Biomedical subjects
Publications and source records attributed to J P Richard.
Mercury pulmonary embolism following intentional or accidental injection of metallic mercury are uncommon. Generally, there are few clinical signs (acute pneumopathy in 50% of the cases). Chest x-ray shows multiple and bilateral point opacities with a metallic density. We present two cases of mercury pulmonary embolism after intentional intravenous mercury injection (one attempted suicide and one HIV+ drug addict).
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Removal of eggs from the nests of incubating birds or substitution of eggs for chicks disrupts incubation behavior and induces changes in the secretion of prolactin and luteinizing hormone (LH). The aim of the present study was to determine how different stimuli, such as physical contact with eggs and tactile, visual, and/or auditory cues from chicks, interact to control the transition between incubation and brooding and to induce changes in prolactin and LH plasma concentrations. Physical contact with chicks, in the presence or absence of eggs, induced brooding behavior and an immediate fall in plasma prolactin concentration and a gradual increase in LH concentration. Vocalizations, particularly clucking and food calls, increased rapidly while incubation and nest attachment disappeared slowly. No change in plasma prolactin or LH concentration was observed in incubating hens which could hear and see or only hear chicks. These incubating hens showed no interest in chicks and continued to incubate persistently. To conclude, tactile stimuli, alone or in combination with visual and/or auditory stimuli from newly hatched chicks, are the only cues that induce the transition from incubation to brooding and the associated decrease in prolactin secretion and increase in LH secretion.
Studies in vertebrates have shown that hormones can influence circadian rhythms of behavior. We investigated whether testosterone could induce rhythmicity in arrhythmic Japanese quail, kept in DD. The animals used were 3 1/2-week-old castrated males from a line of quail selected for the lack of the circadian rhythm of feeding activity. After 3 weeks in DD, 8 birds were implanted with an empty implant and 16 others with a testosterone implant. Two weeks later, the operation was repeated. After implantation, we noticed that 15 out of 16 testosterone-treated birds showed a circadian rhythm of feeding activity, in contrast to the control birds, which remained arrhythmic. The clarity of this rhythm increased significantly after each implantation. A positive correlation was found between the indexes of clarity of the rhythm (autocorrelation coefficient ratio and area of the peak of spectrum) and the plasma testosterone level. The period of the induced free-running rhythm was identical to the specific value of the endogenous circadian rhythm in immature quail. The circadian period showed a significant lengthening with the second implantation. This lengthening looks like the variation previously observed in maturing rhythmic or implanted quail. So, it would appear that testosterone can act on rhythmicity on at least two levels: by inducing the circadian rhythm and increasing its clarity and by modulating its period. To explain these results, several hypotheses can be considered. First, the observed arrhythmy may be the consequence of an internal desynchronization of oscillators, responsible for generating the circadian rhythm of feeding activity, and testosterone could play a role in the coupling of these oscillators. Alternatively, we suggest that testosterone could act on the transcription of genes implicated in the control of the rhythmicity or may regulate by rapid signals the cellular rhythmic activity. The possible functional values of the enhancing of circadian rhythmicity by testosterone at different stages of the bird's life were discussed.
In conditions of constant darkness, interindividual variability in the clarity of circadian rhythmicity was observed in sexually immature young quail, with birds classified as more or less rhythmic or arrhythmic. The relative clarity of this circadian rhythm was observed on the actograms by measurement of the autocorrelation coefficient ratio over 12 cycles. Autocorrelation coefficients were calculated from sequential series of total activity over 12-minute periods. Crosses of selected phenotypes with different clarities of rhythmicity were conducted in order to study the possibility of selection of this characteristic. From a random population (N = 42, twice), pairs of the most rhythmic birds (3 families), and pairs of arrhythmic birds (4 families) were reared. Autocorrelation coefficient ratios of F1 birds from rhythmic families (N = 54) were greater than those of F1 birds (N = 48) from arrhythmic families (t-test, p < .0001). These ratios in offspring were significantly correlated with that of the mean parent of each clutch of siblings (N = 102, r = .35, p = .0003). This result was maintained in a second generation (F2) of birds, for which significant differences in expressed rhythmicity were observed. That is, autocorrelation coefficient ratios of F2 birds from two rhythmic families (N = 30) were greater than those of F2 birds from arrhythmic families (N = 20) (t-test, p = .039). Comparison of F2 outbred and inbred birds from rhythmic pairs showed greater values of autocorrelation coefficient ratios in the case of inbred birds (N = 16) than for outbred birds (N = 30; t-test, p = .036). There was no difference between outbred (N = 20) and inbred birds (N = 15) from arrhythmic pairs. Therefore, selection of a rhythmic strain seems possible, whereas crosses between two arrhythmic birds may also give rise to rhythmic birds. Comparisons between rhythmic birds of different families did not show differences in the free-running period of the circadian rhythm, which is true also for rhythmic birds bred from two arrhythmic parents. Therefore, our selection procedure did not seem to be based on the characteristics of the pacemaker itself, but rather on a downstream event. Although the parents were not selected on the basis of quantity of activity per cycle or on the duration of the active phase, significant differences among the offspring of different families were shown.
