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Latinos form the largest U.S. minority and will account for one quarter of the population by 2050. Immigration trends from 1995-2010 challenged health systems in "new destination" regions such as the southeastern U.S., with Latino population increases of 200-400%, and a minimal bilingual health workforce. Academic medical centers and safety net hospitals are challenged to respond beyond the interpreter paradigm of care delivery to provide efficient, cost-effective and compassionate care that complies with the U.S. Title VI mandates. We describe the design and successful implementation of an academic model in the care of Spanish-speaking patients in the pediatric and adult primary care and subspecialty settings in the University of North Carolina Health Care System. This model leverages a limited bilingual workforce to maximize the extent and quality of language-concordant care for this population The innovative features of the UNC Center for Latino Health (CELAH) is based upon five principles: patient navigation, a medical home, a block-scheduling system, a "virtual clinic" model using existing space, and leveraged cost-neutral resources. Patients are scheduled to specific half-day sessions in specialty clinics and matched with bilingual faculty and staff. This facilitates door-to-door care in Spanish for patients, the majority of whom are immigrants from rural Mexico and Central America with limited English and health literacy. CELAH is considered an academic transition model in anticipation of an adequate bilingual health workforce in 1-2 decades. As a hub, this clinical platform supports unique programs in medical education, translational and health equity research, community outreach, and faculty engagement.

BACKGROUND/OBJECTIVES: Telephone calls after dis-charge from the emergency department (ED) are increasinglyused to reduce 30-day rates of return or readmission, buttheir effectiveness is not established. The objective was todetermine whether a scripted telephone intervention by regis-tered nurses from a hospital-based call center would decrease30-day rates of return to the ED or hospital or of death.DESIGN: Randomized, controlled trial from 2013 to 2016.SETTING: Large, academic medical center in the south-east United States.PARTICIPANTS: Individuals aged 65 and older dis-charged from the ED were enrolled and randomized intointervention and control groups (N = 2,000).INTERVENTION: Intervention included a telephone callfrom a nurse using a scripted questionnaire to identifyobstacles to elements of successful care transitions: medica-tion acquisition, postdischarge instructions, and obtainingphysician follow-up. Control subjects received a satisfac-tion survey only.MEASUREMENTS: Primary outcome was return to theED, hospitalization, or death within 30 days of dischargefrom the ED.RESULTS: Rate of return to the ED or hospital or deathwithin 30 days was 15.5% (95% confidence interval(CI) = 13.2–17.8%) in the intervention group and 15.2%(95% CI = 12.9–17.5%) in the control group (P = .86).Death was uncommon (intervention group, 0; control group, 5 (0.51%), 95% CI = 0.06–0.96%); 12.2% ofintervention subjects (95% CI = 10.1–14.3%) and 12.5%of control subjects (95% CI = 10.4–14.6%) returned tothe ED, and 9% of intervention subjects (95% CI = 7.2–10.8%) and 7.4% of control subjects (95% CI = 5.8–9.0%) were hospitalized within 30 days.CONCLUSION: A scripted telephone call from a trainednurse to an older adult after discharge from the ED didnot reduce ED or hospital return rates or death within 30 days. Clinicaltrials.gov identifier: NCT01893931z.

ACADEMIC EMERGENCY MEDICINE 2011; 18:S92–S96 © 2011 by the Society for Academic Emergency Medicine Abstract Objectives: Despite an increasing number of elderly emergency department (ED) patients, emergency medicine (EM) residency training lacks geriatric-specific curricula. The objective was to determine if a 1-year geriatric curriculum, designed for residents, would affect residents’ attitudes, knowledge, and decision-making for older patients seen in the ED. Methods: The authors created a geriatric curriculum for EM residents composed of six lectures on the following topics: trauma, abdominal pain, transitions of care, medication management, iatrogenic injuries, and confusional states. A second component of the curriculum included seven high-fidelity simulation skills training sessions on aortic aneurysm, salicylate toxicity, drugs of abuse, infection from a posterior pressure ulcer, medication-induced elevated prothrombin time resulting in gastrointestinal bleeding, mesenteric ischemia, and myocardial infarction. Before and after completion of the curriculum, residents were assessed on attitudes toward caring for geriatric patients using a validated survey and knowledge of geriatric principals of care using a 35-question multiple choice test. To determine differences before and after the new curriculum was implemented, the paired t-test was performed on knowledge and attitude scores. ED records were also reviewed for frequency of chemical sedation and urinary catheter placement in patients aged 65 and over, both before and after the educational intervention, as a measure of appropriate decision-making. Appropriateness of urinary catheter placement was determined by two physician reviewers using criteria adapted from the Centers for Disease Control and Prevention indications for appropriate urinary catheter use. Reviewers met to adjudicate any disagreements about appropriateness. Fisher’s exact test was used to examine differences in frequency of chemical sedation and urinary catheter placement. Results: Twenty-nine EM residents underwent the training. There was no measured change in attitudes. Knowledge improved from the pre- to posttest with average scores of 58.5 and 68.0%, respectively (p

