free from http://www.sander.ebi.ac.uk/mview/ subject to copyright restrictions. CONTACT: brown@ebi.ac.uk
Computer Communication Networks↗
[Alternative treatment of dry eyes by punctum plugs--results in 60 patients].
PURPOSE: This is an analysis assessing clinical results after treatment with punctum Plugs in dry-eye patients stubborn to usual treatment. METHOD: We analysed retrospectively data from 60 patients treated by fitting in 1 to 4 silicone plugs in canaliculi. RESULTS: This study allowed to assess the advantages and disadvantages of the method, notably according to the selection criteria of patients and to the number of plugs inserted. CONCLUSION: This revertible treatment of the dry-eye syndrome is an efficient alternative, provided that patients are judiciousely selected.
Dry Eye Syndromes↗
Potassium transport in opossum kidney cells: effects of Na-selective and K-selective ionizable cryptands, and of valinomycin, FCCP and nystatin.
The effects of two ionizable cryptands, the Na-selective (221)C10 and the K-selective (222)C10, and of valinomycin, FCCP and nystatin on K+ fluxes in opossum kidney (OK) cells have been quantified. The Na,K-ATPase (ouabain-sensitive 86Rb influx) was stimulated by nystatin (> or = 20%), and inhibited by the other ionophores (50-80%), by barium (K-channel blocker) (61%) and by amiloride (Na entry blocker) (34%). The Vmax of the Na,K-ATPase phosphatase activity was unmodified by the ionophores, indicating the absence of direct interaction with the enzyme. The ATPi content was unmodified by the inhibitors and nystatin, but was lowered by (221)C10 (47%), (222)C10 (75%), valinomycin (72%) and FCCP (88%). Amiloride was found to partially remove the inhibition caused by (222)C10 (51%) and valinomycin (49%). Rb efflux was stimulated by nystatin (32%), unmodified by valinomycin, and was inhibited by (221)C10 (19%), (222)C10 (19%) and FCCP (10%). Barium (39%) and amiloride (32%) inhibited this efflux and, in their presence, the nystatin effect persisted, whereas that of the other ionophores vanished. At pH 6.4, the Rb efflux decreased by 14% of its value at pH 7.4, with no additional inhibition by cryptands. Cryptands are shown to inhibit the pH-sensitive K+-conductance, probably by inducing a K+-H+ exchange at the plasma membrane, and by uncoupling oxidative phosphorylation by inducing the entry of K+ and H+ (and possibly Ca2+) ions into the mitochondria.
Adenosine Triphosphate↗
GIF-DB, a WWW database on gene interactions involved in Drosophila melanogaster development.
GIF-DB (Gene Interactions in the Fly Database) is a new WWW database (http://www-biol.univ-mrs.fr/ approximately lgpd/GIFTS_home_page. html ) describing gene molecular interactions involved in the process of embryonic pattern formation in the flyDrosophila melanogaster. The detailed information is distributed in specific lines arranged into an EMBL- (or SWISS-PROT-) like format. GIF-DB achieves a high level of integration with other databases such as FlyBase, EMBL and SWISS-PROT through numerous hyperlinks. The original concept of interaction databases examplified by GIF-DB could be extended to other biological subjects and organisms so as to study gene regulatory networks in an evolutionary perspective.
Animals↗
Overexpression of ecto-5'-nucleotidase promotes P-glycoprotein expression in renal epithelial cells.
