beta-hydroxy-beta-methylbutyrate (HMB) kinetics and the influence of glucose ingestion in humans

The dietary supplement, beta-hydroxy-beta-methylbutyrate (HMB), has been shown to decrease muscle proteolysis during the stress of exercise and disease. The aim of this investigation was to determine the time course kinetics of HMB and to determine whether oral glucose ingestion alters the kinetics. In Study 1, eight males (32 +/- 10 yrs) participated in two randomize trials: 1) oral ingestion of 1g of HMB with water in capsule form (HMB), and 2) placebo. Blood samples were obtained prior to ingestion of treatment and at 30, 60, 90, 120, 150, and 180 min for the measurement of plasma HMB. Additional blood samples were obtained at 6, 9, and 12 hr. Urine was collected prior to ingestion and at 3, 6, 9, and 12 h for the measurement of urinary HMB. In Study 2, eight males (25 +/- 6 yrs) followed the same study design and testing procedure as for Study 1. Treatments were 1) modified glucose tolerance test (75 g glucose) (GLU), 2) oral ingestion of 3 g of HMB with water (HMB), and 3) ingestion of 3 g of HMB with 75 g of glucose (HMB+GLU). Blood samples were analyzed for insulin, glucose, and HMB. Additional blood samples were obtained at 24h and 36h for the measurement of HMB. Additional urine samples were collected at 24h and 36h. In Study 1, plasma HMB peaked at 120 nmol/ml at 2.0 +/- 0.4 hr in HMB trial. Half-life was 2.37 +/- 0.1 hr. Following the consumption of 1g of HMB, approximately 14% of the HMB consumed accumulated in the urine. In Study 2, plasma glucose and insulin levels were significantly greater in GLU and HMB+GLU treated subjects compared to HMB treated subject at minutes 30, 60 and 90. Plasma HMB peaked at 487.9 +/- 19.0 nmol/ml at 1.0 +/- 0.1 hr in the HMB treated subjects and at 352.1 +/- 15.3 nmol/ml at 1.94 +/- 0.2 hr when subjects consumed HMB+GLU. The time to reach peak was different (P <0.001) between HMB and HMB+GLU. The plasma HMB half-life was less (P = 0.08) 2.38 +/- 0.1 hr in HMB trial compared to 2.69 +/- 0.2 hr in HMB+GLU trial. Area under the plasma HMB curve during the first 3 hr was less (P = 0.002) in the HMB+GLU trial compared to the HMB trial. From 3 h through 36 h the area under the HMB curve tended to be less (P = 0.106) for the HMB+GLU compared to the HMB alone. HMB accumulation in the urine as well as the area under the curve were similar with both HMB (94875.8 +/- 15159.5 nmol/36 hrs) and HMB+GLU (80678.2 +/- 3863.1 nmol/36 hrs). The percentage of the HMB dose that accumulates in the urine was 27% for HMB+GLU and 29% for HMB alone. In conclusion, HMB plasma levels peak within 60 to 120 min depending on the amount of HMB consumed and whether glucose is consumed with HMB. The plasma half-life is approximately 2.5 hr. Plasma HMB reaches baseline levels at approximately 9 hr following ingestion. However, 70 to 85% of the ingested oral HMB is retained in the body for further metabolism.     

13C enrichment of extracellular neurotransmitter glutamate in rat brain--combined mass spectrometry and NMR studies of neurotransmitter turnover and uptake into glia in vivo.

13C-enrichment analysis of glutamate in the extracellular fluid (GLU(ECF): 2-3 microM) by gas-chromatography/mass-spectrometry (GCMS) was combined with in vivo NMR observation of whole-brain GLU (approximately10 mM) to study neurotransmitter uptake. Brain GLU C5 was 13C-enriched by intravenous [2,5-13C]glucose infusion. GLU(ECF) was collected by microdialysis from the cortico-striatal region of awake rats. The 13C-enrichment of basal dialysate GLU C5 during 0.75-1.25 hr of infusion was 0.263 +/- 0.01, very close to the enrichment of whole-brain GLU C5. The result strongly suggests that dialysate GLU consists predominantly of neurotransmitter GLU. For selective 13C-enrichment of neurotransmitter GLU, the whole-brain 13C-enrichment was followed by [12C]glucose infusion to chase 13C from the small glial GLU pool. This leaves [5-13C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. The uptake of synaptic [5-13C]GLU(ECF) into glia and metabolism to glutamine (GLN) were monitored in vivo by NMR observation of [5-13C,15N]GLN formed during 15NH4Ac infusion. The rate of GLN synthesis, derived from neurotransmitter GLU(ECF) (which provided 80-90% of the substrate) was 6.4 +/- 0.44 micromol/g/hr. Hence, the observed rate represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

