Structure of Fab hGR-2 F6, a competitive antagonist of the glucagon receptor.

The monoclonal antibody hGR-2 F6 has been raised against the human glucagon receptor and shown to act as a competitive antagonist. As a first step in the structural characterization of the receptor, the crystal structure of the Fab fragment from this antibody is reported at 2.1 A resolution. The hGR-2 F6 Fab crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 76.14, b = 133.74, c = 37.46 A. A model generated by homology modelling was used as an aid in the chain-tracing and the Fab fragment structure was subsequently refined (final R factor = 21.7%). The structure obtained exhibits the typical immunoglobulin fold. Complementarity-determining regions (CDRs) L1, L2, L3, H1 and H2 could be superposed onto standard canonical CDR loops. The H3 loop could be classified according to recently published rules regarding loop length, sequence and conformation. This loop is 14 residues long, with an approximate beta-hairpin geometry, which is distorted somewhat by the presence of two trans proline residues at the beginning of the loop. It is expected that this H3 loop will facilitate the design of synthetic probes for the glucagon receptor that may be used to investigate receptor activity.

Structural characterisation of the native fetuin-binding protein Scilla campanulata agglutinin: a novel two-domain lectin.

The three-dimensional structure of a 244-residue, multivalent, fetuin-binding lectin, SCAfet, isolated from bluebell (Scilla campanulata) bulbs, has been solved at 3.3 A resolution by molecular replacement using the coordinates of the 119-residue, mannose-binding lectin, SCAman, also from bluebell bulbs. Unlike most monocot mannose-binding lectins, such as Galanthus nivalis agglutinin from snowdrop bulbs, which fold into a single domain, SCAfet contains two domains with approximately 55% sequence identity, joined by a linker peptide. Both domains are made up of a 12-stranded beta-prism II fold, with three putative carbohydrate-binding sites, one on each subdomain. SCAfet binds to the complex saccharides of various animal glycoproteins but not to simple sugars.

Insights into carbohydrate recognition by Narcissus pseudonarcissus lectin: the crystal structure at 2 A resolution in complex with alpha1-3 mannobiose.

Carbohydrate recognition by monocot mannose-binding lectins was studied via the crystal structure determination of daffodil (Narcissus pseudonarcissus) lectin. The lectin was extracted from daffodil bulbs, and crystallised in the presence of alpha-1,3 mannobiose. Molecular replacement methods were used to solve the structure using the partially refined model of Hippeastrum hybrid agglutinin as a search model. The structure was refined at 2.0 A resolution to a final R -factor of 18.7 %, and Rfreeof 26.7 %. The main feature of the daffodil lectin structure is the presence of three fully occupied binding pockets per monomer, arranged around the faces of a triangular beta-prism motif. The pockets have identical topology, and can bind mono-, di- or oligosaccharides. Strand exchange forms tightly bound dimers, and higher aggregation states are achieved through hydrophobic patches on the surface, completing a tetramer with internal 222-symmetry. There are therefore 12 fully occupied binding pockets per tetrameric cluster. The tetramer persists in solution, as shown with small-angle X-ray solution scattering. Extensive sideways and out-of-plane interactions between tetramers, some mediated via the ligand, make up the bulk of the lattice contacts.A fourth binding site was also observed. This is unique and has not been observed in similar structures. The site is only partially occupied by a ligand molecule due to the much lower binding affinity. A comparison with the Galanthus nivalis agglutinin/mannopentaose complex suggests an involvement of this site in the recognition mechanism for naturally occurring glycans.

Structure of the native (unligated) mannose-specific bulb lectin from Scilla campanulata (bluebell) at 1.7 A resolution.

The X-ray crystal structure of native Scilla campanulata agglutinin, a mannose-specific lectin from bluebell bulbs and a member of the Liliaceae family, has been determined by molecular replacement and refined to an R value of 0.186 at 1.7 A resolution. The lectin crystallizes in space group P21212 with unit-cell parameters a = 70. 42, b = 92.95, c = 46.64 A. The unit cell contains eight protein molecules of Mr = 13143 Da (119 amino-acid residues). The asymmetric unit comprises two chemically identical molecules, A and B, related by a non-crystallographic twofold axis perpendicular to c. This dimer further associates by crystallographic twofold symmetry to form a tetramer. The fold of the polypeptide backbone closely resembles that found in the lectins from Galanthus nivalis (snowdrop) and Hippeastrum (amaryllis) and contains a threefold symmetric beta-prism made up of three antiparallel four-stranded beta-sheets. Each of the four-stranded beta-sheets (I, II and III) possesses a potential saccharide-binding site containing conserved residues; however, site II has two mutations relative to sites I and III which may prevent ligation at this site. Our study provides the first accurate and detailed description of a native (unligated) structure from this superfamily of mannose-specific bulb lectins and will allow comparisons with a number of lectin-saccharide complexes which have already been determined or are currently under investigation.

