Biochemical and spectroscopic characterization of the membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617

Membrane-bound nitrate reductase from Marinobacter hydrocarbonoclasticus 617 can be solubilized in either of two ways that will ultimately determine the presence or absence of the small (Ι) subunit. The enzyme complex (NarGHI) is composed of three subunits with molecular masses of 130, 65, and 20 kDa. This enzyme contains approximately 14 Fe, 0.8 Mo, and 1.3 molybdopterin guanine dinucleotides per enzyme molecule. Curiously, one heme b and 0.4 heme c per enzyme molecule have been detected. These hemes were potentiometrically characterized by optical spectroscopy at pH 7.6 and two noninteracting species were identified with respective midpoint potentials at E _m = +197 mV (heme c) and −4.5 mV (heme b). Variable-temperature (4–120 K) X-band electron paramagnetic resonance (EPR) studies performed on both as-isolated and dithionite-reduced nitrate reductase showed, respectively, an EPR signal characteristic of a [3Fe–4S]⁺ cluster and overlapping signals associated with at least three types of [4Fe–4S]⁺ centers. EPR of the as-isolated enzyme shows two distinct pH-dependent Mo(V) signals with hyperfine coupling to a solvent-exchangeable proton. These signals, called “low-pH” and “high-pH,” changed to a pH-independent Mo(V) signal upon nitrate or nitrite addition. Nitrate addition to dithionite-reduced samples at pH 6 and 7.6 yields some of the EPR signals described above and a new rhombic signal that has no hyperfine structure. The relationship between the distinct EPR-active Mo(V) species and their plausible structures is discussed on the basis of the structural information available to date for closely related membrane-bound nitrate reductases. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Arbuscular mycorrhizal fungal propagules in a salt marsh

The tolerance of indigenous arbuscular mycorrhizal fungi (AMF) to stressful soil conditions and the relative contribution of spores of these fungi to plant colonization were examined in a Portuguese salt marsh. Glomus geosporum is dominant in this salt marsh. Using tetrazolium as a vital stain, a high proportion of field-collected spores were found to be metabolically active at all sampling dates. Spore germination tests showed that salt marsh spores were not affected by increasing levels of salinity, in contrast to two non-marsh spore isolates, and had a significantly higher ability to germinate under increased levels of salinity (20) than in the absence of or at low salinity (10). Germination of salt marsh spores was not affected by soil water levels above field capacity, in contrast to one of the two non-marsh spore isolates. For the evaluation of infectivity, a bioassay was established with undisturbed soil cores (containing all types of AM fungal propagules) and soil cores containing only spores as AM fungal propagules. Different types of propagules were able to initiate and to expand the root colonization of a native plant species, but spores were slower than mycelium and/or root fragments in colonizing host roots. The AM fungal adaptation shown by this study may explain the maintenance of AMF in salt marshes.     

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.     

Somatic embryogenesis in tamarillo (<Emphasis Type="Italic">Cyphomandra betacea</Emphasis>): approaches to increase efficiency of embryo formation and plant development

Somatic embryogenesis induction and somatic embryo development of the solanaceous tamarillo tree were previously established and successfully used for plant regeneration from different explants and varieties. Somatic embryogenesis was induced in Murashige and Skoog medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) or picloram and high sucrose concentrations (0.25 M). The embryogenic tissues were transferred to an auxin-free medium, with reduced sucrose levels, to permit embryo development and conversion into plantlets. This two-step protocol is often impaired by an ineffective transition from the proembryogenic masses to embryo development. In this work, attempts to optimize the somatic embryogenesis system of tamarillo by improving the quality of somatic embryo and embryo conversion were carried out. The results showed that the presence of a high number of abnormal somatic embryos did not significantly inhibit plant conversion, hence indicating that shoot apical meristem development was not affected in abnormal somatic embryos. It was also shown that the manipulation of sucrose concentration in the development medium (0.11 M) and dark conditions before conversion increased the number of morphologically normal somatic embryos. The comparison between mature cotyledonary zygotic and somatic embryos showed an inefficient accumulation of storage compounds, mainly lipids, in somatic embryos. These reduced levels of lipid storage could be responsible for the abnormal patterns of embryo development found in tamarillo somatic embryos.     

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2)

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO₂, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma.     