Objectives Older patients discharged from the emergency department (ED) have difficulty comprehending discharge plans and are at high risk of adverse outcomes. The authors investigated whether a postdischarge telephone call–mediated intervention by a nurse would improve discharge care plan adherence, specifically by expediting post–ED visit physician follow-up appointments and/or compliance with medication changes. The second objectives were to determine if this telephone call intervention would reduce return ED visits and/or hospitalizations within 35 days of the index ED visit and to determine potential cost savings of this intervention. Methods This was a 10-week randomized, controlled trial among patients aged 65 and older discharged to home from an academic ED. At 1 to 3 days after each patient's index ED visit, a trained nurse called intervention group patients to review discharge instructions and assist with discharge plan compliance; placebo call group patients received a patient satisfaction survey call, while the control group patients were not called. Data collection calls occurred at 5 to 8 days and 30 to 35 days after the index ED visits for all three groups. Chi-square or Fisher's exact tests were performed for categorical data and the Kruskal-Wallis test examined group differences in time to follow-up. Results A total of 120 patients completed the study. Patients were 60% female and 72% white, with a mean age of 75 years (standard deviation [SD] ± 7.58 years). Intervention patients were more likely to follow up with medical providers within 5 days of their ED visits than either the placebo or the control group patients (54, 20, and 37%, respectively; p = 0.04). All groups performed well in medication acquisition and comprehension of medication indications and dosage. There were no differences in return visits to the ED or hospital within 35 days of the index ED visit for intervention patients, compared to placebo or control group patients (22, 33, and 27%, respectively; p = 0.41). An economic analysis showed an estimated 70% chance that this intervention would reduce total costs. Conclusions Telephone call follow-up of older patients discharged from the ED resulted in expedited follow-up for patients with their primary care physicians. Further study is warranted to determine if these results translate into improved patient outcomes, decreased return ED visits or hospital admissions, and cost savings resulting from this intervention.

Objectives An estimated 14% to 25% of all scientific studies in peer-reviewed emergency medicine (EM) journals are medical records reviews. The majority of the chart reviews in these studies are performed manually, a process that is both time-consuming and error-prone. Computer-based text search engines have the potential to enhance chart reviews of electronic emergency department (ED) medical records. The authors compared the efficiency and accuracy of a computer-facilitated medical record review of ED clinical records of geriatric patients with a traditional manual review of the same data and describe the process by which this computer-facilitated review was completed. Methods Clinical data from consecutive ED patients age 65 years or older were collected retrospectively by manual and computer-facilitated medical record review. The frequency of three significant ED interventions in older adults was determined using each method. Performance characteristics of each search method, including sensitivity and positive predictive value, were determined, and the overall sensitivities of the two search methods were compared using McNemar's test. Results For 665 patient visits, there were 49 (7.4%) Foley catheters placed, 36 (5.4%) sedative medications administered, and 15 (2.3%) patients who received positive pressure ventilation. The computer-facilitated review identified more of the targeted procedures (99 of 100, 99%), compared to manual review (74 of 100 procedures, 74%; p

Introduction: Triage of patients is critical to patient safety, yet no clear information exists as to the utility of initial vital signs in identifying critically ill older emergency department (ED) patients. The objective of this study is to evaluate a set of initial vital sign thresholds as predictors of severe illness and injury among older adults presenting to the ED. Methods: We reviewed all visits by patients aged 75 and older seen during 2007 at an academic ED serving a large community of older adults. Patients’ charts were abstracted for demographic and clinical information including vital signs, via automated electronic methods. We used bivariate analysis to investigate the relationship between vital sign abnormalities and severe illness or injury, defined as intensive care unit (ICU) admission or ED death. In addition, we calculated likelihood ratios for normal and abnormal vital signs in predicting severe illness or injury. Results: 4,873 visits by patients aged 75 and above were made to the ED during 2007, and of these 3,848 had a complete set of triage vital signs. For these elderly patients, the sensitivity and specificity of an abnormal vital sign taken at triage for predicting death or admission to an ICU were 73% (66,81) and 50% (48,52) respectively (positive likelihood ratio 1.47 (1.30,1.60); negative likelihood ratio 0.54 (0.30,0.60). Conclusion: Emergency provider assessment and triage scores that rely primarily on initial vital signs are likely to miss a substantial portion of critically ill older adults. [West J Emerg Med. 2013;14(5):453–460.]

We constructed a bidirectional Web-based system to transmit critical patient information in real time between referring nursing homes and a university hospital emergency department (ED) to facilitate the care of patients referred to our ED. Our model was inexpensive, improved measures of information transfer, and increased provider satisfaction.

