Fibroblast procollagen production rates in vitro based on [3H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [3H]proline and measuring the subsequent formation of [3H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [3H]proline, and [3H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [14C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45-55]. Procollagen production rates were readily determined by this method using 5 to 20 microCi [3H]proline and approximately 10(6) cells. It was found that 3H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/microgram DNA . h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

海草生物量和初级生产力研究进展

海草床是近岸重要的湿地生态系统,具有较高生物量和生产力。海草的生物量和生产力变化除了受到光照、无机碳源、营养盐、温度、盐度、水动力条件、铁限制和污染物等非生物因素制约外,还受到附生藻类和动物摄食等生物因素影响。非生物因素一般有最适合海草生长的范围,生物因素的影响具有两面性。海草生物量和生产力研究基本处于由定性向定量过渡阶段,准确便捷的方法、现场多因子围隔实验、更大时空尺度上的对比研究是今后研究的重点。     

Measurement of oxygen consumption and carbon dioxide production rates by a tracer-bolus method

A new method for measuring oxygen consumption and carbon dioxide production is described. The method is based on the injection of a helium bolus into the inspired gas for repeated breaths; the helium can be delivered through the mouth or through a nostril (the injection system being controlled by the integrated flow signal or by the signal of a thermistor, respectively). Compared with the conventional gas-collection technique, the tracer-bolus method allows more rapid and frequent measurements. The method does not necessarily require an airtight respiratory circuit, so that it can be conveniently applied in less cooperative subjects, including children. The results of validation studies based on a comparison with the standard gas-collection technique are presented; these results indicate that the measurements by the tracer-bolus method are accurate in normal subjects. In patients with obstructive lung disease, the determinations are associated with a systematic error, which is expected, the tracer-bolus method being based on a single-compartment, uniformly ventilated and perfused lung model; the error is, however, predictable from the degree of functional impairment and can be corrected using equations based on routine pulmonary function tests.     

Statistics of the Underwater Light Field: An Empirical Model

The underwater light field is described as a stochastic process, with the vertical attenuation as a random variable. It is shown that the vertical attenuation integral is well described as a Normal process with uncorrelated increments. The attenuation processes within a water body are found to be quite independent of the incoming irradiance. Thus, the relative light intensity at any depth can be approximated by a Lognormal random variable. Based upon the expectation of this Lognormal variable, and the mean value of the incident irradiance, the irradiance delivered at any depth can be estimated, as well as the statistical distribution of the irradiance. Light data from a number of Norwegian soft-water lakes showed good fit to the model. Two productive lakes included in this study also had a light regime well described by the statistical model. However, the model should be extended to cater for seasonal variations in these applications.     

Fibroblast procollagen production rates in vitro based on [H]hydroxyproline production and procollagen hydroxyproline specific activity

In vitro procollagen production rates can be determined by culturing cells in the presence of [³H]proline and measuring the subsequent formation of [³H]hydroxyproline. Values of actual procollagen production can be calculated if the total radioactivity and the specific activity of the newly synthesized procollagen is known. A simple microanalytical method for measuring procollagen specific activity in order to determine procollagen production by lung fibroblasts in vitro is reported. Confluent fibroblasts (IMR-90) were cultured in fresh medium containing [³H]proline, and [³H]hydroxyproline production and prolyl hydroxylation were measured. Hydroxyproline specific activity of nondialyzable procollagen in culture medium as well as extracellular and intracellular free proline specific activity were determined by an ultramicromethod in which the radiolabeled amino acids were reacted with [¹⁴C]dansyl chloride of known specific activity [Airhart et al. (1979) Anal. Biochem. 96, 45–55]. Procollagen production rates were readily determined by this method using 5 to 20 μCi [³H]proline and approximately 10⁶ cells. It was found that ³H-procollagen production rate into culture medium was constant after a lag of 1.6 h, while procollagen production rate (0.23 pmol/μg DNA · h) was constant from time zero to 9 h. The specific activities of extracellular and intracellular free proline were not constant during the labeling period, nor were they equal to procollagen specific activity. These data indicate that free proline pool specific activities are not a valid measure of procollagen specific activity. The experimental approach described obviates the need to define or characterize the proline precursor pool from which procollagen is synthesized, and may be readily applied to determine fibroblast procollagen production rates in vitro.     