UNLABELLED: P-glycoprotein (P-gp), responsible for multidrug resistance (MDR) of tumoral cells, is also expressed in apical membranes of normal epithelial cells, among which are proximal tubular cells. Ecto-5'-nucleotidase (5'Nu), co-located with P-gp in renal brush border membranes, could be instrumental in the expression of MDR phenotype. P-gp activity [assessed by rhodamine 123 (R123) and [3H]vinblastine (3H-VBL) accumulation] was evaluated in MDCK cell lines in which human 5'Nu was expressed at different levels after retroviral infection: MDCK-5'NU/- cells with a low 5'Nu activity (Vmax < 2 pmol/mg protein/min) and MDCK-5'NU/+ cells, which expressed a high level of 5'Nu (Vmax 150 +/- 18.5 pmol/mg protein/min). MDCK-5'NU/- cells did not display functional expression of MDR. In MDCK-5'NU/+ cells, R123 and 3H-VBL accumulation was significantly lower than in MDCK-5'NU/- cells and was dramatically enhanced by P-gp inhibitors. This high P-gp activity in MDCK-5'NU/+ cells was confirmed by their resistance to colchicine (measured by LDH release and MTT assay) as compared to MDCK-5'NU/- and was accounted for by increased membrane expression of P-gp assessed by Western blot. Neither AMP nor adenosine, the substrate and the product of 5'Nu, respectively, affected P-gp activity. Inhibition of 5'Nu with alpha beta-methylene-adenosine-diphosphate (alpha beta MADP) or with a blocking anti-5'Nu antibody (1E9) did not blunt MDR expression in MDCK-5'NU/+ cells. Conversely, the anti-5'Nu antibody 5F/F9, which did not block the enzymatic site, induced a decrease of P-gp activity. Further, incubation of MDCK-5'NU/- cells with conditioned medium from MDCK-5'NU/+ cells, which contained significant amounts of released 5'Nu, induced MDR phenotype. IN CONCLUSION: (i) expression of ecto-5'Nu promotes multidrug resistance (MDR) activity in renal epithelial cells by enhancement of P-gp expression; (ii) this effect does not involve enzymatic activity of 5'Nu; (iii) supernatants of cells that express 5'Nu conferred P-gp activity to 5'Nu negative cells.
5'-Nucleotidase↗
Parathyroid hormone-induced calcium release from intracellular stores in a human kidney cell line in the absence of stimulation of cyclic adenosine 3',5'-monophosphate production.
PTH-induced mobilization of cytosolic Ca2+ in a human kidney cell line (HEK/W) occurring in the absence of cAMP stimulation was characterized and compared with that obtained in the same cells stably transfected by the PTH/PTH-related peptide (PTHrp) receptor (HEK/T). In both cell lines, N-terminal fragments of PTH and PTHrp induced a concentration-dependent biphasic stimulation in [Ca2+]i: a transient peak followed by a slow linear increase. These increases in [Ca2+]i were inhibited by the PTH antagonist [Nle(8,18),Tyr(34)]bPTH(3-34). The transient peaks were due to calcium release from intracellular stores, as they resisted quenching of calcium in the extracellular buffer and were abolished by prior emptying of intracellular stores. These peaks differed, however, both in latency period and in magnitude, in the two cell lines. The phospholipase C inhibitor U73122 inhibited the PTH-induced increase in [Ca2+]i in HEK/T cells, but not in HEK/W. Similarly, PTH-induced inositol phosphate (InsPs) production was detected in HEK/T but not in HEK/W cells. PTH-induced calcium release in HEK/W cells was inhibited by the simultaneous presence of ryanodine and U73122. Low level PTH/PTHrp receptor messenger RNA expression was demonstrated by ribonuclease protection in HEK/W cells, although no specific binding of [125I]PTHrP(1-34) could be detected. Amplification products for the PTH/PTHrp receptor 1, but no other isoforms, were detected by RT-PCR in HEK/W cells. As expected, HEK/T cells responded to PTH by a 500-fold stimulation in cAMP production and expressed large numbers of PTH/PTHrp receptors, as shown by [125I]PTHrp binding. These results demonstrate that the signal transduction pathways activated by PTH in HEK/W and HEK/T cells are different. Because the major difference in these cell lines is the number of PTH/PTHrp receptors expressed, these results suggest that the transduction of signals by the PTH/PTHrp receptor is controlled by receptor number in such a way that PTH stimulates an increase in intracellular calcium in the absence of stimulation of InsPs and cAMP production in cells expressing low levels of PTH/PTHrp receptor, but stimulates calcium release through an InsPs pathway and induces cAMP production in cells expressing large numbers of PTH/PTHrp receptors. The control of receptor number may be one of the mechanisms through which PTH effects are regulated.
