TGF-β/Smad3 signaling promotes renal fibrosis by inhibiting miR-29

TGF-β/Smad3 signaling promotes fibrosis, but the development of therapeutic interventions involving this pathway will require the identification and ultimate targeting of downstream fibrosis-specific genes. In this study, using a microRNA microarray and real-time PCR, wild-type mice had reduced expression of miR-29 along with the development of progressive renal fibrosis in obstructive nephropathy. In contrast, Smad3 knockout mice had increased expression of miR-29 along with the absence of renal fibrosis in the same model of obstruction. In cultured fibroblasts and tubular epithelial cells, Smad3 mediated TGF-β(1)-induced downregulation of miR-29 by binding to the promoter of miR-29. Furthermore, miR-29 acted as a downstream inhibitor and therapeutic microRNA for TGF-β/Smad3-mediated fibrosis. In vitro, overexpression of miR-29b inhibited, but knockdown of miR-29 enhanced, TGF-β(1)-induced expression of collagens I and III by renal tubular cells. Ultrasound-mediated gene delivery of miR-29b either before or after established obstructive nephropathy blocked progressive renal fibrosis. In conclusion, miR-29 is a downstream inhibitor of TGF-β/Smad3-mediated fibrosis and may have therapeutic potential for diseases involving fibrosis.     

Desmoplastic Reaction Influences Pancreatic Cancer Growth Behavior

Connective tissue growth factor (CTGF), which is regulated by transforming growth factor-ß (TGFß), has recently been implicated in the pathogenesis of fibrotic diseases and tumor stroma. Inasmuch as generation of desmoplastic tissue is characteristic for pancreatic cancer, it is not known whether it gives pancreatic cancer cells a growth advantage or is a reaction of the body to inhibit cancer cell progression. In the present study we analyzed the expression and localization of CTGF and evaluated whether it influences the prognosis of pancreas cancer. Tissue samples were obtained from 25 individuals (6 women, 19 men) undergoing pancreatic resection for pancreatic cancer. Tissue samples from 13 previously healthy organ donors (5 women, 8 men) served as controls. Expression of CTGF was studied by Northern blot analysis. In situ hybridization and immunohistochemistry localized the respective mRNA moieties and proteins in the tissue samples. Northern blot analysis revealed that pancreatic cancer tissue samples exhibited a 46-fold increase in CTGF mRNA expression (p < 0.001) over that of normal controls. In vitro studies confirmed that pancreatic stellate cells are the major source of CTGF mRNA expression and revealed a large variance in basal and TGFß-induced CTGF expression in cultured pancreatic cancer cells. This could also be confirmed by in situ hybridization, indicating that CTGF mRNA signals were located principally in fibroblasts, with only weak signals in the cancer cells. High CTGF mRNA levels in the tissue samples correlated with better tumor differentiation (p < 0.03). In addition, patients whose tumors exhibited high CTGF mRNA levels (> onefold increase above normal controls) lived significantly longer than those whose tumors expressed low CTGF mRNA levels (none to onefold) (p < 0.04 multivariate analysis). Our present data indicate that CTGF, as a downstream mediator of TGFß, is overexpressed in connective tissue cells and to a lesser extent in pancreatic cancer cells. Because patients with high CTGF mRNA expression levels have a better prognosis, our findings indicate that the desmoplastic reaction provides a growth disadvantage for pancreatic cancer cells.     

Maxadilan interacts with receptors for pituitary adenylyl cyclase activating peptide in human SH-SY5Y and SK-N-MC neuroblastoma cells

Receptors for pituitary adenylyl cyclase activating peptide (PACAP) have been identified in human SH-SY5Y neuroblastoma cells with PACAP being 1000-fold more potent than vasoactive intestinal peptide (VIP) in [(125)I]PACAP binding inhibition and stimulation of cAMP accumulation. Maxadilan, a vasodilator peptide from the salivary gland of the sand fly Lutzomyia longipalpis also specifically bound to SH-SY5Y cells, and was equipotent to PACAP in [(125)I]PACAP and [(125)I]maxadilan binding inhibition, and stimulation of cAMP accumulation. Maxadilan and PACAP also increased the cytosolic free calcium concentration. In human SK-N-MC neuroblastoma cells PACAP, VIP and maxadilan equipotently stimulated cAMP accumulation. The maximal effects of VIP and maxadilan were additive and reached those of PACAP alone. In human T47D breast carcinoma cells PACAP and VIP were also equipotent in the stimulation of cAMP accumulation, but maxadilan was inactive. The results are consistent with the interaction of maxadilan with PACAP specific PAC(1)receptors in SH-SY5Y cells, but not with VPAC receptors, not differentiating between VIP and PACAP in T47D cells. Moreover, maxadilan is a PAC(1)receptor specific agonist which allows discrimination of co-expressed PAC(1)and VPAC receptors in SK-N-MC cells.     

