Retinoic acid-induced differentiation into astrocytes and glutamatergic neurons is associated with expression of functional and activable phospholipase D

Phospholipase D (PLD) activity in mammalian cells has been associated with cell proliferation and differentiation. Here, we investigated the expression of PLD during differentiation of pluripotent embryonal carcinoma cells (P19) into astrocytes and neurons. Retinoic acid (RA)-induced differentiation increased PLD1 and PLD2 mRNA levels and PLD activity that was responsive to phorbol myristate acetate. Various agonists of membrane receptors activated PLD in RA-differentiated cells. Glutamate was a potent activator of PLD in neurons but not in astrocytes, whereas noradrenaline and carbachol increased PLD activity only in astrocytes. P19 neurons but not astrocytes released glutamate in response to a depolarizing stimulus, confirming the glutamatergic phenotype of these neurons. These results indicate upregulation of PLD gene expression associated with RA-induced neural differentiation.     

Spatial mapping of phosphorus influx in bean root systems using digital autoradiography

A novel technique was developed to spatially map the phosphorus net influx capacity in intact root systems. The method is based on digital autoradiography and permits the quantification of phosphorus influx at high spatial resolution (2 mm). Roots of 18-d-old common bean plants were exposed to (32)P-labelled orthophosphate, quickly frozen, excised, lyophilized, scanned, and exposed to a storage phosphor screen. Plots of (32)P content versus root length (distance from the root tip or from the base of the root) were obtained for three different root classes: basal, basal laterals, and taproot laterals. Radioactivity detected by filmless autoradiography correlated well (r(2)=0.99) with measurements made by scintillation counting. Basal roots absorbed 2.5 times and 1.9 times more phosphorus than the taproot lateral and basal lateral root classes, respectively, in the first 20 mm from the root apex. External phosphorus markedly affected influx: roots averaged 5, 16, and 34 pmol P min(-1) in the apical 20 mm when exposed to 1, 5, and 10 microM P solutions, respectively. The spatial pattern of phosphorus influx along the root axes of the different root classes was rather homogeneous when measured on a root surface area basis. Phosphorus influx in the older segments of basal roots (those next to the hypocotyl) did not differ from the newer segments close to the root apex. However, a heterogeneous pattern was detected for basal roots when measured on a length basis, indicating that both root class and diameter constitute main factors controlling the spatial pattern of net influx.     

An infant with diaphragmatic eventration and respiratory distress

The authors report a rare case of partial diaphragmatic eventration in a 4-month-old infant with recurrent wheezing and low serum IgA values. Because of persistent respiratory symptoms after therapy with inhaled short-acting beta2 agonists and inhaled nebulized corticosteroids, surgery was undertaken to correct the defect. Despite surgery, the clinical symptoms did not improve. Consequently, gatroesophagel reflux was considered and the diagnosis was confirmed with pH-metry, after which the infant was started on a protonic pump inhibitor therapy (PPI), achieving clinical improvement. Our experience suggests that in infants with congenital diaphragmatic eventration who present with respiratory distress gastro-oesophageal reflux should be suspected, and PPI therapy should be started before planning surgery.     

Arsenic accumulation and thiol status in lichens exposed to As(V) in controlled conditions

Thalli of epiphytic lichen Hypogymnia physodes (L.) Nyl. and terricolous Cladonia furcata (Huds.) Schrad., collected from an area with background arsenic concentrations, were exposed to 0, 0.1, 1 and 10 μg mL⁻¹ arsenate (As(V)) solutions for 24 h. After exposure they were kept in the metabolically active state for 0, 24 and 48 h in a growth chamber. In the freeze dried samples glutathione (GSH), glutathione disulphide (GSSG), cysteine (Cys) and cystine were analysed and induction of phytochelatin (PC) synthesis measured by reversed-phase high-performance liquid chromatography in combination with fluorescence detection or UV spectrometry. Total arsenic content in thalli was measured by instrumental neutron activation analysis (INAA). In H. physodes, which contained higher amounts of arsenic compared to C. furcata, total glutathione content significantly decreased in samples exposed to 10 μg mL⁻¹ As(V), whereas in C. furcata a significant increase was observed. In both species PC synthesis was induced in thalli exposed to 10 μg mL⁻¹.     

