"They just looked at me like I was human": The experiences of parenting women and providers with substance use disorder treatment.

BACKGROUND: The current US addiction treatment system does not effectively meet the needs of pregnant and parenting women with substance use disorder (SUD). The aim of this research was to identify barriers and facilitators to engagement and retention in SUD residential treatment for pregnant and parenting women. This research was part of a co-design process to collaboratively create a more patient-centered long-term residential program. DESIGN AND METHODS: The study conducted semi-structured individual interviews with both parenting women with lived experience (WWLE) in residential SUD treatment and SUD treatment providers. Interviews aimed to elicit participants' experiences either receiving or providing care. The study team analyzed data in NVivo-12 using a deductive codebook based on the six principles of trauma informed care (TIC). RESULTS: We conducted a total of 32 interviews (WWLE =13, SUD providers =19). The study identified four major themes: 1) peer relationships provide inspiration and diminish shame; 2) providing individuals safe space to stumble in recovery creates opportunities for growth and builds self-efficacy; 3) reasonable, clear boundaries create a structured, protective environment for early recovery; 4) nonjudgmental connections facilitate engagement and build trust. We identified small pivotal moments along the continuum of care that showed how the elements in the four themes enhanced engagement and retention in treatment. These interactions, along the care continuum, are either structural (workflow process) or relational (interpersonal). CONCLUSION: This research increases understanding of the interplay of the structural and relational barriers and facilitators to engagement and retention in treatment. These seemingly minor positive or negative interactions along the care continuum are pivotal to fully operationalizing TIC and optimizing women's engagement in treatment. Improvement strategies that integrate the voices of WWLE and collaboratively co-design a more patient-centered system are critical steps to improving engagement in SUD treatment and more equitable SUD treatment services.

Role and Function of the Clinical Tutor in Mental Health Nursing in Ireland.

The clinical tutor (CT) in mental health nursing is a role aimed at supporting the learning needs of mental health nursing students undertaking a 12-month post-registration programme. This paper aims to examine the role of the clinical tutor in mental health nursing in Ireland by describing the experience of nursing students and key service stakeholders. A qualitative descriptive design was employed using focus group discussions and semi-structured interviews. Two focus groups were conducted with 14 nursing students in the final week of their one-year programme. Semi-structured interviews were undertaken with seven service stakeholders and service leaders. Participants reported positive experiences of working with the clinical tutor and valued the role in terms of educational and pastoral support. Participants suggested the role strengthened the link between theory and practice and enhanced the relationship between the higher education institute and clinical sites. However, a lack of clarity existed in terms of role description. Participants suggested the CT role enhanced the link between the university and clinical areas providing benefits to both student and service stakeholders. Implementing similar roles may benefit post-registration mental health nursing students in other jurisdictions. Further investigation on how the role operates from the perspective of those in the post may provide more clarity and enhance the development of such roles in the future.

Genetics of ncHH: from a peculiar inheritance of a novel GNRHR mutation to a comprehensive review of the literature.

BACKGROUND: Normosmic congenital hypogonadotropic hypogonadism (ncHH) is caused by the deficient production, secretion, or action of gonadotropin-releasing hormone (GnRH). Its typical clinical manifestation is delayed puberty and azoospermia. Homozygous and compound heterozygous mutations in the GNRHR gene (4q13.2) are the most frequent genetic causes of ncHH. OBJECTIVES: (i) Characterization at the molecular level (genetic origin and functional effect) of a unique homozygous mutation (p.Gly99Glu) in a ncHH man; (ii) to provide a comprehensive catalog of GNRHR mutations with genotype-phenotype correlation and comparison of in vitro studies vs. in silico prediction tools. MATERIAL AND METHODS: A ncHH man and his parents, in whom we performed the following: (i) Sanger sequencing, qPCR of the GNRHR gene; (ii) chromosome 4 SNP array; and (iii) competition binding assay and inositol phosphate signaling assay. PubMed and Human Genome Mutation Database (HGMD) search for GNRHR mutations. Bioinformatic analysis of 55 reported variants. RESULTS: qPCR showed two GNRHR copies in the index case. SNP array revealed the inheritance of two homologous chromosomes 4 from the mother (maternal heterodisomy; hUPD) with two loss of heterozygosity regions, one of them containing the mutated gene (maternal isodisomy; iUPD). Functional studies for the p.Gly99Glu mutation demonstrated a right-shifted GnRH-stimulated signaling response. Bioinformatic tools show that commonly used in silico tools are poor predictors of the function of ncHH-associated GNRHR variants. DISCUSSION: Functional analysis of the p.Gly99Glu mutation is consistent with severely decreased GnRH binding affinity (a severe partial loss-of-function mutation). Complete LOF variants are associated with severe and severe/moderate phenotype, whereas partial LOF variants show wide range of clinical manifestations. CONCLUSION: This is the first ncHH patient carrying a novel causative missense mutation of GNRHR with proven 'severe pLOF' due to maternal hUPD/iUPD of chromosome 4. Our literature review shows that functional studies remain essential both for diagnostic and potential therapeutic purposes.

