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The problem Medical students graduate underprepared for postgraduate medical training despite years of classroom and clinical training. In this article, a medical student shares her personal perspectives on three factors contributing to this problem in undergraduate medical education: students’ peripheral roles in the clinical environment impede learning, students receive inadequate feedback, and assessments do not measure desired learning outcomes. A solution The authors describe how using entrustable professional activities (EPAs) could address these issues and promote students’ clinical engagement by clarifying their roles, providing them with frequent and actionable feedback, and aligning their assessments with authentic work. These factors combined with grading schemes rewarding improvement could contribute to a growth mindset that reprioritizes clinical skill acquisition. The authors explore how medical schools have begun implementing the EPA framework, highlight insights from these efforts, and describe barriers that must be addressed. The future Incorporating EPAs into medical school curricula could better prepare students for postgraduate training while also alleviating issues that contribute to student burnout by defining students’ roles, improving feedback, and aligning assessments with desired learning outcomes.

Medical students graduate underprepared for postgraduate medical training despite years of classroom and clinical training. In this article, a medical student shares her personal perspectives on three factors contributing to this problem in undergraduate medical education: students' peripheral roles in the clinical environment impede learning, students receive inadequate feedback, and assessments do not measure desired learning outcomes.The authors describe how using entrustable professional activities (EPAs) could address these issues and promote students' clinical engagement by clarifying their roles, providing them with frequent and actionable feedback, and aligning their assessments with authentic work. These factors combined with grading schemes rewarding improvement could contribute to a growth mindset that reprioritizes clinical skill acquisition. The authors explore how medical schools have begun implementing the EPA framework, highlight insights from these efforts, and describe barriers that must be addressed.Incorporating EPAs into medical school curricula could better prepare students for postgraduate training while also alleviating issues that contribute to student burnout by defining students' roles, improving feedback, and aligning assessments with desired learning outcomes.

The development of chronic kidney disease (CKD) following an episode of acute kidney injury (AKI) is an increasingly recognized clinical problem. Inhibition of toll-like receptor 4 (TLR4) protects renal function in animal models of AKI and has become a viable therapeutic strategy in AKI. However, the impact of TLR4 inhibition on the chronic sequelae of AKI is unknown. Consequently, we examined the chronic effects of TLR4 inhibition in a model of ischemic AKI. Mice with a TLR4-deletion on a C57BL/6 background and wild-type (WT) background control mice (C57BL/6) were subjected to bilateral renal artery clamping for 19 min and reperfusion for up to 6 weeks. Despite the acute protective effect of TLR4 inhibition on renal function (serum creatinine 1.6 ± 0.4 mg/dL TLR4-deletion vs. 2.8 ± 0.3 mg/dL·WT) and rates of tubular apoptosis following ischemic AKI, we found no difference in neutrophil or macrophage infiltration. Furthermore, we observed significant protection from microvascular rarefaction at six weeks following injury with TLR4-deletion, but this did not alter development of fibrosis. In conclusion, we validate the acute protective effect of TLR4 signal inhibition in AKI but demonstrate that this protective effect does not mitigate the sequential fibrogenic response in this model of ischemic AKI.

Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used for treatment of pain and arthritis. In the body, acetylsalicylic acid is rapidly deacetylated to form salicylic acid. Both compounds have been proposed as anti-inflammatory agents. Major metabolites of salicylic acid are its acyl and phenolic glucuronide conjugates. Formation of these conjugates, catalyzed by UDP-glucuronosyltransferases (UGTs), decreases the amount of pharmacologically active salicylic acid present. We aimed to identify the UGTs catalyzing the glucuronidation of salicylic acid using both heterologously expressed enzymes and pooled human liver microsomes (HLMs) and to develop a liquid chromatography-tandem mass spectrometry method to quantify glucuronidation activity of UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 Supersomes. All UGTs tested, except 1A4, 2B15, and 2B17, catalyzed salicylic acid phenolic and acyl glucuronidation. Ratios of salicylic acid phenolic to acyl glucuronide formation varied more than 12-fold from 0.5 for UGT1A6 to 6.1 for UGT1A1. These results suggest that all UGTs except 1A4, 2B15, and 2B17 might be involved in the glucuronidation of salicylic acid in vivo. From comparisons of apparent Km values determined in pooled HLMs and in expressed UGTs, UGT2B7 was suggested as a likely catalyst of salicylic acid acyl glucuronidation, whereas multiple UGTs were suggested as catalysts of phenolic glucuronidation. The results of this UGT screening may help target future evaluation of the effects of UGT polymorphisms on response to aspirin in clinical and population-based studies.

Prior investigations have shown that localized infusion by microdialysis of gamma-aminobutyric acid(B) (GABA(B)) agonists into the medial basal hypothalamus of male sheep rapidly increases GnRH and LH pulse amplitude. The objectives of these studies were to determine if infusion of GABA(B) agonists SKF 97541 or baclofen into the medial basal hypothalamus of female sheep would affect basal LH secretion and if infusion of a potent antagonist would alter expression of LH surges induced by injection of estrogen. Infusion of either SKF 97541 (10 or 40 microM) or baclofen (1 mM) into estrogen-treated ovariectomized ewes did not alter basal LH secretory patterns, whereas both drugs significantly elevated mean LH and LH pulse amplitude in ovariectomized ewes during the nonbreeding season. Infusion of the antagonist CGP 52432 (250 or 500 microM) did not affect expression of estrogen-induced LH surges in ovariectomized ewes. These observations support the concept that GABA(B) receptors in the medial basal hypothalamus regulate basal LH secretion but do not regulate the surge mode of LH secretion in the female sheep.

The frequency of pulsatile release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) is high in the breeding season and low in the nonbreeding season. These alterations in the patterns of GnRH and LH release are due to an interaction of daylength and gonadal steroid negative feedback. A vast amount of data indicates that steroid-responsive neural systems may play a role in regulating seasonal changes in GnRH release. One candidate system is neuropeptide Y (NPY). To determine the independent and interactive influences of photoperiod and steroid exposure on NPY mRNA levels, we used hypothalamic tissue from four groups (n = 4 per group) of castrated male sheep that were simultaneously housed in photochambers and exposed to: (1) a 16L:8D photoperiod (LD); (2) LD and implanted with testosterone (LD + T); (3) a 10L:14D photoperiod (SD), and (4) SD + T. Circulating levels of T averaged 2.8 ± 0.2 ng/ml in implanted animals, but were undetectable in nonimplanted males. Mean LH levels were significantly reduced (p < 0.01) in the LD + T group as compared with the other groups which did not differ from each other. The silver grain area per NPY neuron in the arcuate nucleus, as assessed by in situ hybridization, was inversely related to mean LH values, with the grain area per cell being significantly greater (p < 0.05) for LD + T males than for all other groups which did not differ from each other. NPY cell numbers were not significantly different (p > 0.10) among the treatment groups. These results show that NPY mRNA expression is increased in male sheep during a LD photoperiod in a T-dependent manner. Our data are consistent with the idea that NPY is involved in the seasonal regulation of GnRH and LH release in the male sheep.

