Roseobacter-Like Bacteria in Red and Mediterranean Sea Aerobic Anoxygenic Photosynthetic Populations

Bacteriochlorophyll a-containing aerobic anoxygenic phototrophs (AAnP) have been proposed to account for up to 11% of the total surface water microbial community and to potentially have great ecological importance in the world's oceans. Recently, environmental and genomic data based on analysis of the pufM gene identified the existence of α-proteobacteria as well as possible γ-like proteobacteria among AAnP in the Pacific Ocean. Here we report on analyses of environmental samples from the Red and Mediterranean Seas by using pufM as well as the bchX and bchL genes as molecular markers. The majority of photosynthesis genes retrieved from these seas were related to Roseobacter-like AAnP sequences. Furthermore, the sequence of a novel photosynthetic operon organization from an uncultured Roseobacter-like bacterial artificial chromosome retrieved from the Red Sea is described. The data show the presence of Roseobacter-like bacteria in Red and Mediterranean Sea AAnP populations in the seasons analyzed.     

Cracking the nut: geographical adjacency of sister taxa supports vicariance in a polytomic salamander clade in the absence of node support

The urodelan genus Lyciasalamandra, which inhabits a relatively small area along the southern Turkish coast and some Aegean islands, provides an outstanding example of a diverse but phylogenetically unresolved taxon. Molecular trees contain a single basal polytomy that could be either soft or hard. We here use the information of nuclear (allozymes) and mitochondrial (fractions of the 16S rRNA and ATPase genes) datasets in combination with area relationships of lineages to resolve the phylogenetic relationships among Lyciasalamandra species in the absence of sufficient node support. We can show that neither random processes nor introgressive hybridization can be invoked to explain that the majority of pairs of sister taxa form geographically adjacent units and interpret that this pattern has been shaped by vicariant events. Topology discordance between mitochondrial and nuclear trees mainly refers to an affiliation of L. helverseni, a taxon restricted to the Karpathos archipelago, to the western-most and geographically proximate mainland taxon in the nuclear tree, while in the organelle tree it turns out to be the sister lineage to the geographically most distant eastern clade. As this discordance cannot be explained by long-branch attraction in either dataset we suppose that oversea dispersal may have accounted for a second colonization of the Karpathos archipelago. It may have initiated introgression and selection driven manifestation of alien eastern mitochondrial genomes on a western nuclear background. Our approach of testing for area relationships of sister taxa against the null hypothesis of random distribution of these taxa seems to be especially helpful in phylogenetic studies where traditional measures of phylogenetic branch support fail to reject the null hypothesis of a hard polytomy.     

Role of G proteins and ERK activation in hemin-induced erythroid differentiation of K562 cells

Heterotrimeric G proteins which couple extracellular signals to intracellular effectors play a central role in cell growth and differentiation. The pluripotent erythroleukemic cell line K562 that acquires the capability to synthesize hemoglobin in response to a variety of agents can be used as a model system for erythroid differentiation. Using Western blot analysis and RT-PCR, we studied alterations in G protein expression accompanying hemin-induced differentiation of K562 cells. We demonstrated the presence of G(alpha s), G(alpha i2) and G(alpha q) and the absence of G(alpha i1), G(alpha o) and G(alpha 16) in K562 cells. We observed the short form of G(alpha s) to be expressed predominantly in these cells. Treatment of K562 cells with hemin resulted in an increase in the levels of G(alpha s) and G(alpha q). On the other hand, the level of G(alpha i2) was found to increase on the third day after induction with hemin, followed by a decrease to levels lower of those of uninduced cells. The mitogen-activated protein kinase ERK1/2 pathway is crucial in the control of cell proliferation and differentiation. Both Gi- and Gq-coupled receptors stimulate MAPK activation. We therefore examined the phosphorylation of ERK1/2 during hemin-induced differentiation of K562 cells. Using anti-ERK1/2 antibodies, we observed that ERK2 was primarily phosphorylated in K562 cells. ERK2 phosphorylation increased gradually until 48 h and returned to basal values by 96 h following hemin treatment. Our results suggest that changes in G protein expression and ERK2 activity are involved in hemin-induced differentiation of K562 cells.     

