Transforming growth factor-beta induces cytoskeleton and extracellular matrix modifications in FRTL-5 thyroid epithelial cells

The action of transforming growth factor-beta (TGF-beta) on the morphology, cytoskeleton and extracellular matrix was investigated in FRTL-5 thyroid epithelial cells. After treatment with TGF-beta, FRTL-5 cells became flat and developed straight and thick bundles of actin microfilaments. This effect of TGF-beta was observed even in the presence of thyrotropin, which has a strong microfilament disrupting action. TGF-beta also influenced some aspects of the extracellular matrix organization. Immunofluorescence staining of FRTL-5 cells revealed both the appearance of a fibrillar array of fibronectin in association with the basal plasma membrane and a change in the morphology of basally located laminin patches. TGF-beta induced the formation of adhesion structures at the ventral portion of the cell membrane. Vinculin was focally concentrated at the end of stress fibers in areas corresponding to focal adhesions as revealed by interference reflection microscopy (IRM). The ability to modulate cytoskeleton organization and extracellular matrix protein distribution might mediate some of the reported TGF-beta effects on the expression of specific functional properties in thyroid cells.     

Yotiao protein, a ligand for the NMDA receptor, binds and targets cAMP-dependent protein kinase II(1)

A yeast two-hybrid screen revealed that regulatory subunits (RII) of PKAII bind the Yotiao protein. Yotiao interacts with the NR1 subunit of the NMDA receptor. A purified C-terminal fragment of Yotiao binds PKAII, via an RII binding site constituted by amino acid residues 1452-1469, with a dissociation constant (K(d)) between 50 and 90 nM in vitro. A stable complex composed of Yotiao, RII and NR1 was immunoprecipitated from whole rat brain extracts. Immunostaining analysis disclosed that Yotiao, RIIbeta and NR1 colocalize in striatal and cerebellar neurons. Co-assembly of Yotiao/PKAII complexes with NR1 subunits may promote cAMP-dependent modulation of NMDA receptor activity at synapses, thereby influencing brain development and synaptic plasticity.     

Improving Dioxygenase Stability by Gene Chromosome Insertion: Implementation in Immobilized-Cell Systems

The immobilization of recombinant cells by using the unstable 3,4-dihydroxyphenylacetate 2,3-dioxygenase was studied as a model. Dioxygenase activity and cell viability were compared in immobilized-cell systems and cells in suspension. Immobilization increased enzyme stability and the efficient degradation of 3,4-dihydroxyphenylacetate. The stability of the cloned enzyme and the viability of the immobilized recombinant cells were well maintained for at least 15 days. We used the strain Escherichia coli CC118-D in which the hpaB gene from Klebsiella pneumoniae, coding for the subunit of 3,4-dihydroxyphenylacetate 2,3-dioxygenase, was inserted into the chromosome. This study has demonstrated that the implementation of E. coli CC118-D in a pilot-scale bioreactor resulted in a 100% stabilization of dioxygenase activity, and could be a useful tool for bioremediation processes.     

Activation of protein kinase calpha in the lysophosphatidic acid-induced bovine sperm acrosome reaction and phospholipase D1 regulation

Protein kinase C (PKC) has been implicated in the sperm acrosome reaction. In the present study, we demonstrate induction of the acrosome reaction and activation of sperm PKCalpha by lysophosphatidic acid (LPA), which is known to induce signal transduction cascades in many cell types via binding to specific cell-surface receptors. Under conditions by which LPA activates PKCalpha, there is significant stimulation of the acrosome reaction, which is inhibited by PKC inhibitors. Protein kinase Calpha belongs to the Ca(2+)-dependent classical PKC family of isoforms, and indeed we show that its activation depends upon the presence of Ca(2+) in the incubation medium. Protein kinase Calpha is a known regulator of phospholipase D (PLD). We investigated the possible regulatory relationships between PKCalpha and PLD1. Using specific antibodies against PLD1, we demonstrate for the first time its presence in bovine sperm. Furthermore, PLD1 coimmunoprecipitates with PKCalpha and the PKCalpha-PLD1 complex decomposes after treatment of the cells with LPA or 12-O:-tetradecanoyl phorbol-13-acetate, resulting in the translocation of PKCalpha to the plasma membrane and translocation of PLD1 to the particulate fraction. A possible bilateral regulation of PKCalpha and PLD1 activation during the sperm acrosome reaction is suggested.     

ERp57 is essential for efficient folding of glycoproteins sharing common structural domains

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.     