Isolation, characterization, molecular cloning and molecular modelling of two lectins of different specificities from bluebell (Scilla campanulata) bulbs.

Two lectins have been isolated from bluebell (Scilla campanulata) bulbs. From their isolation by affinity chromatography, they are characterized as a mannose-binding lectin (SCAman) and a fetuin-binding lectin (SCAfet). SCAman preferentially binds oligosaccharides with alpha(1,3)- and alpha(1,6)-linked mannopyranosides. It is a tetramer of four identical protomers of approx. 13 kDa containing 119 amino acid residues; it is not glycosylated. The fetuin-binding lectin (SCAfet), which is not inhibited by any simple sugars, is also unglycosylated. It is a tetramer of four identical subunits of approx. 28 kDa containing 244 residues. Each 28 kDa subunit is composed of two 14 kDa domains. Both lectins have been cloned from a cDNA library and sequenced. X-ray crystallographic analysis and molecular modelling studies have demonstrated close relationships in sequence and structure between these lectins and other monocot mannose-binding lectins. A refined model of the molecular evolution of the monocot mannose-binding lectins is proposed.

Crystallization and preliminary structural studies of Scilla campanulata lectin complexed with alpha1-6 mannobiose.

Recent work has shown that Scilla campanulata agglutinin from bluebell bulbs has a strong affinity for alpha(1,3)- and alpha(1,6)-linked mannosyl residues and possesses moderate antiretroviral activity. This lectin has been crystallized by the hanging-drop method of vapour diffusion complexed with the disaccharide mannose-alpha1,6-D-mannose. The crystals are in the space group P21212 with unit-cell dimensions a = 70.63, b = 92.79 and c = 47.25 A, and with a dimer in the asymmetric unit. The crystals diffract X-rays to beyond 1.5 A resolution at 277 K and are stable in an X-ray beam. Data to 1.6 A resolution have been collected using a MAR image-plate system at a synchrotron source and the structure of the complex has been solved by the molecular replacement method.

Scilla campanulata agglutinin crystallized in complex with the trimannoside alpha-D-man-(1-->6)-[alpha-D-man-(1-->3)]-alpha-D-Man.

The monocot mannose-specific lectin, Scilla campanulata agglutinin (SCA), from bluebell bulbs has a strong affinity for alpha1,3- and alpha1,6-linked mannosyl residues. SCA has been co-crystallized with the trisaccharide alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranosyl-(1-->3)-alpha-D -mannopyranoside ¿alpha-D-Man-(1-->6)-[alpha-D-Man-(1-->3)]-alpha-D-Man¿, the core structure of biantennary N-linked oligosaccharides. Crystals of the complex were obtained by the hanging-drop vapour-diffusion technique. A complete data set to 2.5 A resolution has been collected at 100 K, using a MAR image-plate system at a synchrotron source, from crystals which belong to the space group C2 with unit-cell dimensions a = 99.38, b = 119.86, c = 77.10 A and beta = 105.56 degrees. Use of a CCD detector with cryo-cooled crystals improved the resolution to 2.3 A. A molecular replacement solution, with the 2.5 A data set, using the native SCA as a search model was obtained, with six subunits per asymmetric unit.

A "reader's theater" intervention to managing grief: posttherapy reflections by a family and clinical team.

The sudden and accidental death of a child can be one of the most devastating events in the life of a family. This paper describes one couple's reflections of their grief and mourning following the death of their adolescent son as well as the clinical team's reflections of therapy. The uniqueness of this paper is that it offers a "reader's theater" intervention that enabled further change to occur. The clinical team used a belief model, emphasizing that altering constraining beliefs is at the heart of healing from such tragedies as sudden death (Wright, Watson, & Bell, 1996). This approach is operationalized through therapeutic conversations between family members, clinician, and clinical team. Interventions such as reflecting teams, therapeutic letters, and "homework tasks" were used to modify or challenge constraining beliefs of both the family members and the clinical team members. However, the intent to co-author a paper with this couple provided the serendipity intervention of a "reader's theater" that further served to identify, affirm, and solidify facilitating beliefs.

Purification and characterization of cathepsin D from normal human breast tissue.

