Normal acute and chronic inflammatory responses in sphingosine kinase 1 knockout mice

Sphingosine-1-phosophate, generated from the phosphorylation of sphingosine by sphingosine kinase enzymes, is suggested to function as an intracellular second messenger for inflammatory mediators, including formyl peptide, C5a, and Fc. More recently, a role for sphingosine kinases during inflammation has also been proposed. Here we show that sphingosine kinase 1 knockout mice exhibit normal inflammatory cell recruitment during thioglycollate-induced peritonitis and that sphingosine kinase 1-null neutrophils respond normally to formyl peptide. In the collagen-induced arthritis model of rheumatoid arthritis, sphingosine kinase 1 knockout mice developed arthritis with normal incidence and severity. Our findings show that sphingosine kinase 1 is dispensable for inflammatory responses and support the need for more extensive studies of sphingosine kinases in inflammation.     

Oral adsorbent AST-120 decreases serum levels of AGEs in patients with chronic renal failure

Advanced glycation end products (AGEs) are senescent macroprotein derivatives that are formed at an accelerated rate in patients with chronic renal failure (CRF). AGE formation and accumulation in plasma and vascular tissues contribute to accelerated atherosclerosis in this devastating disorder. AST-120 is an oral adsorbent that attenuates the progression of CRF by removing uremic toxins. Recently, AST-120 has been reported to reduce the progression of atherosclerosis as well. However, whether AST-120 decreases serum levels of AGEs and subsequently exerts atheroprotective properties remains to be elucidated. Ten nondiabetic CRF patients were enrolled in this study. All patients were kept on regular therapeutic diet and medications throughout the study. Serum AGE levels before and after AST-120 treatments were measured using enzyme-linked immunosorbent assay. Effects of patient-derived serum on atherosclerosis-related gene expression in cultured human umbilical vein endothelial cells (HUVECs) were analyzed by semiquantitative RT-PCR. Administration of AST-120 (6 g/day) for 3 months significantly decreased serum levels of AGEs in nondiabetic CRF patients, whereas AGE levels remained unchanged in age- and renal function-matched CRF patients without AST-120 treatment (n = 6). Patient serum after AST-120 treatment significantly reduced mRNA levels of receptor for AGEs, monocyte chemoattractant protein-1, and vascular adhesion molecule-1 in HUVECs compared with serum before treatment. Moreover, in vitro, AST-120 was found to adsorb carboxymethyllysine (CML), one of the well-characterized, digested food-derived AGEs. This study suggests that atheroprotective properties of AST-120 can be ascribed, at least in part, to its AGE-lowering ability via absorption of CML.     

Molecular cloning of estrogen receptor alpha of the Nile crocodile

Estrogens are essential for normal reproductive activity in female and male vertebrates. In female reptiles, they are essential for ovarian differentiation during a critical developmental stage. To understand the molecular mechanisms of estrogen action in the Nile crocodile (Crocodylus niloticus), we have isolated cDNA encoding the estrogen receptor alpha (ERalpha) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify Nile crocodile cDNA from the ovary. The full-length Nile crocodile ERalpha cDNA was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the Nile crocodile ERalpha showed high identity to the American alligator ERalpha (98%), caiman ER (98%), lizard ER (82%) and chicken ERalpha (92%), although phylogenetic analysis suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. Expression of ERalpha was observed in the ovary and testis of juvenile Nile crocodiles. These data provide a novel tool allowing future studies examining the regulation and ontogenic expression of ERalpha in crocodiles and expands our knowledge of estrogen receptor evolution.     

Successive analysis of antigen trapping and enzymatic digestion on membrane-immobilized avidin

Avidin from egg white was migrated toward a cathode of nondenaturing electrophoresis and then immobilized on a polyvinylidene difluoride membrane. Adrenocorticotropic hormone (ACTH) was specifically captured after the biotinylated anti-ACTH antibody was bound to the membrane-immobilized avidin, and the captured ACTH was digested by the biotinylated trypsin on the membrane after extraction. The digested polypeptides from the ACTH were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These results indicate that target substances can be specifically trapped and digested on membrane-immobilized avidin.     

Solution structure of kurtoxin: a gating modifier selective for Cav3 voltage-gated Ca(2+) channels

Kurtoxin is a 63-amino acid polypeptide isolated from the venom of the South African scorpion Parabuthus transvaalicus. It is the first and only peptide ligand known to interact with Cav3 (T-type) voltage-gated Ca(2+) channels with high affinity and to modify the voltage-dependent gating of these channels. Here we describe the nuclear magnetic resonance (NMR) solution structure of kurtoxin determined using two- and three-dimensional NMR spectroscopy with dynamical simulated annealing calculations. The molecular structure of the toxin was highly similar to those of scorpion α-toxins and contained an α-helix, three β-strands, and several turns stabilized by four disulfide bonds. This so-called "cysteine-stabilized α-helix and β-sheet (CSαβ)" motif is found in a number of functionally varied small proteins. A detailed comparison of the backbone structure of kurtoxin with those of the scorpion α-toxins revealed that three regions [first long loop (Asp(8)-Ile(15)), β-hairpin loop (Gly(39)-Leu(42)), and C-terminal segment (Arg(57)-Ala(63))] in kurtoxin significantly differ from the corresponding regions in scorpion α-toxins, suggesting that these regions may be important for interacting with Cav3 (T-type) Ca(2+) channels. In addition, the surface profile of kurtoxin shows a larger and more focused electropositive patch along with a larger hydrophobic surface compared to those seen on scorpion α-toxins. These distinct surface properties of kurtoxin could explain its binding to Cav3 (T-type) voltage-gated Ca(2+) channels.     

Accelerating Effect of Soy Peptides Containing Collagen Peptides on Type I and III Collagen Levels in Rat Skin

Two weeks of feeding soy peptides containing 2% collagen peptides increased the levels of type I and III tropocollagen and their mRNAs. In contrast, the diet did not increase the mRNA levels of rat hyaluronan synthases, serine palmitoyltransferase (the rate-limiting enzyme of ceramide synthesis), and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (the key enzyme of cholesterol synthesis). These results suggest that feeding of soy peptides with collagen peptides specifically enhanced the tropocollagen level in the skin.     

Seasonal dynamics of fishes in tidepools on tidal mudflats in the Tama River estuary, central Honshu, Japan

We investigated seasonal dynamics of fish in tidepools on tidal mudflats in the Tama River estuary, Japan. A total of 1,156 individuals of 8 gobiid species were collected from May 2008 to April 2009. The total abundance of fishes peaked in September and October. Of three dominant species, Acanthogobius flavimanus was found only in spring and early summer, whereas Pseudogobius masago and Mugilogobius abei occurred in all seasons. The utilization patterns of tidepools with/without cobbles were different among the latter two species.