Build them up and break them down: Tight junctions of cell lines expressing typical hepatocyte polarity with a varied repertoire of claudins.

Tight junctions (TJs) of cells expressing simple epithelial polarity have been extensively studied, but less is known about TJs of cells expressing complex polarity. In this paper we analyzed, TJs of four different lines, that form bile canaliculi (BC) and express typical hepatocyte polarity; WIF-B9, 11-3, Can 3-1, Can 10. Striking differences were observed in claudin expression. None of the cell lines produced claudin-1. WIF-B9 and 11-3 expressed only claudin-2 while Can 3-1 and Can 10 expressed claudin-2,-3,-4,-5. TJs of these two classes of lines differed in their ultra-stucture, paracellular permeability, and robustness. Lines expressing a large claudin repertoire, especially Can 10, had complex and efficient TJs, that were maintained when cells were depleted in calcium. Inversely, TJs of WIF-B9 and 11-3 were leaky, permissive and dismantled by calcium depletion. Interestingly, we found that during the polarization process, TJ proteins expressed by all lines were sequentially settled in a specific order: first occludin, ZO-1 and cingulin, then JAM-A and ZO-2, finally claudin-2. Claudins expressed only in Can lines were also sequentially settled: claudin-3 was the first settled. Inhibition of claudin-3 expression delayed BC formation in Can10 and induced the expression of simple epithelial polarity. These results highlight the role of claudins in the settlement and the efficiency of TJs in lines expressing typical hepatocyte polarity. Can 10 seems to be the most promising of these lines because of its claudin repertoire near that of hepatocytes and its capacity to form extended tubular BC sealed by efficient TJs.

Uranium interaction with two multi-resistant environmental bacteria: Cupriavidus metallidurans CH34 and Rhodopseudomonas palustris.

Depending on speciation, U environmental contamination may be spread through the environment or inversely restrained to a limited area. Induction of U precipitation via biogenic or non-biogenic processes would reduce the dissemination of U contamination. To this aim U oxidation/reduction processes triggered by bacteria are presently intensively studied. Using X-ray absorption analysis, we describe in the present article the ability of Cupriavidus metallidurans CH34 and Rhodopseudomonas palustris, highly resistant to a variety of metals and metalloids or to organic pollutants, to withstand high concentrations of U and to immobilize it either through biosorption or through reduction to non-uraninite U(IV)-phosphate or U(IV)-carboxylate compounds. These bacterial strains are thus good candidates for U bioremediation strategies, particularly in the context of multi-pollutant or mixed-waste contaminations.

Quantitative evaluation of multi-walled carbon nanotube uptake in wheat and rapeseed.

Environmental contamination with carbon nanotubes would lead to plant exposure and particularly exposure of agricultural crops. The only quantitative exposure data available to date which can be used for risk assessment comes from computer modeling. The aim of this study was to provide quantitative data relative to multi-walled carbon nanotube (MWCNT) uptake and distribution in agricultural crops, and to correlate accumulation data with impact on plant development and physiology. Roots of wheat and rapeseed were exposed in hydroponics to uniformly (14)C-radiolabeled MWCNTs. Radioimaging, transmission electron microscopy and raman spectroscopy were used to identify CNT distribution. Radioactivity counting made it possible absolute quantification of CNT accumulation in plant leaves. Impact of CNTs on seed germination, root elongation, plant biomass, evapotranspiration, chlorophyll, thiobarbituric acid reactive species and H(2)O(2) contents was evaluated. We demonstrate that less than 0.005‰ of the applied MWCNT dose is taken up by plant roots and translocated to the leaves. This accumulation does not impact plant development and physiology. In addition, it does not induce any modifications in photosynthetic activity nor cause oxidative stress in plant leaves. Our results suggest that if environmental contamination occurs and MWCNTs are in the same physico-chemical state than the ones used in the present article, MWCNT transfer to the food chain via food crops would be very low.

Increased intracellular H2O2 availability preferentially drives glutathione accumulation in vacuoles and chloroplasts.

