KATP-channels play a minor role in the protective hypoxic shut-down of cerebellar activity in eider ducks (Somateria mollissima).

Eider duck (Somateria mollissima) cerebellar neurons are highly tolerant toward hypoxia in vitro, which in part is due to a hypoxia-induced depression of their spontaneous activity. We have studied whether this response involves ATP-sensitive potassium (KATP) channels, which are known to be involved in the hypoxic/ischemic defense of mammalian neural and muscular tissues, by causing hyperpolarization and reduced ATP demand. Extracellular recordings in the Purkinje layer of isolated normoxic eider duck cerebellar slices showed that their spontaneous neuronal activity decreased significantly compared to in control slices when the KATP channel opener diazoxide (600 µM) was added (F1,70=92.781, p<0.001). Adding the KATP channel blocker tolbutamide (400 µM) 5 min prior to diazoxide completely abolished its effect (F1,55=39.639, p<0.001), strongly suggesting that these drugs have a similar mode of action in this avian species as in mammals. The spontaneous activity of slices treated with tolbutamide in combined hypoxia/chemical anoxia (95% N2-5% CO2 and 2 mM NaCN) was not significantly different from that of control slices (F1,203=0.071, p=0.791). Recovery from hypoxia/anoxia was, however, slightly but significantly weaker in tolbutamide-treated slices than in control slices (F1,137=15.539, p<0.001). We conclude that KATP channels are present in eider duck cerebellar neurons and are activated in hypoxia/anoxia, but that they do not play a key role in the protective shut-down response to hypoxia/anoxia.

Slow intrinsic oscillations in thick neocortical slices of hypoxia tolerant deep diving seals.

Direct evidence that the mammalian neocortex is an important generator of intrinsic activity comes from isolated neocortical slices that spontaneously generate multiple rhythms including those in the beta, delta and gamma range. These oscillations are also seen in intact animals where they interact with other areas including the hippocampus, thalamus and basal ganglia. Here we show that thick isolated neocortical slices from hooded seals intrinsically generate persistent spontaneous activities, both repetitive non-rhythmic activity with activity states lasting for several minutes, and oscillating activity with rhythms that are much slower (<0.1 Hz) than the rhythms previously described in vitro. These intrinsic activities were very robust and persisted for up to 1 h even in severely hypoxic conditions. We hypothesize that the remarkable hypoxia tolerance of the hooded seal nervous system made it possible to maintain functional integrity in slices thick enough to preserve intact neuronal networks capable of generating these slow oscillations. The observed activities in seal neocortical slices support the notion that mammalian cortical networks intrinsically generate multiple states of activity that include oscillatory activity all the way down to <0.1 Hz. This intrinsic neocortical excitability is an important contributor not only to sleep but also to the default awake state of the neocortex.

Properties of small molecules affecting insulin receptor function.

Small molecules with insulin mimetic effects and oral availability are of interest for potential substitution of insulin injections in the treatment of diabetes. We have searched databases for compounds capable of mimicking one epitope of the insulin molecule known to be involved in binding to the insulin receptor (IR). This approach identifies thymolphthalein, which is an apparent weak agonist that displaces insulin from its receptor, stimulates auto- and substrate phosphorylation of IR, and potentiates lipogenesis in adipocytes in the presence of submaximal concentrations of insulin. The various effects are observed in the 10(-5)-10(-3) M range of ligand concentration and result in partial insulin activity. Furthermore, analogues of the related phenol red and fluorescein molecules fully displace insulin from the IR ectodomain, however, without insulin agonistic effects. The interactions are further characterized by NMR, UV-vis, and fluorescence spectroscopies. It is shown that both fluorescence and UV-vis changes in the ligand spectra induced by IR fragments occur with Kd values similar to those obtained in the displacement assay. Nevertheless, insulin itself cannot completely abolish binding of the small molecules. Determination of the binding stoichiometry reveals multiple binding sites for ligands of which one overlaps with the insulin binding site on the receptor.

