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Post-translational modifications of the histone protein components of eukaryotic chromatin play an important role in the regulation of chromatin structure and gene expression (1). Given the requirement of Rad6/Bre1-dependent ubiquitination of histone H2B for H3 dimethylation (at lysines 4 and 79) and gene silencing (2-7), removal of ubiquitin from H2B may have a significant regulatory effect on transcription. Here we show that a putative deubiquitinating enzyme, Ubp8, is a structurally nonessential component of both the Spt-Ada-Gcn5-acetyltransferase (SAGA) and SAGA-like (SLIK) histone acetyltransferase (HAT) complexes in yeast. Disruption of this gene dramatically increases the cellular level of ubiquitinated-H2B, and SAGA and SLIK are shown to have H2B deubiquitinase activity. These findings demonstrate, for the first time, how the ubiquitin moiety can be removed from histone H2B in a regulated fashion. Ubp8 is required for full expression of the SAGA- and SLIK-dependent gene GAL10 and is recruited to the upstream activation sequence (UAS) of this gene under activating conditions, while Rad6 dissociates. Furthermore, trimethylation of H3 at lysine 4 within the UAS increases significantly under activating conditions, and remarkably, Ubp8 is shown to have a role in regulating the methylation status of this residue. Collectively, these data suggest that the SAGA and SLIK HAT complexes can regulate an integrated set of multiple histone modifications, counteracting repressive effects that alter chromatin and regulate gene expression.

Knowledge of specific neurotransmitters as well as the pathways and mechanisms regulating appetite in ruminants that continually graze, such as sheep, is incomplete. Although fundamentally agouti-related protein (AGRP) has a similar function across species to increase food intake, the regulation of AGRP may vary across grazing and intermittent feeders. To investigate the role of orexigenic peptides in the regulation of feed intake, we first extracted messenger RNA from sheep that were fasted for 3 days, which was then used for PCR followed by cloning and sequencing to demonstrate the presence of hypothalamic AGRP expression. Ovine AGRP was closely related to the bovine, but contained sequence differences with human and mouse AGRP. Analysis of genomic DNA also revealed a similar gene structure to other published species. Secondly, using dual-labeled immunohistochemistry, we determined that there was both increased AGRP immunoreactivity and increased abundance of c-Fos immunoreactivity in AGRP neurons in the arcuate nucleus of fasted sheep. Because AGRP neurons are activated by fasting, we hypothesized that AGRP would stimulate feeding in this ruminant species. Sheep fed ad libitum were injected intracerebroventricularly with concentrations of AGRP at 0.2 and 2.0 nmol/kg. AGRP at 2.0 nmol/kg significantly increased food intake at 4, 6 and 12 h (p < 0.05). A 4th study was done to investigate the interactions of AGRP and neuropeptide Y (NPY) on food intake over a 24-hour period. Intracerebroventricular injections of either AGRP or NPY significantly increased cumulative food intake over saline controls. When AGRP and NPY were injected in combination, food intake was increased over saline controls; however, AGRP did not potentiate the effects of NPY. These results demonstrate that AGRP stimulates food intake in sheep and highlights the important differences between this species and rodent models.

