Peptidase D of Escherichia coli K-12, a metallopeptidase of low substrate specificity

Abstract Peptidase D of Escherichia coli was overproduced from a multicopy plasmid and purified to electrophoretic homogeneity. The pure enzyme was stable at 4°C or −20°C and had a pH optimum at pH 9, and a p I of 4.7; the temperature optimum was at 37°C. As the enzyme was activated by Co²⁺ and Zn²⁺, and deactivated by metal chelators, it appears to be a metallopeptidase. By activity staining of native gels, 11 dipeptides which are preferentially cleaved by peptidase D were identified. Peptidase D activity required dipeptide substrates with an unblocked amino terminus and the amino group in the α or β position. Non-protein amino acids and proline were not accepted in the C-terminal position, whereas some dipeptide amides and formyl amino acids were hydrolyzed. K _m values of 2 to 5 mM indicate a relatively poor interaction of the enzyme with its substrates.     

Characterization of a rice mutant having an increased susceptibility to light stress at high temperature

The effect of light irradiation at high temperature was investigated in a rice mutant, spl-2 , which is sensitive to solar radiation. Dead spots appeared on the mutant leaves when cultured at a high temperature (40°C) under strong white-light illumination (15 W m⁻²). A similar damage was also observed in the wild-type leaves under the same conditions when the plants were preincubated in the dark for one day. Preillumination with weak light (6 W m⁻²) lessened the irradiation-induced damage in the wild-type. These observations suggest that in rice plants the acclimatization to weak light has a protective effect against strong irradiation at high temperature, and the spl-2 locus participates in the mechanisms of the acclimatization. The action spectrum for the irradiation-induced damage at the high temperature (40°C) in the spl-2 leaves indicated that the maximum damage occurred at around 480 nm and, in a lesser extent, at around 680 nm. Activity of the O⁻₂₋ and H₂O₂₋scavenging enzymes in the spl−2 leaves were almost the same or somewhat higher than those in the wild-type after irradiation with strong white light (15 W m⁻²) at 40°C, whereas the content of ascorbic acid in the spl−2 decreased significantly compared with the wild type.     

In vitro-selection of hydroxyproline-resistant cell lines of wheat (Triticum aestivum): accumulation of proline, decrease in osmotic potential, and increase in frost tolerance

Five hundred hydroxyproline-resistant cell lines were selected from cell cultures of wheat ( Triticum aestivum L. cv. Koga II) after plating on 10 to 30 m M hydroxyproline (Hyp) containing solid Gamborg B 5 medium. All selected cell lines from 30 m M Hyp-medium contained increased (up to 17-fold) levels of free proline. Seventy-four cell lines were transferred to Hyp-free medium and subcultivated 25 times, for 12 months altogether, after which 80% still had increased proline levels. Fourteen cell lines with increased proline levels were further investigated in liquid media with regard to their frost tolerance, which was measured by means of electrolyte leakage. Ten of them showed increased fros tolerance, with LT 50 values as low as 2.7°C below that of the wild type (-4.7°C). Besides increased proline levels and increased percentage dry weight, the Hyp-resistant cell lines had lower osmotic potentials. Osmotic potentials correlated better than levels of free proline with the increase in frost tolerance.     

Macromolecular synthesis by yeasts under frozen conditions

Although viable fungi have been recovered from a wide variety of icy environments, their metabolic capabilities under frozen conditions are still largely unknown. We investigated basidiomycetous yeasts isolated from an Antarctic ice core and showed that after freezing at a relatively slow rate (0.8°C min⁻¹), the cells are excluded into veins of liquid at the triple junctions of ice crystals. These strains were capable of reproductive growth at −5°C under liquid conditions. Under frozen conditions, metabolic activity was assessed by measuring rates of [³H]leucine incorporation into the acid-insoluble macromolecular fraction, which decreased exponentially at temperatures between 15°C and −15°C and was inhibited by the protein synthesis inhibitor cycloheximide. Experiments at −5°C under frozen and liquid conditions revealed 2–3 orders of magnitude lower rates of endogenous metabolism in ice, likely due to the high salinity in the liquid fraction of the ice (equivalent of ≈ 1.4 mol l⁻¹ of NaCl at −5°C). The mesophile Saccharomyces cerevisae also incorporated [³H]leucine at −5°C and −15°C, indicating that this activity is not exclusive to cold-adapted microorganisms. The ability of yeast cells to incorporate amino acid substrates into macromolecules and remain metabolically active under these conditions has implications for understanding the survival of Eukarya in icy environments.     

