Using biological-physical modelling for informing sea lice dispersal in Loch Linnhe, Scotland.

Sea lice are a constraint on the sustainable growth of Scottish marine salmonid aquaculture. As part of an integrated pest management approach, farms coordinate procedures within spatial units. We present observations of copepodids being at relatively greater density than nauplii in upper waters, which informs the development of surface layer sea lice transmission modelling of Loch Linnhe, Scotland, for informing farm parasite management. A hydrodynamic model is coupled with a biological particle-tracking model, with characteristics of plankton sea lice. Simulations are undertaken for May and October 2011-2013, forced by local wind data collected for those periods. Particles are continually released from positions representing farm locations, weighted by relative farm counts, over a 2-week period and tracked for a further 5 days. A comparison is made between modelled relative concentrations against physical and biological surveys to provide confidence in model outputs. Connectivity between farm locations is determined in order to propose potential coordination areas. Generally, connectivity depends on flow patterns in the loch and decreases with increased farm separation. The connectivity indices are used to estimate the origins of the sea lice population composition at each site, which may influence medicinal regimens to avoid loss of efficacy.

Multicomponent telerehabilitation programme for older veterans with multimorbidity: a programme evaluation.

INTRODUCTION: Older veterans with multimorbidity experience physical, mental and social factors which may negatively impact health and healthcare access. Physical function, behaviour change skills and loneliness may not be addressed during traditional physical rehabilitation. Thus, a multicomponent telerehabilitation programme could address these unmet needs. This programme evaluation assessed the safety, feasibility and change in patient outcomes for a multicomponent telerehabilitation programme. METHODS: Individuals were eligible if they were a veteran/spouse, age ≥50 years and had ≥3 comorbidities. The telerehabilitation programme included four core components: (1) High-intensity rehabilitation, (2) Coaching interventions, (3) Social support and (4) Technology. Physical therapists delivered the 12-week programme and collected patient outcomes at baseline, 4 weeks, 8 weeks and 12 weeks. Programme evaluation measures included safety events (occurrence and type), feasibility (adherence) and patient outcomes (physical function). Safety and feasibility outcomes were analysed using descriptive statistics. The mean pre-post programme difference and 95% CI for patient outcomes were generated using paired t-tests. RESULTS: Twenty-one participants enrolled in the telerehabilitation programme; most were male (81%), white (72%) and non-Hispanic (76%), with an average of 5.7 (3.0) comorbidities. Prevalence of insession safety events was 3.2% (0.03 events/session). Fifteen (71.4%) participants adhered to the programme (attended ≥80% of sessions). Mean (95% CI) improvements for physical function are as follows: 4.7 (2.4 to 7.0) repetitions for 30 s sit to stand, 6.0 (4.0 to 9.0) and 5.0 (2.0 to 9.0) repetitions for right arm curl and left arm curl, respectively, and 31.8 (15.9 to 47.7) repetitions for the 2 min step test. CONCLUSION: The telerehabilitation programme was safe, feasible and demonstrated preprogramme to postprogramme improvements in physical function measures while addressing unmet needs in a vulnerable population. These results support a randomised clinical trial while informing programme and process adaptations.

Determining the age of individual Lepeophtheirus salmonis (Krøyer, 1837) copepodids by measuring stored lipid volume; proof of principle.

Confocal microscopy has facilitated measurement of stained lipid volume in Lepeophtheirus salmonis copepodid larvae. Quantity of lipid, location and morphology of vesicles may allow an estimate of age and viability.

The pH dependence of the reductive carboxylation of pyruvate by malic enzyme.

The maximum velocity of the malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) reductive carboxylation of pyruvate and V/KCO2 are pH-independent from pH 5.5 to pH 8.5. V/K for pyruvate exhibits pK values values of 6.50 +/- 0.25 and 7.25 +/- 0.25. These data suggest that the binding of pyruvate locks the protonation state of enzyme. In addition, the pK values are within experimental error identical for the pH dependence of V/Kmalate and V/Kpyruvate. Thus, the catalytic groups appear to have reverse protonation states in the two reaction directions. The ratio of (V/Kmalate)/(V/Kpyruvate) is 100, suggesting that the protonation state of enzyme is optimum in the malate oxidative decarboxylation direction. Thus, the group with a pK of about 6 is unprotonated and the group with a pK of 7.5 is protonated for malate decarboxylation, and the opposite is true for pyruvate reductive carboxylation.

Time-resolved fluorescence of O-acetylserine sulfhydrylase.

