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Copyright of Food & Fermentation Industries is the property of Food & Fermentation Industries and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Human kallikrein-kinin system (KKS) is a proteolytic cascade with two serine-protease zymogen couples (Factor XII and prekallikrein (PK) and their activated forms, FXIIa, PKa, respectively), releasing bradykinin by cleavage of native high-molecular-weight kininogen (nHK) into cleaved HK. For KKS investigation in human plasma, this cascade is usually triggered on ice eventually by mixing with purified proteins. It has been established that purified FXIIa, PK, and nHK required a fixed order and timing for mixing protein on ice to ensure reproducibility of testing, we investigated the activation kinetics of both enzymes. The activation process of this in vitro minimal reconstitution of KKS was studied by progress curve analysis, in condition of high enzyme/substrate ratio and by using on natural rather than peptide substrates. FXIIa and PKa were found five-times less active on ice than at 37°C: kcat = 0.133 +- 0.034 and 0.0119 +- 0.0027 s-1, KM = 672 +- 150 and 115 +- 24 nM, respectively. The progress curve analysis of our in vitro KKS reconstitutions differed from a Michaelis--Menten mathematical simulation by a faster initial rate and a slower late rate. These two features were also observed ex vivo by using dextran sulfate-activated plasma and could reinforce the hypothesis of a maximal local effect (bradykinin release) and a minimal systemic consequence (PK preservation) in KKS activation process. Analyzing the complete curve of cold KKS activation would provide valuable information for ex vivo investigation of KKS in samples from patients presenting with hereditary angioedema and other inflammatory conditions. [ABSTRACT FROM AUTHOR]

Although surface with multiple holes (SMHs) is widely used in industrial components, the precise inspection of SMH is a knotty task due to the inefficiency of traditional surface inspection technology. Recently, a five-axis surface sweep scanning approach is emerging and shows great potential to boost the surface inspection efficiency by sensing surface in a continuous sweep scanning way. However, an efficient sweep scanning path should cater to the unique kinematic characteristics of the five-axis inspection system. In this article, we present an approach to automatically generate efficient five-axis sweep scanning paths for inspecting SMH. First, the skeleton of SMH is extracted along with the surface partitioned into several patches, based on which a guiding curve-based sweep scanning path can be defined for each surface patch. By modeling the skeleton of SMH as a directed Euler graph and finding a proper sequence to traverse the graph, a continuous five-axis sweep scanning path is then generated to sweep all patches without any transition among them. The nonsweeping time could be eliminated so that the sweep scanning efficiency is drastically improved in this way. Simulation and physical inspection experiments are conducted on two SMHs, showing that our method significantly outperforms existing approaches, such as the popular zigzag method and the method from the leading commercial software of RENISHAW. Note to Practitioners—This article aims to generate an efficient sweep scanning path of a five-axis coordinate measuring machine (CMM) for inspecting the SMHs of a precision component, such as an engine block or a gearbox cover. Although traditional five-axis CMM path generation methods can be applied to SMH inspection, most of them are designed for the point-by-point inspection mode, which intrinsically suffers from extremely low efficiency. Recently, an emerging five-axis surface sweep scanning technology shows great potential for boosting the efficiency of surface inspection by using a continuous surface sensing stylus mounted on a rapid rotary probe head. The realization of high-speed five-axis surface scanning depends on a proper sweep scanning path which takes advantage of the superior kinematic performance of the rotary probe head as much as possible and accordingly avoids assigning heavy workload to the three linear axes with weaker kinematic performance. However, up to date, only a handful of five-axis sweep scanning path planning methods are reported and mainly focus on the simple and compact surface. Therefore, existing methods are incompetent to generate a suitable sweep scanning path for high-speed inspection of SMH. For these reasons, we present a novel and practical approach to automatically generate a continuous five-axis sweep scanning path for complex SMH. The generated path exactly meets the unique kinematic requirements of high-speed five-axis inspection and eliminates all noninspection operations. In this way, the SMH inspection can be accomplished with high efficiency. [ABSTRACT FROM AUTHOR]

