Resting forms of gram negative chemolithoautotrophic bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum

The haloalkaliphilic chemoautotrophic gram-negative bacteria Thioalkalivibrio versutus, strain AL2, and Thioalkalimicrobium aerophilum, strain AL3, were shown to possess the capacity to produce resting forms, namely cyst-like refractile cells (CRC), whose production was controlled by the level of the d1 extracellular factors, exhibiting the function of anabiosis autoinducers. The conditions were elucidated that promoted the formation of CRC in the developmental cycles of the cultures studied, in condensed cell suspensions undergoing autolysis, and under the action of exogenously introduced chemical analogues of anabiosis autoinducers (alkylhydroxybenzenes). The peculiarities of the fine structure of the resting cells obtained were studied. Distinctions were revealed (with respect to viability and thermotolerance) between the CRC formed under different conditions. The relationship between the growth strategy and survival strategy of extremophilic bacteria is discussed with taking into account the effect of the d1 autoregulatory factors. A new model of CRC formation is proposed: CRC production in the life cycle of bacteria developing under conditions of increased concentration of anabiosis autoinducers.     

Effects of defoliation caused by the leaf miner<Emphasis Type="Italic"> Cameraria ohridella</Emphasis> on wood production and efficiency in<Emphasis Type="Italic"> Aesculus hippocastanum</Emphasis> growing in north-eastern Italy

The leaf miner Cameraria ohridella causes premature defoliation of Aesculus hippocastanum trees. Repeated defoliation has been reported to cause decrease in radial growth of trees and a progressive decline due to reduced production and allocation of photosynthates. Our study represents an attempt to estimate the impact of C. ohridella on annual wood increments and the hydraulic properties of the wood as well as on the dry mass of seeds. Twenty-two adult horse chestnut trees were selected, four of which had been chemically treated to prevent attack (controls). All other trees were heavily infested. The ground cover (GC) of each tree, measured from monthly hemispherical photographs, revealed that infested trees were completely defoliated in September and the slope of the GC-to-measurement dates relationship (named GC decrease index) was positively related to the number of mines per leaf. Anatomical observations showed that infested trees produced more wood per year than controls through more false rings with wider xylem conduits and, hence, with higher conductive area and theoretical flow. In fact, the theoretical flow was positively related to the defoliation intensity. In contrast, the allocation of photosynthates to seeds was strongly reduced in infested trees with respect to controls (up to 50% less). The hypothesis was advanced that horse chestnut trees reacted to C. ohridella attacks by increasing the hydraulic efficiency of the wood, thus ameliorating the water and nutrient supply to leaves between the spring and mid-summer and, therefore, compensating, at least partly, the reduced leaf lifespan.     

Electrochemiluminescent biosensors array for the concomitant detection of choline, glucose, glutamate, lactate, lysine and urate

A multifunctional bio-sensing chip was designed based on the electrochemiluminescent (ECL) detection of enzymatically produced hydrogen peroxide. Six different oxidases specific for choline, glucose, glutamate, lactate, lysine and urate were non-covalently immobilised on imidodiacetic acid chelating beads (glucose oxidase only) or on diethylaminoethyl (DEAE) anion exchanger beads, and spotted on the surface of a glassy carbon foil (25 mm(2) square), entrapped in PVA-SbQ photopolymer. The chip measurement was achieved by applying during 3 min a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference, while capturing a numeric image of the multifunctional bio-sensing chip with a CCD camera. The use of luminol supporting beads (DEAE-Sepharose) included in the sensing layer was shown to enable the achievement of spatially well defined signals, and to solve the hydrogen peroxide parasite signal which appeared between contiguous spots using luminol free in solution. The detection limits of the different biosensor were found to be 1 microM for glutamate, lysine and uric acid, 20 microM for glucose and 2 microM for choline and lactate. The detection ranges were 1-25 microM (uric acid), 1 microM-0.5 mM (glutamate and lysine), 20 microM-2 mM (glucose) and 2 microM-0.2 mM (choline and lactate). The ECL chip was used for the detection of glucose, lactate and uric acid in human serum matrix. Good correlations between measured and expected values were found without the need of internal calibration of the sample, demonstrating the potentiality of the ECL multifunctional bio-sensing chip.     