Genomic resources for flatfish research and their applications

Flatfishes are a group of teleosts of high commercial and environmental interest, whose biology is still poorly understood. The recent rapid development of different 'omic' technologies is, however, enhancing the knowledge of the complex genetic control underlying different physiological processes of flatfishes. This review describes the different functional genomic approaches and resources currently available for flatfish research and summarizes different areas where microarray-based gene expression analysis has been applied. The increase in genome sequencing data has also allowed the construction of genetic linkage maps in different flatfish species; these maps are invaluable for investigating genome organization and identifying genetic traits of commercial interest. Despite the significant progress in this field, the genomic resources currently available for flatfish are still scarce. Further intensive research should be carried out to develop larger genomic sequence databases, high-density microarrays and, more detailed, complete linkage maps, using second-generation sequencing platforms. These tools will be crucial for further expanding the knowledge of flatfish physiology, and it is predicted that they will have important implications for wild fish population management, improved fish welfare and increased productivity in aquaculture.     

The circadian system of ruin lizards: a seasonally changing neuroendocrine loop?

Previous studies have shown that the amplitude of daily melatonin production in cultured ruin lizard pineal organs explanted in the summer is significantly higher than that from organs explanted in the winter. To test whether seasonal photoperiodic changes are decoded autonomously by the pineal gland, pineals explanted in summer were cultured in vitro and exposed to changes between winter and summer photoperiods. The changes in photoperiod duration did not affect the daily profiles of in vitro melatonin production. The discrepancy between the present in vitro results and those from lizards exposed to winter or summer photoperiods before pineal explantation supports the view that circadian information entering the pineal gland via its innervation is involved in determining seasonal changes of melatonin production in ruin lizards. We further examined whether a central component of the circadian system of ruin lizards, specifically the retinae of the lateral eyes, expresses similar seasonal changes in function as does the pineal gland. We did not find any difference between summer and autumn-winter in the effectiveness of either bilateral retinalectomy or optic nerve lesion-at the level of the optic chiasm-in altering circadian locomotor behavior in constant conditions. Both surgical procedures mostly induced a shortening of the free-running period of the locomotor rhythm of similar magnitude in all seasons. Thus, the retinae do not appear to participate in the seasonal reorganization of the circadian system in ruin lizards.     

Telomere dysfunction: a new player in radiation sensitivity

Human individuals often exhibit important differences in their sensitivity to ionising radiation. Extensive literature links radiation sensitivity with impaired DNA repair which is due to a lack of correct functioning in many proteins involved in DNA-repair pathways and/or in DNA-damage checkpoint responses. Given that ionising radiation is an important and widespread diagnostic and therapeutic tool, it is important to investigate further those factors and mechanisms that underlie individual radiosensitivity. Recently, evidence is accumulating that telomere function may well be involved in cellular and organism responses to ionising radiation, broadening still further the currently complex and challenging scenario.     

Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins

The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg(2+)-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg(2+), the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.     

The effect of hydrodynamic shear on 3D engineered chondrocyte systems subject to direct perfusion

Bioreactors allowing direct-perfusion of culture medium through tissue-engineered constructs may overcome diffusion limitations associated with static culturing, and may provide flow-mediated mechanical stimuli. The hydrodynamic stress imposed on cells within scaffolds is directly dependent on scaffold microstructure and on bioreactor configuration. Aim of this study is to investigate optimal shear stress ranges and to quantitatively predict the levels of hydrodynamic shear imposed to cells during the experiments. Bovine articular chondrocytes were seeded on polyestherurethane foams and cultured for 2 weeks in a direct perfusion bioreactor designed to impose 4 different values of shear level at a single flow rate (0.5 ml/min). Computational fluid dynamics (CFD) simulations were carried out on reconstructions of the scaffold obtained from micro-computed tomography images. Biochemistry analyses for DNA and sGAG were performed, along with electron microscopy. The hydrodynamic shear induced on cells within constructs, as estimated by CFD simulations, ranged from 4.6 to 56 mPa. This 12-fold increase in the level of applied shear stress determined a 1.7-fold increase in the mean content in DNA and a 2.9-fold increase in the mean content in sGAG. In contrast, the mean sGAG/DNA ratio showed a tendency to decrease for increasing shear levels. Our results suggest that the optimal condition to favour sGAG synthesis in engineered constructs, at least at the beginning of culture, is direct perfusion at the lowest level of hydrodynamic shear. In conclusion, the presented results represent a first attempt to quantitatively correlate the imposed hydrodynamic shear level and the invoked biosynthetic response in 3D engineered chondrocyte systems.