Effect of naloxone and D-Met2-Pro5-enkephalinamide treatment on the DNA synthesis in the developing rat brain

Effects of a single dose of naloxone and of D-Met²-Pro⁵-enkephalinamide on the DNA synthesis in the forebrain, hypothalamus and cerebellum of 11 day old female rats were studied. As an index of DNA synthesis the rate of incorporation of ³H-thymidine into DNA was measured 30 min after a sc. injection of 40 μCi/100 g b.w.. A time dependent effect of naloxone administration on cerebral DNA synthesis was observed. In the forebrain at 1 and 3 hrs after naloxone injection an increased rate of ³H-thy-midine incorporation into DNA was found followed by a marked decrease at 9 and 12 hrs. The effect in the hypothalamus was similar but the initial increase at 1 hr was absent. On cerebellar DNA synthesis naloxone had no effect. The administration of D-Met²-Pro⁵-enkephalinamide resulted in a marked reduction in the labelling of cerebral and hypothalamic DNA between 1 to 12 hrs. Except a decrease at 1 hr no effect was found in the cerebellum.     

A study of prostaglandin F2α as the luteolysin in swine: III effects of estradiol valerate on prostaglandin F, progestins, estrone and estradiol concentrations in the utero-ovarian vein of nonpregnant gilts

Polyvinyl catheters were placed into the right and left utero-ovarian veins and saphenous vein and artery of three control (C) and four estradiol valerate (EV) treated gilts on Day 9 after onset of estrus. The EV treated gilts received 5mg EV/day on Days 11 through 15 after onset of estrus. On Days 12 through 17 utero-ovarian vein blood samples were collected at 15 min intervals from 0700 to 1000 hr and 1900 to 2200 hr and single samples were taken at 1100 and 2300 hr. Peripheral blood samples (saphenous vein or artery) were taken at 0700, 1100, 1900 and 2300 hr from Day 12 until the control gilts returned to estrus or until Day 25 for EV treated gilts and used to measure plasma steroid hormone concentrations. Utero-ovarian vein prostaglandin F (PGF) concentrations (ng/ml, n=1,177) were measured by RIA. Status (control EV treated gilts) by day interactions were detected (P=.10). Curvilinear day trends were detected for plasma PGF concentrations in control (P<.01) but not EV treated gilts. PGF concentrations ( ) for control and EV treated gilts were 1.20 ± 2.08 and .26 ± .84 ng/ml, respectively. PGF peaks (concentrations greater than + 2 S.D.) occured with greater frequency in control gilts (X² = 4.87; P<.05). The interestrus interval ( ) for control and treated gilts was 19.0 ± .6 and 146.5 ± 74.8 days, respectively. Data indicate that estradiol valerate may exert its luteotrophic effect by preventing PGF release from the uterus.     

A study of prostaglandin F2alpha as the luteolysin in swine: III effects of estradiol valerate on prostaglandin F, progestins, estrone and estradiol concentrations in the utero-ovarian vein of nonpregnant gilts

Polyvinyl catheters were placed into the right and left utero-ovarian veins and saphenous vein and artery of three control (C) and four estradiol valerate (EV) treated gilts on Day 9 after onset of estrus. The EV treated gilts received 5mg EV/day on Days 11 through 15 after onset of estrus. On Days 12 through 17 utero-ovarian vein blood samples were collected at 15 min intervals from 0700 to 1000 hr and 1900 to 2200 hr and single samples were taken at 1100 and 2300 hr. Peripheral blood samples (saphenous vein or artery) were taken at 0700, 1100, 1900 and 2300 hr from Day 12 until the control gilts returned to estrus or until Day 25 for EV treated gilts and used to measure plasma steroid hormone concentrations. Utero-ovarian vein prostaglandin F (gf) concentrations (ng/ml, n-1,177) were measured by RIA. Status (control vs EV treated gilts) by day interactions were detected (P=.10). Curvilinear day trends were detected for plasma PGF concentrations in control (P less than .01) but not EV treated gilts. PGF concentrations (X +/- S.D.) for control and EV treated gilts were 1.20 +/- 2.08 and .26 +/- .84 ng/ml, respectively. PGF peaks (concentrations greater than X + 2 S.D.) occurred with greater frequency in control gilts (X2 =4.87; P less than .05). The interestrus interval (X +/- S.E.) for control and treated gilts was 19.0 +/- .6 and 146.5 +/- 74.8 days, respectively. Data indicate tht t estradiol valerate may exert its luteotrophic effect by preventing PGF release from the uterus.     

