The second Ca(2+)-binding domain of NCX1 binds Mg2+ with high affinity

We report the effects of binding of Mg(2+) to the second Ca(2+)-binding domain (CBD2) of the sodium-calcium exchanger. CBD2 is known to bind two Ca(2+) ions using its Ca(2+)-binding sites I and II. Here, we show by nuclear magnetic resonance (NMR), circular dichroism, isothermal titration calorimetry, and mutagenesis that CBD2 also binds Mg(2+) at both sites, but with significantly different affinities. The results from Mg(2+)-Ca(2+) competition experiments show that Ca(2+) can replace Mg(2+) from site I, but not site II, and that Mg(2+) binding affects the affinity for Ca(2+). Furthermore, thermal unfolding circular dichroism data demonstrate that Mg(2+) binding stabilizes the domain. NMR chemical shift perturbations and (15)N relaxation data reveal that Mg(2+)-bound CBD2 adopts a state intermediate between the apo and fully Ca(2+)-loaded forms. Together, the data show that at physiological Mg(2+) concentrations CBD2 is loaded with Mg(2+) preferentially at site II, thereby stabilizing and structuring the domain and altering its affinity for Ca(2+).     

5-Lipoxygenase and anandamide hydrolase (FAAH) mediate the antitumor activity of cannabidiol, a non-psychoactive cannabinoid

It has been recently reported that cannabidiol (CBD), a non-psychoactive cannabinoid, is able to kill glioma cells, both in vivo and in vitro , independently of cannabinoid receptor stimulation. However, the underlying biochemical mechanisms were not clarified. In the present study, we performed biochemical analysis of the effect of CBD both in vivo , by using glioma tumor tissues excised from nude mice, and in vitro , by using U87 glioma cells. In vivo exposure of tumor tissues to CBD significantly decreased the activity and content of 5-lipoxygenase (LOX, by ∼ 40%), and of its end product leukotriene B₄ (∼ 25%). In contrast cyclooxygenase (COX)-2 activity and content, and the amount of its end product prostaglandin E₂, were not affected by CBD. In addition, in vivo treatment with CBD markedly stimulated (∼ 175%) the activity of fatty acid amide hydrolase (FAAH), the main anandamide-degrading enzyme, while decreasing anandamide content (∼ 30%) and binding to CB₁ cannabinoid receptors (∼ 25%). In vitro pre-treatment of U87 glioma cells with MK-886, a specific 5-LOX inhibitor, significantly enhanced the antimitotic effect of CBD, whereas the pre-treatment with indomethacin (pan-COX inhibitor) or celecoxib (COX-2 inhibitor), did not alter CBD effect. The study of the endocannabinoid system revealed that CBD was able to induce a concentration-dependent increase of FAAH activity in U87 cells. Moreover, a significantly reduced growth rate was observed in FAAH-over-expressing U87 cells, compared to wild-type controls. In conclusion, the present investigation indicates that CBD exerts its antitumoral effects through modulation of the LOX pathway and of the endocannabinoid system, suggesting a possible interaction of these routes in the control of tumor growth.     

Enhancement of cellulase activity by clones selected from the combinatorial library of the cellulose-binding domain by cell surface engineering

To improve the cellulolytic activity of a yeast strain displaying endoglucanase IotaIota (EG II) from Trichoderma reesei, a combinatorial library of the cellulose-binding domain (CBD) of EG II was constructed by using cell surface engineering. When EG II degrades celluloses, CBD binds to cellulose, and its catalytic domain cleaves the glycosidic bonds of cellulose. CBD had a flat face, composed of five amino acids for binding. It was supposed that the three hydrophobic amino acid residues of the five amino acid residues were essential for binding to cellulose. Therefore, by improving the two remaining amino acid residues, construction of mutants with a combinatorial library of the two amino acids in CBD was carried out and binding ability and hydrolysis activity were measured. In the first screening by halo assay using the Congo Red staining method, about 200 of the 2000 colonies formed clear halos, and then five colonies with the clearest halos were finally selected. In the second screening, the binding ability of the five mutants to phosphoric acid-swollen Avicel was measured. In addition, the measurement of hydrolysis activity toward carboxymethylcellulose (CMC) using the screened mutants was carried out. As a result, the mutated EG II exhibiting higher binding ability (1.5-fold) had higher hydrolysis activity (1.3-fold) compared to the parent EG II-displaying yeast cell, demonstrating that CBD has confirmatively some effect on the cellulase activity through its binding ability of the enzyme to cellulose.     

