Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1

SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.     

An antitumor promoter from Moringa oleifera Lam.

In the course of studies on the isolation of bioactive compounds from Philippine plants, the seeds of Moringa oleifera Lam. were examined and from the ethanol extract were isolated the new O-ethyl-4-(α- -rhamnosyloxy)benzyl carbamate (1) together with seven known compounds, 4(α- -rhamnosyloxy)-benzyl isothiocyanate (2), niazimicin (3), niazirin (4), β-sitosterol (5), glycerol-1-(9-octadecanoate) (6), 3-O-(6′-O-oleoyl-β- -glucopyranosyl)-β-sitosterol (7), and β-sitosterol-3-O-β- -glucopyranoside (8). Four of the isolates (2, 3, 7, and 8), which were obtained in relatively good yields, were tested for their potential antitumor promoting activity using an in vitro assay which tested their inhibitory effects on Epstein–Barr virus-early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). All the tested compounds showed inhibitory activity against EBV-EA activation, with compounds 2, 3 and 8 having shown very significant activities. Based on the in vitro results, niazimicin (3) was further subjected to in vivo test and found to have potent antitumor promoting activity in the two-stage carcinogenesis in mouse skin using 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and TPA as tumor promoter. From these results, niazimicin (3) is proposed to be a potent chemo-preventive agent in chemical carcinogenesis.     

MOLECULAR PHYLOGENETIC ANALYSIS OF rbcL IN THE PRYMNESIOPHYTA1

The nucleotide sequences of rbcL genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were determined from six species of Prymnesiophyta to clarify their phylogenetic relationships. Molecular phylogenetic trees were constructed using PAUP (Phylogenetic Analysis Using Parsimony). These analyses suggest that the Prymnesiophyta, except for the Pavlovales, area relatively stable monophyletic group. Pleurochrysis carterae, included in the Isochrysidales, is a sister species of a monophyletic group consisting of other members of the Isochrysidales, Gephyrocapsa oceanica and Emiliania huxleyi, members of the Coccosphaerales, Calyptrosphaera sphaeroidea and Umbilicosphaera sibogae var. foliosa, and a member of the Prymnesiales, Chrysochromulina hirta. The nucleotide sequence of rbcL from G. oceanica was identical to that from E. huxleyi within the region examined. Our trees show that G. oceanica and E. huxleyi are more closely related to C. hirta than to U. sibogae, C. sphaeroidea, and P. carterae. These results suggest that orders in the Prymnesiophyceae, including the above-mentioned genera, should be redefined.     

Insular Gray Matter Volume and Objective Quality of Life in Schizophrenia

Improving quality of life has been recognized as an important outcome for schizophrenia treatment, although the fundamental determinants are not well understood. In this study, we investigated the association between brain structural abnormalities and objective quality of life in schizophrenia patients. Thirty-three schizophrenia patients and 42 age-, sex-, and education-matched healthy participants underwent magnetic resonance imaging. The Quality of Life Scale was used to measure objective quality of life in schizophrenia patients. Voxel-based morphometry was performed to identify regional brain alterations that correlate with Quality of Life Scale score in the patient group. Schizophrenia patients showed gray matter reductions in the frontal, temporal, limbic, and subcortical regions. We then performed voxel-based multiple regression analysis in these regions to identify any correlations between regional gray matter volume and Quality of Life Scale scores. We found that among four subcategories of the scale, the Instrumental Role category score correlated with gray matter volume in the right anterior insula in schizophrenia patients. In addition, this correlation was shown to be mediated by negative symptoms. Our findings suggest that the neural basis of objective quality of life might differ topographically from that of subjective QOL in schizophrenia.     

Relationships between Micro-Vascular and Iodine-Staining Patterns in the Vicinity of the Tumor Front of Superficial Esophageal Squamous Carcinoma

Objective

The aim of the present study was to clarify differences between micro-vascular and iodine-staining patterns in the vicinity of the tumor fronts of superficial esophageal squamous cell carcinomas (ESCCs).

Methods

Ten consecutive patients with ESCCs who were treated by endoscopic submucosal dissection (ESD) were enrolled. At the edge of the iodine-unstained area, we observed 183 sites in total using image-enhanced magnifying endoscopy. We classified the micro-vascular and iodine-staining patterns into three types: Type A, in which the line of vascular change matched the border of the iodine-unstained area; Type B, in which the border of the iodine-unstained area extended beyond the line of vascular change; Type C, in which the line of vascular change extended beyond the border of the iodine-unstained area. Then, by examining histopathological sections, we compared the diameter of intra-papillary capillary loops (IPCLs) in cancerous areas and normal squamous epithelium.

Results

We investigated 160 sites that the adequate quality of pictures were obtained. There was no case in which the line of vascular change completely matched the whole circumference of the border of an iodine-unstained area. Among the 160 sites, type A was recognized at 76 sites (47.5%), type B at 79 sites (49.4%), and type C at 5 sites (3.1%). Histological examination showed that the mean diameter of the IPCLs in normal squamous epithelium was 16.2±3.7μm, whereas that of IPCLs in cancerous lesions was 21.0±4.4μm.

