Do method and species lifestyle affect measures of maximum metabolic rate in fishes?

The rate at which active animals can expend energy is limited by their maximum aerobic metabolic rate (MMR). Two methods are commonly used to estimate MMR as oxygen uptake in fishes, namely during prolonged swimming or immediately following brief exhaustive exercise, but it is unclear whether they return different estimates of MMR or whether their effectiveness for estimating MMR varies among species with different lifestyles. A broad comparative analysis of MMR data from 121 fish species revealed little evidence of different results between the two methods, either for fishes in general or for species of benthic, benthopelagic or pelagic lifestyles.

Measurement and relevance of maximum metabolic rate in fishes.

Maximum (aerobic) metabolic rate (MMR) is defined here as the maximum rate of oxygen consumption (M˙O2max ) that a fish can achieve at a given temperature under any ecologically relevant circumstance. Different techniques exist for eliciting MMR of fishes, of which swim-flume respirometry (critical swimming speed tests and burst-swimming protocols) and exhaustive chases are the most common. Available data suggest that the most suitable method for eliciting MMR varies with species and ecotype, and depends on the propensity of the fish to sustain swimming for extended durations as well as its capacity to simultaneously exercise and digest food. MMR varies substantially (>10 fold) between species with different lifestyles (i.e. interspecific variation), and to a lesser extent (

Resolution of an iridoid synthon, gastrolactol, by means of dynamic acetylation and lipase-catalyzed alcoholysis.

A short synthetic route to asymmetric iridoids was developed. The three key steps were an intramolecular [4 + 2] cycloaddition reaction of an enamine derivative of 8-oxocitral (2), a dynamic acetylation, and an enzymatic resolution of the gastrolactyl acetates 5a and 5b, iridoids with three stereocenters. Some regio- and stereoselective heterogeneous catalytic hydrogenations of double bonds in iridoid aglucones were discussed.

Improved enantioselectivity of a lipase by rational protein engineering.

A model based on two different binding modes for alcohol enantiomers in the active site of a lipase allowed rational redesign of its enantioselectivity. 1-Halo-2-octanols were poorly resolved by Candida antarctica lipase B. Interactions between the substrates and the lipase were investigated with molecular modeling. Unfavorable interactions were found between the halogen moiety of the fast-reacting S enantiomer and a region situated at the bottom of the active site (stereoselectivity pocket). The lipase was virtually mutated in this region and energy contour maps of some variants displayed better interactions for the target substrates. Four selected variants of the lipase were produced and kinetic resolution experiments were undertaken with these mutants. Single point mutations gave rise to one variant with doubled enantioselectivity as well as one variant with annihilated enantioselectivity towards the target halohydrins. An increased volume of the stereoselectivity pocket caused a decrease in enantioselectivity, while changes in electrostatic potential increased enantioselectivity. The enantioselectivity of these new lipase variants towards other types of alcohols was also investigated. The changes in enantioselectivity caused by the mutations were well in agreement with the proposed model concerning the chiral recognition of alcohol enantiomers by this lipase.

Mass transport limitations reduce the effective stereospecificity in enzyme-catalyzed kinetic resolution.

[reaction-see text] The kinetic resolution of seudenol catalyzed by Candida antarctica lipase B in hexane was investigated. Large differences in reaction rate and stereospecificity were observed when different enzyme preparations were used. These differences were ascribed to mass transport limitations which reduced both reaction rate and stereospecificity. Lyophilized enzyme preparations were more apt to give this problem than immobilized preparations. Further, low substrate concentrations enhanced the effect. Thus, high alcohol concentrations and enzyme immobilization can be recommended.

An active-site titration method for lipases.

A method for active-site titration of lipases has been developed based on irreversible inhibition by methyl p-nitrophenyl n-hexylphosphonate. This method was applied to five lipases displaying from minor to pronounced interfacial activation. Soluble and immobilized lipases were successfully titrated in aqueous media. A low concentration of sodium dodecyl sulfate was needed for lipases displaying pronounced interfacial activation. The carrier of some of the immobilized preparations adsorbed part of the produced p-nitrophenolate. This problem could be solved by extracting the p-nitrophenolate after inhibition. The method was extended to apolar organic solvents in the case of immobilized lipase preparations.

Molecular modeling of the enantioselectivity in lipase-catalyzed transesterification reactions.

Two strategies based on the use of subsets for calculating the enantioselectivity in lipase-catalyzed transesterifications using the CHARMM force field were investigated. Molecular dynamics was used in our search for low energy conformations. Molecular mechanics was used for refining these low energy conformations. A tetrahedral intermediate with a rigid central part was used for mimicking the transition state. The energy differences between the transition states of the diastereomeric enzyme-substrate complexes were calculated. The way of defining the subsets was based on two fundamentally different strategies. The first strategy used predefined parts of the enzyme and the substrate as subsets. The second approach formed energy-based subsets, varying in size with the substrates studied. The selection of residues to be included in these energy-based subsets was based on the energy of the interaction between the specific residue or water molecule and the transition state. The reaction studied was the kinetic resolution of secondary alcohols in transesterifications using the Candida antarctica lipase B as chiral biocatalyst. The secondary alcohols used in the study were 2-butanol, 3-methyl-2-butanol, and 3,3-dimethyl-2-butanol.

