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O H Olsen

Publications and source records attributed to O H Olsen.

34 records · Page 2Linked to original sources
PubMed1997

Computational studies of the activation of lipases and the effect of a hydrophobic environment.

We have investigated the activation pathway of three wild type lipases and three mutants using molecular dynamics techniques combined with a constrained mechanical protocol. The activation of these lipases involves a rigid body hinge-type motion of a single helix, which is displaced during activation to expose the active site and give access to the substrate. Our results suggest that the activation of lipases is enhanced in a hydrophobic environment as is generally observed in experiments. The energy gain upon activation varies between the different lipases and depends strongly on the distribution of the charged residues in the activating loop region. In a low dielectric constant medium (such as a lipid environment), the electrostatic interactions between the residues located in the vicinity of the activating loop (lipid contact zone) are dominant and determine the activation of the lipases. Calculations of the pKas qualitatively indicate that some titratable residues experience significant pK shifts upon activation. These calculations may provide sufficient details for an understanding of the origin and magnitude of a given electrostatic effect and may provide an avenue for exploring the activation pathway of lipases.

Binding Sites↗
PubMed1996

Activation of intrinsic and synaptic currents in leech heart interneurons by realistic waveforms.

Leech heart interneurons were voltage-clamped with realistic waveforms to investigate the currents underlying the oscillation in the cells. By estimating the leak current parameters in regions in which there was little contamination by voltage-gated currents, it was possible to measure the Ca2+ current, the persistent Na+ current, Ip, and the hyperpolarization-activated inward current, Ih. The experiments verified a prediction of a computer model of HN cells that the shape of the typical waveform was such that the low-threshold Ca2+ currents were partially inactivated during a slow up-ramp to a plateau potential. A step within the same range of the membrane potential as the realistic waveform produced > 4 times as much Ca2+ current. In two-cell voltage-clamp experiments, the step produced 20 times more graded inhibition than the normal presynaptic waveform. When the presynaptic heart interneuron oscillated with spikes, the graded inhibition was larger. The difference may arise from integration of a slowly decaying component of the spike-mediated inhibition. The persistent Na+ current had a very low threshold. During the most hyperpolarized phase of the waveform, Ip deactivated to 50% of its maximum conductance. A substantial part of Ip, therefore, was effectively contributing to the leak current in the HN cells. The h-current increased for waveforms that had longer periods, whereas increasing the h-current in the model reduced the period. The h-current thus provides negative feedback to perturbations that alter the period of the oscillation.

Action Potentials↗
PubMed1996

Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor.

Tissue-factor-pathway inhibitor (TFPI) is a multivalent inhibitor with three tandemly arranged Kunitz- type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny , W. F., Likert, K. M., Brown, S. G., Miletich, J. R & Broze, G. J. (1989) Nature 338, 518-520] by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPI domain 2 interacts with factor Xa, and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI-(1-161)-peptide and separate recombinant TFPI-KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (Ki = 250 nM), KPI domain 2 inhibited factor Xa (Ki = 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (Ki = 0.1 nM) and chymotrypsin (Ki = 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki = 26 nM) and cathepsin G (Ki = 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three-dimensional models of the proteins derived by comparative molecular-modelling methods. Studies of factor Xa inhibition by intact TFPI (Ki approximately 0.02 nM) suggested that regions other than the contact area of the KPI domain, interacted strongly with factor Xa. Secondary-site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin.

Amino Acid Sequence↗
PubMed1996

Structural changes in factor VIIa induced by Ca2+ and tissue factor studied using circular dichroism spectroscopy.

Factor VIIa (fVIIa) is composed of four discrete domains, a gamma-carboxyglutamic acid (Gla)-containing domain, two epidermal growth factor (EGF)-like domains, and a serine protease domain, all of which appear to be involved, to different extents, in an optimal interaction with tissue factor (TF). All except the second EGF-like domain contain at least one Ca2+ binding site and many properties of fVIIa, e.g., TF and phospholipid binding and amidolytic activity, are Ca(2+)-dependent. A CD study was performed to characterize and locate the conformational changes in fVIIa induced by Ca2+ and TF binding. In addition to intact fVIIa, derivatives lacking the Gla domain or the protease domain were used. Assignment of the Ca(2+)-induced changes in the far-UV region of the fVIIa spectrum to the Gla domain could be made by comparing the CD spectra obtained with these fVIIa derivatives. The changes primarily appeared to reflect a Ca(2+)-induced ordering of alpha-helices existing in the apo state of fVIIa. This was corroborated by models of the apo and Ca2+ forms of fVIIa, obtained as difference spectra between fVIIa derivatives, were very similar to those of isolated Gla peptides from other vitamin K-dependent plasma proteins. The near-UV CD spectrum of fVIIa was dominated by aromatic residues residing in the protease domain and specific bands affected by Ca2+ were indicative of tertiary structural alterations. The formation of a fVIIa:TF complex led to secondary structural changes that appeared to be restricted to the catalytic domain, possibly shedding light on the mechanism by which TF induces an enhancement of fVIIa catalytic activity.

