Effect of freezing and thawing on the biomechanical characteristics of porcine ocular tissues

PurposeTo evaluate the effect of freezing and thawing on the biomechanical properties of ex-vivo porcine ocular tissue.MethodsThirty-six porcine eyes (18 pairs) were obtained fresh from a local abattoir and split into two groups of nine pairs to study the effect of storage at −20 °C and −80 °C. A randomly-selected eye from each pair (Control Group, CG) was tested fresh while the fellow eyes were frozen for 14 days, either at −20 °C and −80 °C (Frozen Group, FG) before thawing and testing. Seventy-two strips were extracted from the corneas and scleras of eye globes and subjected to uniaxial tension tests under loads up to 1.0 N. Following five preconditioning cycles, the load and elongation data obtained experimentally were analysed to derive the tissue’s stress-strain and tangent modulus-strain behaviour.ResultsCorneal tissue subjected to freezing at −20 °C exhibited significant increases in tangent modulus (mechanical stiffness) by 13 ± 17% (p = 0.003) at 1% strain and 14 ± 12% (p < 0.001) at 2% strain. In contrast, the increases in corneal stiffness at −80 °C were insignificant (6 ± 14%, p = 0.099 at 1% strain, 6 ± 15%, p = 0.091 at 2% strain). The corresponding increases in tangent modulus in the sclera were all insignificant (for −20 °C: 4 ± 14%, p = 0.265 at 1% strain, 3 ± 9%, p = 0.186 at 2% strain; for −80 °C: 3 ± 18%, p = 0.537 at 1% strain and 3 ± 18%, p = 0.491 at 2% strain).ConclusionsThe study provided evidence that freezing and thawing led to insignificant changes in ocular tissue stiffness except in corneal tissue that was frozen at −20 °C.     

Effects of freeze-thaw on the biomechanical and structural properties of the rat Achilles tendon

Rodent models are commonly used to investigate tendon healing, with the biomechanical and structural properties of the healed tendons being important outcome measures. Tendon storage for later testing becomes necessary when performing large experiments with multiple time-points. However, it is unclear whether freezing rodent tendons affects their material properties. Thus the aim of this study was to determine whether freezing rat Achilles tendons affects their biomechanical or structural properties. Tendons were frozen at either −20 °C or −80 °C directly after harvesting, or tested when freshly harvested. Groups of tendons were subjected to several freeze-thaw cycles (1, 2, and 5) within 3 months, or frozen for 9 months, after which the tendons were subjected to biomechanical testing. Additionally, fresh and thawed tendons were compared morphologically, histologically and by transmission electron microscopy. No major differences in biomechanical properties were found between fresh tendons and those frozen once or twice at −20 °C or −80 °C. However, deterioration of tendon properties was found for 5-cycle groups and both long-term freezing groups; after 9 months of freezing at −80 °C the tear resistance of the tendon was reduced from 125.4 ± 16.4N to 74.3 ± 18.4N (p = 0.0132). Moreover, tendons stored under these conditions showed major disruption of collagen fibrils when examined by transmission electron microscopy. When examined histologically, fresh samples exhibited the best cellularity and proteoglycan content of the enthesis. These properties were preserved better after freezing at −80 °C than after freezing at −20 °C, which resulted in markedly smaller chondrocytes and less proteoglycan content. Overall, the best preservation of histological integrity was seen with tendons frozen once at −80 °C. In conclusion, rat Achilles tendons can be frozen once or twice for short periods of time (up to 3 months) at −20 °C or −80 °C for later testing. However, freezing for 9 months at either −20 °C or −80 °C leads to deterioration of certain parameters.     