The lysosomal aspartyl protease cathepsin D is present in most mammalian cells and is active in the catabolism of intracellular and endocytosed proteins. It appears to be overexpressed and abnormally secreted in breast cancer cells, and may contribute to the process of tumor metastasis. In the present study, cathepsin D was purified 4500-fold from normal human breast tissue using pepstatin-agarose, DEAE Sephadex, and Sephadex G-75 chromatography. The resulting enzyme on SDS-PAGE contained five protein bands (47, 31, 29, 13, and 12kDa) which were all immunoreactive on western blot analysis using anti-cathepsin D polyclonal antibodies. The isoform profile of purified cathepsin D consisted of three major peaks at approximate pI 7.3, 6.8, and 6.3, and a broad area of lower activity between pI of 5.0 and 2.0. The purified enzyme had a broad pH optimum centered around pH 3.3. Lectin blotting indicated that cathepsin D is a glycoprotein which is recognized by Galanthus nivalis agglutinin and concanavalin A, suggesting the presence of mannose residues. However, Sambucus nigra agglutinin, Tetragonolobus purpureas agglutinin, Triticum vulgaris agglutinin, and Erythrina cristagalli agglutinin failed to recognize cathepsin D, suggesting a lack of lectin-available sialic acid, fucose, N-acetylglucosamine, and galactose residues, respectively.

Crystallization and preliminary X-ray analysis of a novel plant lectin from Calystegia sepium.

A mannose-specific lectin from Calystegia sepium has been crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as precipitant. The needle-shaped crystals are orthorhombic, space group C222(1) with cell dimensions a = 55.2(1), b = 55.9 (1), c = 196.1 (1) A. Fresh crystals diffract to 1.9 A resolution on a synchrotron radiation source. The asymmetric unit contains a dimer of two identical 16 kDa subunits with a packing density of 2.36 A(3) Da(-1). Intensity data have been observed beyond 2.0 A, but reasonable statistics restricted the usable range to 2.0 A.

Purification, crystallization and preliminary X-ray analysis of a mannose-binding lectin from bluebell (Scilla campanulata) bulbs.

Crystals have been grown of a mannose-specific lectin from bluebell (Scilla campanulata) bulbs in a form suitable for X-ray diffraction studies. The crystals, which diffract to high resolution, grew in hanging drops by vapour diffusion, equilibrating with a solution of 70% saturated ammonium sulfate at pH 4.7-4.8 at 293 K, in the absence of any mannose saccharides. Crystals are orthorhombic, P2(1)2(1)2, with unit-cell dimensions a = 70.78, b = 93.69, c = 46.92 A. The functional lectin molecule is organized as a tetramer of four identical 14 kDa subunits, with only two subunits in the asymmetric unit. Data to 1.86 A resolution have been recorded and the structure determined by the molecular replacement method.

Live supervision and family systems nursing: postmodern influences and dilemmas.

Evolving nursing practices in a graduate nursing education programme that offers live clinical supervision in family systems nursing can be conceptualized within a postmodern perspective. Postmodernism is described as a debate about knowledge that questions traditional foundational theories or explanations in science, culture, religion and literature. Practices of a clinical supervision system, i.e. a graduate student, supervisor, and clinical team, illustrate postmodern influences on clinical work with families. Dilemmas posed by contemporaneity of modern and postmodern influences are also explored.

Prognostic significance of increased thallium-201 lung uptake during dipyridamole myocardial scintigraphy: comparison with exercise scintigraphy.

OBJECTIVE: To compare the incidence of cardiac events among patients with increased lung uptake of thallium after dipyridamole stress, matched subjects without such uptake and matched subjects with increased lung uptake during exercise stress. DESIGN: Retrospective case control study based on quantitative and semiquantitative visual consensus analysis of thallium scintigraphy. SETTING: Nuclear cardiology laboratory of a university teaching hospital. PATIENTS: Thirty-six patients with increased lung activity after dipyridamole stress and two groups of matched control subjects identified from 3150 consecutive thallium single photon emission computed tomography studies. INTERVENTIONS: Telephone follow-up and chart reviews to determine the incidence of cardiac events. MAIN RESULTS: Cardiac events were significantly less frequent in the study group with increased lung uptake after dipyridamole stress (two of 36,5%) than in the control group with increased thallium lung uptake at exercise scintigraphy (nine of 36,25%; P = 0.046). CONCLUSIONS: Increased lung uptake of thallium has less ominous short term prognostic significance when observed in association with dipyridamole stress rather than with exercise.

The Canadian exercise technetium 99m-labeled teboroxime single-photon emission computed tomographic study. Canadian Exercise Teboroxime SPECT Study Investigators.