Four studies were designed to determine whether 1) tumor necrosis factor-alpha (TNF) and the Lipopolysaccharide (LPS) binding ligand, CD14, are produced by sheep adipose tissue; 2) nutritional reserves and/or short-term fasting affect circulating concentrations of TNF; 3) there is a relationship between TNF and metabolic factors in sheep; and 4) inflammation alters circulating concentrations of leptin. In Exp. 1 and 2, ewes were assigned, based on ultrasonic assessments of last-rib subcutaneous fat measurements to fat (fat thickness > 1 cm; mean = 1.52 +/- 0.03 cm) or thin (fat thickness < 1 cm; mean = 0.25 +/- 0.03 cm) groups. Fat and thin ewes were assigned to fed or fasted groups for a total of four groups (fed-fat; fasted-fat; fed-thin; fasted-thin). Fed-ewes had ad libitum access to feed, and fasted-ewes were prohibited feed 48 h before initiation of sample collection. In Exp. 1, subcutaneous fat samples were collected from just above the last rib for detection of TNF and CD14 mRNA, and immunoreactivity. Tumor necrosis factor-alpha-like immunoreactivity in adipocytes was sparse, more pronounced in cells in fed-ewes than fasted-ewes, and localized to membranes between adjacent cells in nucleated regions. Immunoreactivity for CD14 was minimally observed but present in adipocytes and widely expressed in infiltrating monocytes and epithelial vascular cells. Leptin was detected in adipocytes. In Exp. 2, plasma samples collected every 6 h for 24 h were analyzed for plasma concentrations of TNF. Fat ewes had greater plasma concentrations of TNF than thin ewes (P = 0.039). In Exp. 3, wethers were injected i.v. with interleukin-1beta or TNF. Blood samples were collected every 15 min for 8 h following injection. Plasma concentration of leptin was not affected by treatment (P > 0.39). In Exp. 4, wethers were injected with LPS. Blood samples were collected every 15 min for 8 h following injection. Plasma concentration of leptin was not altered by LPS (P > 0.20). These results provide evidence: 1) of TNF-like immunoreactivity within fat tissue; 2) that elements within fatty tissues have CD14 that may allow adipocyte function to be directly affected by LPS; 3) that plasma concentrations of leptin are not altered by LPS treatment; and 4) that circulating concentrations of TNF are elevated with obesity in sheep.

Administration of endotoxin suppresses circulating concentration of luteinizing hormone (LH) in a number of species, including rats, sheep, cattle, and non-human primates. Specifically, endotoxin administration decreases circulating concentration of LH and LH pulses frequency in castrated male sheep. Endotoxin could alter circulating concentrations of LH via actions at the hypothalamus through altered GnRH production and/or release, or endotoxin could alter circulating concentrations of LH at the level of the pituitary via inhibition of LH production and release or inhibition of LH in response to GnRH. The site of endotoxin suppression of circulating concentrations of LH as well as possible mediators of endotoxin suppression of circulating concentrations of LH, including cortiocotropin-releasing hormone, arginine vasopressin, glucocorticoids, inflammatory cytokines, prostaglandins, and opioids, are discussed.

Estradiol plus progesterone (EP) implants have been shown to favorably alter the time course or decrease the severity of many of the clinical manifestations associated with coccidiosis and endotoxemia in calves. This study evaluated the effect of EP treatment on plasma tumor necrosis factor-alpha (TNF), thromboxane (TXB), prostacyclin (PRC), nitrite and nitrate (NO[x]), and cortisol. Holstein steer calves were divided into four groups: control, EP, endotoxin (LPS), and EP + LPS (n = five/group). Estradiol/progesterone pellets (Synovex-S) were implanted subcutaneously when calves reached 20 wk of age. One week after implantation, calves were injected i.v. with endotoxin (i.e., lipopolysaccharide; LPS, 0.6 microgram/kg of BW) or nonpyrogenic saline placebo. Body temperature was measured and blood was collected before injection and at 1, 2, 3, 4, 6, and 8 h thereafter. Plasma concentrations of TNF, cortisol, TXB, PRC, NO[x], were measured. Body temperature increased in both LPS and LPS-EP calves, but had returned to normal by 6 h in the LPS-EP group (P < 0.05). Plasma TNF and cortisol increased after LPS (P < 0.01), but were not differentially affected by EP treatment. Likewise, EP did not affect the magnitude of increase in LPS-induced PRC, but EP decreased the magnitude of increase in TXB (P < 0.05). Plasma NO[x]) levels were increased (P < 0.01) in calves after LPS; treatment with EP attenuated the LPS-associated increase in plasma NO[x] levels. These results suggest that EP exerts specific effects on different components of the proinflammatory cytokine cascade. Although the initiation of responses mediated by TNF, cortisol, and PRC do not seem to be differentially affected by EP, components of the nitric oxide- and TXB-axis responses to LPS are decreased in calves pretreated with EP.

