Elevated indole-3-acetic acid peroxidase activity is involved in the cadmium-induced hydrogen peroxide production in barley root tip

All of the analyzed heavy metals significantly inhibited root growth, but in addition to Cd exposure an elevated IAA-POD activity was detected under Co, Cu and Hg treatment, while Ni and Pb inhibited its activity. The Cd-induced IAA-POD activity increased from the root apex towards to the mature region of root tips. However similar or even more severe root growth inhibition was observed by exogenously applied IAA, IAA-POD activity was activated only at high IAA concentrations. Elevated Cd-induced H₂O₂ production was detected both in the absence or in the presence of IAA in the reaction mixture, but in the case of IAA as a possible substrate for PODs the production of H₂O₂ increased markedly just in the Cd-treated roots. Exogenously applied H₂O₂ also activated IAA-POD activity. Our results indicated that in the development of Cd toxicity syndrome the production of ROS during IAA degradation by elevated IAA-POD activity plays a crucial role, mainly under severe Cd stress.     

Effect of heavy metals on root growth and peroxidase activity in barley root tip

In the present work, we investigated the alteration of oxidative and peroxidative activities of peroxidases (PODs) along the longitudinal root axis of barley seedlings during heavy metal (HM; e.g., Cd, Cu, Hg, Ni, Pb) treatment. Analysis of the individual root segments revealed that all of the analyzed HMs caused an increase of guaiacol-POD activity, however to a different extent and spatial distribution. Cd-induced ferulic acid POD activity was observed along the whole root tip (RT), while Cu and Hg caused its increase in the meristematic zone and Ni mainly at the end of the differentiation zone of RT. The activation of coniferyl alcohol POD by HMs was detected along the whole RT. HM-induced hydrogen peroxide-generating POD activity was localized mainly to the elongation zone of RT. Elevated chlorogenic acid POD activity was observed in the meristematic zone and at the end of the differentiation zone of RTs. The activation of several PODs is probably associated with enhanced H₂O₂ production and lignification as a defense response of roots to several HM, to prevent their uncontrolled flux. On the other hand, this defense response is accompanied by root growth inhibition, due to the enhanced rigidification of cell wall and accelerated differentiation of RTs.     

Evolutionary Dynamics of Clustered Irregularly Interspaced Short Palindromic Repeat Systems in the Ocean Metagenome

Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from the Sorcerer II Global Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, <0.0001). We developed a catalogue of elementary CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.Prokaryotes are highly diverse (33). One of the explanations of this diversity is the high extinction rate, due to genetic aggression, which leads to the clearance of ecological niches and, as a result, may allow new prokaryotic species to emerge. In the absence of host defense, viral infection of prokaryotic colonies results in colony extinction or the fixation of a fraction of the invader''s genetic material in the host genome, profoundly affecting the life cycle of the host (32). Thus, bacteria and archaea have developed various kinds of defense mechanisms to resist this pressure; the best studied of these mechanisms is restriction-modification systems (4).Along with well-known prokaryotic defense mechanisms, such as rapid evolution of cell receptors or the use of restriction-modification or toxin-antitoxin systems (see, e.g., references 6, 21, and 25), newly discovered clustered regularly interspaced palindromic repeat (CRISPR) systems seem to play an important role in protecting the cell from archaeal virus or bacteriophage assaults (reviewed in reference 36). A typical CRISPR system is a genetic locus comprising CRISPR-associated (cas) genes coding for proteins of several distinct functional classes (8, 19, 29) and a CRISPR cassette. A CRISPR cassette is formed by almost identical direct repeats with an average length of 32 nucleotides (nt), which are separated by similarly sized, unique spacers. A considerable proportion of spacers is similar to known phage or virus sequences, suggesting that the system is involved in antivirus defense (8, 29, 31). This involvement was experimentally demonstrated when a CRISPR system was shown to be essential for cell survival after invasion by foreign DNA (5). The mechanism is thought to be analogous to eukaryotic RNA interference (29), but it has not been characterized in detail yet.CRISPR cassettes retain information that could be used to reveal the evolutionary history of individual systems. First, it has been shown that CRISPR-associated genes could be divided into eight subtypes according to operon organization and gene phylogeny (19). Second, the repeats of different CRISPR cassettes may be similar, which might indicate a common origin of such cassettes. The first attempt to cluster CRISPR cassettes by the similarity of repeat sequences resulted in 12 clusters (27). In that study, the cassettes were obtained by the application of PILER-CR to completely sequenced genomes. Third, pairwise comparison of spacers could also reveal the specific evolutionary history of individual CRISPR cassettes.So far, most large-scale studies of CRISPR systems have been restricted to well-studied organisms with completely sequenced genomes (5, 9, 20, 28, 30). However, the dynamic interaction between viruses or phages and microorganisms in natural environments is of particular interest (2, 10, 15, 23, 35, 38, 40-42). It may be studied using CRISPRs in a metagenome, that is, sequenced DNA fragments collected in one geographical location and therefore representing one ecological niche with all its inhabitants. This approach is interesting for two reasons. First, metagenomic samples provide a common census of coexisting organisms, i.e., in many cases, both the infecting viruses and phages and their victims. Second, most bacteria and archaea from metagenomic samples cannot be cultivated, and hence little is known about their CRISPR systems.To date, three studies have considered host-virus interactions in metagenomes. One study used two thermophilic Synechococcus isolates from microbial mats in hot springs at Yellowstone National Park to demonstrate fast coevolution of the host and phage genomes (22). Two studies described archaeal and bacterial interactions with viruses and phages, respectively, in acidophilic biofilms (2, 39). All environmental communities analyzed so far are extreme and are dominated by few species. Natural samples containing many diverse coexisting organisms may arguably be more interesting.The largest available metagenome, produced by the Sorcerer II Global Ocean Sampling (GOS) expedition, comprises samples of genetic material collected from more than 50 geographical locations of the Pacific and Atlantic oceans (34). This variety provides an opportunity to study the evolution of phage-host interactions reflected in CRISPRs.Three algorithms, PILER-CR (14), the CRISPR recognition tool (CRT) (7), and CRISPRFinder (18), have been developed as tools for the discovery of new CRISPR cassettes. All these algorithms define candidate CRISPR cassette sequences as short direct repeats separated by short unique spacers; they then use a variety of standard repeat-finding techniques. However, the implementation of specific details is different.PILER-CR constructs local alignments of the input sequence to itself; each hit between two close regions is a candidate for an alignment of a repeat with its neighbor copy. In terms of dynamic programming, taking into account the repeat structure of a CRISPR cassette implies looking for hits only within a relatively narrow band around the main diagonal of the dot plot. This process is followed by several refinement steps.CRT does not use alignments to identify candidate repeats; rather, it derives them directly from the analysis of an input sequence. It is based on finding series of short repeats of a specified length (searching for exact k-mer matches) and then extending these repeats (increasing k-mer length) while allowing for a certain level of mismatches.Finally, CRISPRFinder is based on a suffix-tree-based algorithm for repeat discovery, again with additional refinement.All three algorithms were used for the CRISPR cassette search in this study.     

