Stop-flow: A new technique for measuring axonal transport,and its application to the transport of dopamine-β-hydroxylase

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-β-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3°C, while keeping the remainder at 37°C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37°C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 ± 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

Temperature-dependence of rapid axonal transport in sympathetic nerves of the rabbit.

Stop-flow techniques were used to determine how temperature affected the axonal transport of dopamine-beta-hydroxylase (DBH) activity in rabbit sciatic nerves in vitro. These nerves were cooled locally to 2 degrees C for 1.5 hr, which caused a sharp peak of DBH activity to accumulate above the cooled region. Accumulated DBH was then allowed to resume migration at various temperatures. From direct measurements of the rate of migration, we found that the axonal transport velocity of DBH was a simple exponential function of temperature between 13 degrees C and 42 degrees C. Over this range of temperatures, the results were well described by the equation: V=0.546(1.09)T, where V is velocity in mm/hr, and T is temperature in degrees centigrade. The Q10 between 13 degrees and 42 degrees C was 2.33, and an Arrhenius plot of the natural logarithm of velocity versus the reciprocal of absolute temperature yielded an apparent activation energy of 14.8 kcal. Transport virtually halted when temperature was raised to 47 degrees C, although only about half of the DBH activity disappeared during incubation at this temperature. Another transition occurred at 13 degrees C; below this temperature, velocity fell precipitously. This was not an artifact peculiar to the stop-flow system since the rate of accumulation of DBH activity proximal to a cold-block also decreased abruptly when the temperature above the block was reduced below 13 degrees C.     

Stop-flow: a new technique for measuring axonal transport, and its application to the transport of dopamine-beta-hydroxylase.

An apparatus was devised which utilizes local cooling to reversibly interrupt the axonal transport of dopamine-beta-hydroxylase (DBH) in rabbit sciatic nerves in vitro. Lowering the temperature of a short region of nerve to between 1 and 3 degrees C, while keeping the remainder at 37 degrees C, caused DBH activity to accumulate in and proximal to the cooled region. This accumulation was evident after 0.5 hr of cooling and increased in a nearly linear fashion with time for about 3 hr. The cooling-induced interruption in transport was rapidly reversed when nerves were rewarmed to 37 degrees C. Upon rewarming after local cooling for 1.5 hr, a peak of accumulated DBH activity migrated toward the distal end of the nerve at a velocity of 300 +/- 17 mm/day. This velocity was maintained for as long as the peak could be followed and was four times greater than the average velocity estimated from the rate of accumulation of DBH activity above a ligature at the distal end of these same nerves. It is concluded that ligation experiments grossly underestimate the true velocity of axonal transport of DBH and that the present technique offers great advantages in permitting direct study of the migration of separate axonal compartments of transported materials.     

Direct comparison of the rapid axonal transport of norepinephrine and dopamine-β-hydroxylase activity

Stop-flow techniques were used to examine the rapid axonal transport of norepinephrine in rabbit sciatic nerves. When the midpoint of a nerve incubated in vitro was cooled to 2°C while the remainder was kept at 37°C, norepinephrine accumulated proximal to the cooled region at a rate corresponding to an average transport velocity between 5 and 6 mm/hr in a distal direction. Since only about half of the norepinephrine appeared to be free to move, the mean velocity of the moving fraction was probably twice as great. No norepinephrine accumulated distal to a broad cooled region under conditions in which there would have been a significant accumulation of dopamine-β-hydroxylase activity. Therefore, unlike dopamine-β-hydroxylase, norepinephrine may not be subject to rapid retrograde transport. When nerves that had been locally cooled for 1.5 hr were rewarmed uniformly to 37°C, a wave of norepinephrine moved exclusively in a distal direction. The peak of this wave moved at a velocity of 12.2 ± 0.5 mm/hr or 293 ± 12 mm/day; the front of the wave moved at about 18 mm/hr. or 430 mm/day; and the tail probably moved faster than 6 mm/hr. This spectrum of velocities was virtually identical to the one displayed by the wave of dopamine-β-hydroxylase activity that was generated under the same conditions. Our results are consistent with the conclusion that all axonal structures containing norepinephrine also contain dopamine-β-hydroxylase, but they are not consistent with the converse.     