Effect of adrenalectomy and 5-hydroxytryptophan on phasic release of luteinizing hormone

The effect of 5-hydroxytryptophan (5-HTP) on serum progesterone and the possible role of adrenal progesterone in mediating stimulation by 5-HTP of phasic release of luteinizing. hormone (LH) were investigated in estradiol benzoate (EB)-treated ovariectomized rats. LH surges were induced in long-term (at least two weeks) ovariectomized rats by two injections of EB (20 micrograms/rat, s.c.) with an interval of 72 hrs. Administration of 5-HTP (50 mg/kg, i.p.) at 1000 hr in EB-treated ovariectomized rats resulted in a four-fold increase in serum progesterone within 30 mins, and significantly stimulated the LH surge at 1600 hr. This facilitative effect of 5-HTP on serum LH, but not progesterone, was further potentiated in rats pretreated with P-chlorophenylalanine (PCPA) 72 hrs earlier. Adrenalectomy shortly before 5-HTP administration attenuated the LH surge in saline treated controls, and completely blocked the facilitative effect of 5-HTP on the afternoon surge of LH in rats pretreated with PCPA 72 hrs earlier. On the other hand, chronic adrenalectomy (for 6 days) followed by hydrocortisone (0.2 mg/rat/day) replacement not only had no effect on the LH surge in saline treated controls, but also failed to prevent 5-HTP from facilitating the LH surge in PCPA pretreated rats. On the first day of bleeding, the basal LH value at 1000 hr in sham operated controls was significantly suppressed by PCPA pretreatment 48 hrs earlier. The second dose of 5-HTP administered on the next day failed to potentiate LH surges in either sham operated or adrenalectomized rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

LY227942, an inhibitor of serotonin and norepinephrine uptake: biochemical pharmacology of a potential antidepressant drug

LY227942, (+/-)-N-methyl-3-(1-naphthalenyloxy)-3-(2-thiophene)propanamine ethanedioate, is a new, competitive inhibitor of monoamine uptake in synaptosomal preparations of rat brain. LY227942 inhibits uptake of serotonin (5-hydroxytryptamine, 5HT) and norepinephrine (NE) in cortical synaptosomes and uptake of dopamine (DA) in striatal synaptosomes with inhibitor constants (Ki values) of 8.5, 45 and 300 nM, respectively. Upon administration in vivo, LY227942 lowers 5HT and NE uptake in hypothalamus homogenates to half their respective control activities (ED50) at 0.74 and 1.2 mg/kg s.c., 7 and 12 mg/kg i.p., and 12 and 22 mg/kg p.o., but LY227942 at doses up to 30 mg/kg p.o. does not change DA uptake in striatal homogenates. Lowering of 5HT and NE uptake is demonstrated after 15 min and 6 hr, but has dissipated by 16 hr after oral administration. According to radioligand binding determinations, LY227942 possesses only weak affinity for muscarinic receptors, histamine-1 receptors, adrenergic receptors, dopamine receptors and serotonin receptors. These findings suggest that LY227942 has the pharmacological profile of an antidepressant drug and is useful to study the pharmacological responses of concerted enhancement of serotonergic and noradrenergic neurotransmission.     