Ultrastructural changes arising from freezing of leaf blade cells of rye (Secale cereale): an investigation using freeze-substitution

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale L. cv. Voima), which had not been cold acclimated, was determined by measuring the efflux of ninhydrin-positive substances: 50% of the cells were dead at −4°C (LT₅₀) and none survived at −12°C or below. Examination of ultrastructural changes during cold hardening and freezing injury requires frozen tissues prepared for transmission electron microscopy without thawing. Specimens were prepared from leaf blade segments at room temperature, −4°C or −12°C by plunge freezing at 3 m s⁻¹ into a cooling medium at −170°C followed by freeze-substitution in acetone with OsO₄ fixation. Comparisons of room temperature specimens were made with those prepared by chemical fixation using glutaraldehyde/paraformaldehyde/tannic acid. On freezing to −12°C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken and the cytoplasm and mitochondria disorganized whereas the chloroplasts were little affected. On freezing to −4°C, some cells were as disorganized as those at −12°C, others were relatively intact, and some showed evidence of intracellular ice crystal formation.     

Freezing and desiccation tolerance of imbibed canola seed

Depending on the environmental conditions, imbibed seeds survive subzero temperatures either by supercooling or by tolerating freezing-induced desiccation. We investigated what the predominant survival mechanism is in freezing canola ( Brassica napus cv. Quest) and concluded that it depends on the cooling rate. Seeds cooled at 3°C h⁻¹ or faster supercooled, whereas seeds cooled over a 4-day period to −12°C and then cooled at 3°C h⁻¹ to−40°C did not display low temperature exotherms. Both differential thermal analysis and nuclear magnetic resonance (NMR) spectroscopy confirmed that imbibed canola seeds undergo freezing-induced desiccation at slow cooling rates. The freezing tolerance of imbibed canola seed (LT₅₀) was determined by slowly cooling to −12°C for 48 h, followed with cooling at 3°C h⁻¹ to −40°C, or by holding at a constant −6°C (LD₅₀). For both tests, the loss in freezing tolerance of imbibed seeds was a function of time and temperature of imbibition. Freezing tolerance was rapidly lost after radicle emergence. Seeds imbibed in 100 μ M abscisic acid (ABA), particularly at 2°C, lost freezing tolerance at a slower rate compared with water-imbibed seeds. Seeds imbibed in water either at 23°C for 16 h, or 8°C for 6 days, or 2°C for 6 days were not germinable after storage at −6°C for 10 days. Seeds imbibed in ABA at 23°C for 24 h, or 8°C for 8 days, or 2°C for 15 days were highly germinable after 40 days at a constant −6°C. Desiccation injury induced at a high temperature (60°C), as with injury induced by freezing, was found to be a function of imbibition temperature and time.     

The effect of moisture potential on growth and survival of root nodule bacteria in peat culture and on seed