Assessment of florfenicol as a possible treatment for chlamydiosis in koalas (Phascolarctos cinereus).

OBJECTIVE: Because of limited availability of chloramphenicol to veterinary suppliers, a preliminary study was performed to predict whether an analogue, florfenicol, is an efficacious treatment for chlamydiosis in koalas. METHODS: Florfenicol was administered to koalas with naturally occurring chlamydiosis at 20 mg/kg SC (n = 3) and at 5 mg/kg (n = 3) and 10 mg/kg (n = 3) IV. The estimated areas under the plasma concentration versus time curves (AUC) were compared with the minimum inhibitory concentration to inhibit Chlamydia pecorum. Clinical data were also examined from field trials conducted on koalas (n = 19) with naturally occurring chlamydiosis and treated with florfenicol at a range of dosages (5-20 mg/kg SC and 6-15 mg/kg IV). Florfenicol binding to proteins in plasma was also determined. RESULTS: Florfenicol was not detectable in plasma 24 h post-administration at 20 mg/kg SC. The estimated AUC0-24 h following administration at 10 mg/kg IV suggests florfenicol might be effective against Chlamydia spp. via this route. Florfenicol binding to plasma proteins was 13.0% (± 0.30 SEM). After treatment with florfenicol in field trials, 5 of 19 koalas (26%) were released without further treatment, 4 with no long-term follow-up; 6 (32%) required additional treatment with chloramphenicol to resolve chlamydiosis; 7 (36%) failed to clinically improve, of which 3 had clinical signs and/or necropsy findings suggestive of antibiotic-related gastrointestinal dysbiosis; another koala died within minutes of florfenicol administered IV at 7 mg/kg. CONCLUSION: When administered at dosages tolerable in the field, florfenicol is a problematic treatment for chlamydiosis based on equivocal outcomes and plasma concentrations below those that inhibit the pathogen.

Evaluation of multi-scale mineralized collagen-polycaprolactone composites for bone tissue engineering.

A particular challenge in biomaterial development for treating orthopedic injuries stems from the need to balance bioactive design criteria with the mechanical and geometric constraints governed by the physiological wound environment. Such trade-offs are of particular importance in large craniofacial bone defects which arise from both acute trauma and chronic conditions. Ongoing efforts in our laboratory have demonstrated a mineralized collagen biomaterial that can promote human mesenchymal stem cell osteogenesis in the absence of osteogenic media but that possesses suboptimal mechanical properties in regards to use in loaded wound sites. Here we demonstrate a multi-scale composite consisting of a highly bioactive mineralized collagen-glycosaminoglycan scaffold with micron-scale porosity and a polycaprolactone support frame (PCL) with millimeter-scale porosity. Fabrication of the composite was performed by impregnating the PCL support frame with the mineral scaffold precursor suspension prior to lyophilization. Here we evaluate the mechanical properties, permeability, and bioactivity of the resulting composite. Results indicated that the PCL support frame dominates the bulk mechanical response of the composite resulting in a 6000-fold increase in modulus compared to the mineral scaffold alone. Similarly, the incorporation of the mineral scaffold matrix into the composite resulted in a higher specific surface area compared to the PCL frame alone. The increased specific surface area in the collagen-PCL composite promoted increased initial attachment of porcine adipose derived stem cells versus the PCL construct.