Transport of pipecolic acid in adult and developing mouse brain

In an effort to develop an animal model of hyperpipecolatemia, the uptake of pipecolic acid (PA) in the brain and changes of PA levels in serum following administration ofd,l-PA were studied in the mouse using a new sensitive HPLC-EC method. Following i.p. injections (250 mg/kg) to adult male mice, the brain concentration peaks at 5–10 min (40 nmol/g). The level remains relatively stable up to 5 hrs and then declines slowly to 24 hrs. In serum, the level of PA increases rapidly to reach the maximum value at 10 min and then decreases rapidly in the first hour and continues to decline more slowly to 24 hrs. The net uptake of PA following administration of various amounts ofd,l-PA is saturable at low doses (3.9–15.6 mg/kg), and it increases linearly at higher doses in a dose-dependent manner up to the maximum dose (500 mg/kg) used in the present study. Kinetic analysis suggests the presence of two kinds of transport systems. These findings are in good agreement with the previous results usingd,l-[³H]PA in the mouse (7) andl-[¹⁴C]PA in the rat (13). There were no significant differences between uptake ofd-pipecolic acid andl-pipecolic acid (250 mg/kg, i. p., 10 min), suggesting the absence of stereospecificity for PA uptake in the mouse brain. Developmental changes in net brain uptake of PA following injections ofd,l-PA (250 mg/kg, s.c., 10 min) showed an age-dependent decrease which continues until adult levels are reached at four weeks after birth. The results suggest that the blood brain barrier (BBB) for PA is completed during the first month of life. Following administration ofd,l-PA (250 mg/kg, s.c.) to pregnant mice during the period 19–21 days of gestation, PA level increases in fetal brain to a maximum value at 2 hrs (420 nmol/g). This level is unchanged during 24 hrs. The maximum level of PA in fetal serum is reached at 30 min to 1 hr. The level gradually decreases after 1 hr over 24 hrs. These results indicate that PA taken up by the placenta and into the brain is transported from the fetal circulation. Our results also demonstrate that a higher amount of PA is taken up by the fetal than the adult brain. This finding is important in order to develop an animal model of hyperpipecolatemia in which high brain levels of PA should mimick those of human hyperpipecolatemic patients. Our results strongly support the hypothesis that high levels of PA present in brain during fetal life may exert a devastating effect on the development of the human CNS in hyperpipecolatemic children.     

蛋白激酶C对大鼠缺血海马突触体谷氨酸摄取的调控作用

采用大鼠海马脑片体外缺血模型,观察海马突触体内蛋白激酶Ｃ（ＰＫＣ）活性的变化,以及这种变化对突触体谷氨酸（ＧＬＵ）摄取的影响。结果显示：海马脑片体外“缺血”１０ｍｉｎ,其突触体内ＰＫＣ活性基本不变,而缺血３０ｍｉｎ,突触体内ＰＫＣ活性显著上升（Ｐ＜０．０１,ｎ＝６）;非Ｎ－甲基－Ｄ－天门冬氨酸（ＮＭＤＡ）受体拮抗剂ＤＮＱＸ有效地抑制ＰＫＣ活性的同时,可降低胞外ＧＬＵ的堆积,而ＮＭＤＡ受体阻断剂ＡＰ＿５无作用。进一步实验证明,ＰＫＣ激动剂ＰＤＢ浓度依赖性地抑制突触体对３Ｈ－ＧＬＵ的摄取（ＩＣ５０＝１３１±１０μｍｏｌ／Ｌ）,此抑制作用可由ＰＫＣ抑制剂Ｈ－７（１００μｍｏｌ／Ｌ）抵消。提示脑缺血诱发ＧＬＵ堆积的作用机理可能是：脑缺血引发钙内流导致ＧＬＵ过量释放,ＧＬＵ又通过突触前非ＮＭＤＡ受体激活ＰＫＣ,抑制其自身摄取,正反馈性加重胞外ＧＬＵ的堆积。     

Circadian Variation in the Uptake of Tryptophan by Cortical Synaptosomes of the Rat Brain

The kinetics of the high affinity uptake system for L-tryptophan (L-Try)have been measured over 24 hr in cortical synaptosome preparations of rat brain. Both the K_m and V_max, of the uptake process showed a statistically significant 24 hr variation. The highest K_m value, 6.71 ± 10^-5 M, was measured at the beginning of the light phase and the lowest value, 4.23 ± 10^-5 M, 6 hr into the dark phase. V_max was highest at the end of the dark phase (10.43 nmol/mg/5 min) and lowest (4.80 nmol/mg/5 min) 3 hr into the dark phase. In contrast, there was no variation over 24 hr in the V_max/K_m ratio. These results suggest that the high affinity uptake process serves to ensure a constant rate of L-tryptophan entry into the neuron in the face of circadian or ultradian variations in extracellular concentration of tryptophan.     