Experiments are reported to determine the role of Glu-461 in the beta-D-galactopyranosyl group transfer reaction catalyzed by beta-galactosidase. E461G beta-galactosidase catalyzes the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside through a galactosyl-enzyme intermediate that shows a high reactivity toward the anionic nucleophile azide ion, but no detectable reactivity toward the neutral nucleophile trifluoroethanol. By contrast, the galactosylated wild type enzyme is reactive toward trifluoroethanol but not anions. The change in specificity observed for the E461G mutant can be rationalized by a mechanism in which Glu-461 participates in general acid-base catalysis at the leaving group/nucleophile. The observed low activity of E461G beta-galactosidase for hydrolysis of 2,2,2-trifluoroethyl beta-D-galactopyranoside is due entirely to a wild type enzyme contaminant in our preparation of the mutant enzyme, and the mutant enzyme itself has essentially no catalytic activity for cleavage of this substrate. The substitution of glutamate at position 461 by glycine leads to a more than 500 000-fold reduction in the rate constant for enzymatic cleavage of the glycosidic bond to the strongly basic trifluoroethoxide leaving group (pKa = 12.4), but to a smaller 1300-fold reduction in the rate constant for cleavage of the bond to the more weakly basic 4-nitrophenoxide leaving group (pKa = 7.1). This corresponds to a more than 3.5 kcal/mol greater stabilization by Glu-461 of the transition state for the reaction of the substrate with the more basic trifluoroethoxide leaving group. These data are consistent with the conclusion that Glu-461 provides general acid catalysis of leaving group departure, which is most effective for cleavage of the relatively strong bonds to basic alkoxide leaving groups.
Second-order rate constants for transfer of the beta-D-galactopyranosyl group from the galactosyl-enzyme intermediates of the galactosyl transfer reactions catalyzed by E461G and E461Q beta-galactosidases to anionic nucleophiles have been determined. The second-order rate constant for reaction of the galactosylated E461G enzyme with azide ion is 4900 M-1 s-1. By contrast, there is no detectable reaction of the galactosylated wild type enzyme with azide ion (Richard et al., 1995b), and the E461G mutation leads to a large decrease in the second-order rate constant kcat/Km for catalysis of cleavage of beta-D-galactopyranosyl azide, which is the microscopic reverse of the reaction of azide ion with the galactosyl-enzyme intermediate. These data show that the E461G mutation causes a more than 8000-fold increase in the equilibrium constant for transfer of the beta-D-galactopyranosyl group from beta-galactosidase to azide ion. We propose that this change represents the requirement for the coupling of galactosyl transfer from the native enzyme to the thermodynamically unfavorable protonation of the carboxylate group of Glu-461, but the expression of the full chemical affinity of azide ion for galactosyl transfer from the mutant enzyme which lacks this ionizable side chain at position 461. The reactions of acetate, butyrate and methoxyacetate ions with the galactosylated E461G enzyme and of acetate with the galactosylated E461Q enzyme give both the corresponding beta-galactopyranosyl derivatives and D-galactose, and the formation of the latter represents formal catalysis of the reaction of water with the galactosylated enzyme. However, the reaction of formate ion with the galactosylated E461G enzyme gives only D-galactose. These results suggest that carboxylate anions can take the place of the excised propionate side chain of Glu-461 to provide general base catalysis of the reaction of water with the galactosyl-enzyme intermediates. The relative reactivity of anionic nucleophiles toward the covalent galactosyl-enzyme intermediate of the reactions catalyzed by the E461G enzyme is similar to that observed for partitioning of stable carbocations in water. This suggests that replacement of the anionic side chain of Glu-461 by a hydrogen exposes an enzyme-stabilized oxocarbenium ion intermediate to reaction with external nucleophilic reagents.
