Biomedical subjects
Publications and source records attributed to P Raymond.
Platypnoea-orthodeoxia is a rare syndrome of postural hypoxaemia accompanied by breathlessness. The dyspnoea is induced by upright posture. The definitive diagnosis is usually made by contrast echocardiography. The treatment of choice is surgical closure of the intracardiac communication which may result in dramatic symptomatic improvement.
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To evaluate the relevance of a simple carbon balance model (Seginer et al., 1994, Scientia Horticulturae 60: 55-80) in source-limiting conditions, the dynamics of growth, respiration and carbohydrate reserves of tomato plants were observed in prolonged darkness. Four days prior to the experiments, plants were exposed to high or low light levels and CO(2) concentrations. The concentration of carbohydrates in vegetative organs was 30-50 % lower in plants that were exposed to low carbon assimilation conditions compared with those exposed to high carbon assimilation conditions. During prolonged darkness, plants with low carbohydrate reserves exhibited a lower whole-plant respiration rate, which decreased rapidly to almost zero after 24 h, and carbohydrate pools were almost exhausted in leaves, roots and flowers. In plants with high carbohydrate reserves, the whole-plant respiration rate was maintained for a longer period and carbohydrates remained available for at least 48 h in leaves and flowers. In contrast, fruits maintained fairly stable and identical concentrations of carbohydrates and the reduction in their rate of expansion was moderate irrespective of the pre-treatment carbon assimilation conditions. The time-course of asparagine and glutamine concentrations showed the occurrence of carbon stress in leaves and flowers. Estimation of source and sink activities indicated that even after low carbon assimilation, vegetative organs contained enough carbohydrates to support fruit growth provided their own growth stopped. The time of exhaustion of these carbohydrates corresponded grossly to the maintenance stage simulated by the model proposed by Seginer et al. (1994), thus validating the use of such a model for optimizing plant growth.
We report here the long term follow-up of the first french case, at our knowledge, of alcohol septal transcoronary ablation in March 1998 in Clinique Ambroise Paré in an 76 year-old woman. This patient was in NYHA III-IV functional class and at control outflow gradient was 100 mmHg. She was prior treated with high dosis of beta-blockers then DDD-pacemaker with no effects on symptoms. The technique used was the one described by Sigwart and al., with injection of 3 cm3 of ethyl alcohol in the first septal branch, after checking decrease of gradient during occlusion of septal branche with balloon angioplasty. After alcohol ablation, the gradient decreased immediately to 15 mmHg and disappeared at long-term follow up. Three years and half after the procedure, no complication occurred, the patient remains asymptomatic and the control echocardiography shows interventricular septal reduction of thickness from 22 to 12 mm.
Sequence analysis revealed phospholipase A2 (PLA2) motifs in capsid proteins of parvoviruses. Although PLA2 activity is not known to exist in viruses, putative PLA2s from divergent parvoviruses, human B19, porcine parvovirus, and insect GmDNV (densovirus from Galleria mellonella), can emulate catalytic properties of secreted PLA2. Mutations of critical amino acids strongly reduce both PLA2 activity and, proportionally, viral infectivity, but cell surface attachment, entry, and endocytosis by PLA2-deficient virions are not affected. PLA2 activity is critical for efficient transfer of the viral genome from late endosomes/lysosomes to the nucleus to initiate replication. These findings offer the prospect of developing PLA2 inhibitors as a new class of antiviral drugs against parvovirus infections and associated diseases.
The effects of mannose (Man) and glucose (Glc) on central metabolism, proteolysis, and expression of the root starvation-induced protease (RSIP; F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283-292) were investigated in maize (Zea mays L. cv DEA) root tips. Changes in metabolite concentrations (sugars, ester-phosphates, adenine nucleotides, and amino acids) were monitored using in vivo and in vitro (13)C- and (31)P-NMR spectroscopy, in parallel with the changes in respiration rates, protein contents, proteolytic activities, and RSIP amounts. The inhibition of proteolysis, the decrease in proteolytic activities, and the repression of RSIP expression triggered by Man, at concentrations usually used to study sugar signaling (2 and 10 mM), were found to be related to a drop of energy metabolism, primarily due to a Man-induced Pi sequestration. However, when supplied at low concentration (2 mM) and with the adequate phosphate concentration (30 mM), energy metabolism was restored and Man repressed proteolysis similarly to Glc, when provided at the same concentration. These results indicate that Man should be used with caution as a Glc analog to study signalization by sugars in plants because possible signaling effects may be hindered by Pi sequestration.