Postpartum aggression in mice: Experiential and environmental factors

Six experiments were conducted to assess the influence of duration of lactation, the presence of young, and the stimulus characteristics of intruder animals upon postpartum aggression of mice. The first experiment showed that postpartum aggression toward conspecifics was highest between Day 3 and Day 8, declined between Day 9 and Day 14, and was present toward males but absent toward females between Day 15 and Day 21 of the lactation period. Experiment 2 showed that lactating mice rarely attacked conspecifics to which they had been previously exposed but would readily attack strangers. Experiment 3 and 4 demonstrated that lactating animals never attacked intruders when tested 5 hr after pup removal. However, placement of young behind a wire partition in the home-cage for 5 hr or replacement of the offspring for as little as 5 min following 5 hr of separation restored postpartum aggression. The fifth experiment showed that 1- and 10-day old intruders were seldom attacked while intense aggression was directed against 14- and 20-day old intruders. Finally, Experiment 6 demonstrated that 14-day old intruders whose hair was removed were rarely attacked.     

Study on the role of endogenous polyamines in glucagon, isoproterenol or serum-mediated induction of tyrosine aminotransferase in cultured heart cells

In confluent and serum-starved embryonic heart cell cultures, the addition of serum (10%), glucagon (GLU, 0.1 microM) or isoproterenol (ISO, 10 microM), causes the onset of ornithine decarboxylase (ODC) activity, with a maximum after 5-6 hr. This is paralleled by polyamine accumulation and by the induction of TAT, which, in the case of GLU and ISO, exhibits maximal activity at 4-3 hr respectively, followed by a net decline. Cyclic AMP (cAMP) also accumulates after exposure to GLU or ISO. However, under different conditions of ODC inhibition, serum fails to induce TAT, thus supporting a relevant role of cellular polyamines in serum action. Conversely, cAMP and TAT responses to GLU or ISO are markedly improved under prevention of polyamine accumulation, which also leads to a longer lasting TAT inducibility. The suggestion is made that polyamines are not required in the cAMP-dependent mechanism of TAT induction, but rather in the restoration of the basal activity of the enzyme.     

Divergence and evolution of homologous regions of Bombyx mori nuclear polyhedrosis virus.

Homologous regions (hrs) (hr1,hr2-left,hr2-right,hr3,hr4-left,hr 4-right, and hr5) similar to those found in the Autographa californica nuclear polyhedrosis virus (AcNPV) genome were found in the Bombyx mori NPV (BmNPV) genome. The BmNPV hrs contained two to eight repeats of a homologous nucleotide sequence which were on average about 75 bp long. All of these homologous sequence repeats contained a 26-bp-long palindrome motif with an EcoRI or EcoRI-like site at its core. The consensus sequence of the BmNPV hrs showed 95% conservation with respect to those found in AcNPV. Nucleotide sequence analysis indicated that hr2-left and hr2-right of BmNPV evolved from an ancestor similar to hr2 of AcNPV by inversion, cleavage, and ligation. The polarities of the BmNPV and AcNPV hrs were conserved except for that of hr4-left. Within hr4-right of BmNPV, four repeats of a previously underscribed palindrome motif were found. Bmhr5D, a BmNPV mutant which lacked hr5, replicated at a rate similar to that of wild-type BmNPV in BmN cells and silkworm larvae, indicating that hr5 was not essential for viral replication. After ten passages of Bmhr5D in BmN cells, no detectable changes in its genome were observed by restriction endonuclease analysis. The evolution and divergence of the BmNPV genome are also discussed.     

Plasma variation of atrial natriuretic peptide in central hypervolemia (diurnal or nocturnal) induced by antiorthostatic decubitus at -6 degrees

Plasma levels of ANP were measured during a 4 hrs head-down tilt at -6 degrees in 5 healthy male volunteers (aged 20-22 M.2). The experiments took place from 8 to 14 hrs, (day), and from 22 to 7 hrs (night). The control period was 1 hr. in a seated position (8 to 9 hrs. for day and 22 to 23 hrs. for night). Blood samples were collected at 9 and 23 hrs. and every 20 min. during the first hours, and every hours thereafter. Electroencephalograms were continuously recorded during night. Our results showed a similar increase in ANP during both experimental conditions. During night there was no correlations between ANP and sleep stages. Finally the differences observed in renal responsiveness to central volume expansion during day or night could not be explained by a difference in renin, aldosterone, vasopressin (previously demonstrated in several studies) or ANP secretion.     