One biochemical response to increased H2O2 availability is the accumulation of glutathione disulphide (GSSG), the disulphide form of the key redox buffer glutathione. It remains unclear how this potentially important oxidative stress response impacts on the different sub-cellular glutathione pools. We addressed this question by using two independent in situ glutathione labelling techniques in Arabidopsis wild type (Col-0) and the GSSG-accumulating cat2 mutant. A comparison of in situ labelling with monochlorobimane (MCB) and in vitro labelling with monobromobimane (MBB) revealed that, whereas in situ labelling of Col-0 leaf glutathione was complete within 2 h incubation, about 50% of leaf glutathione remained inaccessible to MCB in cat2. High-performance liquid chromatography (HPLC) and enzymatic assays showed that this correlated tightly with the glutathione redox state, pointing to significant in vivo pools of GSSG in cat2 that were unavailable for MCB labelling. Immunogold labelling of leaf sections to estimate sub-cellular glutathione distribution showed that the accumulated GSSG in cat2 was associated with only a minor increase in cytosolic glutathione but with a 3- and 10-fold increase in plastid and vacuolar pools, respectively. The data are used to estimate compartment-specific glutathione concentrations under optimal and oxidative stress conditions, and the implications for redox homeostasis and signalling are discussed.

Differential redistribution of Ca2+-handling proteins during polarisation of MDCK cells: Effects on Ca2+ signalling.

The spatial organisation of the Ca(2+) signal in microdomains enables the regulation of various processes in specific regions of the cell and is essential for the versatility of cell responses to various stimuli. Ca(2+) signals can be independently regulated in the cytoplasm and in the nucleoplasm. Increases in the concentration of Ca(2+) in the nucleus can have specific effects different from those due to increases of Ca(2+) in the cytoplasm. We investigated the influence of cell polarity on the subcellular distribution of molecules responsible for intracellular Ca(2+) homeostasis (Ca(2+) release channels, Ca(2+) pumps and Ca(2+) binding proteins) and its influence on the intracellular Ca(2+) signal in MDCK cells with respect to its cytoplasmic or nucleoplasmic localisation. The intracellular Ca(2+) store was largely reorganised during cell polarisation, with a differential redistribution of IP3R, Ca(2+)-binding proteins and SERCA between the nuclear envelope and the periphery of the cell. This was accompanied by morphological changes in cell shape, which condense the cytoplasm around the nucleus, and in the shape of the nucleus, resulting in invaginations of the nuclear envelope. This facilitates Ca(2+) exchanges between the cytoplasm and the nucleoplasm, and preserves the ability to generate nucleoplasmic Ca(2+) transients in agonist-stimulated polarised MDCK cells.

In situ identification and imaging of bacterial polymer nanogranules by infrared nanospectroscopy.

We have employed atomic force microscope-based infrared spectroscopy (AFM-IR) to spatially map energy storage polymers inside individual bacteria Rhodobacter capsulatus. AFM-IR allows chemical mapping of sub-cellular features with a spatial resolution of <100 nm. We have used key absorption bands of the energy storage polymer polyhydroxybutyrate (PHB) known from FTIR to spatially map the molecular distribution of PHB inside bacteria. We have also compared FTIR measurements on bulk PHB with AFM-IR measurements of PHB inside bacteria. We observe a shift in the location of the carbonyl absorption peak between bulk PHB and PHB inside bacteria. We have also used finite element analysis to model AFM-IR measurements of PHB granules, allowing for estimation of the real size of the granules. We have also performed transmission electron microscopy (TEM) of R. capsulatus to determine the size distribution of the PHB granules. Sizes measured by AFM-IR correspond well to TEM measurements.

The performance of PEGylated nanocapsules of perfluorooctyl bromide as an ultrasound contrast agent.

The surface of polymeric nanocapsules used as ultrasound contrast agents (UCAs) was modified with PEGylated phospholipids in order to escape recognition and clearance by the mononuclear phagocyte system and achieve passive tumor targeting. Nanocapsules consisted of a shell of poly(lactide-co-glycolide) (PLGA) encapsulating a liquid core of perfluorooctyl bromide (PFOB). They were decorated with poly(ethylene glycol-2000)-grafted distearoylphosphatidylethanolamine (DSPE-PEG) incorporated in the organic phase before the solvent emulsification-evaporation process. The influence of DSPE-PEG concentration on nanocapsule size, surface charge, morphology, hydrophobicity and complement activation was evaluated. Zeta potential measurements, Hydrophobic interaction chromatography and complement activation provide evidence of DSPE-PEG presence at nanocapsule surface. Electronic microscopy reveals that the core/shell structure is preserved up to 2.64 mg of DSPE-PEG for 100 mg PLGA. In vivo ultrasound imaging was performed in mice bearing xenograft tumor with MIA PaCa-2 cells, either after an intra-tumoral or intravenous injection of nanocapsules. Tumor was observed only after the intra-tumoral injection. Despite the absence of echogenic signal in the tumor after intravenous injection of nanocapsules, histological analysis reveals their accumulation within the tumor tissue demonstrating that tissue distribution is not the unique property required for ultrasound contrast agents to be efficient.

