The Central Region of the Drosophila Co-repressor Groucho as a Regulatory Hub

Groucho (Gro) is a Drosophila co-repressor that regulates the expression of a large number of genes, many of which are involved in developmental control. Previous studies have shown that its central region is essential for function even though its three domains are poorly conserved and intrinsically disordered. Using these disordered domains as affinity reagents, we have now identified multiple embryonic Gro-interacting proteins. The interactors include protein complexes involved in chromosome organization, mRNA processing, and signaling. Further investigation of the interacting proteins using a reporter assay showed that many of them modulate Gro-mediated repression either positively or negatively. The positive regulators include components of the spliceosomal subcomplex U1 small nuclear ribonucleoprotein (U1 snRNP). A co-immunoprecipitation experiment confirms this finding and suggests that a sizable fraction of nuclear U1 snRNP is associated with Gro. The use of RNA-seq to analyze the gene expression profile of cells subjected to knockdown of Gro or snRNP-U1-C (a component of U1 snRNP) showed a significant overlap between genes regulated by these two factors. Furthermore, comparison of our RNA-seq data with Gro and RNA polymerase II ChIP data led to a number of insights, including the finding that Gro-repressed genes are enriched for promoter-proximal RNA polymerase II. We conclude that the Gro central domains mediate multiple interactions required for repression, thus functioning as a regulatory hub. Furthermore, interactions with the spliceosome may contribute to repression by Gro.     

Activation of promoter activity of the catalytic subunit of γ-glutamylcysteine ligase (GCL) in brain endothelial cells by insulin requires antioxidant response element 4 and altered glycemic status: implication for GCL expression and GSH synthesis

Our recent finding that insulin increased the expression of the glutamate-cysteine ligase catalytic subunit (GCLc) with coincident increases in GCL activity and cellular glutathione (GSH) in human brain microvascular endothelial cells (IHECs) suggests a role for insulin in vascular GSH maintenance. Here, using IHECs stably transfected with promoter-luciferase reporter vectors, we found that insulin increased GCLc promoter activity, which required a prerequisite increase or decrease in medium glucose. An intact antioxidant response element-4 was essential for promoter activation, which was attenuated by inhibitors of PI3-kinase/Akt/mTOR signaling. Interestingly, only under low-glucose conditions did promoter activation correlate with increased GCLc expression and GSH synthesis. Low tert-butylhydroperoxide (tBH) concentrations similarly mediated promoter activation, but the maximal activation dose was decreased 10-fold by insulin. Insulin-tBH coadministration abrogated the low or high glucose requirement for promoter activation, suggesting possible ROS involvement. ROS production was elevated at low glucose without or with insulin; however, GSH increases were not inhibited by tempol, suggesting that ROS did not achieve the threshold for driving GCLc promoter activation and de novo GSH synthesis. The minor effect of pyruvate also ruled out a major role for hypoglycemia (± insulin)-induced metabolic stress on GSH induction under these conditions.     

Local kallikrein�Ckinin system is involved in podocyte apoptosis under diabetic conditions

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.     

Location of immunization and interferon-γ are central to induction of salivary gland dysfunction in Ro60 peptide immunized model of Sjögren's syndrome

Introduction

Anti-Ro antibodies can be found in the serum of the majority of patients with Sjögren''s syndrome (SS). Immunization with a 60-kDa Ro peptide has been shown to induce SS-like symptoms in mice. The aim of this study was to investigate factors involved in salivary gland (SG) dysfunction after immunization and to test whether the induction of SS could be improved.

Methods

Ro60 peptide immunization was tested in Balb/c mice, multiple antigenic peptide (MAP)-Ro60 and Pertussis toxin (PTX) were tested in SJL/J mice. In addition, two injection sites were compared in these two strains: the abdominal area and the tailbase. Each group of mice was tested for a loss of SG function, SG lymphocytic infiltration, anti-Ro and anti-La antibody formation, and cytokine production in cultured cells or homogenized SG extracts.

Results

Ro60 peptide immunization in the abdominal area of female Balb/c mice led to impaired SG function, which corresponded with increased Th1 cytokines (IFN-γ and IL-12) systemically and locally in the SG. Moreover, changing the immunization conditions to MAP-Ro60 in the abdominal area, and to lesser extend in the tailbase, also led to impaired SG function in SJL/J mice. As was seen in the Balb/c mice, increased IFN-γ in the SG draining lymph nodes accompanied the SG dysfunction. However, no correlation was observed with anti-MAP-Ro60 antibody titers, and there was no additional effect on disease onset or severity.

Conclusions

Effective induction of salivary gland dysfunction after Ro60 peptide immunization depended on the site of injection. Disease induction was not affected by changing the immunization conditions. However, of interest is that the mechanism of action of Ro60 peptide immunization appears to involve an increase in Th1 cytokines, resulting in the induction of SG dysfunction.     

Differential proteomics of plant development

In this mini-review, recent advances in plant developmental proteomics are summarized. The growing interest in plant proteomics continually produces large numbers of developmental studies on plant cell division, elongation, differentiation, and formation of various organs. The brief overview of changes in proteome profiles emphasizes the participation of stress-related proteins in all developmental processes, which substantially changes the view on functional classification of these proteins. Next, it is noteworthy that proteomics helped to recognize some metabolic and housekeeping proteins as important signaling inducers of developmental pathways. Further, cell division and elongation are dependent on proteins involved in membrane trafficking and cytoskeleton dynamics. These protein groups are less prevalently represented in studies concerning cell differentiation and organ formation, which do not target primarily cell division. The synthesis of new proteins, generally observed during developmental processes, is followed by active protein folding. In this respect, disulfide isomerase was found to be commonly up-regulated during several developmental processes. The future progress in plant proteomics requires new and/or complementary approaches including cell fractionation, specific chemical treatments, molecular cloning and subcellular localization of proteins combined with more sensitive methods for protein detection and identification.     

Modulation of synaptic plasticity by stress hormone associates with plastic alteration of synaptic NMDA receptor in the adult hippocampus

Stress exerts a profound impact on learning and memory, in part, through the actions of adrenal corticosterone (CORT) on synaptic plasticity, a cellular model of learning and memory. Increasing findings suggest that CORT exerts its impact on synaptic plasticity by altering the functional properties of glutamate receptors, which include changes in the motility and function of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid subtype of glutamate receptor (AMPAR) that are responsible for the expression of synaptic plasticity. Here we provide evidence that CORT could also regulate synaptic plasticity by modulating the function of synaptic N-methyl-D-aspartate receptors (NMDARs), which mediate the induction of synaptic plasticity. We found that stress level CORT applied to adult rat hippocampal slices potentiated evoked NMDAR-mediated synaptic responses within 30 min. Surprisingly, following this fast-onset change, we observed a slow-onset (>1 hour after termination of CORT exposure) increase in synaptic expression of GluN2A-containing NMDARs. To investigate the consequences of the distinct fast- and slow-onset modulation of NMDARs for synaptic plasticity, we examined the formation of long-term potentiation (LTP) and long-term depression (LTD) within relevant time windows. Paralleling the increased NMDAR function, both LTP and LTD were facilitated during CORT treatment. However, 1-2 hours after CORT treatment when synaptic expression of GluN2A-containing NMDARs is increased, bidirectional plasticity was no longer facilitated. Our findings reveal the remarkable plasticity of NMDARs in the adult hippocampus in response to CORT. CORT-mediated slow-onset increase in GluN2A in hippocampal synapses could be a homeostatic mechanism to normalize synaptic plasticity following fast-onset stress-induced facilitation.