A structural basis for enantioselective inhibition of Candida rugosa lipase by long-chain aliphatic alcohols.

Molecular modeling showed that the enantiomers of heptyl 2-methyldecanoate are productively bound to the active site of Candida rugosa lipase in quite different conformations. The fast-reacting S-enantiomer may well occupy the previously identified acyl-binding tunnel in the active site of the lipase. By contrast, the slow-reacting R-enantiomer must be bound to the active site, leaving the tunnel empty to allow the formation of two catalytically essential hydrogen bonds between His 449 of the catalytic triad and the transition state of the catalyzed reaction. This information enables us to propose a molecular mechanism explaining how long-chain aliphatic alcohols act as enantioselective inhibitors of this lipase in the resolution of 2-methyldecanoic acid. Long-chain aliphatic alcohols may coordinate to the acyl-binding tunnel of the C. rugosa lipase, thereby selectively inhibiting the turnover of the fast-reacting S-enantiomer, thus resulting in a lowered enantioselectivity in the resolution.

A cohort study with regard to the risk of haematological malignancies in patients treated with x-rays for benign lesions in the locomotor system. I. Epidemiological analyses.

Roentgen treatment for painful benign conditions in the locomotor system as arthrosis and spondylosis was in Sweden very common up to the beginning of the 1960s. The mode of treatment differed from the British ankylosing spondylitis series as smaller parts of the red bone marrow were exposed and smaller doses were applied. A cohort of 20,024 such patients treated 1950-1964 at two hospitals in northern Sweden was analysed with regard to the risk of haematological malignancies. Average factors for conversion of prescribed skin doses to mean absorbed red bone marrow doses were estimated on random samples of the different treatment sites and then applied on the cohort in its whole. The standard incidence ratio (SIR) for leukaemia was 1.18 (95% CI: 0.98-1.42) and the standard mortality ratio (SMR) 1.25 (0.99-1.45). In the highest dose group (mean absorbed red bone marrow dose > 0.5 Gy) the corresponding values were 1.40 (1.00-1.92) and 1.50 (1.08-2.04). In the mortality analysis also a slightly increased myeloma risk was noted with SMR = 1.20 (0.99-1.56). Extension of the cohort and nested case-control studies are under progress.

Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa.

The substrate-binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme. Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme-substrate transition-state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.

Enhanced stereoselectivity in pig liver esterase catalysed diester hydrolysis. The role of a competitive nucleophile.

The enantioselectivity of pig liver esterase catalysed hydrolysis of cis-N-benzyl-2,5-bis(methoxy-carbonyl)pyrrolidine (1) has previously been shown to be very dependent on the reaction conditions. Hydrolysis performed in media buffered with tris(hydroxymethyl)aminomethane (Tris) afforded a monoester with much higher optical purity than hydrolysis in media without Tris. Detailed product studies in a Tris-buffered medium have been performed using NMR-techniques and a 13C-labelled ester. The NMR-studies revealed the presence of (2S,5R)-N-benzyl-2-methoxycarbonyl-5-[[[2-hydroxy-1,1- bis(hydroxymethyl)ethyl]amino]carbonyl]pyrrolidine (4) as an intermediate, which together with the isolated product (2S,5R)-N-benzyl-2-carboxy-5-[[[2-hydroxy-1,1-bis(hydroxymethyl) ethyl]amino]carbonyl]pyrrolidine (3) suggested Tris as a competitive nucleophile to water. The increased enantioselectivity seen in the produced (2R,5S)-N-benzyl-2-methoxy-carbonyl-5-carboxypyrrolidine (2) was explained by the preference of Tris to react faster with one of the diastereomeric acyl enzymes over the other.

Perioperative adjuvant chemotherapy in breast cancer. The Scandinavian Adjuvant Chemotherapy Study 1.

In a randomized study of operable primary breast cancer patients initiated January 1965, 507 patients received one single course with cyclophosphamide 5 mg/kg/day for six days, first dose given immediately after mastectomy. The 519 control patients received no adjuvant chemotherapy. In other respects both groups were treated equally. Median follow-up time is 17.1 years. In terms of relapse-free percentage, the difference between the groups after 20 years is 13.5% in favour of the cyclophosphamide group. The difference is statistically highly significant. This benefit is observed in node positive as well as in node negative patients, and over as well as under 50 years. The immediate side effects of the cyclophosphamide course have been very moderate. No late complications have been observed. In a parallel randomized study with 110 patients where the same course was given 2-4 weeks after mastectomy, no benefit could be observed.

Structures, absolute configurations, and syntheses of volatile signals from three sympatric ant-lion species,Euroleon nostras, Grocus bore, andMyrmeleon formicarius (Neuroptera: Myrmeleontidae).