Role of mental retardation-associated dystrophin-gene product Dp71 in excitatory synapse organization, synaptic plasticity and behavioral functions.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by deficient expression of the cytoskeletal protein, dystrophin. One third of DMD patients also have mental retardation (MR), likely due to mutations preventing expression of dystrophin and other brain products of the DMD gene expressed from distinct internal promoters. Loss of Dp71, the major DMD-gene product in brain, is thought to contribute to the severity of MR; however, the specific function of Dp71 is poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: Complementary approaches were used to explore the role of Dp71 in neuronal function and identify mechanisms by which Dp71 loss may impair neuronal and cognitive functions. Besides the normal expression of Dp71 in a subpopulation of astrocytes, we found that a pool of Dp71 colocalizes with synaptic proteins in cultured neurons and is expressed in synaptic subcellular fractions in adult brains. We report that Dp71-associated protein complexes interact with specialized modular scaffolds of proteins that cluster glutamate receptors and organize signaling in postsynaptic densities. We then undertook the first functional examination of the brain and cognitive alterations in the Dp71-null mice. We found that these mice display abnormal synapse organization and maturation in vitro, altered synapse density in the adult brain, enhanced glutamatergic transmission and reduced synaptic plasticity in CA1 hippocampus. Dp71-null mice show selective behavioral disturbances characterized by reduced exploratory and novelty-seeking behavior, mild retention deficits in inhibitory avoidance, and impairments in spatial learning and memory. CONCLUSIONS/SIGNIFICANCE: Results suggest that Dp71 expression in neurons play a regulatory role in glutamatergic synapse organization and function, which provides a new mechanism by which inactivation of Dp71 in association with that of other DMD-gene products may lead to increased severity of MR.

Tree stem phosphoenolpyruvate carboxylase (PEPC): lack of biochemical and localization evidence for a C4-like photosynthesis system.

Here, the kinetic properties and immunolocalization of phosphoenolpyruvate carboxylase (PEPC) and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in young stems of Fagus sylvatica were investigated. The aim of the study was to test the hypothesis that there is a C4-like photosynthesis system in the stems of this C3 tree species. The activity, optimal pH and L-malate sensitivity of PEPC, and the Michaelis-Menten constant (Km) for phosphoenolpyruvate (PEP), were measured in protein extracts from current-year stems and leaves. A gel blot experiment and immunolocalization studies were performed to examine the isozyme complexity of PEPC and the tissue distribution of PEPC and Rubisco in stems. Leaf and stem PEPCs exhibited similar, classical values characteristic of C3 PEPCs, with an optimal pH of c. 7.8, a Km for PEP of c. 0.3 mM and a IC50 for L-malate (the L-malate concentration that inhibits 50% of PEPC activity at the Km for PEP) of c. 0.1 mM. Western blot analysis showed the presence of two PEPC subunits (molecular mass c. 110 kDa) both in leaves and in stems. Immunogold labelling did not reveal any differential localization of PEPC and Rubisco, neither between nor inside cells. This study suggests that C4-type photosynthesis does not occur in stems of F. sylvatica and underlines the importance of PEPC in nonphotosynthetic carbon fixation by most stem tissues (fixation of respired CO2 and fixation via the anaplerotic pathway).

Erwinia carotovora Evf antagonizes the elimination of bacteria in the gut of Drosophila larvae.

