Vaccination of Atlantic salmon with recombinant VP2 of infectious pancreatic necrosis virus (IPNV), added to a multivalent vaccine, suppresses viral replication following IPNV challenge

Atlantic salmon (Salmo salar) pre-smolts vaccinated with Norvax^®Protect (NP) or injected with saline, were demonstrated to have a significantly (P≤0·0001) higher probability of being IPNV-infected when sampled during 12 weeks post challenge (PC) with IPNV (11·7 times and 32·5 times higher, respectively), than fish vaccinated with Norvax^®Protect-IPN (NP-IPN), a vaccine identical to NP except for the presence of recombinant VP2. Furthermore, following experimental challenge with IPNV, an IPNV-specific secondary humoral immune response was detected in the group vaccinated with NP-IPN. Statistical analysis revealed that NP-IPN-vaccinated fish had a significantly (P≤0·0001) higher probability of producing IPNV-specific antibodies during 12 weeks PC with IPNV, compared to NP-vaccinated or saline-injected fish (5·3 times and 7·6 times higher, respectively). The results support efficacy data from field trials in Norway, where NP-IPN has proven successful in the prevention of IPN in Atlantic salmon post-smolts. However, the immunological mechanisms behind the increased IPNV clearance remain unknown.     

Mould growth on building materials under low water activities. Influence of humidity and temperature on fungal growth and secondary metabolism

The influence of relative humidity (RH) and temperature on growth and metabolism of eight microfungi on 21 different types of building material was investigated. The fungi were applied as a dry mixture to the materials, which were incubated at 5°C, 10°C, 20°C and 25°C at three humidity levels in the range 69–95% RH over 4–7 months. The lower limit for fungal growth on wood, wood composites and starch-containing materials was 78% RH at 20–25°C and increased to 90% RH at 5°C. An RH of 86% was necessary for growth on gypsum board. Ceramic materials supported growth at RH >90%, although 95% RH was needed to yield chemically detectable quantities of biomass. Almost exclusively only Penicillium, Aspergillus and Eurotium (contaminant) species grew on the materials. Production of secondary metabolites and mycotoxins decreased with humidity and the quantities of metabolites were insignificant compared with those produced at high RH (RH >95%), except in the case of Eurotium.     

Biological,immunological and biochemical characterization of cleaved prolactin generated by lactating mammary gland

We have previously shown that rat prolactin is proteolytically cleaved in its loop by peripheral tissues of the rat. Of the tissues examined to date, lactating mammary gland exhibits the highest prolactin-cleaving activity. The objective of this study was to characterize cleaved prolactin, biologically, immunologically and chemically. By modifying an established analytical method, we were able to generate large (μg) amounts of cleaved rat prolactin from cell fractions of rat mammary gland which could then be assayed for biological and immunological activity relative to intact hormone. The cleaved product showed no significant difference relative to the intact rat prolactin when assayed for its ability to compete with ¹²⁵I-labelled ovine prolactin for the prolactin receptor and for its ability to stimulate the proliferation of rat Nb2 lymphoma cells. Cleaved rat prolactin, however, did show a 50–60% reduction in activity relative to intact rat prolactin when assayed by radioimmunoassay. Using Edman degradation and partial amino acid analysis, we determined that the second N-terminus of the cleaved rat prolactin begins at amino acid 149. The divergence of biological and immunological activity produced by proteolytic cleavage in the loop of rat prolactin suggests that biological and immunological sites differ in location. The possible physiological implications of a cleaved rat prolactin molecule generated by target tissue with maintained biological activity and reduced immunological activity are discussed.     

Sulphate-reducing bacteria which do not induce accelerated corrosion

The corrosive activity of the recently isolated SRB genera has not previously been reported in the literature. In this investigation, three genera of sulphate-reducing bacteria (SRB). Desulfovibrio vulgaris, Desulfobacter postgatei and Desulfobulbus propionicus were tested for their ability to induce accelerated corrosion of mild steel in laboratory growth media. Desulfovibrio vulgaris, well-recognised for its corrosive activity, caused a 78·2% increase in weight loss compared to the control, uninoculated medium (95% confidence limits +37·0% to +130·6%). Desulfobacter postgatei and Desulfobulbus propionicus had no significant effect on corrosion. For Desulfobacter postgatei the mean rate of corrosion was 10·6% more than the control (95% confidence limits −12·0% to +39·0%). For Desulfobulbus propionicus the mean corrosion rate was increased by 5·8% over control (95% confidence limits −9·8% to +24·2%).     

