Expanded bed chromatography of proteins in small-diameter columns. II. Methods development and scale up

The scaled down system developed in Part I of this series was further validated by using a 1-cm diameter column for method development studies for the separation of two model proteins, alcohol dehydrogenase and -glucosidase, from unclarified yeast homogenate by hydrophobic interaction expanded bed chromatography based on the STREAMLINE matrix. The efficacy of solids removal and establishment of optimal binding and separation condition by stepwise elution were investigated. Equilibration of the EBA column and loading at high salt strengths affected the subsequent recovery of the two target proteins. Although good resolution between the target proteins could be achieved, peak tailing was found to be a consistent problem. The optimised separation protocol was scaled up 25-fold to a column diameter of 5.0 cm. The results were in good agreement with the run conducted in the 1-cm column, indicating the potential of using the small columns as an viable approach for method scouting and development studies.     

Purification of a `double-headed' inhibitor of α-amylase/Proteinase K from wheat germ by expanded bed chromatography

The double-headed inhibitor of -amylase and Proteinase K was purified from wheat germ using Cu(II)-Streamline^TM-chelating resin. The endogenous -amylase could be inactivated by heating. Followed by this step, both packed bed and expanded bed gave similar activity yield of around 83% and fold purification around 23. In the case of expanded bed, it was not necessary to separate precipitated protein before the chromatography. The purified preparation gave a single band on SDS–PAGE and the estimated molecular weight of 21 kDa was in agreement with the reported value for the inhibitor designated as PKI-3 in the literature.     

CHARACTERIZATION AND COMPARISON OF PROKARYOTIC EPIPHYTES ASSOCIATED WITH THREE EAST AFRICAN SEAGRASSES1

Prokaryotic epiphytes on leaves of three seagrass species, Thalassodendron ciliatum, Thalassia hemprichii, and Cymodocea rotundata, from two Kenyan coastal sites, Nyali (a high‐nutrient site) and Vipingo (a low‐nutrient site), were characterized genetically and morphologically. Denaturing gradient gel electrophoresis (DGGE) and clone libraries of PCR‐amplified 16S rRNA gene fragments were used to study prokaryotes associated with these seagrasses. In general, the epiphytic coverage was greater in the high‐nutrient site, while the microbial diversity was linked to seagrass species rather than the study sites. Cytophaga–Flavobacteria–Bacteroides (CFB) were associated with T. ciliatum and T. hemprichii mainly in the nutrient‐poor site, while α‐, β‐, and γ‐proteobacteria were associated with all three species at the two study sites. Some bacteria phylotypes were closely related to sequences of microorganisms previously recovered from wastewaters or other contaminated sources, indicating the influx of land‐based wastes into these coastal lagoon ecosystems. The abundance of potential nitrogen (N₂)‐fixing cyanobacteria on C.  rotundata, particularly in the low‐nutrient site, suggested that this association may have been acquired to meet N demands. Unicellular cyanobacteria were dominant and associated with C. rotundata and T. hemprichii (with those on T. hemprichii being closely related to cyanobacterial symbiotic species), while T. ciliatum was almost devoid of cyanobacterial associations at the same site (Nyali), which suggests specificity in the cyanobacteria–seagrass associations. The abundance of prokaryotic epiphytes was considered to be linked to water depth and tidal exposure.     

Modulator Dynamics Shape the Design Space for Stepwise‐Elution Simulated Moving Bed Chromatographic Separations

For proteins and other biological macromolecules, SMB chromatography is best operated non‐isocratically. However, traditional modes of non‐isocratic SMB operation generate significant mobile‐phase modulator dynamics. The mechanisms by which these modulator dynamics affect a separation's success, and thus frame the design space, have yet to be explained quantitatively. Here, the dynamics of the modulator (e.g., salts in ion exchange and hydrophobic interaction chromatography) are explicitly accounted for. This leads to the elucidation of two new design constraints, presented as dimensionless numbers, which quantify the effects of the modulator phenomena and thus predict the success of a non‐isocratic SMB separation. Consequently, these two new design constraints re‐define the SMB design space. Computational and experimental studies at the boundaries of this design space corroborate the theoretical predictions. The design of efficient and robust operating conditions through use of the new design space is also demonstrated.     

