Responses of a model legume Lotus japonicus to lipochitin oligosaccharide nodulation factors purified from Mesorhizobium loti JRL501.

Lotus japonicus has been proposed as a model legume for molecular genetic studies of symbiotic plant-microbe interactions leading to the fixation of atmospheric nitrogen. Lipochitin oligosaccharides (LCOs), or Nod factors, were isolated from the culture of Mesorhizobium loti strain JRL501 (MAFF303099), an efficient microsymbiont of L. japonicus B-129 cv. Gifu. High-performance liquid chromatography and mass spectrometric analyses allowed us to identify at least five different structures of LCOs that were produced by JRL501. The major component was NodMl-V(C18:1, Me, Cb, AcFuc), an N-acetyl-glucosamine pentamer in which the nonreducing residue is N-acylated with a C18:1 acyl moiety, N-methylated, and carries a carbamoyl group and the reducing N-acetylglucosamine residue is substituted with 4-O-acetyl-fucose. Additional novel LCO structures bearing fucose instead of acetyl-fucose at the reducing end were identified. Mixtures of these LCOs could elicit abundant root hair deformation on L. japonicus roots at a concentration of 10(-7) to 10(-9) M. Spot inoculation of a few nanograms of LCOs on L. japonicus roots induced the formation of nodule primordia in which the early nodulin genes, ENOD40 and ENOD2, were expressed in a tissue-specific manner. We also observed the formation of a cytoplasmic bridge (preinfection thread) in the swollen outermost cortical cells. This is the first description of cytoplasmic bridge formation by purified LCOs alone in a legume-forming determinate nodules.

Progesterone enhances interleukin-15 production in human endometrial stromal cells in vitro.

Interleukin-15 (IL-15) is a novel cytokine that stimulates lymphocyte proliferation and migration via a trimeric receptor sharing the ss and gamma signal-transducing chains with the IL-2 receptor. It is suggested that IL-15 is involved in regulating the proliferation and differentiation of uterine natural killer cells. In the human endometrium, we have recently reported that IL-15 messenger ribonucleic acid (mRNA) levels significantly increased during the secretory phase compared with those during the proliferative phase. In this study we investigated whether the female sex steroids progesterone (P) and estradiol (E(2)) regulate IL-15 messenger RNA (mRNA) and the secretion in human endometrial stromal cells (ESC) in vitro. Northern blot analyses revealed a significant increase in IL-15 mRNA levels in ESC treated with P alone or E(2) plus P compared with vehicle. Furthermore, P is a potent inducer of IL-15 mRNA expression in ESC in a dose-dependent manner. On the other hand, E(2) alone did not increase IL-15 mRNA expression. By enzyme-linked immunosorbent assay, IL-15 protein secretion was stimulated by P and further enhanced by combined treatment with E(2) and P, whereas E(2) alone was ineffective. It is suggested that IL-15 is deeply involved in the hormonal control of the human endometrium by P and E(2).

A saponin from callus tissue of Stauntonia hexaphylla.

A new 30-nortriterpenoid saponin was isolated from the callus tissues of Stauntonia hexaphylla. The structure of the saponin (tentatively named mubenoside A) was elucidated as 3 beta,20 alpha-dihydroxy-30-nor-olean-12-en-28-oic acid 3-O-[beta-D-xylopyranosyl(1----2)-alpha-L-arabinopyranosyl(1----3) ]-beta-D- glucopyranoside by means of spectral experiments.

Retention behavior of positional isomers of disubstituted cyclomalto-oligosaccharide (cyclodextrin) derivatives on an ODS column.

The correlation between hydrophobic effects and structures of three and four positional isomers of 6(1),6n-di-O-triphenylmethyl (trityl)- or 6(1),6n-di-O-tert.- butyldimethylsilyl (tert.-BuMe2Si)-cyclomaltohexaoses (cG6s, alpha-cyclodextrin) (n = 2-4), -cyclomaltoheptaoses (cG7s, beta-cyclodextrin) (n = 2-4), and -cyclomaltooctaoses (cG8s, gamma-cyclodextrin) (n = 2-5) on an ODS column are discussed. Cyclodextrins with two hydrophobic-substituted groups bonded to hydroxyl groups tended to show low retention of positional isomers in which the binding positions of the two substituted groups on the cyclodextrin ring were far apart from each other.