Lack of access to medical information for nursing home residents during emergency department (ED) evaluation is a barrier to quality care. We hypothesized that the quantity of information available in the ED differs based on the funding source of the resident's nursing home.Cross-sectional observational study.Single academic ED.Participants were 128 skilled nursing facility (SNF) residents age 65 or older from 12 SNFs.Emergency physicians documented knowledge of 9 essential information items. SNFs were categorized as accepting or not accepting Medicaid.Questionnaires were completed for 128 patients, of whom 95 (74%) were from 1 of 8 Medicaid-funded SNFs and 33 (26%) were from 1 of 4 SNFs not accepting Medicaid. Patients from SNFs accepting Medicaid were younger (79 versus 87, P.001) and less frequently white (62% versus 97%, P.001). The mean number of 9 possible information items available was lower for patients from SNFs that accept Medicaid (7.13 versus 8.15, P.001). Emergency providers also reported lower satisfaction regarding access to information for residents from SNFs that accept Medicaid (P.05). The association between residence in an SNF that accepts Medicaid and lower ED information scores remained after linear regression with clustering by SNF controlling for age, gender, and race. The most common source of information for residents from both types of SNFs was transfer papers from the SNF.Less information is available to ED providers for patients from SNFs that accept Medicaid than for residents from SNFs that do not accept Medicaid. Further study is needed to examine this information gap.

Objectives: Methods to accurately identify elderly patients with a high likelihood of hospital admission or subsequent return to the emergency department (ED) might facilitate the development of interventions to expedite the admission process, improve patient care, and reduce overcrowding. This study sought to identify variables found among elderly ED patients that could predict either hospital admission or return to the ED. Methods: All visits by patients 75 years of age or older during 2007 at an academic ED serving a large community of elderly were reviewed. Clinical and demographic data were used to construct regression models to predict admission or ED return. These models were then validated in a second group of patients 75 and older who presented during two 1-month periods in 2008. Results: Of 4,873 visits, 3,188 resulted in admission (65.4%). Regression modeling identified five variables statistically related to the probability of admission: age, triage score, heart rate, diastolic blood pressure, and chief complaint. Upon validation, the c-statistic of the receiver operating characteristic (ROC) curve was 0.73, moderately predictive of admission. We were unable to produce models that predicted ED return for these elderly patients. Conclusions: A derived and validated triage-based model is presented that provides a moderately accurate probability of hospital admission of elderly patients. If validated experimentally, this model might expedite the admission process for elderly ED patients. Our models failed, as have others, to accurately predict ED return among elderly patients, underscoring the challenge of identifying those individuals at risk for early ED returns. ACADEMIC EMERGENCY MEDICINE 2010; 17:252–259 © 2010 by the Society for Academic Emergency Medicine

The leucine zipper proteins are a group of transcriptional regulators that dimerize to form a DNA binding domain. It has been proposed that this dimerization results from the hydrophobic association of the alpha-helices of two leucine zipper monomers into a coiled coil. We propose a model for a coiled coil based on a periodic hydrophobic-hydrophilic amino acid motif found in the leucine zipper regions of 11 transcriptional regulatory proteins. This model predicts the symmetrical formation of secondary hydrogen bonds between the polar side chains of one helix and the peptide carbonyls of the opposite chain, supplementing the interactions between hydrophobic side chains. Physical modeling (CPK) and in vacuo molecular mechanics calculations of the stability of the GCN4 leucine zipper coiled coil configured in accordance with this model demonstrate a greater stability for this conformer than for a conformer configured according to a current hydrophobic model. Molecular dynamics simulations show similar stability of the two models in vacuo but a higher stability of the hydrophobic model in water.

Sorting of prohormones and propeptides into secretory vesicles at the trans-Golgi face probably depends on a signal contained within the amino acid sequence of the peptide. To date no consensus sequence has been identified in prohormones or propeptides that might serve such a targeting function. In this report, we have analyzed the amino acid sequences and secondary structures of 15 prohormones and propeptides that have been shown experimentally to be sorted to secretory vesicles when the corresponding cDNA is transfected into mouse pituitary AtT20 cells. From these analyses, we have identified a motif that is shared by all of these diverse propeptides and might serve as a vesicular targeting sequence. This motif is degenerate and consists of two or more leucines occupying one side of a highly amphipathic α-helix with a serine (or rarely threonine) positioned N-terminal to the leucines and projecting to the same side of the helix.

Peptides encoded in the antisense strand of DNA have been predicted and found experimentally to bind to sense peptides and proteins with significant selectivity and affinity. Such sense--antisense peptide recognition has been observed in many systems, most often by detecting binding between immobilized and soluble interaction partners. Data obtained so far on sequence and solvent dependence of interaction support a hydrophobic-hydrophilic (amphipathic) model of peptide recognition. Nonetheless, the mechanistic understanding of this type of molecular recognition remains incomplete. Improving this understanding likely will require expanding the types of characteristics measured for sense--antisense peptide complexes and hence the types of analytical methods applied to such interactions. Understanding the mechanism of sense--antisense peptide recognition also may provide insights into mechanisms of native (sense) peptide and protein interactions and protein folding. Such insight may be helpful to learn how to design macromolecular recognition agents in technology for separation, diagnostics and therapeutics.

Source Citation Poldermans D, Fioretti PM, Forster T, et al. Dobutamine stress echocardiography for assessment of perioperative cardiac risk in patients undergoing major vascular surgery. Circulati...

Source Citation Raby KE, Barry J, Creager MA, et al. Detection and significance of intraoperative and postoperative myocardial ischemia in peripheral vascular surgery. JAMA. 1992 Jul 8:268:222-7.