Solution structure of the satiety factor, CART, reveals new functionality of a well-known fold.

Cocaine and amphetamine regulated transcript (CART) peptide has been shown to be an anorectic peptide that inhibits both normal and starvation-induced feeding and completely blocks the feeding response induced by neuropeptide Y and regulated by leptin in the hypothalamus. The C-terminal part containing the three disulfide bridges CART(48-89) is the biologically active part of the molecule affecting food intake. The solution structure of the active part of CART has a fold equivalent to other functionally distinct small proteins. CART consists mainly of turns and loops spanned by a compact framework composed by a few small stretches of antiparallel beta-sheet common to cystine knots.

New strategy for the design of ligands for the purification of pharmaceutical proteins by affinity chromatography.

A new approach for the identification of ligands for the purification of pharmaceutical proteins by affinity chromatography is described. The technique involves four steps. Selection of an appropriate site on the target protein, design of a complementary ligand compatible with the three-dimensional structure of the site, construction of a limited solid-phase combinatorial library of near-neighbour ligands and solution synthesis of the hit ligand, immobilisation, optimisation and application of the adsorbent for the purification of the target protein. This strategy is exemplified by the purification of a recombinant human insulin precursor (MI3) from a crude fermentation broth of Saccharomyces cerevisiae.

The relationship between insulin bioactivity and structure in the NH2-terminal A-chain helix.

Studies of naturally occuring and chemically modified insulins have established that the NH2-terminal helix of the A-chain is important in conferring affinity in insulin-receptor interactions. Nevertheless, the three-dimensional structural basis for these observations has not previously been studied in detail. To correlate structure and function in this region of the molecule, we have used the solution structure of an engineered monomer (GluB1, GluB10, GluB16, GluB27, desB30)-insulin (4E insulin) as a template for design of A-chain mutants associated with enhanced or greatly diminished affinity for the insulin receptor. In the context of 4E insulin, the employed mutants, i.e. ThrA8-->His and ValA3-->Gly, result in species with 143% and 0.1% biological activity, respectively, relative to human insulin. The high-resolution NMR studies reveal two well-defined structures each resembling the template. However, significant structural differences are evident notably in residues A2-A8 and their immediate environment. In comparison with the template structure, the A8His mutation enhances the helical character of residues A2-A8. This structural change leads to additional exposure of a hydrophobic patch mainly consisting of species invariant residues. In contrast, the A3Gly mutation leads to stretching and disruption of the A2-A8 helix and changes both the dimensions and the access to the hydrophobic patch exposed in the more active insulins. We conclude that the mutations induce small, yet decisive structural changes that either mediate or inhibit the subtle conformational adjustments involved in the presentation of this part of the insulin pharmacophore to the receptor.

A structural switch in a mutant insulin exposes key residues for receptor binding.

Despite years of effort to clarify the structural basis of insulin receptor binding no clear consensus has emerged. It is generally believed that insulin receptor binding is accompanied by some degree of conformational change in the carboxy-terminal of the insulin B-chain. In particular, while most substitutions for PheB24 lead to inactive species, glycine or D-amino acids are well tolerated in this position. Here we assess the conformation change by solving the solution structure of the biologically active (GluB16, GlyB24, desB30)-insulin mutant. The structure in aqueous solution at pH 8 reveals a subtle, albeit well-defined rearrangement of the C-terminal decapeptide involving a perturbation of the B20-23 turn, which allows the PheB25 residue to occupy the position normally taken up by PheB24 in native insulin. The new protein surface exposed rationalizes the receptor binding properties of a series of insulin analogs. We suggest that the structural switch is forced by the structure of the underlying core of species invariant residues and that an analogous rearrangement of the C-terminal of the B-chain occurs in native insulin on binding to its receptor.

[Professional learning and collective knowledge. Learning environment in two Norwegian hospital departments]. / Profesjonslaering og kollektiv kunnskap. Laeringsmiljø i to norske sykehusavdelinger.