A new indolopyridoquinazoline-type alkaloid from phellodendron amurense callus tissues

Callus tissue from the stems of Phellodendron amurense (Rutaceae) produced a new indolopyridoquinazoline-type alkaloid, 7, 8-dihydroxyrutaecarpine (2), together with known 7-hydroxyrutaecarpine (1). Their structures were established using spectroscopic methods.

Progesterone induces the fibulin-1 expression in human endometrial stromal cells.

BACKGROUND: By using microarray analysis with human endometrial stromal cells (ESCs), we previously reported that the mRNA for fibulin-1, an extracellular matrix as well as a plasma glycoprotein, is up-regulated by progesterone. In the present study, we tried to clarify the spatial and temporal regulation mechanism of fibulin-1 in the human endometrium. METHODS AND RESULTS: Quantitative analysis with real-time PCR experiments on human endometrial tissues showed significantly higher fibulin-1 mRNA expressions in secretory phase endometria than in proliferative phase. Immunohistochemical studies revealed that the fibulin-1 protein is expressed in the glandular epithelium in proliferative phase endometria, and that expression switched to the stroma in secretory phase endometria. In culture experiments with ESCs, a significant increase of fibulin-1 mRNA expression was observed in cells treated with 6 alpha-methyl-17 alpha-hydroxy-progesterone acetate (MPA) or 8 bromoadenosine 3':5'-cyclic monophosphate (8-Br-cAMP). MPA stimulated the fibulin-1 mRNA expression in a dose-dependent manner, and a progesterone antagonist, RU-486, inhibited the stimulatory effect almost completely. By contrast, beta-estradiol alone did not increase the fibulin-1 mRNA expression. CONCLUSIONS: These results suggest that fiblin-1 is an important molecule that mediates progesterone action in human ESC differentiation towards implantation.

Quantitative analysis using the star volume method applied to skeleton patterns extracted with a morphological filter.

In this study, a morphological filter was combined with star volume analysis and applied to digital images to determine its potential usefulness in assessing trabecular structure. Three digital "geometric" test patterns (square, rectangle, circle) were created on a CRT (cathode ray tube). Each shape was arranged into five groups by size to yield 15 final "skeletal" patterns that were subsequently assessed with star volume analysis. Also, three digital X-ray images (background, soft tissue, bone block) were processed with a morphological filter to create three sets of 11 skeletal patterns each. These patterns were also assessed with star volume analysis and the ratio of extracted skeletal elements (in pixel numbers) to total pixel numbers was expressed as the pixel percentage. Star volume analysis was then applied to these digital skeletal images to yield the volume of extracted "skeletal" trabecular elements (Vsk) and the volume of nonskeletal (marrow space) elements (Vsp). The Vsk and Vsp were compared for all the different skeletal patterns. The pixel percentages were then compared to the star volume results for the X-ray test patterns. The Vsk decreased and Vsp increased as the number of operations (n) increased for both digital X-ray images and the geometric test patterns when the X-ray images were depicted by pixel percentages. Also, all true bone test patterns were clearly different both visually and quantitatively when compared to the noise skeletons extracted from background and soft tissue. Therefore, as Vsk was increased, so was connectivity. It can be concluded that the application of morphological filters and star volume analysis may be a useful tool in quantitatively determining the characteristics and continuity of trabecular skeletal structures. Further studies involving a larger number of bone samples and using models to compare measurements of calculated versus actual volume should reveal the true potential of this method for evaluating bone structure and its relationship to bone strength and also increase the tools available for evaluating bone diseases such as osteoporosis.

Immunohistochemical demonstration of aberrant glycosylation and epidermal growth factor receptor in tubal metaplasia of the uterine cervix.