Peat from three sources was dried, milled and packed separately in polyethylene bags and sterilized by irradiation. The carrier was impregnated with broth cultures of either Rhizobium leguminosarum bv. trifolii strain WU95, Bradyrhizobium japonicum strain CB1809 or B. lupini strain WU425 and sterile water to provide five moisture potentials in the range > - 1 × 10⁴ - 1 × 10⁶ Pa. The packets were stored at 26°C under conditions which restricted moisture loss. Numbers of root nodule bacteria were counted at intervals up to 12 weeks. No single moisture potential was optimum for all strains in all carriers because of a significant ( P < 0.05) interaction between moisture potential × strain × carrier × time. Where direct comparisons could be made, all strains survived best at - 1 × 10⁴ and/or −3.2 × 10⁴ Pa. Seeds of Trifolium subterraneum and polypropylene beads (used to avoid seed coat toxicity), were inoculated with WU95 prepared in two sources of peat and at each of the above moisture potentials and stored at 15°C. Seed coat toxicity significantly effected the log death rate ( k ) of WU95 on subterraneum clover seed for the period 0–0.25 d ( k 1.796) compared with k - 0.399 for polypropylene beads. In the first 24 h moisture did not affect survival but by 28 d rhizobia grown in Badenoch peat survived best at −3.2 × 10⁴ Pa. In Millicent peat, survival was equally as good at −3.2 × 10⁴ and −1 × 10⁴ Pa.     

Phytochrome A does not mediate reduced stem extension from cool day‐temperature treatments

Stem elongation can be suppressed by a temperature drop at the onset of the photoperiod (DROP) or with a cooler day than night temperature (DT and NT, respectively), commonly described as DIF (DT ‐ NT). To test our hypothesis that phytochrome A (phyA) mediated the reduction of stem elongation caused by −DIF and DROP, we conducted experiments with photomorphogenic mutants of tomato ( Solarium lycopersicon L.) and transgenic potato ( Solarium tuberosum L.). The plants studied were tomato mutants fri ¹ (deficient in phyA) and tri ³ (deficient in phytochrome B1 [phyBl]) and their isogenic wild‐type (WT) cv. Moneymaker, nontransformed potato, and two lines each of antisense phyA (15‐9 and 15‐11) and overexpressed phyA (PS‐2 and PS‐4). Plants were placed in three temperature regimens with a daily mean of 20°C: a constant 20°C (0 DIF), an 8°C DROP for 3 h, and a ‐ 8°C DIF. For all tomato genotypes, −DIF and DROP reduced intemode length by ≥ 21% and stem elongation by 30% compared to that of plants at 0 DIF. Interactions between temperature treatment and genotype were nonsignificant. For potato, −DIF, but not DROP, significantly reduced intemode length of WT (by 39%) and both antisense lines (by 36 or 48%) but only one of the two lines of overexpressed phyA plants (by 18%). The −DIF significantly reduced stem length for only antisense phyA (by 36 or 48%) and WT (by 35%) plants. Thus, at least for tomato and potato, it appears that phyA does not control stem extension in relation to cool‐temperature treatments.     

Cryopreservation of common carp (Cyprinus carpio L.) sperm II. Optimal conditions for fertilization

Experiments were carried out on the cryopreservation of common carp ( Cyprinus carpio L.) sperm. Optimal conditions for fertilization including suitable medium and sperm:egg ratio were determined. Sperm was diluted in modified Kurokura's 'Extender 2'containing DMSO as cyroprotectant in 10% final concentration. The dilution rate was 1:9 (sperm:diluent). Sperm was diluted and equilibrated (10 min) at 2°C. Sperm was then frozen in plastic straws (0.5 ml) at the following rate: 0°C–4°C: 4°C min⁻¹; −4°C–80°C: 11°C min⁻¹; from −80°C they were plunged directly into liquid nitrogen (− 196°C). Frozen samples were thawed in a water bath at 40°C. Fertilization rates achieved were much higher in water than in other solutions. Optimal ratios of frozen sperm:egg:water (1:20:20 in volume) and optimal number of frozen spermatozoa:egg (10⁵ spz: 1 egg) were determined. In such conditions, a strong positive correlation (c =+0.846) was found between the post-thaw motility and the fertility of frozen sperm securing high fertilization (99.6%, percent of control). No significant difference was found between fertilization and hatching rates achieved using frozen-thawed common carp sperm.     