Conditions for learning in two Norwegian hospital departments are explored in this article. 20 in-depth interviews with physicians working on the surgical and medical departments of a large general hospital were carried out in 1994. We focus on the social aspects of learning and apply access to learning situations as an analytical perspective to explain how possibilities for learning are created in the daily activities of the two departments. Access to learning situations is created in a "zone of possibility" between the formal organisation and the more informal interpersonal networks in the hospital. The division of the department into sections is used as an example of how organisational factors determine with which areas one becomes familiar. The notion of "the good apprentice" and the relationship between initiative and invitation illuminate the significance of interpersonal factors for access to learning situations. Finally, we illustrate how time is an important but scarce resource influencing the development of shared knowledge in the department.

[How to learn medical practice? A qualitative study of physicians' perception of their own learning processes]. / Hvordan laeres medisinsk praksis? En kvalitativ studie av legers oppfatning av egne laeringsprosesser.

In this article we explore how physicians develop professional knowledge in their everyday work. 20 indepth interviews with physicians working in the surgical and internal medicine departments of a Norwegian hospital were conducted in 1994. Explanatory models from situated learning theory were applied to analyse the taped interview material. The expert-novice relation emerges as the core of the traditional medical learning institutions. Here the inexperienced physician learns the necessary practical skills, ways of reasoning and standards of diagnosis and treatment in the hospital. In interaction with a more experienced physician the novice transcends what she can do alone and develops as a professional. The quality of learning depends on the dialogue between the novice and the expert in the actual situation. The physicians' descriptions of their own learning process change with increasing competence and position in the hospital hierarchy.

Oxysporidinone: a novel, antifungal N-methyl-4-hydroxy-2-pyridone from Fusarium oxysporum.

Oxysporidinone (1), a novel 3,5-disubstituted N-methyl-4-hydroxy-2-pyridone, was isolated from fermentations of Fusarium oxysporum (CBS 330.95) by counter-current chromatography. The structure was determined by spectroscopic methods including NMR, MS, IR, and UV analysis. Oxysporidinone exhibited growth inhibitory activity against several common plant pathogenic fungi.

Solution structure of an engineered insulin monomer at neutral pH.

Insulin circulates in the bloodstream and binds to its specific cell-surface receptor as a 5808 Da monomeric species. However, studies of the monomer structure and dynamics in solution are severely limited by insulin self-association into dimers and higher oligomers. In the present work we use site-directed mutagenesis of the dimer- and hexamer-forming surfaces to yield the first insulin species amenable for structure determination at neutral pH by nuclear magnetic resonance (NMR) spectroscopy. The preferred insulin mutant, i.e., (B1, B10, B16, B27) Glu, des-B30 insulin retains 47% biological potency and remains monomeric at millimolar concentrations in aqueous solution at pH 6.5-7.5 as judged by NMR and near-UV circular dichroism (CD) spectroscopy. From a series of 2D 1H-NMR spectra collected at pH 6.5 and 34 degrees C, the majority of the resonances are assigned to specific residues in the sequence, and nuclear Overhauser enhancement (NOE) cross-peaks are identified. NOE-derived distance restraints in conjunction with torsion restraints based on measured coupling constants, 3JHNH alpha, are used for structure calculations using the hybrid method of distance geometry and simulated annealing. The calculated structures show that the major part of the insulin mutant is structurally well defined with an average root mean square (rms) deviation between the 25 calculated structures and the mean coordinates of 0.66 A for backbone atoms (A2-A19 and B4-B26) and 1.31 A for all backbone atoms. The A-chain consists of two antiparallel helices, A2-A7 and A12-A19, connected by a loop. The B-chain contains a loop region (B1-B8), an alpha-helix (B9-B19), and a type I turn (B20-B23) and terminates as an extended strand (B24-B29). The B1-B4 and B27-B29 regions are disordered in solution. The structure is generally similar to crystal structures and resembles a crystalline T-state more than an R-state in the sense that the B-chain helix is confined to residues B9-B19.

