Roles of sphingosine-1-phosphate signaling in angiogenesis

Sphingosine-1-phosphate (S1P) is a blood-borne lipid mediator with pleiotropic biological activities. S1P acts via the specific cell surface G-protein-coupled receptors, S1P(1-5). S1P(1) and S1P(2) were originally identified from vascular endothelial cells (ECs) and smooth muscle cells, respectively. Emerging evidence shows that S1P plays crucial roles in the regulation of vascular functions, including vascular formation, barrier protection and vascular tone via S1P(1), S1P(2) and S1P(3). In particular, S1P regulates vascular formation through multiple mechanisms; S1P exerts both positive and negative effects on angiogenesis and vascular maturation. The positive and negative effects of S1P are mediated by S1P(1) and S1P(2), respectively. These effects of S1P(1) and S1P(2) are probably mediated by the S1P receptors expressed in multiple cell types including ECs and bone-marrow-derived cells. The receptor-subtype-specific, distinct effects of S1P favor the development of novel therapeutic tactics for antitumor angiogenesis in cancer and therapeutic angiogenesis in ischemic diseases.     

Construction of a Genetic Map and Development of DNA Markers Linked to the Sex-Determining Locus in the Patagonian Pejerrey (Odontesthes hatcheri)

The process of sex differentiation in fishes is regulated by genetic and environmental factors. The sex of Patagonian pejerrey (Odontesthes hatcheri) appears to be under strong genotypic control (GSD) because the sex ratios are balanced (1:1) between 17°C and 23°C. However, sex ratios become female-biased at <15°C and male-biased at 25°C, which shows that this species also possesses some degree of temperature-dependent sex determination (TSD). Identification of the genetic sex of an individual will help elucidate the molecular basis of sex differentiation in this species. In this study, we used amplified fragment length polymorphism (AFLP) analysis to develop a genetic linkage map for both sexes and a sex-linked DNA marker for Patagonian pejerrey. The AFLP analysis of 23 male and 23 female progeny via 64 primer combinations produced a total of 153 bands. The genetic linkage map consisted of 79 markers in 20 linkage groups and 48 markers in 15 linkage groups for males and females, respectively. One AFLP marker tightly linked to the sex-determining locus was identified: the marker, ACG/CAA-217, amplified to the male-specific DNA fragment. Sequence analysis of this region revealed a single nucleotide polymorphism (SNP) between males and females, which was converted into a SNP marker. This marker provides genetic confirmation that the sex of Patagonian pejerrey is determined genetically and would be useful for the analysis of the molecular basis of GSD and TSD in this species.     

Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins

Background  

The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).     

The Critical Role of Nitric Oxide Signaling,via Protein S-Guanylation and Nitrated Cyclic GMP,in the Antioxidant Adaptive Response

A nitrated guanine nucleotide, 8-nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), is formed via nitric oxide (NO) and causes protein S-guanylation. However, intracellular 8-nitro-cGMP levels and mechanisms of formation of 8-nitro-cGMP and S-guanylation are yet to be identified. In this study, we precisely quantified NO-dependent formation of 8-nitro-cGMP in C6 glioma cells via liquid chromatography-tandem mass spectrometry. Treatment of cells with S-nitroso-N-acetylpenicillamine led to a rapid, transient increase in cGMP, after which 8-nitro-cGMP increased linearly up to a peak value comparable with that of cGMP at 24 h and declined thereafter. Markedly high levels (>40 μm) of 8-nitro-cGMP were also evident in C6 cells that had been stimulated to express inducible NO synthase with excessive NO production. The amount of 8-nitro-cGMP generated was comparable with or much higher than that of cGMP, whose production profile slightly preceded 8-nitro-cGMP formation in the activated inducible NO synthase-expressing cells. These unexpectedly large amounts of 8-nitro-cGMP suggest that GTP (a substrate of cGMP biosynthesis), rather than cGMP per se, may undergo guanine nitration. Also, 8-nitro-cGMP caused S-guanylation of KEAP1 in cells, which led to Nrf2 activation and subsequent induction of antioxidant enzymes, including heme oxygenase-1; thus, 8-nitro-cGMP protected cells against cytotoxic effects of hydrogen peroxide. Proteomic analysis for endogenously modified KEAP1 with matrix-assisted laser desorption/ionization time-of-flight-tandem mass spectrometry revealed that 8-nitro-cGMP S-guanylated the Cys⁴³⁴ of KEAP1. The present report is therefore the first substantial corroboration of the biological significance of cellular 8-nitro-cGMP formation and potential roles of 8-nitro-cGMP in the Nrf2-dependent antioxidant response.     

Sterol-induced Dislocation of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase from Endoplasmic Reticulum Membranes into the Cytosol through a Subcellular Compartment Resembling Lipid Droplets

Sterol-induced binding to Insigs in the endoplasmic reticulum (ER) allows for ubiquitination of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. This ubiquitination marks reductase for recognition by the ATPase VCP/p97, which mediates extraction and delivery of reductase from ER membranes to cytosolic 26 S proteasomes for degradation. Here, we report that reductase becomes dislocated from ER membranes into the cytosol of sterol-treated cells. This dislocation exhibits an absolute requirement for the actions of Insigs and VCP/p97. Reductase also appears in a buoyant fraction of sterol-treated cells that co-purifies with lipid droplets, cytosolic organelles traditionally regarded as storage depots for neutral lipids such as triglycerides and cholesteryl esters. Genetic, biochemical, and localization studies suggest a model in which reductase is dislodged into the cytosol from an ER subdomain closely associated with lipid droplets.     

