NMR solution studies of hamster galectin-3 and electron microscopic visualization of surface-adsorbed complexes: evidence for interactions between the N- and C-terminal domains.

Galectin-3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal domain that includes several repeats of a proline-tyrosine-glycine-rich motif. Earlier work based on a crystal structure of human galectin-3 CRD, and modeling and mutagenesis studies of the closely homologous hamster galectin-3, suggested that N-terminal tail residues immediately preceding the CRD might interfere with the canonical subunit interaction site of dimeric galectin-1 and -2, explaining the monomeric status of galectin-3 in solution. Here we describe high-resolution NMR studies of hamster galectin-3 (residues 1--245) and several of its fragments. The results indicate that the recombinant N-terminal fragment Delta 126--245 (residues 1--125) is an unfolded, extended structure. However, in the intact galectin-3 and fragment Delta 1--93 (residues 94--245), N-terminal domain residues lying between positions 94 and 113 have significantly reduced mobility values compared with those expected for bulk N-terminal tail residues, consistent with an interaction of this segment with the CRD domain. In contrast to the monomeric status of galectin-3 (and fragment Delta 1--93) in solution, electron microscopy of negatively stained and rotary shadowed samples of hamster galectin-3 as well as the CRD fragment Delta 1--103 (residues 104--245) show the presence of a significant proportion (up to 30%) of oligomers. Similar imaging of the N-terminal tail fragment Delta 126--245 reveals the presence of fibrils formed by intermolecular interactions between extended polypeptide subunits. Oligomerization of substratum-adsorbed galectin-3, through N- and C-terminal domain interactions, could be relevant to the positive cooperativity observed in binding of the lectin to immobilized multiglycosylated proteins such as laminin.

Molecular modeling and mutagenesis studies of the N-terminal domains of galectin-3: evidence for participation with the C-terminal carbohydrate recognition domain in oligosaccharide binding.

A model structure (Henrick,K., Bawumia,S., Barboni,E.A.M., Mehul,B. and Hughes, R.C. (1998) Glycobiology:, 8, 45-57) of the carbohydrate recognition domain (CRD, amino acid residues 114-245) of hamster galectin-3 has been extended to include N-terminal domain amino acid residues 91-113 containing one of the nine proline-rich motifs present in full-length hamster galectin-3. The modeling predicts two configurations of the N-terminal tail: in one the tail turns toward the first (SI) and last (S12) beta-strands of the CRD and lies at the apolar dimer interface observed for galectins -1 and -2. In the second folding arrangement the N-terminal tail lies across the carbohydrate-binding pocket of the CRD where it could participate in sugar-binding: in particular tyrosine 102 and adjacent residues may interact with the partly solvent exposed nonreducing N-acetylgalactosamine and fucose substituents of the A-blood group structure GalNAcalpha1,3 [Fucalpha1,2]Galbeta1,4GlcNAc-R. Binding studies using surface plasmon resonance of a recombinant fragment Delta1-93 protein containing residues 94-245 of hamster galectin-3 and a collagenase-derived fragment Delta1-103 containing residues 104-245, as well as alanine mutagenesis of residues 101-105 in Delta1-93 protein, support the prediction that Tyr102 and adjacent residues make significant contributions to oligosaccharide binding.

Kinetic measurements of binding of galectin 3 to a laminin substratum.

Galectin 3, a beta-galactoside binding protein, contains a C-terminal carbohydrate recognition domain (CRD) and an N-terminal segment including multiple repeats of a proline/tyrosine/glycine-rich motif. Previous work has shown that galectin 3 but not the isolated CRD binds to laminin, a multivalent ligand, with positive cooperativety indicating the formation of multiple interactions although the lectin in solution is monomeric. Using surface plasmon resonance, we find that hamster galectin 3 at sub-micromolar concentrations or its isolated CRD at all concentrations binds to a laminin substratum with similar association (k(ass); 10-30,000 M(-1) S(-1)) and dissociation (k(diss); 0.2-0.3 S1(-1)) rates and weak affinity (Ka; 1-3 x 10(5) M(-1)). At higher concentrations of galectin 3 the off rate decreases ten fold leading to increased affinity. Ligation of an N-terminal epitope of galectin 3 with a monoclonal Fab fragment increases association and dissociation rates ten fold. A recombinant protein obtained by deletion of the first 93 N-terminal residues binds to laminin with positive cooperativity and a slowly dissociating fraction (K(diss); 0.002 S(-1)) accumulates on the substratum. The data suggest that homophilic interactions between CRD as well as N terminal domains are implicated in galectin 3 aggregation on the substratum leading to positive binding cooperativity.

Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3.

A model of the carbohydrate recognition domain CRD, residues 111-245, of hamster galectin-3 has been made using homology modeling and dynamics minimization methods. The model is based on the known x-ray structures of bovine galectin-1 and human galectin-2. The oligosaccharides NeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligands for galectin-3, as well as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation found in each case. These studies indicate a putative extended carbohydrate-binding subsite in the hamster galectin-3 involving Arg139, Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 for fucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituents on the primary galactose. Each of these positions is variable within the whole galectin family. Two of these residues, Arg139 and Ser232, were selected for mutagenesis to probe their importance in this newly identified putative subsite. Residue 139 adopts main-chain dihedral angles characteristic of an isolated bridge structural feature, while residue 232 is the C-terminal residue of beta-strand-11, and is followed immediately by an inverse gamma-turn. A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, -2, and -3. Minimized docked models were generated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc, GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc. Correlation of the computed protein-carbohydrate interaction energies for each lectin-oligosaccharide pair with the experimentally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of Arg139 in recognition of the extended sialylated ligand.

Psychiatric symptoms and psychological profile of patients with near fatal asthma: absence of positive findings.

BACKGROUND: We examined the presence of psychiatric symptoms and personality characteristics in patients with asthma and near fatal asthma (NFA). An NFA attack is defined by the presence of one or more of the following symptoms: respiratory arrest, alteration in consciousness, need for mechanical ventilation, Pa CO2 > 50 mm Hg. METHODS: To assess the relevance of a specific psychiatric profile or the difference in personality characteristics existing in patients that survived an NFA attack and asthmatic patients. The authors interviewed a sample of 17 asthmatic patients who experienced one or more NFA attacks. A control group of 17 control patients with asthma who never experienced NFA attacks was enrolled. After a baseline assessment, the patients underwent an interview concerning their personal and familiar psychiatric history and a psychodiagnostic investigation using Hamilton scales for anxiety and depression, Zung scales for anxiety and depression, and Minnesota Multiphasic Personality Inventory. The study was performed in a 6-month period. RESULTS: No significant differences in the results of psychodiagnostic tests between NFA patients and the control group were reported. Psychiatric history was similar in the two groups. CONCLUSIONS: Our results suggest that psychiatric symptoms and personality characteristics are not related to the presence of asthma with or without NFA.

Near fatal asthma and psychopathological characteristics: a group-control study.

Psychological factors may play a role in asthma. In particular, emotional upsets have been correlated with fatal asthma attacks, and an abnormal personality attitude is considered to be a risk factor in fatal asthma. Moreover, some authors have recently reported favourable asthma outcome in patients with severe asthma and psychiatric abnormalities, when psychoactive treatment was initiated. On the understanding that people with fatal and "near fatal asthma" (NFA) are components of the same subset of the asthmatic population, we undertook a study aimed at assessing the importance of personality and psychiatric factors in asthma mortality. Between June 1991 and December 1993, a sample of 17 patients with asthma who had experienced one or more near fatal asthma attacks (respiratory arrest, or need for respiratory assistance, or altered conscious state, or arterial carbon dioxide tension (Pa,CO2) > 6.7 kPa (50 mmHg)), and 17 control patients with asthma who had never experienced such an attack (control group) were enrolled. All patients underwent: 1) an interview concerning their personal and family psychiatric history; 2) a psychodiagnostic investigation by a battery of four of the most widely used psychiatric tools: Hamilton scales for anxiety and depression; Zung scales for anxiety and depression; and Minnesota Multiphasic Personality Inventory. No statistical difference was found in psychodiagnostic tests between study and control groups. The psychiatric history was similar in the two groups. Our results suggest that personality characteristics and psychiatric history are not related to asthma outcome, and that the psychiatric approach is not expected to be useful in preventing mortality in asthma.

Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin.

Streptomyces rochei A2 endoglucanase (eglS) and beta-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgs1 probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis and genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.

Estimation of occlusion pressure during assisted ventilation in patients with intrinsic PEEP.