STOP-FLOW ANALYSIS OF THE AXONAL TRANSPORT OF DOPA DECARBOXYLASE (EC 4.1.1.26) IN RABBIT SCIATIC NERVES

The axonal transport of DOPA-decarboxylase (EC 4.1.1.26) was investigated in rabbit sciatic nerves by means of in vitro stop-flow techniques. Enzyme activity accumulated just proximal to a region that was locally cooled to 5°C in nerves that were elsewhere incubated at 37°C. The accumulation of enzyme activity was linear with time and corresponded to an average orthograde transport velocity of 11 mm/day. Retrograde transport was not detected. When nerves that had been locally cooled for 3 h were rewarmed, the accumulated enzyme activity moved distally along them as a wave with a narrow range of velocities. The front of this wave traveled at a rate of about 150mm/day, and the mean velocity of the wave was about 120 mm/day. These values are much lower than those previously obtained for tyrosine hydroxylase (EC 1.14.16.2), dopamine-β-hydroxylase (EC 1.14.2.1) and norepinephrine in similarly designed experiments. Thus DOPA-decarboxylase appeared to be transported at intermediate velocities, and, since the mean velocity of the moving fraction was about 11 times the average velocity, it is ljkely that only 9% of the enzyme was undergoing transport at any given moment.     

Sperm quality of Colossoma macropomum after room‐temperature and cold storage

The objective of this study was to evaluate the effects of cold and room‐temperature storage on the quality of Colossoma macropomum sperm. The experiment was carried out in December (end of Spring), in Nova Mutum‐MT, Brazil, involving nine C. macropomum males (4 years old; 6.4 ± 1.5 kg average weight). The fish were selected and transferred to masonry tanks (4 m³) in a laboratory (water renewal rate: 10 L/s; average water temperature: 28°C). Subsequently, reproduction was induced using 2.5 mg of crude carp pituitary extract/kg and the semen was harvested 240 degree hours after hormonal induction. The following sperm characteristics were analyzed every 5 hr using Image J/casa software: total motility (MOT), curvilinear velocity (VCL), average path velocity (VAP), straight‐line velocity (VSL), straightness of sperm path (STR), wobble (WOB), progressive motility (PROG), beat cross frequency (BCF) and total number of spermatozoa (NSPZ). A fresh sample of semen from each animal was kept at room temperature (25.3 ± 1.2°C). For analysis of cooled semen, syringes were kept in cooling boxes at an average temperature of 16.9 ± 2.1°C. The reduction (p < 0.05) of MOT in semen kept at room temperature occurred at 10 hr (13.95%); in cooled semen, however, MOT declined at 15 hr (76.87%). At 15 hr, there was practically no MOT in the semen kept at room temperature (0.20%), whereas in the cooled semen this situation was observed only at 35 hr (2.91%) The MOT of cooled sperm was higher (p < 0.05) at all times (except zero time), compared with the semen maintained at room temperature. At 15 hr, the cooled spermatozoa showed higher (p < 0.05) VCL (142.18 μm/s) and BCF (29.72 Hz) than those maintained at room temperature (VCL: 51.18 μm/s; BCF: 19.57 Hz). After 15 hr, only the cooled sperm showed quality. In conclusion, semen cooling allows for extending the viability of C. macropomum spermatozoa from 5 to 10 hr without compromising their quality in most characteristics. At 15 and 25 hr of cooling, sperm viability is still observed, though with decreased quality.     

Low temperature slowing and cold-block of fast axoplasmic transport in mammalian nerves in vitro

1) Fast axoplasmic transport in mammalian nerve in vitro was studied using an isotope labeling technique. The rate of outflow in cat sciatic nerve fibers of 410 mm/day in vitro was reduced at temperatures below 38°C with a Q₁₀ of 2.0 in the range 38–18°C and a Q₁₀ of 2.3 at 38–13°C. 2) At a temperature of 11°C a partial failure of transport occurred. At temperatures below 11°C a complete block of fast axoplasmic transport occurred, a phenomenon termed “cold-block.” No transport at all was seen over the temperature range of 10–0°C for times lasting up to 48 hr. 3) Transport was resumed after a period of cold-block lasting up to 22 hr when the nerves were brought back to a temperature of 38°C. Some deleterious effects due to cold-block were seen in the recovery phase as indicated by a reduction in crest amplitude, change in its form, and slowed rate. 4) The ∼P level (combined ATP and creatine phosphate) remained near control level in nerves kept at low or cold-block temperatures for times as long as 64 hr. The reduction in fast axoplasmic transport rate seen at low temperatures for times up to 22 hr was therefore considered due to a decrease in the utilization of ATP, a concept in accord with the “transport filament” model proposed to account for fast axoplasmic transport. 5) The sloping of the front of the crest over the temperature range of 18–13°C suggests an additonal factor at the lower temperatures. A disassembly of microtubules is discussed as a possible explanation of the cold-block phenomenon.     