Morphological Response of the Halophilic Fungal Genus Wallemia to High Salinity

The basidiomycetous genus Wallemia is an active inhabitant of hypersaline environments, and it has recently been described as comprising three halophilic and xerophilic species: Wallemia ichthyophaga, Wallemia muriae, and Wallemia sebi. Considering the important protective role the fungal cell wall has under fluctuating physicochemical environments, this study was focused on cell morphology changes, with particular emphasis on the structure of the cell wall, when these fungi were grown in media with low and high salinities. We compared the influence of salinity on the morphological characteristics of Wallemia spp. by light, transmission, and focused-ion-beam/scanning electron microscopy. W. ichthyophaga was the only species of this genus that was metabolically active at saturated NaCl concentrations. W. ichthyophaga grew in multicellular clumps and adapted to the high salinity with a significant increase in cell wall thickness. The other two species, W. muriae and W. sebi, also demonstrated adaptive responses to the high NaCl concentration, showing in particular an increased size of mycelial pellets at the high salinities, with an increase in cell wall thickness that was less pronounced. The comparison of all three of the Wallemia spp. supports previous findings relating to the extremely halophilic character of the phylogenetically distant W. ichthyophaga and demonstrates that, through morphological adaptations, the eukaryotic Wallemia spp. are representative of eukaryotic organisms that have successfully adapted to life in extremely saline environments.Hypersaline habitats had long been considered to be populated almost exclusively by prokaryotic organisms and the research on hypersaline environments had consequently been monopolized by bacteriologists. In 2000, the first reports appeared showing that fungi are active inhabitants of solar salterns (20). Until then, fungi able to survive in environments with a low amount of biologically available water (low water activity [a_w]) were only known as contaminants of foods preserved with high concentrations of salt or sugar. Since their first discovery in salterns, many new species have been discovered in natural hypersaline environments around the world, including some species that were previously known only as food-borne contaminants. Due to these discoveries, fungi are now recognized as an integral part of indigenous halophilic microbial communities since they can grow and adjust across the whole salinity range, from freshwater to almost saturated NaCl solutions (49). Most fungi differ from the majority of halophilic prokaryotes (16): they tend to be extremely halotolerant rather than halophilic and do not require salt to remain viable, with the exception of Wallemia spp.The order Wallemiales (Wallemiomycetes, Basidiomycota) was only recently introduced to define the single genus Wallemia, a phylogenetic maverick in the Basidiomycota (49). Until 2005, this genus contained only the species W. sebi. However, taxonomic analyses of isolates from sweet, salty, and dried foods (41) and from hypersaline evaporation ponds in the Mediterranean Sea, the Caribbean, and the Dead Sea (45, 49) have resolved this genus into three species: W. ichthyophaga, W. muriae, and W. sebi. The first two of these three Wallemia spp. require additional solutes in the growth media, and W. ichthyophaga is the most halophilic eukaryote described to date, since it cannot grow without the addition of 9% NaCl (wt/vol), and it still shows growth at a_w of 0.77, equivalent to 30% NaCl (wt/vol) (49).The survival, and especially the growth, of microorganisms in highly saline environments requires numerous adaptations (6, 18, 21, 34). The dominant representatives and the most thoroughly investigated halophilic fungi in hypersaline waters of the salterns are the black yeasts, and particularly the model organism Hortaea werneckii (20). An important level of adaptation of the black yeasts to high salinity is seen in their extremophilic ecotype, which is characterized by a special meristematic morphology and changes in cell wall structure and pigmentation (27). Other fungal osmoadaptations include the accumulation of osmolytes (27, 28, 40), the extrusion of sodium (5), modification of the plasma membrane (44) and the cell wall, and even changes in fungal colony morphology (27).The fungal cell wall is the first line of defense against environmental stress; therefore, adaptation at the cell wall level is expected to have one of the most important roles for successful growth at a low a_w (24, 32). The cell wall is essential for maintaining the osmotic homeostasis of cells, since it protects them against mechanical damage as well as high concentrations of salts (7). The central fibrillar glycan network of the cell wall is embedded in highly flexible amorphous cement, which allows considerable stretching with changing osmotic pressure (14, 29). Its balance between a rigid and a dynamic structure influences the shape of cells (14) and enables growth and hyphal branching (11).Since the species within the genus Wallemia have been recognized only recently (49), little is known about their mechanisms of adaptation to high salinity. To investigate the effects of low and high NaCl concentrations on cell morphology, with particular emphasis on cell wall ultrastructure, we compared W. ichthyophaga, the most halophilic fungal species known thus far, with the related xerophilic W. muriae and W. sebi. Micrographs were prepared by using light, transmission, and scanning electron microscopy. The results reveal how this eukaryotic genus uses adaptations at the cell wall level for thriving in extremely saline environments.     