Seven substituted alkyl beta-D-galactopyranosides 1-OR have been prepared and shown to be fair to excellent substrates for hydrolysis catalyzed by beta-galactosidase (Escherichia coli, lac Z). Brønsted parameters of (beta 1g)k3 = -0.49 +/- 0.13 and (beta 1g)kcat/Km = -0.75 +/- 0.14, respectively, were determined at pH 8.6 for k3 (s-1), the first-order rate constant for cleavage of enzyme-bound 1-OR, and kcat/Km (M-1 s-1), the second-order rate constant for reaction of the free enzyme and 1-OR. There is a weak correlation between log Km and the pKa of the alkyl alcohol leaving group, which is attributed to stabilization of the Michaelis complex by hydrophobic interactions between the enzyme and electron-withdrawing halogen substituents at the alkoxy leaving group. These binding interactions are probably both productive and expressed in the value of kcat/Km and nonproductive and expressed in the value of k3. The negative values of beta 1g are inconsistent with enzymatic catalysis of endocyclic cleavage of the glycosidic bond. The values of beta 1g for enzyme-catalyzed cleavage of alkyl beta-D-galactopyranosides lie between those observed for the spontaneous (beta 1g approximately -1.25) and specific-acid-catalyzed (beta 1g approximately 0) cleavage of acetals, and these pathways are therefore excluded for the enzyme-catalyzed reaction. Removal of the metal cofactor Mg2+ from the enzyme causes a approximately 0.2 unit decrease in (beta 1g)k3 for beta-galactosidase-catalyzed cleavage of 1-OR. The interpretation of this change in beta 1g is unclear. The Brønsted coefficients for the beta-galactosidase-catalyzed reaction are consistent with participation by an essential catalytic residue in concerted general-acid catalysis of cleavage of the glycosidic bond of 1-OR and/or stabilization of developing negative charge at the alkoxy oxygen by interaction with the magnesium ion cofactor.
Velocities for the synthesis of beta-D-galactopyranosyl derivatives by transfer of the galactosyl group from beta-galactosidase to seven alkyl alcohols, glucose, and azide ion have been determined as the difference in the velocities for beta-galactosidase-catalyzed cleavage of 4-nitrophenyl beta-D-galactopyranoside to give 4-nitrophenoxide anion (v PNP) and hydrolysis of this compound to give D-galactose (v Gal). Rate constant ratios kROH/ks (M-1) for partitioning of the galactosylated enzyme between reaction with alkyl alcohols and solvent determined by this method are in good agreement with values of kROH/ks (M-1) determined by analysis of alcohol inhibition of enzyme-catalyzed hydrolysis of the corresponding alkyl beta-D-galactopyranosides. Absolute rate constants kROH (M-1 s-1) for reaction of alkyl alcohols with the galactosylated enzyme intermediate were calculated from the corresponding rate constant ratio kROH/ks (M-1) and ks = 710 s-1. A Brønsted parameter of (beta nuc) ROH = -0.19 +/- 0.10 was determined from the second-order rate constants for the reactions of alcohols with the galactosylated enzyme. The large difference between (beta 1g)kcat/Km = -0.75 +/- 0.14 for cleavage of alkyl beta-D-galactopyranosides to form the galactosylated enzyme and (beta nuc)ROH = -0.19 for the reverse synthesis reaction requires that the equilibrium constants for galactosyl group transfer from alkyl beta-D-galactopyranosides to the enzyme increase sharply with decreasing pKa of the alkyl alcohol leaving group. These data give beta eq = -0.56 +/- 0.05 for the reaction of alkyl beta-D-galactopyranosides with ethanol to form ethyl beta-D-galactopyranoside and alkyl alcohol. Several effects that lead to this increased ease of cleavage of alkyl beta-D-galactopyranosides with decreasing basicity of the alkoxy group are discussed. A second-order rate constant of kGlc = 1.2 x 10(4) M-1 s-1 was determined for reaction of glucose with the galactosylated enzyme. The relatively low reactivity of glucose is surprising, because an earlier observation that the galactosylated enzyme complex generated by the cleavage of lactose undergoes release of glucose and synthesis of allolactose at nearly equal rates suggests that the binding of glucose to the galactosylated enzyme should be partly irreversible and that it takes place near the encounter-controlled limit. The data suggest a significant stabilization of nonproductive complexes formed by binding of glucose to the galactosylated enzyme. beta-Galactosidase catalyzes the hydrolysis of beta-D-galactopyranosyl azide, but not the synthesis of this compound by reaction of azide ion with the galactosylated enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)