Static and time-resolved fluorescence of the internal aldimine of the pyridoxal 5'-phosphate (PLP)-dependent enzyme O-acetylserine sulfhydrylase (OASS) and those of free PLP, and the PLP-L-valine Schiff base have been measured to gain insight into the photophysics of PLP bound to OASS. Exciting at 330 nm, free coenzyme exhibits a band at 415 nm, whereas PLP-valine and OASS (also when excited at their absorbance maxima) exhibit a structured emission with a peak at 420 nm and shoulders at 490 and 530 nm. The emission bands at 420 and 490 nm are attributed to the enolimine and ketoenamine tautomers of the internal aldimine, respectively, while the 530 nm emission might arise from a dipolar species formed upon proton dissociation in the excited state. Time-resolved fluorescence of OASS (PLP-valine), excited at 412 nm (415 nm) and collected at lamda > 470 nm, indicates the presence of two components characterized by lifetimes (tau) of 0.6 (0.08) and 3.8 (1.55) ns with equal fractional intensity (f). In the presence of acetate the slow component dominates OASS emission with f of 0.98. Excitation at 350 nm as a function of emission wavelengths (400-560 nm) shows at least three components. The f of the slow component increases from 400 to 440 nm, then decreases, whereas the f of the intermediate and fast components behave in the opposite way. Results indicate that: (i) the fast component is associated with the emission at 530 nm; (ii) the slow component is associated with the emission at 420 nm; (iii) a fast additive component, characterized by a very short lifetime, is present on the blue side of the emission spectrum; (iv) the intermediate component results from overlapping contributions, including the emission of the band at 490 nm, that could not be resolved; (v) the increased emission at 490 nm, caused by acetate binding, is likely due to the stabilization of the ketoenamine tautomer induced by an increase in polarity of the active site microenvironment and/or a decrease in proton dissociation in the excited state; (vi) excitation at 330 nm, where the enolimine tautomer absorbs, leads to emission decays typical of the ketoenamine.

Equilibrium model in an in vitro poly(ADP-ribose) turnover system.

Poly(ADP-ribose) metabolism plays an important role in numerous DNA-related functions. This homopolymer is synthesized by poly(ADP-ribose) polymerase and is degraded mainly by the poly(ADP-ribose) glycohydrolase. The activities of these two enzymes in the nucleus are closely coordinated. To better understand the interactions between these enzymes, we designed an in vitro system in which both enzymes are present at the same time. In this work, we report a model describing the synthesis and degradation of the poly(ADP-ribose) in turnover conditions. Because the half-life of the polymer in the cell is close to 1 min, we studied the very early kinetic interactions of these two enzymes.

Identification of an allosteric anion-binding site on O-acetylserine sulfhydrylase: structure of the enzyme with chloride bound.

A new crystal structure of O-acetylserine sulfhydrylase (OASS) has been solved with chloride bound at an allosteric site and sulfate bound at the active site. The bound anions result in a new "inhibited" conformation, that differs from the "open" native or "closed" external aldimine conformations. The allosteric site is located at the OASS dimer interface. The new inhibited structure involves a change in the position of the "moveable domain" (residues 87-131) to a location that differs from that in the open or closed forms. Formation of the external aldimine with substrate is stabilized by interaction of the alpha-carboxyl group of the substrate with a substrate-binding loop that is part of the moveable domain. The inhibited conformation prevents the substrate-binding loop from interacting with the alpha-carboxyl group, and hinders formation of the external Schiff base and thus subsequent chemistry. Chloride may be an analog of sulfide, the physiological inhibitor. Finally, these results suggest that OASS represents a new class of PLP-dependent enzymes that is regulated by small anions.

Crystallization and preliminary X-ray data for the A-isozyme of O-acetylserine sulfhydrylase from Salmonella typhimurium.

The A-isozyme of O-acetylserine sulfhydrylase, a pyridoxal phosphate-dependent enzyme isolated from Salmonella typhimurium catalyzes the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The pyridoxal form of the enzyme has been crystallized in two different forms. One form is in the orthorhombic space group P2(1)2(1)2(1) with cell constants a = 144.4 A, b = 96.9 A and c = 54.3 A and contains two monomers each of molecular weight 34,000 per asymmetric unit. The second form is in a hexagonal space group with unit cell dimensions a = b = 115 A, and c = 348 A and contains two 68,000 dimers per asymmetric unit. Complete native enzyme data sets have been collected for both crystal forms using an R-Axis II detector. A search for suitable heavy-atom derivatives is underway. Although both crystal forms diffract X-rays to better than 2.5 A, the orthorhombic form is more suited to a detailed structural analysis due to the extended lifetime in the X-ray beam and the relative size of the unit cell.