THE COMBINED EFFECTS OF LOW TEMPERATURE AND SO2+NO2 POLLUTION ON THE NEW SEASON'S GROWTH AND WATER RELATIONS OF PICEA SITCHENSIS

Picea sitchensis (Bong.) Carr. seedlings were exposed to SO₂, NO₂ and SO₂+ NO₂ during dormancy in controlled environments, and were taken to night temperatures of 4, 0, −5, −10 and −15 °C in a freezer. Conditions in the freezer were carefully monitored during the low–temperature treatments. In two experiments, different photoenvironments and temperature regimes were imposed prior to the cold treatments, and different effects were observed. In the first, only limited frost hardiness was achieved and night temperatures of −15 °C were lethal. Temperatures of −5 and − 10 °C led to poor survival of lateral buds, particularly in plants exposed to 45 ppb SO₂. The poor bud break in plants exposed to SO₂ and to − 5 °C resulted in a loss of the effectiveness of this temperature as a chill requirement. Pressure-volume analysis showed that the shoots of plants exposed to NO₂ had greater elasticity (lower elastic moduli, e), so that loss of turgor occurred at lower relative water contents. In contrast, a hardening period (2 weeks in night/day temperatures of 3/10 °C and 8 h days at 50 μmol m⁻² s⁻¹ PAR) gave decreased elasticity and lower solute potentials of spruce shoots. In the second experiment, exposure to 30 ppb SO₂ and SO₂+ NO₂ led to slight, but consistent, increases in frost injury to the needles of plants frozen to − 5 and − 10 °C. The results suggest that the main interaction of low temperatures and winter pollutants may be on bud survival rather than on needle damage, but that effects are subtle, only occurring with certain combinations of pollutant dose and cold treatment.     

Energy balance and temperature relations of Azorella compacta, a high-elevation cushion plant of the central Andes

The environmental relationships and ecophysiology of Azorella compacta, a giant cushion plant, were investigated in Parque Nacional Lauca, Chile (18°10'–18°25' S and 69°16' W, 4400 m asl). The diurnal temperature range can reach 42 °C on some days of the year. The surface temperature of A. compacta was 13 °C below that of the air temperature of −7 °C at dawn, but from midmorning to late afternoon, the plant surface temperature remained within a few degrees of the air temperature. Soil surface temperatures did not differ between north- and south-facing slopes, but a model showed an increase in radiation reception by north-facing slopes throughout most of the year. Gas exchange measurements of A. compacta measured at the onset of the wet season ranged from −0.6662 to 11.4 μmol·m⁻²·s⁻¹, and maximum stomatal conductance (Gs) was 410 mmol·m⁻²·s⁻¹. The estimated light compensation point was 89 μmol·m⁻²·s⁻¹ and estimated light saturation occurred at about 1280 μmol·m⁻²·s⁻¹. Diurnal water potential measurements for A. compacta ranged from −1.67 to −2.65 MPa. This is one of the first ecophysiological studies of a tropical alpine cushion plant.     

Intergenerational acclimation in aphid overwintering

Abstract.  1. When first instar nymphs and adults of the grain aphid Sitobion avenae (Fabricius) (Hemiptera: Aphidiae) were maintained in long-term cultures (>6 months) at 20 °C and 10 °C, the LT₅₀ decreased from −8 and −8.8 °C to −16.0 and −13.5 °C, respectively.
2. When aphids from the 20 °C culture were transferred to 10 °C, there was a progressive increase in cold tolerance through three successive generations. Transfer of newly moulted pre-reproductive adults reared at 10 °C for three generations back to 20 °C resulted in a rapid loss of cold hardiness in their nymphal offspring.
3. In all generations reared at 10 °C, first born nymphs were more cold hardy than those born later in the birth sequence. The LT₅₀ of nymphs produced on the first day of reproduction in the first, second and third generations maintained at 10 °C were −14.8, −17.0 and −16.6 °C, respectively. Thereafter, nymphal cold hardiness decreased over the subsequent 14 days of reproduction in each generation at 10 °C with mean LT₅₀ values of −10.3, −12.6 and −14.8 °C, respectively. By contrast, the cold tolerance of first born nymphs of aphids reared continuously at 20 °C did not differ in comparison with later born siblings. The LT₅₀ of adult aphids was also unaffected by ageing.
4. The ecological relevance of these findings is discussed in relation to the overwintering survival of aphids such as S. avenae .     