Encapsulation of recombinant <Emphasis Type="Italic">E. coli</Emphasis> expressing cyclopentanone monooxygenase in polyelectrolyte complex capsules for Baeyer–Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one

Recombinant Escherichia coli cells, over-expressing cyclopentanone monooxygenase activity, were immobilized in polyelectrolyte complex capsules, made of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), CaCl₂ and NaCl. More than 90% of the cell viability was preserved during the encapsulation process. Moreover, the initial enzyme activity was fully maintained within encapsulated cells while it halved in free cells. Both encapsulated and free cells reached the end point of the Baeyer–Villiger biooxidation of 8-oxabicyclo[3.2.1]oct-6-en-3-one to 4,9-dioxabicyclo[4.2.1]non-7-en-3-one at the same time (48 h). Similarly, the enantiomeric excess above 94% was identical for encapsulated and free cells.     

Modulation of granulocyte functions by peptide YY in the rat: Age-related differences in Y receptors expression and plasma dipeptidyl peptidase 4 activity

It has been acknowledged that aging exerts detrimental effects on cells of the innate immune system and that neuropeptides, including neuropeptide Y (NPY) and NPY-related peptides fine-tune the activity of these cells through a receptor specific mechanism. The present study investigated the age-dependent potential of peptide YY (PYY) to modulate different granulocyte functions. The PYY reduced the carrageenan-elicited granulocyte accumulation into the air-pouch of aged (24 months) rats, and markedly decreased the phagocytosis of zymosan, as well as the H₂O₂ production, when applied in vivo (20 μg/air-pouch). The anti-inflammatory effect of PYY was less prominent in adult (8 months) and young (3 months) rats. However, the proportions of granulocytes expressing Y1, Y2 and Y5 receptor subtypes were significantly lower in both aged and young rats when compared to adult rats. Furthermore, the aging was found to be associated with the diminished dipeptidyl peptidase 4 (DP4, an enzyme converting the NPY and PYY to Y2/Y5 receptor selective agonists) activity in plasma. In conclusion, the diverse age-related anti-inflammatory effect of PYY in rats originates from different expression levels of Y1, Y2, and Y5 receptor subtypes in addition to different plasma DP4 activity.     

RFLP analysis of cpDNA in the genus <Emphasis Type="Italic">Hypericum</Emphasis>

The chloroplast DNA of 43 species including 16 sections from the genus Hypericum was studied by PCR-RFLP analysis. The PCR-amplified products of four cpDNA regions, trnC-trnD, psbC-trnS, trnL-trnF and rbcL were digested with four restriction endonucleases. A high level of interspecific variation was detected while intraspecific diversity was not observed. The resulting parsimony analysis indicated the monophyletic assemblage of the sections Androsaemum, Olympia, Drosocarpium and Trigynobrathys. Monophyly of Hypericum is weakly supported, but close relationships of H. perforatum and H. maculatum are indicated. The members of Ascyreia are weakly resolved, but clustering of H. kouytchense and H. oblongifolium is well supported, however, H. reptans is nested with Olympia. CpDNA profiles and the positions on the parsimony tree indicate that the chloroplast donor among the putative parents of the hybrid species H. ×inodorum is H. androsaemum.     

A new species of the genus <Emphasis Type="Italic">Allothrombium</Emphasis> (Acari: Trombidiidae) from Montenegro

Allothrombium clavatum sp. n. with reduced inner claw of tarus III, one seta on coxa II, clavate dorsal idiosomal setae and 3-lobed posterior margin of scutum, collected as ectoparasite of an undetermined aphid, is described and illustrated from the central part of Montenegro (Balkan Peninsula). A key to world species of Allothrombium (larva) is presented.     

4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet β-cells,an experimental model of type-2 diabetes

Insulin production in pancreatic β-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the β-cells' glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic β-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an ～ 7-fold improved axial resolution (～ 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary β-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that β-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of β-cell condition, before their transplantations.