Racemisation of drug enantiomers by benzylic proton abstraction at physiological pH

The enantiomers of the aromatase inhibitors 3-(4-aminophenyl)-pyrrolidine-2,5-dione (WSP-3, II ), its N-pentyl derivative ( III ), and the antifungal econazole ( IV ), all possessing a benzylic proton at the chiral centre, are rapidly racemised in vitro in phosphate buffer (0.01 M) at pH 7.4 and 23°C with t_½ values of 7, 6, and 5 h respectively. In vivo studies in rats show that (+)-econazole is racemised after intraperitoneal injection with t_½ = 1.24h. The enantiomers of the antifungal 1-[(benzofuran-2-yl)-4-chlorophenylmethyl] imidazole ( V ) were stable at pH 7.4, attributable to steric hindrance to carbanion formation in the racemisation step. © 1994 Wiley-Liss, Inc.     

Liposomes with Metronidazole for Topical Use: The Choice of Preparation Method and Vehicle

Abstract

Liposomes containing metronidazole were prepared for the treatment of skin disorder Rosacea. To optimize the composition and size of liposomes, natural and synthetic lipids were used in three different preparation methods. Optimal liposomal preparation was incorporated into five dermal vehicles (three O/W emulsion creams and two gels) and vehicles tested for the stability (at 20 and 40 °C) during a storage period of 4 weeks. The evaluation of the vehicles was based on the comparison between the original size distribution of liposomes and liposomes in vehicles (after 4 weeks) and on the rheological behaviour. The best vehicle appears to be the Carbopol? gel, in which the liposomal size stayed unchanged even at 40 °C during the 4 weeks period.     

Catalytic and thermodynamic properties of β-glucosidases produced by Lichtheimia corymbifera and Byssochlamys spectabilis

Abstract

The objective of the present study was to optimize parameters for the cultivation of Lichtheimia corymbifera (mesophilic) and Byssochlamys spectabilis (thermophilic) for the production of β-glucosidases and to compare the catalytic and thermodynamic properties of the partially purified enzymes. The maximum amount of β-glucosidase produced by L. corymbifera was 39?U/g dry substrate (or 3.9?U/mL), and that by B. spectabilis was 77?U/g (or 7.7?U/mL). The optimum pH and temperature were 4.5 and 55?°C and 4.0 and 50?°C for the enzyme from L. corymbifera and B. spectabilis, respectively. β-Glucosidase produced by L. corymbifera was stable at pH 4.0–7.5, whereas the enzyme from B. spectabilis was stable at pH 4.0–6.0. Regarding the thermostability, β-glucosidase produced by B. spectabilis remained stable for 1?h at 50?°C, and that from L. corymbifera was active for 1?h at 45?°C. Determination of thermodynamic parameters confirmed the greater thermostability of the enzyme produced by the thermophilic fungus B. spectabilis, which showed higher values of ΔH, activation energy for denaturation (E_a), and half-life t_(1/2). The enzymes were stable in the presence of ethanol and were competitively inhibited by glucose. These characteristics contribute to their use in the simultaneous saccharification and fermentation of vegetable biomass.     

Distribution of P1 Type Nuclease in the Genus Penicillium

P₁ type nuclease, which hydrolyzes RNA and heat-denatured DNA completely into 5’-mononucleotides and also shows 3’-nucleotidase activity, was widely distributed among various species belonging to the genus Penicillium such as P. expansum, P. notatum, P. steckii and P. meleagrinum. P₁ type nucleases isolated from these strains were produced in a form of complex with malonogalactan when molds were grown on wheat bran. These enzymes showed similar characters in heat-stability (stable at 60°C), temperature optimum (60 to 70°C for RNA and heat denatured DNA, and 70°C for 3’-AMP) and sensitivity to EDTA. The enzymes from P. steckii and P. expansum were immunologically co-related to nuclease P₁.

In addition, many strains of Penicillium produced base-nonspecific RNases forming 3’-mononucleotides via 2’: 3 ’-cyclic nucleotides. These RNases showed similarity in heat-lability (completely inactivated at 60°C), temperature optimum (45 to 50°C), sensitivity to Zn²⁺ and Cu²⁺, and relative hydrolysis rate toward 2’: 3’-cyclic nucleotides (A?C>U?G).     

