A rapid electrochemical method for assimilation test of microorganisms

Summary A microbial electrode consisting of the immobilized microorganisms to be tested and an oxygen electrode was used to study the assimilation characteristics of microorganisms. When a sample solution containing a substrate was injected into the microbial sensor system, the current of the sensor markedly decreased with time if the microorganisms assimilated the substrate. On the other hand, no current decrease was observed if the microorganisms could not assimilate the substrate. Assimilation characteristics of various microorganisms such as molds, yeasts, bacteria, actinomycetes and activated sludges were tested with various substrates. The time required for a test was 30 min per substrate by the pulse method (sample injection period, 5 min). Good correlations were obtained between this electrochemical method and the conventional growth test. The fundamental differences between the two methods and the application of the electrochemical method are discussed.     

Concordant induction of rapid in vivo pulsatile insulin secretion by recurrent punctuated glucose infusions

Insulin is largely secreted as serial secretory bursts superimposed on basal release, insulin secretion is regulated through changes of pulse mass and frequency, and the insulin release pattern affects insulin action. Coordinate insulin release is preserved in the isolated perfused pancreas, suggesting intrapancreatic coordination. However, occurrence of glucose concentration oscillations may influence the process in vivo, as it does for ultradian oscillations. To determine if rapid pulsatile insulin release may be induced by minimal glucose infusions and to define the necessary glucose quantity, we studied six healthy individuals during brief repetitive glucose infusions of 6 and 2 mg x kg(-1) x min(-1) for 1 min every 10 min. The higher dose completely synchronized pulsatile insulin release at modest plasma glucose changes ( approximately 0.3 mM = approximately 5%), with large ( approximately 100%) amplitude insulin pulses at every single glucose induction (n = 54) at a lag time of 2 min (P < 0.05), compared with small (10%) and rare (n = 3) uninduced insulin excursions. The smaller glucose dose induced insulin pulses at lower significance levels and with considerable breakthrough insulin release. Periodicity shift from either 7- to 12-min or from 12- to 7-min intervals between consecutive glucose (6 mg x kg(-1) x min(-1)) infusions in six volunteers revealed rapid frequency changes. The orderliness of insulin release as estimated by approximate entropy (1.459 +/- 0.009 vs. 1.549 +/- 0.027, P = 0.016) was significantly improved by glucose pulse induction (n = 6; 6 mg x kg(-1) x min(-1)) compared with unstimulated insulin profiles (n = 7). We conclude that rapid in vivo oscillations in glucose may be an important regulator of pulsatile insulin secretion in humans and that the use of an intermittent pulsed glucose induction to evoke defined and recurrent insulin secretory signals may be a useful tool to unveil more subtle defects in beta-cell glucose sensitivity.     

Effects of intracerebroventricular administration of D,L-2-amino-5-phosphonovaleric acid, an N-methyl-D-aspartate receptor antagonist, on luteininizing hormone release in ovariectomized lambs.

To determine whether endogenous glutamate and aspartate control LH secretion via N-methyl-D-aspartate (NMDA) receptors in the sheep, we evaluated the effects of the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (AP5) on secretion of LH in ovariectomized lambs. Twelve lambs were ovariectomized and surgically implanted with lateral cerebroventricular cannulae. At the time of the experiment, (38 wk of age) they received intracerebrally 4 injections of either 50 (n = 4), 100 (n = 4), or 200 micrograms (n = 4) of AP5. Blood samples were collected every 10 min for 8 h with animals receiving AP5 at hours 4, 5, 6, and 7. Patterns of LH during the preinjection period were compared to those during the period encompassing AP5 injections. Mean concentrations of LH were lower during AP5 injections than during the preinjection periods, a response that was not influenced by dose (0.87 +/- 0.08 vs. 0.69 +/- .07 ng/ml; p < 0.01). LH pulse amplitude decreased during AP5 treatment relative to the preinjection periods, but this difference was not statistically significant (0.79 +/- 0.11 vs. 0.68 +/- 0.10 ng/ml; p = 0.09). There were no effects of AP5 on LH pulse frequency (1.00 +/- 0.10 vs. 0.83 +/- 0.15 pulses/h for injection and preinjection periods; p > 0.10). A second experiment was done to evaluate a higher dose of AP5. Four animals were chosen to receive 4 injections of 2 mg of AP5 in a design identical to that used in the first experiment.     