Male rats, 30 d old, were treated with the antiestrogen ICI 182,780 (3–150 d) to determine sequences of events leading to testicular atrophy and infertility. Plasma testosterone and LH concentrations were unchanged. ICI 182,780 induced dilation of efferent ductules as early as 3 d post treatment, and the dilation increased over time, resulting in an overall increase of 200% in tubule diameter. A gradual reduction in height of the ductule epithelium was observed; however, the microvilli height increased up to d 73 but subsequently decreased. A transient increase in lysosomes in nonciliated cells was seen from d 15 to d 100. Testicular weight increased by d 45 and seminiferous tubules were dilated by d 52. These effects on testes persisted until d 100, but on d 150 the weight decreased and severe atrophy was observed. These testicular effects were probably owing to accumulation of fluid following inhibition of reabsorption in the efferent ductules, similar to the ER-α knockout mouse. In agreement with this conclusion, there was a decrease in Na+-H+ exchanger-3 mRNA and protein, which is consistent with previous studies showing that ER is required for expression of Na+-H+ exchanger-3 and ultimately fluid reabsorption in the efferent ductules.

The objectives of this study were to determine if the gamma-aminobutyric acid (GABA)(B) agonist, 3-aminopropyl (methyl) phosphinic acid (SKF97541), would increase luteinizing hormone (LH) secretion when infused by microdialysis into the medial basal hypothalamus (MBH) of the castrated ram, and to determine if the action of SKF97541 would be attenuated by co-infusion of the GABA(B) antagonist CGP52432. Initial experiments established that infusion of SKF alone, at concentrations as low as 5 microM, increased mean LH, LH pulse amplitude, and in some cases, pulse interval. In the last experiment, animals were treated with artificial cerebrospinal fluid (CSF) alone, SKF alone (30 microM), 3-[[(3, 4-dichlorophenol) methyl] amino] propyl] diethoxymethyl) phosphinic acid (CGP) alone (500 microM), or SKF plus CGP. SKF increased both mean LH and LH pulse amplitude as compared with CSF. CGP alone had no significant effect on LH, but it attenuated the effect of SKF on mean LH. These observations indicate that the stimulatory effects of GABA(B) agonists on LH pulse patterns are mediated through GABA(B) receptors and provide further evidence that GABA(B) receptors located in the MBH can regulate pulsatile GnRH-LH release.

This study tested the hypothesis that photoperiod affects the ability of testosterone to reduce proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus and luteinizing hormone-releasing hormone (LHRH) mRNA levels in both the preoptic area (POA) or medial basal hypothalamus (MBH). Twenty castrated male sheep were assigned to one of four treatment groups (i): short days (SD; n=5) (ii), short days with testosterone (SD+T; n=5) (iii), long days (LD; n=5), or (iii) long days with testosterone (LD+T; n=5). Blood samples were collected twice weekly for the last 3 weeks of photoperiod treatment and assessed for LH to validate the response to photoperiod. After evaluating LH levels, one animal each from the LD+T and SD+T groups was excluded from the analyses. Mean concentrations of LH were lower (P 0.10) from each other. Neither POA nor MBH LHRH mRNA levels were affected (P>0.10) by treatment. Conversely, POMC mRNA levels were suppressed (P 0.10) from each other. These observations suggest that photoperiod specific, testosterone-induced alterations in LHRH mRNA levels are not a mechanism whereby testosterone suppresses LHRH release, and that increased beta-endorphin synthesis and release do not mediate testosterone-induced seasonal suppression of LHRH release.

Infusion of baclofen, a GABAB agonist, into the medial basal hypothalamus (MBH) of castrated rams rapidly increases LH pulse amplitude without altering pulse frequency. The objectives of this study were to determine whether baclofen infusion increased LH in testosterone (T)-treated and intact rams, the increased LH was due to increased GnRH release, and FSH secretion also was increased. In the first experiment we tested the main effects and interaction of baclofen and T on FSH and LH pulse patterns in castrated rams (n = 7). In the second experiment we determined whether baclofen affected GnRH and LH pulses in intact males. Microdialysis guide cannulae were implanted bilaterally into the MBH. After recovery of the animal from surgery, the MBH was perfused using concentric microdialysis probes (2-mm tip) with artificial cerebrospinal fluid (aCSF) for a 3-h control period followed by either aCSF or 1 mm baclofen for 4 h. Blood samples were taken at 10-min intervals. T suppressed mean LH concentrations (10.4...

This study tested the hypothesis that the effects of the opiate antagonist naloxone on GnRH (and LH) secretion is affected by photoperiod length and testosterone (T) concentrations. The effect of infusing naloxone on GnRH and LH pulse patterns was determined in four groups of orchidectomized sheep: long day (LD) photoperiod treated with T, LD without T (LDC), short day photoperiod (SD) with T, SDC (n = 5-7/group). Hypophyseal-portal and jugular blood samples were collected at 10 min intervals for 4 h before and 4 h during naloxone infusion (1 mg/kg/h). Neither photoperiod nor T affected either mean GnRH or LH whereas naloxone (P0.01) increased both. LD photoperiod (P0.01), T (P0.01) and naloxone (P0.01) all increased LH pulse amplitude whereas only naloxone increased GnRH pulse amplitude (P0.01). There was an interaction (P0.01) between steroid and naloxone on LH, but not GnRH, pulse amplitude. Both LD photoperiod and T increased both LH and GnRH (P0.01) interpulse-interval (IPI). Naloxone decreased GnRH IPI (P0.01). The LH/GnRH pulse amplitude ratio was (P0.02) increased by T--likely a secondary response to the T-induced increase in IPI. These results are interpreted as showing that in the ram the endogenous opiate peptides regulate both GnRH pulse frequency and amplitude, but that their specific role is modulated by photoperiod and T. These results do not support the concept that the opiate peptides are the primary mediators of the negative feedback effects of T.