Brown Spot of Rice is Affected by Photon Irradiance and Temperature

Brown spot, caused by Bipolaris oryzae, is one of the most destructive diseases in rice. In this study, the effect of photon irradiance and temperature on brown spot development was evaluated. The concentration of total soluble sugars (fructose, glucose and sucrose) in rice leaves was also evaluated. Rice plants of cv. ‘Oochikara’ were inoculated with B. oryzae and kept in a greenhouse [20 ± 2°C (night time) and 35 ± 2°C (day time), ≈ 1000 μmol photons/m²/s] or two different mist chambers (25 or 32 ± 2°C, ≈ 15 μmol photons/m²/s at the top canopy). Plants kept in a mist chamber at 32 ± 2°C, under low photon irradiance, showed reduced incubation period (IP) and increase in the rate of lesion expansion. Brown spot severity in rice leaves was 67.8% at 32 ± 2°C, 27.8% at 25 ± 2°C and 11.4% under greenhouse conditions. The highest brown spot severity was found on plants grown under low photon irradiance, in which soluble sugar concentrations were lowest, suggesting that disease development was boosted under these particular growing conditions. Based on the results of this study, a continuous high temperature and low photon irradiance, in the presence of high relative humidity, and low soluble sugars contribute to an increase in brown spot development.     

Giemsa-based cytological assessment of area,shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 μm, the shortest dimension averaged 14.4 ± 1.8 μm and the nucleus averaged 6.3 ± 0.8 μm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 μm² (average 193 ± 50 μm²). The area could be predicted reliably from the longest and shortest dimensions (r² = 0.903). The areas of goblet cell nuclei were 15–58 μm² (average 33 ± μm²) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.     

Effects of storage conditions on the stability and distribution of clinical trace elements in whole blood and plasma: Application of ICP-MS

BackgroundKnowledge of trace element stability during sample handling and preservation is a prerequisite to produce reliable test results in clinical trace element analysis.MethodAn alkaline dissolution method has been developed using inductively coupled plasma mass spectrometry to quantify eighteen trace element concentrations: vanadium, chromium, manganese, cobalt, nickel, copper, zinc, arsenic, selenium, bromine, molybdenum, cadmium, antimony, iodine, mercury, thallium, lead, and bismuth in human blood, using a small sample volume of 0.1 mL. The study evaluated the comparative effects of storage conditions on the stability of nutritionally essential and non-essential elements in human blood and plasma samples stored at three different temperatures (4 °C, −20 °C and −80 °C) over a one-year period, and analysed at multiple time points. The distribution of these elements between whole blood and plasma and their distribution relationships are illustrated using blood samples from 66 adult donors in Queensland.ResultsThe refrigeration and freezing of blood and plasma specimens proved to be suitable storage conditions for many of the trace elements for periods up to six months, with essentially unchanged concentrations. Substantially consistent recoveries were obtained by preserving specimens at −20 °C for up to one year. Ultra-freezing of the specimens at −80 °C did not improve stability; but appeared to result in adsorption and/or precipitation of some elements, accompanied by a longer sample thawing time. A population sample study revealed significant differences between the blood and plasma concentrations of six essential elements and their relationships also varied significantly for different elements.ConclusionBlood and plasma specimens can be reliably stored at 4 °C for six months or kept frozen at −20 °C up to one year to obtain high quality test results of trace elements.     

Effects of radiation,temperature and humidity on photosynthesis,transpiration and water use efficiency of oilseed rape (Brassica campestris L.)

The net photosynthetic rate (F), transpiration rate (Q) and water use efficiency (F/Q) of oilseed rape (Brassica campestris L. cv. Span) was studied under a range of atmospheric conditions by gas exchange techniques. The plants were at the full bloom/pod initiation stage of development at the time of measurement. The environmental conditions consisted of various levels of photosynthetically active radiation (100 to 2800 (μmol m^?2 s^?1 PAK: 400–700 nm), air temperature (10 to 42°C) and vapour pressure deficit (0.7 to 2.1 kPa VPD). The peak values ofF were recorded at 1600 μmol m^?2 s^?1 PAR, 20°C air temperature and 1.2 kPa VPD of air in the chamber. Q increased with increasing PAR, air temperature and VPD. However, theF/Q remained high and almost constant from 600 to 1600 μmol m^?2 s^?1 PAR, but declined at the low and high photon flux densities.F/Q decreased progressively with increase in air temperature and VPD of air in the chamber.     