Calcium↗
Tight connection between choline transport and phosphatidylcholine synthesis in MDCK cells.
In MDCK cells, choline uptake, the first step in the CDP-choline pathway for the biosynthesis of choline-containing phospholipids and osmolytes, occurs via both a transport system highly specific for choline and a non-specific pathway. The specific choline carrier is present at the apical domain of cells grown on dishes and is sodium-independent. Growing the cells on a permeant support results in the preferential localization of the specific choline carrier at the basolateral domain. To characterize the relationships between the choline uptake sites and the synthesis of phosphatidylcholine, MDCK cells were incubated with [Me-3H]choline and/or [Me-14C]choline for various times (up to 36 h) and the incorporation of label into phospholipids and water-soluble molecules was determined. For cells grown on dishes, addition of [Me-3H]choline at the apical side was followed by rapid incorporation of the label into the successive intermediates of the CDP-choline pathway. A comparable situation was found when growing the cells on a permeant support and adding the labelled choline at the basolateral side of the culture. On the other hand, radioactive choline added to the apical bath entered the CDP pathway to only a very low extent. Efflux experiments on cells loaded with choline from either the apical or the basolateral side demonstrate the existence of intracellular pools of choline. Addition of hemicholinium-3, an inhibitor of the specific choline carrier, markedly reduced the metabolism of choline taken up by the cells on the basolateral side but had no effect on that transported at the apical side. These results strongly suggest the existence of a tight connection between the entry of choline through the specific choline carrier and phosphatidylcholine synthesis in MDCK cells.
Animals↗
[Acute necrotizing enterocolitis during a prolonged treatment with neuroleptics].
A 32-year old man treated for several years with phenothiazine for chronic psychosis developed acute necrotizing colitis. The causal relationship with neuroleptics was reinforced by the absence of any other treatment and by histological findings including extensive mucosal necrosis without stenotic lesion and without mesenteric vessels alteration. The patient required emergency total colectomy and was discharged after 7 weeks of hospitalisation in the intensive care unit.
Acute Disease↗
Differential targeting to the plasma membrane of the Torpedo 15-kDa proteolipid expressed in oocytes.
Xenopus laevis oocytes were injected with poly(A)+ RNAs extracted from the electric lobes of Torpedo marmorata, which contain a homogeneous population of cholinergic neurons. These primed oocytes were able to synthesize acetylcholine and to release the neurotransmitter in a calcium-dependent manner. Fractionation of oocyte membranes as well as immunofluorescence experiments showed that the 15-kDa proteolipid, a common subunit of the vacuolar H(+)-ATPase and of a presynaptic membrane protein capable of calcium-dependent acetylcholine translocation called the mediatophore, was located at the oocyte plasma membrane. In contrast, oocytes injected with separate transcripts encoding the 15-kDa proteolipid and choline acetyltransferase were unable to release acetylcholine in spite of an equivalent acetylcholine content and a higher level of 15-kDa proteolipid expression. We observed by immunofluorescence that under these conditions, the 15-kDa proteolipid was expressed in granular cytoplasmic membranes, which were then identified as being Golgi vesicles by cell fractionation. The striking difference in the distribution of the 15-kDa proteolipid expressed in oocytes primed with Torpedo electric lobe mRNA as compared with that seen in oocytes injected with the cRNA alone suggests that another protein endogenous to the electric lobe may be implicated in the localization of the 15-kDa proteolipid at the plasma membrane. Moreover, such a targeting mechanism could contribute to the capacity of electric lobe mRNA-injected oocytes to release acetylcholine.