No Enhancement of 24-Hour Visuomotor Skill Retention by Post-Practice Caffeine Administration

Caffeine is widely consumed throughout the world and appears to indirectly facilitate learning and memory through effects on attention and motivation. Animal work indicates that post-training caffeine administration augments inhibitory avoidance memory, spatial memory, and object memory. In humans, post-training caffeine administration enhances the ability to discern between familiar images and new, similar images. However, the effect of post-training caffeine administration on motor memory has not been examined. Therefore, we tested two groups of low caffeine consumers (average weekly consumption ≤500 mg) in a double-blind, placebo-controlled study involving acquisition of a continuous isometric visuomotor tracking skill. On Day 1, subjects completed 5 blocks (150 repetitions) of training on the continuous isometric visuomotor skill and subsequently ingested either 200 mg of caffeine or placebo. On day 2, subjects completed an additional 5 blocks of training. Day 1 mean performance and performance variability were both similar between groups, suggesting that both groups acquired the motor skill similarly. For mean performance on Day 2, patterns of re-learning, mean performance learning magnitudes, mean performance learning rates, and mean performance retention magnitudes were all similar between groups. For performance variability on Day 2, there was a small trend towards increased variability in the caffeine group during re-learning, but performance variability learning magnitudes and performance variability retention magnitudes did not differ between groups. Because motor skill acquisition can also be conceptualized as a reduction in performance variability, these results suggest that there may be a small negative effect of post-practice caffeine administration on memory of a newly-learned visuomotor skill. Overall, we found no evidence to suggest that post-training caffeine administration enhances 24-hour retention of a newly-learned continuous visuomotor skill, and these results support the notion that memory-enhancing effects of post-training caffeine ingestion may be task-specific.     

Sleep: sequential reduction of paradoxical (REM) and elevation of slow-wave (NREM) sleep by a non-convulsive dose of insulin in rats

Administration of a single non-convulsive dose of insulin (1.0, I.U./kg., I.P.) which produced no observable gross behavioral changes in rats, reduced rapid eye movement (REM) sleep time 100% in the first 3 hrs. and 82% by the 4th hr., reaching control subject levels (saline-treated) by the 6th hr. In contrast, slow-wave sleep (NREM) time in insulin treated animals exceeded control subject levels by 49% by the end of the 2nd hr., returning to normal by the 5th hr. Although there was no difference between insulin and saline treated rats for the total 8 hr. post-injection recording period for total percentage of time awake, or slow-wave sleep time, a 44% reduction in REM sleep time was observed in insulin treated animals compared to that of a saline treated control. The significance of these findings are discussed in terms of known neurochemical changes i.e., an increase of both brain tryptophan and serotonin in rats, induced by a subconvulsive dose of exogenous insulin.     

Protective effect of a caspase inhibitor in models for cerebral ischemia in vitro and in vivo.

In primary neuronal-astrocyte cultures from mouse brain, ischemic conditions were simulated by combined oxygen-glucose deprival (OGD) for 2 hrs. This treatment resulted in near complete neuronal damage 24 hrs. later and was accompanied by DNA degradation and apoptotic nuclear morphology. Since caspases are key enzymes in the propagation and execution of programmed cell death, we evaluated the effect of the caspase inhibitor z-VAD-fmk. Damage following 2 hrs. OGD could be reduced by up to 56% with z-VAD-fmk (p<0.05). DNA-fragmentation and caspase activation has been also reported in an in vivo model of cerebral ischemia imitating human stroke. In this model the middle cerebral artery (MCA) is permanently occluded resulting in focal cerebral ischemia and subsequent infarction. Since z-VAD.fmk does not penetrate the blood-brain barrier it was applied intraventricularly as a bolus injection given 30 min. before MCA occlusion which was followed by 24 hrs. of infusion. Infarct volume was determined 48 hrs. after MCA occlusion by means of in vivo magnetic resonance imaging. Z-VAD.fmk dose dependently reduced infarct volume reaching a significant decrease of the cortical infarct by 45% when given as a 120 ng bolus followed by 40 ng/hr. infusion (p<0.05). In summary, our study supports the concept that caspase inhibitors are beneficial in brain ischemia.     