The Usefulness of DNA Sequencing After Extraction by Whatman FTA Filter Matrix Technology and Phenotypic Tests for Differentiation of Candida albicans and Candida dubliniensis

Since C. dubliniensis is similar to C. albicans phenotypically, it can be misidentified as C. albicans. We aimed to investigate the prevalence of C. dubliniensis among isolates previously identified as C. albicans in our stocks and to compare the phenotypic methods and DNA sequencing of D1/D2 region on the ribosomal large subunit (rLSU) gene. A total of 850 isolates included in this study. Phenotypic identification was performed based on germ tube formation, chlamydospore production, colony colors on chromogenic agar, inability of growth at 45 °C and growth on hypertonic Sabouraud dextrose agar. Eighty isolates compatible with C. dubliniensis by at least one phenotypic test were included in the sequence analysis. Nested PCR amplification of D1/D2 region of the rLSU gene was performed after the fungal DNA extraction by Whatman FTA filter paper technology. The sequencing analysis of PCR products carried out by an automated capillary gel electrophoresis device. The rate of C. dubliniensis was 2.35 % (n = 20) among isolates previously described as C. albicans. Consequently, none of the phenotypic tests provided satisfactory performance alone in our study, and molecular methods required special equipment and high cost. Thus, at least two phenotypic methods can be used for identification of C. dubliniensis, and molecular methods can be used for confirmation.     

Formulation and Optimization of Nonionic Surfactants Emulsified Nimesulide-Loaded PLGA-Based Nanoparticles by Design of Experiments

This investigation aimed to develop nimesulide (NMS)-loaded poly(lactic-co-glycolic acid) (PLGA)-based nanoparticulate formulations as a biodegradable polymeric drug carrier to treat rheumatoid arthritis. Polymeric nanoparticles (NPs) were prepared with two different nonionic surfactants, vitamin E d-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) and poly(vinyl alcohol) (PVA), using an ultrasonication solvent evaporation technique. Nine batches were formulated for each surfactant using a 3² factorial design for optimal concentration of the emulsifying agents, 0.03–0.09% for vitamin E TPGS and 2–4% for PVA. The surfactant percentage and the drug/polymer ratio (1:10, 1:15, 1:20) of the NMS-loaded NPs were investigated based on four responses: encapsulation efficiency, particle size, the polydispersity index, and the surface charge. The response surface plots and linearity curves indicated a relationship between the experiment’s responses and a set of independent variables. The NPs produced with both surfactants exhibited a negative surface charge, and scanning electron micrographs revealed that all of the NPs were spherical in shape. A narrower size distribution and higher drug loadings were achieved in PVA-emulsified PLGA NPs than in the vitamin E TPGS emulsified. Decreasing amounts of both nonionic surfactants resulted in a reduction in the emulsion’s viscosity, which led to a decrease in the particle size of NPs. According to the ANOVA results obtained in this present research, vitamin E TPGS exhibited the best correlation between the independent variables, namely drug/polymer ratio and the surfactant percentage, and the dependent variables (encapsulation efficiency R² = 0.9603, particle size R² = 0.9965, size distribution R² = 0.9899, and surface charge R² = 0.8969) compared with PVA.KEY WORDS: ANOVA, factorial design, nanoparticles, nimesulide, PLGA, PVA, vitamin E TPGS     

Voltage dependence of the Ca<Superscript>2+</Superscript> transient in endocardial and epicardial myocytes from the left ventricle of Goto–Kakizaki type 2 diabetic rats

There is much evidence that a combination of ibuprofen (IBU) and Aspirin (ASA) can antagonize the irreversible inhibition of platelet function. This study was designed to investigate the degree of gastric damage, bleeding time (BT) and fluctuations in the serum levels of prostaglandin E₂ (PGE₂) and thromboxane A₂ (TXA₂) after oral administration of ASA (200 mg/kg) and IBU (50 mg/kg) either alone or in combination in rats in vivo. The stomach was assessed for any damage either after 6 h, 18 h or 6 days and carboxymethylcellulose (1% CMC) served as a vehicle and control. ELISA was used to measure TXA₂ and PGE₂ in serum. Bleeding time was assessed using tail blood. The results show that ASA and IBU either alone or in combination can cause gastric ulceration in 25–100% of the rats at 6 and 18 h. In contrast, gastric ulceration was seen in 50% of rats with a combination of ASA given before IBU only after 6 days of oral administration. BT was unaffected either by ASA or IBU when administered alone except after 18 h for IBU. In contrast, BT was significantly reduced when IBU was administered before ASA after 18 h and 6 days (P < 0.001). Serum PGE₂ levels decreased significantly after ASA administered either alone or in combination with IBU for 6 h, 18 h and 6 days (P < 0.05). Serum TXA₂ levels were significantly reduced after 6 h, 18 h and 6 days following ASA and IBU administration except for IBU alone which caused a significant increase in serum TXA₂ 6 days after its administration (P < 0.01). It can be concluded that ASA and IBU administered either alone or in combination can cause gastric ulcers in the rat stomach after 6 h and 18 h, but less severe after 6 days. IBU either alone or in combination with ASA reduced BT only after 18 h and 6 days of administration. Together, the results show that gastric ulceration correlated well with the inhibition of serum PGE₂ and TXA₂ levels.     