The administration of monosodium-L-glutamate (MSG) during the neonatal period is known to result in central nervous system lesions in the arcuate nucleus of the hypothalamus and the retina. Rodents so treated exhibit behavioral deficts and endocrinopathies including obesity, hypogonadism, hypothyroidism, pituitary atrophy, tail automutilation and diminished locomotor activity. Assessment of endocrine status revealed normal serum levels of glucagon, thyroid-stimulating hormone and luteinizing hormone, and diminished levels of thyroid hormones and growth hormone in MSG-treated rats. Prolactin levels were elevated in the glutamate-treated male rats. Within the brain hypothalamic levels of thyrotropin-releasing hormone, luteinizing hormone-releasing hormone, and somatostatin were unchanged. Measurement of neurotransmitters and neurotransmitter-related enzymes in individual hypothalamic nuclei derived from MSG-treated rats revealed normal levels of norepinephrine, serotonin and glutamic acid decarboxylase, but reduced levels of choline acetyltransferase and dopamine in the arcuate nucleus and median eminence. Histochemical methods for visualization of dopamine and acetylcholinesterase in the mediobasal hypothalamus confirmed these findings. The MSG-treated animals exhibited a normal diurnal rhythm of pineal serotonin N-acetyltransferase activity. These data indicate that the MSG-induced endocrine deficiency syndrome results at least partly from destruction of cholinergic and dopamingeric tuberoinfundibular systems in the hypothalamus.

We studied the relationship between sympathetic neuronal function and left ventricular performance during stress in humans by analysis of endogenous catecholamine levels and systolic time intervals. The results showed that performance on a stressful cognitive task was accompanied by changes in plasma catecholamines, heart rate (HR), blood pressure (BP), and systolic time intervals. The cardiovascular responses were significantly correlated with plasma levels of epinephrine (E) and norepinephrine (NE). The relationship between plasma catecholamines and systolic time intervals is thought to reflect sympatho-adrenomedullary impact on left ventricular performance during stress.

The effect of various ionic manipulations and cationic ionophores on the rates of release and synthesis of serotonin were investigated in brain slices prepared from adult male rats. Two depolarizing, univalent cationic ionophores, gramacidin (10 μg/ml) and valinomycin (10 μ/ml), and the non-depolarizing, calcium-specific ionophore, A23187 (190 μM), stimulated both the release and synthesis of serotonin in this tissue preparation. Electrical field depolarization of the brain slices also stimulated both the release and synthesis of serotonin, effects which were completely blocked by a calcium-free media and 1 mM EGTA or by 10 mM Mg2+. Lithium partially blocked the release of serotonin by the stimulated brain slices, but markedly augmented the rates of serotonin biosynthesis. Although electrical stimulation significantly increased the rate of [3H]tryptophan uptake by the slices, the increased rates of release and synthesis of serotonin following treatment of the tissues with the ionophores and lithium were not associated with increased rates of tryptophan uptake. Furthermore, despite the inhibition of the release and synthesis of serotonin by stimulated slices incubated in the presence of magnesium or in the absence of calcium, rates of tryptophan uptake by the stimulated brain slice remained increased. These results taken together with the results of other studies suggest that release and synthesis of serotonin by the serotonergic neuron is tightly coupled to the ionic events attending depolarization and further argue than transcellular calcium fluxes may be of special importance in this regulation. These results do not support the postulate that regulation of serotonin release and synthesis is dependent upon substrate availability as a major variable.

A peptide a-amidating enzyme was purified to apparent homogeneity from porcine pituitary. This enzyme is a glycoprotein with a mol wt of 64,000, a metal prosthetic group, and a dependence upon ascorbate and molecular oxygen. The purified enzyme has a strong preference for peptides ending in glycine. It also catalyzes the oxidation of valylglycine bonds more rapidly than prolylglycine bonds, and demonstrates a primary isotope effect greater than 5 when the α-hydrogens of glycine are replaced by deuterium. Kinetic analysis is consistent with a ping-pong or double displacement catalytic mechanism in which both the peptide substrate and ascorbate are competitive inhibitors with respect to each other. With respect to its kinetic properties, catalytic mechanism, and cofactor requirements, the purified amidating enzyme is very similar to dopa-mine β-hydroxylase, a finding which supports the previous suggestion that the peptide α-amidating enzyme be classified as a peptidyl glycine monooxygenase. (Endocrinology 1...

In male rats maintained on a 12 h light-dark schedule (6 AM-6 PM), there is a nyctohemeral cycle of plasma prolactin which consists of a nadir at 11:30 AM and an apogee at approximately 11:30 PM. In rats exposed to constant darkness, this rhythm persists for 7 days. Seven days of constant light, however, reverses this diurnal variation such that plasma prolactin levels peak at 11:30 AM and reach a nadir at approximately 11:30 PM. In animals maintained on a 12 h light-dark cycle, ganglionectomy and lateral ventricular injections of 6-OH-dopamine (250 mug) also appear to reverse the diurnal variation of plasma prolactin, whereas a single injection of 6-OH-dopamine (250 mug) into the third ventricle decreases plasma prolactin values at all times intervals but does not alter the diurnal rhythm. Both sites of 6-OH-dopamine administration markedly deplete hypothalamic dopamine and norepinephrine, but injection of 6-OH-dopamine into the lateral ventricle destroys the catecholaminergic terminals in the pineal, whereas injection of 6-OH-dopamine into the third ventricle does not. Pinealectomy slightly increases the early morning values of plasma prolactin, but otherwise has no effect on the diurnal variation of prolactin. Five conclusions appear to be justified: 1) there is a nyctohemeral rhythm of plasma prolactin, which is reversed by constant light; 2) the pineal gland probably plays no role in the diurnal regulation of plasma prolactin secretion; 3) the diurnal rhythm of plasma prolactin is controlled by sympathetic input into the brain via the superior cervical ganglion; 4) a rhythm of plasma prolactin develops in constant light which is the exact opposite of the normal diurnal variation; 5) there appears to be a noradrenergic pathway in the hypothalamus or brainstem which stimulates release of prolactin.