Sperm motility and cryopreservation of spermatozoa in freshwater gobies

Testicular sperm motility and methods for the cryopreservation of spermatozoa in the freshwater goby Rhinogobius sp. CB (Cross Band type) were examined. Spermatozoa were almost immotile upon dilution with 300 mOsm kg⁻¹ of NaCl, KCl and mannitol solutions but began to swim in solutions with concentrations <200 mOsm kg⁻¹. The highest percentage and longest duration of motility was obtained in the 0 and 100–200 mOsm kg⁻¹ solutions, respectively. The highest post-thaw motility, c. 50% of motility before cryopreservation, was obtained when spermatozoa were diluted with an extender of 10% methanol and 90% artificial seminal plasma, cooled at −10·0 ± 1·1° C min⁻¹ (mean ± s . e .) to −50° C and plunged into liquid nitrogen. Spermatozoa were cryopreserved in a 50 μl acrylic haematocrit tube to store the small amount of milt. As the cryopreservation method described above was applicable to the endangered Rhinogobius sp. BI (Bonin Island type), it is probable that this method can be used for other species of freshwater gobies.     

Effect of temperature on swimming performance of sea bass juveniles

At four temperatures ( T= 15, 20, 25 and 28° C) swimming performance of Dicentrarchus labrax was significantly correlated with total length (23–43 mm L _T); r²=0.623–0.829). The relative critical swimming speed ( RU _crit= U _crit L _T⁻¹), where U _crit is the critical swimming speed, was constant throughout the L _T range studied. The significant effect of temperature on the relative critical swimming speed was described binomially: RU _crit=−0.0323T²+ 1.578 T −10.588 (r²=1). The estimated maximum RU _crit (8.69 L _T s⁻¹) was achieved at 24.4° C, and the 90% performance level was estimated between 19.3 and 29.6° C.     

Is tilting behaviour at low swimming speeds unique to negatively buoyant fish? Observations on steelhead trout,Oncorhynchus mykiss,and bluegill,Lepomis macrochirus

When swimming at low speeds, steelhead trout and bluegill sunfish tilted the body at an angle to the mean swimming direction. Trout swam using continuous body/caudal fin undulation, with a positive (head-up) tilt angle ( 0 , degrees) that decreased with swimming speed ( u , cm s⁻¹) according to: 0 =(164±96).u^{(−1.14±0.41)} (regression coefficients; mean±2 s.e. ). Bluegill swimming gaits were more diverse and negative (head down) tilt angles were usual. Tilt angle was −3·0 ± 0.9° in pectoral fin swimming at speeds of approximately 0.2–1.7 body length s⁻¹ (Ls⁻¹; 3–24 cm s⁻¹), −4.5 ±2.6° during pectoral fin plus body/caudal fin swimming at 1·2–1·7 L s⁻¹ (17–24cm s⁻¹), and −5.0± 1.0° during continuous body/caudal fin swimming at 1.6 and 2.5 L s⁻¹ (22 and 35cm s⁻¹). At higher speeds, bluegill used burst-and-coast swimming for which the tilt angle was 0.1±0.6°. These observations suggest that tilting is a general phenomenon of low speed swimming at which stabilizers lose their effectiveness. Tilting is interpreted as an active compensatory mechanism associated with increased drag and concomitant increased propulsor velocities to provide better stabilizing forces. Increased drag associated with trimming also explains the well-known observation that the relationship between tail-beat frequency and swimming speed does not pass through the origin. Energy dissipated because of the drag increases at low swimming speeds is presumably smaller than that which would occur with unstable swimming.     