Statistical discrimination of sex in Melanoides tuberculata (Gastropoda: Thiaridae)

Systematists may rely on morphometric differences among samples of specimens for the recognition of living and fossil species, even though morphometric differentiation may be caused by non-genetic factors, such as ecophenotypy, differential growth rates and taphonomic mixing. When genetic differences between sexes or among closely related species are expressed as differences in the morphology of the individual or population, potentially valuable information becomes available to the systematist for a variety of genetic and ecological investigations. We have studied the morphology of the freshwater snail Melanoides tuberculata (Muller, 1774) in Israel, where males occur in what would otherwise be normally parthenogenetic (all female) populations. In modern M. tuberculata, sex may be determined by observation of gonadal tissue; in fossil specimens, any classification according to sex must be accomplished using only preservable features of the mineralized shell. Previous research confirmed that in large samples, mean shell shape of male and female snails differed significantly, but the degree of difference was too small to identify the sex of any individual specimen. We apply a three stage process that results with a high degree of accuracy in the discrimination of individual M. tuberculata specimens by sex on the basis of continuous morphological characters: (1) measurement of many aspects of shell morphology of individuals of known sex, and stepwise discrimination to discover which of the variables, if any, contribute to the morphometric differentiation of males from females (one time only, for the species); (2) use of these selected variables in a clustering procedure to make a preliminary assignment of each specimen to sex; (3) use of cluster assignments in a discrimination procedure to optimally predict sex. For species that exhibit morphometric differences between two groups, and for which continuous morphometric variation precludes the a priori recognition of discrete clusters, this sequential procedure may be of broad applicability. These objective methods may be applied to the discrimination within any set of specimens for which the hypothesis of two, and only two, constituent groups may be entertained.     

Effects of long-term exposure to 900 megahertz electromagnetic field on heart morphology and biochemistry of male adolescent rats

The pathological effects of exposure to an electromagnetic field (EMF) during adolescence may be greater than those in adulthood. We investigated the effects of exposure to 900 MHz EMF during adolescence on male adult rats. Twenty-four 21-day-old male rats were divided into three equal groups: control (Cont-Gr), sham (Shm-Gr) and EMF-exposed (EMF-Gr). EMF-Gr rats were placed in an EMF exposure cage (Plexiglas cage) for 1 h/day between postnatal days 21 and 59 and exposed to 900 MHz EMF. Shm-Gr rats were placed inside the Plexiglas cage under the same conditions and for the same duration, but were not exposed to EMF. All animals were sacrificed on postnatal day 60 and the hearts were extracted for microscopic and biochemical analyses. Biochemical analysis showed increased levels of malondialdehyde and superoxide dismutase, and reduced glutathione and catalase levels in EMF-Gr compared to Cont-Gr animals. Hematoxylin and eosin stained sections from EMF-Gr animals exhibited structural changes and capillary congestion in the myocardium. The percentage of apoptotic myocardial cells in EMF-Gr was higher than in either Shm-Gr or Cont-Gr animals. Transmission electron microscopy of myocardial cells of EMF-Gr animals showed altered structure of Z bands, decreased myofilaments and pronounced vacuolization. We found that exposure of male rats to 900 MHz EMF for 1 h/day during adolescence caused oxidative stress, which caused structural alteration of male adolescent rat heart tissue.     

Intraspecific variation in defense against a generalist lepidopteran herbivore in populations of Eruca sativa (Mill.)

Populations of Eruca sativa (Brassicaceae) from desert and Mediterranean (Med) habitats in Israel differ in their defense against larvae of the generalist Spodoptera littoralis but not the specialist Pieris brassicae. Larvae of the generalist insect feeding on plants of the Med population gained significantly less weight than those feeding on the desert plants, and exogenous application of methyl jasmonate (MJ) on leaves of the Med plants significantly reduced the level of damage created by the generalist larvae. However, MJ treatment significantly induced resistance in plants of the desert population, whereas the generalist larvae caused similar damage to MJ‐induced and noninduced plants. Analyses of glucosinolates and expression of genes in their synthesis pathway indicated that defense in plants of the Med population against the generalist insect is governed by the accumulation of glucosinolates. In plants of the desert population, trypsin proteinase inhibitor activity was highly induced in response to herbivory by S. littoralis. Analysis of genes in the defense‐regulating signaling pathways suggested that in response to herbivory, differences between populations in the induced levels of jasmonic acid, ethylene, and salicylic acid mediate the differential defenses against the insect. In addition, expression analysis of myrosinase‐associated protein NSP2 suggested that in plants of the desert population, glucosinolates breakdown products were primarily directed to nitrile production. We suggest that proteinase inhibitors provide an effective defense in the desert plants, in which glucosinolate production is directed to the less toxic nitriles. The ecological role of nitrile production in preventing infestation by specialists is discussed.