Effect of liposome-treated red blood cells in an anemic rat model

Context: Liposomes have been shown to improve human red blood cell (RBC) in vitro quality by minimizing membrane damage occurring during 42-d hypothermic storage. Small animal models are necessary to evaluate novel blood products and guide future clinical studies.

Objectives: The aim of this study was to assess the effect of liposome treatments on rat RBC hypothermic storage lesion (HSL) and to examine in vivo outcomes of transfusing liposome treated RBCs in a rat model.

Materials and methods: Unilamellar liposomes were synthesized which contained saturated (DPPC:CHOL, 7:3?mol%), unsaturated (DOPC:CHOL, 7:3?mol%), saturated charged (DPPC:CHOL:PS, 6:3:1?mol%), and unsaturated charged (DOPC:CHOL:PS, 6:3:1?mol%) phospholipids. After liposome treatment, rat RBC quality was assessed by percent hemolysis, deformability, aggregation, hematological indices, microvesiculation, and cholesterol/phospholipid concentrations. An anemic rat model of myocardial ischemia and reperfusion (I/R) was used to evaluate the outcomes of transfusing liposome-treated RBCs.

Results: All four liposome treatments resulted in significant decreases in hemolysis, with the most prominent effect seen with DOPC-liposomes (DOPC: 1.6?±?0.1% versus control: 3.1?±?0.2%, p?=?0.015). RBCs treated with uncharged liposomes had lower hemolysis compared with charged liposomes (3.4?±?0.2% versus 3.9?±?0.4%, p?=?0.010). The in vivo study showed no significant difference in the hemoglobin levels and infarct size (53.3?±?13.1% versus 45.3?±?8.4%, p?=?0.223) between liposome and control groups.

Discussion and conclusion: Liposome treatment improved in vitro quality of stored rat RBCs. However, the changes observed in vitro were not sufficient to improve the in vivo outcomes of myocardial I/R in anemic rats transfused with liposome-treated RBCs.     

Solubilities Studies of Basic Amino Acids

The solbilities of l-basic amino acids in the type of free, monohydro-chloride and dihydrochloride in water were determined, and the results were formulated as follows.

l-Arginine: log S = 0.9770+0.01345t (t is from 0°~70°C)

l-Histidine: log S = 0.3627+0.00905t (t is from 0°~70°C)

l-Arginine monohydrochloride: log S = 1.6532+0.01301t (t is from 0°~70°C)

l-Lysine monohydrochloride dihydrate: log S = 1.6990+0.01294t (t is from 0°~55°C)

l-Lysine monohydrochloride monohydrate: log S = 1.7404+0.01256t (t is from 55°~70°C)

l-Lysine dihydrochloride: log S = 2.2138+0.00409t (t is from 0°~70°C)

l-Histidine dihydrochloride: log S = 1.9085+0.00265t (t is from 0°~70°C)

Three component systems (basic amino acid, hydrochloric acid, water) were studied and the solubilities in mixed solution system of alcohol-water were also investigated.     

Investigations on feasibility of in situ development of amphotericin B liposomes for industrial applications

Amphotericin B (AmB) liposome formulations are very successful in the treatment of fungal infections and leishmaniasis. But higher cost limits its widespread use among people in developing countries. Therefore, we have developed a modified ethanol-injection method for the preparation of AmB liposomes. Two liposomal formulations were developed with dimyristoyl phosphatidylcholine [F-1a] and soya phosphatidylcholine [F-2a], along with egg phosphatidyl glycerol and cholesterol. AmB was dissolved in acidified dimethyl acetamide and mixed with ethanolic lipid solution and rapidly injected in 5% dextrose to prepare liposomes. Liposomes were characterized on the basis of size (~100?nm), zeta (–43.3?±?2.8 mV) and percent entrapment efficiency (>95%). The in vitro release study showed an insignificant difference (P?≥?0.05) for 24-hour release between marketed AmB liposomes (AmBisome) and F-1a and F-2a. Proliposome concentrate, used for the preparation of in situ liposomes, was physically stable for more than 3 months at experimental conditions. Similarly, AmB showed no sign of degradation in reconstituted liposomes stored at 2–8°C for more than 3 months. IC₅₀ value of Ambisome (0.18 µg/mL) was comparatively similar to F-1a (0.17 µg/mL) and F-2a (0.16 µg/mL) against intramacrophagic amastigotes. Under experimental conditions, a novel modified method for AmB liposomes is a great success and generates interest for development as a platform technology for many therapeutic drug products.     