Cyclin-dependent kinases (CDKs) form a conserved superfamily of eukaryotic serine-threonine protein kinases whose activity requires the binding of a cyclin protein. CDKs are involved in many aspects of cell biology and notably in the regulation of the cell cycle. Three cDNAs encoding a C-type CDK, and a member of each B-type CDK subfamily, were isolated from tomato (Lycopsersicon esculentum Mill.) and designated Lyces;CDKC;1 (accession no. AJ294903), Lyces; CDKB1;1 (accession no. AJ297916), and Lyces;CDKB2;1 (accession no. AJ297917). The predicted amino acid sequences displayed the characteristic PITAIRE (CDKC), PPTALRE (CDKB1), and PPTTLRE (CDKB2) motives in the cyclin-binding domain, clearly identifying the type of CDK. The accumulation of all transcripts was associated preferentially with dividing tissues in developing tomato fruit and vegetative organs. In contrast to that of CDKA and CDKBs, the transcription pattern of Lyces;CDKC;1 was shown to be independent of hormone and sugar supply in tomato cell suspension cultures and excised roots. This observation, together with the absence of a patchy expression profile in in situ hybridization experiments, suggests a non-cell cycle regulation of Lyces;CDKC;1. Using a two-hybrid assay, we showed that Lyces;CDKC;1 did not interact with mitotic and G1 cyclins. The role of plant CDKCs in the regulation of cell division and differentiation is discussed with regard to the known function of their animal counterparts.
Early fruit development in tomato (Lycopersicon esculentum Mill.) proceeds from two distinct phases of growth, essentially cell division and cell expansion. In this study, we investigated the expression characteristics of the key cell-cycle regulators, mitotic and G1 cyclins, during tomato fruit development. We isolated six genes designated Lyces;CycA1;1, Lyces;CycA2;1, Lyces; CycA3;1, Lyces;CycB1:1 and Lyces;CycB2;1 encoding tomato mitotic cyclins, and Lyces;CycD3;1 encoding a G1 cyclin. The accumulation of transcripts was predominantly associated with mitotically active organs: developing fruits, young leaves and roots, and with cell-suspension cultures under appropriate sugar feeding conditions. Transcripts for all the isolated cyclin genes could be detected in the epidermis and pericarp of fruit tissues where some slight mitotic activity still remained at the onset of ripening. However, Lyces;CycA3;1 and Lyces;CycD3;1 were expressed in the gel tissue at the late stage of fruit development, suggesting that they are involved in endoreduplication of the differentiated and giant cells of the gel tissue.
Following fruit set, the early development of tomato (Lycopersicon esculentum Mill.) fruit comprises two distinct phases: a cell division phase and a consecutive phase of cell expansion until the onset of ripening. In this study, we analyzed cytological and molecular changes characterizing these early phases of tomato fruit development. First we investigated the spatial and temporal regulation of the mitotic activity during fruit development. The DNA content of isolated nuclei from the different fruit tissues was determined by flow cytometry analysis. The results confirm the data of mitotic activity measurements and show that cell differentiation, leading to expanded cells, is characterized by endoreduplication. Second, we isolated two cDNAs, named Lyces;CDKA1 (accession no. Y17225) and Lyces;CDKA2 (accession no. Y17226), encoding tomato homologs of the cyclin-dependent kinase (CDK) p34(cdc2). Tomato CDKA gene expression was followed at both the transcriptional and translational levels during fruit development. The transcripts for Lyces;CDKA1 and Lyces;CDKA2 and the corresponding CDKA proteins are predominantly accumulated during the phase of cell division between anthesis and 5 d post anthesis (DPA). In whole fruits, the maximum CDK activity was obtained between 5 and 10 DPA. The determination of the kinase activity using protein extracts from the different fruit tissues was in agreement with mitotic activity analysis. It showed the particular disappearance of the activity in the gel tissue as early as 15 DPA. The overall data of CDK activity measurements suggest a strong post-translational regulation of CDK at the temporal and spatial levels during early tomato fruit development.
