The effect of partial dehydration on the developmental capacity of mouse embryos stored in the supercooled state

The effect of partial dehydration on the ability of mouse blastocysts to withstand storage at subzero temperatures without freezing was studied. The embryos were equilibrated with a supercooling medium developed at the Centre for Food and Animal Research, containing 3% (Medium A) or 6% (Medium B) methanol and propanediol, and then with the same medium, A or B, containing 0–0.5 mol sucrose. The embryos were placed in 0.25 ml straws, cooled to −5°C or −10°C and stored for up to 3 days. After storage, the embryos were cultured for 48 h in M16 and their ability to develop into expanded blastocysts was used to gauge their survival in supercooled storage.

The maximal beneficial effect of partial dehydration occurred in media supplemented with 0.3–0.5 mol sucrose: the proportions of dehydrated embryos surviving 24 h storage at −5°C and −10°C were 84–85% and 91–100%, respectively, compared with only 58% and 52% of non-dehydrated, supercooled embryos. The corresponding figures for dehydrated embryos after 48 or 72 h storage at −5°C were 86–92% and 38–58% compared with 13% and 4% of non-dehydrated embryos. Similarly, 75–85% and 47–55% of partially dehydrated embryos survived storage for 48 h or 72 h, respectively, at −10°C, compared with 5% and 0% of non-hydrated embryos. Thus, reducing the water content of early mouse blastocysts improved their ability to withstand subzero storage.     

Circadian rhythm in size and H-uridine incorporation of single puffs of Drosophila salivary glands in vitro

Salivary glands of late third instar larvae of Drosophila melanogaster were kept in a chemically defined medium at 25 °C, under a light-dark cycle (light time from 9–21 h). Puff size and ³H-uridine incorporation of various single puffs changed significantly during a 24 h period in vitro. Maxima occurred at 8 h and 21 h, minima at 24 h and 11 h. In the unpuffed region 3A6-3C2, only one maximum of ³H-uridine incorporation—at 17 h—was observed.     

Production of high level of cellulase-free and thermostable xylanase by a wild strain of Thermomyces lanuginosus using beechwood xylan

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was studied for production of high level of cellulase-free thermostable xylanase at 50°C using xylan. Optimization of the medium composition was carried out on shake-flask level using Graeco-Latin square technique. This increased xylanase production from 527 nkat ml⁻¹ in the original medium to 9168–9502 nkat ml⁻¹ in the optimized medium under optimized culture conditions e.g. initial medium pH (6.0–6.5), culture temperature (50°C) and time (5–6 d). The lag phase was very much shorter in the laboratory reactor compared to which existed in the shake cultures and 7111 nkat of xylanase activity were obtained per ml of culture filtrate at 60 h of cultivation. With a 15 min reaction time, the optimal pH and temperature for the xylanase activity were at 6.5 and 65°C, respectively. The enzyme was almost stable over a broad range of pH 3–9 at 20°C, with an optimum stability at pH 6.5. After 51 h heating at 50°C the enzyme retained 60%, 100% and 90% activity at pH 5.0, 6.5 and 8.0, respectively. The crude enzyme could hydrolyse xylan effectively and in only 6 h 67.3%, 54.0% and 49.2% saccharifications were achieved for 2%, 5% and 10% substrate levels, respectively. The principal product of hydrolysis was xylobiose together with smaller amounts of xylooligosaccharides (degree of polymerization 3–7) and xylose.     