Three-dimensional structure of O-acetylserine sulfhydrylase from Salmonella typhimurium.

The last step in cysteine biosynthesis in enteric bacteria is catalyzed by the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase. Here we report the crystal structure at 2.2 A resolution of the A-isozyme of O-acetylserine sulfhydrylase isolated from Salmonella typhimurium. O-acetylserine sulfhydrylase shares the same fold with tryptophan synthase-beta from Salmonella typhimurium but the sequence identity level is below 20%. There are some major structural differences: the loops providing the interface to the alpha-subunit in tryptophan synthase-beta and two surface helices of tryptophan synthase-beta are missing in O-acetylserine sulfhydrylase. The hydrophobic channel for indole transport from the alpha to the beta active site of tryptophan synthase-beta is, not unexpectedly, also absent in O-acetylserine sulfhydrylase. The dimer interface, on the other hand, is more or less conserved in the two enzymes. The active site cleft of O-acetylserine sulfhydrylase is wider and therefore more exposed to the solvent. A possible binding site for the substrate O-acetylserine is discussed.

Catalytic competence of O-acetylserine sulfhydrylase in the crystal probed by polarized absorption microspectrophotometry.

The reactions of the pyridoxal 5'-phosphate-dependent enzyme O-acetylserine sulfhydrylase with the substrate O-acetyl-L-serine and substrate analogs have been investigated in the crystalline state by single-crystal polarized absorption microspectrophotometry. This approach has allowed us to examine the catalytic competence of the enzyme in different crystalline states, one of which was used to determine the three-dimensional structure; experimental conditions were defined for the accumulation of catalytic intermediates in the crystal suitable for crystallographic analyses.O-Acetyl-L-serine reacts with the enzyme in one of the crystal forms leading via a beta-elimination reaction to the accumulation of the alpha-aminoacrylate Schiff base, absorbing maximally at 320 and 470 nm, as in solution. The dissociation constant for the alpha-aminoacrylate Schiff base is in the millimolar range, 500-fold higher than in solution, suggesting that crystal lattice interactions may oppose functionally relevant conformational changes. The dissociation constant exhibits a bell-shaped dependence on pH centered at pH 7. At this pH the alpha-aminoacrylate species slowly decays with time (30% decrease in 24 hours). The alpha-aminoacrylate intermediate readily reacts with sodium azide, an analog of sulfide, the natural nucleophilic agent, to give a new amino acid and the native enzyme, indicating that the crystalline enzyme catalyzes the overall beta-replacement reaction as in solution. In other crystal forms, including that used for the X-ray investigation, O-acetyl-L-serine either has an even higher dissociation constant or causes crystal damage upon binding. When the crystalline enzyme reacts with either L-cysteine or L-serine, the external aldimine intermediate is formed. The dissociation constants for both substrate analogs are closer to those observed in solution and are modulated by pH as in solution. Findings demonstrate that O-acetylserine sulfhydrylase is catalytically competent in the crystal although some regions of the molecule, likely involved in an open-closed transition induced by O-acetyl-L-serine binding, may have a limited flexibility. The accumulation in the crystal of both the external aldimine and the alpha-aminoacrylate intermediate makes feasible their structural determination and, therefore, the elucidation of the catalytic pathway at the molecular level.

Ligand binding induces a large conformational change in O-acetylserine sulfhydrylase from Salmonella typhimurium.

Covalent binding of L-methionine as an external aldimine to the pyridoxal 5'-phosphate-cofactor in the K41A mutant of O-acetylserine sulfhydrylase from Salmonella typhimurium induces a large conformational change in the protein. Methionine mimics the action of the substrate O-acetyl-L-serine during catalysis. The alpha-carboxylate moiety of L-methionine in external aldimine linkage with the active site pyridoxal 5'-phosphate forms a hydrogen bonding network to the "asparagine-loop" P67-T68-N69-G70 which adopts a different conformation than in the native protein. The side-chain nitrogen of Asn69 moves more than 7 A to make a hydrogen bond to the alpha-carboxylate group of the inhibitor. As the external aldimine is formed, the PLP tilts by 13 degrees along its longitudinal axis such that C4' moves toward the entrance to the active site and the side-chain of the methionine is directed toward the active site entrance. The local rearrangement acts as a trigger to induce a large global conformational change in the protein. A subdomain comprised of beta-strand 4, alpha-helix 3, beta-strand 5 and alpha-helix 4 moves towards the active site by a rotation of 7 degrees. This subdomain movement results in a reduction of the severe twist of its central beta-sheet and reduces the active site entrance to a small hole, giving access only to small molecules like sulfide, the second substrate, or acetate, the first product.

