[The use of lived experiences among psychiatrists: a qualitative empirical exploration]. / Gebruik van ervaringskennis onder psychiaters; kwalitatieve empirische verkenning.

BACKGROUND: Psychiatrists generally make little use of lived experiences in addition to clinical and scientific knowledge, even while its use may make services more humane. AIM: This study aims to explore psychiatrists’ lived experience perspectives and their considerations when integrating the personal into the professional domain. METHODS: As part of a qualitative participatory research approach, peer supervision sessions were followed for 2 years and additional interviews and a focus group were organised which were then analysed thematically. RESULTS: The participating psychiatrists had three main considerations for using their own experiences in clinical practice: personal, professional and clinical relevance. We identified 11 facilitating and 9 hindering factors in working with lived experiences related to clinical practice. In addition to the high workload and responsibility as a practitioner, a barrier is the lack of experience and recognition of this as a type of knowledge within the profession, including misconceptions about possible harmful effects. Facilitating factors included the opportunity to share with peers, a warm working relationship with patients and being able to create openness and destigmatisation among colleagues. CONCLUSION: Psychiatrists appreciated the integration of lived experiences into the professional domain, even though still in its infancy. The peer supervision setting in this study was experienced as a safe space to share personal experiences with vulnerability and explore how they can harness lived experiences in the work context.

Development of the South African wastewater sludge guidelines.

The new South African Wastewater Sludge Guideline Series will replace the document entitled: "Permissible Utilisation and Disposal of Sewage Sludge, Edition 1, 1997" which deals with application of sewage sludge to land. The new guideline series will be published as 5 volumes: Volume 1: Management Options for the Use or Disposal of Wastewater Sludge, Volume 2: Requirements for the agricultural use of wastewater sludge, Volume 3: Requirements for the on site and off-site disposal of wastewater sludge, Volume 4: Requirements for the use of wastewater sludge in the production of commercial products, Volume 5: Requirements for the incineration of wastewater sludge, We envisage that Volumes 1 and 2 will be published at the time of the conference and that work has begun on Volume 3, 4 and 5. The paper aims to communicate the major differences between the previous sludge guidelines and these new sludge guideline series. For example, the paper details the new wastewater sludge classification system and the requirements for the characterisation of the sludge for classification purposes. The paper will also explain the approach followed in the development of Volume 1 and 2 and will elaborate on the proposed approach for Volumes 3, 4 and 5.

Characterizing the mechanisms of progression in multiple sclerosis: evidence and new hypotheses for future directions.

Major advancements have been achieved in our ability to diagnose multiple sclerosis (MS) and to commence treatment intervention with agents that can favorably affect the disease course. Although MS exacerbations and the emergence of disability constitute the more conspicuous aspects of the disease process, evidence has confirmed that most of the disease occurs on a constitutive and occult basis. Disease-modifying therapies appear to be modest in the magnitude of their treatment effects, particularly in the progressive stage of the disease. Therapeutic strategies currently used for MS primarily target the inflammatory cascade. Several potential mechanisms appear to be involved in the progression of MS. Characterizing these mechanisms will result in a better understanding of the various forms of the disorder and how to effectively treat its clinical manifestations. It is our objective within this 2-part series on progression in MS to offer both evidence-based observations and hypothesis-driven expert perspectives on what constitutes the cause of progression in MS. We have chosen areas of inquiry that appear to have been most productive in helping us to better conceptualize the landscape of what MS looks like pathologically, immunologically, neuroscientifically, radiographically, and genetically. We have attempted to advance hypotheses focused on a deeper understanding of what contributes to the progression of this illness and to illustrate new technical capabilities that are catalyzing novel research initiatives targeted at achieving a more complete understanding of progression in MS.

The utility of computerized neuropsychological assessment of cognitive dysfunction in patients with relapsing-remitting multiple sclerosis.

