Cytoplasmic shuttling of protons in anabaena sensory rhodopsin: implications for signaling mechanism

It was found recently that Anabaena sensory rhodopsin (ASR), which possibly serves as a photoreceptor for chromatic adaptation, interacts with a soluble cytoplasmic transducer. The X-ray structure of the transducer-free protein revealed an extensive hydrogen-bonded network of amino acid residues and water molecules in the cytoplasmic half of ASR, in high contrast to its haloarchaeal counterparts. Using time-resolved spectroscopy of the wild-type and mutant ASR in the visible and infrared ranges, we tried to determine whether this hydrogen-bonded network is used to translocate protons and whether those proton transfers are important for interaction with the transducer. We found that the retinal Schiff base deprotonation, which occurs in the M intermediate of the photocycle of all-trans-ASR, results in protonation of Asp217 on the cytoplasmic side of the protein. The deprotonation of the Schiff base induces a conformational change of ASR observed through the perturbation of associated lipids. We suggest that the cytoplasmic shuttling of protons in the photocycle of all-trans-ASR and the ensuing conformational changes might activate the transducer. Consequently, the M intermediate may be the signaling state of ASR.     

uPAR expression under hypoxic conditions depends on iNOS modulated ERK phosphorylation in the MDA-MB-231 breast carcinoma cell line

Urokinase plasminogen activator receptor （uPAR） plays a major role in cancer-invasion and metastasis and uPAR expression is correlated with a poor prognosis in various cancer types. Moreover, the expression of uPAR is increased under hypoxic conditions. Nitric oxide （NO） and its metabolites produced by inducible nitric oxide synthase （iNOS） are important products ofhypoxic stress, and NO may activate or modulate extracellular signal regulated kinase （ERK）. Here, we evaluated uPA, uPAR, and activated ERK levels under hypoxic conditions, and the modulatory effects of iNOS and NO in the MDA-MB-231 human breast cancer cell line. Cells were incubated in a hypoxic or normoxic incubator and treated with PD98059 （a MEK 1/2 inhibitor, which abrogates ERK phosphorylation） and aminoguanidine （a selective iNOS inhibitor）, uPAR expression, ERK phosphorylation, and uPA activity were found to be increased under hypoxic conditions. Moreover, when cells were treated with PD98059 under hypoxic conditions, uPAR was downregulated, whereas aminoguanidine markedly increased ERK phosphorylation in a dose dependent manner. Furthermore, aminoguanidine increased uPAR expression and prevented the inhibition of uPAR expression by PD98059. These results demonstrated that uPAR is induced by hypoxia and that increased uPAR expression is mediated by ERK phosphorylation, which in turn is modulated by iNOS/NO in MDA-MB-231 cells. We conclude that iNOS/NO downregulates the expression of uPAR under hypoxic conditions via ERK pathway modulation.     

Application of human umbilical cord blood-derived mesenchymal stem cells in disease models

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) are regarded as an alternative source of bone marrow-derived mesenchymal stem cells because collection of cord blood is less invasive than that of bone marrow. hUCB-MSCs have recently been studied for evaluation of their potential as a source of cell therapy. In this review, the general characteristics of hUCB-MSCs and their therapeutic effects on various diseases in vitro and in vivo will be discussed.     

Endothelial Nitric Oxide Synthase (eNOS) gene polymorphisms in spontaneously aborted embryos

Endothelium-derived nitric oxide (NO) is synthesized from L-arginine by endothelial nitric oxide synthase (eNOS) encoded by the eNOS gene on chromosome 7. The effects of the eNOS polymorphisms with the risk of spontaneous pregnancy losses are conflicting. In this study, we investigated the association of the eNOS genotypes with spontaneously aborted embryos in Koreans. Case-control studies were performed to evaluate the association between endothelial nitric oxide synthase (eNOS) gene polymorphisms and spontaneously aborted embryos. Ninety-nine spontaneously aborted fetuses at <20 weeks of gestational age and 103 child controls and 282 adult controls. Genotype frequency of three eNOS gene polymorphisms, ?786T>C, VNTR in intron 4 (4a4b), and 894G>T in spontaneously aborted embryos was surveyed. The frequencies of ?786TC and CC genotypes in aborted embryos were significantly higher than in both child and adult controls. The frequencies of 4a4a homozygote of VNTR polymorphism in intron 4 and TT homozygote of 894G>T polymorphisms were also higher in aborted embryos than in adult controls. Haploptype analysis suggested that ?786T>C polymorphism was a possible risk factor for spontaneously aborted embryos. eNOS gene polymorphisms, ?786T>C, VNTR in intron 4 (4a4b), and 894G>T, are associated with the risk of spontaneously aborted fetuses.     