Crystallization of the NAD-dependent malic enzyme from the parasitic nematode Ascaris suum.

The malic enzyme from muscle mitochondria of the parasitic nematode Ascaris suum is a tetramer of 65 kDa monomers that catalyzes the oxidative decarboxylation of malate to pyruvate and CO2 with NAD cofactor as oxidant. This malic enzyme is critical to the nematode for muscle function under anaerobic conditions. Unlike mammalian versions of the enzyme such as that found in rat liver, which require NADP as cofactor, the nematode version is an NAD-dependent enzyme. We report the crystallization of samples of the nematode enzyme at room temperature from pH 7.5 solutions of polyethylene glycol 4000 containing magnesium sulfate, NAD and sodium tartronate. Immediately upon mixing of protein and precipitant solutions, a marked precipitation of the protein occurs. Out of this precipitate, crystals appear almost immediately, most commonly in a truncated cube form that can grow to 0.5 to 0.7 mm on a cube edge in two to three days. The crystals are trigonal, space group P3(1)21 or its enantiomer, with a = b = 131.2(7) A, c = 152.6(9) A, and two monomers per asymmetric unit. Fresh crystals diffract X-radiation from a synchrotron source (lambda = 0.95 A) to about 3.0 A resolution. Rotational analysis of Patterson functions indicates that the malic enzyme tetramer has 222 symmetry.

Role of the divalent metal ion in the NAD:malic enzyme reaction: an ESEEM determination of the ground state conformation of malate in the E:Mn:malate complex.

The conformation of L-malate bound at the active site of Ascaris suum malic enzyme has been investigated by electron spin echo envelope modulation spectroscopy. Dipolar interactions between Mn2+ bound to the enzyme active site and deuterium specifically placed at the 2-position, the 3R-position, and the 3S-position of L-malate were observed. The intensities of these interactions are related to the distance between each deuterium and Mn2+. Several models of possible Mn-malate complexes were constructed using molecular graphics techniques, and conformational searches were conducted to identify conformers of malate that meet the distance criteria defined by the spectroscopic measurements. These searches suggest that L-malate binds to the enzyme active site in the trans conformation, which would be expected to be the most stable conformer in solution, not in the gauche conformer, which would be more similar to the conformation required for oxidative decarboxylation of oxalacetate formed from L-malate at the active site of the enzyme.

Mechanism from isotope effects.

Abstract Isotope effects represent perhaps one of the most versatile tools available to investigators interested in the determination of reaction mechanism, particularly in the case of the mechanistic enzymologist. Interpretation of isotope effect data is somewhat more difficult for enzyme reactions, since the chemical or isotope-dependent step(s) is(are) normally not solely rate-limiting as they are for non-enzyme-catalyzed reactions. One can, however, take advantage of rate-limitation by multiple steps in an enzyme-catalyzed reaction to obtain information on a number of aspects of mechanism. In this paper, simple theory for the application of isotope effects to reaction mechanism is developed, and applied to organic reactions and those catalyzed by enzymes. Techniques used to measure isotope effects depend somewhat on the isotope used, that is radioisotope vs. stable isotope, or hydrogen isotope vs. heavier atoms. Techniques to be discussed include competitive and noncompetitive (or internal discrimination) measurements. In enzymecatalyzed reactions, information can be obtained on the order of addition of reactants and release of products, and this will be illustrated using the 6-phosphogluconate and alcohol dehydrogenase reactions. The use of multiple isotope effects can be used to distinguish between stepwise and concerted reactions, and this will be illustrated with the formate and glucose 6-phosphate dehydrogenase and malic enzyme reactions.

Mechanism from isotope effects.