Traditional paper-and-pencil neuropsychological batteries used to document cognitive deficits in multiple sclerosis (MS) patients lack timing precision. This makes it difficult to accurately measure psychomotor slowing, a central cognitive symptom of MS. Additionally, traditional batteries lack multiple alternate forms necessary to control for practice effects when assessing cognition over time. Finally such batteries are lengthy and expensive. Computerized neuropsychological batteries address many of these shortcomings. They measure response time more precisely, require less administration time, include alternate forms, and are ideal for rapid screening/triage. Although there are normative data on the reliability and validity of computerized measures, there have been no controlled validation studies with MS patients. The current study was designed to validate a computerized neuropsychological battery (ANAM) for use with relapsing-remitting (RR) MS patients. Prior to initiation of interferon-beta-1a (Avonex) treatment, subjects participated in a neuropsychological evaluation consisting of traditional and computerized measures. Moderate-to-high correlations were found between computerized and traditional measures. Computerized tests accurately predicted performance on key traditional tests. The battery was also concordant with traditional measures in identifying RR MS patients with and without neurocognitive impairment. Findings are discussed with respect to increased accuracy and accessibility of neuropsychological evaluations for MS patients.

Chronic muscle pain induced by repeated acid Injection is reversed by spinally administered mu- and delta-, but not kappa-, opioid receptor agonists.

Opioids are commonly used for pain relief clinically and reduce hyperalgesia in most animal models. Two injections of acidic saline into one gastrocnemius muscle 5 days apart produce a long-lasting bilateral hyperalgesia without associated tissue damage. The current study was undertaken to assess the effects of opioid agonists on mechanical hyperalgesia induced by repeated intramuscular injections of acid. Morphine (mu-agonist), [D-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (mu-agonist; DAMGO), 4-[((alpha)R)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (delta-agonist; SNC80), or (1S-trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cylcohexyl]-benzeneacetamide hydrochloride (kappa-agonist; U50,488) were administered intrathecally to activate opioid receptors once hyperalgesia was developed. Mechanical hyperalgesia was assessed by measuring the withdrawal thresholds to mechanical stimuli (von Frey filaments) before the first and second intramuscular injection, 24 h after the second intramuscular injection, and for 1 h after administration of the opioid agonist or vehicle. Morphine, DAMGO, and SNC80 dose dependently increased the mechanical withdrawal threshold back toward baseline responses. The reduction in hyperalgesia produced by morphine and DAMGO was prevented by H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH(2) (CTAP) and that of SNC80 was prevented by naltrindole. U50,488 had no effect on the decreased mechanical withdrawal thresholds. Thus, activation of mu- and delta-, but not kappa-, opioid receptors in the spinal cord reduces mechanical hyperalgesia following repeated intramuscular injection of acid, thus validating the use of this new model of chronic muscle pain.

High-resolution NMR structure of the chemically-synthesized melanocortin receptor binding domain AGRP(87-132) of the agouti-related protein.

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic (appetite-stimulating) activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), contains five disulfide bonds and exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The three-dimensional structure of this domain has been determined by 1H NMR at 800 MHz. The first 34 residues of AGRP(87-132) are well-ordered and contain a three-stranded antiparallel beta sheet, where the last two strands form a beta hairpin. The relative spatial positioning of the disulfide cross-links demonstrates that the ordered region of AGRP(87-132) adopts the inhibitor cystine knot (ICK) fold previously identified for numerous invertebrate toxins. Interestingly, this may be the first example of a mammalian protein assigned to the ICK superfamily. The hairpin's turn region presents a triplet of residues (Arg-Phe-Phe) known to be essential for melanocortin receptor binding. The structure also suggests that AGRP possesses an additional melanocortin-receptor contact region within a loop formed by the first 16 residues of its C-terminal domain. This specific region shows little sequence homology to the corresponding region of the agouti protein, which is an MC1R antagonist involved in pigmentation. Consideration of these sequence differences, along with recent experiments on mutant and chimeric melanocortin receptors, allows us to postulate that this loop in the first 16 residues of its C-terminal domain confers AGRP's distinct selectivity for MC3R and MC4R.

CCR2 and CCR5 receptor-binding properties of herpesvirus-8 vMIP-II based on sequence analysis and its solution structure.