The unidirectional steady state reaction rates of several enzymes and metabolic fluxes of distinct processes were measured simultaneously in hypoxic maize root tips using two-dimensional phosphorus NMR exchange spectroscopy. A single spectrum monitors ATP synthesis and hydrolysis as well as the activities of four enzymes involved in key pathways of central metabolism: UDP-glucose pyrophosphorylase, phosphoglucomutase, hexose-phosphate isomerase, and enolase. The corresponding unidirectional reaction rates and net metabolic fluxes were calculated from spectral intensities. This method provides a unique picture, at enzyme resolution, of how metabolism reacts in a concerted fashion to changes in external parameters such as temperature and oxygen concentration. By increasing hypoxia via an increase in temperature, we measured the expected increase in glycolysis through enolase activity while total ATP synthesis settled. At the same time, we observed a net flux through phosphoglucomutase and UDP-glucose pyrophosphorylase toward carbohydrate synthesis. This result is discussed in relation to the current hypothesis on the turnover of cell walls and sucrose. This reaction also produces a net flux of pyrophosphate, which is needed by pyrophosphate:fructose-6-phosphate 1-phosphotransferase to work as a glycolytic enzyme.
Three-week-old maize (Zea mays L.) plants were submitted to light/dark cycles and to prolonged darkness to investigate the occurrence of sugar-limitation effects in different parts of the whole plant. Soluble sugars fluctuated with light/dark cycles and dropped sharply during extended darkness. Significant decreases in protein level were observed after prolonged darkness in mature roots, root tips, and young leaves. Glutamine and asparagine (Asn) changed in opposite ways, with Asn increasing in the dark. After prolonged darkness the increase in Asn accounted for most of the nitrogen released by protein breakdown. Using polyclonal antibodies against a vacuolar root protease previously described (F. James, R. Brouquisse, C. Suire, A. Pradet, P. Raymond [1996] Biochem J 320: 283-292) or the 20S proteasome, we showed that the increase in proteolytic activities was related to an enrichment of roots in the vacuolar protease, with no change in the amount of 20S proteasome in either roots or leaves. Our results show that no significant net proteolysis is induced in any part of the plant during normal light/dark cycles, although changes in metabolism and growth appear soon after the beginning of the dark period, and starvation-related proteolysis probably appears in prolonged darkness earlier in sink than in mature tissues.
There are conflicting results in the literature concerning the effect of gavage vehicle, corn oil (CO) versus aqueous suspension, on the toxicity of haloalkanes. The purpose of our study was to assess the influence of oral dosing vehicle on the acute hepatotoxicity of CCl4 and nephrotoxicity of CHCl3. Male Sprague-Dawley rats, fed ad libitum, were treated (po) with single doses of CCl4 or CHCl3 using corn oil (CO), or an aqueous preparation (5%) of Emulphor (EL620) or Tween-85 (Tw-85) as vehicle (10 ml/kg). Rats were killed 48 h after treatment. Blood was collected for plasma alanine aminotransferase (ALT) determination and renal cortical slices were prepared for p-aminohippuric acid (PAH) incorporation. The comparison, between gavage vehicles, of the slopes and ED50 of the dose-response curves, although not significantly different, indicated clear trends for enhanced potency with CO for CHCl3 nephrotoxicity but not for CCl4 hepatotoxicity. However, ALT values, a measure of the severity of effect for CCl4, also indicated that CO, when compared to EL620 and Tw-85, tended to enhance CCl4 hepatotoxicity at low toxicity incidence. Furthermore, CO clearly enhanced the severity of effect for CHCl3 nephrotoxicity, as measured by the slice-to-medium PAH ratios, at high dosage. The greater severity of the lesion produced by exposure to these chemicals, when administered in CO, is consistent with the trends observed for their potency (dose-response curves). Our results agree with an increased toxicity of haloalkanes by the gavage vehicle CO reported in the literature. Thus, CO should be considered a potential confounder in hepato- and nephrotoxicity assays.