The Parkinson's gene PINK1 regulates cell cycle progression and promotes cancer-associated phenotypes.

PINK1 (phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced kinase 1), a Parkinson's disease-associated gene, was identified originally because of its induction by the tumor-suppressor PTEN. PINK1 promotes cell survival and potentially metastatic functions and protects against cell stressors including chemotherapeutic agents. However, the mechanisms underlying PINK1 function in cancer cell biology are unclear. Here, using several model systems, we show that PINK1 deletion significantly reduced cancer-associated phenotypes including cell proliferation, colony formation and invasiveness, which were restored by human PINK1 overexpression. Results show that PINK1 deletion causes major defects in cell cycle progression in immortalized mouse embryonic fibroblasts (MEFs) from PINK1(-/-) mice, and in BE(2)-M17 cells stably transduced with short hairpin RNA against PINK1. Detailed cell cycle analyses of MEF cell lines from several PINK1(-/-) mice demonstrate an increased proportion of cells in G2/M and decreased number of cells in G1 following release from nocodazole block. This was concomitant with increased double and multi-nucleated cells, a reduced ability to undergo cytokinesis and to re-enter G1, and significant alterations in cell cycle markers, including failure to increase cyclin D1, all indicative of mitotic arrest. PINK1(-/-) cells also demonstrated ineffective cell cycle exit following serum deprivation. Cell cycle defects associated with PINK1 deficiency occur at points critical for cell division, growth and stress resistance in cancer cells were rescued by ectopic expression of human PINK1 and demonstrated PINK1 kinase dependence. The importance of PINK1 for cell cycle control is further supported by results showing that cell cycle deficits induced by PINK1 deletion were linked mechanistically to aberrant mitochondrial fission and its regulation by dynamin-related protein-1 (Drp1), known to be critical for progression of mitosis. Our data indicate that PINK1 has tumor-promoting properties and demonstrates a new function for PINK1 as a regulator of the cell cycle.

Validation of ultrasonography in detecting structural disease of the urogenital tract of the koala, Phascolarctos cinereus.

A retrospective review of case records of ultrasonography and necropsy outcomes of 62 koalas was used to investigate the accuracy of ultrasonography in assessing koala urogenital tract structural disease at the Port Macquarie Koala Hospital. The results showed high concordance, supporting ultrasonography as an effective tool for evaluating structural disease of the koala urogenital tract, most commonly seen with chlamydiosis. The study also illustrates the advances benefiting animal welfare that can be made by wildlife carer groups through using a scientific, evidence-based approach.

Mandibular reconstruction with a bioactive-coated cementless Ti6Al4V modular endoprosthesis in Macaca fascicularis.

The titanium mandibular modular endoprosthesis fixed with polymethylmethacrylate cement in the medullary space of the mandible has been introduced in previous studies. However, the internal parts of these devices have been found to be prone to loosening and wound dehiscence. The current study introduces a newly designed bioactive-coated cementless modular mandibular endoprosthesis, which was used for reconstruction in Macaca fascicularis. The devices were coated with hydroxyapatite (HA) and hydroxyapatite/bioglass (HA/BG) and implanted in unilateral mandibular segmental defects in nine monkeys for 6 months. Biomechanical testing found the reconstructed mandible to have a mean stiffness value of 110.43 N/mm. Histologically, there were both fibrous capsule and woven bone around the device body, and histomorphology analysis showed 64.17% bone contact to device stem surface. The percentage bone volume calculated from micro-computed tomography analysis around the stem surface was found to be superior to that reported in previous studies of cemented mandibular endoprostheses. Intermodular connection screw loosening has also been resolved with the dovetail interconnection. In conclusion, the current bioactive-coated cementless mandibular endoprosthesis is feasible for use in mandibular segmental reconstruction. However, insufficient load-bearing capability and a high rate of intraoral wound dehiscence were found in the majority of the study animals. Further device modifications and improvements in the surgical technique need to be addressed in future studies.