Temperature dependence of two parameters in a photosynthesis model

The temperature dependence of the photosynthetic parameters V_cmax, the maximum catalytic rate of the enzyme Rubisco, and J_max, the maximum electron transport rate, were examined using published datasets. An Arrehenius equation, modified to account for decreases in each parameter at high temperatures, satisfactorily described the temperature response for both parameters. There was remarkable conformity in V_cmax and J_max between all plants at T_leaf < 25 °C, when each parameter was normalized by their respective values at 25 °C (V_cmax0 and J_max0), but showed a high degree of variability between and within species at T_leaf > 30 °C. For both normalized V_cmax and J_max, the maximum fractional error introduced by assuming a common temperature response function is < ± 0·1 for most plants and < ± 0·22 for all plants when T_leaf < 25 °C. Fractional errors are typically < ± 0·45 in the temperature range 25–30 °C, but very large errors occur when a common function is used to estimate the photosynthetic parameters at temperatures > 30 °C. The ratio J_max/V_cmax varies with temperature, but analysis of the ratio at T_leaf = 25 °C using the fitted mean temperature response functions results in J_max0/V_cmax0 = 2·00 ± 0·60 (SD, n = 43).     

Separate and combined effects of temperature and hypoxia on breathing pattern in the domestic fowl

Summary Minute ventilation (V _E), tidal volume (V _T), respiratory frequency (f) and clavicular air sac gas composition were measured in conscious domestic fowl breathing air and hypoxic gas mixtures at neutral (18±1°C) and raised (33±1°C) air temperatures. Increases inV _E caused by inhalation of 10%, 8% or 6.5% O₂ in N₂, respectively, were independent of temperature although at each level the absoluteV _E was ca. 21·min⁻¹ greater in the panting birds. Changes in respiratory pattern during hypoxia were markedly dependent on temperature. At 18°C almost all of the increasedV _E resulted from increasedf. At 33°C hypoxia led to a strong suppression off and increase inV _T. It is concluded that hyperthermia and hypoxia are additive and non-interactive in their effects on ventilatory drive, in agreement with previously reported effects of hypercapnia and physical exercise on breathing in panting fowl.     

Photosynthetic capacity and temperature responses of photosynthesis of rubber trees (Hevea brasiliensis Müll. Arg.) acclimate to changes in ambient temperatures

The aim of this study was to assess the temperature response of photosynthesis in rubber trees (Hevea brasiliensis Müll. Arg.) to provide data for process-based growth modeling, and to test whether photosynthetic capacity and temperature response of photosynthesis acclimates to changes in ambient temperature. Net CO₂ assimilation rate (A) was measured in rubber saplings grown in a nursery or in growth chambers at 18 and 28°C. The temperature response of A was measured from 9 to 45°C and the data were fitted to an empirical model. Photosynthetic capacity (maximal carboxylation rate, V _cmax, and maximal light driven electron flux, J _max) of plants acclimated to 18 and 28°C were estimated by fitting a biochemical photosynthesis model to the CO₂ response curves (A–C _i curves) at six temperatures: 15, 22, 28, 32, 36 and 40°C. The optimal temperature for A (T _opt) was much lower in plants grown at 18°C compared to 28°C and nursery. Net CO₂ assimilation rate at optimal temperature (A _opt), V _cmax and J _max at a reference temperature of 25°C (V _cmax25 and J _max25) as well as activation energy of V _cmax and J _max (E _aV and E _aJ) decreased in individuals acclimated to 18°C. The optimal temperature for V _cmax and J _max could not be clearly defined from our response curves, as they always were above 36°C and not far from 40°C. The ratio J _max25/V _cmax25 was larger in plants acclimated to 18°C. Less nitrogen was present and photosynthetic nitrogen use efficiency (V _cmax25/N _a) was smaller in leaves acclimated to 18°C. These results indicate that rubber saplings acclimated their photosynthetic characteristics in response to growth temperature, and that higher temperatures resulted in an enhanced photosynthetic capacity in the leaves, as well as larger activation energy for photosynthesis.     

