Studies onC-glycosides in higher plants

Sixteen species including 1 variety and 1 form from 9 genera belonging to Saxifragaceae were examined for bergenin content. Bergenin was found only in Saxifragoideae, no bergenin being present in Hydrangeoideae and Ribesioideae. The amount of bergenin was shown to range from 0.1 to 1 mg per g fresh weight, depending on the organs tested, and the highest amounts were usually observed in rhizomes. The contents of bergenin in a leaf ofSaxifraga stolonifera increased continuously with the leaf expansion, while the level on a fresh weight basis remained constant during the leaf growth.     

TGF-β1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition

Epithelial–mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis in many types of malignancies, including colorectal cancer. Although the importance of EMT is also considered in colorectal neuroendocrine carcinoma (NEC), its regulatory mechanisms have not been elucidated. We recently established a human colorectal NEC cell line, SS-2. In this study, we aimed to clarify whether these cells were sensitive to transforming growth factor beta 1 (TGF-β1) and whether EMT could be induced through TGF-β1/Smad signaling, with the corresponding NEC cell-specific changes in invasiveness. In SS-2 cells, activation of TGF-β1 signaling, as indicated by phosphorylation of Smad2/3, was dose-dependent, demonstrating that SS-2 cells were responsive to TGF-β1. Analysis of EMT markers showed that mRNA levels changed with TGF-β1 treatment and that E-cadherin, an EMT marker, was expressed in cell-cell junctions even after TGF-β1 treatment. Invasion assays showed that TGF-β1-treated SS-2 cells invaded more rapidly than non-treated cells, and these cells demonstrated increased metalloproteinase activity and cell adhesion. Among integrins involved in cell-to-matrix adhesion, α2-integrin was exclusively upregulated in TGF-β1-treated SS-2 cells, but not in other colon cancer cell lines, and adhesion and invasion were inhibited by an anti-α2-integrin blocking antibody. Our findings suggest that α2-integrin may represent a novel therapeutic target for the metastasis of colorectal NEC cells.     

Pine wilt disease as promising causal agent of the mass mortality of Pinus armandii Franch. var. amamiana (Koidz.) Hatusima in the field

The causal agent of the mass mortality of field populations of Pinus armandii Franch. var. amamiana (Koidz.) Hatusima (PAA) was investigated with special respect to the involvement of pine wilt disease. Wood chips, branches and/or increment cores for detecting the pinewood nematode, feeding marks of the vector insect and environmental stress in the past, respectively, were taken from live and dead PAA trees grown in three locations, Yaku-shima and Tanega-shima Islands and a plantation in Kagoshima City, from 1997 to 1998. Five trees died after the spring of 1996 and, of these, four were inhabited by the pinewood nematode. Feeding marks of the vector insects were found on the branches of all dead trees and most of the live trees investigated. These results suggest that the infection of pine wilt disease in PAA trees occurs in the field. Annual ring growth of the sample trees showed neither intervention nor growth reduction, which implies strong environmental stress that may cause mortality in PAA trees.     

Influence of the introduced parasitoid Torymus sinensis (Hymenoptera: Torymidae) on T. koreanus and T. beneficus as indigenous parasitoids of the chestnut gall wasp Dryocosmuskuriphilus (Hymenoptera: Cynipidae) on chestnut trees in Nagano Prefecture, Japan

We describe here the parasitoid wasps Torymus sinensis Kamijo and T. beneficus Yasumatsu & Kamijo (early-spring and late-spring strains), which are introduced and indigenous natural enemies of the chestnut gall wasp Dryocosmus kuriphilus Yasumatsu, an invasive chestnut pest in Japan. We recently discovered specimens of T. koreanus Kamijo, endemic in Korea, among Torymus parasitoids collected from D. kuriphilus galls in a Japanese chestnut orchard. In this study we compare the composition of Torymus parasitoids emerging from D. kuriphilus galls before and after the release of T. sinensis. Before the release of T. sinensis, early-spring and late-spring strains of T. beneficus predominated (58.3 and 20.8% of specimens collected). However, a few years after the release, both T. beneficus strains had been almost completely displaced by T. sinensis. In contrast to the rapid decrease in T. beneficus, T. koreanus did not decrease drastically before and even after the release of T. sinensis (approximately 10–20% of specimens collected). These results suggest that not a few T. koreanus were present in the Japanese chestnut orchard investigated at least several years after the release of T. sinensis, although both the T. beneficus strains were rapidly displaced by T. sinensis during this period.     

