Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells. (65/155046)

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.  (+info)

Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. (66/155046)

ZO-2, a member of the MAGUK family, was thought to be specific for tight junctions (TJs) in contrast to ZO-1, another MAGUK family member, which is localized at TJs and adherens junctions (AJs) in epithelial and nonepithelial cells, respectively. Mouse ZO-2 cDNA was isolated, and a specific polyclonal antibody was generated using corresponding synthetic peptides as antigens. Immunofluorescence microscopy with this polyclonal antibody revealed that, similarly to ZO-1, in addition to TJs in epithelial cells, ZO-2 was also concentrated at AJs in nonepithelial cells such as fibroblasts and cardiac muscle cells lacking TJs. When NH2-terminal dlg-like and COOH-terminal non-dlg-like domains of ZO-2 (N-ZO-2 and C-ZO-2, respectively) were separately introduced into cultured cells, N-ZO-2 was colocalized with endogenous ZO-1/ZO-2, i.e. at TJs in epithelial cells and at AJs in non-epithelial cells, whereas C-ZO-2 was distributed along actin filaments. Consistently, occludin as well as alpha catenin directly bound to N-ZO-2 as well as the NH2-terminal dlg-like portion of ZO-1 (N-ZO-1) in vitro. Furthermore, immunoprecipitation experiments revealed that the second PDZ domain of ZO-2 was directly associated with N-ZO-1. These findings indicated that ZO-2 forms a complex with ZO-1/occludin or ZO-1/alpha catenin to establish TJ or AJ domains, respectively.  (+info)

JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells. (67/155046)

To understand the role of redox-sensitive mechanisms in vascular smooth muscle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase activation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-derived growth factor BB-induced ERK2 activation. In contrast, DPI only inhibited agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family along with the AP-1-dependent reporter gene revealed that AP-1 with c-Fos and JunB composition exhibited a higher transactivating activity than AP-1 with other compositions tested. All three agonists significantly stimulated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesis in VSMC. These results also suggest a role for NADH/NADPH oxidase activity in some subset of early signaling events such as p38 MAP kinase activation and c-Fos and JunB induction, which appear to be important in agonist-induced AP-1 activity and DNA synthesis in VSMC.  (+info)

Effects of lipopolysaccharide on production of interleukin-1 and interleukin-6 by bovine mammary epithelial cells in vitro. (68/155046)

This investigation was performed to determine the effect of lipopolysaccharide (LPS) on production of interleukin (IL)-1 and IL-6 by bovine mammary epithelial cells in vitro. After confluence, the cells were stimulated with LPS (0.1, 1.0 or 10 micrograms/ml) for 4, 8, 24, and 48 hr. LPS increased production of both IL-1 and IL-6 production from mammary cells in a dose dependent manner. The expression of mRNA for IL-1 receptor antagonist (IL-1ra) was demonstrated by reverse transcription-polymerase chain reaction in bovine mammary epithelial cells.  (+info)

Reverse transcription-nested polymerase chain reaction for detecting p40 RNA of Borna disease virus, without risk of plasmid contamination. (69/155046)

Several methods for the detection of Borna disease virus (BDV) RNA have been reported, one being the reverse transcription-nested polymerase chain reaction (RT-nested PCR) method. However, due to the possibility of contamination of the cloned DNA in a reaction tube, false-positive results might be obtained by RT-nested PCR. To detect only BDV RNA without anxiety of contamination, we developed an RT-nested PCR system using "mRNA selective PCR kit". Using this system, cDNA of BDV p40 in the plasmid (up to 5 x 10(7) molecules) was not amplified. BDV specific sequence was amplified from total RNA (more than 50 pg) of MDCK/BDV cells, which were persistently infected with BDV. These results indicate that this mRNA selective RT-nested PCR system can specifically amplify target RNA as distinguished from plasmid contaminated.  (+info)

A genetic approach to visualization of multisynaptic neural pathways using plant lectin transgene. (70/155046)

The wiring patterns among various types of neurons via specific synaptic connections are the basis of functional logic employed by the brain for information processing. This study introduces a powerful method of analyzing the neuronal connectivity patterns by delivering a tracer selectively to specific types of neurons while simultaneously transsynaptically labeling their target neurons. We developed a novel genetic approach introducing cDNA for a plant lectin, wheat germ agglutinin (WGA), as a transgene under the control of specific promoter elements. Using this method, we demonstrate three examples of visualization of specific transsynaptic neural pathways: the mouse cerebellar efferent pathways, the mouse olfactory pathways, and the Drosophila visual pathways. This strategy should greatly facilitate studies on the anatomical and functional organization of the developing and mature nervous system.  (+info)

Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog. (71/155046)

Cerebellar granule cells are the most abundant type of neuron in the brain, but the molecular mechanisms that control their generation are incompletely understood. We show that Sonic hedgehog (Shh), which is made by Purkinje cells, regulates the division of granule cell precursors (GCPs). Treatment of GCPs with Shh prevents differentiation and induces a potent, long-lasting proliferative response. This response can be inhibited by basic fibroblast growth factor or by activation of protein kinase A. Blocking Shh function in vivo dramatically reduces GCP proliferation. These findings provide insight into the mechanisms of normal growth and tumorigenesis in the cerebellum.  (+info)

Augmentation is a potentiation of the exocytotic process. (72/155046)

Short-term synaptic enhancement is caused by an increase in the probability with which synaptic terminals release transmitter in response to presynaptic action potentials. Since exocytosed vesicles are drawn from a readily releasable pool of packaged transmitter, enhancement must result either from an increase in the size of the pool or an elevation in the fraction of releasable vesicles that undergoes exocytosis with each action potential. We show here that at least one major component of enhancement, augmentation, is not caused by an increase in the size of the readily releasable pool but is instead associated with an increase in the efficiency with which action potentials induce the exocytosis of readily releasable vesicles.  (+info)