Neuropeptide Y (NPY) provides an important hypothalamic link between nutritional status and neuroendocrine mechanisms regulating growth and reproduction. The objective of the following series of experiments was to determine the effects of single or continuous administration of NPY on secretion of luteinizing hormone (LH) and (or) growth hormone (GH). In experiment 1, four ovariectomized (OVX) ewes and four OVX + estrogen-treated ewes each received, in a 4 x 4 Latin Square arrangement of treatments, a single injection of 0, 0.5, 5, or 50 microg NPY via an intracerebroventricular (i.c.v.) cannulae to determine the effects on secretion of GH. NPY significantly elevated serum GH at the 50 microg dose regardless of estrogen exposure (P = 0.003). In experiment 2, eight OVX ewes were infused i.c.v. with NPY or saline (n = 4/trmt) continuously for 20 h in a linearly increasing dose, ending at 50 microg/h NPY. Blood samples were collected via jugular cannulae every 10 min during hour -4-0 (interval 1, pre-treatment), hour 6-10 (interval 2) and hour 16-20 (interval 3) relative to the initiation of infusion (0 h). Mean LH and LH pulse frequency were lower in NPY- versus saline-infused ewes during intervals 2 and 3 (P < 0.01), but NPY had no discernable effect on serum GH (P > 0.10). In experiment 3, four OVX ewes were continuously infused with NPY as in experiment 2, except that the maximum 50 microg/h dose was achieved after only 10 h of infusion. Blood samples were collected every 10 min, beginning 4 h before and continuing until 4h after the NPY infusion. Mean serum LH changed significantly over time (P = 0.0001), decreasing below pre-treatment levels by hour 3 of NPY infusion (P < 0.01), and returning to pre-treatment concentrations following the end of infusion (P > 0.15). Serum GH also changed significantly over time (P < 0.001). Mean GH levels tended to be greater than pre-treatment levels by hour 2 of infusion (P < 0.08), but thereafter returned to basal levels. Serum GH also increased following the end of NPY infusion (P < 0.03). From these data we conclude that NPY exerts a persistent inhibitory effect on secretion of LH, and may stimulate the secretion of GH during the initiation and cessation of infusion of NPY. These observations support a role for NPY in mediating the effects of undernutrition on both LH and GH, and also provide evidence for potential mechanisms by which leptin, acting through NPY, may stimulate the secretion of GH., (Copyright 2002 Elsevier Science Inc.)

Obesity has been a major concern in the horse industry for many years, and the recent discovery of leptin and leptin receptors in numerous nonequine species has provided a basis for new approaches to study this problem in equine. The objectives were to: 1) clone a partial sequence ofthe equine leptin and leptin receptor genes so as to enable the design of primers for RT-PCR determination of leptin and leptin receptor gene presence and distribution in tissues, 2) develop a radioimmunoassay to quantify peripheral concentrations of leptin in equine, 3) determine if peripheral concentrations of leptin correlate with body condition scores in equine, and 4) determine if changing body condition scores would influence peripheral concentrations of leptin in equine. In Experiment 1, equine leptin (GenBank accession number AF179275) and the long-form of the equine leptin receptor (GenBank accession number AF139663) genes were partially sequenced. Equine leptin receptor mRNA was detected in liver, lung, testis, ovary, choroid plexus, hypothalamus, and subcutaneous adipose tissues using RT-PCR. In Experiment 2, 71 horses were categorized by gender, age, and body condition score and blood samples were collected. Sera were assayed for leptin using a heterologous leptin radioimmunoassay developed for equine sera. Serum concentrations of leptin increased in horses with body condition score (1 = thin to 9 = fat; r = 0.64; P = 0.0001). Furthermore, serum concentrations of leptin were greater in geldings and stallions than in mares (P = 0.0002), and tended to increase with age of the animal (P = 0.08). In Experiment 3, blood samples, body weights, and body condition scores were collected every 14 d from 18 pony mares assigned to gain or lose weight over a 14-wk interval based on initial body condition score. Although statistical changes (P = 0.001) in body condition scores were achieved, congruent statistical changes in peripheral concentrations of leptin were not observed, likely due to the small range of change that occurred. Nonetheless, serum concentrations of leptin tended to be greater in fat-restricted mares than in thin-supplemented mares (P = 0.09). We conclude that leptin and leptin receptors are present in equine tissues and that peripheral concentrations of leptin reflect a significant influence of fat mass in equine.