Properties of the Biotin Transport System in Bifidobacterium breve N4

Binding of biotin to resting cells of Bifidobacterium breve N4, which grew in a biotin-deficient medium, was independent of pH from 1 to 9 and of temperature below 50 C. It was not inhibited by metabolic inhibitors including sulfhydryl reagents, but it was inhibited by treatment with 80% ethanol or 5% trichloroacetic acid. It was also competitively inhibited by biotin-sulfone, but not by tetrahydrothiophene nor dethiobiotin. The binding constant was calculated to be 3.3 × 10⁸m^?1. The amount of biotin unextractable with hot water, representing part of the transported biotin, increased gradually for 20 min, this increase was inhibited by NaF, hydroxylamine and low temperature. ¹⁴C-biotin on the cells was displaced by cold biotin and biotin-sulfone; the displacement was not inhibited by metabolic inhibitors, but it was dependent on temperature. A few minutes after binding, the biotin was released to the medium. The release was dependent on pH and temperature, was affected by energy sources and was inhibited by metabolic inhibitors, e.g. NaF, p-chloromercuribenzoic acid and hydroxylamine. It could be stopped at any time by cooling to 0 C or by adding NaF, and the amount of accumulated biotin did not increase under those conditions. These results suggest that the binding sites on the cell surface decreased in number or in their binding affinity for biotin through an energy-dependent process.     

Alicyclic acid metabolism in plants

When the epicotyls of etiolated pea seedlings were fed with 40 mM potassium quinate solution in the dark for 24 hr, a marked accumulation of shikimic acid occurred in the tissue. This effect was much more pronounced in epicotyls preliminarily starved in a phosphate solution for 24 hr. On the other hand, supplying shikimate to the epicotyls brought about no significant accumulation of quinic acid. Tracer studies with¹⁴C-shikimate have shown that, in the epicotyls, shikimic acid was rapidly metabolized and fairly high radioactivities were observed in the amino acid fraction. However, feeding of¹⁴C-shikimate together with unlabeled alicyclic acids resulted in a reduction of shikimate utilization. When³H-quinate was fed to the epicotyls, radioactivities were retained mostly in an acidic fraction, indicating the sluggish conversion of quinic acid. In starved epicotyls, however, nearly half of the absorbed radioactivity was consumed. In tracer experiments the conversion of quinate to shikimate was clearly observed, whereas the reverse reaction was not. From these findings the metabolic role of quinic acid in quinate-less pea seedlings is discussed.     

Kinetic studies of three different molecular forms of urokinase for the activation of native human plasminogen

The kinetic parameters of three different molecular forms of urokinase (UK) for the activation of native Glu-plasminogen were compared. The apparent Michaelis constant (Km. app.) of each UK was almost of the same order of magnitude (31-38 microM), but the catalytic constants (kc) were observed to be different: UKh (high molecular weight form, molecular weight 53,000), 2.4 +/- 0.2 s-1; UK+ (low molecular weight form, molecular weight 33,000), 0.83 +/- o.10 s-1, and UKl (trypsin-digested form, molecular weight 36,000), 0.91 +/- 0.18 s-1. The overall second order rate constant, kc/Km calculated for UKh was 7.7 X 10(4) M-1 s-1, higher than for UKl (2.2 X 10(4) M-1 s-1) or UKt (2.4 X 10(4) M-1 s-1), indicating the possibility of a much higher degree of enzymatic specificity and efficiency.     

Biodistribution of 125I-labeled polymeric vaccine carriers after subcutaneous injection

Polymeric nanoparticles (NPs) comprised of hydrophilic poly(γ-glutamic acid) in the main chain and hydrophobic phenylalanine in the side chain (γ-PGA-Phe) are a promising vaccine carrier for various kinds of diseases. However, little is known about the fate of subcutaneously administered γ-PGA-Phe NPs. Therefore, we newly synthesized γ-PGA graft phenylalanine and tyrosine conjugates (γ-PGA-Phe-Tyr), and then γ-PGA-Phe-Tyr NPs were labeled with ¹²⁵I for monitoring their biodistribution (γ-PGA-Phe-Tyr(¹²⁵I) NPs). Dynamic light scattering (DLS) measurements showed that γ-PGA-Phe-Tyr(¹²⁵I) NPs showed 200 nm in diameter and a negative ζ-potential, which was comparable to those of their precursors. γ-scintigraphic images showed that in mice, subcutaneously injected γ-PGA-Phe-Tyr(¹²⁵I) NPs were mainly observed at the site of injection (SOI), but not other organs 1 h after administration. However, γ-PGA-PheTyr(¹²⁵I) NPs were almost undetectable at the SOI and other organs at 11 days postinjection. Similar results were observed when γ-PGA-Phe-Tyr(¹²⁵I) NPs were subcutaneously injected into rats. Furthermore, at 11 days postinjection, 73 ± 3% of the injected dose of γ-PGA-Phe-Tyr(¹²⁵I) NPs was detected in the feces (14 ± 1%) and urine (59 ± 1%). These results clearly showed that subcutaneously injected γ-PGA-Phe-Tyr(¹²⁵I) NPs were cleared from the body, and γ-PGA-Phe NPs were safe and effective vaccine carriers.