Browsing by Author "VERGARA, M"

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    CHEMICAL MODIFICATION OF ESSENTIAL ARGININES IN PIG-LIVER PHOSPHOMEVALONATE KINASE
    (1982) VERGARA, M; ALVEAR, M; CARDEMIL, E; JABALQUINTO, AM; EYZAGUIRRE, J
    Phosphomevalonate [MVAP] kinase, an enzyme of the polyisoprenoid biosynthesis pathway, catalyzes the transfer of phosphate from ATP to MVAP, with the formation of pyrophosphomevalonate and ADP. The pig liver enzyme, a monomer of MW 22,000, possesses 1 cysteinyl residue, which is essential for catalysis. By means of chemical modification of a partially purified preparation, the participation of arginine residues in the enzyme active site was studied. Butanedione and phenylglyoxal were chosen as group-specific reagents. The kinetics of inactivation by both reagents is rather complex, suggesting that several arginine residues, directly or indirectly related to the active site, are being modified. Both substrates, MVAP and Mg-ATP, protect against inactivation but to a different extent, depending on the modifier used. With butanedione, almost total protection is achieved with Mg-ATP. Better protection with MVAP is observed for the modification with phenylglyoxal, but with this reagent, Mg-ATP protects strongly only at very high concentrations. A reversible inactivation mechanism is followed with butanedione, while for phenylglyoxal this mechanism appears to be irreversible, in agreement with findings by other authors. MVAP kinase probably presents 2 or more arginyl residues in or near its active site, one of them being involved in the binding of Mg-ATP, and at least another located in the neighborhood of the MVAP binding site. A more precise determination of the number of essential residues requires its measurement by chemical methods, utilizing a homogeneous enzyme preparation.
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    CIRCADIAN VARIATION OF RECTAL TEMPERATURE IN NEWBORN SHEEP
    (1987) RECABARREN, SE; VERGARA, M; LLANOS, AJ; SERONFERRE, M
    In adult animals, body temperature shows a 24 h rhythm that is endogenously generated. We examined the existence of 24 h rhythms of temperature in 10 newborn sheep. Four newborns, aged 5 to 28 days were kept with their mothers under nycthemeral conditions, and the remaining 6 lambs, aged 21 to 43 days, were kept with their mothers in constant light from day 3 after birth. Experiments were performed with both groups of lambs in the laboratory. Additional experiments were performed with the 6 lambs kept under constant light while they were in the pen with their mothers to rule out artifacts due to manipulation or artificial feeding. During the experiments done in the laboratory, the lambs were kept blindfolded in a canvas sling and were fed baby formula approximately every four hours (lambs kept under nycthemeral conditions) or every hour (constant light lambs). Lights were on in the room during the whole experiment. Temperature in the room was maintained at 18 .+-. 0.1.degree. C (mean .+-. SEM). In the experiments done in the pen, animals remained with the mother and room temperature was not controlled. In all experiments, rectal temperature was hourly measured for 24 h with a thermocouple inserted in the lamb''s rectum and connected to a Philipp Schenk digital recorder. Lambs kept under nycthemeral conditions show a variation of mean rectal temperature (t.degree.) with a period of 24 h, that fits a cosine function (P < 0.001): Rectal t.degree. (.degree.C)=40.6 + 0.4 cos [15 (t-16.22)]. The mesor is 40.6, the amplitude 0.4, and the acrophase expressed in h is 16.22 (n=4). Lambs kept under constant light show a variation of rectal temperature with a period of 24 h, independently of whether the measurements were done in the laboratory or in the pens. The acrophases varied widely in these animals, when the acrophase were synchronized so .theta.=2400, mean rectal temperature showed a variation with a period of 24 h that fits the equation (P < 0.001): Rectal t.degree. (.degree.C)=39.5 + 0.18 cos [15 (t-0.23)]. The presence of a 24 h rhythm of rectal t.degree. in lambs kept under nycthemeral conditions, and its persistence in lambs kept under constant light suggests that the rhythm of rectal temperature observed in the newborn lamb is a true circadian rhythm. From these data it can be inferred therefore, that the part of the circadian system controlling core body temperature is functional in the newborn lamb.