Human herpesvirus-8 (HHV-8) is the infectious agent responsible for Kaposi's sarcoma and encodes a protein, macrophage inflammatory protein-II (vMIP-II), which shows sequence similarity to the human CC chemokines. vMIP-II has broad receptor specificity that crosses chemokine receptor subfamilies, and inhibits HIV-1 viral entry mediated by numerous chemokine receptors. In this study, the solution structure of chemically synthesized vMIP-II was determined by nuclear magnetic resonance. The protein is a monomer and possesses the chemokine fold consisting of a flexible N-terminus, three antiparallel beta strands, and a C-terminal alpha helix. Except for the N-terminal residues (residues 1-13) and the last two C-terminal residues (residues 73-74), the structure of vMIP-II is well-defined, exhibiting average rmsd of 0.35 and 0.90 A for the backbone heavy atoms and all heavy atoms of residues 14-72, respectively. Taking into account the sequence differences between the various CC chemokines and comparing their three-dimensional structures allows us to implicate residues that influence the quaternary structure and receptor binding and activation of these proteins in solution. The analysis of the sequence and three-dimensional structure of vMIP-II indicates the presence of epitopes involved in binding two receptors CCR2 and CCR5. We propose that vMIP-II was initially specific for CCR5 and acquired receptor-binding properties to CCR2 and other chemokine receptors.

Chemical synthesis of lymphotactin: a glycosylated chemokine with a C-terminal mucin-like domain.

The synthesis of a 93-residue chemokine, lymphotactin, containing eight sites of O-linked glycosylation, was achieved using the technique of native chemical ligation. A single GalNAc residue was incorporated at each glycosylation site using standard Fmoc-chemistry to achieve the first total synthesis of a mucin-type glycoprotein. Using this approach quantities of homogeneous material were obtained for structural and functional analysis.

Functional contributions of noncysteine residues within the cystine knots of human chorionic gonadotropin subunits.

Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys-X(1)-Gly-X(2)-Cys and Cys-X(3)-Cys. Previously, we characterized the contributions that cystine residues of the hCG subunit cystine knots make in folding, assembly, and bioactivity. Here, we determined the contributions that noncysteine residues make in hCG folding, secretion, and assembly. When the X(1), X(2), and X(3) residues of hCG-alpha and -beta were substituted by swapping their respective cystine knot motifs, the resulting chimeras appeared to fold correctly and were efficiently secreted. However, assembly of the chimeras with their wild type partner was almost completely abrogated. No single amino acid substitution completely accounted for the assembly inhibition, although the X(2) residue made the greatest individual contribution. Analysis by tryptic mapping, high performance liquid chromatography, and SDS-polyacrylamide gel electrophoresis revealed that substitution of the central Gly in the Cys-X(1)-Gly-X(2)-Cys sequence of either the alpha- or beta-subunit cystine knot resulted in non-native disulfide bond formation and subunit misfolding. This occurred even when the most conservative change possible (Gly --> Ala) was made. From these studies we conclude that all three "X" residues within the hCG cystine knots are collectively, but not individually, required for the formation of assembly-competent hCG subunits and that the invariant Gly residue is required for efficient cystine knot formation and subunit folding.

Intracellular folding pathway of the cystine knot-containing glycoprotein hormone alpha-subunit.

Three of the five disulfide bonds in the glycoprotein hormone alpha-subunit (GPH-alpha) form a cystine knot motif that stabilizes a three-loop antiparallel structure. Previously, we described a mutant (alpha(k)) that contained only the three knot disulfide bonds and demonstrated that the cystine knot was necessary and sufficient for efficient GPH-alpha folding and secretion. In this study, we used alpha(k) as a model to study the intracellular GPH-alpha folding pathway. Cystine knot formation proceeded through a 1-disulfide intermediate that contained the 28-82 disulfide bond. Formation of disulfide bond 10-60, then disulfide bond 32-84, followed the formation of 28-82. Whether the two non-cystine knot bonds 7-31 and 59-87 could form independent of the knot was also tested. Disulfide bond 7-31 formed rapidly, whereas 59-87 did not form when all cysteine residues of the cystine knot were converted to alanine, suggesting that 7-31 forms early in the folding pathway and that 59-87 forms during or after cystine knot formation. Finally, loop 2 of GPH-alpha has been shown to be very flexible, suggesting that loop 2 does not actively drive GPH-alpha folding. To test this, we replaced residues 36-55 in the flexible loop 2 with an artificially flexible glycine chain. Consistent with our hypothesis, folding and secretion were unaffected when loop 2 was replaced with the glycine chain. Based on these findings, we describe a model for the intracellular folding pathway of GPH-alpha and discuss how these findings may provide insight into the folding mechanisms of other cystine knot-containing proteins.

Efficacy of inpatient and home treatment in psychiatrically disturbed children and adolescents. Follow-up assessment of the results of a controlled treatment study.