Differential effect of chromium compounds on the immune response of the African mouth breeder Oreochromis mossambicus (Peters)

The objective of this study was to investigate the effect of trivalent (chromic chloride) and hexavalent (potassium dichromate) forms of chromium in the African mouth breeder Oreochromis mossambicus (Peters), with reference to the humoral immune response and lymphoid cells/organs. The 96 h LD₅₀for hexavalent and trivalent chromium was found to be 75 and 1000 μg fish⁻¹, respectively. Groups of fishes were injected intraperitoneally with 10, 1, 0·1 and 0·01% LD₅₀hexavalent and trivalent forms of chromium and subsequently immunised with bovine serum albumin (5 mg in 0·2 ml physiological saline). Both forms of chromium suppressed the antibody response, with hexavalent chromium being more suppressive than trivalent chromium. Reduction in spleen weight, splenocyte number and the percentage of blood lymphocytes was observed following administration of both forms of chromium. The possible immunological mechanisms behind the differential suppression of the antibody response and the reduction in spleen weight, splenocyte and lymphocyte counts are discussed.     

In vitro studies of the effect of Environmental Conditions on the anthacnose pathogen of bananas, Colletotrichum musae

In vitro studies were undertaken to determine the effect of pH, temperature, water availability and carbon dioxide (CO₂) concentration on germination and growth of Colletotrichum musae, the causal pathogen of anthracnose of bananas. The optimum pH for germination and growth varied between 4·0 and 5·0 depending on temperature. At low pH (< 3·0) and 15°C, both germination and growth were significantly reduced, with a marked increase in the lag time, in days, prior to growth. C. musae germinated and grew over a wide range of water activities (a_w; 0·995−0·94 and 0·995−0·92, respectively) at 20, 25 and 30°C. In all cases where germination occurred appresoria were subsequently produced. Optimum growth occurred at 30°C and 0·995 a_w, although this changed to 0·98 a_w at 35°C. Increasing CO₂ concentration to 15% or reducing oxygen concentration to 1% resulted in a significant (P < 0·05) reduction in growth, but did not inhibit growth completely.     

Growth and protein synthesis of barramundi, Lates calcarifer, fed lupin as a partial protein replacement

Protein synthesis is an essential growth process in all animals. Little information is available on post-prandial protein synthesis and even less where different protein sources are compared. Protein synthesis was measured at 4 and 24 h after feeding juvenile barramundi in order to determine the effect of using lupin as a partial protein replacement for fish meal on the post-prandial protein metabolism. Juvenile barramundi (4.3 ±0.6 g) were held in a recirculation system (27 °C, salinity 10‰ and 24 h light) for 15 days. Fish were fed one of two isonitrogenous isoenergetic diets (40% crude protein, 16% lipid and 18.5 GE MJ kg^− 1). One diet was formulated with 100% fish meal as the protein source while the other had 45% of the protein replaced with lupin ingredients (lupin kernel meal (Lupinus angustifolius) and lupin protein concentrate). All fish were fed a ration of 6%·d^− 1 and feed intake was not significantly different between the two diets. Specific growth rate (SGR) and growth efficiency (in relation to protein (PPV) and energy (PEV)) were 6.5 ± 0.14%·d^− 1, 43.8 ± 2.72% and 38.31 ± 1.56%, respectively, and were not significantly different between the two diets. There was no significant difference in protein synthesis between the two diets at 4 and 24 h after feeding, however protein synthesis was significantly higher 4 h after feeding than at 24 h (p = 0.02). Neither growth performance nor protein metabolism was altered by replacing 45% of the protein with lupin protein and indicated this to be a suitable protein source for barramundi feeds.     