Modulation of luteinizing hormone pulse amplitude by the frequency of luteinizing hormone-releasing hormone stimulation and by testosterone in castrated, hypothalamic-lesioned male rats

The frequency of spontaneous luteinizing hormone (LH) pulses is thought to be a direct result of the frequency of luteinizing hormone-releasing hormone (LHRH) pulses from the hypothalamus. By contrast, the amplitude of spontaneous LH pulses may be controlled by several factors other than the amplitude of LHRH pulses. We tested two hypotheses: 1) that LH pulse amplitude is determined in part by the frequency of LHRH pulses of constant magnitude, and 2) that testosterone (T) exerts a direct feedback effect on the pituitary gland to regulate LH pulse amplitude. Gonadal feedback was eliminated by castrating adult male rats (n = 20). Endogenous LHRH secretion was eliminated by lesioning the medial basal hypothalamus. Serum LH levels (0.19 +/- 0.04 ng/ml RP-2, mean +/- SEM) and T levels (0.15 +/- 0.02 ng/ml), measured several weeks after hypothalamic lesioning, confirmed the hypogonadotropic hypogonadal state of the animals. During a 8-h period, unanesthetized, unrestrained animals were injected with 40-ng pulses of LHRH via catheters into the jugular vein, and blood samples for LH measurement were drawn at 10-min intervals. The LHRH pulse interval was 20 min during the first 4 h in all animals. The pulse interval was doubled to 40 min in half of the animals (n = 10) during the next 4 hours; in the other 10 animals, the pulse interval was maintained constant at 20 min throughout the study. Within both of these groups, one-half of the animals (n = 5) were infused with T to achieve a physiological level of T in serum (2.46 +/- 0.36 ng/ml at 4 h), while the other half received vehicle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron spin echo studies on chloroplasts. Spectral characteristics of electron transport components and light-induced transients

Electron-spin resonance echoes are used to study the complex overlapping ESR spectra of whole chloroplasts. By varying the repetition rate of the microwave pulse sequence, delay time, and pulse width, signals with different longitudinal and transverse relaxation times were extracted. We have identified the echo signals due to plastocyanin and ferredoxins. In addition, we have found a strong signal at g = 4.3, that possibly arises from distorted cytochrome, and weak signals in the region g = 6-9. The strong echo signal at g = 2.0047 (Signal II), is made up of at least three "dark" components that differ in their relaxation times. Upon illumination at 1.2 K several of the echo signals including Signal II show reversible light-induced components. The kinetics of these transients depend on the addition of 3(3,4-dichlorophenyl)-1,1-dimethyl urea. Part of the transients are believed to arise from cyclic electron flow around Photosystem I.     

31P-NMR studies on the energy metabolism of the living rat brain using a surface coil method

We have examined the changes of the energetic metabolic state of rat and mouse brains under hypoxic hypoxia or ischemia using a 31P-NMR spectrometer with a surface coil. The NMR spectrometer has a super-conducting magnet providing a homogeneous magnetic field of 6.3 tesla. A probe was remodelled to accommodate an experimental animal in it. The animals were anesthetized with 1.0% or 1.5% halothane throughout the experiments. The optimal measurement conditions were a 90 degrees pulse width of 20 microsecond, and a 2 sec pulse repetition time. 200 acquisitions of FIDs was required for high spatially resolved signals. The Pcr/ATP ratio of the live, anesthetized rat brains was 1.76 +/- 0.46 (n = 8) for cerebra and 1.63 +/- 0.11 (n = 4) for cerebella. That of gerbil brains was 1.23 +/- 0.09 (n = 4). The Pcr/ATP ratio did not show any significant changes under both the conditions of hypoxic hypoxia or ischemia. The value of Pcr/Pi ratio decreased in the hypoxic conditions. The level of Pcr. of rat cerebrum decreased by 76.8 +/- 10.5% at 10% oxygen and by 57.3 +/- 15.7% at 5% oxygen compared with the value of 20% oxygen. The ATP level in the rat brain also decreased according to the degree of hypoxia. Cerebral ischemia was produced in the gerbil by ligation of the common carotid artery. The levels of Pcr. and ATP were severely depressed in the ischemic hemisphere but those of the intact side remained normal.     