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The present study examines a putative effect of exogenous melatonin on the circadian organization of the mink. Two approaches were used to determine first whether entrainment of free-running rhythms of locomotor activity in constant darkness can be obtained by daily melatonin injections, thus demonstrating a control of melatonin on the clock generating circadian rhythms, the suprachiasmatic nucleus of the hypothalamus. Entrainment was never obtained in the 8 vehicle-injected females and 7 out of the 8 melatonin injected-ones. In 3 females free-running in constant darkness, a phase advance followed by a few days of transient effect was observed when melatonin injections coincided with the onset of activity. However, the comparison of the regression of the daily activity onset related to successive days by covariance analysis revealed that true entrainment was effective in only 1 female. Second, we examined the distribution of melatonin binding sites within the brain of juvenile and adult mink using an in vitro autoradiographic procedure with [125I]2-iodomelatonin. No binding sites were observed in the suprachiasmatic nucleus of any of the animals. However, all animals displayed a high density of melatonin binding sites in the pars tuberalis of the pituitary. The relation between a modulatory control of melatonin on the circadian clock and the presence and density of melatonin binding sites in the clock is discussed. In mink, melatonin does not seem to act as an internal Zeitgeber.
Transurethral prostatic resection using a 1.5% glycine solution causes a well known clinical and metabolic syndrome called TURP-syndrome. Recent development of percutaneous renal surgery is responsible of several similar accidents. In a prospective study of 150 patients (mean age: 35 +/- 10) subjected to a percutaneous nephrolithotomy, the natremia and the amino acid content of the plasma were measured preoperatively and immediately postoperatively by chromatography. The study shows that there is a post-nephrolithotomy syndrome in two per cent. This syndrome contains a hemodilution with hyponatremia and reabsorption of irrigation fluid. Glycolemia, serinemia and threoninemia increase significantly. These modifications have a good correlation between them except for the natremia. Variability of results in this study and in the literature is explained by difficulty and duration of surgery, volume of glycol used, increasing intrarenal pressures and sudden opening of vessels peroperatively. The gravity of post-nephrolithotomy syndrome requires to change the irrigate solute and use normal saline solution when it is possible.
Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)
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The efficacy and tolerance of flumazenil were assessed in a double-blind randomized multicentre trial on 120 ASA I or II patients aged 40.3 +/- 13.9 years. They were anaesthetized with flunitrazepam (25.1 +/- 10.5 micrograms.kg-1.h-1), fentanyl (4.4 +/- 1.9 micrograms.kg-1.h-1) and either vecuronium or pancuronium; residual neuromuscular blockade was antagonized at the end of surgery. 61 patients received flumazenil and 59 a placebo. Sedation comprehension and temporo-spatial orientation were scored at 0, 5, 15, 30, 60, 120 and 240 min after the administration of flumazenil or placebo. Local and general tolerances were evaluated 1 h and 24 h after administration. At the 24th h, the observer's assessment of consciousness, pain, respiration, coughing and vomiting were noted, as well as his identification, or not, of flumazenil or the placebo and their efficacy. Both groups were statistically homogeneous and comparable. Significant and marked efficacy was noted between the 5th and 30th min. There was no difference, at 24 h, between the flumazenil and placebo groups. In most cases, flumazenil was identified by the observer and its efficacy felt to be excellent. No major untoward effect of flumazenil was noted; however a mild and short lasting anxiety occurred in three patients. Tolerance was deemed to be excellent.
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The authors describe a technique of constant flow perfusion of several anaesthetic drugs, using a simple formula to adjust the perfusion rate to the weight of the patients. Calculation of dilutions is easy: the drug concentration is equal to ten times the rate, per hour and per kg, in practical cases. The flow rate is then a tenth of the patient's weight. This method is easy to apply to several drugs, of which pharmacokinetic parameters justify this administration mode: alfentanil, etomidate, vecuronium bromide... but clinical and instrumental observations should be taken into account for the adjustment of the flow.
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