Displacement and Return Movement of Chloroplasts in the Marine Dinophyte Pyrocystis noctiluca. Experiments with Optical Tweezers

Infrared laser traps (optical tweezers) were used to study laser-induced organelle movements in the marine alga Pyrocystis noctiluca (Dinophyta). These cells are highly suitable for optical micromanipulation due to their large size and extensive vacuole. Experiments were done with plastids held by optical tweezers and moved from the nuclear area into the vacuole. The subsequent retraction movement was analysed for speed. The displaced organelles remained connected to their original position by a thin cytoplasmic strand, often less than 1 μm in diameter. When the organelles were released they rapidly returned at an initial rate of 81.7 ± 7.8 μm . s^?1 (overall displacement 50 μm, measured distance 20 μm, 25 °C ± 1 °C, number of cells 22), slowing down with progressive retraction of the connecting strand. The return movement was reduced to 4.2 ± 0.2 μ .s^?1 (n = 10) when the organelles were displaced and held for 1 min. Displacement to a longer distance increased the rate of return movement. A change from a high to a low environmental temperature significantly reduced movement from 94.5 ± 9.0 . s^?1 (30 °C ± 1 °C, n = 22) to 34.5 ± 2.7 μm .s^?1 (5°C ± 1 °C, n = 22). Nocodazole and N-ethylmaleimide (NEM), inhibitors of microtubules and acto-myosin, respectively, did not affect the retraction of the connecting strand, but at high concentrations of NEM it became increasingly difficult to move organelles away from the nuclear area. We suggest that the return movement of organelles within laser-induced artificial strands mainly depends on the viscoelastic properties of the tonoplast. The quantification of these properties by optical tweezers allows determination of reactions of plant cells to temperature changes.     

High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.     

Cryopreservation of Bangladeshi ram semen using different diluents and manual freezing techniques

A study was conducted to establish a sustainable and effective manual freezing technique for cryopreservation of Bangladeshi ram semen. Three diluents and freezing techniques were tested, both as treatment combinations (diluent × freezing technique) and fixed effects (diluent or freezing technique) on post-thaw sperm motility (SM), viability (SV), plasma membrane integrity (SPMI) and acrosome integrity (SAI). Ten rams were selected, based on semen evaluation. Eight ejaculates were used for each treatment combination. Semen samples were diluted using a two-step protocol for home-made Tris-based egg yolk (20%, v/v) diluents: D1 (7% glycerol, v/v) and D2 (5% glycerol, v/v), and one-step for commercial diluent: D3 (Triladyl^®, consists of bi-distilled water, glycerol, tris, citric acid, fructose, spectinomycin, lincomycin, tylosin and gentamycin) at 35 °C. Fraction-A (without glycerol) was added at 35 °C, and following cooling of sample to 5 °C (−0.30 °C/min), Fraction-B (with glycerol) was added. The diluted semen samples were aspirated into 0.25 ml French straws, sealed, and equilibrated at 5 °C for 2 h. The straws were frozen in liquid nitrogen (LN) vapour, in a Styrofoam box. The freezing techniques were; One-step (F1): at −15.26 °C/min from +5 °C to −140 °C; Two-step (F2): at −11.33 °C/min from +5 °C to −80 °C, and −30 °C/min from −80 °C-140 °C; and Three-step (F3): at −11.33 °C/min from +5 °C to −80 °C, at −26.66 °C/min from to −80 °C to −120 °C, and at −13.33 °C/min from −120 °C to −140 °C. Two semen straws from each batch were evaluated before and after freezing. The group F3D3 exhibited significantly higher (p < 0.05) post-thaw SM 63.1 ± 2.5%, SV 79.0 ± 2.1% and SPMI 72.9 ± 1.7%, whereas SAI 72.9 ± 1.7% was significantly higher (p < 0.05) in group F3D2. The freezing technique F2 and F3 had significantly higher (p < 0.05) post-thaw sperm values compared to F1. The post-thaw SM and SV were above 50% and 65% with the freezing technique F2 and F3 but differed non-significant. The SPMI 67.6 ± 2.0% and SAI 76.1 ± 1.4% were significantly higher (p < 0.05) with F3. Likewise, the diluent D2 and D3 had significantly higher (p < 0.05) post-thaw sperm values compared to D1. The post-thaw SM, SV and SPMI were above 50%, 65% and 55% with the diluents D2 and D3 but differed non-significant. The SAI 76.1 ± 1.1% was significantly higher (p < 0.05) with D3. We concluded that the use of a simple home-made Tris-based diluent containing 20% (v/v) egg yolk and 5% glycerol (v/v), two-step dilution and a three-step freezing technique is a sustainable and effective method for freezing ram semen. For further validation, the fertility of ewes artificially inseminated with the frozen semen will be observed.     