High midday temperature stress has stronger effects on biomass than on photosynthesis: A mesocosm experiment on four tropical seagrass species

The effect of repeated midday temperature stress on the photosynthetic performance and biomass production of seagrass was studied in a mesocosm setup with four common tropical species, including Thalassia hemprichii, Cymodocea serrulata, Enhalus acoroides, and Thalassodendron ciliatum. To mimic natural conditions during low tides, the plants were exposed to temperature spikes of different maximal temperatures, that is, ambient (29–33°C), 34, 36, 40, and 45°C, during three midday hours for seven consecutive days. At temperatures of up to 36°C, all species could maintain full photosynthetic rates (measured as the electron transport rate, ETR) throughout the experiment without displaying any obvious photosynthetic stress responses (measured as declining maximal quantum yield, Fv/Fm). All species except T. ciliatum could also withstand 40°C, and only at 45°C did all species display significantly lower photosynthetic rates and declining Fv/Fm. Biomass estimation, however, revealed a different pattern, where significant losses of both above‐ and belowground seagrass biomass occurred in all species at both 40 and 45°C (except for C. serrulata in the 40°C treatment). Biomass losses were clearly higher in the shoots than in the belowground root–rhizome complex. The findings indicate that, although tropical seagrasses presently can cope with high midday temperature stress, a few degrees increase in maximum daily temperature could cause significant losses in seagrass biomass and productivity.     

Dynamique et èvolution de l'herbier à Posidonia oceanica en Méditerranée

Abstract

The natural growth of a Posidonia oceanica meadow is the result of a long process of development (series of events), but the dynamics are often difficult to establish because of the lack of a zero state and of sufficiently long chronological series. In addition, while it is sometimes possible to observe major regressions, the phenomena of recolonisation and of small-scale fluctuations are rarely detected. Nevertheless, with the emergence of new investigatory techniques (lepidochronology, microcartography, monitoring networks, accurate dating techniques, etc.,) considerable progress has been made in this field. The development in space and time of the seagrass beds is dependant on hydrodynamic action and sedimentary deposits. This means that in sheltered bays Posidonia oceanica may form original structures such as barrier reefs or micro-atolls, while in turbulent waters one observes the formation of spectacular structures resulting from erosion (intermatte, “rivière de retour”). A disequilibrium in sedimentation may result in dramatic regression in some seagrass beds. The multiplicity of prevailing environmental conditions results in the occurrence of a range of meadow types: “herbiers de plaines”, “herbiers de collines”, “herbiers tigrés”, “herbiers en escalier”. The impact of human pressure over the past few decades has not been insignificant. Coastal development constructions that have altered the direction of currents and the sedimentation, sewage outfalls that have resulted in an increase in the turbidity of the water and the mechanical action of anchors or trawl nets which have damaged certain areas of seagrass meadow have caused extensive regression. Conversely, the rehabilitation of previously degraded areas has made it possible to monitor the process of recolonisation.     

New insights into DNA barcoding of seagrasses

Taxonomists find some plant genera challenging because of the few morphological differences or unclear characters among closely related species, which leads to the misidentification of taxa. DNA barcoding is an approach to identify species by using short orthologous DNA sequences, known as ‘DNA barcodes’. Concatenated rbcL and matK sequences are considered DNA barcodes for seagrasses. However, these markers are not applicable to all members of seagrasses at the species level, especially within the genus Halophila. Our previous studies indicated that the internal transcribed spacer (ITS) showed higher species resolution than the concatenated rbcL and matK sequences in the case of Halophila ovalis and closely related species. In this study, 26 ITS, two rbcL and two matK consensus sequences from 18 seagrass taxa belonging to four families collected in India, Vietnam, Germany, Croatia and Egypt were processed. Molecular ITS analysis resolved five clades. The results also indicate that the Cymodoceaceae family might be a non-monophyletic group. In conclusion, ITS could be applied as a DNA barcode for seagrasses instead of the rbcL/matK system previously proposed.     

Endoplasmic Reticulum (ER) Stress-Suppressive Compounds from Scrap Cultivation Beds of the Mushroom Hericium erinaceum

Four compounds were isolated from scrap cultivation beds of the mushroom, Hericium erinaceum. Compounds 1–4 were identified as methyl 4-hydroxy-3-(3-methylbutanoyl) benzoate, 2-chloro-1,3-dimethoxy-5-methylbenzene, methyl 4-chloro-3,5-dimethoxybenzoate, and 4-chloro-3,5-dimethoxybenzaldehyde by an interpretation of the NMR and MS data, respectively. This is the first reported isolation of 1 from a natural source. All the compounds showed protective activity against endoplasmic reticulum stress-dependent cell death.     

气垫床在预防压疮中的效果评价

目的：探讨气垫床在预防压疮中的应用,为减少住院患者压疮的发生提供科学依据,提高患者生存质量。方法：选择我院2011年6月-2012年6月住院患者116例为研究对象,对患者进行褥疮风险评估后将116例患者随机分为两组,一组患者使用气垫床,另一组患者普通褥疮预防和护理,对两组患者发生褥疮的情况进行比较。结果：普通组患者发生压疮26例,对照组患者发生压疮5例,经卡方比较,对照组好于普通组,P〈0.05。结论：气垫床的使用不仅可以很好的杜绝压疮的发生,还可以提高患者的生存质量。