The effects of treatment for 5 or 9 days with varying doses of thyrotropin-releasing hormone (TRH) or the linear beta-alanine TRH congener (pGlu-His-Pro-beta-Ala-NH2) on serum levels of TSH, T3, and T4 were studied in mice and rats. At low doses in rats treatment with TRH for 9 days significantly increased serum levels of T3 but not serum T4 whereas a higher dose of TRH (10 mg/kg) reduced serum T3 levels. beta-Ala TRH (0.1--10 mg/kg IP) treatment for 9 days in rats significantly reduced serum T4 levels whereas serum T3 levels were only depressed at higher doses (1--10 mg/kg IP) of the peptide. In mice treatment for 5 days with TRH (1 and 10 mg/kg IP) significantly reduced serum levels of T3 and T4. In addition, TRH (0.1--10 mg/kg IP) or beta-Ala TRH treatment (1.0--10 mg/kg IP) for 9 days significantly reduced serum TSH levels in rats. TRH (10 mg/kg IP for 9 days) also significantly reduced serum GH levels in rats. No alteration in hypothalamic content of TRH and LHRH was observed after chronic TRH treatment. Some, but not all, of our findings support the hypothesis that treatment with high doses of TRH reduce pituitary-thyroid axis functions by a direct effect on hypophysial TRH receptors.

Antisera were raised to a tridecapeptide, Ser-Asp-Val-Thr-Lys-Arg-Gln-His-Pro-Gly-Arg-Arg-Phe, that was synthesized based on the sequence (residues 166-178) of a proposed cDNA for pro-TRH reported by Lechan et al. With this antiserum, immunostaining of Western blots of rat brain extracts revealed two major proteins with mol wt (Mr = 39,000 and 52,000) considerably larger than that of the largest protein (Mr = 29,000) that could be encoded by the cDNA of Lechan et al. Because these observations suggested the possibility of novel TRH precursors, we studied the immunocytochemical distribution of pro-TRH (39-52K) in rat brain. Our anatomical findings were 4-fold. 1) The distributions of 29K pro-TRH and 39-52K pro-TRH are not identical. 2) TRH is found only in regions containing 29K pro-TRH, 39-52K pro-TRH, or both. 3) There are regions that contain both 29K pro-TRH and 39-52K pro-TRH, but no TRH. 4) Regions containing only 39-52K pro-TRH do not contain 29K pro-TRH mRNA as mapped by Segerson et al. From these electrophoretic and anatomical observations, we postulate the existence of at least one and possibly two additional precursors that can be processed to TRH in rat brain.

Current assays for glycine-directed, peptide-amidating enzymes have several shortcomings. In this report, we describe a rapid, sensitive microassay for amidating activity which overcomes these disadvantages. Tissue homogenates are incubated in the presence of D-Tyr-Val-Gly-OH and the product, D-Tyr-Val-NH2, is measured by radioimmunoassay using an antiserum with an affinity for the product, D-Tyr-Val-NH2 (Kaff 2 X 10(8) L/M), 4 orders of magnitude higher than for the substrate, D-Tyr-Val-Gly-OH (Kaff 4 X 10(4) L/M), and 3 orders of magnitude higher than for the deamidated product D-Tyr-Val-OH (Kaff 8 X 10(5) L/M). Addition of N-ethylmaleimide (0.5 mM) to the enzyme incubates prevents the degradation of D-Tyr-Val-NH2 and permits the measurement of enzymatic activity in crude homogenates. This assay is sensitive enough to permit the measurement of amidating activity in crude rat brain homogenates containing as little as 4-8 micrograms protein.

An abiotic system is described which chemically catalyzes the formation of less than Glu-His-Pro-NH2 (thyrotropin-releasing hormone) from less than Glu-His-Pro-amino acid in the presence of copper, ascorbate, and molecular oxygen. Evidence is presented to support the participation of hydroxyl and carbon radicals as reaction intermediates in the production of a peptide amide and an aldehyde or ketone. The characteristics of this model system closely mimic the characteristics of enzymatic peptide amidation, and an oxidative, free-radical mechanism for enzymatic peptide amidation is proposed as an alternative to the mechanism for enzymatic amidation offered by Bradbury et al. (Bradbury, A. F., Finnie, M. D. A., and Smyth, D. G. (1982) Nature 298, 686-688).