Nanoporous protein matrix made of amyloid fibrils of β2‐microglobulin

Amyloid fibrils are considered as novel nanomaterials because of their nanoscale width, a regular constituting structure of cross β‐sheet conformation, and considerable mechanical strength. By using an amyloidogenic protein of β₂‐microglobulin (β₂M) related to dialysis‐related amyloidosis, nanoporous protein matrix has been prepared. The β₂M granules made of around 15 monomers showed an average size of 23.1 nm. They formed worm‐like fibrils at pH 7.4 in 20 mM sodium phosphate containing 0.15 M NaCl following vigorous nondirectional shaking incubation, in which they became laterally associated and interwound to generate the porous amyloid fibrillar matrix with an average pore size of 30–50 nm. This nanoporous protein matrix was demonstrated to be selectively disintegrated by reducing agents, such as tris‐(2‐carboxyethyl) phosphine. High surface area with nanopores on the surface has been suggested to make the matrix of β₂M amyloid fibrils particularly suitable for applications in the area of nanobiotechnology including drug delivery and tissue engineering. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Proteome-Wide Profiling of the MCF10AT Breast Cancer Progression Model

Background

Mapping the expression changes during breast cancer development should facilitate basic and translational research that will eventually improve our understanding and clinical management of cancer. However, most studies in this area are challenged by genetic and environmental heterogeneities associated with cancer.

Methodology/Principal Findings

We conducted proteomics of the MCF10AT breast cancer model, which comprises of 4 isogenic xenograft-derived human cell lines that mimic different stages of breast cancer progression, using iTRAQ-based tandem mass spectrometry. Of more than 1200 proteins detected, 98 proteins representing at least 20 molecular function groups including kinases, proteases, adhesion, calcium binding and cytoskeletal proteins were found to display significant expression changes across the MCF10AT model. The number of proteins that showed different expression levels increased as disease progressed from AT1k pre-neoplastic cells to low grade CA1h cancer cells and high grade cancer cells. Bioinformatics revealed that MCF10AT model of breast cancer progression is associated with a major re-programming in metabolism, one of the first identified biochemical hallmarks of tumor cells (the “Warburg effect”). Aberrant expression of 3 novel breast cancer-associated proteins namely AK1, ATOX1 and HIST1H2BM were subsequently validated via immunoblotting of the MCF10AT model and immunohistochemistry of progressive clinical breast cancer lesions.

Conclusion/Significance

The information generated by this study should serve as a useful reference for future basic and translational cancer research. Dysregulation of ATOX1, AK1 and HIST1HB2M could be detected as early as the pre-neoplastic stage. The findings have implications on early detection and stratification of patients for adjuvant therapy.     

Autophagy and KRT8/keratin 8 protect degeneration of retinal pigment epithelium under oxidative stress

Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.     

Synthesis of Flavonoid O-Pentosides by Escherichia coli through Engineering of Nucleotide Sugar Pathways and Glycosyltransferase

Plants produce two flavonoid O-pentoses, flavonoid O-xyloside and flavonoid O-arabinoside. However, analyzing their biological properties is difficult because flavonoids are not naturally produced in sufficient quantities. In this study, Escherichia coli was used to synthesize the plant-specific flavonoid O-pentosides quercetin 3-O-xyloside and quercetin 3-O-arabinoside. Two strategies were used. First, E. coli was engineered to express components of the biosynthetic pathways for UDP-xylose and UDP-arabinose. For UDP-xylose biosynthesis, two genes, UXS (UDP-xylose synthase) from Arabidopsis thaliana and ugd (UDP-glucose dehydrogenase) from E. coli, were overexpressed. In addition, the gene encoding ArnA (UDP-l-Ara4N formyltransferase/UDP-GlcA C-4″-decarboxylase), which competes with UXS for UDP-glucuronic acid, was deleted. For UDP-arabinose biosynthesis, UXE (UDP-xylose epimerase) was overexpressed. Next, we engineered UDP-dependent glycosyltransferases (UGTs) to ensure specificity for UDP-xylose and UDP-arabinose. The E. coli strains thus obtained synthesized approximately 160 mg/liter of quercetin 3-O-xyloside and quercetin 3-O-arabinoside.