One-electron redox reactions of free radicals in solution. Rate of electron transfer processes to quinones

A large number of biologically-important organic and inorganic free radicals have been produced in aqueous solutions, using the fast-reaction technique of pulse radiolysis and kinetic absorption spectrophotometry. The reactions of these free radicals with menaquinone (vitamin K₃, E₀ = 0.42 V) were followed by observing the formation kinetics of the semiquinone radical anion of menaquinone, •MK⁻. The absorption spectrum of •MK⁻ has maxima at 395 nm and 300 nm, with extinction coefficients of 1.1·10⁴ and 1.25·10⁴ M⁻¹·cm⁻¹, respectively. The pK_a of the radical •MK⁻-H⁺ is 4.6±0.1. The free radicals were produced by a one-electron oxidation or reduction of various compounds by hydroxyl radicals and solvated electrons, e_aq⁻. Alcohols, sugars, carboxylic acids, amino acids, peptides, aliphatic amines and amides, aromatic and heterocyclic molecules, pyridine derivatives (nicotinamide, NAD⁺), and transition metal ions have been examined. Significant differences have been observed in both the efficiency (expressed in percentage) and the rate constants of the electron transfer reactions from these free radicals to menaquinone. Absolute rates of electron transfer from approx. 5·10⁸–5·10⁹ M⁻¹·s⁻¹ have been observed for most of the free radicals studied. Information relating to the nature of the radicals and the acid-base properties of these radicals for effective one-electron redox reactions with quinones is indicated.     

Factors affecting the repair of sublethal freeze-thaw damage in mammalian cells : I. Suboptimal temperature and hypoxia

The repair of sublethal freeze-thaw damage has been demonstrated by exposing cells to two freeze-thaw cycles separated by various times at 37 °C. The influence of suboptimal temperatures and hypoxia on this repair process was investigated. Repair was found to be temperature sensitive, decreasing as the temperature was lowered from 37 °C, and was virtually nonexistent at 5 °C. Lowering the oxygen concentration in the medium to 6.6 μM or to 0.66 μM had no significant effect on repair. The repair system then appears to involve an enzymatic process which is not greatly dependent on the oxygen concentration in the range of 277 (aerobic) to 0.66 μM O₂ in the medium.     

In vitro excystment of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae)

1986. In vitro excystrnent of the metacercaria of Plagiorchis species 1 (Trematoda, Plagiorchiidae). International Journal for Parasitology 16: 641–645. An optimal hatching success of Plagiorchis species 1 metacercariae (100% excystment, active metacercariae, mean hatching speed 2–10 min, lowest variance of the mean speed) was observed after pretreatment in an HCl-pepsin solution at pH 2.0 and 42°C for 60–70 min, and incubation in a hatching medium at 42 °C and pH 7.3–8.0 with a bile salt (Nacholate), NaHCO₃, and a reductant (cysteine with 100% N₂). The minimum conditions for nearly 100% excystment with lower hatching speeds and higher variances were the presence of NaHCO₃, an oxygen concentration reduced to about 3% in the gas phase, pH> 7.3 and a temperature near 30°C if Na-cholate was absent, or in the presence of the bile salt, a phosphate buffer at pH> 5.0 and room temperature only. Obviously some hatching factors acted interchangeably with compensation for missing stimuli by others. The effect of the bile salt was comparable with that of other surfactants. The metacercariae excysted in nonenzymatic media, which implies an active hatching mechanism.     

Protistan bacterivory in an oligomesotrophic lake: Importance of attached ciliates and flagellates

Seasonal and depth variations of the abundance, biomass, and bacterivory of protozoa (heterotrophic and mixotrophic flagellates and ciliates) were determined during thermal stratification in an oligomesotrophic lake (Lake Pavin, France). Maximal densities of heterotrophic flagellates (1.9 × 10³ cells ml^–1) and ciliates (6.1 cells ml^–1) were found in the metalimnion. Pigmented flagellates dominated the flagellate biomass in the euphotic zone. Community composition of ciliated protists varied greatly with depth, and both the abundance and biomass of ciliates was dominated by oligotrichs. Heterotrophic flagellates dominated grazing, accounting for 84% of total protistan bacterivory. Maximal grazing impact of heterotrophic flagellates was 18.9 × 10⁶ bacteria 1^–1h^–1. On average, 62% of nonpigmented flagellates were found to ingest particles. Ciliates and mixotrophic flagellates averaged 13% and 3% of protistan bacterivory, respectively. Attached protozoa (ciliates and flagellates) were found to colonize the diatom Asterionella formosa. Attached bacterivores had higher ingestion rates than free bacterivorous protozoa and may account for 66% of total protozoa bacterivory. Our results indicated that even in low numbers, epibiotic protozoa may have a major grazing impact on free bacteria. Correspondence: C. Amblard.     

Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.     

The Soil Flagellate Heteromita globosa Accelerates Bacterial Degradation of Alkylbenzenes through Grazing and Acetate Excretion in Batch Culture

The impact of grazing by soil flagellates Heteromita globosa on aerobic biodegradation of benzene by Pseudomonas strain PS+ was examined in batch culture. Growth of H. globosa on these bacteria obeyed Monod kinetics (_max, 0.17 ± 0.03 h^–1; K_s, 1.1 ± 0.2 × 10⁷ bacteria mL^–1) and was optimal at a bacteria/ flagellate ratio of 2000. Carbon mass balance showed that 5.2% of total [ring-U-¹⁴C]benzene fed to bacteria was subsequently incorporated into flagellate biomass. Growth-inhibiting concentrations (IC₅₀) of alkylbenzenes (benzene, toluene, ethylbenzene) were inversely related with their octanol/ water partitioning coefficients, and benzene was least toxic for bacteria and flagellates with IC₅₀ values of 4392 (± 167) M and 2770 (± 653) M, respectively. The first-order rate constant for benzene degradation (k₁, 0.48 ± 0.12 day^–1) was unaffected by the presence or absence of flagellates in cultures. However, the rate of benzene degradation by individual bacteria averaged three times higher in the presence of flagellates (0.73 ± 0.13 fmol cell^–1 h^–1) than in their absence (0.26 ± 0.03 fmol cell^–1 h^–1). Benzene degradation also coincided with higher levels of dissolved oxygen and a higher rate of nitrate reduction in the presence of flagellates (p < 0.02). Grazing by flagellates may have increased the availability of dissolved oxygen to a smaller surviving population of bacteria engaged in the aerobic reactions initiating benzene degradation. In addition, flagellates may also have increased the rate of nitrate reduction through the excretion of acetate as an additional electron donor for these bacteria. Indeed, acetate was shown to progressively accumulate in cultures where flagellates grazed on heat-killed bacteria. This study provided evidence that grazing flagellates stimulate bacterial degradation of alkylbenzenes and provide a link for carbon cycling to consumers at higher trophic levels. This may have important implications for bioremediation processes.     

Interactions of temperature and pH on the regulatory properties of pyruvate kinase from organs of a marine mollusc

The effects of low temperature assay (5 °C) on the properties of the aerobic (low phosphate) vs. anoxic (high phosphate) forms of pyruvate kinase (PK) from foot muscle and gill of the whelk Busycon canaliculatum (L.) were assessed at two pH values, pH 7.00 and 7.25, and compared to control conditions of 20 °C and pH 7.00 (all assayed in imidazole buffer). When pH was held constant at 7.00, the decrease in assay temperature to 5 °C had large effects on the measured kinetic parameters of all PK forms, as compared to 20 °C and pH 7.00. However, when assay pH was allowed to rise, from 7.00 to 7.25, with the temperature decrease to 5 °C there were fewer alterations of kinetic parameters and quantitatively smaller changes to enzyme properties. It appears, then, that when pH rises with decreasing temperature following alphastat predictions, kinetic properties of PK are largely conserved. Low temperature, at either pH value, had several significant effects on PK properties. For example, low temperature raised the S_0.5 for phosphoenolpyruvate of PK-anoxic from gill by 3–6 fold and decreased the I₅₀ Mg · ATP for PK-anoxic from foot by the same amount. Arrhenius plots of PK activity for the gill PK forms showed a distinct break at 10 °C; > 10 °C Q₁₀ was 2.5 whereas < 10 °C Q₁₀ was 8.4. Temperature-dependent changes in all cases affected enzyme properties in a manner that would restrict enzyme function at low temperature.     