Association of β-amyloid peptide fragments with neuronal nitric oxide synthase: Implications in the etiology of Alzheimers disease

Neuronal nitric oxide synthase (nNOS) was purified on DEAE-Sepharose anion-exchange in a 38% yield, with 3-fold recovery and specific activity of 5 µmol.min^?1.mg^?1. The enzyme was a heterogeneous dimer of molecular mass 225?kDa having a temperature and pH optima of 40°C and 6.5, K_m and V_max of 2.6 μM and 996 nmol.min^?1.ml^?1, respectively and was relatively stable at the optimum conditions (t_½?=?3?h). β-Amyloid peptide fragments Aβ_17–28 was the better inhibitor for nNOS (K_i?=?0.81 µM). After extended incubation of nNOS (96?h) with each of the peptide fragments, Congo Red, turbidity and thioflavin-T assays detected the presence of soluble and insoluble fibrils that had formed at a rate of 5?nM.min^?1. A hydrophobic fragment Aβ_17–21 [Leu₁₇ – Val₁₈ – Phe₁₉ – Phe₂₀ – Ala₂₁] and glycine zipper motifs within the peptide fragment Aβ_17–35 were critical in binding and in fibrillogenesis confirming that nNOS was amyloidogenic catalyst.     

Kinetics of Thermal Inactivation and Molecular Asymmetry of Urease from Dehusked Pigeongea (Cajanus cajan L.) Seeds

The purified urease from pigeonpea was moderately stable at ?10°C. The shelf-life of the enzyme on storage in 0.1 M Tris-acetate buffer, pH 6.5, at ?10°C showed a single exponential decay with a t_1/2 of approx. 30 days. In the presence of additives like 5mM dithiothreitol the t_1/2 increased to 223 days at the same temperature, in a single exponential decay. The Arrhenius plot of the kinetics of the pigeonpea urease catalysed urea hydrolysis over the temperature range 27 to 77°C, was linear. The Q₁₀ value was found to be 1.46. The energy of activation calculated from the Arrhenius equation was found to be 5.1 kcal/mole active site. The thermal denaturation of pigeopea urease at 65 and 70°C was found to obey biphasic kinetics in which half of the activity is destroyed faster than the remaining half. The time course of thermal inactivation can be described by the following equation, consisting of two first order terms: A_t = A_fast.e^-k _fast + A_slow.e ^-kslow^.t where, A_t is the residual activity at time t, A_fast and A_slow, k_fast and k_slow are the amplitudes and the first-order rate constants of the fast and the slow phases, respectively. The data suggests the existence of site-site heterogeneity in oligomeric urease molecule from pigeonpea.     

Activation and stabilization of cardiac adenylate cyclase by GTP analog and fluoride.

The rate of cyclic AMP formation by rabbit heart membrane particles decreased at assay temperatures greater than 30 °C. Adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity (assayed at 24 °C) decreased exponentially with time of preincubation at 30 or 37 °C, providing evidence for the instability of this enzyme. The half-life, t_1/2, of the enzyme at 37 °C was 9.9 min in the absence and 4.4 min in the presence of MgCl₂. The activity was most labile in the presence of 50 m m Mg²⁺ and 1 m m ATP, having t_1/2 = 1.3min. Prior incubation of membranes with the GTP analog, guanyl-5′-yl imidodiphosphate [Gpp(NH)p], 0.1 m m, for 30 min at 37 °C produced maximal activation of adenylate cyclase; the rate of activation was temperature dependent and was increased in the presence of isoproterenol. The Gpp(NH)p-activated enzyme had increased thermal stability, t_1/2 = 170 min, and was also markedly more stable in the presence of Mg-ATP, t_1/2 = 72min, than nonactivated enzyme. Preactivation with F? (30 min at 24 °C) also stabilized the activity; t_1/2 > 70 min in the absence or presence of Mg-ATP. The Mg²⁺ concentration required for maximal activity was reduced from approximately 60 m m for nonactivated enzyme to 10 m m for the Gpp(NH)p- and F?activated enzyme.     

