Comparison of Proteolytic Activities Produced by Entomopathogenic Photorhabdus Bacteria: Strain- and Phase-Dependent Heterogeneity in Composition and Activity of Four Enzymes

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (~74, ~55, ~54, and ~37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokházi, G. Kóczán, F. Hudecz, L. Gráf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.     

The mapping of linear B‐cell epitope regions in the extracellular parts of the desmoglein 1 and 3 proteins: recognition of immobilized peptides by pemphigus patients' serum autoantibodies

Desmosomal transmembrane glycoproteins desmogleins (Dsg) 1 and 3 are targets of life‐threatening autoimmune blistering disorders such as Pemphigus vulgaris (PV) and Pemphigus foliaceus (PF). In these diseases, pemphigus autoantibodies are produced against Dsg1 and Dsg3 proteins. The autoantibodies bind to these transmembrane elements leading to a loss of desmosomal cell–cell adhesion and clinically, to the presence of blisters and erosions. Identification, characterization, and detailed analysis of the binding sites of autoantibodies have an outstanding importance in understanding the immunopathology of the disease and also in the design of novel diagnostics. Here, we describe the localization of the B‐cell epitope regions of Dsg1 and Dsg3 proteins' extracellular parts recognized by IgG‐type serum autoantibodies of patients with PV and PF. In our study, overlapping pentadecapeptides were synthesized on hydroxypropyl methacrylate pins based on the results of in silico predictions. To detect the interaction between the serum autoantibodies and the immobilized synthetic peptides, modified Enzyme Linked Immunosorbent Assay (ELISA) was performed with pin‐attached peptides testing the serum samples of ten patients and four healthy donors. We identified five possible epitope regions (aa86‐110, aa196‐220, aa226‐250, aa326‐340, and aa486‐520) within the extracellular part of the Dsg1 and four possible epitope regions (aa64‐78, aa330‐344, aa375‐399, and aa446‐460) within that of the Dsg3 protein sequence using these methods. Our data showed that serum autoantibodies of patients, previously identified as Dsg1 and Dsg3 positive, are able to recognize continuous linear epitope regions of both Dsg1 and Dsg3 proteins using pin‐bound overlapping peptides in modified ELISAs. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Role of PCNA and TLS polymerases in D-loop extension during homologous recombination in humans

Homologous recombination (HR) is essential for maintaining genomic integrity, which is challenged by a wide variety of potentially lethal DNA lesions. Regardless of the damage type, recombination is known to proceed by RAD51-mediated D-loop formation, followed by DNA repair synthesis. Nevertheless, the participating polymerases and extension mechanism are not well characterized. Here, we present a reconstitution of this step using purified human proteins. In addition to Pol δ, TLS polymerases, including Pol η and Pol κ, also can extend D-loops. In vivo characterization reveals that Pol η and Pol κ are involved in redundant pathways for HR. In addition, the presence of PCNA on the D-loop regulates the length of the extension tracks by recruiting various polymerases and might present a regulatory point for the various recombination outcomes.     

Changes in the photosynthetic efficiency of winter wheat in response to abiotic stress

The assessment of heat and drought tolerance is of primary importance in breeding programmes designed to improve heat and drought tolerance in cereals. Three winter wheat varieties grown in controlled growth chambers were exposed to heat (H) and drought (D) stress singly and in combination (H+D). The combined effects of H and D stress were much more severe than those of individual treatments for both physiological and yield parameters during grain filling. The chlorophyll content, effective quantum yield of PSII, net assimilation rate, transpiration, stomatal conductance and intercellular CO₂ concentration were greatly reduced by H, D and their interaction. Grain yield decreased to a greater extent (48.3%) in Plainsman V, averaged over the stress treatments, than in Mv Magma (67.8%) and Fatima 2 (53.7%). The least decline was found in grain number, except in Plainsman V. Mv Magma tolerated heat stress better than Fatima 2. In terms of photosynthetic activity, Plainsman V showed better drought tolerance than Mv Magma. The results showed that changes in physiological properties during stress treatment are not always associated with changes in yield parameters, so a combination of methods may be needed to give a more precise picture of the stress tolerance of wheat varieties.     

Influence of the Dabcyl group on the cellular uptake of cationic peptides: short oligoarginines as efficient cell-penetrating peptides

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4–((4–(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity.