Infectious disease processes cause physiological adaptations in animals to reorder nutrient partitioning and other functions to support host survival. Endocrine, immune and nervous systems largely mediate this process. Using endotoxin injection as a model for catabolic disease processes (such as bacterial septicemia), we have focused our attention on regulation of growth hormone (GH) and luteinizing hormone (LH) secretion in sheep. Endotoxin produces an increase in plasma GH and a decrease in plasma LH concentrations. This pattern can be reproduced, in part, by administration of various cytokines. Antagonists to both interleukin-1 (IL-1) and tumor necrosis factor (TNF) given intravenously (IV) prevented the endotoxin-stimulated increase in GH. Since endotoxin will directly stimulate GH and LH release from cultured pituitary cells, the data suggest a pituitary site of action of the endotoxin to regulate GH. Studies with portal vein cannulated sheep indicated that gonadotropin releasing hormone was inhibited by endotoxin, suggesting a central site of action of endotoxin to regulate LH. However, other studies suggest that endotoxin may also regulate LH secretion at the pituitary. Thus, IL-1 and TNF regulate GH release from the pituitary gland while endotoxin induces a central inhibition of LH release.

This study was designed to determine the effect of feeding or fasting of fat or thin ewes on 24-h leptin profiles. Ewes were assigned, based on ultrasonic assessments of last-rib subcutaneous fat measurements, into fat (fat thickness > 1 cm; mean = 1.52 +/- 0.03 cm; range 1.14 to 2.18 cm) or thin (fat thickness < 1 cm; mean = 0.25 +/- 0.03 cm; range 0.03 to 0.84 cm) groups. Fat and thin ewes were then assigned to either fed or fasted (deprived of feed) groups consisting of five ewes per group. Thus, four groups existed and were designated as fat-fed, fat-fasted, thin-fed, and thin-fasted. Fed ewes had ad libitum access to feed throughout the study. Fasted ewes were prohibited access to feed beginning 48 h preceding the experiment. Plasma samples were collected for leptin analysis from ewes every 15 min for 24 h beginning 48 h after the initiation of feed restriction or the congruent interval in fed ewes. Data were subjected to CLUSTER pulse analysis procedures. Profiles of plasma concentrations of leptin were episodic in nature and did not differ in a diurnal manner. Fed ewes had greater mean concentrations of leptin, area under the curve, number of peaks, peak height, peak nadir, and a shorter interval between peaks than fasted ewes (P < or = 0.05). Fat ewes had greater mean concentrations of leptin, area under the curve, number of peaks, peak height, peak nadir, and a shorter interval between peaks than thin ewes (P < 0.02). There also was a tendency for a body condition x treatment interaction for number of peaks (P = 0.073) and interval between peaks (P = 0.056). These results provide evidence that plasma concentrations of leptin are episodic in nature and are influenced by nutritive state and fat thickness over the ribs, but display no circadian variation.

A series of studies was designed to identify methods of improving out-of-season breeding success in ewes. In Experiment 1, 190 mature ewes were assigned to receive in April, either: (A) a control ration of 0.3 kg corn twice daily for 8 d before ram introduction (control ewes n=49), (B) the control ration containing 0.125 mg of melengestrol acetate (MGA) in 0.3 kg corn (MGA8a ewes n=46), (C) the control ration or 7.5 d followed by 1 feeding of 0.5 mg MGA in 0.3 kg corn (MGA1 ewes n=48), or (D) the control ration plus a 20 mg i.m. injection of progesterone on D 8 (P ewes n=47). Ewes were exposed to rams for 21 d. A greater percentage of MGA8a and P ewes lambed than did control ewes (P < 0.04). The lambing rate was greatest among MGA8a (P < 0.02 vs. control), intermediate among P ewes (P < 0.19 vs. control) and least among MGA1 and control ewes (P > 0.79). In Experiment 2, 70 mature ewes were assigned to receive in June, either: (A) a control ration of 0.3 kg of corn twice daily for 8 d before to ram introduction (control ewes n=25), (B) the control ration containing 0.125 mg of MGA per 0.3 kg corn (MGA8b ewes n=21), or (C) the control ration and simultaneous treatment of ewes with a progesterone-containing controlled internal-drug releasing device (CIDR ewes n=24). Ewes were exposed to rams for 21 d. Both CIDR and MGA8b ewes exhibited estrus earlier than did control ewes (P < 0.01). The CIDR ewes exhibited estrus earlier than did the MGA8b ewes (P < 0.05). A greater percentage of ewes treated with CIDR or MGA8b lambed than did control ewes (P < 0.01), with more CIDR ewes lambing than MGA8b ewes (P < 0.01). The lambing rate was greater in CIDR ewes than in control ewes (P < 0.04). These data provide evidence that several options exist to improve pregnancy success in ewes bred out of season and that success varies with method of treatment.