The neural crest is a multipotent cell population that migrates extensively to play important roles during embryonic development. After acquiring motility, trunk neural crest cells delaminate from the spinal cord and migrate to various regions of the body. Several cellular adhesion molecules, such as vinculin, are involved in the regulation of neural crest delamination and migration. In the present study, we found that draxin could inhibit delamination and migration of neural crest cells from the chick spinal cord and abnormal aggregation of the migrating neural crest cells. In the presence of draxin, the resuspended neural crest regained its adhesive ability such that it was significantly increased. Overexpression of draxin caused increased vinculin expression in vivo. Our data indicate that draxin might control delamination and migration of chick trunk neural crest by increasing cell adhesion. [ABSTRACT FROM AUTHOR]

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyse a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O2 and cleavage of the glycosidic bonds in polysaccharides. The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes. [ABSTRACT FROM AUTHOR]

Cellobiohydrolase Cel7A is an industrial important enzyme that breaks down cellulose by a complex processive mechanism. The enzyme threads the reducing end of a cellulose strand into its tunnel-shaped catalytic domain and progresses along the strand while sequentially releasing the disaccharide cellobiose. While some molecular details of this intricate process have emerged, general structure-function relationships for Cel7A remain poorly elucidated. One interesting aspect is the occurrence of particularly strong ligand interactions in the product binding site. In this work, we analyze these interactions in Cel7A from Trichoderma reesei with special emphasis on the Arg251 and Arg394 residues. We made extensive biochemical characterization of enzymes that were mutated in these two positions and showed that the arginine residues contributed strongly to product binding. Specifically, ∼50% of the total standard free energy of product binding could be ascribed to four hydrogen bonds to Arg251 and Arg394, which had previously been identified in crystal structures. Mutation of either Arg251 or Arg394 lowered production inhibition of Cel7A, but at the same time altered the enzyme product profile and resulted in ∼50% reduction in both processivity and hydrolytic activity. The position of the two arginine residues closely matches the two-fold screw axis symmetry of the substrate, and this energetically favors the productive enzyme-substrate complex. Our results indicate that the strong and specific ligand interactions of Arg251 and Arg394 provide a simple proofreading system that controls the step length during consecutive hydrolysis and minimizes dead time associated with transient, non-productive complexes. [ABSTRACT FROM AUTHOR]

Most of humankind live in cities. They therefore play a central role in development processes. Local authorities, through their efforts to attract external investors, try to actively strengthen these processes. Nowadays, it is important for development to be sustainable, both in terms of the environment and citizenship. This article presents the delocalisation processes from three perspectives: the company, the place where the processes are located (i.e. country, region or municipality) and the inhabitant (social capital). The processes are subject to investor acceptance. This win-win strategy, which benefits all three parties, will become the common feature of all delocalisation investment projects which involve the use of available human resources at the place of investment. At present, more invaluable than the low labour cost is the quality of human/social capital, both in context of adaptation to the needs of a specific entrepreneur and one expressed by social and civic awareness. The author defines the theory of stakeholders as the key determinant that should guide municipalities in attracting investors and implementing a sustainable development strategy. Additionally, the paper uses the method of analysis of the literature on the subject, legal documents and available reports. [ABSTRACT FROM AUTHOR]

The hydrolysis of cellulose by processive cellulases, such as exocellulase TrCel7A from Trichoderma reesei, is typically characterized by an initial burst of high activity followed by a slowdown, often leading to incomplete hydrolysis of the substrate. The origins of these limitations to cellulose hydrolysis are not yet fully understood. Here, we propose a new model for the initial phase of cellulose hydrolysis by processive cellulases, incorporating a bound but inactive enzyme state. The model, based on ordinary differential equations, accurately reproduces the activity burst and the subsequent slowdown of the cellulose hydrolysis and describes the experimental data equally well or better than the previously suggested model. We also derive steady‐state expressions that can be used to describe the pseudo‐steady state reached after the initial activity burst. Importantly, we show that the new model predicts the existence of an optimal enzyme‐substrate affinity at which the pseudo‐steady state hydrolysis rate is maximized. The model further allows the calculation of glucose production rate from the first cut in the processive run and reproduces the second activity burst commonly observed upon new enzyme addition. These results are expected to be applicable also to other processive enzymes. The hydrolysis of cellulose by processive cellulases is characterized by an initial burst of high activity followed by a significant reduction of hydrolysis rate, limiting efficient degradation of the substrate. A new model accurately reproducing the activity burst and the subsequent slow‐down is introduced. The model also provides expressions for the pseudo‐steady state reached after the initial activity burst, and predicts the existence of an optimal enzyme‐substrate affinity at which the pseudo‐steady state hydrolysis rate is maximized. [ABSTRACT FROM AUTHOR]