Measurement of fractionated water loss and CO2 production using triply labelled water

The doubly labelled water (DLW) method for measuring CO2 production has recently been the subject of much interest since no other technique gives integrated values for CO2 production over long periods by free-living subjects. The importance of evaporative water loss and fractionation factors to the calculation of CO2 production using this technique is described. Present methods of estimating evaporative water loss and the use of fractionation factors are summarized together with a discussion of their limitations. A novel technique is proposed whereby water labelled with three isotopes can be used to measure evaporative water loss and CO2 production in completely free-living subjects, and the feasibility of the method is tested in simulations using experimental data. This technique has three advantages over existing methods of estimating evaporative water loss: (1) it can be used in completely free-living subjects without any additional experimental procedures (e.g. water-balance studies or physical trapping of water vapour); (2) it gives a direct estimate of fractionated evaporative water loss in each subject, since non-fractionated water lost as vapour is automatically compensated for; and (3) the routes of water loss do not have to be known. The appropriate calculations are presented together with a discussion of the difficulties of measuring oxygen-17 by mass spectrometry. It is estimated that the maximum theoretical error on calculated CO2 production is +/- 0.3%. Practical ways of achieving this theoretical level of accuracy are suggested. We conclude that the proposed technique will allow correction for evaporative water loss to be made more exactly, thereby increasing the accuracy of the heavy water technique for measuring CO2 production in free-living subjects.     

模拟水流条件下初级生产力及光动力学参数

为探讨水动力作用及其他物理因子改变对湖泊初级生产力的影响 ,1999年 5月 8日～ 6月 2 4日在中国科学院太湖湖泊生态系统研究站大型生态实验槽内进行模拟水动力实验 ,分 3种水流状态 2种光强测定初级生产力及其他相关参数。分析了初级生产力、光合速率的垂直分布 ,光合速率随光强的变化 ,并借助光动力学模型拟合得到光动力学参数。结果表明 ,在静止状态下 ,当水表面光强大于 5 0 0μmol/ (m2 · s)时 ,0～ 0 .4 m处存在光抑制现象 ,最大初级生产力出现在 0 .4～ 0 .6 m,此后由于动力作用使水体悬浮物增加 ,改变了水下光照条件 ,致使最大初级生产力呈向上移动的趋势 ,出现在 0～ 0 .2 m间 ;光合速率在静止状态下随深度递减缓慢 ,而到大水流状态则递减极为迅速 ,大水流状态下的平均光合速率明显低于静止状态和小水流状态 ;基于 2种类型的光动力学模型进行非线性拟合得到的 P- I曲线相关性很好 ,2种模型模拟的结果比较接近 ,基本上能够反映太湖光合速率随光强变化的实际情况 ;在太湖这种大型浅水湖泊 ,水动力的作用使得水体中悬浮物增加 ,造成光强的迅速衰减 ,这可能会大大降低湖泊的初级生产过程     

Reconstructed long-term time series of phytoplankton primary production of a large shallow temperate lake: the basis to assess the carbon balance and its climate sensitivity

Aiming at building the carbon budget for further climate change impact research in the large and shallow northern temperate Lake Võrtsjärv, the present paper focuses on reconstructing the full phytoplankton primary production (PP) data series for the lake for the period of 1982–2009 covered by disconnected measurements, and testing the uncertainties involved both in the PP measurements and bio-optical modelling. During this 28-year period, in situ PP was measured in Võrtsjärv in 18 years with ¹⁴C-assimilation technique. We reconstructed the full time series using a semi-empirical PP simulation model based on continuously measured PAR irradiance and interpolated values of monthly measured chlorophyll a (C _chl). The modelling results, which proved highly reliable during the calibration phase, correlated rather weakly with the annual PP estimates for the 18 years, which were based on 2-h incubations at midday, 1–2 times per month. Being based on continuous irradiance data, the modelled PP can be considered more reliable than the sparse measurements, especially for short to medium term studies. We demonstrate that in the long-term, the bio-optical method can be biased if changes in water colour or water level alter the light climate causing adaptive responses in the cellular chlorophyll content of light-limited phytoplankton.     

