Calcium-Binding Property of Dephosphorylated Caseins

Whole casein, α_s-casein and k-casein were dephosphorylated with a phosphoprotein phosphatase prepared from beef spleen and their calcium-binding capacities were compared with those of respective native caseins by a ultracentrifugal method.

The bindings of the calcium to 94% dephosphorylated whole casein and to 97 % dephosphorylated α_s-casein at neutral pH were approximately one third of those to respective native caseins. The decrease of calcium-binding capacity of k-casein due to dephosphorylation was also significant.

The effect of pH on the state and the calcium-binding capacity of dephosphorylated caseins was also examined and the role of organic phosphate groups of casein as calcium-binding sites was discussed.     

Climate change creates rapid species turnover in montane communities

Recent decades have seen substantial changes in patterns of biodiversity worldwide. Simultaneously, climate change is producing a widespread pattern of species’ range shifts to higher latitudes and higher elevations, potentially creating novel assemblages as species shift at different rates. However, the direct link between species’ turnover as a result of climate‐induced range shifts has not yet been empirically evaluated. We measured rates of species turnover associated with species’ range shifts in relatively undisturbed montane areas in Asia, Europe, North America, South America, and the Indo‐Pacific. We show that species turnover is rapidly creating novel assemblages, and this can be explained by variable changes in species’ range limits following warming. Across all the areas we analyzed, mean species’ turnover was 12% per decade, which was nearly balanced between the loss of existing co‐occurrences and the gain of novel co‐occurrences. Turnover appears to be more rapid among ectothermic assemblages, and some evidence suggests tropical assemblages may be responding at more rapid rates than temperate assemblages.     

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl^- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl^- from proteoliposomes following the addition of the K⁺ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H⁺/Cl^- exchanger, as a model system. The efflux assay can be utilized to study any Cl^- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.     

Temperature during early development has long-term effects on microRNA expression in Atlantic cod

Background

Environmental temperature has serious implications in life cycle of aquatic ectotherms. Understanding the molecular mechanisms of temperature acclimation and adaptation of marine organisms is of the uttermost importance for ecology, fisheries, and aquaculture, as it allows modeling the effects of global warming on population dynamics. Regulatory molecules are major modulators of acclimation and adaptation; among them, microRNAs (miRNAs) are versatile and substantial contributors to regulatory networks of development and adaptive plasticity. However, their role in thermal plasticity is poorly known. We have asked whether the temperature and its shift during the early ontogeny (embryonic and larval development) affect the miRNA repertoire of Atlantic cod (Gadus morhua), and if thermal experience has long-term consequences in the miRNA profile.

Results

We characterized miRNA during different developmental stages and in juvenile tissues using next generation sequencing. We identified 389 putative miRNA precursor loci, 120 novel precursor miRNAs, and 281 mature miRNAs. Some miRNAs showed stage- or tissue-enriched expression and miRNAs, such as the miR-17 ~ 92 cluster, myomiRs (miR-206), neuromiRs (miR-9, miR-124), miR-130b, and miR-430 showed differential expression in different temperature regimes. Long-term effect of embryonic incubation temperature was revealed on expression of some miRNAs in juvenile pituitary (miR-449), gonad (miR-27c, miR-30c, and miR-200a), and liver (let-7 h, miR-7a, miR-22, miR-34c, miR-132a, miR-192, miR-221, miR-451, miR-2188, and miR-7550), but not in brain. Some of differentially expressed miRNAs in the liver were confirmed using LNA-based rt-qPCR. The effect of temperature on methylation status of selected miRNA promoter regions was mostly inconclusive.

Conclusions

Temperature elevation by several degrees during embryonic and larval developmental stages significantly alters the miRNA profile, both short-term and long-term. Our results suggest that a further rise in seas temperature might affect life history of Atlantic cod.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1503-7) contains supplementary material, which is available to authorized users.     

microRNA序列和表达模式的多样性

microRNA(miRNA)在后生动植物细胞分化、生长发育、正常生理功能的维持、疾病的发生发展和转归中发挥重要功能.通常认为,已知miRNA对应的序列就是存储于miRBase数据库中相应的成熟miRNA序列.然而,人们忽视了miRNA序列长度和碱基的可变性,低估了来源于同一miRNA基因、非主要表达的miRNA的多样性及其功能的重要性.由于测序技术的快速发展和广泛使用,人们利用二代测序技术数据发现了miRNA序列和miRNA表达水平的多样性.基于miRNA序列和表达的多样性,本文介绍了miRNA序列5′端、3′端和内部的变异体、miRNA变异体的时空差异性表达和miRNA优势表达臂转换等方面近年来的研究进展.     

ADAR1 is required for differentiation and neural induction by regulating microRNA processing in a catalytically independent manner

Adenosine deaminases acting on RNA (ADARs) are involved in adenosine-to-inosine RNA editing and are implicated in development and diseases. Here we observed that ADAR1 deficiency in human embryonic stem cells (hESCs) significantly affected hESC differentiation and neural induction with widespread changes in mRNA and miRNA expression, including upregulation of self-renewal-related miRNAs, such as miR302s. Global editing analyses revealed that ADAR1 editing activity contributes little to the altered miRNA/mRNA expression in ADAR1-deficient hESCs upon neural induction. Genome-wide iCLIP studies identified that ADAR1 binds directly to pri-miRNAs to interfere with miRNA processing by acting as an RNA-binding protein. Importantly, aberrant expression of miRNAs and phenotypes observed in ADAR1-depleted hESCs upon neural differentiation could be reversed by an enzymatically inactive ADAR1 mutant, but not by the RNA-binding-null ADAR1 mutant. These findings reveal that ADAR1, but not its editing activity, is critical for hESC differentiation and neural induction by regulating miRNA biogenesis via direct RNA interaction.