We conducted a study to assess the validity of the occlusion pressure (P0.1) measured during activation of the trigger mechanism (P0.1(aw)trig) in patients showing variable levels of PEEPi during pressure-support ventilation. We first compared P0.1(aw)trig and P0.1 measured with the conventional method (i.e., the airway pressure drop after the first 100 ms of an occluded inspiration) in 16 patients with chronic obstructive pulmonary disease (COPD). We observed good agreement and a highly significant correlation (r = 0.99; bias = 0.3 +/- 0.5 cm H20) between the two methods. In a second part of the study, we compared, in 17 patients, P0.1(aw)trig with (P0.1(es)), measured as the depression generated on the esophageal pressure tracing in the first 100 ms of the inspiratory negative swing, and with P0.1 measured on the P(es) tracing simultaneously with P(aw)trig (P0.1(es-synchro)). Our results showed a good correlation and good agreement between P(aw)trig and P0.1(es) (r = 0.92; bias = 0.3 +/- 0.5 cm H20); P(aw)trig and P0.1(es-synchro) (r = 0.97; bias = 0.1 +/- 0.2 cm H20); and P0.1(es) and P0.1(es-synchro) (r = 0.95, bias = 0.2 +/- 0.4 cm H20), respectively. This study suggests that reliable measurements of inspiratory drive can be obtained easily, on a breath-by-breath basis, from airway pressure tracings during pressure-support ventilation in patients with variable levels of PEEPi.

Normal spatial learning despite regional inhibition of LTP in mice lacking Thy-1.

The process of learning involves stable changes in synaptic efficacy for which long-term potentiation (LTP) provides a widely adopted mammalian model. Synaptic modification induced by learning or LTP may involve the action of cell adhesion molecules. One such candidate is the ubiquitous neuronal glycoprotein Thy-1. In mice in which the gene encoding Thy-1 has been inactivated, we find a regionally selective impairment of LTP in vivo in the hippocampal formation: LTP is normal in area CA1 but strongly inhibited in the dentate gyrus. Spatial learning by Thy-1-deficient mice, as assessed in the watermaze, is unimpaired. Thus LTP in the cortical input to the dentate gyrus seems not to be required for spatial learning.

The glycophosphatidylinositol anchor affects the conformation of Thy-1 protein.

Thy-1 has the structure of a single variable-type immunoglobulin domain anchored to the external face of the plasma membrane via a glycophosphatidylinositol moiety. When the lipid is removed from this anchor by either phospholipase C or D, the reactivity of the delipidated Thy-1 for a range of antibodies, including those known to be determined by amino acid residues, is impaired. We have investigated in detail the effect of delipidation on the reaction with the OX7 monoclonal antibody, determined by the allelic variant residue Arg 89. Analysis of the kinetics of OX7 binding shows that delipidation affects primarily the dissociation of antibody, increasing the dissociation rate constant kdiss from 0.27 x 10(-3) s-1 to 2.39 x 10(-3) s-1. Addition of phospholipase to preformed antibody-antigen complex causes an immediate change from the slow to the faster dissociation rate, implying that delipidation induces a conformational change in the Thy-1 protein that is sufficiently strong to dissociate bound antibody. This conformational change can be demonstrated directly by the circular dichroism spectrum of human Thy-1 that detects changes in the environment of Tyr residues located near the antigenic epitopes. Molecular dynamics studies suggest that, on delipidation, a conformational change occurs in the glycan chain that affects the protein in the region of the antigenic epitopes. This study thus demonstrates that the glycophosphatidylinositol anchor strongly influences the conformation of Thy-1 protein by a mechanism that could occur generally with membrane proteins of this class.

Emergency treatment of bronchospastic attacks in an emergency medicine department: effects of a quality improvement project.

New insights have recently changed the therapeutical approach to bronchospastic attack (BA). In this paper we present the results of a quality assurance study devised to: (1) verify the extent to which these new insights are put into practice in our Emergency Medicine Department (EMD); (2) evaluate changes in EMD medical staff behaviour faced with BA after discussion and distribution of medical practice guidelines on the treatment of BA (a document produced by a group of experts in pulmonary medicine working in the EMD on the basis of current literature and personal experiences); (3) assess the impact of such guidelines on quality care. A retrospective analysis demonstrated an incorrect treatment of BA in the EMD. After the introduction of the guidelines we observed: (1) an improvement in physician behaviour that completely agreed with guidelines in 56% of instances after discussion and distribution of the document, in 8% before; (2) an improvement in the outcome of patients treated for BA in the EMD (13 versus 36% relapses for patients treated in the EMD and discharged).

Improving quality in emergency services to reduce hospital admission.