Copyright of Al-Anbar Journal of Veterinary Sciences is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Monosaccharides and oligosaccharides produced by agarose degradation exhibit potential in the fields of bioenergy, medicine, and cosmetics. Mangrove sediments (MGSs) provide a special environment to enrich enzymes for agarose degradation. However, representative investigations of the agarlytic genes in MGSs have been rarely reported. In this study, agarlytic genes in MGSs were researched in detail from the aspects of diversity, abundance, activity, and location through deep metagenomics sequencing. Functional genes in MGSs were usually incomplete but were shown as results, which could cause virtually high number of results in previous studies because multiple fragmented sequences could originate from the same genes. In our work, only complete and nonredundant (CNR) genes were analyzed to avoid virtually high amount of the results. The number of CNR agarlytic genes in our datasets was significantly higher than that in the datasets of previous studies. Twenty-one recombinant agarases with agarose-degrading activity were detected using heterologous expression based on numerous complete open-reading frames, which are rarely obtained in metagenomics sequencing of samples with complex microbial communities, such as MGSs. Aga2, which had the highest crude enzyme activity among the 21 recombinant agarases, was further purified and subjected to enzymatic characterization. With its high agarosedegrading activity, resistance to temperature changes and chemical agents, Aga2 could be a suitable option for industrial production. The agarase ratio with signal peptides to that without signal peptides in our MGS datasets was lower than that of other reported agarases. Six draft genomes, namely, Clusters 1-6, were recovered from the datasets. The taxonomic annotation of these genomes revealed that Clusters 1, 3, 5, and 6 were annotated as Desulfuromonas sp., Treponema sp., Ignavibacteriales spp., and Polyangiaceae spp., respectively. Meanwhile, Clusters 2 and 4 were potential new species. All these genomes were first reported and found to have abilities of degrading various important polysaccharides. The metabolic pathway of agarose in Cluster 4 was also speculated. Our results showed the capacity and activity of agarases in the MGS microbiome, and MGSs exert potential as a repertory for mining not only agarlytic genes but also almost all genes of the carbohydrate-active enzyme family. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave glycosidic bonds in polysaccharides. The ability of these copper enzymes to boost the degradation of lignocellulose has greatly stimulated research efforts and biocatalytic applications within the biorefinery field. Initially found as oxidizing recalcitrant substrates, such as chitin and cellulose, it is now clear that LPMOs cleave a broad range of oligo- and poly-saccharides and make use of various electron-donating systems. Herein, substrate specificities and electron-donating systems of fungal LPMOs are summarized. A closer look at LPMOs as part of the fungal enzyme machinery might provide insights into their role in fungal growth and plant-pathogen interactions to further stimulate the search for novel LPMO applications. [ABSTRACT FROM AUTHOR]

Abstract: We have measured activity and substrate affinity of the thermostable cellobiohydrolase, Cel7A, from Rasamsonia emersonii over a broad range of temperatures. For the wild type enzyme, which does not have a Carbohydrate Binding Module (CBM), higher temperature only led to moderately increased activity against cellulose, and we ascribed this to a pronounced, temperature induced desorption of enzyme from the substrate surface. We also tested a “high affinity” variant of R. emersonii Cel7A with a linker and CBM from a related enzyme. At room temperature, the activity of the variant was similar to the wild type, but the variant was more accelerated by temperature and about two‐fold faster around 70 °C. This better thermoactivation of the high‐affinity variant could not be linked to differences in stability or the catalytic process, but coincided with less desorption as temperature increased. Based on these observations and earlier reports on moderate thermoactivation of cellulases, we suggest that better cellulolytic activity at industrially relevant temperatures may be attained by engineering improved substrate affinity into enzymes that already possess good thermostability. [ABSTRACT FROM AUTHOR]