A comparative study of in situ hybridization procedures using cRNA applied to Drosophila hydei salivary gland chromosomes

The effect of different denaturation and hybridization procedures on the efficiency of in situ ³H-cRNA hybridization with DNA in the polytene chromosomes of Drosophila hydei was investigated.Denaturation of the DNA in the squash preparations with 90% formamide in 0.1 × SSC at 65 °C for 2.5 h gave a significantly higher retention of radioactivity following in situ hybridization than did denaturation by 30 sec incubation in boiling 0.1 × SSC.A comparison of the effect of various SSC concentrations in the hybridization mixture revealed that among the SSC concentrations tested, 3 × SSC or 4 × SSC gave the highest efficiency of hybrid formation.Hybridization in 50% formamide at 20 °C resulted in continuing hybrid formation over a period of 3.5 h, the majority of the cRNA/DNA hybrids being formed within the first 10 min of the incubation period. The thermal dissociation profile of in situ cRNA/DNA hybrids formed in 50% formamide, 4 × SSC at 20 °C, as determined in 0.1 × SSC indicated a T_m of 66 °C. The shape of the profile and the results of competition experiments suggested a high fidelity of base-matching in the in situ ³H-cRNA/DNA hybrids.Non-chromosomal background labeling in autoradiographs of polytene chromosomes hybridized with ³H-cRNA was effectively reduced by adding a 200–1000 fold excess of cold 28S + 18S RNA.     

Rate of RNA synthesis as a function of temperature in Chinese hamster lung fibroblasts (V-79)

The rates of intracellular RNA synthesis at various temperatures between 33 and 41 °C were determined in Chinese hamster lung fibroblasts by measuring average amounts of [³H]uridine incorporated per cell per unit of time. The energy of activation and Q₂₀ for intracellular RNA synthesis were calculated from the slopes of the relative rates of RNA synthesis in hamster fibroblasts vs time, plotted on Arrhenius coordinates. The incorporation of uridine into RNA is characterized by an energy of activation of 19 200 calories/mole and a Q₁₀ of 2.71. The absolute rates of RNA synthesis were determined at various temperatures, with values ranging from 1.55 to 0.60 × 10⁻¹⁵ g RNA/min/cell at 41 to 33 °C, respectively.     

Characterization of two extracellular proteinases from Aspergillus terreus and their role in the formation of low molecular weight endoglucanases

Two extracellular proteolytic activities from the wood degrading fungus Aspergillus terreus have been characterized. Proteinase I (serine thiol-dependent enzyme) was active over a broad pH range (7·0–10·0) and at 55°C. The second proteinase (metalloproteinase) showed optimal activity at pH 6·0–7·0 and at 65–70°C. Both proteins had isoelectric points at acid pH and contained carbohydrate moieties. The metalloproteinase possessed a uniquely high content of serine and threonine and an extremely low percentage of glutamate and aspartate. The metalloproteinase was involved in the formation of the low molecular mass endoglucanases of A. terreus.     

Quantitative micro-assay for RNA/DNA hybrids in the study of nucleolar RNA from Chironomus tentans salivary gland cells

