Molecular integrity of monoclonal antibodies produced by hybridoma cells in batch culture and in continuous-flow culture with integrated product recovery

The molecular integrity of monoclonal antibodies (MCAB) produced by murine hybridoma cell line TB/C3 was studied in batch and continuous-flow cultures. In batch culture, one band of MCAB was detected initially by Western blotting of sodium dodecyl sulfate (SDS)-polyacrylamide gels run under unreduced conditions, but heterogenous MCAB bands appeared as the culture aged. The latter were due to the degradation of MCAB by proteases active at the neutral pH of the culture. The deleterious effect of proteases was minimized in the continuous-flow cultures which were integrated for product recovery. The MCAB of high quality was purified over 26 days from a culture grown at a dilution rate of 0.025 h(-1) (experiment 1). However, at a lower dilution rate of 0.015 h(-1) (experiment 2), the integrity of MCAB was compromised after the initial 13 days of culture. This was shown to be due to the variation in the carbohydrate content of MCAB produced, as judged by the increased sialylation of heavy chains and the varied reactivity of MCAB with lectins (Maackia amurensis agglutinin, Galanthus nivalis agglutinin, and Datura stramonium agglutinin) as the age of the culture increased. The concentration of the purified MCAB samples by enzyme-linked immunosorbent assay (ELISA) (used normally) was usually higher than that estimated by absorbance at 280 nm. Best correlation between the two methods (ELISA-280 nm ratio of 1.02-1.25) was obtained with experiment 1 samples. This ratio increased in experiment 2 and batch culture samples as the heterogeneity of MCAB produced increased, being 1.03-2.94 and 2.53-4.62, respectively. Therefore, ELISA overestimated MCAB concentration when the molecular integrity of the latter was compromised. The ELISA-A(280) nm ratio might hence provide a useful indicator for assessing the quality of MCAB produced. Comparison of SDS-polyacrylamide gels stained with Coomassie Brilliant Blue R and silver showed that the former correlated better with the MCAB activity stain, whereas the silver stained both the protein- and carbohydrate-rich components. Comparison of the patterns produced with these two stains might therefore offer another parameter to monitor the overall integrity of MCAB produced. Finally, the data presented have important implications on the validity of using long-term and intensive cultures for generating MCAB because such cultures would be subjected to the additive effects reported for batch and continuous modes of growth. (c) 1993 John Wiley & Sons, Inc.     

Emulsion liquid membrane extraction of lactic acid from aqueous solutions and fermentation broth

Studies on the batch extraction of lactic acid using an emulsion liquid membrane system are reported. The membrane phase consists of the tertiary amine carrier Alamine 336 and the surfactant Span 80 dissolved in n-heptane/paraffin and aqueous solutions of sodium carbonate in the internal phase. The effects of internal phase reagent, extraction temperature, and initial external phase pH on the extraction efficiency and the emulsion swelling are examined. A statistical factorial experiment on extraction from clarified lactic acid fermentation broth was carried out to obtain knowledge of the performance of the extraction system from a broth. The extraction efficiency from the fermentation broth is found to be lower as compared to aqueous solutions of pure lactic acid. The effect of pH and the presence of other ionic species on selectivity are discussed. (c) 1993 John Wiley & Sons, Inc.     

Development of a recovery and recycle process for a pseudomonas lipase used for large-scale enzymatic synthesis

Enzymes are potential catalysts for a wide range of large-scale chemical synthesis steps, particularly when the creation of a specific chiral center is desired. The efficient recycling of the enzyme catalyst and the removal of carryover impurities were crucial factors in the improvement of a stereoselective ester hydrolysis step used in the synthesis of a selective leukotriene antagonist. In this enzymatic reaction step, the substrate and product were both largely insoluble, while the enzyme was soluble in the aqueous reaction mixture. Microfiltration and ultrafiltration of the slurry reaction mother liquor indicated near 100% enzyme protein recovery, while activity recovery was about 70% to 80%. These activity losses might be accounted for by enzyme degradation (1 to 2 mg/L . h) during the 40-hour reaction period. Dissolved impurities, principally a diacid byproduct, in the enzyme recycling stream were reduced 60% to 70% by either lowering the solution pH to 4.0 or raising the solution ionic strength to 1 M. (c) 1993 John Wiley & Sons, Inc.     