Cannabidiol,a Non-Psychoactive Cannabinoid Compound,Inhibits Proliferation and Invasion in U87-MG and T98G Glioma Cells through a Multitarget Effect

In the present study, we found that CBD inhibited U87-MG and T98G cell proliferation and invasiveness in vitro and caused a decrease in the expression of a set of proteins specifically involved in growth, invasion and angiogenesis. In addition, CBD treatment caused a dose-related down-regulation of ERK and Akt prosurvival signaling pathways in U87-MG and T98G cells and decreased hypoxia inducible factor HIF-1α expression in U87-MG cells. Taken together, these results provide new insights into the antitumor action of CBD, showing that this cannabinoid affects multiple tumoral features and molecular pathways. As CBD is a non-psychoactive phytocannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anti-cancer drug in the management of gliomas.     

RNA decay machines: The exosome

The multisubunit RNA exosome complex is a major ribonuclease of eukaryotic cells that participates in the processing, quality control and degradation of virtually all classes of RNA in Eukaryota. All this is achieved by about a dozen proteins with only three ribonuclease activities between them. At first glance, the versatility of the pathways involving the exosome and the sheer multitude of its substrates are astounding. However, after fifteen years of research we have some understanding of how exosome activity is controlled and applied inside the cell. The catalytic properties of the eukaryotic exosome are fairly well described and attention is now drawn to how the interplay between these activities impacts cell physiology. Also, it has become evident that exosome function relies on many auxiliary factors, which are intensely studied themselves. In this way, the focus of exosome research is slowly leaving the test tube and moving back into the cell.The exosome also has an interesting evolutionary history, which is evident within the eukaryotic lineage but only fully appreciated when considering similar protein complexes found in Bacteria and Archaea. Thus, while we keep this review focused on the most comprehensively described yeast and human exosomes, we shall point out similarities or dissimilarities to prokaryotic complexes and proteins where appropriate.The article is divided into three parts. In Part One we describe how the exosome is built and how it manifests in cells of different organisms. In Part Two we detail the enzymatic properties of the exosome, especially recent data obtained for holocomplexes. Finally, Part Three presents an overview of the RNA metabolism pathways that involve the exosome. This article is part of a Special Issue entitled: RNA Decay mechanisms.     

Improvement in enzymatic desizing of starched cotton cloth using yeast codisplaying glucoamylase and cellulose-binding domain

To utilize glucoamylase-displaying yeast cells for enzymatic desizing of starched cotton cloth, we constructed yeast strains that codisplayed Rhizopus oryzae glucoamylase and two kinds of Trichoderma reesei cellulose-binding domains (CBD1, CBD of cellobiohydrolase I (CBHI); and CBD2, CBD of cellobiohydrolase II (CBHII)). In this study, we aimed to obtain a high efficiency of enzymatic desizing of starched cotton cloth. Yeast cells that codisplayed glucoamylase and CBD had higher activity on starched cotton cloth than yeast cells that displayed only glucoamylase. Glucoamylase and double CBDs (CBD1 and CBD2) codisplaying yeast cells exhibited the highest activity ratio (4.36-fold), and glucoamylase and single CBD (CBD1 or CBD2) codisplaying yeast cells had higher relative activity ratios (2.78- and 2.99-fold, respectively) than glucoamylase single-displaying cells. These results indicate that the glucoamylase activity of glucoamylase-displaying cells would be affected by the binding ability of CBD codisplayed on the cell surface to starched cotton cloth. These novel strains might play useful roles in the enzymatic desizing of starched cotton cloth in the textile industry.     

Alteration of a single tryptophan residue of the cellulose-binding domain blocks secretion of the Erwinia chrysanthemi Cel5 cellulase (ex-EGZ) via the type II system

Cel5 (formerly known as endoglucanase Z) of Erwinia chrysanthemi is secreted by the Out type II pathway. Previous studies have shown that the catalytic domain (CD), linker region (LR) and cellulose-binding domain (CBD) each contain information needed for secretion. The aim of this work was to further investigate the secretion-related information present in the CBD(Cel5). Firstly(, )deleting a surface-exposed flexible loop had no effect on secretion. This indicated that some structural freedom is tolerated by the type II system. Secondly, mutation of a single tryptophan residue, previously shown to be important for binding to cellulose, i.e. Trp43, was found also to impair secretion. This indicated that the flat cellulose-binding surface of CBD(Cel5 )contains secretion-related information. Thirdly, CBD(Cel5) was substituted by the CBD(EGG) of Alteromonas haloplanctis endoglucanase G, yielding a hybrid protein CD(Cel5)-LR(Cel5)-CBD(EGG) that exhibited 90 % identity with Cel5, including the Trp43 residue. The hybrid protein was not secreted. This indicated that the Trp43 residue is necessary but not sufficient for secretion. Here we propose a model in which the secretion of Cel5 involves a transient intramolecular interaction between the cellulose-binding surface of CBD(Cel5) and a region close to the entry into the active site in CD(Cel5). Once secreted, the protein may then open out to allow the cellulose-binding surface of CBD(Cel5 )to interact with the surface of the cellulose substrate. An implication of this model is that protein molecules fold to a specific secretion-competent conformation prior to secretion that is different from the folding state of the secreted species.     