The high resolution solution structure of the insulin monomer determined by NMR.

Studies of naturally occurring and chemically modified insulins indicate that relatively few of the 51 amino acid residues may be assigned specific roles in insulin-receptor interactions. Most of the insulin X-ray structural information is derived from aggregated species (notably hexamers). Because insulin exerts its physiological effect as a 5808 Dalton monomeric species, it is necessary to consider whether crystal-packing forces have modified the structure from that required for biological action. Insulin aggregation in solution complicates high resolution NMR studies of the monomer. However, site-directed mutagenesis can be used to generate biologically active mutants (e.g., B16-Tyr--> His) that remain monomeric at millimolar concentrations in aqueous solution at low pH. The resulting homogeneous and monomeric samples are suitable for structure determination by NMR methods. The high resolution solution structure of B16--Tyr--> His insulin resembles crystal structures, notably molecule 1 of T6 insulin. Side-chain conformation in some biologically important motifs, however, shows subtle differences between solution and crystal structures.

High-resolution structure of an engineered biologically potent insulin monomer, B16 Tyr-->His, as determined by nuclear magnetic resonance spectroscopy.

Site-directed mutagenesis is used in conjunction with 1H nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy in order to find an insulin species amenable for structure determination in aqueous solution by NMR spectroscopy. A successful candidate in this respect, i.e., B16 Tyr-->His mutant insulin, is identified and selected for detailed characterization by two-dimensional 1H NMR. This mutant species retains 43% biological potency and native folding stability, but in contrast to human insulin it remains monomeric at millimolar concentration in aqueous solution at pH 2.4. The resulting homogeneous sample allows high-quality 2D NMR spectra to be recorded. The NMR studies result in an almost complete assignment of the 1H resonance signals as well as identification of NOE cross peaks. NOE-derived distance restraints in conjunction with torsion restraints based on measured coupling constants, 3JHNH alpha, are used for structure calculations using the hybrid method of distance geometry and simulated annealing. The calculated structures show that the major part of the insulin monomer is structurally well-defined with an average rms deviation between the 20 calculated structures and the mean coordinates of 0.89 A for all backbone atoms, 0.46 A for backbone atoms (A2-A19 and B4-B28), and 1.30 A for all heavy atoms. The structure of the A-chain is composed of two helices from A2 to A7 and from A12 to A19 connected by a short extended strand. The B-chain consists of a loop, B1-B8, an alpha-helix, B9-B19, a beta-turn, B20-B23, and an extended strand from B24 to B30.(ABSTRACT TRUNCATED AT 250 WORDS)

Engineering stability of the insulin monomer fold with application to structure-activity relationships.

To evaluate the possible relationship between biological activity and structural stability in selected regions of the insulin molecule, we have analyzed the guanidine hydrochloride induced reversible unfolding of a series of mutant insulins using a combination of near- and far-UV circular dichroism (CD). The unfolding curves are reasonably described on the basis of a two-state denaturation scheme; however, the observation of subtle differences between near- and far-UV CD detected unfolding indicates that intermediates may be present. Three regions of the insulin molecule are analyzed in detail with respect to their contribution to folding stability, i.e., the central B-chain helix, the NH2-terminal A-chain helix, and the B25-B30 extended chain region. Considerable enhancement of folding stability is engineered by mutations at the N-cap of the central B-chain helix and at the C-cap of the NH2-terminal A-chain helix. Mutations that confer increased stability in these regions are identical to those that lead to enhanced biological activity. In contrast, for insulin species modified in the B25-B30 region of the molecule, we observe no correlation between global folding stability and bioactivity. Mutations in the three regions examined are found to affect stability in a nearly independent fashion, and stabilizing mutations are generally found to enhance the cooperativity of the unfolding transition. We conclude that highly potent insulins (i.e., HisA8, ArgA8, GluB10, and AspB10) elicit enhanced activity because these mutations stabilize structural motifs of critical importance for receptor recognition.