Conclusions

The development of iodine-unstained areas tends to precede any changes in the vascularity of the esophageal surface epithelium.     

Cholesterol-lowering effects of maitake (Grifola frondosa) fiber, shiitake (Lentinus edodes) fiber, and enokitake (Flammulina velutipes) fiber in rats

The effects of mushroom fibers on serum cholesterol and hepatic low-density lipoprotein (LDL) receptor mRNA in rats were investigated. Rats were fed a cholesterol-free diet with 50 g/kg cellulose powder (CP), 50 g/kg maitake (Grifola frondosa) fiber (MAF), 50 g/kg shiitake (Lentinus edodes) fiber (SF), or 50 g/kg enokitake (Flammulina velutipes) fiber (EF) for 4 weeks. There were no significant differences in the body weight, food intake, liver weight, cecum weight, and cecum pH among the groups. Cecal acetic acid, butyric acid, and total short-chain fatty acid (SCFA) concentrations in the SF and EF groups were significantly higher than those in the other groups. The serum total cholesterol concentration in the CP group was significantly higher than that in the MAF and EF groups. The very LDL (VLDL) + intermediate-density lipoprotein (IDL) + LDL-cholesterol concentration in the CP group was significantly higher than that in the MAF, SF, and EF groups, whereas the high-density lipoprotein (HDL)-cholesterol concentration in the EF group was significantly lower than that in the other groups at the end of the 4-week feeding period. The hepatic LDL receptor mRNA level in the EF group was significantly higher than that in the CP group. The fecal cholesterol excretion in the MAF, SF, and EF groups was significantly higher than that in the CP group. The results of this study demonstrate that MAF and EF lowered the serum total cholesterol level by enhancement of fecal cholesterol excretion, and in particular, by enhancement of hepatic LDL receptor mRNA in EF group.     

Effective Trapping of Fruit Flies with Cultures of Metabolically Modified Acetic Acid Bacteria

Acetoin in vinegar is an attractant to fruit flies when combined with acetic acid. To make vinegar more effective in attracting fruit flies with increased acetoin production, Komagataeibacter europaeus KGMA0119 was modified by specific gene disruption of the acetohydroxyacid isomeroreductase gene (ilvC). A previously constructed mutant lacking the putative ligand-sensing region in the leucine-responsive regulatory protein (KeLrp, encoded by Kelrp) was also used. The ilvC and Kelrp disruptants (KGMA5511 and KGMA7203, respectively) produced greater amounts of acetoin (KGMA5511, 0.11%; KGMA7203, 0.13%) than the wild-type strain KGMA0119 (0.069%). KGMA7203 produced a trace amount of isobutyric acid (0.007%), but the other strains did not. These strains produced approximately equal amounts of acetic acid (0.7%). The efficiency of fruit fly attraction was investigated with cultured Drosophila melanogaster. D. melanogaster flies (approximately 1,500) were released inside a cage (2.5 m by 2.5 m by 1.5 m) and were trapped with a device containing vinegar and a sticky sheet. The flies trapped on the sticky sheet were counted. The cell-free supernatant from KGMA7203 culture captured significantly more flies (19.36 to 36.96% of released flies) than did KGMA0119 (3.25 to 11.40%) and KGMA5511 (6.87 to 21.50%) cultures. Contrastingly, a 0.7% acetic acid solution containing acetoin (0.13%) and isobutyric acid (0.007%), which mimicked the KGMA7203 supernatant, captured significantly fewer flies (0.88 to 4.57%). Furthermore, the KGMA0119 supernatant with additional acetoin (0.13%) and isobutyric acid (0.007%) captured slightly more flies than the original KGMA0119 supernatant but fewer than the KGMA7203 supernatant, suggesting that the synergistic effects of acetic acid, acetoin, isobutyric acid, and unidentified metabolites achieved the efficient fly trapping of the KGMA7203 supernatant.     

Oxidative Modification of Cysteine 111 Promotes Disulfide Bond-Independent Aggregation of SOD1

Converging evidence indicates that SOD1 aggregation is a common feature of mutant SOD1-linked fALS, and seems to be directly related to the gain-of-function toxic property. However, the mechanism inducing the aggregation is not understood. To study the contribution of oxidative modification of cysteine residues in SOD1 aggregation, we systematically examined the redox state of SOD1 cysteine residues in the G37R transgenic mouse model at different stages of the disease and under oxidative stress induced by H₂O₂. Our data suggest that under normal circumstance, cysteine 111 residue in SOD1 is free; however, under oxidative stress, it is prone to oxidative modification by providing the thiolate anion (S−). With the progression of the disease, increased levels of oxidative insults facilitated the oxidation of thiol groups of cysteine residues; human mutant SOD1 could generate an upper shift band in reducing SDS-PAGE, which turned out to be a Cys111-peroxidized SOD1 species. We also detected the formation of SOD1 multimers at different stages of the disease, and found that accumulated oxidative stress facilitated the formation of aggregates, which were not mediated by disulfide bond. This oxidative modification of cysteine 111 therefore promotes the formation of disulfide bond-independent aggregation of SOD1.