《生物多样性公约》对遗传资源国际交流政策的影响

《生物多样性公约》于1992年在巴西里约热内卢签订,1993年生效。该公约提出了“国家对生物遗传资源拥有主权、获取生物遗传资源须事先得到资源所有者的知情同意、利用生物遗传资源所产生的利益应由资源所有者和开发者公平分享”三项基本原则,其生效与实施促进了各国生物多样性保护的立法行动,在维护资源原产国利益方面发挥了积极的作用,同时也给遗传资源的国际交流增添了一些障碍。本文介绍了《生物多样性公约》中有关生物遗传资源获取和利益分享的条款及其对国际交流政策的影响,并提出了加强遗传资源国际交换的对策和建议。     

Structural and dynamic aspects of Ca2+ and Mg2+ binding of the regulatory domains of the Na+/Ca2+ exchanger

Intracellular Ca2+ regulates the activity of the NCX (Na+/Ca2+ exchanger) through binding to the cytosolic CBD (Ca2+-binding domain) 1 and CBD2. In vitro studies of the structure and dynamics of CBD1 and CBD2, as well as studies of their kinetics and thermodynamics of Ca2+ binding, greatly enhanced our understanding of NCX regulation. We describe the fold of the CBDs in relation to other known structures and review Ca2+ binding of the different CBD variants from a structural perspective. We also report on new findings concerning Mg2+ binding to the CBDs and finally we discuss recent results on CBD1-CBD2 interdomain interactions.     

Pattern recognition and associative memory as dynamical processes in a synergetic system

We first present numerical results for the decomposition procedure for complex scenes described in Part I of these papers. Part II then mainly deals with a formalism that allows a formulation of our approach to pattern recognition and associative memory that is simultaneously invariant against translation, rotation, and scaling, Part II thus contains an explicit elaboration of ideas of Part I.     

Development of a variational scheme for model inversion of multi-area model of brain. Part II: VBEM method

In Part I and Part II of these two companion papers (henceforth called Part I and Part II), we develop and evaluate a variational Bayesian expectation maximization (VBEM) method for model inversion of our multi-area extended neural mass model (MEN). In this paper, we develop the VBEM method to estimate posterior distributions of parameters of MEN. We choose suitable prior distributions for the model parameters in order to use properties of a conjugate-exponential model in implementing VBEM. Consequently, VBEM leads to analytically tractable forms. The proposed VBEM algorithm starts with initialization and consists of repeated iterations of a variational Bayesian expectation step (VB E-step) and a variational Bayesian maximization step (VB M-step). Posterior distributions of the model parameters are updated in the VB M-step. Distribution of the hidden state is updated in the VB E-step. We develop a variational extended Kalman smoother (VEKS) to infer the distribution of the hidden state in the VB E-step and derive the forward and backward passes of VEKS, analogous to the Kalman smoother. In Part I, we evaluate and validate the VBEM method using simulation studies.     

The projection analysis of NMR chemical shifts reveals extended EPAC autoinhibition determinants

EPAC is a cAMP-dependent guanine nucleotide exchange factor that serves as a prototypical molecular switch for the regulation of essential cellular processes. Although EPAC activation by cAMP has been extensively investigated, the mechanism of EPAC autoinhibition is still not fully understood. The steric clash between the side chains of two conserved residues, L273 and F300 in EPAC1, has been previously shown to oppose the inactive-to-active conformational transition in the absence of cAMP. However, it has also been hypothesized that autoinhibition is assisted by entropic losses caused by quenching of dynamics that occurs if the inactive-to-active transition takes place in the absence of cAMP. Here, we test this hypothesis through the comparative NMR analysis of several EPAC1 mutants that target different allosteric sites of the cAMP-binding domain (CBD). Using what to our knowledge is a novel projection analysis of NMR chemical shifts to probe the effect of the mutations on the autoinhibition equilibrium of the CBD, we find that whenever the apo/active state is stabilized relative to the apo/inactive state, dynamics are consistently quenched in a conserved loop (β2-β3) and helix (α5) of the CBD. Overall, our results point to the presence of conserved and nondegenerate determinants of CBD autoinhibition that extends beyond the originally proposed L273/F300 residue pair, suggesting that complete activation necessitates the simultaneous suppression of multiple autoinhibitory mechanisms, which in turn confers added specificity for the cAMP allosteric effector.     