This study was designed to determine what effects the birth process would have on development of the somatotrophic axis in neonatal pigs. Eight crossbred sows were selected (n = 4 natural birth and n = 4 Caesarian section) for the present study. Blood and tissue samples from 38 pigs were collected at birth. Twenty pigs were maintained with natural birth sows until sacrificed for blood and tissue collection at 2 wk of age. Gestational age at birth did not differ (P > 0.16) between natural birth and C-section pigs. Average daily gain (ADG) from birth until 2 wk of age was reduced (P < 0.0001) by 39.3% in the C-section pigs as compared to the natural birth pigs. Serum growth hormone (GH) did not differ (P > 0.86) at birth, but was greater (P < 0.024) at 2 wk in C-section pigs. Serum insulin-like growth factor 1 (IGF-1) was greater at birth (P < 0. 0025) and at 2 wk of age (P < 0.035) in the natural birth pigs. Serum concentration of IGF-2 did not differ at birth (P > 0.8) but was greater (P < 0.043) in natural birth pigs at 2 wk of age. Pituitary content of GH mRNA and GH-releasing hormone receptor mRNA did not differ (P > 0.90) between groups regardless of age; however, expression of both mRNAs declined (P < 0.0003) from birth until 2 wk of age. There tended to be a birth type X age interaction (P < 0. 082) for liver IGF-1 mRNA such that C-section pigs had a greater expression at 2 wk of age. Liver IGF-1 mRNA expression increased (P < 0.0001) in both groups from birth to 2 wk of age. Liver expression of GH receptor mRNA was greater in C-section pigs at birth (P < 0. 04) and 2 wk of age (P < 0.03). These data provide evidence that the natural birth process affects postnatal development and/or function of the somatotrophic axis in the neonatal pig.

The objective was to determine the effect of central infusion of insulin and (or) glucose on hypothalamic expression of leptin receptor and pituitary secretion of LH in the ewe. Twenty-two ovariectomized ewes (32 wk of age) were fitted with two lateral cerebroventricular (LCV) cannulae and fed 33% of NRC requirements for 8 wk. Ewes (n> or =5/group) were then infused, via LCV cannulae, with artificial cerebrospinal fluid (aCSF) or aCSF containing physiological concentrations of insulin (INS), glucose (GLU), or INS + GLU; the mass of each increasing linearly from Day 0 (mass = 0 units/h) to Day 8 (mass of INS = 80 mIU/hr and GLU = 10 mg/hr). Jugular serum was collected every 12 min for 4 hr on Days 0, 2, and 4. Ewes treated with INS or INS + GLU had greater (P<0.06) mean concentrations of LH than aCSF treated ewes on Day 2 (13.8+/-1.8 and 12.5+/-1.3 > 8.0+/-3.3 ng/ml). Furthermore, on Day 4, concentrations of LH in INS treated ewes exceeded that (P<0.07) of aCSF treated ewes (14.8+/-2.0 > 7.4+/-3.0 ng/ml). Expression of NPY mRNA did not differ between treatments (P = 0.87). Leptin receptor mRNA expression was dramatically reduced (P<0.0002) in INS+GLU versus aCSF treated ewes. These data provide evidence to suggest that insulin may be an important component of hypothalamic mechanisms regulating secretion of LH and expression of leptin receptors in undernourished ruminants.