Distribution of flower morphs, ploidy level and sexual reproduction of the invasive weed Oxalis pes-caprae in the western area of the Mediterranean region

Background and Aims: Oxalis pes-caprae is a widespread invasive weed in regions witha Mediterranean climate. In its native habitat (southern Africa)this species has been reported as heterostylous with trimorphicflowers and a self- and morph-incompatible reproductive system.In most of the areas invaded, only a pentaploid short-styledmorphotype that reproduces mainly asexually by bulbils is reported,but this has only been confirmed empirically. This study aimsto analyse the floral morph proportions in a wide distributionarea, test the sexual female success, and explain the causesof low sexual reproduction of this species in the western areaof the Mediterranean Basin. Methods: Fifty-five populations of O. pes-caprae were sampled in theIberian Peninsula and Morocco to evaluate the floral morph ratioand individual fruit set. In plants from a dimorphic population,hand-pollination experiments were performed to evaluate theeffect of the pollen source on pollen tube growth through thestyle. The ploidy level and genome size of individuals of eachfloral morph were analysed using flow cytometry. Key Results: From the populations studied 89·1 % were monomorphic,with most of them containing the short-styled (SS) floral morph,and 10·9 % were dimorphic containing long-styled(LS) and SS morphs. In some of these, isoplethy was verifiedbut no fruit production was observed in any population. A sterileform was also recorded in several populations. Hand-pollinationexperiments revealed that pollen grains germinated over recipientstigmas. In intermorph crossings, pollen tubes were able todevelop and fruit initiation was observed in some cases, whilein intramorph pollinations, pollen tube development was sporadicand no fruit initiation was observed. All individuals withineach floral form presented the same DNA ploidy level: SS plantswere pentaploid and LS and the sterile form were tetraploid. Conclusions: The low or null sexual reproduction success of this speciesin the area of invasion studied seems related with the highfrequency of monomorphic populations, the unequal proportionof floral morphs in dimorphic populations and the presence ofdifferent ploidy levels between SS and LS morphs. The discoveryof the occurrence of an LS floral morph and a sterile form,whose invading capacity in these areas is as yet unknown, willbe valuable information for management programmes.     

Day-night differences in membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing activity in rat hypothalamus, pituitary, and retina

Membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing enzyme activity was analyzed fluorometrically in the anterior hypothalamus, pituitary, and retina of adult male rats to investigate day-night differences. Six groups (n=6 per group) were assessed—three during the light span and three during the dark span—under a standard 12 h-12 h light-dark cycle (light on from 07:00 to 19:00 h) and controlled temperature environment, with food and water available ad libitum. In the hypothalamus, enzyme activity levels were higher for time points of the dark than the light period. In contrast, the pituitary and retina exhibited the highest levels at the time points of the light period. The pituitary and retina also exhibited significant differences between the clock-hour means of the light period. Day-night differences in membrane-bound pyroglutamyl-2-naphthylamide-hydrolyzing activity may reflect differences in its susceptible endogenous substrates.     

PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms

Cell migration is dependent on the control of signaling events that play significant roles in creating contractile force and in contributing to wound closure. We evaluated wound closure in fibroblasts from mice overexpressing (TgPED) or lacking ped/pea-15 (KO), a gene overexpressed in patients with type 2 diabetes. Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts. This difference was observed both in the absence and in the presence of mytomicin C, an inhibitor of mitosis. In time-lapse experiments, TgPED fibroblasts displayed about twofold lower velocity and diffusion coefficient, as compared to controls. These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques. At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). Inhibition of ERK1/2 activity by PD98059 restored RhoA activation, cytoskeleton organization and cell motility, and almost completely rescued wound closure of TgPED fibroblasts. Interestingly, skin fibroblasts isolated from KO mice displayed an increased wound closure ability. In vivo, healing of dorsal wounds was delayed in TgPED and accelerated in KO mice. Thus, PED/PEA-15 may affect fibroblast motility by a mechanism, at least in part, mediated by ERK1/2.     

Organ-specific cellular requirements for in vivo dendritic cell generation

Bone marrow-derived dendritic cell (DC) precursors seed peripheral organs, where they encounter diverse cellular environments during their final differentiation into DCs. Flt3 ligand (Flt3-L) is critical for instructing DC generation throughout different organs. However, it remains unknown which cells produce Flt3-L and, importantly, which cellular source drives DC development in such a variety of organs. Using a novel BAC transgenic Flt3-L reporter mouse strain coexpressing enhanced GFP and luciferase, we show ubiquitous Flt3-L expression in organs and cell types. These results were further confirmed at the protein level. Although Flt3-L was produced by immune and nonimmune cells, the source required for development of the DC compartment clearly differed among organs. In lymphoid organs such as the spleen and bone marrow, Flt3-L production by hemopoietic cells was critical for generation of normal DC numbers. This was unexpected for the spleen because both immune and nonimmune cells equally contributed to the Flt3-L content in that organ. Thus, localized production rather than the total tissue content of Flt3-L in spleen dictated normal splenic DC development. No differences were observed in the number of DC precursors, suggesting that the immune source of Flt3-L promoted pre-cDC differentiation in spleen. In contrast, DC generation in the lung, kidney, and pancreas was mostly driven by nonhematopoietic cells producing Flt3-L, with little contribution by immune cells. These findings demonstrate a high degree of flexibility in Flt3-L-dependent DC generation to adapt this process to organ-specific cellular environments encountered by DC precursors during their final differentiation.