The formation constants of dimethylthallium(III)-glutathione complexes in aqueous solution

The complexes formed in the dimethylthallium(III) (Me₂Tl⁺), glutathionate (EGC³⁻) and hydrogen ion system in aqueous solution at 37 °C and I = 150 mmol dm⁻³ (NaCl) have been characterised by means of glass-electrode potentiometry. Glutathione protonation constants were found to be 9.123 ± 0.007, 17.42 ± 0.01, 20.78 ± 0.02, and 22.93 ± 0.02. Formation constants for the complexes [(Me₂Tl)EGCH]⁻ and [(·Me₂Tl) EGC]²⁻ were found to be 11.19 ± 0.03 and 2.39 ± 0.02, respectively. Particular attention has been paid to the evaluation of the effect of possible systematic errors on the constant values determined. Reliable standard deviation estimations have been made by applying a Monte Carlo calculation technique.     

Survival of Heleomyza borealis (Diptera, Heleomyzidae) larvae down to − 60°C

Heleomyza borealis Boh. (Diptera, Heleomyzidae) overwinters as larvae in Arctic habitats, where they may experience winter temperatures below ? 15°C. The larvae freeze at c.? 7°C but in acclimation experiments 80% survived when exposed to ? 60°C. Of the larvae exposed to between ? 4 and ? 15°C, only 3% pupated. However, when cooled to ? 20°C this increased to 44%, with 4% emerging as adults. Larvae maintained at 5°C contained low levels of glycerol, sorbitol and trehalose, which did not increase with acclimation to low temperatures. However, levels of fructose increased from 6.1 μg mg^?1 fw in control animals to 17 μg mg^?1 fw when exposed to ? 2°C for 1 week. Larval body water (2.2 ± 0.1 g/g dw, mean ± SD, n = 100) and lipid content (0.22 ± 0.002 g/g fw, mean ± SE) showed no significant change during acclimation to low temperatures. Larvae maintained at a constant 5°C survived for over 18 months with little loss of body mass (from 7.5 ± 1.2 to 7.0 ± 1.2 mg fw, mean ± SD, n = 20), but none pupated. Heleomyza borealis larvae appear to feed and grow until they reach a body mass of about 7.5 mg and then become dormant. They remain in this state until they experience a low temperature stimulus (< ? 15°C) followed by a warm period (≈ 5°C). This ensures that the larvae pupate and adults emerge in early summer, allowing the maximum growing period before the following winter. Heleomyza borealis are adapted to survive long winters in a dormant larval state. They have a low metabolic rate, can conserve body water even at subzero temperatures but do not synthesize large quantities of cryoprotectants.     

THE UPTAKE OF BIOGENIC MONOAMINES BY THE ISOLATED AURICLE OF THE SNAIL (HELIX POMATIA)

—The uptake of [³H]5HT, [³H]dopamine, [³H]noradrenaline and [³H]octopamine into the auricle of Helix pomatia was studied. When tissues were incubated at 25°C in media containing radioactive amines, tissue:medium ratios of about 49:1, 14:1 and 5:1 for 5-HT, dopamine, noradrenaline, and octopamine respectively were obtained after a 20–30 min incubation time. Tissues incubated at 25°C in media containing radioactive amines for 20–30 mins showed that almost all (96%) the radioactivity was present as unchanged [³H]5-HT, [³H]dopamine, [³H]octopamine or [³H]noradrenaline. The high tissue:medium ratios for 5-HT and dopamine, but not for noradrenaline and octopamine, showed saturation kinetics which were dependent upon temperature and sodium ions. From the Lineweaver–Burk plots, two uptake mechanisms for 5-HT at 25°C were resolved; the high affinity uptake process having a K_m1 value of 6.0 ± 10^?8m and a V_m1 value of 0.115 nmol/g/min while the lower affinity process had a K_m2 value of 1.04 ± 10^?6m and a V_m2 value of 0.66nmol/g/min. At 0°C a single uptake mechanism for 5-HT occurred which gave a K_m value of 5.02 ± 10^?8m and a V_m value of 0.0165 nmol/g/min. In the case of dopamine, the Lineweaver–Burk plot at 25°C showed a single uptake process with values for K_m and V_m of 1.55 ± 10^?7m and 0.086 nmol/g/min respectively. This process did not function at 0°C. The effect of various agents and ions upon the accumulation processes for all amines was also studied, and the data indicate that the same neurons probably accumulate more than one amine type. It is concluded that 5-HT and dopamine uptake in the auricle is a mechanism for inactivating these substances at 25°C and that an uptake mechanism for 5-HT also functions at 0°C. The results are discussed from the point of view of 5-HT's being the cardioexcitatory substance in the snail heart.     