The thoracic gland of the ant-lionEuroleon nostras was found to contain nerol oxide (1a) and (Z)-6-undecen-2-ol (nostrenol,3) while the speciesGrocus bore contained 10-homonerol oxide (1b) and nostrenol (3). Nerol (2a) and 10-homonerol (2b) were found in a third species,Myrmeleon formicarius. 10-Homonerol, racemic 10-homonerol oxide, and racemic as well as (R)- and (S)-nostrenol were synthesized. The nerol oxide ofE. nostras and the 10-homonerol oxide ofG. bore were found to be racemic, while both species contained optically pure (R)-nostrenol (28).

Variation of enantiomeric composition of α-pinene in norway spruce,Picea abies, and its influence on production of verbenol isomers byIps typographus in the field.

The enantiomeric composition of α-pinene in individual Norway spruce trees [Picea abies (L.) Karst.] was determined on a chiral GC column after stereoselective hydroboration-oxidation followed by a reaction with isopropyl isocyanate to form the carbamate derivative. The enantiomeric composition varied considerably between trees of different genetic origin. There was a strong correlation between the chirality of α-pinene in host spruce trees and thecis/trans ratio of verbenols found in the hindguts of the bark beetleIps typographus (L.) infesting the trees.

Differences in attraction to semiochemicals present in sympatric pine shoot beetles,Tomicus minor andT. piniperda.

The chemical ecology of host- and mate-finding in the pine shoot beetles,Tomicus minor andT. piniperda, was studied in southern Sweden. Beetles were collected in the field from defined attack phases on Scots pine. Using gas chromatography-mass spectroscopy, a number of oxygen-containing monoterpenes, e.g., 3-carene-10-ol, myrtenol,trans-verbenol, and verbenone, were identified from hindgut extracts of both sexes of both species. Compared toT. minor,T. piniperda contained additional compounds and in larger amounts. The amounts were highest in both species at the time when the beetles had bored into contact with the resin-producing xylem-phloem tissue. The synthesis of (1S,6R)-3-carene-10-ol by photooxidatipn of (+)-(1S,6R)-3-carene is described. In comparative electroantennogram (EAG) measurements on males and females of both species, the most active of the tested compounds wastrans-verbenol. Laboratory bioassays of walking beetles showed thatT. piniperda was attracted to uninfestèd pine logs.T. minor was more strongly attracted to pine logs infested with females than to uninfested pine logs, indicating a female-produced aggregation pheromone. Field tests confirmed thatT. piniperda was strongly attracted to pine logs. The attraction ofT. minor to logs was significant only when logs were combined with racemictrans-verbenol and (1S,6R)-3-carene-10-ol.T. minor was also attracted to a combination of these monoterpene alcohols alone. We suggest that host and mate location inT. piniperda is achieved by means of a kairomone composed of host monoterpenes, whileT. minor utilizes a primitive pheromone synergized by host odors. Evolution of host colonization strategies of the two beetles are discussed.

Methylbutynol effectively replaces methylbutenol, a pheromone component ofIps typographus (L.) (Coleoptera: Scolytidae).

In field experiments in Sweden, the constituent 2-methyl-3-buten-2-ol of the aggregation pheromone of the spruce bark bettleIps typographus (L.) was effectively replaced by 2-methyl-3-butyn-2-ol.

Treatment of node-negative breast cancer patients with short course of chemotherapy immediately after surgery.

In our first study, one short course of chemotherapy (cyclophosphamide, 5 mg/kg/day for 6 days iv) was given perioperatively to 507 patients with operable primary breast cancer; there were 519 control subjects. Randomization was done by telephone from the operating theater, and stratification was by hospital only. The side effects were negligible. With 20-year follow-up, the relapse rate was 60.5% in the control group and 48% in the treatment group (P less than 0.001). The overall survival benefit was marginally significant, but with correction for deaths not related to cancer, P was less than 0.02. A total of 309 patients in the treatment group and 301 in the control group were histologically node negative. The observed benefit in this subgroup was as good as in the node-positive subgroup, but the relapses were too few for testing of meaningful significance. In our second study, all patients received one short perioperative course, and the node-positive patients were randomized between control or continued chemotherapy for 1 year. This improved the beneficial results but also considerably increased the side effects.

Short perioperative versus long-term adjuvant chemotherapy.

A single course of cyclophosphamide IV 5 mg/kg daily for 6 days was given immediately after mastectomy to 507 patients (519 randomized controls). The relapse-free rates were significantly increased, and after 16 years the difference was 12%. In a parallel series the same adjuvant course was given 2-4 weeks after mastectomy to 52 patients (58 randomized controls). No effect of this delayed course was found. In a second study a short multidrug course was given to all patients immediately after mastectomy. One-half of the axillary node-positive cases were randomized to continue with IV CMF for 1 year. The preliminary observations show that the prolonged treatment improved the relapse-free rates significantly during the first few years. One year after mastectomy the difference was 10%; after 2 years, 9%; after 3 years, 11%; and after 4 years, 9%. The side-effects of the short course were negligible, but the side-effects of prolonged treatment were considerable and increased with increasing treatment duration. More trials are needed to find the optimum duration of adjuvant chemotherapy.