Changes in serum luteinizing hormone (LH) concentrations in response to luteinizing hormone releasing hormone (LHRH) in bull calves that attained puberty early or late

The objectives of this study were to determine if the response to luteinizing hormone releasing hormone (LHRH) could be used to select bull calves capable of early sexual maturation and to establish the optimum route and dose of LHRH. In Trial 1, at 4, 10 and 20 week of age, 20 calves were treated iv with 2 microg/kg body weight of LHRH 1 and 5h after commencing a 9-h period of blood sampling. Bulls were separated into early and late maturing (n=10), based on age at puberty (scrotal circumference (SC) of >or=28 cm). At 4 and 20 week of age, peak serum LH concentrations and area under the LH response curve in response to LHRH were lower (P<0.05) in early- versus late-maturing bulls. In Trial 2, calves at 20 week of age were given LHRH as follows: 2 microg/kg body weight iv (n=6), im (n=6) or sc (n=6); 5 microg/kg im (n=6), or ischio-rectally (ir, n=6) or sc (n=6); and 10 microg/kg im (n=6) or sc (n=6). Serum LH concentrations were at a plateau from 30 to 165 min after treatment with 5 microg/kg of LHRH (im or ir; P>0.05). We concluded that the LH responses to LHRH in calves at 4 and 20 week of age could facilitate the development of a simple test (one blood sample prior to treatment with LHRH and a second during the period of sustained response to LHRH) to select early-maturing bulls.     

Effect of PGE2 on uterine contractility and tone in mares

A technique for transvaginal, ultrasound-guided intrauterine injection was developed. After preliminary study using different approaches, the procedure was successful in 24 of 25 (96%) mares, based on detecting fluid in the uterine lumen during and after the injection. The technique was used to study the effect of PGE2, reportedly produced by the embryonic vesicle, on uterine contractility on Day 12 (Day 0 = ovulation). Uterine contractility was scored (1 = minimal, 4 = maximal) every 10 min for 1 h and every 30 min for the next hour by a continuous 1-min ultrasound examination of a longitudinal section of the uterine body without knowledge of group. In Experiment 1, the main effect of group (1-mL vehicle, n = 6; 0.25 microgram PGE2, n = 7) tended to be significant (P < 0.09), and the effect of time was significant (P < 0.008). The mean score was higher for the PGE2 group (2.0 +/- 0.1) than for the vehicle group (1.7 +/- 0.1). An increase in contractility occurred between 0 and 5 min in the vehicle group (P < 0.0004) and between 0 and 10 min in the PGE2 group (P < 0.04). In Experiment 2, there was a tendency (P < 0.08) for effect of group (control without injection, n = 6; 1-mL vehicle, n = 6; 0.025 microgram PGE2, n = 6). The PGE2 group (2.0 +/- 0.1) was different from the vehicle group (1.6 +/- 0.1) and the control group (1.6 +/- 0.1). An increase in contractility occurred between 0 and 20 min in the PGE2 group, and the changes were not significant in the other groups. However, scores were higher in the PGE2 group before treatment, and there were no significant effects when data were converted to percentage changes. The results for an effect of intrauterine treatment of PGE2 on uterine contractility are considered uncertain because of the transient increase in contractility from vehicle injections in Experiment 1 and the higher score in the PGE2 group before treatment, with no significant differences in percentages in Experiment 2. Indirectly, however, an effect of PGE2 was suggested by a shorter (P < 0.05) period of detectability of intrauterine fluid in the PGE2 groups (21 +/- 31 min) than in the vehicle groups (50 +/- 42 min). The shorter period was attributable to greater dispersion of the fluid as a result of increased contractility. In Experiment 3, PGE2 (10 mg, n = 5) and vehicle (4 mL, n = 5) were given intravenously. In addition to uterine contractility, uterine tone was scored (1 = minimal, 4 = maximal) by transrectal digital compression. The main effect of group was significant (P < 0.03) for uterine contractility score, which increased between 0 and 20 min after PGE2 injection. The time effect and interaction were highly significant (P < 0.0001) for uterine tone score, and tone increased in the PGE2 group between 0 and 20 min after injection. The results indicated that PGE2 should be considered as a potential stimulator of both uterine contractions and uterine tone during the time of embryo mobility in mares.     