Production enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Halogeometricum borinquense,characterization of the bioplastic and desalination of the bioreactor effluent

Polyhydroxyalkanoates (PHAs) are a replacement of conventional single-use plastics. Bioprocess conditions of the extreme halophilic archaeon Halogeometricum borinquense strain RM-G1 were selected resulting in the synthesis of 66.80 ± 1.69 % PHA (of cell dry mass) in 72 h using glycerol and tryptone as carbon and nitrogen sources respectively, yielding volumetric productivity of 0.206 ± 0.006 gL⁻¹ h⁻¹ in a repeated batch process in a small-scale bioreactor where 20 % of the production medium was used as the inoculum for the subsequent batch. The purified PHA was characterized as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with 10.21 mol% 3-hydroxyvalerate content possessing glass transition temperature -12.6 °C, degradation temperature 285 °C, number average molecular weight 156,899 Da, weight average molecular weight 288,723 Da, polydispersity index 1.8 and melting temperatures 139.1 °C and 152.5 °C. Maximum (21.7 ± 0.6 L m^-2 h⁻¹) and average (17.2 ± 0.6 L m^-2 h⁻¹) flux values were their respective highest and crystallization time was its least (3.0 ± 0.16 h) when ΔT was 90 °C and polytetrafluoroethylene membrane was applied for desalination of the bioreactor effluent by Direct Contact Membrane Distillation. While using polyvinylidene fluoride membrane, maximum 25.5 ± 0.5 L m^-2 h⁻¹ and average 18.6 ± 0.2 L m^-2 h⁻¹ fluxes were obtained and crystallization time decreased (3.25 ± 0.16 h) even when ΔT was lowered by 20 °C.     

Variations in leaf respiration across different seasons for Mediterranean evergreen species

Leaf respiration (R _L) of evergreen species co-occurring in the Mediterranean maquis developing along the Latium coast was analyzed. The results on the whole showed that the considered evergreen species had the same R _L trend during the year, with the lowest rates [0.83 ± 0.43 μmol(CO₂) m^?2 s^?1, mean value of the considered species] in winter, in response to low air temperatures. Higher R _L were reached in spring [2.44 ± 1.00 μmol(CO₂) m^?2 s^?1, mean value] during the favorable period, and in summer [3.17 ± 0.89 μmol(CO₂) m^?2 s^?1] during drought. The results of the regression analysis showed that 42% of R _L variations depended on mean air temperature and 13% on total monthly rainfall. Among the considered species, C. incanus, was characterized by the highest R _L in drought [4.93 ± 0.27 μmol(CO₂) m^?2 s^?1], low leaf water potential at predawn (Ψ_pd= ?1.08 ± 0.18 MPa) and midday (Ψ_md = ?2.75 ± 0.11 MPa) and low relative water content at predawn (RWC_pd = 80.5 ± 3.4%) and midday (RWC_md = 67.1 ± 4.6%). Compared to C. incanus, the sclerophyllous species (Q. ilex, P. latifolia, P. lentiscus, A. unedo) and the liana (S. aspera), had lower R _L [2.72 ± 0.66 μmol(CO₂) m^?2 s^?1, mean value of the considered species], higher RWC_pd (91.8 ± 1.8%), RWC_md (82.4 ± 3.2%), Ψ_pd (?0.65 ± 0.28 MPa) and Ψ_md (?2.85 ± 1.20 MPa) in drought. The narrow-leaved species (E. multiflora, R. officinalis, and E. arborea) were in the middle. The coefficients, proportional to the respiration increase for each 10°C rise (Q₁₀), ranging from 1.49 (E. arborea) to 1.98 (A. unedo) were indicative of the different sensitivities of the considered species to air temperature variation.     

