Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3',5'-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT-PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP(6-38), had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.     

Purification and characterization of a novel lectin from a freshwater cyanobacterium, Oscillatoria agardhii

In the survey of 14 species of laboratory-cultured cyanobacteria for hemagglutinins, we newly detected the activity in two species, Oscillatoria agardhii, strain NIES-204, and Phormidium foveolarum, strain NIES-503. From the extract of O. agardhii, which showed the highest activity with trypsin-treated erythrocytes of rabbit, a lectin was purified to homogeneity by the combination of precipitation with (NH4)2SO4, gel filtration, hydrophobic chromatography and reverse phase chromatography. The purified lectin, designated OAA, was a monomeric protein with an apparent molecular weight of 13,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 16,000 on gel filtration. The amino acid composition was rich in glycine and acidic amino acids. The hemagglutination activity was inhibited by glycoproteins such as yeast mannan, but not by any of the monosaccharides tested. The activity was stable over a wide range of pH (4-11) and at a high temperature of 80 degrees C, and independent on the presence of divalent cations. The features of OAA resembled those of many of lectins from marine macroalgae. The sequence of amino-terminal residues of OAA was determined as ALYNVENQWGGSSAPWNEGG, which was highly homologous to those of lectins from macroalgae of the genus Eucheuma and that of a myxobacterium Myxococcus xanthus hemagglutinin.     

Mutant luciferase enzymes from fireflies with increased resistance to benzalkonium chloride

Benzalkonium chloride (BAC), used to extract intracellular ATP, interferes with subsequent firefly luciferase-luciferin assays. There was a significant difference among wild-type luciferases with respect to BAC resistance. Luciola lateralis luciferase (LlL) was the most tolerant, followed by Luciola cruciata luciferase (LcL) and Photinus pyralis luciferase. Random mutagenesis of thermostable mutants of LcL showed that the Glu490Lys mutation contributes to improved resistance to BAC. The corresponding Glu490Lys mutation was introduced into thermostable mutants of LlL by site-directed mutagenesis. Kinetic analysis demonstrated that the resultant LlL-217L490K mutant, having both an Ala217Leu and a Glu490Lys mutation, showed the highest resistance to BAC, with an initial remaining bioluminescence intensity of 87.4% and a decay rate per minute of 29.6% in the presence of 0.1% BAC. The Glu490Lys mutation was responsible for increased resistance to inactivation but not inhibition by BAC. The LlL-217L490K had identical thermostability and pH stability to the parental thermostable mutant. From these results, it was concluded that the LlL-217L490K enzyme is advantageous for hygiene monitoring and biomass assays based on the ATP-bioluminescence methodology. This is the first report demonstrating improved resistance to BAC of the firefly luciferase enzyme.     

Decoupling Analysis of Four Selected Countries

We examine decoupling conditions of domestic extraction of materials, energy use, and sulfur dioxide (SO₂) emissions from gross domestic product (GDP) for two BRIC (Brazil, Russia, India and China) countries (i.e., China and Russia) and two Organisation for Economic Co‐operation and Development (OECD) countries (Japan and the United States) during 2000–2007, using a pair of decoupling indicators for resource use (D_r) and waste emissions (D_e) and the decoupling chart, which can distinguish between absolute decoupling, relative decoupling, and non‐decoupling. We find that (1) during 2000–2007, decoupling between environmental indicators and GDP was higher in the two OECD countries as compared with the two BRIC countries. The key reason is that these countries were in different development stages with different economic growth rates. (2) Changes in environmental policies can significantly influence the degree of decoupling in a country. (3) China, Japan, and the United States were more successful in decoupling SO₂ emissions from GDP than in decoupling material and energy use from GDP. The main reason is that, unlike resource use, waste emissions (e.g., SO₂ emissions) can be reduced by effective end‐of‐pipe treatment. (4) The decoupling indicator is different from the changing rate of resource use and waste emissions. If two countries have different GDP growth rates, even though they may have similar values using the decoupling indicator, they may show different rates of change for resource use and waste emissions.     

Hydrocarbon composition of newly isolated strains of the green microalga Botryococcus braunii

Four new strains of Botryococcus braunii were isolated from Japanese waters and cultured under defined conditions. Their hydrocarbon content and composition were analyzed and compared with those of the Darwin and Berkeley strains. The Yamanaka strain produced only alkadienes characteristic of the A race, whereas the others, the Yayoi, Kawaguchi-1 and -2 strains as well as the Darwin and Berkeley strains, produced botryococcenes peculiar to the B race. The hydrocarbon content of the Yamanaka strain was 16.1 % dry weight and that of the B race strains ranged from 9.7 to 37.9%. Botryococcene composition of the Japanese strains differed from each other as well as from the Darwin and Berkeley strains. More than 50% of the hydrocarbons in the Yayoi, Darwin, and Berkeley strains were composed of C₃₄H₅₈, but the main components were different from one another as isomers. The Kawaguchi-1 and -2 strains did not have a high level of C₃₄ botryococcenes, C₃₂ ones being the main components. In these strains significant amounts of squalene-related compounds were detected.     