Multifunctional acetyl-CoA carboxylase 1 is essential for very long chain fatty acid elongation and embryo development in Arabidopsis

Acetyl-CoA carboxylase (ACCase) catalyses the carboxylation of acetyl-CoA, forming malonyl-CoA, which is used in the plastid for fatty acid synthesis and in the cytosol in various biosynthetic pathways including fatty acid elongation. In Arabidopsis thaliana, ACC1 and ACC2, two genes located in a tandem repeat within a 25-kbp genomic region near the centromere of chromosome 1, encode two multifunctional ACCase isoforms. Both genes, ACC1 and ACC2, appear to be ubiquitously expressed, but little is known about their respective function and importance. Here, we report the isolation and characterisation of two allelic mutants disrupted in the ACC1 gene. Both acc1-1 and acc1-2 mutations are recessive and embryo lethal. Embryo morphogenesis is impaired and both alleles lead to cucumber-like structures lacking in cotyledons, while the shortened hypocotyl and root exhibit a normal radial pattern organisation of the body axis. In this abnormal embryo, the maturation process still occurs. Storage proteins accumulate normally, while triacylglycerides (TAG) are synthesised at a lower concentration than in the wild-type seed. However, these TAG are totally devoid of very long chain fatty acids (VLCFA) and consequently enriched in C18:1, like all lipid fractions analysed in the mutant seed. These data demonstrate, in planta, the role of ACCase 1 in VLCFA elongation. Furthermore, this multifunctional enzyme also plays an unexpected and central function in embryo morphogenesis, especially in apical meristem development.     

Evidence for the presence of a cellulase gene in the last common ancestor of bilaterian animals

Until recently, the textbook view of cellulose hydrolysis in animals was that gut-resident symbiotic organisms such as bacteria or unicellular eukaryotes are responsible for the cellulases produced. This view has been challenged by the characterization and sequencing of endogenous cellulase genes from some invertebrate animals, including plant-parasitic nematodes, arthropods and a mollusc. Most of these genes are completely unrelated in terms of sequence, and their evolutionary origins remain unclear. In the case of plant-parasitic nematodes, it has been suggested that their ancestor obtained a cellulase gene via horizontal gene transfer from a prokaryote, and similar suggestions have been made about a cellulase gene recently discovered in a sea squirt. To improve understanding about the evolution of animal cellulases, we searched for all known types of these enzymes in GenBank, and performed phylogenetic comparisons. Low phylogenetic resolution was found among most of the sequences examined, however, positional identity in the introns of cellulase genes from a termite, a sea squirt and an abalone provided compelling evidence that a similar gene was present in the last common ancestor of protostomes and deuterostomes. In a different enzyme family, cellulases from beetles and plant-parasitic nematodes were found to cluster together. This result questions the idea of lateral gene transfer into the ancestors of the latter, although statistical tests did not allow this possibility to be ruled out. Overall, our results suggest that at least one family of endogenous cellulases may be more widespread in animals than previously thought.     

Utilizing hydrolases of opposite enantiopreference for the preparation of both enantiomers of (1R,7aR)-(-)- and (1S,7aS)-(+)-3,6,7,7a-tetrahydro-1-hydroxy-7a-methyl-1H-inden-5(2H)-one

Four racemic esters of (1R*,7aR*)-3,6,7,7a-tetrahydro-1-hydroxy-7a-methyl-1H-inden-5(2H)-one were prepared and subjected to hydrolysis with two types of hydrolases, including alcalase and three lipases. Alcalase and lipase showed opposite enantiopreference on these esters. Based on this result, we developed a gram-scale procedure using butanoate as the substrate, which was treated consecutively with alcalase and lipase from Candida rugosa (CRL), to give both enantiomers of the title compound in high yields and high enantiomeric excess.     