Current methods utilized for serial blood collection in the young pig are limited due to the stress and/or discomfort to which the pig is exposed. Thus, we have developed a non-surgical, minimally invasive cannulation technique which allows jugular vein catheter placement in the young pig without causing extended discomfort or stress. The procedure described is rapid (approximately 8 min/pig) and relatively simple, requiring only minimal anaesthesia for immobilization of the pig during the procedure. Routinely, 2-week-old piglets are standing in their pens within 15-20 min from initiation of the procedure. Piglets recover rapidly from the procedure and display no clinical indications of pain or discomfort. Serum concentrations of cortisol, a standard indicator of stress and/or discomfort, are asymptomatic within 2 h of completing the procedure (k = 26.14+/-3.03 ng/ml). Stress is limited to the initial immobilization of the piglets. With this technique of cannulation, we routinely maintain catheter patency for 2 days, and often for as long as 5 days.

Eight crossbred sows were selected for the present study (n = 4 vaginal delivery and n = 4 Caesarian section [C-section]). Gestation length did not differ between vaginal delivery and C-section pigs (113.6 +/- .1 and 113.2 +/- .3 d, respectively; P > .16). Blood and tissue samples from 38 pigs were collected at birth. All remaining pigs were sustained with vaginal-delivery sows until 2 wk of age (n = 39). At 2 wk of age, remaining pigs were catheterized for blood sample collection to assess pituitary-adrenal responsiveness to an injection of corticotropin-releasing hormone (CRH; 10 microg/kg). Blood samples were collected at -30, -15, 0, 5, 10, 20, 40, 60, and 90 min; pigs received CRH or saline at time 0. Pigs were killed and tissue samples were collected immediately following the last blood sample. Serum concentrations of ACTH and cortisol (CS) were measured. Total RNA was isolated from the pituitary and adrenal glands to evaluate gene expression for mRNA specific for pro-opiomelanocortin (POMC) and for the ACTH receptor. Centrifuged clot:blood ratio was reduced in the C-section pigs at birth (P < .001) and at 2 wk of age (P < .043), compared with the vaginally delivered pigs. Basal serum concentration of ACTH was greater in C-section than in vaginally delivered pigs at birth (P = .01) but did not differ at 2 wk of age (P = .42). Basal serum concentration of CS was not different at birth (P = .86) but was greater in C-section pigs than in vaginally delivered pigs at 2 wk of age (P < .04). Serum concentration of ACTH was not different (P > .99) between the two groups of pigs following the CRH challenge. However, serum concentration of CS was greater (P < .05) in C-section pigs following the CRH challenge. Expression of ACTH receptor mRNA tended to be greater in C-section pigs at birth (P < .063) and at 2 wk of age (P < .016). There was no difference in POMC mRNA between treatments (P > .73); however, there was a developmental increase (P < .001) from birth to 2 wk of age. These data indicate that the birth process plays an important role in postnatal function of the hypothalamic-pituitary-adrenal axis in young pigs.

Infertility associated with suboptimal nutrition is a major concern among livestock producers. Undernourished prepubertal animals will not enter puberty until they are well fed; similarly, adult, normally cyclic females will stop cycling when faced with extreme undernutrition. Work in our laboratory has focused on how body fat (or adiposity) of an animal can communicate to the brain and regulate reproductive competence. In 1994, the discovery in rodents of the obese (ob) gene product leptin, secreted as a hormone from adipocytes, provided a unique opportunity to understand and hence regulate whole body compositional changes. There is now evidence that similar mechanisms are functioning in livestock species in which food intake, body composition, and reproductive performance are of considerable economic importance. Leptin has been reported to be a potent regulator of food intake and reproduction in rodents. There is evidence indicating that at least some of the effects of leptin occur through receptor-mediated regulation of the hypothalamic protein neuropeptide Y (NPY). NPY is a potent stimulator of food intake, is present at high concentrations in feed-restricted cattle and ewes, and is an inhibitor of LH secretion in these livestock species. In our investigations in sheep, we have cloned a partial cDNA corresponding to the ovine long-form leptin receptor, presumably the only fully active form, and have localized the long-form leptin receptor in the ventromedial and arcuate nuclei of the hypothalamus. Leptin receptor mRNA expression was colocalized with NPY mRNA-containing cell bodies in those regions. We have also determined that hypothalamic leptin receptor expression is greater in feed-restricted ewes than in well-fed ewes. These observations provide a foundation for future investigations into the nutritional modulators of reproduction in livestock.

