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1.
Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program.  相似文献   
2.
Richard A. Gill 《Plant and Soil》2014,374(1-2):197-210

Background and aims

Drivers of ecosystem dynamics that are under human influence range from local, land-management decisions to global processes such as warming temperatures and N deposition. The goal of this study was to understand how multiple, potentially interacting factors influence net primary production, N mineralization, and water and soil CO2 fluxes.

Methods

Here I report on a three-year experiment that manipulated air temperature using ITEX passive warming cones and N deposition in a mountain meadow ecosystems that were historically grazed or protected from grazing.

Results

The strongest and most consistent effect was due to the legacy of grazing, with previously grazed sites having lower primary production, lower soil respiration rates, lower soil moisture, and lower soil C and N stocks than historically ungrazed sites. Warming increased soil respiration, but the effect was transient, and decreased over the 3-year study. Nitrogen addition increased primary production in the second and third year of the experiment but had no significant effect on soil respiration. The effect of historical grazing on primary production was approximately double the effect of N addition. Temperature and N deposition rarely interacted except for increasing N availability during the warm, wet growing season of 2004.

Conclusions

These findings indicate that the legacies of land use, with their influence on plant community composition and hydrologic processes, are locally more important than short-term step changes in temperature and nutrient availability.  相似文献   
3.

Aims

This study investigated Cu uptake and accumulation as well as physiological and biochemical changes in grapevines grown in soils containing excess Cu.

Methods

The grapevines were collected during two productive cycles from three vineyards with increasing concentrations of Cu in the soil and at various growth stages, before and after the application of Cu-based fungicides. The Cu concentrations in the grapevine organs and the macronutrients and biochemical parameters in the leaf blades were analyzed.

Results

At close to the flowering stage of the grapevines, the concentration and content of Cu in the leaves were increased. However, the Cu concentrations in the roots, stem, shoots and bunches did not correlate with the metal concentrations in the soil. The application of Cu-based fungicides to the leaves increased the Cu concentrations in the shoots, leaves and rachis; however, the effect of the fungicides on the Cu concentration in the berries was not significant. The biochemical analyses of the leaf blades demonstrated symptoms of oxidative stress that correlated with the Cu concentrations in soil.