Molecular Dynamics Simulations of Phospholipid Bilayers

Abstract

Molecular dynamics (MD) simulations at 37°C have been performed on three phospholipid bilayer systems composed of the lipids DLPE, DOPE, and DOPC. The model used included 24 explicit lipid molecules and explicit waters of solvation in the polar head group regions, together with constant-pressure periodic boundary conditions in three dimensions. Using this model, a MD simulation samples part of an infinite planar lipid bilayer. The lipid dynamics and packing behavior were characterized. Furthermore, using the results of the simulations, a number of diverse properties including bilayer structural parameters, hydrocarbon chain order parameters, dihedral conformations, electron density profile, hydration per lipid, and water distribution along the bilayer normal were calculated. Many of these properties are available for the three lipid systems chosen, making them well suited for evaluating the model and protocols used in these simulations by direct comparisons with experimental data. The calculated MD behavior, chain disorder, and lipid packing parameter, i.e. the ratio of the effective areas of hydrocarbon tails and head group per lipid (a_t/a_h), correctly predict the aggregation preferences of the three lipids observed experimentally at 37°C, namely: a gel bilayer for DLPE, a hexagonal tube for DOPE, and a liquid crystalline bilayer for DOPC. In addition, the model and conditions used in the MD simulations led to good agreement of the calculated properties of the bilayers with available experimental results, demonstrating the reliability of the simulations. The effects of the cis unsaturation in the hydrocarbon chains of DOPE and DOPC, compared to the fully saturated one in DLPE, as well as the effects of the different polar head groups of PC and PE with the same unsaturated chains on the lipid packing and bilayer structure have been investigated. The results of these studies indicate the ability of MD methods to provide molecular-level insights into the structure and dynamics of lipid assemblies.     

Characterization of a thermostable Deconica castanella Laccase and application toward pentachlorophenol degradation

Abstract

Pentachlorophenol (PCP) is an organochlorine pesticide whose toxicity led it to be banned in several countries. In Brazil however, this compound is widely accessible, and its indiscriminate use leads to extensive soil contamination, which requires henceforth, the development of new approaches to manage PCP presence in environment. Considering that PCP is susceptible to undergo enzymatic degradation, this investigation is thence, aimed at the purification, and characterization, of a thermostable fungal enzyme (i.e. Laccase of Deconica castanella (Dc-Lac), and assess its role in PCP in vitro biodegradation. Results evidenced that, molecular mass of the partially purified Dc-Lac was estimated to be of 64?kDa, presenting apparent K_m of 0.47?µmol, and V_max of 11.56?U mg^?1. The optimum temperature and pH were 55?°C and 2.5, respectively. The T½ verified at 55, 60, and 80?°C were 19 and 17?hr, and 47?min, respectively. The highest PCP biodegradation was of 23% at pollutant concentration of 100?µg L^?1, which evidenced that Dc-Lac is a thermostable enzyme that acted directly in PCP degradation and may be a useful asset to remediate this pollutant.     

Mitochondrial response in the apical and lateral flower buds of the Hanfu apple to cold stress during the dormancy stage

In order to study the different physiological bases of cold tolerance in the apical flower buds (AFB) and the lateral flower buds (LFB) of the Hanfu apple (Malus domestica Borkh), we used 4-year-old grafted Hanfu plants as material and evaluated the physiological characteristics of mitochondria in the flower buds, such as electron transport chains (cytochrome pathway and alternative pathway), H₂O₂ content, mitochondrial membrane permeability transition (mPT), and MDA content. AFBs and LFBs showed different changes in total respiratory rate (V_t) during low-temperature stress, except that both reached the lowest V_ts at ?30 °C. The AFB V_t increased to a peak at ?25 °C and decreased sharply to its minimal value at ?30 °C, and then remained relatively low. In contrast, the LFB V_t decreased to its minimal value at ?30 °C and increased sharply to a peak at ?35 °C and then decreased again. In both AFBs and LFBs, the cytochrome pathway was still the main electron transport chain throughout the whole process, and the contributions of the cytochrome pathway (ρV_cyt/V_t) and of the alternative pathway (ρV_alt/V_t) showed similar tendencies to those of V_t as temperature changed. Changes in the AFB mPT were different from those of AFB V_t. LFB mPT zigzagged from peaks at ?25 °C and 35 °C. The H₂O₂ content of the LFBs increased from ?10 °C to ?30 °C, then decreased slightly from ?30 °C to ?35 °C, and then increased again. H₂O₂ content in AFBs went up steadily throughout the whole process. During the early stage of low-temperature treatment, before temperatures reached ?35 °C, LFB MDA content remained relatively low and later increased. MDA content in AFBs began to increase from the beginning of treatment. It can be concluded that the higher cold tolerance of LFBs relative to AFBs could be closely related to their higher V_t and ρV_alt/V_t, which may aid adaptations to stress by supplying energy and metabolic substrates under low-temperature stress conditions.     