The Age-Related Eye Disease Study (AREDS) was initially conceived as a long-term multicenter, prospective study of the clinical course of age-related macular degeneration (AMD) and age-related cataract. Data on progression rates and risk factors from the study will increase understanding of the clinical course of both conditions, generate hypotheses about etiology, and aid in the design of clinical trials of potential interventions. In addition to collecting natural history data, AREDS includes a clinical trial of high-dose vitamin and mineral supplements for AMD and a clinical trial of high-dose vitamin supplements for cataract. The clinical trials were initiated largely because of the widespread public use in the United States of commercially available pharmacologic doses of vitamins and minerals to treat these two eye conditions and the absence of definitive studies on the safety and efficacy of their use. Important design issues for the clinical trials include: defining cataract and AMD, estimating event rates, determining the type and dosage of vitamins and minerals to be tested for each condition, and identifying the parameters necessary for monitoring safety and efficacy. This paper describes the AREDS design, including the study rationale and operational structure, and the approach adopted to combine, for two diseases, clinical trials with a natural history study. Control Clin Trials 1999;20:573–600

We tested the hypothesis that rapidly expressed inhibitory effects of estradiol (E) on luteinizing hormone (LH) release in the male are attributable, in part, to suppression of luteinizing hormone-releasing hormone (LHRH) release. Hypophyseal-portal cannulated, castrated male sheep were infused with E (15 ng/kg/hr) or vehicle. Portal and jugular blood samples were collected at 10-min intervals for 4 hr before, and for either 12 hr (E, n = 4; vehicle, n = 4) or 24 hr (E, n = 8; vehicle, n = 3) after the start of infusion. In animals sampled for 16 hr, temporal changes in both LHRH and LH were assessed. In animals sampled for 28 hr, only LH data were analyzed. Before either the 12-hr or 24-hr infusion, LHRH and/or LH mean concentrations, pulse amplitude and interpulse interval (IPI) did not differ between E- and vehicle-infused animals. In animals sampled for 16 hr, no effects of time or steroid × time interactions were detected for mean LHRH and LHRH pulse amplitude; however, both were greater (P < 0.01) in vehicle-infused than in E-infused males. LHRH IPI was unaffected by infusion. In contrast, both mean LH and LH pulse amplitude declined (P < 0.01) within 4–8 hr after the start of E infusion, whereas mean LH IPI was unaffected. In animals sampled for 28 hr, an effect of time (P < 0.01) and a steroid × time interaction (P < 0.01) was detected for mean LH, and there was an effect of time (P < 0.01) on LH pulse amplitude. Mean LH IPI was not affected. Our results show that in male sheep E rapidly reduces LH release in the absence of a detectable change in LHRH release.

We investigated the effects of microdialyzing γ-aminobutyric acid (GABA) receptor antagonists into either the medial preoptic area (mPOA) or the arcuate-ventromedial region (ARC-VMR) on LH secretion. Bicuculline methiodide (BMI, GABAA receptor antagonist), and either 2-hydroxysaclofen (SAC)

Testosterone (T) inhibits LH secretion partly by acting at unknown sites within the brain to inhibit GnRH secretion. We tested the hypothesis that the preoptic area (POA) and arcuate-ventromedial region (ARC/VMR), areas rich in androgen and estrogen (E) receptors, are neural sites at which T and the T metabolites, dihydrotestosterone (DHT) and estrogen (E), act to suppress LH secretion. Bilateral guide cannulae were surgically implanted into either the POA or ARC/VMR of castrated male sheep. Experiments were conducted under a long day photoperiod to maximize the inhibitory effect of the steroids. In Exp 1, all sheep (n = 6/site) sequentially received bilateral implants of cholesterol (CHOL), T, or E at each site. Jugular blood samples were taken at 10-min intervals for 4 h both immediately before implant insertion and 5 days later. In Exp 2, all sheep (n = 6/site) sequentially received bilateral implants of CHOL, DHT, or E at each site according to a latin square design. Blood samples were taken before an...

The final common pathway controlling reproductive function in vertebrates is the GnRH neuron and its projection to the median eminence (ME), site of peptide release into the pituitary portal system. GnRH neurons are widely distributed; therefore we sought to test the hypothesis that those projecting to the ME are located in specific regions. We used as a model the sheep, a species in which a great deal of information regarding the physiology of GnRH secretion is known. To identify cells projecting to the ME (i.e., neuroendocrine neurons), ewes (n = 10) received injections into the ME of neuronal tract-tracing compounds: cholera toxin-beta subunit (CT-beta) or one of two fluorescent compounds (rhodamine isothiocyanate or fluorescein-conjugated dextran). Forty-eight h later, animals were perfused intracranially and their brains were processed for immunocytochemical localization of GnRH and CT-beta using a dual-immunofluorescent procedure or by single-label immunofluorescent visualization of GnRH combined with direct visualization of fluorescent tracers. Small, well-circumscribed injections into the ME were made successfully in 6 of 10 animals, and these overlapped the location of GnRH terminals and fibers. Neuroendocrine GnRH neurons (those GnRH neurons containing retrogradely transported tracer) were identified throughout their previously reported range: within the diagonal band of the Broca/medial septal region, medial preoptic area (MPOA), anterior hypothalamic area, and medial basal hypothalamus. Although the absolute number of neuroendocrine GnRH neurons varied by region, the percentage of the total GnRH population within each of these areas that was retrogradely labeled did not differ (p > 0.05). Injections placed unilaterally within the ME labeled a similar proportion of GnRH cells both ipsilateral and contralateral to the injection site in all areas except the MPOA, where ipsilaterally labeled cells were approximately twice as numerous as those labeled contralaterally. Injections that missed the ME and were placed either into the third ventricle or into the arcuate nucleus labeled only 0.5% and 4-11% of GnRH neurons, respectively. These results do not support the hypothesis that in the ewe, GnRH neurons projecting to the ME are localized to specific regions. Thus, we postulate that GnRH release into the hypophyseal portal system reflects the output of GnRH neurons located in multiple areas.

The mechanism whereby testosterone (T) reduces pulsatile LHRH and LH release is unknown. We tested the hypothesis that hypothalamic levels of LHRH mRNA decrease and proopiomelanocortin (POMC) mRNA increase coincident with reduced LHRH release induced by either long-term or short-term T treatment in male sheep. Experiment 1 examined the effect of long-term T exposure on LHRH and LH release and LHRH and POMC mRNA levels. Yearling Suffolk rams were castrated and assigned to one of four treatments: 1) castrated (n = 4); 2) castrated, portal cannula (n = 5); 3) castrated+T (n = 4) and 4) castrated+T, portal cannula (n = 4). T-treated males received ten 10-cm silastic T-implants immediately after castration. Surgical placement of devices for collecting hypophyseal-portal blood occurred 2 to 3 months after castration. Seven to 10 days after surgery, blood samples were collected at 10-min intervals for 8 h from portal cannulated males or for 5 h from non-cannulated males to assess pulsatile LHRH and/or LH release. Immediately after blood sample collection, hypothalamic tissue was collected for in situ measurement of LHRH or POMC mRNA. T-treatment decreased (P0.01) mean LHRH and LH and decreased (P0.01) LHRH and LH pulse frequency. T did not significantly affect (P0.10) silver grain area per LHRH neuron, but decreased (P0.01) silver grain area per POMC neuron. Portal cannulation tended to decrease (P = 0.057) silver grain area per LHRH neuron without significantly affecting (P0.10) LHRH cell numbers while reducing (P0.01) silver grain area per POMC neuron and POMC cell numbers. A second experiment examined the effect of 72 h of T-infusion on LHRH and POMC mRNA levels. Castrated yearling males were assigned to receive either vehicle (n = 4) or T (768 ug/kg/day; n = 4). Blood samples were collected at 10 min intervals for 4 h prior to and during the final 4 h of infusion. Infusion of T decreased (P0.01) mean LH and LH pulse frequency. T did not significantly affect (P0.10) silver grain area per LHRH neuron or LHRH cell numbers. T reduced (P0.01) silver grain area per POMC neuron without affecting (P0.10) POMC cell number. We reject our hypothesis and conclude that reduced LHRH or heightened POMC gene expression are not mechanisms whereby T reduces pulsatile LHRH release in male sheep.

