Rhodopsin signaling and organization in heterozygote rhodopsin knockout mice

Rhodopsin (Rho) resides within internal membrane structures called disc membranes that are found in the rod outer segments (ROS) of photoreceptors in the retina. Rho expression is essential for formation of ROS, which are absent in knockout Rho-/- mice. ROS of mice heterozygous for the Rho gene deletion (Rho+/-) may have a lower Rho density than wild type (WT) membranes, or the ROS structure may be reduced in size due to lower Rho expression. Here, we present evidence that the smaller volume of ROS from heterozygous mice is most likely responsible for observed electrophysiological response differences. In Rho+/- mice as compared with age-matched WT mice, the length of ROS was shorter by 30-40%, and the average diameter of ROS was reduced by approximately 20%, as demonstrated by transmission and scanning electron microscopy. Together, the reduction of the volume of ROS was approximately 60% in Rho+/- mice. Rho content in the eyes was reduced by approximately 43% and 11-cis-retinal content in the eye was reduced by approximately 38%, as determined by UV-visible spectroscopy and retinoid analysis, respectively. Transmission electron microscopy of negatively stained disc membranes from Rho+/- mice indicated a typical morphology apart from the reduced size of disc diameter. Power spectra calculated from disc membrane regions on such electron micrographs displayed a diffuse ring at approximately 4.5 nm(-1), indicating paracrystallinity of Rho. Atomic force microscopy of WT and Rho+/- disc membranes revealed, in both cases, Rho organized in paracrystalline and raftlike structures. From these data, we conclude that the differences in physiological responses measured in WT and Rho+/- mice are due to structural changes of the whole ROS and not due to a lower density of Rho.     

Mac-1(low) early myeloid cells in the bone marrow-derived SP fraction migrate into injured skeletal muscle and participate in muscle regeneration

Recent studies have shown that bone marrow (BM) cells, including the BM side population (BM-SP) cells that enrich hematopoietic stem cells (HSCs), are incorporated into skeletal muscle during regeneration, but it is not clear how and what kinds of BM cells contribute to muscle fiber regeneration. We found that a large number of SP cells migrated from BM to muscles following injury in BM-transplanted mice. These BM-derived SP cells in regenerating muscles expressed different surface markers from those of HSCs and could not reconstitute the mouse blood system. BM-derived SP/Mac-1(low) cells increased in number in regenerating muscles following injury. Importantly, our co-culture studies with activated satellite cells revealed that this fraction carried significant potential for myogenic differentiation. By contrast, mature inflammatory (Mac-1(high)) cells showed negligible myogenic activities. Further, these BM-derived SP/Mac-1(low) cells gave rise to mononucleate myocytes, indicating that their myogenesis was not caused by stochastic fusion with host myogenic cells, although they required cell-to-cell contact with myogenic cells for muscle differentiation. Taken together, our data suggest that neither HSCs nor mature inflammatory cells, but Mac-1(low) early myeloid cells in the BM-derived SP fraction, play an important role in regenerating skeletal muscles.     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

Overexpressed GM1 suppresses nerve growth factor (NGF) signals by modulating the intracellular localization of NGF receptors and membrane fluidity in PC12 cells

Ganglioside GM1 has been considered to have a neurotrophic factor-like activity. To analyze the effects of endogenously generated GM1, the rat pheochromocytoma cell line PC12 was transfected with the GM1/GD1b/GA1 synthase gene and showed increased expression levels of GM1. To our surprise, GM1+-transfectant cells (GM1+ cells) showed no neurite formation after stimulation with nerve growth factor (NGF). Autophosphorylation of NGF receptor TrkA and activation of ERK1/2 after NGF treatment were scarcely detected in GM1+ cells. Binding of 125I-NGF to PC12 cells was almost equivalent between GM1+ cells and controls. However, dimer formation of TrkA upon NGF treatment was markedly suppressed in GM1+ cells in both cross-linking analysis with Bis(sulfosuccinimidyl)suberate 3 and 125I-NGF binding assay. The sucrose density gradient fractionation of the cell lysate revealed that TrkA primarily located in the lipid raft fraction moved to the non-raft fraction in GM1+ cells. p75NTR and Ras also moved from the raft to non-raft fraction in GM1+ cells, whereas flotillin and GM1 persistently resided in the lipid raft. TrkA kinase activity was differentially regulated when GM1 was added to the kinase assay system in vitro, suggesting suppressive/enhancing effects of GM1 on NGF signals based on the concentration. Measurement of fluorescence recovery after photobleaching revealed that the membrane fluidity was reduced in GM1+ cells. These results suggested that overexpressed GM1 suppresses the differentiation signals mediated by NGF/TrkA by modulating the properties of the lipid raft and the intracellular localization of NGF receptors and relevant signaling molecules.     