Erwinia Virulence Factor (Evf) has been identified in Erwinia carotovora carotovora 15 (Ecc15) as a virulence factor that promotes colonization of the Drosophila larval gut and provokes the triggering of a systemic immune response. Here we have analysed how Evf promotes persistence and colonization of bacteria inside the larval gut. Erwinia evf mutants do not persist in immune-deficient Drosophila, indicating that Evf does not act by counteracting immunity. The results indicated that Evf is not a toxin because various gram-negative bacteria expressing evf can persist without affecting viability of Drosophila larvae. Evf did not appear to be a factor antagonizing a host-specific reaction because in vitro assays failed to reveal detoxifying enzymatic activities against various compounds thought to contribute to the hostile environment of the gut. These findings were corroborated by the observation that Evf is not required for survival in midgut organ cultures. By contrast, bacteria expressing evf allow persistence in trans of bacteria lacking evf indicating that Evf promotes the accumulation of gram-negative bacteria in the anterior midgut by affecting gut physiology.

Folate-conjugated iron oxide nanoparticles for solid tumor targeting as potential specific magnetic hyperthermia mediators: synthesis, physicochemical characterization, and in vitro experiments.

New folate-conjugated superparamagnetic maghemite nanoparticles have been synthesized for the intracellular hyperthermia treatment of solid tumors. These ultradispersed nanosystems have been characterized for their physicochemical properties and tumor cell targeting ability, facilitated by surface modification with folic acid. Preliminary experiments of nanoparticles heating under the influence of an alternating magnetic field at 108 kHz have been also performed. The nanoparticle size, surface charge, and colloidal stability have been assessed in various conditions of ionic strength and pH. The ability of these folate "decorated" maghemite nanoparticles to recognize the folate receptor has been investigated both by surface plasmon resonance and in folate receptor expressing cell lines, using radiolabeled folic acid in competitive binding experiments. The specificity of nanoparticle cellular uptake has been further investigated by transmission electron microscopy after incubation of these nanoparticles in the presence of three cell lines with differing folate receptor expression levels. Qualitative and quantitative determinations of both folate nanoparticles and nontargeted control nanoparticles demonstrated a specific cell internalization of the folate superparamagnetic nanoparticles.

Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena.

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.

Interaction between wall deposition and cell elongation in dark-grown hypocotyl cells in Arabidopsis.

A central problem in plant biology is how cell expansion is coordinated with wall synthesis. We have studied growth and wall deposition in epidermal cells of dark-grown Arabidopsis hypocotyls. Cells elongated in a biphasic pattern, slowly first and rapidly thereafter. The growth acceleration was initiated at the hypocotyl base and propagated acropetally. Using transmission and scanning electron microscopy, we analyzed walls in slowly and rapidly growing cells in 4-d-old dark-grown seedlings. We observed thick walls in slowly growing cells and thin walls in rapidly growing cells, which indicates that the rate of cell wall synthesis was not coupled to the cell elongation rate. The thick walls showed a polylamellated architecture, whereas polysaccharides in thin walls were axially oriented. Interestingly, innermost cellulose microfibrils were transversely oriented in both slowly and rapidly growing cells. This suggested that transversely deposited microfibrils reoriented in deeper layers of the expanding wall. No growth acceleration, only slow growth, was observed in the cellulose synthase mutant cesA6(prc1-1) or in seedlings, which had been treated with the cellulose synthesis inhibitor isoxaben. In these seedlings, innermost microfibrils were transversely oriented and not randomized as has been reported for other cellulose-deficient mutants or following treatment with dichlorobenzonitrile. Interestingly, isoxaben treatment after the initiation of the growth acceleration in the hypocotyl did not affect subsequent cell elongation. Together, these results show that rapid cell elongation, which involves extensive remodeling of the cell wall polymer network, depends on normal cellulose deposition during the slow growth phase.

Visualization of insulin-loaded nanocapsules: in vitro and in vivo studies after oral administration to rats.