Aluminum as an endocrine disruptor in female Nile tilapia (Oreochromis niloticus)

The effects of aluminum on plasma ion, lipid, protein and steroid hormone concentration were evaluated in Oreochromis niloticus broodstock females. Lipid and protein concentrations from the gonads and liver were also measured. Experiments were performed at neutral and acidic water pH. Four groups of fish were tested for 96 h: 1) control conditions at neutral water pH; 2) control conditions at acidic water pH (CTR-Ac); 3) aluminum at neutral water pH (Al-N); and 4) aluminum at acidic water pH (Al-Ac). Aluminum and acidic water pH exposure caused no ionoregulatory disturbances. Total lipid concentration increased in the mature gonads and decreased in the liver, suggesting an acceleration of lipid mobilization to the ovaries in animals exposed to aluminum. However, a decreased protein concentration in ovaries was also observed. Exposure of control fish to acidic water pH caused an increased concentration of plasma 17α-hydroxyprogesterone. However, females exposed to aluminum at acidic water pH showed a decreased of plasma 17α-hydroxyprogesterone and cortisol. No differences in plasma 17β-estradiol were observed. The physiological mechanisms underlying the disturbances observed are discussed focusing on reproduction. We suggest that aluminum can be considered an endocrine disrupting compound in mature O. niloticus females.     

Role of sensory cues on food searching behavior of a captive Manta birostris (Chondrichtyes,Mobulidae)

This study reports on the first experimental research designed specifically for Manta birostris behavior. The authors attempted to learn about the feeding behavior and environmental cues influencing this behavior, as well as general cognitive ability. The preconditioned Manta's ability to identify food, on the basis of a fraction of the ordinary food signal complex, was tested. The opening of cephalic fins was considered a good indicator of feeding motivation level. The study subject animal used its biological clock to predict time and also associated a specific location with food, suggesting an ability to build up a cognitive map of its environment. Both underwater visual stimuli and olfactory stimuli had a very intense effect on food searching behavior over a 30 m distance, in contrast to visual signs from above the water surface. In addition, although an underwater visual signal resulted in a more intense response than from an olfactory signal, the specimen did not discriminate between different objects tested on the basis of visual sensation. It could therefore be suggested that food searching behavior of Mantas are governed by triggering stimuli, including smell or visual recognition, and modulated by the cognitive spatial map stored in their long‐term memory. These findings will hopefully prove useful while devising protecting policies in the natural environment and/or while keeping these animals in captivity. Zoo Biol 27:294–304, 2008. © 2008 Wiley‐Liss, Inc.     

Protected areas buffer the Brazilian semi‐arid biome from climate change

The Caatinga is a botanically unique semi‐arid ecosystem in northeast Brazil whose vegetation is adapted to the periodic droughts that characterize this region. However, recent extreme droughts events caused by anthropogenic climate change have challenged its ecological resilience. Here, we evaluate how deforestation and protection status affect the response of the Caatinga vegetation to drought. Specifically, we compared vegetation responses to drought in natural and deforested areas as well as inside and outside protected areas, using a time‐series of satellite‐derived Normalized Difference Vegetation Index (NDVI) and climatic data for 2008–2013. We observed a strong effect of deforestation and land protection on overall vegetation productivity and in productivity dynamics in response to precipitation. Overall, deforested areas had significantly lower NDVI and delayed greening in response to precipitation. By contrast, strictly protected areas had higher productivity and considerable resilience to low levels of precipitation, when compared to sustainable use or unprotected areas. These results highlight the importance of protected areas in protecting ecosystem processes and native vegetation in the Caatinga against the negative effects of climate change and deforestation. Given the extremely small area of the Caatinga currently under strict protection, the creation of new conservation areas must be a priority to ensure the sustainability of ecological processes and to avoid further desertification.     

The Use of Bayesian Latent Class Cluster Models to Classify Patterns of Cognitive Performance in Healthy Ageing

The main focus of this study is to illustrate the applicability of latent class analysis in the assessment of cognitive performance profiles during ageing. Principal component analysis (PCA) was used to detect main cognitive dimensions (based on the neurocognitive test variables) and Bayesian latent class analysis (LCA) models (without constraints) were used to explore patterns of cognitive performance among community-dwelling older individuals. Gender, age and number of school years were explored as variables. Three cognitive dimensions were identified: general cognition (MMSE), memory (MEM) and executive (EXEC) function. Based on these, three latent classes of cognitive performance profiles (LC1 to LC3) were identified among the older adults. These classes corresponded to stronger to weaker performance patterns (LC1>LC2>LC3) across all dimensions; each latent class denoted the same hierarchy in the proportion of males, age and number of school years. Bayesian LCA provided a powerful tool to explore cognitive typologies among healthy cognitive agers.     

Molecular characterization of bla(IMP-5), a new integron-borne metallo-beta-lactamase gene from an Acinetobacter baumannii nosocomial isolate in Portugal

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0-17.9 per microg circular DNA using 10(7) protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.