To determine whether the photoperiodic responses of reproductive and prolactin (PRL) rhythms in the ewe requires an intact suprachiasmatic nucleus (SCN) driving the pineal rhythm of melatonin secretion, four groups of ovary-intact ewes over a 6-year period were subjected to bilateral (n = 40) or sham lesions (n = 15) of the SCN. Animals were exposed to an alternating 90–120 day photoregimen of 9L:15D and 16L: 8D photoperiods. Blood samples taken twice weekly were assayed for prolactin and for progesterone to monitor oestrous cycles. On several occasions blood samples also were taken at hourly intervals for 24 h and analyzed for melatonin. Melatonin concentrations in sham lesioned ewes were basal during the lights-on period and rose robustly during darkness. Those sheep bearing unilateral lesions of the SCN (n = 13) or where the lesion spared the SCN entirely (n = 8) had patterns of melatonin secretion similar to sham ewes. The remaining ewes, having complete (n = 9) or incomplete bilateral (n = 8) destruction of the SCN, with one exception, had disrupted patterns of melatonin secretion. The nature of this disruption varied from complete suppression to continuously elevated levels. In lesioned ewes where melatonin secretion was not affected the onset and cessation of ovarian cycles were similar to sham ewes; stimulation of oestrous cycles under 9L:15D and cessation of oestrous cycles under 16L:8D. In contrast, 13 of 17 ewes with disrupted melatonin secretion also exhibited disrupted patterns of ovarian activity. In these animals oestrous cycles were no longer entrained by photoperiod but still occurred in distinct clusters, that is, groups of cycles began and ended spontaneously. Sheep with normal melatonin patterns showed low levels of PRL secretion during short days and elevated PRL levels during long days. However, 8 of 13 ewes with disrupted melatonin showed patterns of PRL secretion that were no longer entrained by photoperiod. A minority of ewes with disrupted melatonin patterns still showed reproductive (n = 4) and PRL (n = 5) responses similar to those of sham-lesioned ewes. These results show that bilateral destruction of the SCN in the ewe disrupts the circadian pattern of melatonin secretion and that this disruption usually, but not always, is associated with altered photoperiodic responses. These results strongly suggest that the SCN are important neural elements within the photoperiod time–keeping system in this species. A role for the SCN in the generation of endogenous transitions in reproductive activity (refractoriness) and prolactin secretion is not supported.

In the rat, p53 promotes tubular apoptosis after ischemic AKI. Acute pharmacologic inhibition of p53 is protective in this setting, but chronic inhibition enhances fibrosis, demonstrating that the role of p53 in ischemic AKI is incompletely understood. Here, we investigated whether genetic absence of p53 is also protective in ischemic AKI. Surprisingly, p53-knockout mice (p53(-/-)) had worse kidney injury, compared with wild-type mice, and exhibited increased and prolonged infiltration of leukocytes after ischemia. Acute inhibition of p53 with pifithrin-α in wild-type mice mimicked the observations in p53(-/-) mice. Chimeric mice that lacked p53 in leukocytes sustained injury similar to p53(-/-) mice, suggesting an important role for leukocyte p53 in ischemic AKI. Compared with wild-type mice, a smaller proportion of macrophages in the kidneys of p53(-/-) and pifithrin-α-treated mice after ischemic injury were the anti-inflammatory M2 phenotype. Ischemic kidneys of p53(-/-) and pifithrin-α-treated mice also showed reduced expression of Kruppel-like factor-4. Finally, models of peritonitis in p53(-/-) and pifithrin-α-treated mice confirmed the anti-inflammatory role of p53 and its effect on the polarization of macrophage phenotype. In summary, in contrast to the rat, inflammation characterizes ischemic AKI in mice; leukocyte p53 is protective by reducing the extent and duration of this inflammation and by promoting the anti-inflammatory M2 macrophage phenotype.

The extent to which inhibitory effects of testosterone (T) on LH secretion in the ram are mediated by its metabolite dihydrotestosterone (DHT) is unknown. Our objective was to determine the effect of inhibiting 5a-reductase activity on pulsatile patterns of LH release in castrated, T-treated male sheep. Nine Dorset and six Hampshire castrated male sheep were allocated equally to one of three treatment groups: 1) infusion of T (768 tLg/kg/day), 2) infusion of the reductase inhibitor (RI) L-651,723 (0.6 mg/kg/day), and 3) T+RI infusion. Treatments were continuously infused for 3 days. Blood samples were collected via an indwelling jugular catheter at 10-min intervals for 4 h immediately prior to (Day 0) and during the final 4 h of infusion (Day 3). Changes in mean LH, LH pulse amplitude, LH interpulse interval (IPI), T, 17B-estradiol (E), and DHT were derived for each animal by subtracting values for Day 0 from Day 3. Data were subjected to one-way analysis of variance. The increase in T and E after infusion of T was similar (p > 0.10) in T- and T+RI-treated males and greater (p 0.10) for T+RI- and RI-treated males. T decreased mean LH more (p 0.10) males. Changes in LH pulse amplitude were similar (p > 0.10) for T- and RI-treated males and for T+RI- and RI-treated males. However, LH pulse amplitude was increased (p < 0.05) by T+RI treatment compared to T treatment alone. We conclude that the conversion of T to DHT by 5a-reductase represents a physiologically important step whereby T inhibits LH secretion in the ram.

In this study we examined the relationship between GnRH, LH, and testosterone (T) in intact rams during the nonbreeding season, the breeding season, and the transition between breeding and nonbreeding season. Blood was collected from the hypophyseal portal and jugular veins at 10-min intervals for 12 h from 3 groups of rams in May, September, and March. Individual LH pulses in May and September occurred at relatively regular intervals whereas in March, groups of pulses were separated by relatively long intervals without pulses. No significant effect of season on GnRH pulse parameters was found, but there was a significant effect of season on LH (p < 0.04) and T (p < 0.001) pulse amplitude and on mean T concentrations (p < 0.001). LH pulse amplitude was highest in March, and T pulse amplitude was highest in September. Simple indices of pituitary and testicular responsiveness were obtained by calculating the ratios of LH to GnRH and T to LH. The ratio of LH to GnRH pulse amplitude was significantly higher in March than in September (p < 0.01) or May (p < 0.05). The ratio of T to LH pulse amplitude was highest (p < 0.01) in September. Release of LH in response to exogenous GnRH also varied significantly (p < 0.01) with month, being higher in March than in September (p < 0.01) or May (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The objective was to determine the effect of chronic testosterone (T) treatment on GnRH and LH secretion in wethers. Rams were either castrated only or castrated and immediately treated with Silastic implants containing T. Several weeks later, a device for collecting hypophyseal-portal blood was surgically implanted. Six to seven days later, blood samples were collected simultaneously and continuously from the portal vessels and jugular vein of pairs of conscious animals. Samples were divided at 10-min intervals for 6-12 h. One hour before the end of collection, all animals received i.v. injections of 250 ng of GnRH. In samples collected simultaneously from 6 pairs of animals, T reduced the frequency of both GnRH pulses (1.8 +/- 0.2 vs. 0.9 +/- 0.3/h, p less than 0.03) and LH pulses (1.6 +/- 0.1 vs. 0.8 +/- 0.3/h, p less than 0.03). T did not alter amplitude of either GnRH or LH pulses. Testosterone reduced mean GnRH (9.7 +/- 0.6 vs. 7.9 +/- 0.5 pg/ml, p less than 0.05), whereas mean LH was not significantly reduced (9.6 +/- 1.4 vs. 6.1 +/- 1.8 ng/ml, p = 0.16). These results support the hypothesis that T reduces GnRH pulse frequency.