A dilatometric investigation of the effects of general anaesthetics,alcohols and hydrostatic pressure on the phase transition in smectic mesophases of dipalmitoyl phosphatidylcholine

The effect of general anaesthetics, alcohols and hydrostatic pressure on the thermal transition in dipalmitoyl phosphatidylcholine multilayer liposomes has been measured using dilatometry. The volume increasse at the transition (ΔV_t) is 0.0350 ± 0.0003 ml/g. the transition temperature (T_t) 41.84 ± 0.09°C and the width of the transition 1.025 ± 0.18°C. ΔH calculated by the Clapeyron-Clausius equation is 8.4 kcal/mol. The n-alcohols C₃C₅ reduced the transition temperature without affecting the transition width which was however, increased by n-hexanol. Trichloroethylene, the fluorescent probe N-phenyl-1-naphthyl-amine, and methoxyflurane all increased the transition width (reduced the cooperativity of the transition) with a simultaneous depression of T_t. Methoxyflurane caused a two-stage transition expansion. Diethyl ether's effect has similarities with both the C₃ and C₆ alcohols. Generally ΔV_t was unaffected by the agents.Pressure increased T_t by 0.0238°C/atm linearly over the range 1–300 atm in both treated and untreated liposomes, and therefore cannot be said to antagonize anaesthetics. In both treated and untreated liposomes ΔV_t and the width of the transition were unaffected by pressure. Pressure thus reverses the effects of anaesthetics on T_t but not their spread of the transition width.     

A new kumamolisin-like protease from Alicyclobacillus acidocaldarius: an enzyme active under extreme acidic conditions

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45?kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH?2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t_1/2 at 80°C, 10?h, pH?2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions.     

Kinetic and microstructural characterization of immobilized penicillin acylase from Streptomyces lavendulae on Sepabeads EC-EP

Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier^?1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t_1/2=625 h vs. t_1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity.     

Supercooling ability and cold hardiness of the pollen beetle Meligethes aeneus

Supercooling point (SCP) and cold‐hardiness of the pollen beetle Meligethes aeneus (Fabricius) (Coleoptera: Nitidulidae) were investigated. Mature eggs from the oviduct were supercooled on average to ?28.0 °C and from oilseed rape buds to ?24.4 °C; first instars were supercooled to ?21.0 °C and second instars to ?16.8 °C. Despite their high supercooling ability, none of the eggs survived 24 h exposure to ?2.5 °C. The supercooling ability of adults varied significantly among feeding and non‐feeding beetles: high SCPs prevailed during the whole warm period, being about ?12 °C; low values of SCP of ?20 °C dominated in non‐feeding beetles. In spring and autumn, beetles displayed the same acclimation efficiency: after 1 week of exposure at 2.0 °C with no access to food their SCPs were depressed equally by about 3 °C. Meligethes aeneus beetles have a different response to low temperatures depending on the season. The lowest tolerance was found in reproductively active beetles after emergence from overwintering sites; the time needed to kill 50% of individuals (Ltime₅₀) was 56.2 h at ?7 °C and the lower lethal temperature needed to kill 50% (Ltemp₅₀) after 24 h exposure was ?8.6 °C. Cold hardiness increased from midsummer to midwinter; Ltime₅₀ was 80 h in August, 182.8 h in September, and 418.1 h in January. Lethal temperature after 24 h exposure was ?9.1 °C in August and ?9.8 °C in September. In February, after diapause, the beetles started to loose their cold tolerance, and Ltemp₅₀ was slightly increased to ?9.5 °C. Hibernating beetles tolerated long exposure at ?7 °C well, but mortality was high after short exposure if the temperature dropped below ?9 °C for 24 h. Despite the season, the beetles died at temperatures well above their mean SCP; consequently, SCP is not a suitable index for cold hardiness of M. aeneus.     