Various cellulases consist of a catalytic domain connected to a carbohydrate-binding module (CBM) by a flexible linker peptide. The linker if often strongly O-glycosylated and typically has a length of 20-50 amino acid residues. Functional roles, other than connecting the two folded domains, of the linker and its glycans, have been widely discussed, but experimental evidence remains sparse. One of the most studied cellulose degrading enzymes is the multi-domain cellobiohydrolase Cel7A from Hypocrea jecorina. Here, we designed variants of Cel7A with mutations in the linker region to elucidate the role of the linker. We found that moderate modification of the linker could result in significant changes in substrate affinity and catalytic efficacy. These changes were quite different for different linker variants. Thus, deletion of six residues near the catalytic domain had essentially no effects on enzyme function. Conversely, a substitution of four glycosylation sites near the middle of the linker reduced substrate affinity and increased maximal turnover. The observation of weaker binding provides some support of recent suggestions that linker glycans may be directly involved in substrate interactions. However, a variant with several inserted glycosylation sites near the CBM also showed lower affinity for the substrate compared to the wild-type and we suggest that substrate interactions of the glycans depend on their exact location as well as other factors such as changes in structure and dynamics of the linker peptide. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in Tm, ranging from 2 to 9°C. Combining the positive mutations yielded additional improvements (up to 19°C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12°C increase in Tm and was able to retain and was able to retain no less than 60% of its activity after heat treatment. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes discovered within the last ten years. They oxidatively cleave polysaccharides (chitin, lignocellulose, hemicellulose and starch-derived), presumably making recalcitrant substrates accessible to glycoside hydrolases. Recently, the first crystal structure of an LPMO-substrate complex was reported, giving insights into the interaction of LPMOs with β-linked substrates (Frandsen et al., 2016). The LPMOs acting on α-linked glycosidic bonds (family AA13) display binding surfaces that are quite different from those of LPMOs that act on β-linked glycosidic bonds (families AA9-AA11), as revealed from the first determined structure (Lo Leggio et al., 2015), and thus presumably the AA13s interact with their substrate in a distinct fashion. Here, several new structures of the same AA13 enzyme, Aspergillus oryzae AA13, are presented. Crystals obtained in the presence of high zinc-ion concentrations were used, as they can be obtained more reproducibly than those used to refine the deposited copper-containing structure. One structure with an ordered zinc-bound active site was solved at 1.65 Å resolution, and three structures from crystals soaked with maltooligosaccharides in solutions devoid of zinc ions were solved at resolutions of up to 1.10 Å. Despite similar unit-cell parameters, small rearrangements in the crystal packing occur when the crystals are depleted of zinc ions, resulting in a more occluded substrate-binding surface. In two of the three structures maltooligosaccharide ligands are bound, but not at the active site. Two of the structures presented show a His-ligand conformation that is incompatible with metal-ion binding. In one of these structures this conformation is the principal one (80% occupancy), giving a rare atomic resolution view of a substantially misfolded enzyme that is presumably rendered inactive. [ABSTRACT FROM AUTHOR]

An obituary of a pioneer in the fields of molecular neurobiology and endocrinology, Peter H. Seeburg is presented.

Lytic polysaccharide monooxygenases (PMO) discovered several years ago are enzymes classified as oxidoreductases. In nature, they participate in microbial degradation of cellulose together with cellulases that belong to the hydrolytic type of enzymes (class of hydrolases). Three PMO from ascomycetes- Thielavia terrestris, Trichoderma reesei, and Myceliophthora thermophila-were isolated and purified to homogeneous state using various types of chromatography. The first two enzymes are recombinant proteins heterologously expressed by the Penicillium verruculosum fungus, while the third is a native PMO secreted by M. thermophila. When acting on microcrystalline cellulose, all these PMOs displayed synergism with the cellulase complex of the P. verruculosum fungus. Replacing 10% of cellulases (by protein concentration) with PMO in the presence of 6.25 mM gallic acid or 2.5 μM of cellobiose dehydrogenase from M. thermophila, used as electron donors for PMO, resulted in the 17-31% increase in the yield of reducing sugars after 24-48 h of the enzymatic reaction. [ABSTRACT FROM AUTHOR]

Cell adhesion molecules of the immunoglobulin (Ig) superfamily represent the biggest group of cell adhesion molecules. They have been analyzed since approximately 40 years ago and most of them have been shown to play a role in tumor progression and in the nervous system. All members of the Ig superfamily are intensively posttranslationally modified. However, many aspects of their cellular functions are not yet known. Since a few years ago it is known that some of the Ig superfamily members are modified by ubiquitin. Ubiquitination has classically been described as a proteasomal degradation signal but during the last years it became obvious that it can regulate many other processes including internalization of cell surface molecules and lysosomal sorting. The purpose of this review is to summarize the current knowledge about the ubiquitination of cell adhesion molecules of the Ig superfamily and to discuss its potential physiological roles in tumorigenesis and in the nervous system. [ABSTRACT FROM AUTHOR]