In vivo, aspirin (acetylsalicylic acid) is rapidly deacetylated to form salicylic acid, which then undergoes primary or secondary glucuronidation catalyzed by UDP-glucuronosyltransferases (UGTs). The variant UGT1A6*2 (T181A, R184S) is associated with altered enzyme function. Our objective was to compare salicylic acid glucuronidation in individuals with different UGT1A6 genotypes.Following orally dosing with 650 mg aspirin, saliva and urine samples were collected over a period of 24 h from healthy individuals with homozygous wild-type UGT1A6 *1/*1 (n=19) and homozygous variant UGT1A6 *2/*2 (T181A, R184S) (n=9) genotypes.No statistically significant differences were observed in salivary pharmacokinetic parameters. Urinary excretion of the sum of aspirin and its metabolites (salicyluric acid, salicyluric acid phenolic glucuronide, salicyl phenolic glucuronide, salicyl acyl glucuronide, salicylic acid) during the early period of 2-4 h of collection was significantly lower in UGT1A6 *1/*1 than in UGT1A6 *2/*2 individuals. Further, UGT1A6 *1/*1 individuals excreted a lower percentage of aspirin and its metabolites in the first 12 h and a greater percentage after 12 h than UGT1A6 *2/*2 individuals.The variant UGT1A6*2 or polymorphisms in other UGTs that are in linkage disequilibrium with UGT1A6*2 may confer more rapid glucuronidation of salicylic acid than the wild-type UGT1A6 *1/*1.

Nonsteroidal anti-inflammatory drugs (NSAIDs), used for the treatment of pain and inflammation, are eliminated primarily through conjugation with polar sugar moieties to form glucuronides. Glucuronidation is catalyzed by the UDP-glucuronosyltransferases (UGT) superfamily. An inverse relationship may exist between glucuronidation activity and NSAID efficacy; however, specific UGTs catalyzing conjugation of the structurally diverse NSAIDs have yet to be identified systematically. Therefore, NSAID glucuronidation activity by 12 individually expressed UGTs was investigated by liquid chromatography-tandem mass spectrometry. The relative rates of NSAID glucuronidation varied among UGT enzymes examined, demonstrating specificity of the individual UGTs toward selected NSAIDs. Kinetic parameters were determined for expressed UGT Supersomes and compared with parameters determined in pooled human liver microsomes (HLMs). Comparison of K(m) values suggested roles for UGTs 1A3 and 2B7 in indene glucuronidation and UGTs 1A9, 2B4, and 2B7 in profen glucuronidation. Inhibitory studies in pooled HLMs support the role of UGTs 1A1, 1A3, 1A9, 2B4, and 2B7 in the glucuronidation of ibuprofen, flurbiprofen, and ketoprofen. Bilirubin did not inhibit indomethacin or diclofenac glucuronidation, suggesting that UGT1A1 was not involved in catalysis. Imipramine did not inhibit glucuronidation of sulindac, sulindac sulfone, indomethacin, or naproxen in pooled HLMs, suggesting that UGT1A3 was not a principal hepatic catalyst. Nevertheless, multiple UGT enzymes, most notably UGTs 1A1, 1A9, 2B4, and 2B7, seem to be involved in the hepatic catalysis of NSAID glucuronidation.

trans-3'-Hydroxycotinine is the major nicotine metabolite excreted in the urine of smokers and other tobacco or nicotine users. On average, about 30% of the trans-3'-hydroxycotinine in urine is present as a glucuronide conjugate. The O-glucuronide of trans-3'-hydroxycotinine has been isolated from smokers urine and appears to be the major glucuronide conjugate of trans-3'-hydroxycotinine in urine. However, nicotine and cotinine are both glucuronidated at the nitrogen atom of the pyridine ring. We report here that human liver microsomes catalyze both the N-glucuronidation and the O-glucuronidation of trans-3'-hydroxycotinine. The N-glucuronide was purified by HPLC, and its structure was confirmed by NMR. Both N- and O-glucuronidation of trans-3'-hydroxycotinine were detected in 13 of 15 human liver microsome samples. The ratio of N-glucuronidation to O-glucuronidation was between 0.4 and 2.7. One sample only catalyzed N-glucuronidation, and one sample did not catalyze either reaction. The rates of N-glucuronidation varied more than 6-fold from 6 to 38.9 pmol/min/mg protein; rates of O-glucuronidation ranged from 2.8 to 23.4 pmol/min/mg protein. The rate of trans-3'-hydroxycotinine N-glucuronidation by human liver microsomes correlated well with both the rate of nicotine and the rate of cotinine N-glucuronidation. trans-3'-Hydroxycotinine O-glucuronidation correlated with neither nicotine nor cotinine N-glucuronidation. These results suggest that the same enzyme(s) that catalyzes the N-glucuronidation of nicotine and cotinine may also catalyze the N-glucuronidation of trans-3'-hydroxycotinine in the human liver but that a separate enzyme catalyzes trans-3'-hydroxycotinine O-glucuronidation.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a lung carcinogen in the rat and believed to be a causative agent for lung cancer in smokers. To exert its carcinogenic potential NNK is metabolically activated by cytochrome P450-catalyzed alpha-hydroxylation. Nicotine and NNK share metabolic pathways. The purpose of this study was to determine whether nicotine alters the fraction of NNK undergoing activation to carcinogenic metabolites. Rats received acute (36 h) or chronic (2 week) s.c. infusions of nicotine at rates producing serum nicotine concentrations 2-3 times the usual venous nicotine concentrations measured in smokers. A single dose of [5-3H]-NNK was administered i.p. 24 h prior to the end of each infusion. Urine was collected for 24 h thereafter and NNK metabolites quantified by radioflow HPLC. Neither acute nor chronic nicotine infusion had any effect on the extent of NNK metabolism by alpha-hydroxylation. These data suggest that nicotine infusion, at rates simulating the higher doses of nicotine replacement therapy now being studied for smoking cessation, neither induces nor inhibits NNK metabolism appreciably and therefore should not alter the formation of carcinogenic NNK metabolites.