Calcium↗
PubMed1996

Theoretical investigation of the dynamics of the active site lid in Rhizomucor miehei lipase.

Interfacial activation of Rhizomucor miehei lipase is accompanied by a hinge-type motion of a single helix (residues 83-94) that acts as a lid over the active site. Activation of the enzyme involves the displacement of the lid to expose the active site, suggesting that the dynamics of the lid could be of mechanistic and kinetic importance. To investigate possible activation pathways and to elucidate the effect of a hydrophobic environment (as would be provided by a lipid membrane) on the lid opening, we have applied molecular dynamics and Brownian dynamics techniques. Our results indicate that the lipase activation is enhanced in a hydrophobic environment. In nonpolar low-dielectric surroundings, the lid opens in approximately 100 ns in the BD simulations. In polar high-dielectric (aqueous) surroundings, the lid does not always open up in simulations of up to 900 ns duration, but it does exhibit some gating motion, suggesting that the enzyme molecule may exist in a partially active form before the catalytic reaction. The activation is controlled by the charged residues ARG86 and ASP91. In the inactive conformation, ASP91 experiences repulsive forces and pushes the lid toward the open conformation. Upon activation ARG86 approaches ASP61, and in the active conformation, these residues form a salt bridge that stabilizes the open conformation.

Binding Sites↗
PubMed1995

[Exposure to anesthetic gases].

In recent years there has been a growing awareness of possible hazards caused by anaesthetic gases in operating theatres. Our study monitored ambient nitrous oxide (N2O) levels in the operating theatres and recovery rooms at the University Hospital in Tromsø. The results show that exposure to waste anaesthetic gases occurs because of leaks in the anaesthetic equipment. The three major sources of leaks are masks, high pressure fittings and exhalation valves. Prevention of leakage from equipment is very important, and a leakage testing programme should be an essential part of the daily control strategy. Good working practices and tracheal intubation results in low exposure to nitrous oxide in the breathing zone of the anesthetic personnel. During mask anaesthesia we have observed nitrous oxide concentrations above 1500 ppm, owing to mask leakage. A closely fitting scavenging mask and good working practices lead to in lower nitrous oxide concentrations. Nitrous oxide exhalation from the patient in the recovery room is only a minor problem.

Air Pollutants, Occupational↗
PubMed1995

Heartbeat control in the medicinal leech: a model system for understanding the origin, coordination, and modulation of rhythmic motor patterns.

We have analyzed in detail the neuronal network that generates heartbeat in the leech. Reciprocally inhibitory pairs of heart interneurons form oscillators that pace the heartbeat rhythm. Other heart interneurons coordinate these oscillators. These coordinating interneurons, along with the oscillators interneurons, form an eight-cell timing oscillator network for heartbeat. Still other interneurons, along with the oscillator interneurons, inhibit heart motor neurons, sculpting their activity into rhythmic bursts. Critical switch interneurons interface between the oscillator interneurons and the other premotor interneurons to produce two alternating coordination states of the motor neurons. The periods of the oscillator interneurons are modulated by endogenous RFamide neuropeptides. We have explored the ionic currents and graded and spike-mediated synaptic transmission that promote oscillation in the oscillator interneurons and have incorporated these data into a conductance-based computer model. This model has been of considerable predictive value and has led to new insights into how reciprocally inhibitory neurons produce oscillation. We are now in a strong position to expand this model upward, to encompass the entire heartbeat network, horizontally, to elucidate the mechanisms of FMRFamide modulation, and downward, to incorporate cellular morphology. By studying the mechanisms of motor pattern formation in the leech, using modeling studies in conjunction with parallel physiological experiments, we can contribute to a deeper understanding of how rhythmic motor acts are generated, coordinated, modulated, and reconfigured at the level of networks, cells, ionic currents, and synapses.

Animals↗
PubMed1995

Modeling the leech heartbeat elemental oscillator. I. Interactions of intrinsic and synaptic currents.