In two German child and adolescent psychiatric treatment and research centers, a controlled treatment study was conducted in which two randomized treatment groups (in-patient treatment and home treatment) were compared. Subjects were children and adolescents with severe psychiatric disorders, for whom normal outpatient treatment was not sufficient (mean age of the patients was 11 years and 9 months at the beginning of treatment). The results showed no differences in therapy outcome between the two treatment modalities. In a further study, the results of which are presented here, a follow-up assessment (average follow-up interval: 3 years and 8 months) of the two treatment groups (follow-up sample of the inpatient treatment group: n = 33; home treatment group: n = 35) was undertaken in order to investigate the course of the psychiatric disturbances and the long-term effects of the treatments. As measurement categories for the outcome "adaption at school" and "number of marked symptoms" were used in pre-, post- and follow-up assessment. The most important results are 1) The number of marked psychiatric symptoms and the adaptation at school or work exhibit the same type of course over time. Post-treatment scores are much better when compared to pre-treatment scores, but decline slightly upon follow-up, although they remain significantly better than the pre-treatment scores. Thus, the study shows that improvements relating to the psychiatric symptoms are quite stable after several years. 2) There were no relevant differences between the treatment modalities "inpatient treatment" and "home treatment" in terms of effect-size upon follow-up, and in inferential analysis. Any tendency towards difference was in favor of home treatment. So the results give strong support to the conclusion that at least for a specific group of patients (about 15% of those patients usually treated in an inpatient setting) residential treatment can be replaced by home treatment and that the long-term therapeutic outcome of home treatment is stable and persistent. Thus, in terms of psychiatric care and clinical practice, our results provide empirical support to the idea that home treatment should be used more frequently and much more broadly in the future.

Comparison of the structure of vMIP-II with eotaxin-1, RANTES, and MCP-3 suggests a unique mechanism for CCR3 activation.

Herpesvirus-8 macrophage inflammatory protein-II (vMIP-II) binds a uniquely wide spectrum of chemokine receptors. We report the X-ray structure of vMIP-II determined to 2.1 A resolution. Like RANTES, vMIP-II crystallizes as a dimer and displays the conventional chemokine tertiary fold. We have compared the surface topology and electrostatic potential of vMIP-II to those of eotaxin-1, RANTES, and MCP-3, three CCR3 physiological agonists with known three-dimensional structures. Surface epitopes identified on RANTES to be involved in binding to CCR3 are mimicked on the eotaxin-1 and MCP-3 surface. However, the surface topology of vMIP-II in these regions is markedly different. The results presented here indicate that the structural basis for interaction with the chemokine receptor CCR3 by vMIP-II is different from that for the physiological agonists eotaxin-1, RANTES, and MCP-3. These differences on vMIP-II may be a consequence of its broad-range receptor recognition capabilities.

Kaposiform hemangioendothelioma of the thymus.

Kaposiform hemangioendothelioma is a rare pediatric neoplasm that presents most commonly in the soft tissues. We report the case of a 1-month-old infant who presented with stridor and was found to have a diffusely infiltrating tumor in the thymus that extended into the pericardium and up the carotid sheaths. Histologic examination revealed a vascular tumor infiltrating among the lobules of the lymphocyte-depleted thymus. The lesion had features of both a capillary hemangioma and Kaposi sarcoma. Immunoperoxidase studies on formalin-fixed, paraffin-embedded tissue demonstrated the neoplastic endothelial cells to be positive for vascular markers CD31 and CD34. Antibody to factor VIII-related antigen labeled feeding vessels, but failed to stain the lobules of tumor. Although these tumors have been treated in a fashion similar to capillary hemangiomas in the past, it may be important to differentiate Kaposiform hemangioendotheliomas because of their association with Kasabach-Merritt syndrome and recent success with more aggressive chemotherapy regimens.

Sexual dimorphism in diverse metazoans is regulated by a novel class of intertwined zinc fingers.

Sex determination is regulated by diverse pathways. Although upstream signals vary, a cysteine-rich DNA-binding domain (the DM motif) is conserved within downstream transcription factors of Drosophila melanogaster (Doublesex) and Caenorhabditis elegans (MAB-3). Vertebrate DM genes have likewise been identified and, remarkably, are associated with human sex reversal (46, XY gonadal dysgenesis). Here we demonstrate that the structure of the Doublesex domain contains a novel zinc module and disordered tail. The module consists of intertwined CCHC and HCCC Zn(2+)-binding sites; the tail functions as a nascent recognition alpha-helix. Mutations in either Zn(2+)-binding site or tail can lead to an intersex phenotype. The motif binds in the DNA minor groove without sharp DNA bending. These molecular features, unusual among zinc fingers and zinc modules, underlie the organization of a Drosophila enhancer that integrates sex- and tissue-specific signals. The structure provides a foundation for analysis of DM mutations affecting sexual dimorphism and courtship behavior.