BACKGROUND: 99mTc-labeled teboroxime undergoes rapid washout from the myocardium. For this reason, its use has been favored in conjunction with pharmacologic stress, which enables patient positioning before tracer administration, and multidetector single-photon emission computed tomography (SPECT), which enables rapid acquisition. We evaluated treadmill exercise 99mTc-labeled teboroxime SPECT with single-detector systems for the detection of coronary artery disease. METHODS AND RESULTS: Treadmill exercise 99mTc-labeled teboroxime SPECT was compared with analogous 201Tl-labeled imaging in 108 patients. Teboroxime was injected first during exercise and then at rest. Nine myocardial segments per study were scored with respect to uptake of activity during stress and at rest (teboroxime) or after redistribution (201Tl). Perfusion was defined as normal, reversible, or fixed. Overall agreement of 201Tl versus teboroxime segmental perfusion (normal vs abnormal) was 772/961 (80.3%; chi 2 = 258; p < 0.001; kappa = 0.51) or (normal vs reversible vs fixed) 711/961 (74.0%; chi 2 = 296; p < 0.001; kappa = 0.42). Fifty-six patients also underwent selective coronary angiography. Stenoses greater than 50% of lumenal diameter were drawn on individualized coronary artery diagrams applied to polar plots of scintigraphic segments to compare detection of coronary artery disease. Sensitivity and accuracy of teboroxime were 0.80 and 0.79, respectively, compared with 0.84 and 0.82 for 201Tl (difference not significant). Mean imaging procedure time was 113.6 minutes for teboroxime and 240.5 minutes for 201Tl (p < 0.001). CONCLUSIONS: 99mTc-labeled teboroxime is amenable to simple modifications of routine treadmill exercise SPECT myocardial perfusion imaging protocols with widely available single-detector SPECT systems. This modality provides results similar to those of exercise 201Tl SPECT and is significantly faster.

A nontraditional approach to family violence.

Family violence has often been conceptualized as a linear phenomenon in which perpetrators commit acts intended to hurt victims. Intervention in these circumstances involves treating the perpetrator and the victim individually. In contrast, this article presents a Systemic Belief Approach to the situation of mutual family violence. A case example illustrates the influence of beliefs on the occurrence of violent acts between family members (in this case, sole-parent mother and adolescent daughter). Family systems nursing interventions such as reflective questions and reflecting teams are used to challenge the family's constraining beliefs, which enables the coevolution of facilitative beliefs that invite healing.

A family systems nursing approach to hypertension.

This article focuses on a family systems nursing approach for essential hypertension. A case example is presented that describes the approach with a hypertensive woman with agoraphobia symptoms. A clinically significant decrease in the client's blood pressure occurred following the family sessions. Clinical observations of improved family relationships and symptom reduction corroborate research findings on the variables of perceived stress, anxiety levels and family coping resources. Interventions such as split-opinions, reframing, and rituals are described.

Western blotting and enzymatic activity analysis of cathepsin D in breast tissue and sera of patients with breast cancer and benign breast disease and of normal controls.

Increased total antigen amounts of cathepsin D in breast tissue have been reported to be associated with increased disease recurrence, more frequent metastasis, and increased mortality in breast cancer patients. In the present study, Western blotting analysis has been used for the first time to determine the relative amounts of precursor and processed forms of cathepsin D in sera and breast tissue of patients with breast cancer, benign breast disease, and normal controls. Sera gave similar blots for breast cancer patients and controls with two major forms of cathepsin D (M(r) 52,000 and 27,000). Malignant breast tissue contained the two forms of cathepsin D found in sera and an additional M(r) 31,000 form which was found in significantly increased (P < 0.001) relative amounts in breast tissue from 43 breast cancer patients [24 +/- 12% (SD)] when compared to 51 benign breast disease patients (13 +/- 8.9%) and 23 normal controls (1.8 +/- 4.4%). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.41) in relative amounts of the M(r) 31,000 form of cathepsin D between proliferative-type and non-proliferative-type fibrocystic breast disease. A cathepsin D assay has been optimized for human breast tissue and used to demonstrate for the first time significantly increased (P < 0.001) amounts of pepstatin-inhibitable, cathepsin D-specific activity in breast tissue from 36 breast cancer patients (2.2 +/- 1.4 units/mg of protein) when compared to 47 benign breast disease patients (0.63 +/- 0.43) and 23 normal controls (0.24 +/- 0.21). Preliminary analysis of subgroups of benign breast disease patients suggested no significant difference (P = 0.21) in pepstatin-inhibitable, cathepsin D-specific activity between proliferative-type and nonproliferative-type fibrocystic breast disease. The positive correlation (r = 0.82) of increased amounts of the M(r) 31,000 form of cathepsin D and increased pepstatin-inhibitable, cathepsin D enzymatic activity in malignant breast tissue suggests that the M(r) 31,000 form is the proteolytically active form of the enzyme which may be involved in the development and/or metastatic spread of breast cancer.