Seagrass ecosystem multifunctionality under the rise of a flagship marine megaherbivore

Large grazers (megaherbivores) have a profound impact on ecosystem functioning. However, how ecosystem multifunctionality is affected by changes in megaherbivore populations remains poorly understood. Understanding the total impact on ecosystem multifunctionality requires an integrative ecosystem approach, which is especially challenging to obtain in marine systems. We assessed the effects of experimentally simulated grazing intensity scenarios on ecosystem functions and multifunctionality in a tropical Caribbean seagrass ecosystem. As a model, we selected a key marine megaherbivore, the green turtle, whose ecological role is rapidly unfolding in numerous foraging areas where populations are recovering through conservation after centuries of decline, with an increase in recorded overgrazing episodes. To quantify the effects, we employed a novel integrated index of seagrass ecosystem multifunctionality based upon multiple, well-recognized measures of seagrass ecosystem functions that reflect ecosystem services. Experiments revealed that intermediate turtle grazing resulted in the highest rates of nutrient cycling and carbon storage, while sediment stabilization, decomposition rates, epifauna richness, and fish biomass are highest in the absence of turtle grazing. In contrast, intense grazing resulted in disproportionally large effects on ecosystem functions and a collapse of multifunctionality. These results imply that (i) the return of a megaherbivore can exert strong effects on coastal ecosystem functions and multifunctionality, (ii) conservation efforts that are skewed toward megaherbivores, but ignore their key drivers like predators or habitat, will likely result in overgrazing-induced loss of multifunctionality, and (iii) the multifunctionality index shows great potential as a quantitative tool to assess ecosystem performance. Considerable and rapid alterations in megaherbivore abundance (both through extinction and conservation) cause an imbalance in ecosystem functioning and substantially alter or even compromise ecosystem services that help to negate global change effects. An integrative ecosystem approach in environmental management is urgently required to protect and enhance ecosystem multifunctionality.     

Fire affects wood formation dynamics and ecophysiology of Pinus pinaster Aiton growing in a dry Mediterranean area

The increase in frequency and intensity of wildfires is seriously affecting forest ecosystems, especially in drought-prone areas. Trees’ recovery after fire is related to direct tree damage and is influenced by climate conditions, such as warm temperature and water shortage. In this study, we evaluate the post-fire effects on a Pinus pinaster Aiton forest growing in a hot and dry area of the Mediterranean region by comparing burned trees with severe crown reduction against unburned and not-defoliated trees. Inter-annual analyses of dendrochronology and stable isotopes in tree rings were combined with xylogenesis monitoring to investigate the effects of fire on tree growth, ecophysiological processes and wood formation. Tree-ring and isotope data showed a growth reduction and a decrease in photosynthetic activity in the burned trees, compared to control individuals, in the three years after fire. Further, the monitoring of cambial activity demonstrated a negative influence of warm and dry periods on wood formation, low xylem production, a delay in phenology and a reduction in xylem plasticity in burned trees. Our findings suggest that substantial photosynthetic limitations caused by crown defoliation and recurrent drought events could lead to severe growth decrease and reduction of trees ability to regain the pre-disturbance productivity rates.     

MORTALITY OF SEA OTTERS IN PRINCE WILLIAM SOUND FOLLOWING THE EXXON VALDEZ OIL SPILL

Abstract: This paper presents an estimate of the total number of sea otters that died as a direct consequence of the oil spill that occurred when the T/V Exxon Valdez grounded in Prince William Sound, Alaska on 24 March 1989. We compared sea otter counts conducted from small boats throughout the Sound during the summers of 1984 and 1985 to counts made after the spill during the summer of 1989. We used ratio estimators, corrected for sighting probability, to calculate otter densities and population estimates for portions of the Sound affected by the oil spill. We estimated the otter population in the portion of Prince William Sound affected by the oil was 6,546 at the time of the spill and that the post-spill population in the summer of 1989 was 3,898, yielding a loss estimate of approximately 2,650. Bootstrapping techniques were used to approximate confidence limits on the loss estimate of about 500–5,000 otters. The wide confidence limits are a result of the complex scheme required to estimate losses and limitations of the data. Despite the uncertainty of the loss estimate it is clear that a significant fraction of the otters in the spill zone survived. We observed otters persisting in relatively clean embayments throughout the oil spill zone suggesting that the highly convoluted coastline of Prince William Sound produced refuges that allowed some sea otters in the oil spill area to survive.     