Primary structure of barwin: a barley seed protein closely related to the C-terminal domain of proteins encoded by wound-induced plant genes.

Barwin is a basic protein with pI above 10 and molecular mass 13.7 kDa isolated from aqueous extracts of barley seed. The complete amino acid sequence of 125 residues has been determined by a combination of conventional protein sequencing, plasma desorption mass spectrometry, and 1H nuclear magnetic resonance spectroscopy. Three disulfide bridges have been localized as Cys31-Cys63, Cys52-Cys86, and Cys66-Cys123 both by 1H nuclear magnetic resonance spectroscopy and by plasma desorption mass spectrometry. The N-terminal residue was identified as pyroglutamate. Barwin is closely related to a peptide segment of 122 residues at the C-terminal region of the proteins encoded by two wound-induced genes in potato plants, win1 and win2, and a protein encoded by the hevein gene of rubber tree. In 77 sequence positions of 125 the barwin, win1, win2, and hevein protein sequences have amino acid sequence identity, when two gaps--one of two residues allowing for the insert of Gly23 and Ala24 and one allowing for the insert of Thr97 in the barwin sequence--are introduced in the latter three. The close sequence similarity with the proteins encoded by the wound-induced potato and rubber tree genes and the ability of the protein to bind saccharides suggest that barwin might belong to a group of proteins involved in a common defense mechanism in plants.

Secondary structure in solution of barwin from barley seed using 1H nuclear magnetic resonance spectroscopy.

Barwin, a basic protein from barley seed of 125 amino acid residues, has been studied by two-dimensional 1H nuclear magnetic resonance spectroscopy. This protein is closely related to the C-terminal domain of proteins whose synthesis is induced by wounding, the so-called win proteins. These proteins may, therefore, have a role in the defense against fungal attack. Full assignment of the 1H nuclear magnetic resonances has been obtained for 104 amino acid residues, and 18 amino acid spin systems were partially assigned. Sequence-specific assignment using nuclear Overhauser spectroscopy has been achieved for 122 of the 125 residues. This has revealed that the secondary structure of the protein is dominated by a large four-stranded antiparallel beta-sheet consisting of the strands Gln2-Thr9, Lys65-Asn71, Gln77-Arg81, and His113-Val121, a small parallel beta-sheet of the strands Trp48-Cys52 and Asp84-Ala87, which together account for a third of the protein. Sequential effects indicate the presence of three small alpha-helices, Tyr30-Lys38, Leu40-Tyr46, and Thr97-Asp103. The secondary structure in other regions of the sequence is characterized mainly by loops and turns and regions where no regular secondary structure arrangement could be identified. A large number of long-range nuclear Overhauser effects has been identified, and these have been used, together with sequential and intranuclear Overhauser effects, for a calculation of the protein's three-dimensional structure.

Three-dimensional structure in solution of barwin, a protein from barley seed.

The solution structure of a 125-residue basic protein, barwin, has been determined using 1H nuclear magnetic resonance spectroscopy. This protein is closely related to domains in proteins encoded by wound-induced genes in plants. Analysis of the 1H nuclear Overhauser spectrum revealed the assignment of more than 1400 nuclear Overhauser effects. Twenty structures were calculated based on 676 nontrivial distance restraints, 152 torsion angle restraints (92 phi, 56 chi 1, and 4 omega for proline), and stereospecific assignments of 38 chiral centers, using distance geometry, simulated annealing, and restrained energy minimization. None of the distance restraints was violated by more than 0.5 A in any of the 20 structures, and none of the torsion angle restraints was violated by more than 1 degree in any of the structures. The RMS difference between the calculated and target interproton distance restraints is 0.033 A, and the average atomic RMS differences between the 20 structures and their geometric average are 1.23 A for backbone atoms and 1.73 A for all heavy atoms. The dominating structural feature of the protein is a well-defined four-stranded antiparallel beta-sheet, two parallel beta-sheets packed antiparallel to each other and four short alpha-helices. The binding site of barwin to the tetramer N-acetylglucosamine has been qualitatively investigated, and the dissociation constant of the complex has been determined using one-dimensional 1H nuclear magnetic resonance spectroscopy.