Problems in developing the biotechnology of algal biomass production

Summary The effects of environmental conditions (solar irradiance and temperature) and population density on the production of Spirulina biomass with brackish water are reported for cultures grown in outdoor ponds. Higher specific growth rates were observed at lower population densities. Lower growth rates were associated with limitation by light in dense cultures under optimum conditions in the summer. Seasonal variation in productivity was observed. In summer, light was the limiting factor, whereas in winter the low daytime temperature appeared to constitute the major limitation. The oxygen concentration in the culture can serve as a useful indicator of limiting factors and can also be used to estimate the extent of such limitations.     

Highly predictable photosynthetic production in natural macroalgal communities from incoming and absorbed light

Photosynthesis–irradiance relationships of macroalgal communities and thalli of dominant species in shallow coastal Danish waters were measured over a full year to test how well community production can be predicted from environmental (incident irradiance and temperature) and community variables (canopy absorptance, species number and thallus metabolism). Detached thalli of dominant species performed optimally at different times of the year, but showed no general seasonal changes in photosynthetic features. Production capacity of communities at high light varied only 1.8-fold over the year and was unrelated to incident irradiance, temperature and mean thallus photosynthesis, while community absorptance was a highly significant predictor. Actual rates of community photosynthesis were closely related to incident and absorbed irradiance alone. Community absorptance in turn was correlated to canopy height and species richness. The close relationship of community photosynthesis to irradiance is due to the fact that (1) large differences in thallus photosynthesis of individual species are averaged out in communities composed of several species, (2) seasonal replacement of species keeps communities metabolically active, and (3) maximum possible absorptance at 100% constrains the total photosynthesis of all species. Our results imply that the photosynthetic production of macroalgal communities is more predictable than their complex and dynamic nature suggest and that predictions are possible over wide spatial scales in coastal waters by measurements of vegetation cover, incoming irradiance and canopy absorptance.     

Effects of light history on primary productivity in a phytoplankton community dominated by the toxic cyanobacterium Cylindrospermopsis raciborskii

1. The effects of instantaneous irradiance and short‐term light history on primary production were determined for samples from a subtropical water reservoir dominated by the toxic cyanobacterium Cylindrospermopsis raciborskii. ¹⁴C‐bicarbonate uptake incubations were conducted on water samples from the reservoir, for irradiance (photosynthetically active radiation) ranging from 0 to 1654 μmol quanta m⁻² s⁻¹. Prior to the ¹⁴C incubations, cells were pre‐treated at irradiance levels ranging from 0 to 1006 μmol quanta m⁻² s⁻¹. 2. The average irradiance experienced by cells during the 2–2.5 h pre‐treatment incubations affected the productivity–irradiance (P–I) parameters: exposure to high light in pre‐treatment conditions caused a substantial decrease in maximum rate of primary production P_max and the photoinhibition parameter β when compared to cells pre‐treated in the dark. 3. While the data collected in this study were not sufficient to develop a full dynamic model of C. raciborskii productivity, P_max and β were modelled as a function of pre‐treatment irradiance, and these models were applied to predict the rate of primary production as a function of both instantaneous and historical irradiance. The results indicated that while cells with a history of exposure to high irradiance will be the most productive in high irradiance, production rates will be highest overall for dark‐acclimated cells in moderate irradiance. 4. Our results may explain why optically‐deep mixing favours C. raciborskii. If the mixing depth z_m exceeds the euphotic depth z_eu, cells will be dark‐acclimated, which will increase their rate of production when they are circulated through the euphotic zone. These results also predict that production rates will be higher during morning hours than for the same irradiance in the afternoon, which is consistent with other phytoplankton studies. 5. Since the rate of production of C. raciborskii‐dominated systems cannot be described by a single P–I curve, accurate estimates of production rates will require measurements over the daily light cycle.     

High algal production rates achieved in a shallow outdoor flume

A mass culture of Tetraselmis suecica grown in seawater enriched with only inorganic nutrients and CO(2) in a shallow outdoor flume containing foil arrays to effect systematic vertical mixing achieved average daily production rates of over 40 g ash-free dry wt (AFDW)/m(2) over periods as long as one month when grown on a three-day dilution cycle. Photosynthetic efficiencies associated with these high production rates averaged 8-11% based on visible irradiance. Operation of the system in a one-, two-, or four-day dilution cycle resulted in lower photosynthetic efficiencies of 6-7%. A remarkable feature of the three-day dilution cycle results was the fact that production on the third day after dilution averaged 60-70 g AFDW/m(2), and corresponding photosynthetic efficiencies averaged 13-19%. The high production rates and photosynthetic efficiencies achieved on the third day after dilution may have reflected the nonequilibrium nature of the production cycle and, in particular, the fact that the adaptation of the cells to changing light condition lagged behind light condition in the culture.     