Twenty-five nonlactating ewes were used to test the hypothesis that methscopolamine bromide (MB) blocks secretion of growth hormone (GH) by affecting hypothalamic rather than pituitary mechanisms. Ewes were randomly assigned to receive a s.c. injection of 96 mg of MB or 2 mL of saline at min = 0. Saline-treated ewes were assigned to receive a subsequent (at +60 min) i.v. injection of 10 microg of growth hormone-releasing hormone (GHRH) to test pituitary responsiveness or .3 mg of clonidine to test hypothalamic responsiveness. Methscopolamine bromide-treated ewes were assigned to receive a subsequent (at +60 min) i.v. injection of 10 microg of GHRH, .3 mg of clonidine, or 2 mL of saline. Jugular blood samples were collected at 10-min intervals from -120 min to +240 min, and serum concentrations of GH were quantified with a RIA. No difference was detected in serum concentrations of GH with respect to MB vs saline treatment (P = .20). Concentrations of GH increased in saline-pretreated ewes following injections of clonidine or GHRH (P < .01). Treatment of ewes with MB, however, limited the ability of clonidine-induced mechanisms to increase concentrations of GH, but did not affect pituitary responsiveness to GHRH (P < .01). These data support the hypothesis that MB inhibits hypothalamic and not pituitary mediated mechanisms that regulate the secretion of GH.

To determine whether transesophageal echocardiography (TEE) is useful in ruling out the presence of atrial thrombus, we performed TEE in 20 patients immediately before valve replacement or valve repair and within 3 days of an autopsy in one patient. Mitral stenosis was the predominant lesion in three patients, mitral regurgitation was seen in 11 patients, five patients had mitral prosthesis malfunction, one patient had a tricuspid prosthesis malfunction, and one patient had aortic stenosis. Eight patients were in atrial fibrillation. Four patients demonstrated spontaneous contrast in the associated atria. Nine patients were receiving oral anticoagulation. Mean left atrial diameter was 5.3 +/- 1.3 mm. TEE revealed no evidence for atrial thrombus in 18 of the 21 patients; this finding was confirmed by careful inspection of the atria including the appendages. TEE demonstrated a left atrial thrombus in two patients and a right atrial thrombus in another (confirmed at the time of surgery or at autopsy). In all cases transthoracic echocardiography was negative. Our data suggest that TEE is useful in ruling out atrial thrombus, and therefore may be a useful test preceding interventions associated with an increased risk of embolism from the atria such as cardioversion, mitral valvuloplasty, or valve replacement.

To determine if transesophageal echocardiography provides better visualization of valvular vegetations than transthoracic echocardiography, we used both methods to evaluate 24 consecutive patients (mean age, 54 years; 15 female patients and nine male patients) referred for symptoms suggestive of infectious endocarditis. Ten of the 24 patients had one or more valvular prostheses. Echocardiograms were classified as positive or negative based on visualization of valvular vegetations or abscesses. Of ten patients with a final diagnosis of infectious endocarditis on extended follow-up, transthoracic echocardiography was positive in five patients. Transesophageal echocardiography not only yielded abnormal findings in all ten of these patients, but also revealed additional information in four of the five patients with abnormal transthoracic echocardiographic examinations. Among the 14 patients who, on subsequent follow-up, were found not to have infectious endocarditis, transthoracic echocardiography was normal in 13 and falsely abnormal in one. Transesophageal echocardiography revealed no evidence of infectious endocarditis in any of these patients. The ten patients who were determined to have infectious endocarditis all had positive blood cultures and no alternative cause for their clinical presentation; in seven patients in this group who underwent operative or postmortem evaluation, infectious endocarditis was confirmed. All patients without infectious endocarditis were demonstrated to have other causes for their clinical presentation. We conclude that transesophageal echocardiography is a highly valuable test in the work-up of patients with suspected infectious endocarditis, especially those patients with inconclusive or normal transthoracic echocardiograms. In addition, transesophageal echocardiography may be of benefit to patients with previously documented infectious endocarditis and a complicated clinical course in whom additional cardiac lesions are suspected but not demonstrated by transthoracic echocardiography.