Estimation of intracellular pH in muscle of fishes from different thermal environments

A technique based on homogenisation of rapidly frozen tissue was used to investigate the regulation of intracellular pH (pH_i) in freshwater and marine fish from diverse environmental temperatures. The following species were held at ambient temperatures of ca. 1°C (Notothenia coriiceps; Antarctica), 5°C (Pleuronectes platessa, Myoxocephalus scorpius; North Sea), and 26°C (Oreochromis niloticus; African lakes). The effects of seasonal acclimatisation to 4, 11 and 18°C were also examined in rainbow trout in the winter, autumn and summer, respectively. Extracellular (whole blood) pH (pH_e) did not follow the constant relative alkalinity relationship, where pH⁺=pOH⁻ for any particular temperature, over a range of 1–26°C (overall δpH_e/δT=0.009±0.002 U °C⁻¹; P<0.001), apparently being regulated by ionic fluxes and ventilation. Intracellular pH (pH_i) was also regulated independently of pN(=0.5 pK water) in all species of fish examined. The inverse relationship between pH_i and environmental temperature gave an overall δpH_i/δT of −0.010±0.001 U °C⁻¹ (for both white and red muscle) and −0.004±0.003 U °C⁻¹ (cardiac muscle). However, between 1 and 11°C δpH_i/δT was much higher (P<0.001), −0.022±0.003 U °C⁻¹ (white muscle) and −0.022±0.004 U °C⁻¹ (red muscle). The possible adaptive roles for these different acid–base responses to environmental temperature variation among tissues and species, and the potential difficulties of estimating pH_i, are discussed.     

Identification, Development, and Regional Distribution of Ribonucleotide Reductase in Adult Rat Brain

The development and regional distribution of ribonucleotide reductase (EC 1.17.4.1) were determined in rat brain. Ribonucleotide reductase was partially purified by ammonium sulfate fractionation (20-40% saturation). Enzyme activity was measured by a specific radiochemical assay. This method involved the reduction of [¹⁴C]cytidine diphosphate (CDP) to [¹⁴C]deoxy-cytidine diphosphate with subsequent hydrolysis and separation of the product ([¹⁴C]deoxycytidine) from substrate ([¹⁴C]cytidine) by Dowex-1-borate ion-exchange chro-matography. The specific activity of ribonucleotide reductase in whole brain of newborn rats was 3.78 ± 0.55 units (pmol/h)/mg protein (SEM; n = 6) and declined to 0.17 ± 0.01 units/mg protein (n = 7) at 10-12 weeks of age, with a further decline to 0.11 ± 0.01 units/mg protein (n = 3) at 1 year. Ribonucleotide reductase activity in rat liver decreased from 4.58 ± 0.62 units/mg protein (n = 3) in newborn animals to 0.06 ± 0.01 units/mg protein (n = 7) at 10-12 weeks and was present at trace levels at 6 months of age. The decline in specific activity with age was not due to a change in the K_m for CDP. The K_m for CDP in brain of newborn and adult rats was 80-90 μM. In 10- to 12-week-old rats, the specific activity of ribonucleotide reductase was similar in the various regions of the brain tested except for the brainstem, which had 50% lower specific activity than the whole brain. These results indicate that ribonucleotide reductase activity is present and widely distributed in adult rat brain.     

High-performance liquid chromatography with electrochemical detection for the determination of 7-monohydroxyethylrutoside in plasma

MonoHER (7-monohydroxyethyl rutoside) is a semisynthetic flavonoid, which can be used as a modulator for doxorubicin-induced cardiotoxicity. To study the pharmacokinetics of monoHER in mice and human an HPLC procedure was developed to measure the level of monoHER in plasma. After extraction of monoHER with methanol, the supernatant was equally diluted (v/v) with 25 mM phosphate buffer (pH 3.33). This solution was analysed by HPLC, using a reversed-phase ODS column, with a mobile phase consisting of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogen phosphate (pH 3.4), 10 mM acetic acid and 36μM EDTA. The retention time of monoHER was about 5.2 min. The lower limit of quantification of monoHER was set at 0.3 μM and the calibration line was linear up to 75 μM. The within-day accuracy and precision of the quality control samples (0.45, 1.0, 10 and 40 μM) were better than 15 and 13%, respectively. The between-day accuracy and precision were less than 3, 20%, respectively. The recovery of monoHER (using quality control concentrations) was concentration independent and ranged from 90.5 to 95.3% except for the lowest quality control, 0.45 μM, of which the recovery was 85%. The concentration of monoHER in plasma decreased with 10% when stored at −80°C for one month and with 20% when stored at −20°C for 3 weeks. The repeated injection of monoHER in aliquots of 10 μM, stored in the autosampler tray (4°C), showed a consistent decrease during a run: 15% over 24 h. To compensate for this decrease, sample duplicates were analysed in a mirror image sequence.     