Complex carbohydrates are ubiquitous in all kingdoms of life. As major components of the plant cell wall they constitute both a rich renewable carbon source for biotechnological transformation into fuels, chemicals and materials, and also form an important energy source as part of a healthy human diet. In both contexts, there has been significant, sustained interest in understanding how microbes transform these substrates. Classical perspectives of microbial polysaccharide degradation are currently being augmented by recent advances in the discovery of lytic polysaccharide monooxygenases (LPMOs) and polysaccharide utilization loci (PULs). Fundamental discoveries in carbohydrate enzymology are both advancing biological understanding, as well as informing applications in industrial biomass conversion and modulation of the human gut microbiota to mediate health benefits. [ABSTRACT FROM AUTHOR]

Modern technologies for the enzyme hydrolysis of cellulose-containing raw materials allow the production of sugars from which alcohols (biofuel), organic and amino acids, biopolymers, feed additives, and other value-added products can be obtained via microbiological conversion. Three types of cellulolytic enzymes are required for the bioconversion of cellulose containing materials: endoglucanase, cellobiohydrolase, and ß-glucosidase. The prospects for improving the hydrolytic capabilities of the enzyme complex secreted from Penicillium verruculosum are investigated in this work by means of genetic engineering to add different combinations and ratios of homologous and heterologous cellulases: endoglucanase IV (EGIV) of Trichoderma reesei, endoglucanase II (EGII), and cellobiohydrolase I (CBHI) of P. verruculosum, along with ß-glucosidase (ß-GLU) of Aspergillus niger. The optimum ratio of components is determined and the catalytic activity of enzymatic complexes is increased by as much as 100%. [ABSTRACT FROM AUTHOR]

The effect of polysaccharide monooxygenase (endoglucanase IV) from the fungus Trichoderma reesei on the hydrolysis of polysaccharide substrates by cellulases secreted by the fungus Penicillium verruculosum has been investigated. Supplementation of the enzyme complex from P. verruculosum by endoglucanase IV from T. reesei has been shown to elevate the efficiency of cellulose hydrolysis by 45%. [ABSTRACT FROM AUTHOR]

The gene ABK52392 from the thermophilic bacterium Acidothermus cellulolyticus 11B was predicted to be endoglucanase and classified into glycoside hydrolase family 12. ABK52392 encodes a protein containing a catalytic domain and a carbohydrate binding module. ABK52392 was cloned and functionally expressed in Escherichia coli. After purification by Ni-NTA agarose affinity chromatography and Q-Sepharose® Fast Flow chromatography, the properties of the recombinant protein (AcCel12B) were characterized. AcCel12B exhibited optimal activity at pH 4.5 and 75 °C. The half-lives of AcCel12B at 60 and 70 °C were about 90 and 2 h, respectively, under acidic conditions. The specific hydrolytic activities of AcCel12B at 70 °C and pH 4.5 for sodium carboxymethylcellulose (CMC) and regenerated amorphous cellulose (RAC) were 118.3 and 104.0 U.mg-1, respectively. The Km and Vmax of AcCel12B for CMC were 25.47 mg·mL-1 and 131.75 U·mg-1, respectively. The time course of hydrolysis for RAC was investigated by measuring reducing ends in the soluble and insoluble phases. The total hydrolysis rate rapidly decreased after the early stage of incubation and the generation of insoluble reducing ends decreased earlier than that of soluble reducing ends. High thermostability of the cellulase indicates its potential commercial significance and it could be exploited for industrial application in the future. [ABSTRACT FROM AUTHOR]