Evaluation of Molecular Test for the Discrimination of “Naked” DNA from Infectious Parvovirus B19 Particles in Serum and Bone Marrow Samples

Low levels of parvovirus B19 (B19V) DNA can be detected in the circulation and in different tissue of immunocompetent individuals for months or years, which has been linked to inflammatory diseases such as cardiomyopathy, rheumatoid arthritis, hepatitis, and vasculitis. However, the detection of B19V DNA does not necessarily imply that infectious virions are present. This study aimed to evaluate the method based on the Benzonase^® treatment for differentiation between the infectious virions from “naked” DNA in serum and bone marrow (BM) samples to be useful for the B19V routine diagnosis. In addition, we estimated the period of viremia and DNAemia in the sera and bone marrow of nonhuman primates experimentally infected with B19V. Serum samples from ten patients and from four cynomolgus monkeys experimentally infected with B19V followed up for 60 days were used. Most of the human serum samples became negative after pretreatment; however, only decreased viral DNA loads were observed in four patients, indicating that these samples still contained the infectious virus. Reduced B19V DNA levels were observed in animals since 7th dpi. At approximately 45th dpi, B19V DNA levels were below 10⁵ IU/mL after Benzonase^® pretreatment, which was not a consequence of active B19V replication. The test based on Benzonase^® pretreatment enabled the discrimination of “naked DNA” from B19V DNA encapsidated in virions. Therefore, this test can be used to clarify the role of B19V as an etiological agent associated with atypical clinical manifestations.     

Glucagon-like peptide-1 receptor dimerization differentially regulates agonist signaling but does not affect small molecule allostery

The glucagon-like peptide-1 receptor (GLP-1R) is a family B G protein-coupled receptor and an important drug target for the treatment of type II diabetes, with activation of pancreatic GLP-1Rs eliciting glucose-dependent insulin secretion. Currently, approved therapeutics acting at this receptor are peptide based, and there is substantial interest in small molecule modulators for the GLP-1R. Using a variety of resonance energy transfer techniques, we demonstrate that the GLP-1R forms homodimers and that transmembrane helix 4 (TM4) provides the primary dimerization interface. We show that disruption of dimerization using a TM4 peptide, a minigene construct encoding TM4, or by mutation of TM4, eliminates G protein-dependent high-affinity binding to GLP-1(7-36)NH₂ but has selective effects on receptor signaling. There was <10-fold decrease in potency in cAMP accumulation or ERK1/2 phosphorylation assays but marked loss of intracellular calcium mobilization by peptide agonists. In contrast, there was near-complete abrogation of the cAMP response to an allosteric agonist, compound 2, but preservation of ERK phosphorylation. Collectively, this indicates that GLP-1R dimerization is important for control of signal bias. Furthermore, we reveal that two small molecule ligands are unaltered in their ability to allosterically modulate signaling from peptide ligands, demonstrating that these modulators act in cis within a single receptor protomer, and this has important implications for small molecule drug design.     

The School Dropout

This paper provides a commentary on Philip Cushman's “A Burning World, an Absent God: Midrash, Hermeneutics, and Relational Psychoanalysis.” Cushman creatively uses the rabbinic midrash as a model hermeneutic and dialogic tradition for contemporary psychoanalysis as an alternative to fundamentalist interpretation. I agree with Cushman's goal, but I find that he places greater emphasis on absence over presence, narrative over law, transcendence over immanence, change over continuity, revolution over evolution, construction over discovery, and individual freedom and autonomy over community and tradition. I believe that it is just this leaning to one side that leaves him defensive about the collapse to relativism. Holding these poles in greater tension and valuing both sides of these polarities offers a dialectical model that serves contemporary psychoanalysis better as a hermeneutic, dialogical, contextual, and dialectical model for interpretation.