Conclusions

The increased availability of Cu in soil had a slight effect on the levels and accumulation of Cu in mature grapevines during the productive season and did not alter the nutritional status of the plant. However, increased Cu concentrations were observed in the leaves. The evidence of oxidative stress in the leaves correlated with the increased levels of Cu in soil.  相似文献   
4.
The delivery of proteins instead of DNA into plant cells allows for a transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing, because it can avoid DNA (transgene) integration into the genome and generate precisely modified “nontransgenic” plants. In this work, we explore direct protein delivery to plant cells using mesoporous silica nanoparticles (MSNs) as carriers to deliver Cre recombinase protein into maize (Zea mays) cells. Cre protein was loaded inside the pores of gold-plated MSNs, and these particles were delivered by the biolistic method to plant cells harboring loxP sites flanking a selection gene and a reporter gene. Cre protein was released inside the cell, leading to recombination of the loxP sites and elimination of both genes. Visual selection was used to select recombination events from which fertile plants were regenerated. Up to 20% of bombarded embryos produced calli with the recombined loxP sites under our experimental conditions. This direct and reproducible technology offers an alternative for DNA-free genome-editing technologies in which MSNs can be tailored to accommodate the desired enzyme and to reach the desired tissue through the biolistic method.Introducing DNA-modifying enzymes rather than DNA-based expression cassettes is an attractive alternative for genetic engineering and genome-editing applications such as gene targeting or site-specific recombination. It offers a transient presence of the enzymes, and the process can be coordinated with high levels of enzymatic activity at the time and sites of the desired DNA recombination events. Many DNA-metabolizing enzymes (endonucleases, transposases, and topoisomerases), when delivered in an unrestrained manner, show adverse effects on cell viability. Delivery in the form of protein or RNA may help to mitigate these effects (Cui et al., 2011; Sander et al., 2011; Watanabe et al., 2012). In addition, by introducing proteins, one can avoid the need to remove the protein-encoding DNA fragments from the engineered plant genome. This may help shorten the time from laboratory to field for future improved germplasms.Site-specific recombinases such as Cre or FLP have been widely used in genetic engineering applications (Sorrell and Kolb, 2005). The 38-kD Cre enzyme specifically binds to and recombines the 34-bp loxP sequences, allowing the removal, integration, or inversion of the DNA fragment flanked by these sequences (for review, see Wang et al., 2011). There are a number of established methodologies designed to provide the Cre recombinase activity for site-specific recombination in eukaryotic cells that do not involve the delivery of DNA. These methods include lipofection (Baubonis and Sauer, 1993), microinjection of protein or mRNA (de Wit et al., 1998; Luckow et al., 2009), electroporation of protein or mRNA (Kolb and Siddell, 1996; Ponsaerts et al., 2004), or using modified microorganisms for Cre delivery to their host cells (Vergunst et al., 2000; Koshy et al., 2010). Another strategy that has been used is the incubation or injection of tissues/cell cultures with cell-permeant Cre, a modified Cre protein fused to protein transduction domains or cell-penetrating peptides (Jo et al., 2001; Will et al., 2002; Lin et al., 2004; Nolden et al., 2006).For biotechnological applications in plant sciences, protein delivery systems have been developed, including microinjection (Wymer et al., 2001), protein immobilization to gold particles (Wu et al., 2011), and protein transduction through cell-penetrating peptides (for review, see Chugh et al., 2010). The cell-penetrating peptides were shown to enable intracellular delivery of the Cre recombinase protein to rice (Oryza sativa) callus tissues (Cao et al., 2006). Nanobiotechnology is offering an attractive alternative, since nanoparticles can be precisely tailored to deliver a particular biomolecule to the cell, tissue, or organism of interest when needed (for review, see Du et al., 2012). Mesoporous silica nanoparticles (MSNs) are particularly suited for this purpose. These porous nanoparticles are formed by a matrix of well-ordered pores that confers high loading capacity of molecules like proteins (for review, see Popat et al., 2011). Additionally, surfaces of MSNs can be readily modified, permitting the customization of nanoparticles to particular experimental needs (for review, see Trewyn et al., 2007). In our previous studies, it was shown that MSNs can be used for the codelivery of DNA and chemicals (Torney et al., 2007) as well as DNA and proteins (Martin-Ortigosa et al., 2012a) to plant cells via biolistics. To improve MSN performance as a projectile, gold plating of MSN surfaces was performed, increasing nanoparticle density and, subsequently, the ability to pass through the plant cell wall upon bombardment (Martin-Ortigosa et al., 2012b).In this work, the Cre recombinase enzyme was loaded into the pores of gold-plated MSNs and delivered through the biolistic method to maize (Zea mays) cells containing loxP sites integrated into chromosomal DNA (Lox-corn; Fig. 1A). Lox-corn expressed the glyphosate acetyltransferase gene (gat) and the Anemonia majano cyan fluorescent protein gene (AmCyan1) flanked by loxP sites. The MSN-released Cre enzyme recombined the loxP sites, thus removing the DNA fragment flanked by these sequences. Such excisions led to the expression of a variant of Discosoma sp. red fluorescent protein gene (DsRed2) and the loss of the selectable marker gene (Fig. 1A). Visual selection was used to recover the recombination events. Subsequently, fertile maize plants were regenerated from the recombined events and DNA analyses confirmed the recombination events. To our knowledge, this is the first time that MSNs have been used for the delivery of a functional recombinase into plant tissues, leading to successful genome editing.Open in a separate windowFigure 1.A, Schematic representation of the MSN-based bombardment technology. Cre protein is loaded into the pores of gold-plated MSN (Cre-6x-MSN) and subsequently bombarded onto immature embryos of a transgenic maize line carrying a loxP construct (Lox-corn). The parental transgenic Lox-corn tissues are blue fluorescence and herbicide resistant because they harbor a cassette with the glyphosate acetyltransferase (gat) selection gene and the AmCyan1 (cyan) marker gene flanked by the loxP sites. The DsRed2 (dsred) gene for the expression of a red fluorescent protein is placed downstream of the cassette. Once Cre recombinase is released inside the cell, it performs the recombination, excising gat-AmCyan1 genes and leading to the expression of the DsRed2 gene, switching the cell fluorescence pattern from blue to red. P, Promoter; T, terminator. UBINTRF, CYANF, and DSRED2R are primers for DNA analysis. B, Transmission electron microscope image showing the typical hexagonal shape and the well-ordered pore structure of a 6x-MSN. C, Scanning electron microscope image showing gold nanoparticle deposition (white dots) in all surfaces of 6x-MSN. D, Western blot showing Cre protein loading and release dynamics from 6x-MSN. The protein loading is almost immediate, even though some protein can be detected in the buffer even after 1 h of loading. For the release, some Cre protein can be observed after 24 h of incubation. Most of the protein remains in the 6x-MSN pellet. C+, 400 ng of Cre protein; Empty, a lane with no protein loading. The bands observed in the Empty lane were the spillover from the neighboring Pellet lane, which represents Cre-loaded 6x-MSN after the release experiment resuspended in Laemmli loading buffer (see “Materials and Methods”).  相似文献   