AXONAL TRANSPORT OF PHENYLETHANOLAMINE-N-METHYLTRANSFERASE IN TOAD SCIATIC NERVE

—The presence of phenylethanolamine-N-methyltransferase (EC 2.1.1.-) and dopamine-β-hydroxylase (EC 1.14.2.1) activities was demonstrated in the sciatic nerve of the toad, Bufo marinus. The rates of accumulation of phenylethanolamine-N-methyltransferase (PNMT) and dopamine-β-hydroxylase (DBH) proximal to a ligation of the sciatic nerve were studied. DBH accumulated proximal to the ligation at a more than 10-fold faster rate than PNMT. By measuring the rate of loss of enzyme activity distal to a ligation, an estimate of per cent clearance of each enzyme was made. Based on the per cent of enzyme activity free to move, the absolute transport rates for each enzyme were estimated to be: PNMT, 3.6 mm/24 h; DBH, 102 mm/24 h. PNMT activity (89 per cent) was recovered in the soluble fraction of sciatic nerve homogenates with no change occurring in the subcellular distribution of the enzyme proximal to ligations. In contrast, 43 per cent of DBH activity was found in the soluble fraction of sciatic nerve homogenates; but a disproportionate increase in paniculate DBH activity was found proximal to sciatic nerve ligations. Reduction of toad body temperature to 4°C resulted in a complete but totally reversible block of the axonal transport of both PNMT and DBH.     

Germination and emergence of Sorghum bicolor. genotypic and environmentally induced variation in the response to temperature and depth of sowing

Abstract The germination of Sorghum bicolor seeds of 9 genotypes was tested at temperatures between 8°C and 48°C on a thermal gradient plate. Samples were tested from three regions of the panicle expected to differ in temperature during grain filling. Seeds of a tenth genotype, SPV 354, produced in controlled-environment glasshouses at different panicle temperatures, were tested similarly. In addition, the emergence of SPV 354 was measured from planting depths of 2 and 5 cm at mean soil temperatures of 15, 20 and 25°C. Four methods of calculating mean germination rate for the nine genotypes were compared. Germination characters like base, optimum and maximum temperature (T_b, T_o, T_m), thermal time (θ)and the germination rate at T_o(R_max showed only small differences between methods. There was a range of genotypic variation in all characters: T_b 8.5–11.9°C; T_o, 33.2–37.5°C; T_m, 46.8–49.2°C; θ, 23.4–38.0°Cd; R_max, 0.69–1.14-d_-1. In contrast, mean germinability (G) was between 90% and 100% over the temperature range 13–40°C. Panicle temperature had no effect on any germination character in SPV 354. However, deeper burial increased θ for emergence and decreased G, irrespective of soil temperature except at 5 cm. Increasing panicle temperature, by reducing seed size, reduced G and increased θ by about 10% only at 15°C and 5 cm depth.     

Changes in exercise and post-exercise core temperature under different clothing conditions

 This study evaluates the effect of different levels of insulation on esophageal (T _es) and rectal (T _re) temperature responses during and following moderate exercise. Seven subjects completed three 18-min bouts of treadmill exercise (75% VO_2max, 22°C ambient temperature) followed by 30 min of recovery wearing either: (1) jogging shoes, T-shirt and shorts (athletic clothing); (2) single-knit commercial coveralls worn over the athletic clothing (coveralls); or (3) a Canadian Armed Forces nuclear, bacteriological and chemical warfare protective overgarment with hood, worn over the athletic clothing (NBCW overgarment). T _es was similar at the start of exercise for each condition and baseline T _re was ∼0.4°C higher than T _es. The hourly equivalent rate of increase in T _es during the final 5 min of exercise was 1.8°C, 3.0°C and 4.2°C for athletic clothing, coveralls and NBCW overgarment respectively (P<0.05). End-exercise T _es was significantly different between conditions [37.7°C (SEM 0.1°C), 38.2°C (SEM 0.2°C and 38.5°C (SEM 0.2°C) for athletic clothing, coveralls and NBCW overgarment respectively)] (P<0.05). No comparable difference in the rate of temperature increase for T _re was demonstrated, except that end-exercise T _re for the NBCW overgarment condition was significantly greater (0.5°C) than that for the athletic clothing condition. There was a drop in T _es during the initial minutes of recovery to sustained plateaus which were significantly (P<0.05) elevated above pre-exercise resting values by 0.6°C, 0.8°C and 1.0°C, for athletic clothing, coveralls, and NBCW overgarment, respectively. Post-exercise T _re decreased very gradually from end-exercise values during the 30-min recovery. Only the NBCW overgarment condition T _re was significantly elevated (0.3°C) above the athletic clothing condition (P<0.05). In conclusion, T _es is far more sensitive in reflecting the heat stress of different levels of insulation during exercise and post-exercise than T _re. Physiological mechanisms are discussed as possible explanations for the differences in response. Received: 30 June 1998 / Accepted: 19 February 1999     

