Hypoxia-Induced Retinal Neovascularization in Zebrafish Embryos: A Potential Model of Retinopathy of Prematurity

Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2–4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression.     

Differential effect of sorbitol and polyethylene glycol on antioxidant enzymes in rice leaves

Polyethylene glycol (PEG) and sorbitol (ST) have each been used inosmotically induced water stress studies in plants, however, these osmotica maynot have equivalent effects in plants. The present study was designed to examinewhether antioxidant enzyme responses in rice leaves are different for PEG and STof osmotic potential –1.5 MPa. As judged by relative watercontent, PEG treatment resulted in a higher degree of water stress in riceleaves than ST treatment. PEG treatment markedly increased lipid peroxidation,judged by malondialdehyde content, in rice leaves. However, ST treatment had noeffect on lipid peroxidation. An increase in peroxidase (POX), ascorbateperoxidase (APX) and glutathione reductase (GR) activities was observed in riceleaves treated with ST. PEG treatment had no effect on POX and APX activitiesand decreased GR activity in rice leaves. The decrease in superoxide dismutaseactivity induced by PEG was more pronounced than by ST. Cycloheximide blockedthe enhanced activities of POX, APX and GR by ST, indicating de novo synthesisof the enzymes. Results suggest that ST but not PEG treatment can up-regulateantioxidant system in rice leaves.     

miR-144 suppresses cell proliferation and migration in colorectal cancer by targeting NRAS

Colorectal cancer (CRC) is a type of malignant cancer that has become particularly prevalent worldwide. It is of crucial importance to CRC treatment that the underlying molecular mechanism of CRC progression is determined. The NRAS gene is an important small G protein that is involved in various biological processes, including cancers. NRAS is an oncogene in many neoplasms but its function and regulation in CRC have seldom been investigated. In this study, it was uncovered that the NRAS protein was significantly upregulated in CRC tissues. According to a bioinformatics prediction, we identified that miR-144 may target NRAS to suppress its expression. In vitro experiments indicated that miR-144 decreased NRAS expression in different CRC cell lines (SW480, LoVo, and Caco2). By inhibiting NRAS, miR-144 repress SW480 cell proliferation and migration. Moreover, miR-144 decelerated the growth of SW480 xenograft tumors in vivo by targeting NRAS. In summary, our results identified a novel miR-144-NRAS axis in CRC that could promote the research and treatment of CRC.     

Localization of phospholipid-rich zones in rat gastric mucosa: possible origin of a protective hydrophobic luminal lining

Mammalian gastric mucosa is unusually hydrophobic or nonwettable, which may be an essential biophysical characteristic of the gastric mucosal barrier. Since this property may be attributable to an adsorbed layer of surface-active phospholipids (SAPL), we investigated the distribution of SAPL in rat oxyntic mucosa. Ferric hematoxylin (FH) and iodoplatinate (IP), selective histochemical stains for phospholipids (as confirmed by spot tests), were used to detect SAPL in frozen sections and aldehyde-fixed tissue, respectively. Using FH staining in conjunction with extraction procedures that either solvate or preserve SAPL, we determined that positive reactivity was the greatest in the apical third of the oxyntic mucosa between the glandular neck region and the surface. IP reactivity appeared to parallel the FH staining pattern. Mucous cells, especially the surface epithelial cells, were heavily stained. Electron microscopic examination revealed that these cells contain inclusion bodies associated with various subcellular organelles, e.g., nuclear envelope, endoplasmic reticulum, Golgi apparatus and its vesicles, and mucous secretory granules. Vesicles and myelin figures, which resembled those found in lung surfactant, were observed extracellularly in close association with the surface mucous cells. Our findings suggest that mucous cells are actively involved in synthesis and storage of SAPL, which may be an essential component of the stomach's protective hydrophobic lining.     

A locking mechanism regulates RNA synthesis and host protein interaction by the hepatitis C virus polymerase

Mutational analysis of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) template channel identified two residues, Trp(397) and His(428), which are required for de novo initiation but not for extension from a primer. These two residues interact with the Delta1 loop on the surface of the RdRp. A deletion within the Delta1 loop also resulted in comparable activities. The mutant proteins exhibit increased double-stranded RNA binding compared with the wild type, suggesting that the Delta1 loop serves as a flexible locking mechanism to regulate the conformations needed for de novo initiation and for elongative RNA synthesis. A similar locking motif can be found in other viral RdRps. Products associated with the open conformation of the HCV RdRp were inhibited by interaction with the retinoblastoma protein but not cyclophilin A. Different conformations of the HCV RdRp can thus affect RNA synthesis and interaction with cellular proteins.     

Identification of a conserved Rac-binding site on NADPH oxidases supports a direct GTPase regulatory mechanism

The NADPH oxidases (Noxs) are a family of superoxide-generating enzymes implicated in a variety of biological processes. Full activity of Nox1, -2, and -3 requires the action of a Rac GTPase. A direct regulatory interaction of Rac with Nox2 has been proposed as part of a two-step mechanism for regulating electron transfer during superoxide formation. Using truncation analysis of Rac binding to the cytoplasmic tail of Nox2, along with peptides derived from this region in cell-free assays, we identify a Rac interaction site within amino acids 419-430 of Nox2. This region is required for binding Rac2 but not p47(phox) or p67(phox) cytosolic regulatory factors. A cell-permeant version of the peptide encompassing amino acids 419-430 specifically inhibits NADPH oxidase activation in intact human neutrophils. Mutational analysis of the putative Rac-binding site revealed specific residues, particularly Lys-421, Tyr-425, and Lys-426, individually required for Rac-dependent NADPH oxidase activity that are conserved in the Rac-regulated Nox1, Nox2, and Nox3 enzymes but not in Nox4 or Nox5. Mutation of the conserved residues in the Rac-binding site of Nox1 also result in the loss of Rac-dependent activity. Our data identify a functional Rac interaction site conserved in Rac-dependent Noxs and support a direct regulatory interaction of Rac GTPases to promote activation of these NADPH oxidases.