Hormonal and ovarian responses to a 5-day progesterone treatment in anoestrous dairy cows in the third week post-partum

Primiparous cows with low body condition at calving have an extended anovulatory period. Induction of ovulation and oestrus is possible with progesterone treatment but the response to this treatment differs between Friesian and Jersey breeds. The objective of this study was to describe changes in pulsatile LH secretion and the synchrony of developing ovarian follicles that occur during a progesterone treatment period of 5 days in primiparous anovulatory cows. The experimental model compared the progesterone treatment with spontaneous post-partum changes as well as a breed comparison in a factorial design.Thirty-six cows (Jersey n=19 and Friesian n=17) were managed to calve with a low body condition score (BCS<4. 5). Daily changes in ovarian follicle size were observed with transrectal ultrasonography in each cow from 8 days post-partum. Thirty of these cows were diagnosed to be anovulatory at 12-18 days post-partum (day 0) and allocated to a treatment (n=16) or a control group (n=14), balanced for breed. Each treated cow had a progesterone-releasing controlled internal drug-releasing (CIDR) device inserted vaginally for 5 days while control cows were left untreated. Changes in plasma LH concentrations were measured with intensive blood sampling over 8 h on days -1, 1, and 4. Blood samples were also collected daily (06:00 h) for determination of plasma progesterone as well as oestradiol concentrations on days 6 and 8.Treatment with progesterone was associated with a transient initial decrease (day 1) in both LH pulse frequency and mean LH concentrations after device insertion, but both had returned to pre-treatment levels by day 4. Jersey cows had a greater pulse frequency, but there was no breed difference in mean LH concentrations. Patterns of ovarian follicle growth were affected by progesterone treatment with an increase in diameter of the dominant follicle (DF) identified after treatment initiation. This followed an earlier emergence of a new DF after device insertion. Follicular response to progesterone was dependent on the diameter of the DF present at treatment initiation. Those follicles >/=9 mm were replaced by a new DF during treatment such that the DF observed at the time of device removal was large (>/=9 mm) and growing in 13/16 cases.Progesterone was not effective for the induction of an LH surge, ovulation and oestrus in anovulatory cows with a low BCS. However, treatment was associated with synchronous development of a DF so that it was large and growing at the end of the treatment period in most cases. This synchronous development may be due to the transient suppression of LH and the presence of an LH-dependent DF.     