Acetylcholinesterase (AChE) activity has been demonstrated ultrastructurally in neurons of the arcuate nucleus and associated with fibers in the arcuate nucleus neuropil and the median eminence (ME) of the rat. In addition, the effects of neonatal monosodium glutamate (MSG) treatment and Halász deafferentation on the AChE staining and localization have been studied. Neonatal MSG-treatment resulted in loss of the majority of AChE-positive neurons in the arcuate nucleus while leaving neuropil staining intact. Halász deafferentation caused a loss of arcuate neuropil activity while leaving the neuronal staining unaltered. These observations are consistent with previous biochemical results suggesting the existence of a cholinergic tuberoinfundibular system with nerve cells in the arcuate nucleus and terminals in the median eminence. In addition, the deafferentation experiments indicated that extra-hypothalamic cholinergic fibers may innervate the arcuate nucleus. Supporting evidence from other biochemical studies and the curious paucity of histochemical and biochemical AChE activity in the ME are also discussed.

The effects of acute stress on norepinephrine (NE) and dopamine (DA) concentrations and of repeated stress on tyrosine hydroxylase (TH) activity were measured in individual hypothalamic nuclei and other rat brain regions. A microdissection technique and radioisotopic enzymatic assays enables these studies to be performed. NE and DA concentrations were reduced and TH activity increased selectively in the arcuate nucleus. These results suggest that the arcuate nucleus may be selectively involved in the stress response and support the hypothesis that catecholamines in the medial basal hypothalamus mediate certain of the neuroendocrine changes observed in stress.

The tryptophan hydroxylase activity of individual nuclei of the limbic system, hypothalamus, and midbrain was determined after various endocrine manipulations in an attempt to identify specific endocrine-responsive serotonergic structures in the rat brain. Following adrenalectomy, thyroidectomy, castration, or treatment with pharmacological doses of dexamethasone, testosterone, or thyroxine, no changes in tryptophan hydroxylase activity occurred in any of the areas of brain examined. Furthermore, in large sections of the hypothalamus and midbrain, total tryptophan hydroxylase activity was unaltered, and no changes in Km for substrate or co-factor were found after adrenalectomy. This study, therefore, has failed to detect any hormonally responsive tryptophan hydroxylase activity in the rat brain. These findings suggest that endocrineinduced alterations in serotonin turnover, if they occur, do so without measurable changes in the activity or kinetic properties of tryptophan hydroxylase. (Endocrinology 98: 7...

Using a recently developed microdissection method, individual hypothalamic nuclei were removed from the brains of adult male rats, and the tyrosine hydroxylase (TH) and dopamine-β- hydroxylase (DBH) activities in each nucleus were determined 9 days after adrenalectomy, thyroidectomy or gonadectomy. Following gonadectomy there was a significant rise in the TH activity in only the median eminence (ME). This increased TH activity was partially reversed by treatment of gonadectomized animals with testosterone. Thyroidectomy induced a significant increase in the TH activity in the ME, the dorsomedial (NDM), the periventricular (NPE) and the arcuate (NARC) nuclei. Twelve hr after administration of thyroxine, the TH activity in the ME of thyroidectomized rats was significantly reduced to levels below those of controls, whereas the TH activities of the other nuclei were returned to control values. Adrenalectomy produced a significant fall in TH activity in the ME, whereas dexamethasone significantly increased the...

The purpose of the present study was to examine the role of substrate availability in the regulation of the release and synthesis of serotonin by brain slices. Electrical field depolarization of the brain slices stimulated the synthesis and release of serotonin in the absence of changes in intracellular tryptophan concentration, in the absence of tryptophan in the incubation bath, and in the absence of changes in total tryptophan uptake. Furthermore, electrical stimulation decreased the apparent Km for tryptophan required for synthesis of serotonin by the slices. Several conclusions were drawn. (1) Rates of serotonin release and synthesis in brain slices may increase independently of the tissue tryptophan concentration or tryptophan uptake. (2) There is little difference in the synthetic rate of serotonin in tissues exposed to pargyline or not at all. (3) Blockade of monoamine oxidase by pargyline appears to give estimates of the synthetic rates of serotonin which are comparable to estimates derived from [3H]-tryptophan incorporation. (4) Newly synthesized serotonin is preferentially released. (5) Only in tissue pretreated with pargyline do increasing concentrations of tryptophan increase the releasable pool of 5-HT. (6) Electrical depolarization may allosterically activate tryptophan hydroxylase; an effect which may be mimicked by homogenization of the tissue.

The purpose of this study was to investigate the chemical nature of the TRH-like bioactivity and immunoreactivity in extracts of human placenta. Methanolic extracts of human placenta contained nearly 36 ng TRH-like bioactivity/g dried tissue. The immunoreactivity of this extract was only 3 ng/g dried tissue. Two molar acetic acid extracts of human placenta yielded 9 ng TRH-like bioactivity/g dried tissue. The immunoreactivity of these acetic acid extracts, however, was 5.5 ng/g dried tissue. When these placental extracts were subjected to gel filtration chromatography, the bioactivity was found to reside in two fractions which were distinct from synthetic TRH. Furthermore, the immunoreactivity present in these placental extracts was also chromatographically distinct from that of synthetic TRH. In conclusion, these experiments confirm the presence of substantial quantities of materials in placenta which possess TRH-like immunoreactivity and bioactivity. These results also argue that the immunoreactive fractions are not bioactive and provide firm evidence that neither the TSH-releasing substances nor the TRH-like immunoreactivity found in human placenta are identical to pyroglu-His-Pro-NH2.