The effect of chronic treatment with zidovudine (AZT) on the inflammatory response was examined in the rat. AZT was administered orally for 36 days. On day 35, inflammation was induced by hindpaw injection of 1% carrageenan lambda. Paw edema over a 24-hour period was used as a marker of the local inflammatory reaction. On day 36, quantification of immunoreactive T-kininogen and alpha 1-inhibitor-3 in liver and serum was used to assess the systemic inflammatory response. Albumin was selected as a protein whose concentration is modified only slightly or not at all during the acute-phase response. Animals treated with AZT transiently exhibited significantly greater (18%) paw edema 3 hours after carrageenan injection. AZT treatment alone induced a 1.8-fold increase in serum T-kininogen concentration, but it had no effect on albumin and alpha 1-inhibitor-3. In rats with inflamed paws, AZT administration led to a significant increase in liver (3.4-fold) and serum (1.8-fold) immunoreactive T-kininogen content. Dot blot analysis of total RNA isolated from liver correlated with the protein measurements. Our results indicate that chronic treatment with AZT potentiates the nonspecific local and the systemic inflammatory responses in the rat.
Two different cDNAs encoding lactate dehydrogenase (LDH) were isolated from a library of hypoxically treated tomato roots and sequenced. The use of gene-specific probes on northern blots showed that Ldh2 mRNA was predominant in well-oxygenated roots and levels remained stable upon oxygen deficit; in contrast, Ldh1 mRNA accumulated to high levels within 2 h of hypoxia or anoxia. Immunoblot analyses of native gels using a polyclonal antiserum raised against an LDH1 fusion protein indicated that LDH2 homotetramer was the major isoform present in aerobic roots. Levels of both LDH1 and LDH2 subunits increased during an 18 h hypoxic treatment, together with a 5-fold rise in activity. These results suggest that the regulation of ldh1 expression is primarily at the transcriptional level while that of ldh2 is post-transcriptional. Increases in Ldh1 mRNA and LDH activity were not correlated with lactic acid production, which was maximal at the onset of anoxia in unacclimated roots and then declined. Taken together, our results indicate that LDH2 present in aerobic roots is principally responsible for lactic acid production occurring transiently upon imposition of anoxia. Possible physiological roles for LDH1 are discussed.
The role of angiotensin-converting enzyme (ACE) in the metabolism of bradykinin (BK) has been studied in several tissues. However, and contrary to angiotensin I, the metabolism of BK at the cardiac level has not been investigated. In this study, we define the participation of ACE in the carboxy-terminal degradation of BK in heart membranes of the dog, human, rabbit, and rat. The calculation of the kinetic parameters characterizing the metabolism of BK and the generated des-Arg9-BK can be summarized as follows: the half-life (t1/2) of BK [dog (218 +/- 32 s) > human (143 +/- 9 s) = rat (150 +/- 4 s) > rabbit (22 +/- 2 s)] and of des-Arg9-BK [dog (1,042 +/- 40 s) > human (891 +/- 87 s) > rat (621 +/- 65 s) > rabbit (89 +/- 8 s)] both showed significant differences according to species. Enalaprilat, an ACE inhibitor, significantly prevented the rapid degradation of BK and des-Arg9-BK in all species studied, whereas retrothiorphan, a neutral endopeptidase inhibitor, and losartan, an angiotensin II type I receptor antagonist, did not affect this metabolism. The relative importance of ACE in the cardiac metabolism of BK was species related: dog (68.4 +/- 3.2%) = human (72.2 +/- 2.0%) > rabbit (47.7 +/- 5.0%) = rat (45.3 +/- 3.9%). ACE participation in the metabolism of des-Arg9-BK was as follows: rabbit (57.0 +/- 4.0%) > dog (39.9 +/- 8.8%) = human (25.4 +/- 5.5%) = rat (36.0 +/- 7.0%). The participation of cardiac kininase I (carboxypeptidase M) in the transformation of BK into des-Arg9-BK was minor: human (2.6 +/- 0.1%) > dog (0.9 +/- 0.1%) = rabbit (1.0 +/- 0.1%) = rat (1.0 +/- 0.1%). These results demonstrate that ACE is the major BK-degrading enzyme in cardiac membranes. However, the metabolism of exogenous BK by heart membranes is species dependent. Our observations could explain some discrepancies regarding the contribution of kinins in the cardioprotective effects of ACE inhibitors.