Antitumor photochemotherapy: biochemical bases, therapeutic uses and perspectives]

The photodynamic therapy of tumors (PDT) is a recent and promising technique for the treatment of tumors which can be reached by the light (directly or by endoscopic illumination). Excellent results are now obtained with hematoporphyrin derivatives such as Photofrin II, provided the concerned tumors are small and well delimited. Porphyrins are transported in blood mainly by lipoproteins, and the low density lipoprotein (LDL) receptor-mediated pathway is probably one of the important factors involved in the selective accumulation of porphyrins by tumor tissues, as cancer cells generally express much more LDL receptors than normal cells. In the present paper, after a brief presentation of the biochemical basis of the light-dependent cytotoxicity of porphyrins, we shall examine the role of lipoproteins, especially LDL, in the transport and the cellular uptake of these compounds. We shall also present recent approaches for the improvement of the PDT efficiency.     

Development of a variational scheme for model inversion of multi-area model of brain. Part I: simulation evaluation

We previously developed an integrated model of the brain within a single cortical area for functional Magnetic Resonance Imaging (fMRI), electroencephalography (EEG), and magnetoencephalography (MEG) using an extended neural mass model (ENMM). We then extended ENMM from a single-area to a multi-area model to develop a neural mass model of the entire brain. To this end, we derived a nonlinear state-space representation of the multi-area model. In Parts I and II of these two companion papers (henceforth called Part I and Part II), we develop and evaluate a variational Bayesian expectation maximization (VBEM) method to estimate parameters of multi-area ENMM (MEN) using E/MEG data. In Part I, we derive a state-space representation of MEN and use VBEM method for model inversion (parameter estimation). We evaluate and validate performance of VBEM method for model inversion of MEN using simulation studies in various signal-to-noise ratios. Details of VBEM method are presented in Part II. The proposed approach provides a useful technique for analyzing effective connectivity using non-invasive EEG and MEG methods.     

Scale-up engineering in animal cell technology: Part II

In the first part of this article we defined what is meant by scale-up. We also introduced the idea that engineers, in seeking to scale up bioprocesses, look at parameters that limit that process. If the parameters are independent of scale or reactor configuration they may enable the calculation of engineering data to predict behavior at large scales. In this exercise, dimensional analysis is one of the engineer's most important tools.In Part II, we will examine the application of the approach outlined in Part I to the analysis of scale-up in situations where shear or mass transfer may be limiting factors, situations which are particularly likely to arise in animal cell culture.     

Uncertainty in life cycle costing for long-range infrastructure. Part II: guidance and suitability of applied methods to address uncertainty

Life cycle costing (LCC) is the state-of-the-art method to economically evaluate long-term projects over their life spans. However, uncertainty in long-range planning raises concerns about LCC results. In Part I of this series, we developed a holistic framework of the different types of uncertainty in infrastructure LCCs. We also collected methods to address these uncertainties. The aim of Part II is to evaluate the suitability of methods to cope with uncertainty in LCC. Part I addressed two research gaps. It presented a systematic collection of uncertainties and methods in LCC and, furthermore, provided a holistic categorization of both. However, Part I also raised new issues. First, a combined analysis of sources and methods is still outstanding. Such an investigation would reveal the suitability of different methods to address a certain type of uncertainty. Second, what has not been assessed so far is what types of uncertainty are insufficiently addressed in LCC. This would be a feature to improve accuracy of LCC results within LCC, by suggesting options to better cope with uncertainty. To address these research gaps, we conducted a systematic literature review. Part II analyzed the suitability of methods to address uncertainties. The suitability depends on data availability, type of data (tangible, intangible, random, non-random), screened hotspots, and tested modeling specifications. We identified types of uncertainties and methods that have been insufficiently addressed. The methods include probabilistic modeling such as design of experiment or subset simulation and evolutionary algorithm and Bayesian modeling such as the Bayesian latent Markov decision process. Subsequently, we evaluated learning potential from other life cycle assessment (LCA) and life cycle sustainability assessment (LCSA). This analysis revealed 28 possible applications that have not yet been used in LCC. Lastly, we developed best practices for LCC practitioners. This systematic review complements prior research on uncertainty in LCC for infrastructure, as laid out in Part I. Part II concludes that all relevant methods to address uncertainty are currently applied in LCC. Yet, the level of application is different. Moreover, not all methods are equally suited to address different categories of uncertainty. This review offers guidance on what to do for each source and type of uncertainty. It illustrates how methods can address both based on current practice in LCC, LCA, and LCSA. The findings of Part II encourage a dialog between practitioners of LCC, LCA, and LCSA to advance research and practice in uncertainty analysis.     