Nicotine is considered the major addictive agent in tobacco. Tobacco users extensively metabolize nicotine to cotinine. Both nicotine and cotinine undergo N-glucuronidation. Human liver microsomes have been shown to catalyze the formation of these N-glucuronides. However, which UDP-glucuronosyltransferases contribute to this catalysis has not been identified. To identify these enzymes, we initially measured the rates of glucuronidation by 15 human liver microsome samples. Fourteen of the samples glucuronidated both nicotine and cotinine at rates ranging from 146 to 673 pmol/min/mg protein and 140 to 908 pmol/min/mg protein, respectively. The rates of nicotine glucuronidation and cotinine glucuronidation by these 14 samples were correlated, r = 0.97 (p < 0.0001). The glucuronidation of nicotine and cotinine by heterologously expressed UGT1A3, UGT1A4, and UGT1A9 was also determined. All three enzymes catalyzed the glucuronidation of nicotine. However, the rate of catalysis by UGT1A4 Supersomes was more than 30-fold greater than that by either UGT1A3 Supersomes or UGT1A9 Supersomes. Interestingly, when expressed per UGT1A protein, measured by a UGT1A specific antibody, cell lysate from V79-expressed UGT1A9 catalyzed nicotine glucuronidation at a rate 17-fold greater than did UGT1A9 Supersomes. UGT1A4 Supersomes also catalyzed cotinine N-glucuronidation, but at one-tenth the rate of nicotine glucuronidation. Cotinine glucuronidation by either UGT1A3 or UGT1A9 was not detected. Both propofol, a UGT1A9 substrate, and imipramine, a UGT1A4 substrate, inhibited the glucuronidation of nicotine and cotinine by human liver microsomes. Taken together, these data support a role for both UGT1A9 and UGT1A4 in the catalysis of nicotine and cotinine N-glucuronidation.

Infusion of baclofen, a GABA(B) agonist, into the medial basal hypothalamus (MBH) of castrated rams rapidly increases LH pulse amplitude without altering pulse frequency. The objectives of this study were to determine whether baclofen infusion increased LH in testosterone (T)-treated and intact rams, the increased LH was due to increased GnRH release, and FSH secretion also was increased. In the first experiment we tested the main effects and interaction of baclofen and T on FSH and LH pulse patterns in castrated rams (n = 7). In the second experiment we determined whether baclofen affected GnRH and LH pulses in intact males. Microdialysis guide cannulae were implanted bilaterally into the MBH. After recovery of the animal from surgery, the MBH was perfused using concentric microdialysis probes (2-mm tip) with artificial cerebrospinal fluid (aCSF) for a 3-h control period followed by either aCSF or 1 mM baclofen for 4 h. Blood samples were taken at 10-min intervals. T suppressed mean LH concentrations (10.4 +/- 1.3 vs. 3.3 +/- 1.3 ng/ml) such that LH pulses were undetectable in some T-treated animals during the control period. The change (control period vs. drug infusion period) in mean LH was greater in response to baclofen than in response to aCSF and was not altered by T. The baclofen x T interaction was nonsignificant. Mean FSH was decreased by T, but was not altered by baclofen. In the second experiment hypophyseal portal blood was collected coincident with microdialysis. Infusion of baclofen into the MBH of intact males (n = 7) resulted within 1 h in the onset of frequent and robust GnRH pulses (0.10/h before baclofen vs. 1.57/h after baclofen) that were followed either immediately or gradually by coincident LH pulses. One interpretation is that baclofen acts downstream of the site of action of T. GABA(B) receptors may regulate pulse amplitude in both the presence and absence of T and regulate pulse frequency by modulating the inhibitory effect of T.

This study tested the hypothesis that photoperiod affects the ability of testosterone to reduce proopiomelanocortin (POMC) mRNA levels in the arcuate nucleus and luteinizing hormone-releasing hormone (LHRH) mRNA levels in both the preoptic area (POA) or medial basal hypothalamus (MBH). Twenty castrated male sheep were assigned to one of four treatment groups (i): short days (SD; n=5) (ii), short days with testosterone (SD+T; n=5) (iii), long days (LD; n=5), or (iii) long days with testosterone (LD+T; n=5). Blood samples were collected twice weekly for the last 3 weeks of photoperiod treatment and assessed for LH to validate the response to photoperiod. After evaluating LH levels, one animal each from the LD+T and SD+T groups was excluded from the analyses. Mean concentrations of LH were lower (P0.01) in the LD+T group than in the other treatment groups, which did not differ (P0.10) from each other. Neither POA nor MBH LHRH mRNA levels were affected (P0.10) by treatment. Conversely, POMC mRNA levels were suppressed (P0.01) in the LD+T males compared with the other treatment groups which did not differ (P0.10) from each other. These observations suggest that photoperiod specific, testosterone-induced alterations in LHRH mRNA levels are not a mechanism whereby testosterone suppresses LHRH release, and that increased beta-endorphin synthesis and release do not mediate testosterone-induced seasonal suppression of LHRH release.

We investigated the effects of gamma-aminobutyric acid (GABA) agonists muscimol and baclofen (GABA(A) and GABA(B) agonists, respectively) and antagonists bicuculline methiodide (BMI, GABA(A) antagonist) or 2-hydroxysaclofen (SAC) and CGP 55845A (GABA(B) antagonists) on prolactin (PRL) secretion in castrated rams. The drugs were applied by microdialysis into either the medial preoptic area (mPOA) or ventromedial hypothalamus (VMH). Dialysis of baclofen into the mPOA significantly increased mean PRL (p0.05), whereas SAC caused a small, but significant decrease (p0.01). Dialysis of either muscimol or BMI into the mPOA had no effect on prolactin. In the VMH, baclofen significantly increased (p0.01) mean PRL but SAC and CGP 55845A were ineffective, whereas dialysis of either muscimol or BMI increased mean prolactin (p0.01). These results show that infusion into the mPOA of drugs that affect GABA(B) receptor alter PRL release, whereas infusion of a GABA(A) agonists and antagonist was without effect on PRL release. In contrast, infusion of both GABA(A) and GABA(B) agonists and a GABA(A) antagonist into the VMH altered PRL secretion. This suggest that GABAergic neurons in both regions participate in regulating PRL secretion, but by different receptor systems.

