Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O--D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

Serotonin Stimulates Both Cytosolic and Membrane-Bound Guanylate Cyclase in NG108–15 Cells

The cyclic GMP (cGMP) content was rapidly (greater than 30 s) increased by serotonin [5-hydroxytryptamine (5-HT)] (EC50 = 10 microM), and the increase lasted for greater than 10 min in NG108-15 cells. The 5-HT-induced elevation of cGMP level (EC50 = 10 microM) at 20 s ("fast" elevation) was inhibited by ICS 205-930 or MDL 72,222 and by Ca2+ deficiency in the reaction medium but not by organic Ca2+ antagonists. The 5-HT effect at 10 min ("slow" elevation) was not inhibited by several antagonists for 5-HT receptors of the 1A, 1B, 1C, 1D, 2, and 3 subtypes and was independent from external Ca2+ concentration. The fast and slow effects of 5-HT were similar to the effects of bradykinin and atrial natriuretic peptide (ANP), respectively, in aspects of both Ca2+ dependency and time course of the effects. Bradykinin transiently stimulated formation of inositol phosphates as well as accumulation of cGMP, a finding suggesting that intracellular Ca2+ is involved in bradykinin-induced cGMP accumulation as shown in the fast response to 5-HT. ANP, an activator of membrane-associated guanylate cyclase (mGC), slowly (approximately 60 s) increased the cGMP content (EC50 = 10 nM), a result lasting for greater than 10 min, and the effects were independent from external Ca2+, as shown in the slow response to 5-HT. 5-HT and ANP did not induce formation of inositol phosphates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructure and Chemical Analysis of the Cell Wall of Pythium debaryanum1

The ultrastructure and component polysaccharides of the cell wall of Pythium debaryanum IFO-5919 were investigated. From results obtained by means of acid, alkali, Schweitzer reagent and β-1, 3-glucanase treatments and electron microscopy, it was concluded that 1) the acid-extracted fraction was a 1,3-linked branched glucan, 2) the alkali-extracted fraction was a mixture of 1,3-, 1,6-, and 1,3,6-linked highly branched two glucans, 3) the Schweitzer reagent-extracted fraction was a β-1, 4-linked glucan, 4) the cell wall was constructed from two types of cullulosic microfibrils, as a frame and as a finer network, and amorphous β-1, 3-glucan including β-1, 6-linkage, 5) cellulosic microfibrils were covered by matrix material consisting of a mixture of amorphous β-1, 3-linked and β-1, 6-linked branching glucans.     

Circadian Yin-Yang Regulation and Its Manipulation to Globally Reprogram Gene Expression

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Assessment of methylation events during colorectal tumor progression by absolute quantitative analysis of methylated alleles

To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm(2) of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R >or= 0.966, P<0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P<0.0001 by Kolmogorov-Smirnov test). Single-PCR AQAMA provided accurate methylation level measurement. Variable MINT locus methylation level changes occur during malignant progression of colorectal adenoma. Combining AQAMA with on-slide SBM provides a sensitive assay that allows detailed histology-oriented analysis of DNA methylation levels and may give new, accurate insights into understanding development of epigenetic aberrancies in colorectal cancer progression.     

Regulation of excitation energy in Nannochloropsis photosystem II

Photosynthesis Research - Recently, we isolated a complex consisting of photosystem II (PSII) and light-harvesting complexes (LHCs) from Nannochloropsis granulata (Umetani et al. Photosynth Res...     

Cardiotrophin-1 stimulates endothelin-1 via gp130 in vascular endothelial cells

Endothelin-1 (ET-1) is a vasoconstricting and mitogenic peptide released from vascular endothelial cells under normal and pathophysiological conditions, and synthesis and secretion of ET-1 are stimulated by cytokines. Cardiotrophin-1 (CT-1) is a new member of the interleukin-6-type cytokines that induce biological actions through the glycoprotein (gp) 130. The present study was designed to determine the presence of CT-1 and the gp130 cytokine system in vascular endothelial cells and to investigate whether CT-1 stimulates synthesis and secretion of ET-1 in the vascular endothelial cells. We first sought to determine gene expression and immunoreactivity of CT-1, gp130 and ET-1 in cultured canine aortic endothelial cells (CAECs) using Northern blot analysis and immunocytochemistry, which revealed the presence of CT-1 and gp130 together with ET-1 in CAECs. CT-1 increased ET-1 gene expression in CAECs, and stimulated ET-1 secretion from CAECs in a dose-dependent manner. Furthermore, inhibition of gp130 by monoclonal antibody attenuated ET-1 secretion from CAECs, suggesting that actions of CT-1 on the secretion of ET-1 are mediated through gp130 receptor system. The present study, therefore, reports the presence of CT-1 and gp130 in vascular endothelial cells and mechanisms of secretion of ET-1 related to this cytokine system.     

Ventricular cardiotrophin-1 activation precedes BNP in experimental heart failure

Both cardiotrophin-1 (CT-1) and B-type or brain natriuretic peptide (BNP) are activated by cardiomyocyte stretch, and gene expression of CT-1 and BNP are augmented in the heart in experimental and human congestive heart failure (CHF). The goal of this study was to define cardiac gene expression of CT-1 and BNP by Northern blot analysis in normal (n=5), early left ventricular dysfunction (ELVD, n=5) and overt CHF dogs (n=5), in which ventricular function is progressively decreased. CT-1 mRNA was detected in both atria and ventricles in normal dogs. Ventricular CT-1 mRNA production increased in ELVD, and it further increased in overt CHF. Ventricular BNP mRNA remained below or at the limit of detection in normal and ELVD models, and it markedly increased in overt CHF. This study reports differential regulation of gene expression of CT-1 and BNP in the heart during the progression of CHF, and demonstrates that ventricular CT-1 gene activation precedes ventricular BNP gene activation.     

Decrease of cholesterol in mouse melanoma causes secretion of lysosomal enzymes

We examined the change in the subcellular distribution of a lysosomal enzyme, beta-glucuronidase (beta-G), caused by decreased cholesterol levels in mouse melanoma cells using an HMG-CoA reductase inhibitor, lovastatin and lipoprotein-deficient serum (LDS). There was a decrease in the cholesterol content of the cells and increased secretion of the mature form of beta-G located in lysosomes, as documented by Percoll density gradient fractionation, digitonin permeabilization and immunoprecipitation. Furthermore, another lysosomal enzyme, cathepsin H, was found to be released in the medium from cells treated with lovastatin. Both the precursor and mature forms of cathepsin H were detected in the medium of treated cells. Next, when cells were treated with LDS without lovastatin, concomitantly with the decrease in the levels of cholesterol and beta-G activity in the cells, beta-G activity in the medium increased. Also, the ratio of beta-G (3.2-fold) released in the medium from cells treated with Dulbecco's modified Eagle medium (D-MEM) containing lovastatin and LDS was higher than that (2.3-fold) on treatment with D-MEM containing LDS without lovastatin. From these results, it was suggested that the exocytosis of mature enzymes from lysosomes into the medium or mis-sorting of the lysosomal precursor forms to the medium was caused by the lovastatin- and/or LDS-induced decrease in the cholesterol content of the cells, although the mechanism of secretion by lysosomal enzymes differed somewhat.