The Determination of Phosphorus in Haemophilus influenzae Type b Conjugate Vaccines by Inductively Coupled Plasma-Atomic Emission Spectrometry

This study describes a method for the determination of phosphorus in lyophilized Haemophilus influenzae type b conjugate vaccines by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The concentration of polysaccharide is directly related to the concentration of phosphorus as measured in the laboratory. Phosphorus is present in the polyribosyl-ribitol phosphate (PRP) group of the Haemophilus influenzae type b conjugate vaccine. The repeating unit of PRP is 3-B-D ribose[1-1]ribitol-5-phosphate. Phosphorus in the final container is measured in μ g per dose. The amount of PRP is calculated from this and reported in μ g per dose. The Haemophilus influenzae type b conjugate vaccine was analyzed for phosphorus content within the range of 1·34 to 2·02 μg phosphorus per ml. The relative difference of phosphorus concentrations determined by the ICP-AES method from the phosphorus concentrations determined by the traditional colorimetric molybdate method ranged from 2·2 to 10·6%. Phosphorus spike recovery for the vaccine ranged from 93 to 99% (1·93±0·13 μ g P/ml). The phosphorus determination of NIST SRM 3139 phosphorus spectrometric solution differed by 3·0% from the certified phosphorus value (10·00 mg P/ml).     

Germination responses of Discopodium penninervium Hochst. to temperature and light

The germination responses of Discopodium penninervium were tested at different constant and alternating temperature regimes as well as under various light conditions both in the laboratory and glasshouse. Seeds incubated at 10, 15, 20, 25 and 30 °C failed to germinate. When the seeds were incubated at alternating temperatures of 20/12 °C and 30/12 °C under continuous light, germination was 89 and 61%, indicating that the species requires alternating temperatures as a cue for germination. However, germination declined as the amplitude of alternating temperatures increased from 8 °C and was completely inhibited at an amplitude of 23 °C, suggesting that the optimum amplitude is around 8 °C. Germination was less than 10% in light and nil in darkness at 20 °C in the laboratory. In contrast, seeds incubated at 20/12 °C germinated to 96 and 86% in light and darkness, respectively. Seeds incubated under leaf shade in the glasshouse failed to germinate whereas those incubated under direct daylight and darkness germinated to 44 and 50%, respectively, 30 days after sowing. When seeds incubated under leaf shade and in darkness were exposed afterwards to light, final percent germination was 83% from seeds incubated initially under direct daylight, 79% from those incubated under leaf shade and 86% from those incubated in darkness. The requirement for alternating temperatures and light rich in red:far red ratio to break the dormancy of seeds of D. penninervium could restrict germination to gaps in the vegetation. The results conform with the ecology of the species.     

Seed germination and dormancy in the medicinal woodland herbs Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae)

We used a double germination phenology or “move-along” experiment (sensu Baskin and Baskin, 2003) to characterize seed dormancy in two medicinal woodland herbs, Collinsonia canadensis L. (Lamiaceae) and Dioscorea villosa L. (Dioscoreaceae). Imbibed seeds of both species were moved through the following two sequences of simulated thermoperiods: (a) 30/15 °C→20/10 °C→15/6 °C→5 °C→15/6 °C→20/10 °C→30/15 °C, and (b) 5 °C→15/6 °C→20/10 °C→30/15 °C→20/10 °C→15/6 °C→5 °C. In each sequence, seeds of both species germinated to high rates (>85%) at cool temperatures (15/6 and 20/10 °C) only if seeds were previously exposed to cold temperatures (5 °C). Seeds kept at four control thermoperiods (5, 15/6, 20/10, 30/15 °C) for 30 d showed little or no germination. Seeds of both species, therefore, have physiological dormancy that is broken by 12 weeks of cold (5 °C) stratification. Morphological studies indicated that embryos of C. canadensis have “investing” embryos at maturity (morphological dormancy absent), whereas embryos of D. villosa are undeveloped at maturity (morphological dormancy present). Because warm temperatures are required for embryo growth and cold stratification breaks physiological dormancy, D. villosa seeds have non-deep simple morphophysiological dormancy (MPD). Neither species afterripened in a 6-month dry storage treatment. Cold stratification treatments of 4 and 8 weeks alleviated dormancy in both species but C. canadensis seeds germinated at slower speeds and lower rates compared to seeds given 12 weeks of cold stratification. In their natural habitat, both species disperse seeds in mid- to late autumn and germinate in the spring after cold winter temperatures alleviate endogenous dormancy.     