We investigated the effects of microdialyzing alpha-aminobutyric acid (GABA) receptor antagonists into either the medial preoptic area (mPOA) or the arcuate-ventromedial region (ARC-VMR) on LH secretion. Bicuculline methiodide (BMI, GABA(A) receptor antagonist), and either 2-hydroxysaclofen (SAC) or CGP 55845A (CGP, GABA(B) receptor antagonists) were used. In experiment 1, castrated rams received 4-h dialysis into either the mPOA (n = 5) or ARC-VMR (n = 4) of artificial cerebrospinal fluid (aCSF) followed by 4 h of either BMI (aCSF-BMI, 375 microM in mPOA, 1 mM in the ARC-VMR for 2-1/2 h), or aCSF-SAC (5 mM). In experiment 2, castrated rams received dialysis only in the ARC-VMR (n = 5) of aCSF-aCSF, aCSF-BMI (375 microM), or aCSF-CGP (50 microM). In experiment 3, untreated or testosterone (T)-treated castrated rams (n = 6/group) received dialysis only in the ARC-VMR of aCSF-aCSF, aCSF-BMI (375 microM), or aCSF-CGP (500 microM). Jugular blood was collected at 10-min intervals. In experiment 1, BMI suppressed mean plasma LH (p0.05) and increased interpulse interval (IPI, p0.05) at both sites. In experiment 2, BMI significantly reduced mean LH and increased IPI (p0.01). In experiment 3, BMI reduced mean LH in both the presence (p0.05) and absence of T (p0.01) and increased IPI (p0.01) in the absence of T. SAC, CGP, and aCSF did not affect LH in any experiment. These results show that dialysis of BMI, into either the mPOA or the ARC-VMR of either castrated or T-treated castrated rams decreased LH release, whereas dialysis of GABA(B) antagonists at these sites was without detectable effect.

Testosterone (T) inhibits LH secretion partly by acting at unknown sites within the brain to inhibit GnRH secretion. We tested the hypothesis that the preoptic area (POA) and arcuate-ventromedial region (ARC/VMR), areas rich in androgen and estrogen (E) receptors, are neural sites at which T and the T metabolites, dihydrotestosterone (DHT) and estrogen (E), act to suppress LH secretion. Bilateral guide cannulae were surgically implanted into either the POA or ARC/VMR of castrated male sheep. Experiments were conducted under a long day photoperiod to maximize the inhibitory effect of the steroids. In Exp 1, all sheep (n = 6/site) sequentially received bilateral implants of cholesterol (CHOL), T, or E at each site. Jugular blood samples were taken at 10-min intervals for 4 h both immediately before implant insertion and 5 days later. In Exp 2, all sheep (n = 6/site) sequentially received bilateral implants of CHOL, DHT, or E at each site according to a latin square design. Blood samples were taken before and 7 days after implant insertion. In Exp 3, which followed the same design as Exp 2, implants of E, T, or DHT were placed only in the ARC/VMR. In the final experiment, the effects of T and CHOL implants in the ARC/VMR were compared. Neither T, DHT, nor CHOL implants at either site affected LH secretion. In contrast, E treatment in the ARC/VMR suppressed mean plasma LH levels (P0.01), primarily due to an increase in interpulse interval (P0.01). Estrogen implants in the POA caused a small, but nonsignificant (P0.05), decrease in mean LH levels in the first experiment and an increase in LH interpulse interval (P0.05) in the second experiment. These results suggest that the ARC/VMR and possibly the POA are sites at which E acts to reduce GnRH secretion in male sheep.

The final common pathway controlling reproductive function in vertebrates is the GnRH neuron and its projection to the median eminence (ME), site of peptide release into the pituitary portal system. GnRH neurons are widely distributed; therefore we sought to test the hypothesis that those projecting to the ME are located in specific regions. We used as a model the sheep, a species in which a great deal of information regarding the physiology of GnRH secretion is known. To identify cells projecting to the ME (i.e., neuroendocrine neurons), ewes (n = 10) received injections into the ME of neuronal tract-tracing compounds: cholera toxin-beta subunit (CT-beta) or one of two fluorescent compounds (rhodamine isothiocyanate or fluorescein-conjugated dextran). Forty-eight h later, animals were perfused intracranially and their brains were processed for immunocytochemical localization of GnRH and CT-beta using a dual-immunofluorescent procedure or by single-label immunofluorescent visualization of GnRH combined with direct visualization of fluorescent tracers. Small, well-circumscribed injections into the ME were made successfully in 6 of 10 animals, and these overlapped the location of GnRH terminals and fibers. Neuroendocrine GnRH neurons (those GnRH neurons containing retrogradely transported tracer) were identified throughout their previously reported range: within the diagonal band of the Broca/medial septal region, medial preoptic area (MPOA), anterior hypothalamic area, and medial basal hypothalamus. Although the absolute number of neuroendocrine GnRH neurons varied by region, the percentage of the total GnRH population within each of these areas that was retrogradely labeled did not differ (p0.05). Injections placed unilaterally within the ME labeled a similar proportion of GnRH cells both ipsilateral and contralateral to the injection site in all areas except the MPOA, where ipsilaterally labeled cells were approximately twice as numerous as those labeled contralaterally. Injections that missed the ME and were placed either into the third ventricle or into the arcuate nucleus labeled only 0.5% and 4-11% of GnRH neurons, respectively. These results do not support the hypothesis that in the ewe, GnRH neurons projecting to the ME are localized to specific regions. Thus, we postulate that GnRH release into the hypophyseal portal system reflects the output of GnRH neurons located in multiple areas.

Since Chiral’95, where certain features of our new method of producing chiral materials were introduced [1], our manufacturing capabilities have increased dramatically resulting in a large selection of novel materials which have been partially characterised. We now wish to reveal all details of this method. We call our new method of producing chiral materials the Supported Microfilament Helix (SMFH) Technique [2]. The method involves winding fine conductive or resistive filaments around a supporting mandrel line. This wound continuous line serves as a chiral precursor from which a wide variety of configurations can be made. The different electromagnetic, size and strength characteristics of the mandrel line coupled with the large variety of conductive filaments which can be wound around the line to produce chiral precursors, enlarges the scope of novel chiral materials. This should provide a better understanding of the phenomenon as well as offering a more efficient method of producing these materials for science and engineering applications.

We investigated the effects on LH secretion of infusing gamma-aminobutyric acid (GABA) agonists muscimol and baclofen (GABAA and GABAB receptor agonists, respectively) into either the medial preoptic area (mPOA) or the arcuate-ventromedial region (ARC-VMR) of the hypothalamus of castrated rams during the nonbreeding season. Bilateral microdialysis of artificial cerebrospinal fluid for 4 h followed by treatment with artificial cerebrospinal fluid, baclofen (1 mM), or muscimol (1 mM in the ARC-VMR, 250 microM in the mPOA) for 4 h was carried out on three separate occasions in random order. Simultaneously, jugular venous blood was collected at 10-min intervals, and plasma later was assayed for LH. The estimated dose of baclofen delivered to each unilateral microdialysis site was 7.9 micrograms; for muscimol, it was 1.1 micrograms for the mPOA and 4.5 micrograms for the ARC-VMR. In the mPOA, baclofen had no detectable effect, whereas muscimol had a delayed suppressive effect on mean LH (P0.01). In the ARC-VMR muscimol rapidly reduced mean LH (P0.01). In contrast, baclofen increased mean LH (P = 0.01) and LH pulse amplitude (P = 0.05) without altering the LH interpulse interval (P0.10). These results support the involvement of GABAA receptors in the mPOA in regulating LH secretory patterns. More importantly, both GABAA and GABA(B) receptors in the ARC-VMR appear to differentially modulate LH and, presumably, GnRH release. Whether GABA acts directly on the GnRH neuron or indirectly through other neural systems remains to be determined.