Effects of a rapid change in muscle glycogen availability on metabolic and hormonal responses during exercise

To evaluate the metabolic and hormonal adaptations following a rapid change in muscle glycogen availability, 14 subjects had their muscle glycogen content increased in one leg (IG) and decreased in the other (DG). In group A (n = 7), subjects exercised on a bicycle ergometer at 70% maximal oxygen uptake for 20 min using the DG leg. Without resting these same subjects exercised another 20 min using the IG leg. Subjects in group B (n = 7) followed the same single-leg exercise protocol but in the reverse order. In order to get some information on the time sequence of these possible adaptations, blood samples were collected at rest and at the beginning and the end of each exercise period (min 5, 20, 25, and 40). Results indicated that 5 min after the switch from the DG leg to the IG leg, transient increases in plasma free fatty acids (1.20 to 1.39 meq X 1(-1)) and serum insulin (10.1 to 12 mu X 1(-1)) concentrations occurred. Between minute 25 and 40 of exercise, the DG to IG switch was accompanied by a decrease in free fatty acids and glycerol concentrations as well as an increase in lactate levels. An opposite response was observed in the IG to DG condition during the same time span. Plasma norepinephrine, epinephrine, glucagon, and serum cortisol concentrations were not significantly affected by the leg change. These results suggest a rapid preferential use of muscle glycogen when available and a time lag in the response of the extramuscular substrate mobilization factors.     

Fasting-induced suppression of pulsatile luteinizing hormone secretion is related to body energy status in ovariectomized goats

The effect of energy status on the response of luteinizing hormone (LH) pulse frequency to acute short-term energy deficiency created by fasting in estradiol-treated ovariectomized Shiba goats was studied in two experiments. In experiment 1, eight goats whose mean body weight (BW) was 25.6 +/- 5.8 (mean +/- S.D.)kg were fed 500 g hay cubes daily for 1 week. Then they were fasted for 3 days. Blood samples were collected for 4 h at 6 min intervals on the last day of feeding, first, second and third day of fasting for LH analysis. The goats were divided into light (<24 kg, n = 4) and heavy (> or =24 kg, n = 4) groups for data analysis. There was no difference in LH pulse frequency between the last day of feeding and each day of fasting in the heavy group. LH pulse frequency was significantly (P < 0.05) suppressed on the second day (3.3 +/- 1.3 pulses/4 h) and on the third day (2.3 +/- 1.9 pulses/4 h) relative to the day prior to fasting (4.8 +/- 1.5 pulses/4 h) in the light group. In experiment 2, BW plus a body mass index (gBMI: (body weight (kg)/withers height (m)/body length (m)) x 10) were measured to define energy status. Nine goats (BW, 25.6 +/- 5.8 kg) were fed 500 g hay cubes daily for a week and then fasted for 3 days. Then they were divided into two groups offered either a maintenance (n = 4) or a restricted (n = 5) level of feeding for 4 weeks. The restricted level of feeding was 30% of maintenance requirement based on the BW recorded weekly. The feeding level was then adjusted to maintain BW for a further week followed by 3 day fasting for restricted animals. Blood samples were collected for 6 h at 10 min intervals on the day prior to fasting and on third day of fasting before and after the dietary manipulation. BW (26.6 +/- 2.2 to 26.8 +/- 3.8 kg) and gBMI (8.4 +/- 0.4 to 7.8 +/- 0.3) remained constant over the period prior to fasting for the maintenance animals but were significantly lower (P < 0.05) after 4 weeks for the restricted goats (BW, 26.3 +/- 2.1 to 21.5 +/- 2.4 kg; gBMI, 8.4 +/- 0.9 to 6.9 +/- 0.7). There was no significant difference in the LH pulse frequency between feeding and fasting day in both sampling periods in the maintenance group. In the restricted group, LH pulse frequency was not suppressed by fasting in the first sampling period (6.8 +/- 2.9 to 5.2 +/- 2.5 pulses/6 h), whereas it tended to be suppressed (4.8 +/- 3.1 to 1.6 +/- 2.3 pulses/6 h; P < 0.06) and was significantly (P < 0.05) correlated to body weight (r = 0.70) and gBMI (r = 0.81) after the dietary manipulation. These results suggest that the suppressive effect of short-term energy restriction (fasting) on pulsatile LH secretion is related to body energy status.     