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    ONTOGENY OF THE CIRCADIAN VARIATION OF PLASMA PROLACTIN IN SHEEP
    (1989) VERGARA, M; PARRAGUEZ, VH; RIQUELME, R; FIGUEROA, JP; LLANOS, AJ; SERONFERRE, M
    The ontogeny of circadian rhythms is unknown. The newborn sheep has a circadian rhythm of temperature; to study the ontogeny of other rhythms, we examined the 24-h variation of plasma prolactin concentration in fetal and newborn sheep. To this effect, we measured plasma prolactin concentration in chronically catheterized fetuses (n = 7) and in newborn lambs raised under short day nycthemeral (12 light:12 dark n = 13) or constant light conditions (n = 5). Indwelling catheters were implanted into the jugular vein and carotid artery of late gestation fetuses (0.9 gestation) and newborns (5-29 days old). Experiments were performed 4 or more days after surgery. Lambs were kept in a canvas sling and were fed cow''s milk either by mouth or through a nasogastric catheter at established time intervals. Haematocrit, pH, and blood gases were measured before and after the experiments in all cases and remained within normal values. Lights were on and room temperature was maintained constant during the whole experiment. Samples were obtained every 1-2 h for 24 h in fetuses and newborn lambs under nycthemeral conditions and every hour for 48 h in newborn lambs kept under constant light. Plasma prolactin was measured by radioimmunoassay. The presence of a 24 h rhythm was determined by Cosinor analysis. Fetuses, aged 129 .+-. 6 days (SD) n = 7, showed a variation in plasma prolactin concentration with a period of 24 h that fits the equation: plasma prolactin (ng ml-1) = 97.0 + 15.4 cos 15 (t-23.0), P = 0.035. Newborn lambs, aged 7.9 .+-. 2.3 days old (n = 11), kept under nycthemeral conditions showed a variation of plasma prolactin with a period of 24 h that fits the equation: plasma prolactin (ng ml-1) = 75.3 + 12.9 cos 15 (t-12.0), P = 0.036. This rhythm disappeared in newborns of 23.4 .+-. 3.8 days of age (n = 7). Newborn lambs, aged 22.0 .+-. 2.7 days (n = 5), raised under constant light, showed a 24 h variation of plasma prolactin in the individual data. These newborns showed a tendency to higher prolactin values and a wider dispersion of plasma prolactin concentration than the two other groups. To normalize the variance the group mean was calculated after log transformation. When log values were synchronized considering the acrophase of the theoretical function as 24 h a rhythm that fits the equation: log plasma prolactin (ng ml-1) = 1.8 + 0.29 cos 15 (t-0.5), P = 0.031, was observed. This indicates that the rhythm of prolactin in the newborn sheep free-runs under constant light. The presence of a rhythm in plasma prolactin with a period of 24 h in fetuses and in the newborn, and the existence of this free running rhythm in newborns kept under constant light, suggest that the prolactin rhythm observed is a circadian rhythm endogenously generated. In addition, the disappearance of the rhythm in older newborns raised under short nycthemeral conditions, and its maintenance in newborns of similar age kept under constant light, suggests that the newborn sheep is sensitive to photoperiod.
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    ONTOGENY OF THE CIRCADIAN-RHYTHM OF CORTISOL IN SHEEP
    (1989) PARRAGUEZ, VH; VERGARA, M; RIQUELME, R; RAIMANN, R; LLANOS, AJ; SERONFERRE, M
    In this work we investigated the ontogeny of the rhythm of plasma cortisol in sheep. Plasma cortisol was measured by radioimmunoassay in blood samples obtained every 1 or 2 h, for periods of 24 or 48 h, in 13 fetal sheep (124-140 days of gestation; 130.6 .+-. 1.5, mean .+-. SE) and in 23 newborn (5-39 days of age). To this end, indwelling polyvinyl catheters were implanted into the femoral artery and vein in all animals. The presence of rhythm was determined by Cosinor Analysis. Newborns were separated into four groups. Group 1, newborns younger than 15 days of age (7.9 .+-. 0.7 days), and Group 2, newborns older than 15 days of age (25.4 .+-. 2.3), were raised under nyctohemeral conditions (12L:12D). Group 3, newborns younger than 15 days of age (11.4 .+-. 0.9 days), and Group 4, newborns older than 15 days of age (22.0 .+-. 1.2 days), were raised under constant light conditions. A 24-h rhythm of plasma cortisol (F) was observed in newborns over 15 days of age under both nyctohemeral: F (ng/ml) = 16.1 .+-. 7.6 cos [15 (t-12.9)], (p = 0.01, n = 8) and constant llight conditions: F (ng/mL) = 17.1 + 3.9 cos [15 (t-7.9)], (p = 0.02, n = 5). No rhythm was observed in fetal sheep or in newborn sheep younger than 15 days of age under nyctohemeral or constant light conditions.