Cell weight kinetics simulation in chemostat and batch culture of the rhodophyte Porphyridium cruentum

Experimental observations of cell size variations in the proliferating rhodophyte Porphyridium cruentum cultured under fully controlled conditions showed significant decreases from inoculation to a steady state in the chemostat with 0.23 d(-1) dilution rate and to a minimum in batch, dropping in size by ratios of over 10. To numerically simulate these variations, we assumed that the cell is made up of two categories of components that behave differently during the interphase and mitosis. These have been called essential (EC) and accessory (AC) components. It is assumed that the cell divides once the EC have doubled in size, regardless of the AC's state. The experimental cell weight time courses were correctly simulated by a model of synchronous cell kinetics based on these assumptions. The EC's specific growth rate was 1.5 times that of the whole cell, when no limitation occurred. The increase in cell weight observed during batch cultures after nutrient exhaustion was suitably simulated by assuming that EC growth stops when a limiting nutrient is exhausted. Several parameters characterizing the cell kinetics were defined, particularly the minimum minimorum EC or cell weight (26 and 15 pg for chemostat and batch, respectively), which was influenced by the cultivation method, and the maximum whole cell weight (224 to 244 pg), which depended on the inoculum's age. The influence of culture conditions on the amount of essential and accessory components contained in a cell was examined. A new approach was developed with respect to these compartments to determine the most suitable strategy and conduct a predictive approach for valuable molecule production.     

Redox-induced structural dynamics of Fe-heme ligand in myoglobin by X-ray absorption spectroscopy

The Fe(III) --> Fe(II) reduction of the heme iron in aquomet-myoglobin, induced by x-rays at cryogenics temperatures, produces a thermally trapped nonequilibrium state in which a water molecule is still bound to the iron. Water dissociates at T > 160 K, when the protein can relax toward its new equilibrium, deoxy form. Synchrotron radiation x-ray absorption spectroscopy provides information on both the redox state and the Fe-heme structure. Owing to the development of a novel method to analyze the low-energy region of x-ray absorption spectroscopy, we obtain structural pictures of this photo-inducible, irreversible process, with 0.02-0.06-A accuracy, on the protein in solution as well as in crystal. After photo-reduction, the iron-proximal histidine bond is shortened by 0.15 A, a reinforcement that should destabilize the iron in-plane position favoring water dissociation. Moreover, we are able to get the distance of the water molecule even after dissociation from the iron, with a 0.16-A statistical error.     

Disparate aggrecan gene expression in chondrocytes subjected to hypotonic and hypertonic loading in 2D and 3D culture

The effects of hypotonic (180 mOsm) and hypertonic (580 mOsm) medium loading on chondrocyte aggrecan gene expression in 2D monolayer and 3D hydrogel culture (agarose or alginate) were studied. Aggrecan promoter activity was monitored using a luciferase reporter gene assay and transient transfection. Osmotic loading was observed to differentially affect promoter activity, with hypotonic loading generally producing at least a 40% elevation in promoter activity, except for the case of alginate where a 50% suppression was observed. Hypertonic loading produced at least a 35% decrease in activity for all cultures. Similar osmolality-induced changes to aggrecan mRNA levels were observed in monolayer cells using qPCR. Deletion of exon 1 blocked the sensitivity of monolayer cells to hypertonic but not hypotonic medium changes. Confocal microscopy measurements suggested that the degree of hypotonic swelling in cells encapsulated in 3D matrix was restricted compared to monolayer cells whereas the degree of hypertonic shrinking was similar under both culture conditions.     

Extremely rapid turnover of S-adenosylmethionine decarboxylase in Crithidia fasciculata

The activity of S-adenosylmethionine decarboxylase (AdoMetDC) in Crithidia fasciculata was shown to be correlated to the growth of the parasite. An increase in activity was observed during exponential growth. Inhibition of protein synthesis induced an extremely rapid decay of AdoMetDC activity. The half-life of the enzyme was estimated to be about 3 min, which is the shortest half-life ever recorded for an eukaryotic AdoMetDC. The reduction in AdoMetDC activity was correlated with a decrease in AdoMetDC protein content, demonstrating a rapid turnover of the enzyme. No polyamine-mediated feedback regulation of AdoMetDC was observed in the parasite.     

Targeting of adenovirus via genetic modification of the viral capsid combined with a protein bridge

A potential barrier to the development of genetically targeted adenovirus (Ad) vectors for cell-specific delivery of gene therapeutics lies in the fact that several types of targeting protein ligands require posttranslational modifications, such as the formation of disulfide bonds, which are not available to Ad capsid proteins due to their nuclear localization during assembly of the virion. To overcome this problem, we developed a new targeting strategy, which combines genetic modifications of the Ad capsid with a protein bridge approach, resulting in a vector-ligand targeting complex. The components of the complex associate by virtue of genetic modifications to both the Ad capsid and the targeting ligand. One component of this mechanism of association, the Fc-binding domain of Staphylococcus aureus protein A, is genetically incorporated into the Ad fiber protein. The ligand is comprised of a targeting component fused with the Fc domain of immunoglobulin, which serves as a docking moiety to bind to these genetically modified fibers during the formation of the Ad-ligand complex. The modular design of the ligand solves the problem of structural and biosynthetic compatibility with the Ad and thus facilitates targeting of the vector to a variety of cellular receptors. Our study shows that targeting ligands incorporating the Fc domain and either an anti-CD40 single-chain antibody or CD40L form stable complexes with protein A-modified Ad vectors, resulting in significant augmentation of gene delivery to CD40-positive target cells. Since this gene transfer is independent of the expression of the native Ad5 receptor by the target cells, this strategy results in the derivation of truly targeted Ad vectors suitable for tissue-specific gene therapy.     

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.