Tubal metaplasia (TM) of the uterine cervix may be confused with endocervical glandular dysplasia or adenocarcinoma of the uterine cervix in histologic specimens. TM is characterized histologically by architecturally normal endocervical glands containing ciliated or clear cells, nonciliated cells, and intercalated or peg cells, resembling normal tubal mucosa. Although several characteristics of TM are still undetermined, TM is considered a benign lesion. In this study, the expressions of epithelial-specific antigen (ESA), epidermal growth factor receptor (EGFR), and fucosyl SSEA1 (Le(y)) antigens were examined by an immunohistochemical method in 14 cases of TM in the uterine cervix (5 with normal endocervix, 5 with endocervical glandular dysplasia, 3 with adenocarcinoma in situ, and 1 with microinvasive adenocarcinoma). In the normal endocervical cells, expression of the EGFR antigen was rarely observed in the cytoplasm, and ESA staining was generally restricted to the basolateral membrane. Le(y) expression was occasionally observed in the cytoplasm of subcolumnar reserve cells. On the other hand, expression of EGFR, ESA, and Le(y) was observed in the cytoplasm of TM as well as in adenocarcinomas of the uterine cervix. These results suggest that TM in the uterine cervix might possess a neoplastic character.

Synthesis of novel heterobranched beta-cyclodextrins from 4(2)-O-beta-D-galactosyl-maltose and beta-cyclodextrin by the reverse action of pullulanase, and isolation and characterization of the products.

From the mixture of 4(2)-O-beta-D-galactosyl-maltose (Gal-G2) and beta-cyclodextrin (betaCD), novel heterobranched betaCDs, (Gal-G2)-betaCD and (Gal-G2)2-betaCDs, were synthesized by the reverse action of debranching enzyme. The optimum conditions for the production of (Gal-G2)2-betaCDs were examined. A mixture of (Gal-G2)2-betaCDs was produced in about 4% yield when Aerobacter aerogenes pullulanase (64 units per 1 g of Gal-G2) was incubated with 1.6 M Gal-G2 and 0.16 M betaCD at 50 degrees C for 4 days. The reaction products, (Gal-G2)2-betaCDs, were separated into three peaks by HPLC analysis on a Hypercarb S column. Their structures were analyzed by fast atom bombardment mass spectroscopy and NMR spectroscopies, and confirmed by comparison of their hydrolyzates by beta-galactosidase with the authentic (G2)2 -betaCDs. The structures of (Gal-G2)-betaCD and three components of (Gal-G2)2-betaCDs were identified as 6-O-(GalG2)-betaCD, 6(1),6(2)-, 6(1),6(3)- and 6(1),6(4)-di-O-(Gal-G2)2-betaCD, respectively.

Synthesis of novel heterobranched beta-cyclodextrins from alpha-D-mannosylmaltotriose and beta-cyclodextrin by the reverse action of pullulanase, and isolation and characterization of the products.

Alpha-D-mannosyl-maltotriose (Man-G3) were synthesized from methyl alpha-mannoside and maltotriose by the transfer action of alpha-mannosidase. (Man-G3)-betaCD and (Man-G3)2-betaCD were produced in about 20% and 4% yield, respectively when Aerobacter aerogenes pullulanase (160 units per 1 g of Man-G3) was incubated with the mixture of 1.6 M Man-G3 and 0.16 M betaCD at 50 degrees C for 4 days. The reaction products, (Man-G3)-betaCD were separated to three peaks by HPLC analysis on a YMC-PACK A-323-3 column and (Man-G3)2-betaCD were separated to several peaks by HPLC analysis on a Daisopak ODS column. The major product of (Man-G3)-betaCDs was identified as 6-O-alpha-(6(3)-O-alpha-D-mannosylmaltotriosyl)-betaCD by FAB-MS and NMR spectroscopies. The structures of (Man-G3)2-betaCDs were analyzed by TOF-MS and NMR spectroscopies, and confirmed by comparison of elution profiles of their hydrolyzates by alpha-mannosidase and glucoamylase on a graphitized carbon column with those of the authentic di-glucosyl-betaCDs. The structures of three main components of (Man-G3)2-betaCDs were identified as 6(1),6(2)-, 6(1),6(3)- and 6(1),64-di-O-(63-O-alpha-D-mannosyl-maltotriosyl)-betaCD.