Pro-Leu-Gly-NH2 (PLG), which is the C-terminal tripeptide tail of oxytocin, has been reported to possess melanocyte-stimulating hormone (MSH)-release-inhibiting activity. Although it has been isolated from bovine hypothalamus, little is known about the CNS distribution of this peptide in other species. In this report, we describe the development of a radioimmunoassay which can be used to measure both PLG and oxytocin following chromatographic separation by high pressure liquid chromatography (HPLC). Using this method, we are unable to demonstrate the presence of any endogenous PLG in rat hypothalamus, preoptic area, pituitary, or eye tissue. However, synthetic PLG, which is added to tissue homogenates as an internal standard, is consistently recovered from all areas. We conclude that the PLG tripeptide is not present in the rat brain and thus cannot be the physiological regulator of MSH secretion.

Dissatisfaction with current methods for the production of immunogens for raising antisera to TRH stimulated us to synthesize the hapten, GABA-His-Pro-NH2. Coupling of this hapten to bovine serum albumin at a molar ratio of 18:1 by means of a water-soluble carbodiimide produced an immunogen which stimulated the rapid production in New Zealand white rabbits of antisera with an affinity (2.42 +/- 0.3 X 10(9) l/mol) for TRH, some 8-fold higher than that of antisera (0.33 +/- 0.03 X 10(9) l/mol) raised by immunization with a conjugate produced by the currently accepted bis-diazotized-benzidine bridging technique. These higher affinity antibodies when used in a standard TRH radioimmunoassay permitted the detection of less than 1/pg of TRH per assay tube and showed an extremely low affinity for the two major metabolites of TRH, p-Glu-His-Pro-COOH and His-Pro diketopiperazine (Ka 4.84 X 10(4) and 4.0 X 10(4) l/mol, respectively). Application of this newer radioimmunoassay to the measurement of TRH in brain tissue yielded measurements of TRH content similar to those determined by current RIA methods. Chromatography of whole crude brain extracts revealed one major immunoreactive peak corresponding to authentic TRH. We conclude that immunization of rabbits with this hapten rapidly produces antisera with a high affinity for TRH suitable for the development of a very sensitive TRH radioimmunoassay.

The enzymes catalyzing the breakdown of TRH are soluble and dependent upon active sulfhydryl groups. One of these enzymes is a pyroglutamyl aminopeptidase (EC 3.4.11.8) (apparent molecular weight 60,000 daltons) with a specificity restricted to pyroglutamyl peptide bonds. The second enzyme is a prolyl endopeptidase (apparent molecular weight 70,000 daltons) with a specificity restricted to proline peptide bonds wherein the proline is preceded by an alpha adjacent amino acid possessing a blocked N-terminus. Substrate requirements for this latter enzyme are identical to those reported previously for the TRH deamidase of rat brain, the prolyl endopeptidase of rabbit brain and the post proline cleaving enzyme of rat brain. We conclude that this enzymatic activity variously described as TRH deamidase, post proline cleaving enzyme, and prolyl endopeptidase is that of a single enzyme best denoted as a prolyl endopeptidase (EC 3.4.22.16, proposed). The pyroglutamyl aminopeptidase has a Km for TRH of 45 micro M as compared to a Km for TRH of 2400 micro M for the prolyl endopeptidase. Total enzymatic activity of the prolyl endopeptidase, however, is approximately 3500 nmol/h/rat brain with the total enzymatic activity of the pyroglutamyl aminopeptidase being approximately 600 nmol/h/rat brain. The 5- to 6-fold higher affinity of the pyroglutamyl aminopeptidase for TRH suggests that of these two catabolic pathways, removal of the N-terminal pyroglutamyl moiety is likely to be the more important pathway for the initial catabolism of TRH in rat brain. Analysis of substrate specificity permitted the synthesis of several effective competitive inhibitors of the two enzymes. Of these, the most effective inhibitors of the pyroglutamyl aminopeptidase were p-glu-NH2 (Ki 185 micro M) and p-glu-his-OCH3 (Ki 16.5 micro M). The most effective inhibitors of the prolyl endopeptidase were Ac-gly-pro-NH2 (Ki 1143 micro M) and Z-gly-pro-NH2 (Ki 442 micro M).

To study the possibility of glycine-directed amidation in rat brain, we synthesized the substrate p-Glu-His-Pro-Gly-OH. Adult and neonatal rat brain and adult rat pituitary were sonicated, frozen and thawed, and fractionated by gel permeation chromatography, and fractions from each tissue were assayed for enzymatic activity capable of converting this model substrate into thyrotropin-releasing hormone. We report the presence in rat brain and rat pituitary of two enzymes catalyzing conversion of p-Glu-His-Pro-Gly-OH into thyrotropin-releasing hormone. Based on the differing chemical and physical properties of these two enzymes and their differing affinities for a number of p-Glu-His-Pro-aa-OH analogs (in which aa = glycine, beta-alanine, gamma-butyric acid, and delta-aminovaleric acid), we conclude that there are two distinct enzymatic processes for the terminal amidation of peptides in brain and that COOH-terminal extensions other than glycine are capable of directing COOH-terminal amidation.