Factors associated with early neonatal attendance to a paediatric emergency department.

AIM: To examine the demographic and perinatal factors involved in the presentation of newborn babies to a paediatric emergency department (PED) and outcome following attendance. METHODS: Term babies who attended the PED of the Royal Belfast Hospital for Sick Children (RBHSC) in the first 2 weeks of life, during two separate 3-month periods in summer and winter 2010-2011 were identified retrospectively from the PED electronic database. Perinatal and demographic data were also obtained on all babies born in the Royal Jubilee Maternity Hospital (RJMH) during the same time period. RESULTS: A total of 223 attendances to the PED involving 208 babies were identified with almost equal distribution during summer and winter months. Almost two thirds (n=139, 62%) of babies presented out-of-hours. Over half of babies were self-referred by parent/carer. The most common presentation was feeding difficulty, vomiting or faltering growth, accounting for 36%. Significant factors associated with attendance to PED included birth weight <2500 g, deprivation and postnatal stay more than 2 days. Sixty-one babies (24%) presenting to PED were admitted to hospital. Significant factors for admission included age ≤ 48 h and presentation during the standard working day. Overall, a third of babies admitted stayed less than 24 h (34%). CONCLUSIONS: Large numbers of babies attend the PED in the first 2 weeks of life, commonly out of hours, from deprived areas and with feeding difficulties. A quarter of babies attending are admitted to hospital, with one-third discharged following an overnight stay. Services should be reevaluated, particularly in this current financial climate, in an attempt to find new models of care for these young babies.

Computed tomography-based tissue-engineered scaffolds in craniomaxillofacial surgery.

INTRODUCTION: Tissue engineering provides an alternative modality allowing for decreased morbidity of donor site grafting and decreased rejection of less compatible alloplastic tissues. METHODS: Using image-based design and computer software, a precisely sized and shaped scaffold for osseous tissue regeneration can be created via selective laser sintering. Polycaprolactone has been used to create a condylar ramus unit (CRU) scaffold for application in temporomandibular joint reconstruction in a Yucatan minipig animal model. Following sacrifice, micro-computed tomography and histology was used to demonstrate the efficacy of this particular scaffold design. RESULTS: A proof-of-concept surgery has demonstrated cartilaginous tissue regeneration along the articulating surface with exuberant osseous tissue formation. Bone volumes and tissue mineral density at both the 1 and 3 month time points demonstrated significant new bone growth interior and exterior to the scaffold. CONCLUSION: Computationally designed scaffolds can support masticatory function in a large animal model as well as both osseous and cartilage regeneration. Our group is continuing to evaluate multiple implant designs in both young and mature Yucatan minipig animals.

Dietetic guidelines on food and nutrition in the secondary prevention of cardiovascular disease - evidence from systematic reviews of randomized controlled trials (second update, January 2006).

AIM: To update dietetic guidelines based on systematic review evidence on dietary advice to prevent further events in people with existing cardiovascular disease (CVD) (secondary prevention). METHODS: The Cochrane Library, MEDLINE and EMBASE were comprehensively searched to January 2005 for systematic reviews on aspects of diet and heart health. Reviews were included if they searched systematically for randomized controlled trials relating to diet and secondary prevention of CVD. Each review was critically appraised by at least two members of the UK Heart Health and Thoracic Dietitians Group. The quality and results of each review were discussed and summarized at a group meeting. RESULTS: Evidence-based strategies that reduce cardiovascular events in those with CVD include reduction in saturated fat and substitution with unsaturated fats. Individuals who have suffered a myocardial infarction may also benefit from adopting a Mediterranean type diet and increasing intake of omega 3 fats, but it is not clear whether they are beneficial for all patients with CVD. There is no systematic review evidence to support the use of antioxidant vitamins supplements, low glycaemic index diets, or homocysteine lowering therapies in this group. CONCLUSION: There remains good evidence that reducing saturated fat reduces morbidity in patients with CVD. This advice is consistent for most manifestations of CVD, with the addition of Mediterranean dietary advice and increased omega 3 fats for those who have had a myocardial infarction.