Differential responsiveness to replacement therapy of pre- and postpubertally castrated mice with respect to intermale aggression

Male mice castrated before Day 6 of postnatal life differ from adult castrates in that they do not exhibit normal patterns of intermale aggression following replacement therapy as adults. Two experiments sought to determine the effect castration after Day 6, but before puberty, would have on this response to adult replacement therapy. It was found that adult castrates showed some increases in fighting as early as six hr after a subcutaneous (s.c.) injection of 2 mg testosterone propionate (TP). At 30 hr after treatment 14/15 mice fought, while at 72 hr 15/15 fought and the number of fights in 10 min was significantly higher than at 30 hr. In the second experiment, the response to adult treatment with 2 mg TP (s.c.) in mice castrated in Day 10 or Day 50 was compared. Different groups were tested at 16, 40, 64, and 88 hr after TP treatment. The latency to respond to TP was significantly less and the level of fighting obtained was significantly greater in Day 50 castrates than in Day 10 castrates at each time of testing. The proportion of Day 10 (14/15) and Day 50 (15/15) castrates fighting was equal by 88 hr, while the mean fighting frequency was significantly lower in Day 10 castrates (M = 4.93 ± 4.78) than in Day 50 castrates (M = 8.06 ± 1.63). Day 50 castrates fought significantly more often than controls at 40 hr, whereas the level of fighting attained by Day 10 castrates was not significant until 64 hr. These results suggest that even after the organizational period for aggression, testosterone is necessary for maintenance and/or preparation of the substrate essential to the elicitation of aggression.     

Prolactin messenger ribonucleic acid levels, prolactin synthesis, and radioimmunoassayable prolactin during the estrous cycle in the golden Syrian hamster

The purpose of this study was to observe the molecular dynamics of pituitary prolactin (PRL) gene expression during the estrous cycle of the Golden Syrian hamster. PRL messenger ribonucleic acid (mRNA) levels, PRL synthesis (3H-PRL in the incubation media or incubated pituitary after a 3 hr incubation with 3H-leucine), and radioimmunoassayable (RIA) PRL (in the incubation media or incubated pituitary after the 3 hr incubation) were measured in the morning (0930-1100 hr) on each day of the cycle. We observed that all of these PRL indices declined or did not change from Day 2 to Day 3 of the cycle. From Day 3 to Day 4 (proestrus), however, PRL mRNA levels increased 33-38% and media 3H-PRL increased 32-42%, while there were no significant changes in pituitary 3H-PRL, or RIA-PRL in the media or pituitary. From Day 4 to Day 1 (estrus) there was a reciprocal change in the levels of 3H-PRL in the pituitary vs. the media, with the former increasing 37-50% and the latter decreasing 25-32%. Pituitary RIA-PRL also increased 45-64% from Day 4 to Day 1 while media RIA-PRL did not change. These data are consistent with the following hypothesis: On the morning of proestrus (Day 4) in the hamster, PRL mRNA levels are elevated compared to those on Day 3, signaling an increase in PRL synthesis. This newly synthesized PRL is shunted into a "readily releasable" pool on the morning of Day 4 (contributing to the afternoon surge of serum PRL), and into a "preferentially stored" pool by the morning of Day 1 (for release in response to cervical stimulation and use as a luteotrophin to maintain early pregnancy should fertilization occur).     

Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by (13)C NMR and microdialysis combined with gas-chromatography/mass-spectrometry. Brain GLU C5 was (13)C enriched by intravenous [2,5-(13)C]glucose infusion, followed by [(12)C]glucose infusion to chase (13)C from the small glial GLU pool. This leaves [5-(13)C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the (13)C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU(ECF)) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5-(13)C]GLU(ECF) was monitored in vivo through the formation of [5-(13)C,(15)N]GLN during (15)NH(4)Ac infusion. From the rate of [5-(13)C,(15)N]GLN synthesis (1.7 +/- 0.03 micromol/g/h), the mean (13)C enrichment of extracellular GLU (0.304 +/- 0.011) and the (15)N enrichment of precursor NH(3) (0.87 +/- 0.014), the rate of synthesis of GLN (V'(GLN)), derived from neurotransmitter GLU(ECF), was determined to be 6.4 +/- 0.44 micromol/g/h. Comparison with V(GLN) measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 +/- 0.44 micromol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.