Testosterone modulates the differential release of luteinizing hormone and follicle-stimulating hormone that occurs in response to changing gonadotropin-releasing hormone pulse frequency in the male monkey, Macaca fascicularis

Selective elevations of plasma follicle-stimulating hormone (FSH) levels are characteristic of some physiological conditions, such as the early stages of human puberty, and in some disorders of testicular function, such as idiopathic oligospermia. We tested the hypotheses that a slow gonadotropin-releasing hormone (GnRH) pulse frequency favors a selective elevation of plasma FSH and that this is influenced by the circulating steroidal milieu. We administered exogenous GnRH at frequencies of once every 90 min (q 90 min) and once every 240 min (q 240 min) to castrated prepubertal male monkeys who had received either empty (sham) or testosterone (T)-filled Silastic capsules at the time of castration. At the end of each experimental frequency period, mean plasma levels of luteinizing hormone (LH) and FSH were measured. Plasma T levels were also measured. Animals with T implants had plasma levels of this hormone that were in the adult range (approximately equal to 8 ng/ml), whereas those with sham implants had plasma T levels in the prepubertal range (less than or equal to 4 ng/ml). In animals with sham implants, mean plasma FSH levels were markedly elevated at the slower GnRH pulse frequency (39.5 +/- 3.6 ng/ml following GnRH q 240 min compared with 23.7 +/- 2.8 ng/ml following GnRH q 90 min). This selective FSH elevation was not apparent in animals with T implants. Mean plasma LH levels were similar (approximately equal to 8 micrograms/ml) at the two GnRH pulse frequencies, in both T-treated and sham-implanted animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of mating stimuli and oxytocin on plasma cortisol concentration in gilts

The involvement of oxytocin (OT) in the regulation of glucocorticoid secretion during stress reaction, parturition, and suckling has been documented in various species. In this study four in vivo experiments were conducted on gilts (1) to demonstrate the influence of mating stimuli on plasma cortisol concentration, (2) to test the effect of OT alone and (3) OT combined with OT-antagonist on cortisol secretion and (4) to clarify the role of progesterone and estradiol in cortisol response to exogenous OT. In experiment 1, plasma cortisol concentration in gilts (n=4) increased (p<0.05) from 16.1 +/- 5.3 ng ml(-)1 (control period: 30 min before mating) to 42.8 +/- 11.6 ng ml(-1) and 46.6 +/- 9.6 ng ml(-1) at the time of leaving the pen and during the first visual and olfactory contact with the boar, respectively. During coitus the elevation was maintained (48.8 +/- 9.8 ng ml(-1); p<0.05 vs. control). The plasma cortisol concentration returned to pre-mating levels within 30 min after mating. In experiment 2, gilts (n=7) were treated, according to Latin square design, with saline (2 ml; i.v.) and OT (10, 20, and 30 IU; i.v.). The magnitude of cortisol response (area under cortisol curve) was higher (p<0.01) only after treatments with 20 and 30 IU OT vs. control period (30 min before OT). Gilts (n=3) of experiment 3 were infused with OT-antagonist (Atosiban; 25 mg per gilt per 2 hours; i.v.) and then were injected with OT (20 IU; i.v.) 60 min after the beginning of Atosiban administration. Blockage of OT receptors by Atosiban reversed the stimulatory effect of OT on cortisol secretion. In experiment 4, ovariectomized gilts (n=25) primed (i.m.) with corn oil (n=7), progesterone (P4; n=7), estradiol benzoate (EB; n=4) or EB+P4 (n=7) were treated with OT (20 IU; i.v.). Plasma cortisol concentrations were increased following OT administration in all gilts of experiment 4. The highest cortisol response to OT was noted in gilts primed with EB+P4 (p<0.01 vs. other groups). In conclusion: (1) leaving the pens, visual and olfactory contact with the boar as well as coitus, increased plasma cortisol concentrations in gilts to similar levels; (2) exogenous OT (20 and 30 IU per gilt) increased cortisol plasma concentration, (3) this effect was abolished by OT-antagonist and (4) E2+P4 elevated cortisol response to OT. Oxytocin may be included to secretagogues of the hypothalamus-pituitary-adrenocortical axis in pigs.     