Cell viability and protein turnover in nongrowingBacillus megaterium at sporulation suppressing temperature

InBacillus megaterium, a temperature that suppresses sporulation (43°C) only slightly exceeds both the optimum growth temperature and the temperature still permitting sporulation (40–41°C). Here we show that, when cells grown at 35°C and transferred to a sporulation medium, were subjected to shifts between 35°C and the sporulation suppressing temperature (SST, 43°C), their development and proteolytic activities were deeply affected. During the reversible sporulation phase that took place at 35°C for 2–3 h (T₂–T₃), the cells developed forespores and their protein turnover was characterized by degradation of short-lived proteins and proteins made accessible to the proteolytic attack because of starvation. During the following irreversible sporulation phase refractile heat-resistant spores appeared at T₄–T₅. Protein turnover rate increased again after T₂ and up to T₈ 60–70% prelabelled proteins were degraded. The SST suppressed sporulation at its beginning; at T₃ no asymmetric septa were observed and the amount of heat-resistant spores at T₈ was by 4–5 orders lower than at 35°C. However, the cells remained viable and were able to sporulate when transferred to a lower temperature. Protein degradation was increased up to T₃ but then its velocity sharply dropped and the amount of degraded protein at T₈ corresponded to slightly more than one-half of that found at 35°C. The cytoplasmic proteolytic activity was enhanced but the activity in the membrane fraction was decreased. When a temperature shift to SST was applied at the beginning of the irreversible sporulation phase (T_2.5), the sporulation process was impaired. A portion of forespores lyzed, the others were able to complete their development but most spores were not heat-resistant and their coats showed defects. Protein degradation increased again because an effective proteolytic system was developed during the reversible sporulation phase but the amount of degraded protein was slightly lower than at 35°C. A later (T₄) shift to SST had no effect on the sporulation process.     

Impact of two different types of heat stress on chloroplast movement and fluorescence signal of tobacco leaves

Although the chloroplast movement can be strongly affected by ambient temperature, the information about chloroplast movement especially related to high temperatures is scarce. For detailed investigation of the effects of heat stress (HS) on tobacco leaves (Nicotiana tabacum L. cv. Samsun), we used two different HS treatments in dark with wide range of elevated temperatures (25–45°C). The leaf segments were either linearly heated in water bath at heating rate of 2°C min⁻¹ from room temperature up to maximal temperature (T _m) and then linearly cooled down to 25°C or incubated for 5 min in water bath at the same T _m followed by 5 min incubation at 25°C (T-jump). The changes in light-induced chloroplast movement caused by the HS pretreatment were detected after the particular heating regime at 25°C using a method of time-dependent collimated transmittance (CT) and compared with the chlorophyll O–J–I–P fluorescence rise (FLR) measurements. The inhibition of chloroplast movement started at about 40°C while the fluorescence parameters responded generally at higher T _m. This difference in sensitivity of CT and FLR was higher for the T-jump than for the linear HS indicating importance of applied heating regime. A possible influence of chloroplast movement on the FLR measurement and a physiological role of the HS-impaired chloroplast movement are discussed.     