Effect of temperature on the life-history traits of <Emphasis Type="Italic">Neoseiulus californicus</Emphasis> (Acari: Phytoseiidae) fed on <Emphasis Type="Italic">Panonychus ulmi</Emphasis>

The developmental rate and reproductive biology of Neoseiulus californicus, a generalist predator on spider mites and small insects, was investigated in the laboratory at five constant temperatures: 15, 20, 25, 30, and 34°C. The European red mite, Panonychus ulmi, an important pest in Korean apple orchards, was used as prey. Mean developmental time and adult longevity were inversely related to temperature from 15 to 30°C. Lifetime fecundity was greatest at 25°C, whereas daily fecundity was highest at 30°C. The sex ratio (female to male) was highest (0.77) at 25°C and lowest (0.67) at 34°C. Survivorship during immature development varied from 74.3 to 92.9%, with the lowest rate at 34°C. Life table parameters were analyzed and pseudo-replicates for the generation time (t _G ), the intrinsic rate of natural increase (r _m), finite rate of increase (λ), net reproductive rate (R ₀), and doubling time (t _D ) were generated using the Jackknife method. Generation time (t _G ) was lowest (10.7 days) at 34°C, R ₀ was highest (49.2) at 25°C, and both r _m (0.29) and λ (1.34) were highest at 30°C. In conclusion, the development and adult life-history traits obtained for N. californicus fed on P. ulmi indicated significant potential for biological control.     

Physiological flexibility: the key to success and survival for Antarctic fairy shrimps in highly fluctuating extreme environments

1. The anostracan fairy shrimp Branchinecta gaini inhabits one of the most hostile environments on earth, living in pools and lakes in Antarctica. Between January 2002 and January 2003 temperatures in two pools where B. gaini are extremely abundant on Adelaide Island ranged from ?18.6 to ?15.7 °C in winter, to 19.4 to 17.1 °C in summer, whilst air temperatures ranged from ?34 to 6.3 °C. 2. Branchinecta gaini survives winter as cysts, but endures large summer temperature fluctuations as adults. Cysts froze between ?24.4 and ?25.7 °C. In experiments adults survived 0–10 °C with no mortality for 1 week, 25 °C for nearly 48 h with 50% mortality, and at 32 °C complete mortality occurred in <1 h. 3. Oxygen consumption (M?O₂) in B. gaini approximately doubled for every 10 °C temperature rise (Q₁₀ = 2.04) up to 20 °C where it reached a peak. Females had, on average 19% higher M?O₂ than males. Females also had greater metabolic scopes, (maximum–minimum M?O₂ across temperatures was ×3.6 for females, ×3.1 for males). 4. Ventilation frequency increased linearly with temperature, and did not decline at 25 °C, indicating animals were ‘trying’ progressively harder to supply oxygen to tissues, and oxygen deficiency was the probable cause of death. Females had a higher ventilation frequency than males (8.6–17.1% higher) and they also exhibited greater scope to raise ventilation frequency (×2.4 for females versus ×1.5 for males). 5. Great metabolic flexibility allows B. gaini to exploit extreme, highly fluctuating environments, and larger ventilatory and respiratory scopes allow females to survive higher temperatures than males. Because of this flexibility their prospects for coping with physical environmental change are high.     

Effects of decomposition and storage conditions on the δ13C and δ15N isotope values of killer whale (Orcinus orca) skin and blubber tissues

Several different factors in the collection and preservation of whale skin and blubber samples were examined to determine their effect on the results obtained by stable nitrogen and carbon isotope (δ¹⁵N and δ¹³C) analysis. Samples of wet killer whale skin retained their original stable isotope values for up to 14 d at 4°C or lower. However, decomposition significantly changed the δ¹⁵N value within 3 d at 20°C. Storage at ?20°C was as effective as ?80°C for the preservation of skin and blubber samples for stable isotope analysis for at least a year. By contrast, once a skin sample had been freeze‐dried and lipid extracted, the stable isotope values did not change significantly when it was stored dry at room temperature for at least 12 mo. Preservation of whale skin samples for a month in DMSO‐salt solution, frozen or at room temperature, did not significantly change the δ¹⁵N and δ¹³C values of lipid extracted tissues, although the slight changes seen could influence results of a study if only small changes are expected.