Transesophageal color flow Doppler findings are reported in 36 patients with a St. Jude Medical mechanical mitral valve prosthesis who had no auscultatory evidence for prosthetic valve dysfunction. Multiple jets consistent with mitral regurgitation originating from the central and lateral portion of the prosthesis were found in all patients. Maximum jet length ranged from 11 to 51 mm (mean 21 +/- 9 mm). Maximum jet area ranged from 0.2 to 4.1 cm3 (mean 1.2 +/- 0.9 cm2). The color M-mode Doppler interrogation showed two distinct components of the regurgitant jet: brief early systolic flow consistent with valve closure followed by holosystolic regurgitant flow consistent with transvalvular leakage. Four patients (11%) had a maximum regurgitant jet length exceeding 30 mm and absence of early systolic closure regurgitant flow by M-mode color imaging, suggesting clinically silent paravalvular leakage. Two pin-sized paravalvular suture line defects were confirmed in one patient at cardiac transplantation. We conclude that transesophageal echocardiography is a highly sensitive method for detection of mitral regurgitation in the St. Jude Medical mitral prosthesis. Clinically silent paravalvular leakage should be suspected if the maximum jet length exceeds 30 mm and color M-mode interrogation fails to demonstrate an early systolic closure regurgitant flow component.

The differentiation of left ventricular aneurysm from diffuse left ventricular dilation and hypokinesia may have important therapeutic consequences. Thus the diagnostic accuracy of wide angle two dimensional echocardiography for the detection of left ventricular aneurysm was evaluated in a prospective study of 26 consecutive patients with the clinical suspicion of left ventricular aneurysm referred over a 10 month period. Every patients was examined with two dimensional echocardiography and left ventricular cineangiography, and findings were interpreted by two independent observers. A dilated hypokinetic left ventricle without aneurysm formation on cineangiography in nine patients was identified in all with two dimensional echocardiography. A left ventricular aneurysm on cineangiography in 17 patients was correctly identified in 14 with the two dimensional study, as were the site and extent of the lesion (apical in 12, anterior in 1 and inferior in 1). One apical aneurysm was interpreted on the two dimensional study as apical dyskinesia; one anterior and one posterobasal aneurysm were missed with this technique. Mural thrombi were correctly identified with two dimensional echocardiography in seven of seven patients. It is concluded that two dimensional echocardiography is an accurate noninvasive method that allows differentiation of left ventricular aneurysm from diffuse left ventricular dilation in the majority of patients. It provides information regarding the resectability of the aneurysm and may obviate cineangiography in many cases.

Five patients with pulmonary embolism, in whom right-sided intracardiac thromboembolus was detected by echocardiography and confirmed by either angiography, surgery or postmortem examination, are described. One of these patients died from massive pulmonary embolism after right heart catheterization. In two patients treated medically, either partial or total lysis of the thromboembolus was demonstrated echocardiographically; in another two patients, the right atrial thromboembolus was successfully removed surgically. Typical locations and echocardiographic characteristics of right-sided thromboemboli are described. The potential usefulness of two-dimensional echocardiography in both the diagnosis and the management of patients with right-sided intracardiac thromboembolism is discussed.

Nine patients with proved left ventricular pseudoaneurysm after transmural myocardial infarction were studied by two-dimensional echocardiography. In all patients two-dimensional echocardiography successfully displayed the pseudoaneurysm. The unique two-dimensional echocardiographic characteristics of pseudoaneurysm include: a sharp discontinuity of the endocardial image at the site of the pseudoaneurysm communication with the left ventricular cavity, and the presence of a relatively narrow orifice in comparison with the maximum diameter of the pseudoaneurysm fundus; visualization of the maximum diameter of the pseudoaneurysm fundus frequently required a slightly different tomographic view than that required for demonstration of the orifice. The distinctive echocardiographic features of pseudoaneurysm in these patients and technical implications for optimal visualization are described. Most of the pseudoaneurysms we encountered and many of those previously described were located posteriorly. We found the use of inferior angulated view modified from the standard apical four-chamber view extremely helpful in detecting the orifice in patients with posterior or posterolateral pseudoaneurysms. We conclude that two-dimensional echocardiography is an important technique for diagnosis of left ventricular pseudoaneurysm.