     

A New Zearalenone Biodegradation Strategy Using Non-Pathogenic Rhodococcus pyridinivorans K408 Strain

Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17β-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17β-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects.     

A grid-based, satellite-image supported, multi-attributed vegetation mapping method (MÉTA)

In this paper we present the main characteristics of a new, grid-based, landscape-ecology-oriented, satellite-image supported, field vegetation mapping method, called MÉTA (MÉTA stands for Magyarországi Él?helyek Térképi Adatbázisa: GIS Database of the Hungarian Habitats). The goals of the MÉTA method based vegetation mapping program (MÉTA mapping) include the following: (1) to map the actual (semi-)natural vegetation of Hungary; (2) to evaluate Hungarian (semi-)natural vegetation heritage for conservation purposes; (3) to evaluate the present state of Hungarian landscapes from a vegetation point of view; (4) to collect vegetation and landscape ecological data for the prognosis of future changes of vegetation and the landscape. Spatial resolution, mapped attributes and mapping methods were developed to meet these goals. The MÉTA method uses a hexagon grid with cells of 35 hectares. In the hexagons, habitat types are listed, then the area, naturalness-based habitat quality, spatial pattern in the hexagon, effect of the neighbourhood, connectedness, and threats are recorded for each habitat type. Other attributes are recorded in the hexagons: potential natural vegetation, area occupied by invasive plant species, area of old fields, land use of grasslands, and landscape health status (naturalness and regeneration potential of the landscape in general). One hundred hexagons form a quadrat — mainly for practical, organizational reasons, but also for collecting certain vegetation data at this spatial scale. For standardization of mapping, three different pre-printed data sheets and two different kinds of guides have been composed (Mapping Guide and Habitat Guide) and field trainings were organized. For standardization of estimation of naturalness-based habitat quality and regeneration potential field examples were prepared for each habitat type and each category of these attributes.     

Efficient synthesis of an (aminooxy) acetylated‐somatostatin derivative using (aminooxy)acetic acid as a ‘carbonyl capture’ reagent

Owing to the high chemoselectivity between an aminooxy function and a carbonyl group, oxime ligation is one of the most preferred procedures for the preparation of peptide conjugates. However, the sensitivity of (aminooxy)acetylated peptides to ketones and aldehydes makes their synthesis and storage difficult. In our study, we established the efficient synthesis of an (aminooxy)acetylated‐somatostatin derivative in the presence of free (aminooxy)acetic acid, which was used as a ‘carbonyl capture’ reagent in the final cleavage step. This (aminooxy)acetylated compound was further used for the chemoselective ligation (oxime bond formation) with daunorubicin and 4‐fluorobenzaldehyde leading to the formation of conjugates with potential applications in targeted cancer chemotherapy and positron emission tomography. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Phagocytosis of cells dying through autophagy induces inflammasome activation and IL-1β release in human macrophages

Phagocytosis of naturally dying cells usually blocks inflammatory reactions in host cells. We have recently observed that clearance of cells dying through autophagy leads to a pro-inflammatory response in human macrophages. Investigating this response further, we found that during engulfment of MCF-7 or 293T cells undergoing autophagic death, but not apoptotic or anoikic ones, caspase-1 was activated and IL-1β was processed, then secreted in a MyD88-independent manner. Autophagic dying cells were capable of preventing some LPS-induced pro-inflammatory responses, such as TNFα, IL-6 and IL-8 induction, but synergized with LPS for IL-1β production. Caspase-1 inhibition prevented macrophage IL-1β release triggered by the dying cells and also other pro-inflammatory cytokines which were not formed in the presence of IL-1 receptor antagonist anakinra either. IL-1β secretion was also observed using calreticulin knock down or necrostatin treated autophagic MCF-7 cells and it required phagocytosis of the dying cells which led to ATP secretion from macrophages. Blocking K (+) efflux during phagocytosis, the presence of apyrase, adding an antagonist of the P2X7 receptor or silencing the NOD-like receptor protein NALP3 inhibited IL-1β secretion. These data suggest that during phagocytosis of autophagic dying cells ATP, acting through its receptor, initiates K (+) efflux, inflammasome activation and secretion of IL-1β, which initiates further pro-inflammatory events. Thus, autophagic death of malignant cells and their clearance may lead to immunogenic response.