At the Emergency Department of Udine General Hospital (Italy) a programme to reduce admissions to the Internal Medicine Department was introduced in 1991. The majority of these admissions come from the Emergency Department, where many people, often without acute conditions, claim medical care. The programme consisted in organizational, professional and economic changes. At the end of 1991, the overall number of admissions to Udine General Hospital, as compared to 1990, decreased by 7.1%, but admissions to the Internal Medicine Department showed an 11.2% reduction. Finally, examinations for internal medical complaints in the emergency ward, not followed by hospitalization, increased by 15.5%. These results showed a reduction in admissions to the Internal Medicine Department greater than previously planned, with an increase in the number of outpatient examinations in the emergency room not followed by admission. Further targets were planned for 1992 to increase the quality of the service.

Selective inhibition of neurite outgrowth on mature astrocytes by Thy-1 glycoprotein.

THY-1, the smallest member of the immunoglobulin superfamily, is a major cell-surface component expressed by several tissues. The protein, carbohydrate and gene structures of this molecule are known, yet its function is not. It is highly expressed in nervous tissue, where it appears on virtually all neurons after the cessation of axonal growth. Here we show that expression of Thy-1 by a neural cell line inhibits neurite outgrowth on mature astrocytes, but not on other cellular substrata which include Schwann cells and embryonic glia. This inhibition of neurite extension on astrocytes can be reversed by low concentrations (nanomolar) of soluble Thy-1. If a similar interaction between neuronal Thy-1 and astrocytes occurs in vivo, it could stabilize neuronal connections and suppress axonal regrowth after injury in the astrocyte-rich areas of adult central nervous system.

Activation of T lymphocytes by cross-linking of glycophospholipid-anchored Thy-1 mobilizes separate pools of intracellular second messengers to those induced by the antigen-receptor/CD3 complex.

T cells can be activated, not only by the conventional (antigen-receptor/CD3 complex) route, but also by cross-linking any one of their lipid-anchored surface glycoproteins. We have compared early transmembrane signalling events mediated through CD3 with those mediated through Thy-1, a lipid-linked surface glycoprotein, on the human lymphoid cell line Jurkat and transfectants expressing higher levels of Thy-1. Cross-linking of Thy-1 causes immediate phosphatidylinositol (PI) turnover and an influx of extracellular Ca2+, while releasing very little Ca2+ from intracellular stores. CD3 activation, on the other hand, causes PI turnover which releases intracellular Ca2+, and only secondarily induces an influx of extracellular ions. The Thy-1 response is detectable at very low levels of surface Thy-1, and is not mimicked by enzymatic removal of lipid-linked proteins from the cell surface. The Thy-1-induced Ca2+ influx is more sensitive to L channel blockers than the CD3-mediated flux. These results indicate that the initial stages of Thy-1-mediated activation involve the rapid and extensive mobilization of the intracellular second messengers, PI and Ca2+, by mechanisms separate to those activated by the antigen-receptor/CD3 complex.

Sulphate-stimulate liver glycogen phosphorylase b: new evidence of changes in the conformation of the enzyme.

It has been known for same time that sulphate ions stimulate liver glycogen phosphorylase b, both in the presence and in the absence of AMP. In the present paper we describe some observations (like a modified method of purification of the enzyme after sulphate treatment of the animals) suggesting that actual changes of the physical properties of the enzyme occur after intravenous injection of sodium sulphate. In order to avoid formation of phosphorylase a these studies were performed on enzyme from phosphorylase-b-kinase deficient (gsd/gsd) rats.

The purification of acid phosphatase from honey bee venom (Apis mellifica).

Acid phosphatase from bee venom was purified by a combination of saturated ammonium sulphate precipitation, gel filtration and ion exchange chromatography. The final product which is a glycoprotein contained less than 0.1% phospholipase A2 or hyaluronidase activity and existed in two molecular weight (96,000 and 45,000) forms. Acid phosphatase is a potent allergen, in bee venom allergic patients, which is capable of releasing histamine from sensitized human basophils and of inducing a wheal and flare reactions in sensitized human skin.

Inhibitors binding to L-aromatic amino acid decarboxylase.

The effect of a number of inhibitors of L-aromatic amino acid decarboxylase activity on the absorption spectrum of the enzyme-bound coenzyme has been studied. It has been observed that the compounds tested, even if devoid of the amino function and therefore unable to form the Schiff base with the coenzyme, modify significantly the enzyme spectrum, indicating their binding to the coenzyme active site. Spectral modifications suggest that at least two kinds of binding of inhibitors to L-aromatic amino acid decarboxylase may occur, depending on their structural features. Moreover, from the spectra obtained at different concentrations of the inhibitors their affinity constants have been determined: data indicate that the cathecol ring gives the largest contribution to the binding, while the presence of the carboxyl group, the aminic group and the aliphatic chain are responsible for a decrease in the binding, which could be relevant for the efficiency of the catalysis.