Engineering craniofacial scaffolds.

OBJECTIVE: To develop an integrated approach for engineering craniofacial scaffolds and to demonstrate that these engineered scaffolds would have mechanical properties in the range of craniofacial tissue and support bone regeneration for craniofacial reconstruction. EXPERIMENTAL VARIABLE: Scaffold architecture designed to achieve desired elasticity and permeability. Scaffold external shape designed to match craniofacial anatomy. OUTCOME MEASURE: Final fabricated biomaterial scaffolds. Compressive mechanical modulus and strength. Bone regeneration as measured by micro-CT scanning, mechanical testing and histology. SETTING: Departments of Biomedical Engineering, Oral/Maxillofacial Surgery, and Oral Medicine, Pathology and Oncology at the University of Michigan. RESULTS: Results showed that the design/fabrication approach could create scaffolds with designed porous architecture to match craniofacial anatomy. These scaffolds could be fabricated from a wide range of biomaterials, including titanium, degradable polymers, and degradable calcium phosphate ceramics. Mechanical tests showed that fabricated scaffolds had compressive modulus ranging 50 to 2900 MPa and compressive strength ranging from 2 to over 56 MPa, within the range of human craniofacial trabecular bone. In vivo testing of designed scaffolds showed that they could support bone regeneration via delivery of BMP-7 transduced human gingival fibroblasts in a mouse model. Designed hydroxyapatite scaffolds with pore diameters ranging from 400 to 1200 microns were implanted in minipig mandibular defects for 6 and 18 weeks. Results showed substantial bone ingrowth (between 40 and 50% at 6 weeks, between 70 and 80% at 18 weeks) for all scaffolds, with no significant difference based on pore diameter. CONCLUSION: Integrated image-based design and solid free-form fabrication can create scaffolds that attain desired elasticity and permeability while fitting any 3D craniofacial defect. The scaffolds could be manufactured from degradable polymers, calcium phosphate ceramics and titanium. The designed scaffolds supported significant bone regeneration for all pore sizes ranging from 300 to 1200 microns. These results suggest that designed scaffolds are clinically applicable for complex craniofacial reconstruction.

Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

Role of aspartate7.32(302) of the human gonadotropin-releasing hormone receptor in stabilizing a high-affinity ligand conformation.

Mammalian gonadotropin-releasing hormone (GnRH) receptors preferentially bind mammalian GnRH, which has Arg in position eight. The Glu(7.32(301)) residue, which determines selectivity of the mouse GnRH receptor for Arg(8)-containing GnRH, is Asp(7.32(302)) in the human GnRH receptor. We have confirmed that Asp(7.32(302)) confers selectivity of the human GnRH receptor for Arg(8) of GnRH and investigated the mechanism of this specificity using site-directed mutagenesis and ligand modification. We find that although Arg(8) and Asp(7.32(302)) are required for high-affinity binding of GnRH, conformationally constrained peptides, with D-amino acid substitutions in position six or with a 6,7 gamma-lactam, bind the human GnRH receptor with high affinity, which is independent of the presence of Asp(7.32(302)) in the receptor or Arg(8) in the ligand. The ability of the ligand constraints to compensate for the absence of both Arg(8) and Asp(7.32(302)) indicates that these residues both have roles in stabilizing a high affinity ligand conformation and that their roles are complementary. This suggests that the Arg(8) and Asp(7.32(302)) side chains interact to induce a high affinity conformation of native GnRH. Thus, Asp(7.32(302)) of the human GnRH receptor determines selectivity for mammalian GnRH by its ability to induce a high affinity conformation of its native ligand. However, this initial interaction seems not to contribute to the final ligand-receptor complex. We propose that Arg(8) interacts transiently with Asp(7.32(302)) to induce a high-affinity ligand conformation of GnRH, which then interacts with a binding pocket that is common for both constrained and unconstrained analogs of GnRH.

An optical fibre sensor for in situ measurement of external species in fluids based on artificial neural network pattern recognition.