Quantitation of adenovirus DNA and virus particles with the PicoGreen fluorescent Dye.

A microplate assay for the rapid quantitation of adenovirus DNA has been developed using the fluorescent dye PicoGreen, which selectively binds double-stranded DNA. The method was first applied to extracted adenoviral DNA and then extended to samples of intact, purified adenovirus after lysis of the viral capsid with the ionic detergent SDS. Utilizing the stoichiometric relationship between adenovirus DNA and intact particles, a physical particle count of intact virus is then derived for the sample. This PicoGreen-based assay has excellent reproducibility, linearity, and sensitivity. In its present form, this assay has a limit of quantitation of 10.3 ng/ml viral DNA, predicted to correspond to 2.6 x 10(8) virus particles/ml. This procedure was compared to a widely utilized spectroscopic method, in which samples are lysed with SDS and absorbance is read at 260 nm, and found to be 10- to 20-fold more sensitive. The dye binding assay also uses considerably less sample volume (<20%) than that needed for the spectroscopic method. Particle count values generated by the PicoGreen procedure are consistently lower (typically 1.5- to 2-fold) than this spectroscopic method. The applications and limitations of this method in the analysis of adenovirus samples are discussed.     

Citrus auraptene targets translation of MMP-7 (matrilysin) via ERK1/2-dependent and mTOR-independent mechanism

Matrix metalloproteinase (MMP)-7 is considered to play essential roles in cancer progression. We examined the efficacy of auraptene, a citrus coumarin derivative, for suppressing MMP-7 expression in the human colorectal adenocarcinoma cell line HT-29. Auraptene remarkably inhibited the production of proMMP-7 protein, without affecting its mRNA expression level. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), showed similar results, suggesting that auraptene suppresses mTOR-dependent proMMP-7 translation. Interestingly, however, auraptene showed no effects on the activation of Akt/mTOR signaling, whereas the phosphorylation levels of 4E binding protein (4EBP)1 and eukaryotic translation initiation factor (eIF)4B were substantially decreased. In addition, auraptene remarkably dephosphorylated constitutively activated extracellular signal-regulated kinase (ERK)1/2. Transfection of ERK1/2 siRNA led to a significant reduction of proMMP-7 protein production as well as of the phosphorylation of eIF4B. These results demonstrate that auraptene targets the translation step for proMMP-7 protein synthesis by disrupting ERK1/2-mediated phosphorylation of 4EBP1 and eIF4B.     

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human -interferon (-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.Abbreviation HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

15-Deoxy-Δ¹²,¹⁴ prostaglandin J₂ reduces the formation of atherosclerotic lesions in apolipoprotein E knockout mice

Aim

15-Deoxy-Δ^12,14 Prostaglandin J₂ (15d-PGJ₂) is a ligand of peroxisome proliferator-activated receptor γ (PPARγ) having diverse effects such as the differentiation of adipocytes and atherosclerotic lesion formation. 15d-PGJ₂ can also regulate the expression of inflammatory mediators on immune cells independent of PPARγ. We investigated the antiatherogenic effect of 15d-PGJ₂.

Methods

We fed apolipoprotein (apo) E-deficient female mice a Western-type diet from 8 to 16 wk of age and administered 1 mg/kg/day 15d-PGJ₂ intraperitoneally. We measured atherosclerotic lesions at the aortic root, and examined the expression of macrophage and inflammatory atherosclerotic molecules by immunohistochemical and real-time PCR in the lesion.

Results

Atherosclerotic lesion formation was reduced in apo E-null mice treated with 15d-PGJ₂, as compared to in the controls. Immunohistochemical and real-time PCR analyses showed that the expression of MCP-1, TNF-α, and MMP-9 in atherosclerotic lesions was significantly decreased in 15d-PGJ₂ treated mice. The 15d-PGJ₂ also reduced the expression of macrophages and RelA mRNA in atherosclerotic lesions.

Conclusion

This is the first report 15d-PGJ₂, a natural PPARγ agonist, can improve atherosclerotic lesions in vivo. 15d-PGJ₂ may be a beneficial therapeutic agent for atherosclerosis.