The effect of acute high light and low temperature stresses on the ascorbate–glutathione cycle and superoxide dismutase activity in two Dunaliella salina strains

Dunaliella species accumulate carotenoids and their role in protection against photooxidative stress has been investigated extensively. By contrast, the role of other antioxidants in this alga, has received less attention. Therefore, the components of the ascorbate–glutathione cycle, along with superoxide dismutase (E.C. 1.15.1.1) and peroxidase (E.C. 1.11.1.11) activity were compared in two strains of Dunaliella salina. Strain IR‐1 had two‐fold higher chlorophyll and β‐carotene concentration than Gh‐U. IR‐1 had around four‐fold higher superoxide dismutase, ascorbate peroxidase and pyrogallol peroxidase activities than Gh‐U on a protein basis. Ascorbate and glutathione concentrations and redox state did not differ between strains and there was little difference in the activity of ascorbate–glutathione cycle enzymes (monodehydroascorbate reductase [E.C. 1.6.5.4], dehydroascorbate reductase [E.C. 1.8.5.1] and glutathione reductase [E.C. 1.8.1.7]). The response of these antioxidants to high light and low temperature was assessed by transferring cells from normal growth conditions (28°C, photon flux density of 100 μmol m^?2 s^?1)to 28°C/1200 μmol m^?2 s^?1; 13°C/100 μmol m^?2 s^?1; 13°C/1200 μmol m^?2 s^?1 and 28°C/100 μmol m^?2 s^?1 for 24 h. Low temperature and combined high light‐low temperature decreased chlorophyll and β‐carotene in both strains indicating that these treatments cause photooxidative stress. High light, low temperature and combined high light‐low temperature treatments increased the total ascorbate pool by 10–50% and the total glutathione pool by 20–100% with no consistent effect on their redox state. Activities of ascorbate–glutathione cycle enzymes were not greatly affected but all the treatments increased superoxide dismutase activity. It is concluded that D. salina can partially adjust to photooxidative conditions by increasing superoxide dismutase activity, ascorbate and glutathione.     

Determination of monohydroxyethylrutoside in heart tissue by high-performance liquid chromatography with electrochemical detection

7-Monohydroxyethylrutoside (monoHER) is one of the components of the registered drug Venoruton. It showed a good protection against the cardiotoxic effects of doxorubicin. The analysis of monoHER was developed to study the pharmacokinetic profile of the drug in heart tissue. MonoHER was extracted from heart tissue homogenate with methanol. The supernatant was diluted 1:1 (v/v) with 25 mM phosphate buffer and injected onto a reversed-phase ODS column. The mobile phase consisted of 49% methanol and 51% of an aqueous solution containing 10 mM sodium dihydrogenphosphate (pH 3.4), 10 mM acetic acid and 36 μM EDTA. The retention time of monoHER was about 5.2 min and no endogenous peaks were interfering. The lower limit of quantification was 0.072 nmol g⁻¹ wet heart tissue. The calibration line was linear up to 24 nmol g⁻¹. The within-day accuracy and precision of the quality controls (0.12, 1.2 and 12.0 nmol g⁻¹) were smaller than 17 and 19%, respectively. The between-day accuracy and precision were better than 6 and 11%, respectively. The recovery of monoHER from heart tissue ranged from 104.1 to 114.3% and was concentration independent. MonoHER was stable in heart tissue when stored at −80°C for 6 months. Repeated injection of monoHER from aliquots of 7.2 nmol g⁻¹ placed on the sample tray at 4°C for 24 h showed a decrease in the concentration of 30.3%. Analyzing sample duplicates in a mirror image sequence could compensate for the influence of this gradual decrease. The small sample volume allowed one to measure monoHER in the hearts of mice.