喉罩通气下七氟醚全凭吸入麻醉在小儿先天性心脏病介入手术的临床麻醉效果

目的:探讨喉罩通气下七氟醚全凭吸入麻醉在小儿先天性心脏病介入手术的临床麻醉效果。方法:选取2017年4月～2019年5月期间我院收治的行先天性心脏病介入手术患儿98例,根据随机数字表法将其分为对照组(n=49)和研究组(n=49)。对照组给予氯胺酮诱导,全凭丙泊酚维持,面罩吸氧;研究组给予全凭七氟醚诱导、维持,喉罩通气。比较两组患儿麻醉前(T0)、切皮前(T1)、切皮后1 min(T2)、切皮后30 min(T3)、术后(T4)的血流动力学指标[平均动脉压(MAP)、心率(HR)]及应激反应指标[血糖、皮质醇],记录两组患儿手术时间、麻醉诱导时间、术后苏醒时间等围术期指标情况。记录两组围术期不良反应发生情况。结果:研究组手术时间、麻醉诱导时间、术后苏醒时间均短于对照组(P0.05)。两组T0时间点血糖、MAP、皮质醇、HR比较差异无统计学意义(P0.05);对照组T1～T4时间点MAP、血糖、皮质醇、HR均较T0升高(P0.05);研究组T1～T4时间点血糖、MAP、皮质醇、HR与T0时间点比较无差异(P0.05);研究组T1～T4时间点血糖、MAP、皮质醇、HR低于对照组(P0.05)。两组不良反应发生率比较无差异(P0.05)。结论:小儿先天性心脏病介入手术中应用喉罩通气下七氟醚全凭吸入麻醉,诱导迅速且安全、术后苏醒快、手术时间短,可有效维持血流动力学稳定,减少应激反应。     

Role of frequency and amplitude of repetitive HCG stimulations for sustained progesterone secretion from the bovine corpus luteum in vitro.

This investigation aimed at evaluating a role for frequencies and amplitudes of repeated HCG stimulations for the optimal maintenance of progesterone (P4) secretion from the bovine corpus luteum in vitro. Slices (100-120 mg) of midluteal bovine corpora lutea were perifused with medium M199 (0.05% BSA, pH 7.2, 38.5 degrees C) and the perifusion effluent collected at 15 minute intervals for 20-29 hours. Unstimulated P4 release (n = 5) was distinctly pulsatile (by Pulsar pulse algorithm), with pulses occurring every 90 +/- 6 minutes (mean +/- SEM) and pulse amplitudes of 14.4 +/- 1.1 ng. Conversely, no pulses were detected in two control perifusions. Unstimulated P4 release increased during the first 5 perifusion hours (from 39.3 +/- 4.6 to 50.3 +/- 5.6 ng/15 min, p less than 0.01), but then appeared to decline (to 29.3 +/- 1.3 ng/15 min, p less than 0.05) towards the end of the perifusion periods. Hourly pulses of HCG (6.7 mM) did not change the P4 pulse amplitudes (16.6 +/- 2.0 ng), the pulse periodicities (105 +/- 15 min) and overall release rates (34.7 +/- 5.7 ng/15 min), nor did they prevent the decline in P4 secretion towards the end of perifusions (n = 5). In contrast, 2-hourly HCG stimulations maintained stable P4 release rates throughout the perifusion periods (34.7 +/- 6.8 ng/15 min), with P4 pulses of similar amplitudes (14.7 +/- 1.7 ng), but of lower periodicities (135 +/- 2 min, p less than 0.05) than during unstimulated conditions (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of intensity and pulse width of different moderators for designing a new diffractometer for protein crystals with large unit cells in J-PARC/MLF