Using the microdissection method of Palkovits and a sensitive microassay for dopamine (DA) and norepinephrine (NE), the CNS projections of the nigral A8, A9 and A10 dopaminergic cell bodies have been determined in rat brain. Following bilateral electrolytic lesions of the A8-A9-A10 region, DA levels fell significantly in the following areas: dorsal septal nucleus (85%),olfactory tubercle (75%), lateral amygdaloid nucleus (75%), hebenula (75%), parietal cortex (75%), cingulate cortex (70%), cerebellum (50%), thalamic paraventricular nucleus (50%), and preoptic suprachiasmatic nucleus (45%). Of particular interest was the finding of a 40% fall in the dopamine levels of the median eminence after such lesions. Concentrations of NE in these same areas following lesions of the A8-A9-A10 region were unchanged. These findings suggest that the nigral A8, A9 and A10 dopaminergic cell bodies, project diffusely to many different areas of cortex, hypothalamus, and brain stem, and, more importantly, provide evidence for a previously unsuspected dopaminergic pathway from substantia nigra to median eminence, an area intimately concerned withneuroendocrine regulation.

The mechanism for the post-translational conversion of glutamine to pyroglutamic acid on the N terminus of newly synthesized peptides and proteins is unknown. An assay is reported that permits measurement of the rate of conversion of Gln-His-Pro-NH2 to pyroGlu-His-Pro-NH2 (TRH). Using this assay, we demonstrate that the spontaneous cyclization of the N-terminal glutamine of this peptide occurs only slowly under physiological conditions. Furthermore, we describe the presence in rat brain, porcine pituitary, and human B lymphocytes of an enzyme(s) which converts Gln-His-Pro-NH2 into pyroGlu-His-Pro-NH2. The enzyme(s) appears to be a glycoprotein, is maximally active at neutral pH, has a Mr of 55,000, and contains catalytically significant sulfhydryl groups. The product of the enzymatic reaction was confirmed by high resolution fast atom bombardment-mass spectrometry. In preliminary studies, we find that over 90% of the enzyme in bovine adrenal medulla is contained in the soluble chromaffin vesicle fraction. These findings indicate that in vivo the post-translational conversion of a glutaminyl-peptide into a pyroglutamyl-peptide is neither spontaneous nor abiotic as has been previously proposed.

—A specific and sensitive, radioisotopic microassay for tryptophan hydroxylase (EC 1.14.36) is described, which is capable of determining enzymatic activity in as little as 5 μg of crude brainstem homogenate. 5-Hydroxytryptophan, the immediate product of hydroxylation of tryptophan is enzymatically converted to N-acetylserotonin. A radioisotopic label is then introduced by the enzymatic methylation of N-acetylserotonin in the presence of [3H]methyl-S-adenosyl-methionine. The [3H]-melatonin thus formed is isolated by extraction and counted. With this assay, the activity in individual hypothalamic nuclei (arcuate nucleus, median eminence, suprachiasmatic nucleus, and medial forebrain bundle) has been measured.

Adult male rats were either castrated, thyroidectomized, or treated with haloperidol and the rates of turnover of dopamine (DA) and norepinephrine (NE) in the median eminence (ME), the arcuate and dorsomedial nuclei of the hypothalamus were estimated from the rate of decay of DA and NE concentrations as determined by radioenzymatic assay following blockade of catecholamine synthesis by alpha-methyl-p-tyrosine. The ME of animals similarly prepared was also examined for changes in the total activity and kinetic properties of tyrosine hydroxylase (TH). Four days following the administration of haloperidol (400 microgram/kg) or 10 days after castration, there was a significant increase in the rate of turnover of DA but not NE in the ME accompanied by an increase in the Vmax but not Km for the substrate or cofactor of TH. Furthermore, the administration of haloperidol to hypophysectomized rats also significantly increased the TH activity in the ME, indicating that such changes may occur independently of any changes in serum prolactin levels. Ten days after thyroidectomy, or three weeks after treatment with prophylthiouracil, there was a significant increase in the turnover rate of DA in both the ME and dorsomedial nucleus but not in the arcuate nucleus. No changes in the turnover rates of NE in any of the three areas were observed following thyroidectomy. In the ME, the increase in turnover of DA was accompanied by an increase in the total TH activity (Vmax) as welll as a decrease in Km for tetrahydrobiopterin but not tyrosine. From these results 4 conclusions were drawn: (1) following halperidol, castration, and thyroidectomy there are increases in the activity of dopaminergic terminals within the ME; (2) castration, haloperidol and thyroidectomy may influence the activity of dopaminergic terminals within the ME by different mechanisms; (3) changes in tyrosine hydroxylase and turnover of catecholamines within the ME may occur independently of changes in prolactin levels; and (4) local recurrent afferent circuits may exist in the arcuate nucleus region of the hypothalamus.