In plants, the vascular system, specifically the phloem, functions in delivery of small RNA ( sRNA) to exert epigenetic control over developmental and defense-related processes. Although the importance of systemic sRNA delivery has been established, information is currently lacking concerning the nature of the protein machinery involved in this process. Here, we show that a PHLOEM SMALL-RNA BINDING PROTEIN 1 (PSRP1) serves as the basis for formation of an sRNA ribonucleoprotein complex ( sRNPC) that delivers sRNA (primarily 24 nt) to sink organs. Assembly of this complex is facilitated through PSRP1 phosphorylation by a phloem-localized protein kinase, PSRPK1. During long-distance transport, PSRP1- sRNPC is stable against phloem phosphatase activity. Within target tissues, phosphatase activity results in disassembly of PSRP1- sRNPC, a process that is probably required for unloading cargo sRNA into surrounding cells. These findings provide an insight into the mechanism involved in delivery of sRNA associated with systemic gene silencing in plants. [ABSTRACT FROM AUTHOR]

In plants, small interfering RNAs (siRNA) and microRNAs move to distant tissues where they control numerous developmental and physiological processes such as morphogenesis and stress responses. Grafting techniques and transient expression systems have been employed to show that sequence-specific siRNAs with a size of 21-24 nucleotides traffic to distant organs. We used inverted-repeat constructs producing siRNA targeting the meiosis factor DISRUPTED MEIOTIC cDNA 1 ( DMC1) and GFP to test whether silencing signals move into meiotically active tissues. In grafted Nicotiana tabacum, a transgenic DMC1 siRNA signal made in source tissues preferably entered the anthers formed in the first flowers. Here, the DMC1 siRNA interfered with meiotic progression and, consequently, the flowers were at least partially sterile. In agro-infiltrated N. benthamiana plants, a GFP siRNA signal produced in leaves was allocated and active in most flower tissues including anthers. In hypocotyl-grafted Arabidopsis thaliana plants, the DMC1 silencing signal consistently appeared in leaves, petioles, and stem, and only a small number of plants displayed DMC1 siRNA signals in flowers. In all three tested plant species the systemic silencing signal penetrated male sporogenic tissues suggesting that plants harbour an endogenous long-distance small RNA transport pathway facilitating siRNA signalling into meiotically active cells. [ABSTRACT FROM AUTHOR]

The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Cellulolytic fungi represent a promising group of organisms, as they have evolved complex systems for adaptation to their natural habitat. The filamentous fungus Myceliophthora thermophila constitutes an exceptionally powerful cellulolytic microorganism that synthesizes a complete set of enzymes necessary for the breakdown of plant cell wall. The genome of this fungus has been recently sequenced and annotated, allowing systematic examination and identification of enzymes required for the degradation of lignocellulosic biomass. The genomic analysis revealed the existence of an expanded enzymatic repertoire including numerous cellulases, hemicellulases, and enzymes with auxiliary activities, covering the most of the recognized CAZy families. Most of them were predicted to possess a secretion signal and undergo through post-translational glycosylation modifications. These data offer a better understanding of activities embedded in fungal lignocellulose decomposition mechanisms and suggest that M. thermophila could be made usable as an industrial production host for cellulolytic and hemicellulolytic enzymes. [ABSTRACT FROM AUTHOR]

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source-specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat-1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass. [ABSTRACT FROM AUTHOR]

Plant C2 domain proteins play important roles in diverse cellular processes including growth, development, and membrane targeting, as well as in abiotic and biotic stress adaptations by sensing intracellular Ca signals. In this study, we isolated a novel C2 domain protein gene, TaERG3, from wheat infected by Puccinia striiformis f. sp. tritici. TaERG3 was predicted to encode a 144 amino acid protein with molecular mass of 15.68 kD and isoelectric point of 3.93. Analysis of the deduced amino acid sequence of TaERG3 using InterProScan revealed the presence of an N-terminal calciumdependent phospholipid-binding module (C2 domain, 5 to 103). Transient expression analysis showed that the TaERG3 protein was predominately and uniformly localized in the plasmalemma and nucleus of onion epidermal cells. Quantitative real-time PCR analyses indicated that TaERG3 transcript was differentially induced in both incompatible and compatible interactions, as well as by applied abscisic acid (ABA) and CaCl. However, the significant transcript changes induced by methyl jasmonate, ethylene, and salicylic acid treatments were not as dramatic as those induced by ABA. TaERG3 was also up-regulated by environmental stimuli including low temperature and high salinity. These results imply that TaERG3 might be involved in wheat defence responses against stripe rust and abiotic stresses in an ABA-dependent signalling pathway. [ABSTRACT FROM AUTHOR]