Recovery of aSasa tsuboiana population after mass flowering and death

The recovery process of aSasa tsuboiana population after a mass flowering and death in 1977 was investigated by 15 years of observation in the Hira Mountains, Kinki district, western Japan. Seed production was high (6600–13 800 seeds m⁻² inSasa plots and 3900 seeds m⁻² in a forest plot) but emergent seedling density was low (14–21 seedlings m⁻²), probably because of seed predation byMicrotus montebelli occurring between seed shedding and the next spring. The seedling density had decreased further by the next year and theS. tsuboiana population recovered from only a limited number of seedlings. In spite of such a low initial density, theS. tsuboiana population was able to regenerate successfully and attained the previous full stand height in 7–16 years.Miscantbus sinensis invaded and delayed the recovery ofS. tsuboiana in one plot, butS. tsuboiana became dominant as it caught up with the height ofM. sinensis. Seedling growth patterns, such as frequent tillering, the onset of rhizome extension in the early stage of seedling growth and frequent culm production from rhizomes, played important roles in the successful regeneration ofS. tsuboiana.     

内蒙古高寒露天煤矿排土场植被恢复研究

探究不同恢复年限对于露天煤矿植被群落变化和植物个体生长的影响,进一步加强生态环境保护与生态修复治理措施。以内蒙古高寒露天煤矿排土场恢复4年(2018-2021年)的植物群落为研究对象,采用样方调查方法,对样地内植物群落进行调查,设置草本样方(1 m×1 m)和灌木样方(5 m×5 m),记录植物物种组成、高度及盖度等指标,计算Shannon-Wiener多样性指数、Simpon指数、Pielou均匀度指数和物种重要值。探讨不同的恢复年限植物群落变化特征和优势植物的养分吸收,为矿区人工植被恢复提供科学依据。结果表明:(1)矿区排土场植物群落物种数、生物量和群落多样性均随着恢复年限的增加而显著增加。总物种数从恢复第1年的16种增加到恢复第4年的31种,主要是由于非人工种植植物从6种增加到19种导致,特别是在恢复第2年增加显著,然后趋于平稳,这说明恢复第2年是物种增加关键的时期;(2)随着恢复年限的增加,生物量和多样性指数显著增加;矿区植被优势物种多以禾本科披碱草(Elymus dahuricus)、无芒雀麦(Bromus inermis);豆科植物苜蓿(Medicago sativa)、草木樨(Melilotus officinalis)、沙打旺(Astragalus adsurgens)、锦鸡儿(Caragana sinica);菊科植物大籽蒿(Artemisia sieversiana);十字花科油菜(Brassica napus)和胡颓子科沙棘(Hippophae rhamnoides)为主。(3)比较不同优势植物叶片、茎和根的氮(N)、磷(P)和钾(K)含量发现,草本优势种中菊科和豆科植物显著高于禾本科植物。灌木优势种中沙棘茎的N含量和P含量显著高于锦鸡儿。研究认为,在排土场植被恢复初期(4年)的物种选择上,豆科植物和菊科植物是草本植物首选的先锋物种,另外,沙棘是灌木首选的物种。     

The effect of charcoal production on carbon cycling in African biomes

Using biomass for charcoal production in sub-Saharan Africa (SSA) may change carbon stock dynamics and lead to irreversible changes in the carbon balance, yet we have little understanding of whether these dynamics vary by biome in this region. Currently, charcoal production contributes up to 7% of yearly deforestation in tropical regions, with carbon emissions corresponding to 71.2 million tonnes of CO₂ and 1.3 million tonnes of CH₄. With a projected increased demand for charcoal in the coming decades, even low harvest rates may throw the carbon budget off-balance due to legacy effects. Here, we parameterized the dynamic global vegetation model LPJ-GUESS for six SSA biomes and examined the effect of charcoal production on net ecosystem exchange (NEE), carbon stock sizes and recovery time for tropical rain forest, montane forest, moist savanna, dry savanna, temperate grassland and semi-desert. Under historical charcoal regimes, tropical rain forests and montane forests transitioned from net carbon sinks to net sources, that is, mean cumulative NEE from −3.56 ± 2.59 kg C/m² to 2.46 ± 3.43 kg C/m² and −2.73 ± 2.80 kg C/m² to 1.87 ± 4.94 kg C/m² respectively. Varying charcoal production intensities resulted in tropical rain forests showing at least two times higher carbon losses than the other biomes. Biome recovery time varied by carbon stock, with tropical and montane forests taking about 10 times longer than the fast recovery observed for semi-desert and temperate grasslands. Our findings show that high biomass biomes are disproportionately affected by biomass harvesting for charcoal, and even low harvesting rates strongly affect vegetation and litter carbon and their contribution to the carbon budget. Therefore, the prolonged biome recoveries imply that current charcoal production practices in SSA are not sustainable, especially in tropical rain forests and montane forests, where we observe longer recovery for vegetation and litter carbon stocks.