We plan to design a high-resolution biomacromolecule neutron time-of-flight diffractometer, which allows us to collect data from crystals with unit cells above 250 Å, in the materials and life science experimental facility at the Japan Proton Accelerator Research Complex. This new diffractometer can be used for a detailed analysis of large proteins such as membrane proteins and supermolecular complex. A quantitative comparison of the intensity and pulse width of a decoupled moderator (DM) against a coupled moderator (CM) considering the pulse width time resolution indicated that the DM satisfies the criteria for our diffractometer rather than the CM. The results suggested that a characteristic feature of the DM, i.e., narrow pulse width with a short tail, is crucial for the separation of Bragg reflections from crystals with large unit cells. On the other hand, it should be noted that the weak signals from the DM are buried under the high-level background caused by the incoherent scattering of hydrogen atoms, especially, in the case of large unit cells. We propose a profile-fitting integration method combined with the energy loss functions and a background subtraction method achieved by employing the statistics-sensitive nonlinear iterative peak-clipping algorithm.     

Pulsatility of growth hormone secretion and its relation to growth.

84 short children were submitted to nocturnal spontaneous growth hormone (GH) secretion tests and to provocative insulin-arginine tests. Discrepancies between the GH peak under the provocative test (I-AP) and nocturnal GH maximal peak (PA) and mean concentration (MC) were frequently observed, despite significant statistical correlation between I-AP and PA (r = 0.47; p < 0.02) and between I-AP and MC (r = 0.42; p < 0.02). Night profiles were evaluated by time analysis: 31 fitted a theoretical model, consisting of a cosine function of time (modelizable profiles). Spectral analysis, from Fourier transformation, indicated predominant periods after cluster analysis. The major predominant period in modelizable (n = 9) and in nonmodelizable (n = 28) profiles was close to 180 min and a secondary period was on average 122 min in modelizable (n = 20) and 105 min in nonmodelizable (n = 23) profiles. Two modelizable and two nonmodelizable profiles escaped this classification. The general, auxological and GH secretory status did not differ significantly between patients with modelizable and nonmodelizable profils. Growth velocity correlated with GH mean concentration (r = 0.36; p < 0.001), but not with plasma insulin-like growth factor-I levels nor with any of the pulsatility indices: number of peaks, main period, and pulse height index = mean GH peak/mean GH concentration. The relevance of GH pulsatility to growth is, therefore, unclear in humans.     

Laser photobleaching leads to a fluorescence grade adenosine deaminase

The enzyme adenosine deaminase (adenosine aminohydrolase EC 3.5.4.4) from calf intestinal mucosa is commercially available at high purity grade yet, at the sensitivity at which fluorescence studies may be undertaken, a nonpeptidic fluorescence is detectable at lambda exmax = 350 nm and lambda emmax = 420 nm. A sevenfold decrease of this nonpeptidic fluorescence was obtained upon irradiation by the third harmonic (355 nm) of a Nd:YAG laser for 16 min, at 5 mJ/pulse, with a pulse width of 6 ns at a repetition rate of 10 Hz. The decline of fluorescence was accompanied by a negligible loss of enzymatic activity. Moreover, the integrity of the protein was ascertained by (i) its fluorescence (lambda exmax = 305 nm, lambda emmax = 335 nm) and lifetime distribution and (ii) its kinetics in the presence of the substrate adenosine and two inhibitors, all of which remained essentially unaltered. Laser photobleaching is a simple way to achieve a fluorescence grade adenosine deaminase.     

A new bioassay method for quantitative analysis of tetracyclines

A new method is described for quantitative analysis of tetracycline, based on the decrease in external pH of bacterial suspensions after the addition of a glucose pulse. The decrease in external pH of these suspensions was inversely proportional to the concentration of tetracycline. The correlation coefficient of standard response lines derived from the bioassay was 0.99. Tetracycline potency was determined in six tetracycline HCl samples by the sugar pulse bioassay and a turbidimetric method. The turbidimetric assay result varied from the glucose-pulse data by no more than 7 and 3% at 3 and 7 min, respectively. The procedure is rapid, precise and quantitative, and requires minimal preparation and use of media, with savings in laboratory resources and time.