Characterisation of the extractable pectins and hemicelluloses of the cell wall of glasswort, Salicornia ramosissima

Cell walls of glasswort (Salicornia ramosissima Woods), a halophytic Chenopodiaceae, prepared as alcohol-insoluble solids, were found to be rich in arabinose, galacturonic acid, glucose and proteins, and contained 0·7% ferulic acid and 3·8% acetic acid. Pectic and hemicellulosic polysaccharides were extracted by cyclohexanediaminotetraacetic acid, hot dilute acid, cold dilute alkali and concentrated alkali (twice), with yields of 2·9, 19·1, 4·7, 7·4 and 1·9% of the alcohol-insoluble solids, respectively. Protein-rich material precipitated upon dialysis. The dialysed fractions were fractionated by ion-exchange chromatography, and the main fractions were analysed by gel-filtration and glycosyl linkage analysis. The hot acid extract contained 46·2% arabinose and 28·9% galacturonic acid, with high degrees of methylation and acetylation (65 and 45, respectively). It could be fractionated into a low-molecular-weight arabinan rich in ferulic acid, and a pectic fraction still relatively rich in neutral sugars. The concentrated alkali extracts were rich in xylose (33·4 and 23·6%, respectively). They were separated by ion-exchange chromatography into a fucogalactoxyloglucan and a glucuronoarabinoxylan.     

Studies on rat renal cortical cell kallikrein I. Separation and measurement

A technique has been developed to separate and measure kallikrein in a heterogeneous population of rat renal cortical cells in suspension. After rat kidneys were perfused in situ in anaesthetized rats, viable, counted cortical cell suspensions were obtained.Cells were suspended in a sucrose/Tris buffer containing 0.5% deoxycholate, homogenized, centrifuged, dialyzed, and gel filtered on Sephadex G-25. Column chromatography on DEAE-cellulose resulted in a single peak of esterase activity between 0.20 to 0.25 M NaCl/sodium phosphate buffer. Subsequent elution yielded an alkaline esterase which was identical to kallikrein isolated from rat urine, insofar as pH optimum, effects of inhibitors, bioassay activity and immunological properties were concerned. Calculated yields were about 70% of the total esterase activity present in the parent cell homogenates. Recoveries of a purified rat urinary kallikrein added to the cell homogenates, the DEAE-cellulose columns, or the eluates from the columns ranged from 83–108% (mean 96%). Using this technique, it was found that the amount of kallikrein activity present in non-incubated renal cortical cells ranged from 0.6 · 10⁻² to 4.6 · 10⁻²α-N-tosyl-l-arginine methyl ester (Tos-Arg-OMe) esterase units per 10⁸ cells. However, cells incubated in a nutrient medium at 37°C for 3–8 h contained no measurable kallikrein activity, whereas the surounding medium had kallikrein activity which could be significanyly decreased by aldosterone and decreased by spironolactone.     

Late Albian dinoflagellate cyst paleobiogeography as indicator of asymmetric sea surface temperature gradient on both hemispheres with southern high latitudes warmer than northern ones

Late Albian temperature sensitive dinoflagellate cysts are characterized in order to better understand mid-Cretaceous sea surface temperature gradients. Distribution maps of thirty-seven species recovered from one hundred fifty outcrops and deep sea drilling holes (ODP, DSDP) Sites located from low and high paleolatitudes over the two hemispheres (75°N–70°S) are encountered. Fifty years of published data available in eighty-seven articles have been considered and synthesized using a database coupled with a Geographical Information System (GIS). The continuous and disjoint biogeographic patterns of dinocyst species along latitudes define seven climatic belts, four in the northern hemisphere (high, mid-high, mid-low, low latitude belts) and three in the southern hemisphere (low, mid-low, mid-high latitude belts). Dinocysts restricted along latitudes are temperature sensitive species. Limit ranges of temperature sensitive dinocysts of mid-low and mid-high latitude belts reveal mixing belts, located at 40–45°N and 50°–70°S. They represent major palaeofrontal systems as paleosubtropical fronts with strongly mixed water column. The northern frontal system (40–45°N) was located as in the modern ocean. The large southern frontal system (50°S–70°S) was 10°–20° poleward to Antarctica. Semi-quantitative temperature range limits for mid-Cretaceous dinoflagellates and SST gradients in the two hemispheres are suggested by setting dinocyst climatic belts against estimated δ¹⁸O temperature curve from fish teeth. A paleoecological classification is suggested. Latitudinal distribution of extant temperature sensitive dinoflagellate cysts follows the asymmetric modern temperature gradient. Asymmetric latitudinal ranges of Late Albian dinocyst species of mid-low latitude belts restricted between 45°N and 70°S demonstrate asymmetric temperature gradients with southern high latitudes being warmer than northern high latitudes.     