Trait anxiety and prior exposure to non-stressful stimuli: effects on psychophysiological arousal and anxiety.

Arousal and anxiety responses to stressful stimulation are products of multiple factors that may include the physiological, behavioral, cognitive, affective, trait, and state components of our six-system model, as well as mediational and non-mediational perspectives. Within the context of this model, the present experiment examined the effects of prior exposure to neutral stimuli on arousal and anxiety responses to stress. High and low trait-anxious participants were randomly assigned to one of three prior exposure conditions in which they were exposed to stressful stimuli following exposure to: (a) neutral, non-stressful stimulation in the same modality (the Intramodality Prior Exposure or IPE experimental condition); (b) neutral stimuli in a different modality (the Cross-modality Prior Exposure or CPE control condition); or (c) a rest period (the Stress Only or SO control condition). Results showed that low trait anxious subjects had consistently larger arousal and anxiety responses to stress than did highs and that prior exposure to certain same-modality neutral stimuli reduced responses to subsequent stressors. In addition, arousal was greater in response to cognitive than to affective and in response to non-mediational than mediational conditions. Results are discussed in terms of the inverted-U arousal function and of the six-system model and its implications for understanding anxiety and arousal.

The chemokine receptor antagonist AOP-RANTES reduces monocyte infiltration in experimental glomerulonephritis.

UNLABELLED: The chemokine receptor antagonist AOP-RANTES reduces monocyte infiltration in experimental glomerulonephritis. BACKGROUND: This study was designed to evaluate the role of the novel chemokine receptor antagonist amino-oxypentane RANTES (AOP-RANTES), which blocks the binding of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and RANTES to the chemokine receptor-5 (CCR-5) on the infiltration of monocytes in experimental glomerulonephritis. METHODS: Rats were treated twice daily with 12.5 microg AOP-RANTES following an induction of anti-rat-thymocyte antibody-mediated glomerulonephritis. The white blood cell count, glomerular monocyte infiltration, chemokine expression, and collagen type IV deposition were assessed. RESULTS: The induction of glomerulonephritis increased glomerular monocyte/macrophage (M/M) infiltration at 24 hours and at 5 days was still higher than in controls. AOP-RANTES prevented glomerular M/M infiltration at 24 hours and at 5 days. This was paralleled by reduced glomerular collagen type IV deposition as a fibrotic marker in nephritic animals. CONCLUSION: These data show that the CCR-5 chemokine receptor antagonist AOP-RANTES ameliorates M/M infiltration and improves glomerular pathology in experimental glomerulonephritis. The use of chemokine receptor antagonists may offer a new therapeutic option in inflammatory renal injuries.

Unusual breast lesions: radiologic-pathologic correlation.

Unusual lesions of the breast can present a diagnostic challenge. These lesions include systemic diseases, benign tumors, and primary and metastatic malignancies. Lymphadenopathy is the most common mammographic finding associated with collagen vascular disease. Wegener granulomatosis may manifest as an irregular, high-density mass simulating breast cancer. Diabetic fibrous mastopathy manifests at mammography as very dense breast tissue and at ultrasonography (US) as an irregular, hypoechoic mass with striking posterior acoustic shadowing simulating malignancy. Fibromatosis simulates malignancy at mammography as an irregularly shaped, uncalcified, high-density mass and at US as an irregular, hypoechoic mass with posterior acoustic shadowing. At US, granular cell tumor may manifest as a solid, poorly marginated mass with marked posterior acoustic shadowing or may appear more benign. At mammography, hamartomas are typically well-circumscribed, round to oval masses with a thin, radiopaque pseudocapsule; at US, they manifest as a sharply defined, heterogeneous oval mass or as normal glandular tissue. Phyllodes tumor manifests at mammography as a large, well-circumscribed oval or lobulated mass; at US, it usually manifests as an inhomogeneous, solid-appearing mass. At mammography, primary breast lymphoma manifests as a relatively circumscribed mass or a solitary, indistinctly marginated, uncalcified mass. Metastatic lesions may manifest mammographically as single or multiple masses or as diffuse skin thickening; at US, they tend to have circumscribed margins with low-level internal echoes. Radiologists should be familiar with the characteristic mammographic appearances of these lesions and should consider benign and systemic causes in the differential diagnosis when malignant-appearing findings are encountered.