PURPOSE: Biodegradable poly(isobutylcyanoacrylate) nanocapsules have been recognized as a promising carrier for oral administration of peptides and proteins. In the present study, we investigate the fate of insulin-loaded nanocapsules by fluorescence and transmission electron microscopy (TEM) after intragastric force-feeding to rats. METHODS: Insulin-, Texas-red-labeled insulin, or gold-labeled insulin-loaded nanocapsules were first characterized. Rats received a single dose of nanocapsules (diameter 60-300 nm, 57 IU insulin/kg) by intragastric force-feeding. After 90 min, ileum was isolated and prepared for fluorescence and transmission electron microscopy. RESULTS: Nanocapsules were observed on both sides of the gut epithelium and in blood capillaries. In M-cell-free epithelium, apparently intact nanocapsules could be seen in the underlying tissue, suggesting they could cross the epithelium and carry the encapsulated peptide. In M-cell-containing epithelium, nanocapsules appeared degraded in the vicinity of macrophages. It is noteworthy that intestinal absorption of nanocapsules was observed without artifacts forcing the nanocapsules to stay in the gut. CONCLUSIONS: Based on TEM observations, this study shows the intestinal absorption of biodegradable nanocapsules leading to the transport of insulin across the epithelium mucosa. The fate of the nanocapsules appeared different depending on the presence or the absence of M cells in the intestinal epithelium.

The sperm roller: a modified testicular duct linked to giant sperm transport within the male reproductive tract.

The male reproductive tracts of Drosophila display considerable variation in the relative size of their components, notably of the testes, but there are few structural differences between species. Here we report a remarkable coiled structure separating the testes from the seminal vesicles in the giant sperm species Drosophila bifurca. This evolutionary novelty, known as the 'sperm roller', seems to be an exaggeration in the size of the testicular duct as revealed by light and electron microscopic observations. It consists of a tubular monocellular epithelium lying on the basal laminae and muscle and conjunctive cells. The lumen of the roller contains crypts. The apical membrane of the epithelial cells presents numerous long microvilli protruding into the lumen. The sperm roller structure is probably involved in managing sperm during their transit through the male genital tract, because sperm are seen in bundles at the base of the testis, whereas they are singly rolled up by the time they enter the seminal vesicles. The hypercoiling of the individual spermatozoon within the roller probably occurs as the result of an osmotic process produced by features of the epithelial wall and the dramatically increased exchange surface. This is the first report of a specialized device of this type in Drosophila or, more generally, in insects.

In Vitro Development of Rat Cerebellar Neurons of Early Embryonic Origin. An Anatomical and Electrophysiological Study.

The development of the major morphological and electrophysiological properties of presumptive Purkinje cells (PCs) was studied in primary cultures of rat cerebellum dissociated on the 14th embryonic day, when PCs are minimally differentiated and migrate in vivo. PCs were identified with a specific antibody to calbindin D-28K (CaBP), which allowed visualization of the different morphological types of PCs between 3 and 29 days in vitro (DIV). CaBP-immunopositive cells were first detected at 3 DIV. Thereafter, the shape of these cells resembled some of those described in vivo. After 20 DIV, 95% of the CaBP-immunopositive cells had characteristic PC dendritic trees, although they were very atrophic. Glial cells immunopositive for the glial fibrillary acidic protein (GFAP) were first seen at 3 DIV. Thereafter GFAP-immunopositive cells resembled Bergmann cells or velate astrocytes. Neurons regarded as PCs were studied electrophysiologically using the patch-clamp whole-cell configuration. Voltage-dependent, tetrodotoxin-sensitive fast inward currents were virtually absent at 2 - 4 DIV, but increased between 7 and 14 DIV to reach two-thirds of the amplitude obtained after 15 DIV. These currents were large enough to give rise to overshooting spikes as early as 7 DIV in the current-clamp mode. This time schedule is in keeping with that of PCs developed in situ. The tetraethylammonium-sensitive, slowly inactivating outward currents had reached two-thirds of the amplitude obtained after 15 DIV by 3 - 4 DIV. Their amplitude remained stable between 4 and 7 DIV, and increased to their maximal value during 7 - 14 DIV, with a marked shortening of action potentials. 4-Aminopyridine-sensitive, fast-inactivating outward currents might also be associated with development, since they were present in 66% of the cells between 7 and 14 DIV but in only 39% from 15 to 29 DIV; however, their amplitude did not vary with time. Presumptive PCs bore l-glutamate-activated receptors, which preceded the emergence of kynurenate-sensitive, spontaneous synaptic currents at 7 DIV. These currents were sometimes intermingled with inhibitory currents, although presumptive PCs were sensitive to gamma-aminobutyrate at 7 DIV. The present model represents some unequivocal features of PC development, although the PCs used had undergone minimal differentiation in vivo and were cultured in a very disturbed cellular environment.