A standard nomenclature that concisely describes any disaster is currently lacking. This article describes a model taxonomy system. Instead of the term "disaster," a root word "PICE," "potential injury-creating event," is used. Descriptive modifiers to account for all possible scenarios surround this root word, as illustrated. [table: see text] A modifier is chosen from each column and a stage is assigned to each PICE. Column A describes the potential for additional casualties. Column B describes whether resources are overwhelmed and, if so, whether they must simply be augmented (disruptive) or they must first be reconstituted (paralytic). Column C describes the extent of geographic involvement. "Stage" refers strictly to the likelihood that outside medical assistance will be needed. Stage 0 means there is little chance, stage I means there is a small chance (place outside help on "alert"), stage II means there is a moderate chance (place on "standby"), and stage III means local medical resources are clearly overwhelmed (immediately dispatch outside resources, commit personnel, prepare remote hospitals). For example, a multiple vehicle crash in a large city would be a "static, controlled, local PICE, stage 0." In conclusion, a new nomenclature for describing disasters is reported. A short phrase describes the incident and communicates the need for outside assistance. The model may be useful for disaster planning, management, and research.

The areas of the brain involved in photoperiodic control of reproduction are not well defined. The objective of this study was to determine whether anterior hypothalamic area (AHA) lesions in the ewe affected the responses of the reproductive system to shifts in the length of the daily photoperiod and development of photorefractoriness to a constant short day photoperiod. Eleven intact ewes received bilateral radiofrequency lesions of the AHA (AHAX), and five received sham lesions (sham). The ewes then were placed in photochambers and exposed alternately to two approximately 90-day periods of long [16 h of light, 8 h of darkness (16L:8D)] and short (10L:14D) days and then to 10L:14D for an additional 165 days. Blood samples were collected twice weekly to monitor plasma profiles of progesterone, PRL, and total T4, and during the second 16L:8D photoperiod, hourly for one 24-h period to assess melatonin release. Lesions increased (P0.001) the interval between the start of long days and cessation of estrous cycles during both long day periods, but did not affect the interval between the start of short days and the onset of estrous cycles for either the first (P = 0.08) or second (P0.10) short day period. Consequently, the durations of both anestrous periods were shorter (P0.001) for AHAX than for sham ewes. AHA lesions did not affect (P0.10) diurnal patterns of melatonin release. No effects (P0.10) of lesions were evident on plasma patterns of PRL or total T4 for any short or long day photoperiod. Development of photorefractoriness to constant short days either did not occur or was markedly delayed in five of nine AHAX (P0.01) ewes, whereas the other four AHAX ewes became refractory at a time similar (P0.10) to that in sham ewes. Responses to inhibitory long day photoperiods and constant short days were highly (P0.05) correlated (r = 0.74) and appeared dependent upon the extent of the AHA lesion. These results suggest that AHA lesions disrupt neuronal pathways mediating the effects of shifts in photoperiod on reproductive activity and development of photorefractoriness to constant short days. Our results suggest that the effects of AHA lesions are confined to the termination of reproductive activity, and that different neural pathways participate in photostimulation vs. photosuppression or photorefractoriness.

The extent to which inhibitory effects of testosterone (T) on LH secretion in the ram are mediated by its metabolite dihydrotestosterone (DHT) is unknown. Our objective was to determine the effect of inhibiting 5 alpha-reductase activity on pulsatile patterns of LH release in castrated, T-treated male sheep. Nine Dorset and six Hampshire castrated male sheep were allocated equally to one of three treatment groups: 1) infusion of T (768 micrograms/kg/day), 2) infusion of the reductase inhibitor (RI) L-651,723 (0.6 mg/kg/day), and 3) T+RI infusion. Treatments were continuously infused for 3 days. Blood samples were collected via an indwelling jugular catheter at 10-min intervals for 4 h immediately prior to (Day 0) and during the final 4 h of infusion (Day 3). Changes in mean LH, LH pulse amplitude, LH interpulse interval (IPI), T, 17 beta-estradiol (E), and DHT were derived for each animal by subtracting values for Day 0 from Day 3. Data were subjected to one-way analysis of variance. The increase in T and E after infusion of T was similar (p0.10) in T- and T+RI-treated males and greater (p0.01) than in RI-treated males. The increase of DHT was greater (p0.01) in T-treated than either T+RI- or RI-treated males whereas the change was similar (p0.10) for T+RI- and RI-treated males. T decreased mean LH more (p0.01) than RI. T+RI suppressed mean LH more (p0.01) than RI but not as much (p0.01) as T alone.(ABSTRACT TRUNCATED AT 250 WORDS)

9578 Background: To determine the efficacy and safety of neoadjuvant chemotherapy for local tumour control and overall survival in patients with high-risk soft-tissue sarcomas (tumour size ≥ 5 cm, G II/III, extracompartimental and deep localisation) we have analyzed the clinical outcome in 25 patients. Methods: Patients with high-risk soft-tissue sarcomas were treated with four cycles of etoposide 125 mg/m2/day 1+4, ifosfamide 1500 mg/m2/day 1–4 and doxorubicin 50 mg/m2/day 1 (EIA regimen) followed by definitive surgery with or without postoperative radiotherapy and adjuvant chemotherapy. 21 patients received chemotherapy in a combined neoadjuvant/adjuvant clinical setting; eighteen of them completed adjuvant chemotherapy. Four patients received chemotherapy in an adjuvant setting only. Results: The objective response rate of neoadjuvant chemotherapy assessable in 21 patients was 43%. Including NED (n = 7) and partial remissions (n = 3), the radiographic response rate was 47.6% with additional 42.9% stable diseases (n = 9). Surgery was performed in two patients before completing the four neoadjuvant chemotherapy cycles because of disease progression. Median overall survival for all patients from the time of first diagnosis was 21.6+ months [range: 8 - 50] and from the start of treatment 20.5+ months [range: 8 - 49]; median progression-free survival after start of treatment was 18.6+ months [range: 1 - 49]. After completion of chemotherapy, R0-resection could be performed in 13 of 21 patients (60%). At completion of entire treatment, 21 of 25 patients (84%) had NED after R0- and R1-resection. Two patients showed no disease progression after R2-resection and are alive with disease (AWD) 21 and 18 months after start of treatment. Two patients died of disease (DOD) ten and eight months after first diagnosis due to metastases. Conclusions: The high proportion of R0-resections supports the idea of a tumour downstaging after neoadjuvant treatment. Our data support the hypothesis that neoadjuvant chemotherapy might be beneficial as an integral part in the treatment strategy of high-risk soft-tissue sarcoma patients. No significant financial relationships to disclose.