A microassay for RNA/DNA hybrids has been designed for the study of RNA from different nuclear components of Chironomus tentans salivary gland cells. The procedure comprises a scale reduction of the conventional filter method for hybridization, using ultraviolet microphotometry for quantitation of RNA and DNA. Hybridization is performed in 0.3 μl of 2 × SSC containing 1–2 × 10^-2 μg DNA, immobilized on a 0.2 mm² ‘micro-filter’, and 0.5–5 × 10⁻² μg RNA, with a specific activity of more than 10⁶ cpm/μg. Results obtained by the microtechnique are found to agree with results obtained by a large-scale, standard procedure. The applicability of the microtechnique is demonstrated in saturation and presaturation-competition experiments. RNA from micro-isolated nucleoli hybridizes a maximum of 0.22% of Chironomus tentans DNA, which corresponds to about 100 cistrons for the 38S ribosomal precursor in the haploid genome. The hybrids show a steep thermal dissociation profile with a T_m of 79 °C, close to the value expected for hybrids with a G + C content of 42%. Presaturation of filter-bound DNA by total unlabelled nucleolar RNA prevents 80% of the subsequent hybridization by labeled nucleolar Presaturation by RNA from one of the two nucleolar organizers prevents to a similar degree the subsequent hybridization by RNA from the other nucleolar organizer. This result indicates a sequence similarity of RNA transcribed in different nucleolar organizers. Further applications of the microtechnique are presented in the accompanying paper where the hybridization properties of chromosomal and nuclear sap RNA are investigated.     

Nutritional characteristics of a mixotrophic nanoflagellate,Ochromonas sp.

Autotrophic and heterotrophic growth characteristics of a nano-flagellate were investigated. The flagellate,Ochromonas sp., was isolated from the northern Baltic Sea. Autotrophic growth was poor. Axenically pregrown flagellates did not increase significantly in cell number during incubation in different inorganic media. The number of flagellates remained constant 3–5 weeks in cultures kept in the light (100mol m^–2 sec^–1), whereas in the dark, a high mortality rate was found. Uptake of inorganic¹⁴C into an acid-stable fraction indicated thatOchromonas had a functional photosynthetic apparatus. Heterotrophic growth in both liquid medium and medium containing bacteria was rapid. The maximum growth rate corresponded to a generation time of 5.3 hours. Light had no effect on heterotrophic growth. Cells pregrown onEscherichia coli minicells survived without additional bacteria as food when kept in the light, but rapid death occurred in darkness. In conclusion, heterotrophy is the major mechanism to support growth in this species ofOchromonas, but under poor environmental conditions photoautotrophy might be a strategy for survival rather than growth.     

The potential use of tree-rings to reconstruct streamflow and estuarine salinity in the Valdivian Rainforest eco-region, Chile

Water availability is one of the main limitations for future development and economic activity in many regions of the world. This applies even to areas of relatively high annual rainfall, such as the Valdivian Rainforest eco-region in Chile (35–48°S). Streamflow and water availability are crucial for several economic activities in the eco-region including hydroelectricity, irrigation, salmon farming, sports fishing of introduced trout and tourism. Scientific research focused on the spatial and temporal patterns of streamflow is a key element for planning the future development within the eco-region. In this paper we explore the potential of tree-rings for streamflow reconstruction in this region. Our preliminary results indicate a significant correlation (r=0.59 P<0.001, 1929–2000) between prior summer Río Bueno streamflow (January–April) and the average between a composite tree-ring chronology using Pilgerodendron uviferum from Chile and two composite Austrocedrus chilensis chronologies from Argentinean Patagonia. Similarly, there is a significant correlation (r=0.57, P<0.001, 1943–2002), between the Austrocedrus chronology (40°44′S) at Centinela in Argentinean Patagonia and the previous spring through early autumn (November–April) streamflow for Río Puelo. The tree-ring records used to correlate with Río Bueno and Río Puelo discharges show low-frequency variability and therefore the potential to reconstruct this variability in streamflow for the last 500–780 years. We also found a significant correlation between the composite Pilgerodendron standard ring-width chronology and the current summer water salinity (January–April) at the Reloncavi Estuary (r²=0.60, P<0.01, 1992–2000). Water salinity is a crucial determinant of the carrying capacity of salmon farming. Future research should provide reconstructions of streamflow, water salinity and other water quality attributes from tree-rings. These data will provide inputs to modeling scenarios of future water availability and are crucial to decision-making and planning of resource management and socio-economic development in the Valdivian Rainforest eco-region.     