Protocols for hyperlactatemia induction in the lactate minimum test adapted to swimming rats

The lactate minimum test (LACmin) has been considered an important indicator of endurance exercise capacity and a single session protocol can predict the maximal steady state lactate (MLSS). The objective of this study was to determine the best swimming protocol to induce hyperlactatemia in order to assure the LACmin in rats (Rattus norvegicus), standardized to four different protocols (P) of lactate elevation. The protocols were P1: 6 min of intermittent jumping exercise in water (load of 50% of the body weight — bw); P2: two 13% bw load swimming bouts until exhaustion (tlim); P3: one tlim 13% bw load swimming bout; and P4: two 13% bw load swimming bouts (1st 30 s, 2nd to tlim), separated by a 30 s interval. The incremental phase of LACmin beginning with initial loads of 4% bw, increased in 0.5% at each 5 min. Peak lactate concentration was collected after 5, 7 and 9 min (mmol L^− 1) and differed among the protocols P1 (15.2 ± 0.4, 14.9 ± 0.7, 14.8 ± 0.6) and P2 (14.0 ± 0.4, 14.9 ± 0.4, 15.5 ± 0.5) compared to P3 (5.1 ± 0.1, 5.6 ± 0.3, 5.6 ± 0.3) and P4 (4.7 ± 0.2, 6.8 ± 0.2, 7.1 ± 0.2). The LACmin determination success rates were 58%, 55%, 80% and 91% in P1, P2, P3 and P4 protocols, respectively. The MLSS did not differ from LACmin in any protocol. The LACmin obtained from P4 protocol showed better assurance for the MLSS identification in most of the tested rats.     

Formulation of a Streptomyces Biocontrol Agent for the Suppression of Rhizoctonia Damping-off in Tomato Transplants

Formulations of a Streptomyces biological control agent for Rhizoctonia damping-off in tomato seedlings were developed for the first time from vegetative propagules obtained from actively growing, nonsporulating liquid cultures. Alginate beads, durum flour (starch) granules, and talcum powder formulation of this new actinomycetous antagonist (Streptomyces sp. Di-944) isolated from the rhizosphere of field-grown tomato (Lycopersicon esculentum) suppressed damping-off caused by Rhizoctonia solani in tomato plug transplants (cv. Bonny Best) in a peat-based, soilless potting mix under greenhouse conditions. For formulations, vegetative biomass of Streptomyces sp. Di-944 from 3-day-old liquid fermentation in yeast extract–malt extract–glucose broth was lyophilized and pulverized to obtain fragments of viable vegetative filaments. The pulverized biomass had an initial viable count of 2 × 10⁷colony forming units/g and retained 100% viability for 2 weeks when stored at 4°C. Formulations stored at 4°C had a longer shelf life than those stored at 24°C based on viability at 2-week intervals over a 6-month storage period. In addition, dual culture tests showed declining efficacy for surviving Streptomyces propagules in formulations during this storage period. At 4°C, the powder and granular formulations were found to be the most stable and were shown to be 100% viable after 14 and 10 weeks of storage, respectively. However, at the end of 24 weeks, the number of viable propagules in the powder and granular formulations declined to 1.2 × 10⁵ and 7 × 10³ colony forming units/g, respectively. Alginate beads were the least stable in storage. Even at 4°C, 6.9 × 10⁴ and 7.3 × 10² viable propagules/g formulation were detected at the end of 12 and 24 weeks, respectively. The talcum powder formulation delivered to tomato seeds as a seed-coating was the most effective biocontrol treatment. It suppressed damping-off in 10-day-old tomato transplants by almost 90% compared to 30 and 22% damping-off reduction when alginate beads or starch granules were delivered concomitantly with tomato seeds. Seed-coating with powder formulation of the biocontrol agent was as effective as drench application of the fungicide, oxine benzoate (No-Damp), in controlling Rhizoctonia damping-off and superior to the commercial biocontrol agent, Streptomyces griseoviridis (Mycostop), applied to tomato seeds as seed-coating.     