Chronic Beryllium Disease: Revealing the Role of Beryllium Ion and Small Peptides Binding to HLA-DP2

Chronic Beryllium (Be) Disease (CBD) is a granulomatous disorder that predominantly affects the lung. The CBD is caused by Be exposure of individuals carrying the HLA-DP2 protein of the major histocompatibility complex class II (MHCII). While the involvement of Be in the development of CBD is obvious and the binding site and the sequence of Be and peptide binding were recently experimentally revealed [1], the interplay between induced conformational changes and the changes of the peptide binding affinity in presence of Be were not investigated. Here we carry out in silico modeling and predict the Be binding to be within the acidic pocket (Glu26, Glu68 and Glu69) present on the HLA-DP2 protein in accordance with the experimental work [1]. In addition, the modeling indicates that the Be ion binds to the HLA-DP2 before the corresponding peptide is able to bind to it. Further analysis of the MD generated trajectories reveals that in the presence of the Be ion in the binding pocket of HLA-DP2, all the different types of peptides induce very similar conformational changes, but their binding affinities are quite different. Since these conformational changes are distinctly different from the changes caused by peptides normally found in the cell in the absence of Be, it can be speculated that CBD can be caused by any peptide in presence of Be ion. However, the affinities of peptides for Be loaded HLA-DP2 were found to depend of their amino acid composition and the peptides carrying acidic group at positions 4 and 7 are among the strongest binders. Thus, it is proposed that CBD is caused by the exposure of Be of an individual carrying the HLA-DP2*0201 allele and that the binding of Be to HLA-DP2 protein alters the conformational and ionization properties of HLA-DP2 such that the binding of a peptide triggers a wrong signaling cascade.     

From analysis to synthesis: new ligand binding sites on the lactate dehydrogenase framework. Part II

In Part I of this article (published in the March issue of TIBS1), substrate-binding and catalysis in lactate dehydrogenase were examined by genetic modification of the protein structure and analysis of the functional consequences. In Part II, the conclusions are used in the design and synthesis of two modified forms of the enzyme; one in which the substrate specificity is shifted to produce a more effective malate dehydrogenase than that isolated from the host organism and one which no longer requires its allosteric activator (fructose 1,6-bisphosphate).     

From analysis to synthesis: new ligand binding sites on the lactate dehydrogenase framework. Part I

In Part I of this article, the naturally evolved protein framework of lactate dehydrogenase is investigated by genetically introduced modifications which reveal the structural basis of its catalytic and substrate-binding properties. In Part II (to be published in the April issue of TIBS), this analytical information is exploited in the design of two modified forms of the enzyme; one which is specific for a new substrate and one which lacks allosteric regulation.     

SUMO Modification of DNA topoisomerase II: Trying to get a CENse of it all

DNA topoisomerase II (topo II) is an essential determinant of chromosome structure and function, acting to resolve topological problems inherent in recombining, transcribing, replicating and segregating DNA. In particular, the unique decatenating activity of topo II is required for sister chromatids to disjoin and separate in mitosis. Topo II exhibits a dynamic localization pattern on mitotic chromosomes, accumulating at centromeres and axial chromosome cores prior to anaphase. In organisms ranging from yeast to humans, a fraction of topo II is targeted for SUMO conjugation in mitotic cells, and here we review our current understanding of the significance of this modification. As we shall see, an emerging consensus is that in metazoans SUMO modification is required for topo II to accumulate at centromeres, and that in the absence of this regulation there is an elevated frequency of chromosome non-disjunction, segregation errors, and aneuploidy. The underlying molecular mechanisms for how SUMO controls topo II are as yet unclear. In closing, however, we will evaluate two possible interpretations: one in which SUMO promotes enzyme turnover, and a second in which SUMO acts as a localization tag for topo II chromosome trafficking.