Seasonal changes in vertical distribution, biomass and faecal production of tubificids in the profundal region of a shallow Japanese lake

Two tubificid species Limnodrilus hoffmeisteri and L. claparedeianus formed more than 93% of the total number of oligochaetes in the profundal. Limnodrilus spp. worms were found down to 33 cm in the sediment but in great numbers in the upper zone in June and October. Worms confined to the top 15 cm of sediment accounted for 53-92% of the total number. There were two annual maxima in population density and biomass, one in late spring (66000 inds m⁻², 17 g wet wt m⁻²) and the other in mid autumn (97000 inds m⁻², 176 g wet wt m⁻²). Two regression lines describing the effect of temperature on faecal production rate were obtained; Log F = 0.0604 T (°C) −0.7660 (below 15°C), Log F = 0.0266 T – 0.2170 (above 15°C). In total 26.8 kg dry wt m⁻² of sediment was defecated annually by Limnodrilus spp. The sediment in the 0–10 cm stratum may pass through the guts of the worms 2.3 times a year. Sedimentation rates in profundal region were very low with respect to the faecal production rates of the tubificids.     

Uptake of amino acids and peptides by developing barley embryos

Developing embryos of barley ( Hordeum vulgare L. cv. Bomi) detached 21–27 days after anthesis took up 1 mM [¹⁴C]-glutamine at pH 5 and 30°C at a rate of about 20 nmol embryo^−l h⁻¹ (5 μol g⁻¹h⁻¹). The uptake was inhibited by about 50% by di-nitrophenol and by about 80% by 300 m M unlabelled glutamine or alanine. The bulk of the uptake appeared, therefore, to be due to carrier-mediated active transport. The pH optimum of the uptake was 4.5. Leucine, proline, lysine, arginine and as-paragine were taken up at approximately similar rates as glutamine, and they also inhibited the uptake of glutamine. This, suggests that the uptake of glutamine was at least partly due to an unspecific carrier(s) also shared by other amino acids. The embryos also took up the dipepti.de glycykarcosine; the rate was about 6 nmol embryo⁻¹h⁻¹ (1.5 μol g⁻¹h⁻¹) (2 mM glycylsarcosine, pH 4.5, 30°C). The uptake was inhibited by about 70% by dinitrophenol or by 300 m M glycylglycine. This indicates that the bulk of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was about 4.5.
The rates of glutamine and glycylsarcosine uptake increased during the early and middle stages of embryo development (until day 28 after anthesis), but decreased towards the end of the maturation of the grain. These changes, as well as the relatively high activities, suggest that carrier-mediated active uptake of amino acids, and possibly also that of peptides, plays a role in the nutrition of the developing embryo.     

The comparative effects of nitrogen and oxygen on the microflora of beef steaks in carbon dioxide-containing modified atmosphere vacuum skin-packaging (MA-VSP) systems

The effects of 80% oxygen–20% carbon dioxide (O₂–CO₂) and 80% nitrogen–20% carbon dioxide (N₂–CO₂) atmospheres were compared with respect to the microbial and sensory characteristics of vacuum skin-packaged grain-fed beef steaks stored at −1 and 4 °C. In both N₂–CO₂ and O₂–CO₂ atmospheres, lactobacilli were predominant over Brochothrix , pseudomonads, enterobacteria and yeasts and moulds. The results of the current investigation showed that the O₂–CO₂ atmospheres did not yield total viable counts in excess of 10⁵ cfu cm⁻² on beef steaks after 4 weeks of storage. However, the sensory analysis and thiobarbituric acid (TBA) values (as a measure of oxidative rancidity) of the products were unacceptable at this time. In contrast, the N₂–CO₂ atmospheres yielded maximum total viable counts of approximately 10⁷ cfu cm⁻² and the sensory analysis and TBA values of the product were judged to be acceptable after 4 weeks of storage at −1 °C. These results indicate that sensory effects of the product were influenced to a greater extent by the chemical effects of high concentration of O₂ on rancidity than by the high levels of lactobacilli.