Relations between the thermocline meso and microzooplankton,chlorophyll a and primary production distributions in Lancaster Sound

Summary Primary productivity in Lancaster Sound was estimated from chlorophyll and light irradiance profiles and light saturation measurments. Mesozooplankton concentrations were measured using an in situ zooplankton counter mounted on a Batfish and with the BIONESS. Microzooplankton concentrations were estimated from samples taken with the BIONESS. The horizontal and vertical distribution of the two groups of zooplankton are described and related to the distribution of chlorophyll primary production and physical features of the Sound. A westward intrusion of Baffin Bay water along the north shore of the sound which mixed with Arctic water flowing easterly resulted in a complex physical environment in the sound. The admixing of these water masses resulted in a highly variable biological structure. The thermocline was the main physical feature that affected the depth of the chlorophyll layer and the levels of primary production, with a shallow thermocline resulting in higher primary production. The admixing of Baffin Bay water with Arctic water at different areas of the sound combined with the different times of appearance of open water resulted in the populations of Calanus hyperboreus and C. glacialis showing different age structures, the youngest being in the Arctic water at the western end of the Sound. Microzooplankton populations were concentrated at various depths depending on the species. Oithona juveniles preferred the upper 10 m when the thermocline was near the surface. Most macrozooplankton were found below the thermocline and the primary production maxium. The largest concentrations of microzooplankton were found at the western end of the Sound in the Arctic water believed to be still in the biological spring.     

Cinétique de la rhizogenèse adaptative à la sécheresse. Relation avec I'évolution des paramètres hydriques et morphologiques chez le Sinapis alba, à deux niveaux d'énergie lumineuse

Kinetics of adaptive drought rhizogenesis, as related to growth and water deficits in Sinapis alba, at two irradiance levels .
The rhizogenic activity in Sinapis alba L., subjected to progressively increasing water deficit during growth, shows a sigmoidal pattern of kinetics with three phases: initiation or latent phase, rapid exponential increase, and stationary phase. This pattern remains unchanged if the level of irradiance during growth is reduced. The same number of short tuberized roots is obtained as under high irradiance, although initiation of rhizogenesis appears to be delayed by a few days. However, this delay is not observed if the rhizogenesis is analyzed as a function of the water potential of aerial organs or as a function of the soil water deficit. Since the rate of dehydration depends upon the evaporative demand, which is modulated by high or low irradiance, drought rhizogenesis seems to be directly related to water deficits in the soil-plant system. The onset of drought rhizogenesis, starting at the end of the growth period of the aerial organs, appears concomitant with the transpiration decline which is induced by water stress at both irradiance levels.     

Managing water resources for crop production

Increasing crop production to meet the food requirements of the world''s growing population will put great pressure on global water resources. Given that the vast freshwater resources that are available in the world are far from fully exploited, globally there should be sufficient water for future agricultural requirements. However, there are large areas where low water supply and high human demand may lead to regional shortages of water for future food production. In these arid and semi-arid areas, where water is a major constraint on production, improving water resource management is crucial if Malthusian disasters are to be avoided. There is considerable scope for improvement, since in both dryland and irrigated agriculture only about one-third of the available water (as rainfall, surface, or groundwater) is used to grow useful plants. This paper illustrates a range of techniques that could lead to increased crop production by improving agricultural water use efficiency. This may be achieved by increasing the total amount of water available to plants or by increasing the efficiency with which that water is used to produce biomass. Although the crash from the Malthusian precipice may ultimately be inevitable if population growth is not addressed, the time taken to reach the edge of the precipice could be lengthened by more efficient use of existing water resources. <br>     

Microcystin production by Microcystis aeruginosa: Direct regulation by multiple environmental factors

Effects of environmental variations on microcystin (MCYST) production were tested by evaluating results obtained from batch culture experiments with Microcystis aeruginosa MASH-01A19. Time series of cell and MCYST concentrations were evaluated independently with two models, a static linear and a dynamic non-linear model. Significant influences of irradiance, sulphur and phosphorus (P_i) supply on MCYST production were identified and notably, these effects occurred independently of influences on growth rate. Specifically, effects on the two main parameters intrinsic growth rate and MCYST production coefficient were inversely correlated. With increasing irradiance and P_i supply MCYST production was reduced. By contrast, MCYST production was lowered when sulphur was depleted. Since MCYST production was influenced by more than one environmental variable it is suggested that MCYST is under multiple regulation. A well-distinguishable control of MCYST production by one environmental factor can be excluded. All the observed effects on MCYST production do not disagree with earlier findings that MCYST may be involved in processes linked to photosynthesis.     