An optical fibre sensor system capable of detecting contaminants (e.g. particles, inorganic or organic species) in water and other fluids is reported. In this article experimental results are presented for a single optical fibre sensor located at a distance of 150 m from the transmitter/receiver of the system. The fibre is addressed using an optical time domain reflectometer (OTDR) in order to achieve the spatial resolution (along the fibre length) necessary for this investigation. Novel signal processing techniques involving artificial neural networks and pattern recognition have been applied to the signals arising from the sensor in order to allow cross-sensitivity effects, e.g. from fouling due to calcification, to be extracted from the real measurand, e.g. alcohol content.

Insertional mutagenesis of the arginine cage domain of the gonadotropin-releasing hormone receptor.

The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a structural "arginine cage" domain. Previous computational and mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this domain. We have investigated the functional importance of the intrahelical position and orientation of the arginine cage using insertional mutagenesis. Introduction of a single Ala proximal to the conserved Asp-Arg of this domain caused loss of detectable ligand binding. Inserting a second Ala, however, restored high-affinity agonist binding. Further insertion of three or four Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant receptors confirms that the structure required in this region for efficient signaling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that specific orientations of this segment can stabilize high-affinity receptor conformations that are uncoupled from signal transduction.

Systematic analysis of oxidative degradation of polysaccharides using PAGE and HPLC--MS.

Oxidation of polysaccharides yields hydroxyaldehydes and hydroxycarboxylic acids. Aldehydes and carboxylic acids were separately conjugated to 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) or tyrosine t-butyl ester (TBT). The ANTS-labeled derivatives were separated by molecular size on PAGE gels and detected by fluorescence. TBT-labeled derivatives were separated by reverse phase chromatography on a C18-HPLC column and analyzed by positive ion electrospray mass spectroscopy (HPLC--MS). This combination of procedures allowed a systematic analysis of carbohydrate oxidation products.

A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists. Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants.

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The chicken GnRH receptor had high affinity for mammalian GnRH (K(i) 4.1 +/- 1.2 nM), similar to the human receptor (K(i) 4.8 +/- 1.2 nM). But, in contrast to the human receptor, it also had high affinity for chicken GnRH ([Gln(8)]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K(i) 5.3 +/- 0.5 and 0.6 +/- 0.01 nM). Three mammalian receptor antagonists were also pure antagonists in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellular loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.

Multiple interactions of the Asp(2.61(98)) side chain of the gonadotropin-releasing hormone receptor contribute differentially to ligand interaction.

Mutation of Asp(2.61(98)) at the extracellular boundary of transmembrane helix 2 of the gonadotropin-releasing hormone (GnRH) receptor decreased the affinity for GnRH. Using site-directed mutagenesis, ligand modification, and computational modeling, different side chain interactions of Asp(2.61(98)) that contribute to high-affinity binding were investigated. The conservative Asp(2. 61(98))Glu mutation markedly decreased the affinity for a series of GnRH analogues containing the native His(2) residue. This mutant showed smaller decreases in affinity for His(2)-substituted ligands. The loss of preference for His(2)-containing ligands in the mutant receptor shows that Asp(2.61(98)) determines the specificity for His(2). Analysis of the affinities of a series of position 2-substituted ligands suggests that a hydrogen bond forms between Asp(2.61(98)) and the delta NH group of His(2) and that Asp(2. 61(98)) forms a second hydrogen bond with the ligand. Substitution of Asp(2.61(98)) with an uncharged residue further decreased the affinity for all ligands and also decreased receptor expression. Computational modeling indicates an intramolecular ionic interaction of Asp(2.61(98)) with Lys(3.32(121)) in transmembrane helix 3. The uncharged, Lys(3.32(121))Gln mutation also markedly decreased agonist affinity. The modeling and the similar phenotypes of mutants with uncharged substitutions for Asp(2.61(98)) or Lys(3.32(121)) are consistent with the presence of this helix 2-helix 3 interaction. These studies support a dual role for Asp(2.61(98)): formation of an interhelical interaction with Lys(3.32(121)) that contributes to the structure of the agonist binding pocket and an interaction with His(2) of GnRH that helps stabilize agonist complexing.