Positive theta-angles in proteins by nuclear magnetic resonance spectroscopy.

Non-glycine residues with positive theta-angles have been identified in four proteins, barley serine proteinase inhibitor CI-2, bacterial ribonuclease (barnase) of Bacillus amyloliquefaciens, hen egg white lysozyme and a basic protein from barley seed (barwin) by use of nuclear magnetic resonance spectroscopy. By accurate measurements of the coupling constant (3)JHNHalpha and integration of the nuclear Overhauser HN-Halpha cross peak, positive theta-angles could be determined reliably to 60 degrees +/- 30 degrees, in full agreement with the crystal structures for lysozyme, barnase and serine proteinase inhibitor CI-2. The work emphasizes that positive theta-angles can also occur in non-glycine residues and in the four proteins, positive theta-angles have been observed for the residue types aspartic acid, asparagine, arginine, serine, glutamine, histidine, tyrosine, tryptophan and phenylalanine. The measured (3)JHNHalpha coupling constants and the intensity of the intraresidue HN-Halpha NOEs agree well with the solution structures of three of the proteins, using the existing parametrization of the Karplus curve (Pardi, A., Billeter, M. and Wuthrich, K. (1984) J. Mol. Biol., 180, 741-751; Ludvigsen, S. Andersen, K.V. and Poulsen, F.M. (1991) J Mol. Biol., 217, 731-736).

Refinement of the three-dimensional solution structure of barley serine proteinase inhibitor 2 and comparison with the structures in crystals.

The three-dimensional structure of barley serine proteinase inhibitor, CI-2, has been determined using nuclear magnetic resonance spectroscopy. The present structure determination is a refinement of the structure previously determined by us, using in the present case stereo-specific assignments, and a virtually complete set of assignments of the two-dimensional nuclear Overhauser spectrum. The structure determination is based on the identification of more than 1300 nuclear Overhauser effects, of which 961 were used in the structure calculation as distance restraints, and on 94 dihedral angle restraints, of which 31 are for chi 1 angles in defined chiral centers. These have been used to calculate a series of 20 three-dimensional structures using a combination of distance geometry, simulated annealing and restrained molecular dynamics. Each of the 20 structures was in agreement within less than 0.5 A of each of the distance restraints and with all dihedral angle restraints. When compared to the geometric average structure of the 20 refined structures the root-mean-square differences for the backbone atoms were 0.8 (+/- 0.2) A and for all atoms were 1.6 (+/- 0.2) A. By comparison, the values obtained for the structures determined previously were 1.4 (+/- 0.2) A and 2.1 (+/- 0.1) A, respectively. The structures were also compared to the structure determined in the crystalline state by X-ray diffraction showing root-mean-square differences of 1.6 (+/- 0.2) A and 2.8 (+/- 0.2) A for the backbone and all atoms, respectively. Common features of the solution structure and the two crystal structures are the four-stranded beta-structure, composed of a pair of parallel strands, and three pairs of antiparallel beta-strands flanked on one side by a 12-residue alpha-helix and on the other side by a loop containing the serine proteinase binding site. The new analysis of the structure has revealed an additional pair of antiparallel beta-strands, consisting of residues 65 to 67 and 81 to 83, that was not seen in either of the crystal structures or the previous solution structure. Identification of this was based on nuclear magnetic resonance evidence for the hydrogen bond (67HN to 81CO) not reported previously. Also the presence of a bifurcated hydrogen bond involving Phe69 CO and HN atoms of Ala77 and Gln78 was observed in solution but not in crystals. Minor differences between the two structures were observed in the phi-angles of residues Met59 and Glu60 in the inhibitory site.