Finger temperatures during military field training at 0 to −29 °C

1. Skin and rectal temperatures were recorded continuously in 70 measurements during typical tasks of infantry and artillery training at 0 to −29 °C. The duration of the measurements varied from 55 min to 9.5 h.

2. The distribution of finger skin temperatures was quite similar at ambient temperature ranges 0 to −10 °C and −10 to −20 °C, while at −20 to −30 °C the finger temperatures were clearly lower.

3. At different ambient temperature ranges, 20–69% of finger temperatures were low enough to cause cold thermal sensations.

4. Sensation of cold was experienced at a finger temperature of 11.6±3.7 °C (mean±SD).     

Lipase-catalyzed acylation of naringin with palmitic acid in highly concentrated homogeneous solutions

Acylation reactions of naringin with palmitic acid were performed by a lipase after formation of highly concentrated homogeneous solutions. Their initial naringin concentration was 840–950 mM, which is 20–60 times greater than that in organic solvent media. The overall productivity of highly concentrated solutions was more than 15 times greater than those of organic phase media. The addition of DMSO (20–40%, w/w) to substrate mixtures lowered the melting temperature of a naringin–palmitic acid mixture (1:1 molar ratio) to about 40 °C. Reactions at 80 °C apparently followed Michaelis–Menten kinetics despite extremely high substrate concentrations. As the temperature increased from 60 °C to 80 °C, the apparent viscosity of the highly concentrated solution decreased remarkably from 4.31 Pa s to 0.063 Pa s. An activation energy of 7.65 kcal/mol obtained in a range of 60–75 °C suggests a diffusion-control. On the other hand, an activation energy of 17.09 kcal/mol in a range of 75–90 °C indicates a reaction-control. The highest product conversion yield of 33% (mol/mol) was obtained in a 10 h reaction at 80 °C. Addition of activated molecular sieves to the highly concentrated solution increased the product conversion yield by 7% (mol/mol), suggesting that the original equilibrium was disrupted by removing water and then a new equilibrium was reached.     

Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions

A uricase-producing bacterium was isolated from soil with a medium containing uric acid as the only carbon source. Based on its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the genus Microbacterium. After heat treatment at 70 °C for 30 min, the uricase retained about 100% of the initial activity. The enzyme activity remained largely unchanged when it was stored in borate buffer at pH 8.5 at 37 °C for 40 days. The effects of different factors on the enzyme production were studied. Maize milk was the best C and N resources, and the uric acid showed to be an inducer for uricase production. When the strain was cultured at 30 °C at pH 7.5 for 30–36 h, the uricase activity peaked at 1.0 U/ml.     

Influence of incubation temperature and time on resistant starch type III formation from autoclaved and acid-hydrolysed cassava starch

Raw cassava starch, having 74.94 and 0.44 g/100 g resistant starch type II and III (RS II and RS III), respectively, was autoclaved at 121 °C in water, 1, 10 or 100 mmol/L lactic acid. The formation of RS III was evaluated in relation to variable incubation temperature (−20 to 100 °C), incubation time (6–48 h) and autoclaving time (15–90 min). Negligible to low quantities of RS III (0.59–2.42 g/100 g) were formed from autoclaved starch suspended in 100 mmol/L lactic acid, whereas intermediate to high quantities (2.68–9.97 g/100 g) were formed from autoclaved starch suspended in water, 1 or 10 mmol/L lactic acid, except for treatments with water or 10 mmol/L lactic acid incubated at 100 °C for 6 h (1.74 g/100 g). Autoclaving times corresponding to maximum RS III contents were 15 and 45 min for water and 10 mmol/L lactic acid, respectively. Whereas, the RS III fractions from cassava starch suspended in water had melt transitions between 158 and 175 °C with low endothermic enthalpies (0.2–1.6 J/g), the thermal transitions of the acid treated samples were indistinct.