Effect of Temperature on the Life History of Cirrospilus sp. near lyncus (Hymenoptera: Eulophidae), a Parasitoid of Phyllocnistis citrella (Lepidoptera: Gracillariidae)

The effect of four constant temperatures on the life history of Cirrospilus sp. near lyncus was examined in the laboratory. This species is one of the most abundant generalist indigenous parasitoids of the citrus leafminer, Phyllocnistis citrella Stainton, in Spain. Adult lifespan of C. sp. near lyncus decreased from 50.2 to 9.1 days as temperatures increased from 15 to 30°C, respectively. Both gross fecundity and host-feeding were highest at 20°C (170.48 eggs and 32.33 hosts). Oviposition rates were optimal at higher temperatures (5.22 eggs per day at 25°C and 4.79 eggs per day at 30°C) and were dependent on female age. In contrast, host-feeding rates for a given temperature did not depend on age. Generation time decreased with increasing temperatures from 68.05 days at 15°C to 12.19 days at 30°C. Net reproduction peaked at 20°C (68.86 viable females per female). Intrinsic rate of increase doubled from 15°C (0.059 females per female per day) to 20°C (0.127 females per female per day) and almost doubled again from 20 to 30°C (0.210 females per female per day). Given these parameters, C. sp. near lyncus could perform optimally in the area occupied by P. citrella in the Mediterranean region.     

Interaction of cell-membrane prolactin receptor with its antibody.

Antisera against a partially purified prolactin-receptor preparation derived from pregnant-rabbit mammary glands were generated in guinea pigs. On double immuno-diffusion, each antiserum produced a single precipitin line with the prolactin receptors. The anti-receptor sera also specifically inhibited the binding of 125I-labelled sheep prolactin to membrane particles as well as to highly purified prolactin receptors derived from the rabbit mammary glands. The same antisera, however, had no effect on the binding of 125I-labelled insulin to the same membranes. These antisera did not bind or destroy prolactin. Moreover, the binding of 125I-LABELLED PROLACTIN TO MEMBRANE PARTICLES DErived from different tissues from a number of species was also inhibited by the antisera, thus suggesting that the immunological determinants of the prolactin receptors are similar in various tissues derived from different species. The factors in the antisera that were responsible for inhibiting the binding of 125I-labelled prolactin to its receptors were found to be associated with the gamma-globulin fraction. In addition, 131I-labelled gamma-globulins derived from one antiserum were shown to bind to membrane particles derived from mammary glands, and an increase in binding of gamma-globulin was accompanied by a decrease in binding of prolactin. Kinetic analyses of inhibition of 125I-labelled prolactin binding by antisera by using the methods of Lineweaver & Burk [J. Am. Chem. Soc. (1934) 56, 658-666] and Dixon [Biochem. J. (1953) 55, 170-171], revealed that the mechanism is a hyperbolic competitive inhibition. The demonstration of hormone-receptor-antibody complexes further favours this mechanism. The availability of anti-receptor sera should facilitate studies on the functional role as well as other biochemical, immunological and physiological properties of the prolactin receptors.     