An endopeptidase (designated RSIP, for root-starvation-induced protease) was purified to homogeneity from glucose-starved maize roots. The molecular mass of the enzyme was 59 kDa by SDS/PAGE under reducing conditions and 62 kDa by gel filtration on a Sephacryl S-200 column. The isoelectric point of RSIP was 4.55. The purified enzyme was stable, with no auto-proteolytic activity. The enzyme activity was strongly inhibited by proteinaceous trypsin inhibitors, di-isopropylfluorophosphate, 3,4-dichloroisocoumarin and PMSF, suggesting that the enzyme is a serine protease. The maximum proteolytic activity against different protein substrates occurred at pH 6.5. With the exception of succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin, no hydrolysis was detected with synthetic tryptic, chymotryptic or peptidylglutamate substrates. The determination of the cleavage sites in the oxidized B-Chain of insulin showed specificity for hydrophobic residues at the P2 and P3 positions, indicating that RSIP is distinct from other previously characterized maize endopeptidases. Both subcellular fractionation and immuno-detection in situ indicated that RSIP is localized in the vacuole of the root cells. RSIP is the first vacuolar serine endopeptidase to be identified. Glucose starvation induced RSIP: after 4 days of starvation, RSIP was estimated to constitute 80% of total endopeptidase activity in the root tip. These results suggest that RSIP is implicated in vacuolar autophagic processes triggered by carbon limitation.
Previous results in male Sprague-Dawley rats indicate that acetone (A), methyl ethyl ketone (MEK), and methyl isobutyl ketone (MiBK) pretreatments (3 d, p.o.) at a dosage of 6.8 mmol/kg potentiate CCl4 hepatotoxicity. The potentiation potency profile observed was MiBK > A > MEK. In the present study, male Sprague-Dawley rats were treated for 3 d with 6.8 mmol/kg (p.o.) of A, MEK, or MiBK using Emulphor as vehicle (10 ml/kg). Rats were either killed 18 h after the last pretreatment or treated with CCl4 (prepared in corn oil) and then killed 48 h later. Livers were perfused; purified plasma membrane (PM), sinusoidal (SM) and basal canalicular membrane (BCM) fractions were prepared. Membrane fluidity was monitored by fluorescence polarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). The following membrane enzymes were measured to monitor membrane purity and treatment effects: 5'-nucleotidase (5N), leucine aminopeptidase (LAP), and alkaline phosphatase (AP). Our results suggest that CCl4 modifies membrane integrity as indicated by a decrease in liver membrane 5N, LAP, and AP activity. CCl4 also increased the fluidity of the lipid and protein portions of the liver membranes as measured by the DPH and TMA-DPH fluorescence probes, respectively. Of the three ketones, only A altered CCl4 effects on plasma membrane enzymes and decreased BCM fluidity. The data only partially support increased susceptibility of liver membranes by ketone pretreatment as a factor implicated in the mechanism of potentiation of CCl4-induced hepatotoxicity.
Kinin antagonists at B1 and B2 receptors were examined on liver and serum concentrations of immunoreactive T-kininogen and its gene expression in a rat model of carrageenan-induced hindpaw edema. Whereas the B2 antagonist, HOE140, dose-dependently inhibited the paw edema induced by intraplantar injection of carrageenan, the B1 receptor agonist [Sar(D-Phe8)des-Arg9]BK and antagonist [Lys(Leu8)des-Arg9]BK were ineffective. On its own, HOE140 (3.25 x 10(-7) mol/ paw, intraplantar) had no effect on liver and serum T-kininogen levels but it significantly enhanced liver T-kininogen concentrations in rats pretreated with carrageenan at 8 and 24 h postinjection. In the liver, the most pronounced effect was seen at 24 h (treated 248 +/- 7 micrograms/g vs. untreated 113 +/- 9 micrograms/g). The same dose of HOE140 increased serum T-kininogen from 1255 +/- 57 to 1696 +/- 83 micrograms/ml at 24 h. HOE140 did not affect tissue albumin content during the same period. Transcript measurements revealed that the steady-state level of liver T2-kininogen mRNA was specifically increased by HOE140 during inflammation. In carrageenan-treated rats, the B1 antagonist [Lys(Leu8)des-Arg9]BK also significantly increased liver T-kininogen at 24 h. The present results support a role for B2 kinin receptors in the early phase of acute phase protein synthesis and of both B2 and B1 receptors in the late phase (24 h). Hence, systemic effects of kinins should be taken into account in the pharmacology and physiopathology of B1 and B2 kinin receptors in inflammation.