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.     

PDGF-BB enhances alpha1beta1 integrin-mediated activation of the ERK/AP-1 pathway involved in collagen matrix remodeling by rat mesangial cells

Platelet-derived growth factor-BB (PDGF-BB) has been implicated in the pathogenesis of progressive glomerulonephritis (GN). Previous studies have reported that PDGF-BB stimulates mesangial cells (MCs)-induced collagen matrix remodeling through enhancement of alpha1beta1 integrin-dependent migratory activity. To determine the cell signaling pathway responsible for abnormal MC-related mesangial matrix remodeling in progressive GN, we studied the involvement of the extracellular signal-regulated kinase (ERK)/activator protein-1 (AP-1) pathway in PDGF-BB-enhanced collagen gel contraction. Western blotting and gel shift assay revealed that MC-induced gel contraction resulted in ERK activation in parallel with that of AP-1 binding, peaking at 4 h and lasting at least for 24 h. Application of the MEK inhibitor, U0126, and the c-jun/AP-1 inhibitor, curcumin, inhibited gel contraction and AP-1 activity, respectively, dose dependently. PDGF-BB enhanced not only gel contraction but ERK phosphorylation and AP-1 activity by MCs. Marked inhibitory effects on PDGF-BB-induced gel contraction and ERK/AP-1 activity were observed in the presence of either function blocking anti-alpha1- or anti-beta1-integrin antibody or U0126. Consistently, AP-1-inactive MCs expressing a dominant-negative mutant of c-jun showed a significant decrease of PDGF-BB-induced gel contraction as compared with mock-transfected MCs. Finally, migration assay showed that ERK/AP-1 activity is required for PDGF-BB-stimulated alpha1beta1 integrin-dependent MC migration to collagen I. These results indicated that PDGF-BB enhances alpha1beta1 integrin-mediated collagen matrix reorganization through the activation of the ERK/AP-1 pathway that is crucial for MC migration. We conclude that the ERK/AP-1 pathway plays an important role in PDGF-BB-induced alpha1beta1 integrin-dependent collagen matrix remodeling; therefore, the inhibition of its pathway may provide a novel approach to regulate abnormal collagen matrix remodeling in progressive GN.     

Inositol 1,4,5-trisphosphate receptor contains multiple cavities and L-shaped ligand-binding domains

Calcium concentrations are strictly regulated in all biological cells, and one of the key molecules responsible for this regulation is the inositol 1,4,5-trisphosphate receptor, which was known to form a homotetrameric Ca(2+) channel in the endoplasmic reticulum. The receptor is involved in neuronal transmission via Ca(2+) signaling and for many other functions that relate to morphological and physiological processes in living organisms. We analysed the three-dimensional structure of the ligand-free form of the receptor based on a single-particle technique using an originally developed electron microscope equipped with a helium-cooled specimen stage and an automatic particle picking system. We propose a model that explains the complex mechanism for the regulation of Ca(2+) release by co-agonists, Ca(2+), inositol 1,4,5-trisphosphate based on the structure of multiple internal cavities and a porous balloon-shaped cytoplasmic domain containing a prominent L-shaped density which was assigned by the X-ray structure of the inositol 1,4,5-trisphosphate binding domain.     

The effect of high glucose on NO and O2- through endothelial GTPCH1 and NADPH oxidase

Although endothelial dysfunction deteriorates diabetic angiopathy, the mechanisms are obscure. We revealed that high glucose augmented eNOS through stimulation of eNOS mRNA in cultured BAECs. NO was decreased and O2- was increased simultaneously. NOS inhibitor, inhibited O2- release, so did NADPH oxidase inhibitor. The effects were synergistic. Both intracellular BH4 level and GTPCH1 activity were decreased by high glucose, in line with decrease of GTPCH1 mRNA. HMG-CoA reductase inhibitor, atorvastatin increased GTPCH1 mRNA and activity, and BH4 level. Conclusively, high glucose leads to eNOS dysfunction by inhibiting BH4 synthesis and atorvastatin stimulate BH4 synthesis directly, and it may work as atherogenic process.