We have developed a biophysical model of a pair of reciprocally inhibitory interneurons comprising an elemental heartbeat oscillator of the leech. We incorporate various intrinsic and synaptic ionic currents based on voltage-clamp data. Synaptic transmission between the interneurons consists of both a graded and a spike-mediated component. By using maximal conductances as parameters, we have constructed a canonical model whose activity appears close to the real neurons. Oscillations in the model arise from interactions between synaptic and intrinsic currents. The inhibitory synaptic currents hyperpolarize the cell, resulting in activation of a hyperpolarization-activated inward current Ih and the removal of inactivation from regenerative inward currents. These inward currents depolarize the cell to produce spiking and inhibit the opposite cell. Spike-mediated IPSPs in the inhibited neuron cause inactivation of low-threshold Ca++ currents that are responsible for generating the graded synaptic inhibition in the opposite cell. Thus, although the model cells can potentially generate large graded IPSPs, synaptic inhibition during canonical oscillations is dominated by the spike-mediated component.

Animals↗
PubMed1995

Modeling the leech heartbeat elemental oscillator. II. Exploring the parameter space.

In the previous paper, we described a model of the elemental heartbeat oscillator in the leech. Here, the parameters of our model are explored around the baseline canonical model. The maximal conductances of the currents and the reversal potential of the leak current are varied to reveal the effects of individual currents and the interaction between synaptic and intrinsic currents in the model. The model produces two distinct modes of oscillation as the parameters are varied, S-mode and G-mode. These two modes are defined, their origin is identified, and the parameter space is mapped into S-mode and G-mode oscillation and no oscillation. Finally, we will make predictions for how the period can be modulated in heart interneurons.

Animals↗
PubMed1994

Cavity mutants of Savinase. Crystal structures and differential scanning calorimetry experiments give hints of the function of the buried water molecules in subtilisins.

The subtilisin molecule possesses several internal water molecules, which may be characterised as an integral part of the protein structure. We have introduced specific mutations (T71I, T71S, T71V, T71A and T71G) at position 71 in the subtilisin variant Savinase from Bacillus lentus. This position is involved in a hydrogen bonded network with several internal water molecules, forming a water channel. The water channel and most of the other internal water molecules are positioned in the interface between two half-domains of the subtilisin molecule. The data presented here indicate that the internal water molecules are structural, and may be the result of trapping during the folding process.

Amino Acids↗
PubMed1993

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.

Amino Acid Sequence↗
PubMed1993

Quantifying periodic activity in central pattern generators: the crayfish swimmeret.

Experimental recordings from neurons are generally difficult to quantify. Spike patterns and membrane potential cycles are often compared qualitatively and are hence limited by human resolution and judgement. This paper describes a statistical method for quantifying individual spike patterns and membrane potential fluctuations in oscillating systems, allowing quantification of successive 'individual' bursts. The neuronal activities of the network can therefore be subject to more extensive analyses and comparisons. Principal-component analysis was used to extract the principal components from a sequence of bursts recorded in vitro from the crayfish (Pacifasticus leniusculus) swimmeret. The principal component coefficients were correlated with intuitive biological concepts such as burst displacement, number of spikes in a burst, and burst width. The bursting of both interneurones and motor neurones were found to be modulated by some unidentified oscillator that displaced the bursts in time. The amplitude of the time displacements was significant, i.e., of the order of the bursting period (1.5 s), and the period of the oscillation was approximately 200 bursts. The strength of the method is that this feature was detected without any prior suspicion of its existence.

Action Potentials↗
PubMed1988

Protein secondary structure and homology by neural networks. The alpha-helices in rhodopsin.

Neural networks provide a basis for semiempirical studies of pattern matching between the primary and secondary structures of proteins. Networks of the perceptron class have been trained to classify the amino-acid residues into two categories for each of three types of secondary feature: alpha-helix or not, beta-sheet or not, and random coil or not. The explicit prediction for the helices in rhodopsin is compared with both electron microscopy results and those of the Chou-Fasman method. A new measure of homology between proteins is provided by the network approach, which thereby leads to quantification of the differences between the primary structures of proteins.

Amino Acid Sequence↗
PubMedDate not supplied

A model of a segmental oscillator in the leech heartbeat neuronal network.

We modeled a segmental oscillator of the timing network that paces the heartbeat of the leech. This model represents a network of six heart interneurons that comprise the basic rhythm-generating network within a single ganglion. This model builds on a previous two cell model (Nadim et al., 1995) by incorporating modifications of intrinsic and synaptic currents based on the results of a realistic waveform voltage-clamp study (Olsen and Calabrese, 1996). Due to these modifications, the new model behaves more similarly to the biological system than the previous model. For example, the slow-wave oscillation of membrane potential that underlies bursting is similar in form and amplitude to that of the biological system. Furthermore, the new model with its expanded architecture demonstrates how coordinating interneurons contribute to the oscillations within a single ganglion, in addition to their role of intersegmental coordination.

Animals↗