Impact of inertial period internal waves on fixed-depth primary production estimates

Fixed-depth subthermocline phytoplankton production estimateswere compared to variable-depth estimates calculated along thesimulated trajectory of an internal wave. The mean of 5000 MonteCarlo simulations, varying wave phase and amplitude, was notsignificantly different than the fixed-depth estimate for averageLake Michigan internal waves. Differences were significant,however, for wave amplitudes >5 m but only at some depths.Differences between the two estimates were related to differencesin irradiance received and the portion of the photosynthesisirradiance(P-I) curve controlling production. Oscillating communitiesalways receive more irradiance than fixed-depth communitiesand the magnitude of this increase is related to the extinctioncoefficient and the amplitude of the internal waves. Productionwas also estimated along an individual isotherm trajectory (isotherm-derived)and compared to fixed-depth production. Larger differences betweenthese isotherm-derived and fixed-depth estimates were notedin some cases and were related to differences in the mean isothermdepth and the sampled (fixed) depth. If one accounts for thetrajectory of the sampled community, fixed-depth estimates arereliable; however, if the trajectory is unknown or unaccountedfor, any individual fixed-depth production estimate may notadequately measure in situ production. ¹Contribution No. 493, Great Lakes Environmental Research Laboratory     

Temperature significantly affects retroviral vector production and deactivation rates, and thereby determines retroviral titers

The retroviral titer obtained from the pMFG/ψCRIP producer cell line is determined by a dynamic interplay of vector production and deactivation rates. Both these rates are influenced by temperature. It was determined that; (i) the retroviral half-lives are strongly influenced by temperature and the temperature dependency can be described by the Arrhenius equation with an activation energy of 39 kcal/gmol; (ii) the actual retroviral vector productivity per cell is highest at 37?°C with retroviral production rate of 24.4(±7.0; ±?standard deviation) colony forming unit (CFU)/cell/day; (iii) the dynamic interplay of these two factors produces an optimal temperature of 34?°C for pMFG/ψCRIP cells under the culture conditions used; and (iv) the cellular growth rate is highest at 37?°C at 26.8 hr doubling time. Taken together, these parameters can be used to optimize a two-step retroviral production protocol, where the cells are first grown under optimal growth conditions (37?°C) and second, the virus is produced at 34?°C to yield the highest titer. These results have significant implications for optimal retroviral production protocols.     

Patterns and controls of the dynamics of net primary production by understory macroalgal assemblages in giant kelp forests

Macroalgae are important primary producers in many subtidal habitats, yet little information exists on the temporal and spatial dynamics of net primary production (NPP) by entire subtidal assemblages. This knowledge gap reflects the logistical challenges in measuring NPP of diverse macroalgal assemblages in shallow marine habitats. Here, we couple a simple primary production model with nondestructive estimates of taxon‐specific biomass on subtidal reefs off Santa Barbara, California to produce a 4‐year time series of net primary production by intact assemblages of understory macroalgae in giant kelp forests off Santa Barbara, California, USA. Daily bottom irradiance varied significantly throughout the year, and algal assemblages were on average exposed to saturating irradiance for only 1.3–4.5 h per day, depending on the time of year. Despite these variable light‐limiting conditions, biomass rather than irradiance explained the vast majority of variation observed in daily NPP at all times of the year. Measurements of peak biomass in spring and summer proved to be good predictors of NPP for the entire year, explaining as much as 76% of the observed variation. In contrast, bottom irradiance was a poor predictor of NPP, explaining <10% of the variation in NPP when analyzed seasonally and ~2% when evaluated annually. Our finding that annual NPP by macroalgal assemblages can be predicted from a single, nondestructive measurement of biomass should prove useful for developing time series data that are necessary to evaluate natural and anthropogenic changes in NPP by one of the world's most productive ecosystems.