Isotope effects represent perhaps one of the most versatile tools available to investigators interested in the determination of reaction mechanism, particularly in the case of the mechanistic enzymologist. Interpretation of isotope effect data is somewhat more difficult for enzyme reactions, since the chemical or isotope-dependent step(s) is(are) normally not solely rate-limiting as they are for non-enzyme-catalyzed reactions. One can, however, take advantage of rate-limitation by multiple steps in an enzyme-catalyzed reaction to obtain information on a number of aspects of mechanism. In this paper, simple theory for the application of isotope effects to reaction mechanism is developed, and applied to organic reactions and those catalyzed by enzymes. Techniques used to measure isotope effects depend somewhat on the isotope used, that is radioisotope vs. stable isotope, or hydrogen isotope vs. heavier atoms. Techniques to be discussed include competitive and noncompetitive (or internal discrimination) measurements. In enzyme-catalyzed reactions, information can be obtained on the order of addition of reactants and relase of products, and this will be illustrated using the 6-phosphogluconate and alcohol dehydrogenase reactions. The use of multiple isotope effects can be used to distinguish between stepwise and concerted reactions, and this will be illustrated with the formate and glucose 6-phosphate dehydrogenase and malic enzyme reactions.

Rapid development of polyclonal antisera against infectious salmon anaemia virus and its optimization and application as a diagnostic tool.

Infectious salmon anaemia is an important disease of Atlantic salmon. One of the current methods of diagnosis is the indirect fluorescent antibody test (IFAT), using a monoclonal antibody specific to the haemagglutinin of the virus. The conformationally dependent nature of this antibody could be a drawback in its usefulness in other tests. This study describes the development and optimization of a polyclonal antiserum against infectious salmon anaemia virus, including a method of separating virus from cell culture components within culture supernatant. The antiserum was subsequently optimized for use in a variety of immunological diagnostic tests, including IFAT and an alkaline phosphatase-based immunoassay, and Western blot.

Kinetic studies to determine the mechanism of regulation of bovine liver glutamate dehydrogenase by nucleotide effectors.

A combination of kinetic and isotope effect studies in the presence and absence of the effectors ADP and GTP was used to elucidate the mechanism of regulation of bovine liver glutamate dehydrogenase. ADP at low concentrations of glutamate competes with TPN for free enzyme. GTP exhibits a similar effect at high concentrations (100 microM and above). When ADP binds at its allosteric site, it increases the off rates of both alpha-ketoglutarate and TPNH from their product complexes. This results in a decrease in V/K for both substrates, an increase in V, and an increase in the deuterium isotope effects for all three parameters so that they are all about 1.3. The rate of release of glutamate from E-TPNH-glutamate is also apparently enhanced since no substrate inhibition by glutamate is observed in the presence of ADP. The effect of GTP is in opposition to that of ADP in that GTP decreases the off rates for both TPN and glutamate from E-TPN-glutamate as well as the off rates for alpha-ketoglutarate and TPNH. This results in an increase in the V/K's for both substrates, a decrease in V, and a decrease in the deuterium isotope effects for all three parameters to a value of 1. Substrate inhibition by glutamate is also eliminated by GTP probably by preventing any significant accumulation of E-TPNH to which glutamate binds as an inhibitor.

Isotope partitioning in the adenosine 3',5'-monophosphate dependent protein kinase reaction indicates a steady-state random kinetic mechanism.

Isotope partitioning beginning with the binary E.MgATP and E.N-acetyl-Leu-Arg-Arg-Ala-Ser-Leu-Gly (Ser-peptide) complexes indicates that the kinetic mechanism for the adenosine 3',5'-monophosphate dependent protein kinase is steady-state random. A total of 100% of the initial radioactive E.MgATP complex is trapped as phospho-Ser-peptide at infinite Ser-peptide concentration at both low and high concentration of uncomplexed Mg2+, suggesting that the off-rate of MgATP from the E.MgATP.Ser-peptide complex is slow relative to the catalytic steps. Km for Ser-peptide in the trapping reaction decreases from 17 microM at low Mg2+ to 2 microM at high Mg2+, indicating that Mg2+ decreases the off-rate for MgATP from the E.MgATP complex. A total of 100% of the radioactive E.Ser-peptide complex is trapped as phospho-Ser-peptide at low Mg2+, but only 40% is trapped at high Mg2+ in the presence of an infinite concentration of MgATP, suggesting that the off-rate for Ser-peptide from the central complex is much less than catalysis at low but not at high Mg2+. In support of this finding, the Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly (Ala-peptide) increases from 0.27 mM at low Mg2+ to 2.4 mM at high Mg2+. No trapping was observed at either high or low Mg2+ for the E.MgADP complex up to a phospho-Ser-peptide concentration of 5 mM. Thus, it is likely that in the slow-reaction direction the kinetic mechanism is rapid equilibrium.(ABSTRACT TRUNCATED AT 250 WORDS)