Acetylcholine↗
In vitro expression of the 15 kDa subunit of the mediatophore and functional reconstitution of acetylcholine release.
The mediatophore is a presynaptic oligomeric protein purified from the presynaptic plasma membrane of Torpedo synaptosomes on the basis of its ability to mediate a calcium-dependent acetylcholine release when solubilized and reconstituted into proteoliposomes. We investigated the ACh translocating activity of the 15 kDa proteolipid subunit of the mediatophore when expressed in Xenopus oocytes and reconstituted into proteoliposomes loaded with ACh. 1. A calcium-dependent ACh translocation was observed when oocytes were injected with polyadenylated mRNAs extracted from the electric lobe of the Torpedo brain or with an in vitro transcribed RNA encoding the 15 kDa subunit. 2. No release response was obtained when oocytes were non-injected or injected with Torpedo liver mRNAs. 3. This ACh translocation mechanism showed calcium-dependent activation and desensitisation and was inhibited by cetiedil, sharing these properties with the release of ACh observed at the synapse. 4. The ACh translocating activity of an N terminal deleted mediatophore 15 kDa subunit was strongly reduced and the deleted proteolipid appeared less sensitive to the action of cetiedil (alpha-cyclohexyl-alpha-(3-thienyl)-acetate of perhydroazepinyl-alpha-ethyl citrate monohydrate). 5. A significant ACh release response was observed when the 15 kDa proteolipid of the H(+)-ATPase from bovine chromaffin granules was tested. 6. These results show that this ACh translocating activity could be induced in the oocyte membranes by the expression of the 15 kDa subunit alone.
Acetylcholine↗
Stimulation by recombinant human growth hormone of growth and development of remaining bowel after subtotal ileojejunectomy in rats.
The impact of human recombinant growth hormone (GH) after massive small bowel resection was studied in 38 weaning female Wistar rats (65 +/- 5 days old; 193 +/- 26 g). Animals underwent a 80% small bowel resection, leaving in place similar lengths of jejunum and ileum. Animals were assigned to four groups: group A (n = 9), small bowel resection only; group B (n = 10), resection and treatment with 0.2 GH units (GHU) s.c. every other day; group C (n = 9), resection and 0.4 GHU; and group D (n = 10), laparotomy without intestinal resection. Twenty-eight days later, weight gain (percentage of initial weight) was 1 +/- 3 in group A, 12 +/- 8 in group B, 12 +/- 9 in group C, and 16 +/- 7 in group D; p < 0.001, groups B-D. Time to recover initial weight was 26.2 +/- 3.3 days in group A; 11.7 +/- 5.4 days in group B (p < 0.001); and 16 +/- 6.1 days in group C (p < 0.001 vs. A). The size of the intestinal remnant after the rats were killed was 1.3 +/- 0.6 cm (13 +/- 10% of initial length) in A; 5.15 +/- 2.4 cm (37 +/- 18%) in B (p < 0.01); 4.2 +/- 2.3 cm (33 +/- 20%) in C (p < 0.01 vs. A); and 4.4 +/- 3.5 cm (5.8 +/- 4.9%) in D (p < 0.001 vs A). Villus height and diameter, average number of mitosis per field, and muscular layer and wall thickness were greater in groups A, B, and C than in group D (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
A precise investigation on the TL behavior of LiF: Mg, Cu, P (GR-200A).
LiF: Mg, Cu, P is a TL material presenting unique dosimetric features. The TL sensitivity of this material was studied as a function of the annealing temperature and of the repeated cycles of annealing-irradiation-readout. A fading study was carried out over a period of 40 days with the purpose of checking the stability of the stored dosimetric information as a function of different annealing temperatures. A detailed statistical analysis of sets of data, obtained from repeated measurements on a group of ten dosimeters, is presented.
Fluorides↗