Adaptation of soil microbial growth to temperature: Using a tropical elevation gradient to predict future changes

Terrestrial biogeochemical feedbacks to the climate are strongly modulated by the temperature response of soil microorganisms. Tropical forests, in particular, exert a major influence on global climate because they are the most productive terrestrial ecosystem. We used an elevation gradient across tropical forest in the Andes (a gradient of 20°C mean annual temperature, MAT), to test whether soil bacterial and fungal community growth responses are adapted to long‐term temperature differences. We evaluated the temperature dependency of soil bacterial and fungal growth using the leucine‐ and acetate‐incorporation methods, respectively, and determined indices for the temperature response of growth: Q₁₀ (temperature sensitivity over a given 10oC range) and T_min (the minimum temperature for growth). For both bacterial and fungal communities, increased MAT (decreased elevation) resulted in increases in Q₁₀ and T_min of growth. Across a MAT range from 6°C to 26°C, the Q₁₀ and T_min varied for bacterial growth (Q_10–20 = 2.4 to 3.5; T_min = ?8°C to ?1.5°C) and fungal growth (Q_10–20 = 2.6 to 3.6; T_min = ?6°C to ?1°C). Thus, bacteria and fungi did not differ significantly in their growth temperature responses with changes in MAT. Our findings indicate that across natural temperature gradients, each increase in MAT by 1°C results in increases in T_min of microbial growth by approximately 0.3°C and Q_10–20 by 0.05, consistent with long‐term temperature adaptation of soil microbial communities. A 2°C warming would increase microbial activity across a MAT gradient of 6°C to 26°C by 28% to 15%, respectively, and temperature adaptation of microbial communities would further increase activity by 1.2% to 0.3%. The impact of warming on microbial activity, and the related impact on soil carbon cycling, is thus greater in regions with lower MAT. These results can be used to predict future changes in the temperature response of microbial activity over different levels of warming and over large temperature ranges, extending to tropical regions.     

The role of temperature in enhancing the insecticidal activity of Bacillus thuringiensis against Spodoptera littoralis larvae

The present study scrutinised how far temperature would affect the velocity of the insecticidal activity of Bacillus thuringiensis, as the rapidity of pest control achievements is of a great concern. Third instar Spodoptera littoralis larvae were treated with Bt at three concentration levels under five different temperatures (15°C, 20°C, 25°C, 30°C and 35°C). LT₅₀s were evaluated in each case. The LT₅₀ values showed various levels of reductions as temperature and/or Bt concentration increased, indicating that the velocity of mortality (1/LT₅₀) and/or the rapidity of Bt activity was almost temperature dependant. However, relatively high and low reduction percentages in the LT₅₀ values on the elevation of 5°C were obtained at lower and higher temperature ranges, respectively. The temperature coefficient, Q ₁₀ values, determined within narrow ranges (5°C) showed great reductions when temperature increased from 15°C to 20°C at all Bt concentrations. Raising temperature by 5°C above 20°C or 25°C almost caused similar Q ₁₀ values indicating constant increase in the response of Bt activity within 20–30°C temperature range. Q ₁₀ values over 30°C were comparatively very low. This proved that decrease in Q ₁₀ values due to the rise of temperature was dependant on the starting temperature.     

Heterotrophic activity of bacterioplankton along the gradients of the chlorophyll <Emphasis Type="Italic">a</Emphasis> concentration and water temperature in marine and limnetic ecosystems

The dependence of the heterotrophic activity of bacterioplankton (V, μg C L^–1 h^–1) on the concentration of chlorophyll a (Chl, μg L^–1) and the water temperature (T) was examined for lakes (37°29′–80°36′ N) and marine polar waters (69°16′–80°36′ N). It was shown that ~76% of the V variations was related to changes in Chl and T.     

Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus

Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (T_m) of 77.3 ± 0.1°C (4.6°C higher than the wild-type) and a half-life of over 13 hr at 65°C (7.9-fold better than the wild-type), with unchanged kinetic parameters. Stabilizing mutations Q11L and A191S were incorporated into PFEI variant L30P, previously described to be enantioselective in the hydrolysis of the (−)-enantiomer of the Vince lactam. The final variant Q11L/L30P/A191S showed a significant improvement in thermal stability (T_m of 80.8 ± 0.1°C and a half-life of 65 min at 75°C), while retaining enantioselectivity (E > 100). Structural studies revealed that A191S establishes a hydrogen bond network between a V-shaped hairpin and the α/β hydrolase domain that leads to higher rigidity and thus would contribute to explaining the increase in stability.