A novel alpha-Proteobacterium resides in the mitochondria of ovarian cells of the tick Ixodes ricinus

An intracellular bacterium from Ixodes ricinus ticks collected in Italy was characterized by electron microscopy (EM), PCR sequencing of the 16S rRNA gene, molecular phylogenetic analysis, and in situ hybridization (ISH). This bacterium was shown by EM to be present in the cytoplasm, as well as in the mitochondria of ovarian cells. When universal 16S rRNA bacterial primers were used, PCR amplification of ovarian DNA followed by cloning and sequencing resulted in the same sequence being found in each sample. Phylogenetic analysis of this sequence showed that the bacterium from which it was derived, tentatively designated IricES1, is part of a novel clade in the alpha subdivision of the Proteobacterium: ISH and PCR assays of various tissues performed with oligonucleotides specific for the IricES1 16S rRNA showed that IricES1 is restricted to ovarian cells. Based on the results obtained, we inferred that the bacteria seen by EM in ovarian cells are a single type of bacteria, corresponding to IricES1. PCR screening of 166 ticks from various parts of Italy and one site in England showed that IricES1 was present in 96% of adult females and 44% of nymphs (unsexed). No adult males were found to be infected. Despite the apparent parasitism of host mitochondria by IricES1, the available information suggests that the bacterium has an obligate relationship with its host, although this must be confirmed.     

Effect of maprotiline on Ca(2+) movement in human neuroblastoma cells

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2+ concentrations ([Ca2+]i) was explored by using the Ca2+-sensitive probe fura-2. Maprotiline at concentrations greater than 100 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50 = 200 microM). Maprotiline-induced [Ca2+]i rise was reduced by 50% by removal of extracellular Ca2+. Maprotiline-induced [Ca2+]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of maprotiline on [Ca2+]i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2+]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.     

A preliminary phylogeny of the Pterasteridae (Echinodermata,Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium

The Pterasteridae comprises a diversified group of extant largely deep-sea starfishes. Generic diagnoses have been based classically on soft tissue characters and skeletal architecture. A preliminary phylogeny of sixteen extant species is here worked out by cladistic analysis. The resulting tree suggests monophyly of extant genera and the validity of dissociated plates for identification of genera. Fossil remains of Pterasteridae are here described for the first time. By comparison with extant species, all the skeletal remains from the lower Upper Campanian of Belgium and from the lower Maastrichtian of Germany are tentatively assigned to the genusPteraster. The fossil record of starfishes is poor, but the present Late Cretaceous pterasterids provide one more piece of evidence of the high diversity of starfishes during the Mesozoic. Known Late Cretaceous and Paleogene fossils are broadly similar, which suggests the end-Cretaceous extinction event did not cause major turnover in asteroid faunal composition. As suggested for other starfish groups, both the fossil record of deep-sea Pterasteridae in shelf settings and tree topology imply an onshore-offshore evolutionary trend.      

Determination of glutathionyl-hemoglobin in human erythrocytes by cation-exchange high-performance liquid chromatography

Since glutathionyl-hemoglobin has been suggested to be a clinical marker of oxidative stress in human blood and given the growing biological relevance of oxidative stress as a pathogenic factor in several diseases, we describe a method to measure glutathionyl-hemoglobin concentration in erythrocytes, by using cation-exchange high-pressure liquid chromatography with UV detection. The glutathionyl-hemoglobin peak has been identified on the basis of the following findings: (a) the peak increased when the sample was incubated with oxidized glutathione; (b) the peak disappeared when the sample was reduced with dithiothreitol, with the simultaneous increase of that corresponding to hemoglobin A(0); (c) the peak could be detected by incubating hemoglobin A(0) with reduced glutathione; (e) deconvoluted mass spectrum of the glutathionyl-hemoglobin peak showed a 16172.0-Da molecular mass, corresponding to hemoglobin beta bound to glutathione. Glutathionyl-hemoglobin concentration has been determined in erythrocytes of 40 healthy subjects, with a mean value of 2.58+/-0.7%, calculated as the percentage of its peak area ratio to that of total hemoglobin (HbA(0)+HbA(2)+HbA(1C)+glutathionyl-hemoglobin). The availability of a simple and reproducible method to detect glutathionyl-hemoglobin concentration in blood could be useful in monitoring oxidative stress, and for investigating the efficacy of antioxidant therapies in clinical trials.