Anxiety and arousal: tests of a new six-system model.

The present experiment was a test of a new six-system model of anxiety, which includes physiological, behavioral, cognitive, affective, trait, and state components of anxiety and also differentiates between direct and mediated responses. The State-Trait Anxiety Inventory was used to screen 795 undergraduates at the University of Maryland. Of the 52 subjects chosen, half were high trait anxious and half low. The two groups were further divided into high and low situational stress conditions. Subjects in the high-stress condition were exposed to two types of stressful cognitive and two types of stressful affective tasks. Subjects in the low-stress condition were exposed to the same four tasks with the stressful aspect removed. Prior state anxiety and cognitive or affective sensitivity were also considered. It was found that the most influential factor in resultant arousal was situational stress. Trait anxiety, state anxiety, and cognitive vs. affective sensitivity also significantly influenced both direct and mediated physiological and subjective anxiety responses. In addition, rather than leading to increased arousal, as hypothesized, the presence of trait and state anxiety reduced arousal under certain conditions.

Aminooxypentane-RANTES, an inhibitor of R5 human immunodeficiency virus type 1, increases the interferon gamma to interleukin 10 ratio without impairing cellular proliferation.

Studies have demonstrated that the beta-chemokines RANTES, MIP-1alpha, and MIP-1beta suppress human immunodeficiency type 1 (HIV-1) replication in vitro. Infection with HIV-1 requires expression of CD4 antigen and the chemokine receptor CXCR4 (X4) or CCR5 (R5) on the surface of target cells. The engagement of these receptors with the viral surface proteins is essential for the membrane fusion process. This study investigated the anti-HIV-1 activity of a derivative of RANTES, the CCR5 antagonist aminooxypentane (AOP)-RANTES, on R5 HIV-1 isolates in peripheral blood mononuclear cells. In drug exposure experiments, AOP-RANTES efficiently inhibited viral replication of HIV-1 R5 strains, with a viral breakthrough observed after the withdrawal of the compound. The HIV-1-specific proliferative capacity was maintained under all conditions when compared with controls. An increase in IFN-gamma production accompanied by a parallel decrease in the generation of IL-10 was observed following the in vitro exposure of cells to AOP-RANTES in the presence of three of four HIV-1 R5 isolates. These experiments confirmed that the chemokine receptor antagonist AOP-RANTES was effective as an inhibitor of HIV-1 R5 strain infectivity in peripheral blood mononuclear cells. The capacity of this compound to maintain HIV-1-specific proliferative activity with a shift toward a type 1 cytokine profile makes this compound a unique molecule, one adopting an immunological pathway to limit HIV-1 infection.

NMR structure of a minimized human agouti related protein prepared by total chemical synthesis.

The structure of the chemically synthesized C-terminal region of the human agouti related protein (AGRP) was determined by 2D 1H NMR. Referred to as minimized agouti related protein, MARP is a 46 residue polypeptide containing 10 Cys residues involved in five disulfide bonds that retains the biological activity of full length AGRP. AGRP is a mammalian signaling molecule, involved in weight homeostasis, that causes adult onset obesity when overexpressed in mice. AGRP was originally identified by homology to the agouti protein, another potent signaling molecule involved in obesity disorders in mice. While AGRP's exact mechanism of action is unknown, it has been identified as a competitive antagonist of melanocortin receptors 3 and 4 (MC3r, MC4r), and MC4r in particular is implicated in the hypothalamic control of feeding behavior. Full length agouti and AGRP are only 25% homologous, however, their active C-terminal regions are approximately 40% homologous, with nine out of the 10 Cys residues spatially conserved. Until now, 3D structures have not been available for either agouti, AGRP or their C-terminal regions. The NMR structure of MARP reported here can be characterized as three major loops, with four of the five disulfide bridges at the base of the structure. Though its fold is well defined, no canonical secondary structure is identified. While previously reported structural models of the C-terminal region of AGRP were attempted based on Cys homology between AGRP and certain toxin proteins, we find that Cys spacing is not sufficient to correctly determine the 3D fold of the molecule.