Constitutively Active Inflammasome in Human Melanoma Cells Mediating Autoinflammation via Caspase-1 Processing and Secretion of Interleukin-1��

Interleukin-1β (IL-1β) is a pleiotropic cytokine promoting inflammation, angiogenesis, and tissue remodeling as well as regulation of immune responses. Although IL-1β contributes to growth and metastatic spread in experimental and human cancers, the molecular mechanisms regulating the conversion of the inactive IL-1β precursor to a secreted and active cytokine remains unclear. Here we demonstrate that NALP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 in human melanoma cells. Late stage human melanoma cells spontaneously secrete active IL-1β via constitutive activation of the NALP3 inflammasome and IL-1 receptor signaling, exhibiting a feature of autoinflammatory diseases. Unlike human blood monocytes, these melanoma cells require no exogenous stimulation. In contrast, NALP3 functionality in intermediate stage melanoma cells requires activation of the IL-1 receptor to secrete active IL-1β; cells from an early stage of melanoma require stimulation of the IL-1 receptor plus the co-stimulant muramyl dipeptide. The spontaneous secretion of IL-1β from melanoma cells was reduced by inhibition of caspase-1 or the use of small interfering RNA directed against ASC. Supernatants from melanoma cell cultures enhanced macrophage chemotaxis and promoted in vitro angiogenesis, both prevented by pretreating melanoma cells with inhibitors of caspases-1 and -5 or IL-1 receptor blockade. These findings implicate IL-1-mediated autoinflammation as contributing to the development and progression of human melanoma and suggest that inhibiting the inflammasome pathway or reducing IL-1 activity can be a therapeutic option for melanoma patients.     

Elastin peptides prepared from piscine and mammalian elastic tissues inhibit collagen‐induced platelet aggregation and stimulate migration and proliferation of human skin fibroblasts

We obtained pure elastin peptides from bovine ligamentum nuchae, porcine aorta, and bonito bulbus arteriosus. The inhibitory activity of these elastin peptides on platelet aggregation induced by collagen and the migratory and proliferative responsivenesses of human skin fibroblasts to these elastin peptides were examined. All of bonito, bovine, and porcine elastin peptides found to inhibit platelet aggregation, but bonito elastin peptides showed a higher inhibitory activity than bovine and porcine elastin peptides did. All elastin peptides enhanced the proliferation of fibroblasts 3.5‐ to 4.5‐fold at a concentration of 10 µg/ml. Bovine and porcine elastin peptides stimulated the migration of fibroblasts, with the optimal response occurring at 10^?1 µg/ml, while maximal response was at 10² µg/ml for bonito elastin peptides. Furthermore, pretreatment of fibroblasts by lactose depressed their ability to migrate in response to all elastin peptides, suggesting the involvement of elastin receptor in cell response. These results suggest that both mammalian and piscine elastin peptides can be applied as useful biomaterials in which elasticity, antithrombotic property, and the enhancement of cell migration and proliferation are required. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Site‐directed mutagenesis of rat thioltransferase: Effects of essential cysteine residues for the protection against oxidative stress

A cDNA of rat liver thioltransferase was cloned and then expressed using pMAL‐c expression vector in Escherichia coli. Recombinant rat liver thioltransferase was expressed as a fusion protein with maltose‐binding protein and then purified by amylose resin column chromatography to be homogeneity on 12.5% SDS‐polyacrylamide gel electrophoretic analysis. The expressed proteins were shown as two bands at around 53 and 41 kDa, suggesting that the high molecular one was a fusion protein of recombinant thioltransferase (11.7 plus 41 kDa) and the other (smaller one) was a maltose‐binding protein (41 kDa). A recombinant thioltransferase catalyzed a thiol/disulfide exchange reaction in the same way as thioltransferases purified from various sources. Compared with wild type, the mutants C23A, C26A, C79A, and C83A showed 0%, 17%, 82%, and 86% in the enzymatic activity, respectively. In addition, wild‐type‐transfected bacteria expressed in bacterial cells showed a strong resistance to H₂O₂ treatment as well as the case of active mutants (C79A and C83A), but inactive mutants (C23A and C26A) showed no resistance to H₂O₂ treatment as same as mocktransfection. Thioltransferase can be important for survival of bacterial cells under oxidative stress. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:60–65, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20312     

Determination of platinum derived from cisplatin in human tissues using electrospray ionization mass spectrometry

Determination of platinum (Pt) derived from cisplatin in tissues was performed by electrospray ionization mass spectrometry (ESI-MS) using silver (Ag) as the internal standard. Pt and Ag reacted with diethyldithiocarbamate (DDC), and were extracted using isoamylalcohol and acidified with oxalic acid. The compounds were termed Pt(DDC)(3)(+) and Ag(DDC)(2)(+), based on their m/z values exhibiting the highest peaks at m/z 639 and m/z 405, respectively. The limit of detection was 30 pg and the quantitation range was from 100 to 10,000 pg using 5 mg tissue. The present method allowed the determination of Pt in wet-ashed tissue in 10 min.     

Increased leptin may be involved in the short life span of ayu (Plecoglossus altivelis)

It is well known that ayu (Plecoglossus altivelis) die after spawning and have a life span of only 1 year. The determinants for such a short life span are probably connected with spawning and related changes in hormonal homeostases. One of these changes is that the ayu's feeding activity decreases both during and after spawning. We investigated the relationships among leptin, one of the regulators of food intake, and two other major hormones, 17 beta-estradiol and prolactin (PRL). Ir-leptin levels were significantly higher during spawning, and were associated with a decrease in appetite. Ir-leptin levels were also synchronized with levels of 17 beta-estradiol and PRL-like protein. Therefore, one possible explanation for the decrease in appetite during ayu spawning is that the elevation of 17 beta-estradiol homeostasis induced the secretion of Ir-leptin. The inability to decrease leptin to the basal levels because of high estrogen after spawning could be in part responsible for the short life span of ayu.