Single crystals of dextran: High temperature polymorph

Lamellar single crystals of a high temperature polymorph of synthetic dextran were prepared at temperatures ranging from 120 to 200°C in a mixture of water and polyethylene glycol. Individual crystals with lath-like shapes gave well resolved electron diffraction diagrams from which the reciprocal unit cell parameters a^*, b^* and γ^* could be measured. The direct cell parameters were then determined from a series of electron diffraction diagrams obtained by sequential tilting of the crystal about the b^* axis. This gave a = 0·922 ± 0·001 nm, b = 0·922 ± 0·001 nm, c (chain axis) = 0·78 ± 0·01 nm, α = γ = 90° and β = 91·3° ± 0·5°. The crystal symmetry was P2₁ with b as the unique monoclinic axis. These data coupled with the observed density of the crystals, indicated that the unit cell contained two antiparallel dextran chains of two residues each. When the crystals were grown at temperatures between 90 and 120°C, a percentage of crystals containing both low and high temperature polymorphs were obtained. These mixed crystals had most likely grown in syntaxy.     

Thermal sensitivity of uncoupling protein expression in polar and temperate fish

Uncoupling proteins (UCP), capable of increasing proton leakage across the inner mitochondrial membrane, may play a role in the temperature-dependent setting of energy turnover in animals (and their mitochondria). Therefore, the genes and expression of fish UCP were investigated in the Antarctic eelpout Pachycara brachycephalum and a temperate confamilial species, the common eelpout Zoarces viviparus. UCP full-length cDNA was amplified from liver and muscle using RT–PCR and rapid amplification of cDNA ends (RACE). The fish UCP mRNA consists of 1906 bp in P. brachycephalum and of 1876 bp in Z. viviparus. Both zoarcid sequences contain open reading frames of 939 bp, encoding 313 amino acids, with 98% and 99% identity, respectively. Protein sequences of zoarcid UCP are closest related to fish and mammalian UCP2. For analysis of temperature-dependent expression common eelpouts were cold-acclimated from 10 °C to 2 °C and Antarctic eelpouts were warm-acclimated from 0 °C to 5 °C. Identical cDNA probes for both species were developed to investigate fish UCP mRNA expression, and protein expression levels were detected by Western Blot in the enriched membrane fraction. During cold-acclimation in Z. viviparus, mRNA levels increased by a factor up to 2.0, protein levels increased up to 1.5, in line with mitochondrial proliferation during cold-acclimation. Despite decreased mitochondrial protein content, in Antarctic eelpout UCP levels rose upon warm acclimation by a factor up to 2.0 (mRNA) and 1.6 (protein), respectively. Besides the ongoing discussion of UCP function in vertebrates, the data are indicative of a significant role of fish UCP in thermal adaptation of fish mitochondria.     

An amino acidic adjuvant to augment cryoinjury of MCF-7 breast cancer cells

One of the major challenges in cryosurgery is to minimize incomplete cryodestruction near the edge of the iceball. In the present study, the feasibility and effectiveness of an amino acidic adjuvant, glycine was investigated to enhance the cryodestruction of MCF-7 human breast cancer cell at mild freezing/thawing conditions via eutectic solidification. The effects of glycine addition on the phase change characteristics of NaCl–water binary mixture were investigated with a differential scanning calorimeter and cryo-macro/microscope. The results confirmed that a NaCl–glycine–water mixture has two distinct eutectic phase change events – binary eutectic solidification of water–glycine, and ternary eutectic solidification of NaCl–glycine–water. In addition, its effects on the cryoinjury of MCF-7 cells were investigated by assessing the post-thaw cellular viability after a single freezing/thawing cycle with various eutectic solidification conditions due to different glycine concentrations, end temperatures and hold times. The viability of MCF-7 cells in isotonic saline supplemented with 10% or 20% glycine without freezing/thawing remained higher than 90% (n = 9), indicating no apparent toxicity was induced by the addition of glycine. With 10% glycine supplement, the viability of the cells frozen to −8.5 °C decreased from 85.9 ± 1.8% to 38.5 ± 1.0% on the occurrence of binary eutectic solidification of glycine–water (n = 3 for each group). With 20% glycine supplement, the viability of the cells frozen to −8.5 °C showed similar trends to those with 10% supplement. However, as the end temperature was lowered to −15 °C, the viability drastically decreased from 62.5 ± 2.0% to 3.6 ± 0.7% (n = 3 for each group). The influences of eutectic kinetics such as nucleation temperature, hold time and method were less significant. These results imply that the binary eutectic solidification of water–glycine can augment the cryoinjury of MCF-7 cells, and the extent of the eutectic solidification is significant.