5.
Opium poppy (Papaver somniferum) is one of the world’s oldest medicinal plants and remains the only commercial source for the narcotic analgesics morphine, codeine and semi-synthetic derivatives such as oxycodone and naltrexone. The plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties including the vasodilator papaverine, the cough suppressant and potential anticancer drug noscapine and the antimicrobial agent sanguinarine. Opium poppy has served as a model system to investigate the biosynthesis of benzylisoquinoline alkaloids in plants. The application of biochemical and functional genomics has resulted in a recent surge in the discovery of biosynthetic genes involved in the formation of major benzylisoquinoline alkaloids in opium poppy. The availability of extensive biochemical genetic tools and information pertaining to benzylisoquinoline alkaloid metabolism is facilitating the study of a wide range of phenomena including the structural biology of novel catalysts, the genomic organization of biosynthetic genes, the cellular and sub-cellular localization of biosynthetic enzymes and a variety of biotechnological applications. In this review, we highlight recent developments and summarize the frontiers of knowledge regarding the biochemistry, cellular biology and biotechnology of benzylisoquinoline alkaloid biosynthesis in opium poppy.  相似文献   
6.
New data have been acquired on the biology, morphological features and distribution of Norwegian (Atlantic) pollock Theragra finnmarchica in the Barents Sea. Two individuals of this rare species gadoid (Gadidae) were caught in June and July 2012 in the south-eastern part of the Barents Sea, indicating a wider distribution area of this species than previously thought. It has been confirmed that a number of morphological features of Norwegian pollock is different from T. chalcogramma, and that it feeds on macroplankton (Euphausiidae, Hyperiidae).  相似文献   
7.
Eukaryotic ribosome biogenesis involves ∼200 assembly factors, but how these contribute to ribosome maturation is poorly understood. Here, we identify a network of factors on the nascent 60S subunit that actively remodels preribosome structure. At its hub is Rsa4, a direct substrate of the force-generating ATPase Rea1. We show that Rsa4 is connected to the central protuberance by binding to Rpl5 and to ribosomal RNA (rRNA) helix 89 of the nascent peptidyl transferase center (PTC) through Nsa2. Importantly, Nsa2 binds to helix 89 before relocation of helix 89 to the PTC. Structure-based mutations of these factors reveal the functional importance of their interactions for ribosome assembly. Thus, Rsa4 is held tightly in the preribosome and can serve as a “distribution box,” transmitting remodeling energy from Rea1 into the developing ribosome. We suggest that a relay-like factor network coupled to a mechano-enzyme is strategically positioned to relocate rRNA elements during ribosome maturation.  相似文献   
8.
Cell migration is mediated by the dynamic remodeling of focal adhesions (FAs). Recently, an important role of endosomal signaling in regulation of cell migration was recognized. Here, we show an essential function for late endosomes carrying the p14–MP1 (LAMTOR2/3) complex in FA dynamics. p14–MP1-positive endosomes move to the cell periphery along microtubules (MTs) in a kinesin1- and Arl8b-dependent manner. There they specifically target FAs to regulate FA turnover, which is required for cell migration. Using genetically modified fibroblasts from p14-deficient mice and Arl8b-depleted cells, we demonstrate that MT plus end–directed traffic of p14–MP1-positive endosomes triggered IQGAP1 disassociation from FAs. The release of IQGAP was required for FA dynamics. Taken together, our results suggest that late endosomes contribute to the regulation of cell migration by transporting the p14–MP1 scaffold complex to the vicinity of FAs.  相似文献   
9.
During epithelial cell polarization, Yurt (Yrt) is initially confined to the lateral membrane and supports the stability of this membrane domain by repressing the Crumbs-containing apical machinery. At late stages of embryogenesis, the apical recruitment of Yrt restricts the size of the apical membrane. However, the molecular basis sustaining the spatiotemporal dynamics of Yrt remains undefined. In this paper, we report that atypical protein kinase C (aPKC) phosphorylates Yrt to prevent its premature apical localization. A nonphosphorylatable version of Yrt dominantly dismantles the apical domain, showing that its aPKC-mediated exclusion is crucial for epithelial cell polarity. In return, Yrt counteracts aPKC functions to prevent apicalization of the plasma membrane. The ability of Yrt to bind and restrain aPKC signaling is central for its role in polarity, as removal of the aPKC binding site neutralizes Yrt activity. Thus, Yrt and aPKC are involved in a reciprocal antagonistic regulatory loop that contributes to segregation of distinct and mutually exclusive membrane domains in epithelial cells.  相似文献   
10.
Autophagy, the process by which proteins or organelles are engulfed by autophagosomes and delivered for vacuolar/lysosomal degradation, is induced to ensure survival under starvation and other stresses. A selective autophagic pathway for 60S ribosomal subunits elicited by nitrogen starvation in yeast—ribophagy—was recently described and requires the Ubp3-Bre5 deubiquitylating enzyme. This discovery implied that an E3 ligases act upstream, whether inhibiting the process or providing an initial required signal. In this paper, we show that Ltn1/Rkr1, a 60S ribosome-associated E3 implicated in translational surveillance, acts as an inhibitor of 60S ribosomal subunit ribophagy and is antagonized by Ubp3. The ribosomal protein Rpl25 is a relevant target. Its ubiquitylation is Ltn1 dependent and Ubp3 reversed, and mutation of its ubiquitylation site rendered ribophagy less dependent on Ubp3. Consistently, the expression of Ltn1—but not Ubp3—rapidly decreased after starvation, presumably to allow ribophagy to proceed. Thus, Ltn1 and Ubp3-Bre5 likely contribute to adapt ribophagy activity to both nutrient supply and protein translation.  相似文献   
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