Isolation, DNA topoisomerase-II inhibition, and cytotoxicity of three new terpenoids from the bark of Macaranga tanarius

One new pimarane-type diterpenoid (1) and two new taraxerane-based triterpenoids (2 and 3) were isolated from the bark of Macaranga tanarius, along with seven known compounds. Their structures were identified by spectroscopic methods including NMR and MS analyses. Compounds 1-5 were tested for their in vitro inhibition of DNA topoisomerase II, as well as for their cytotoxicities against human lung carcinoma A549 cells (Table 3). The triterpenoids 2-5 showed strong activities in both assays, but the diterpenoid 1 was only moderately active.     

Purification and characterization of a high molecular weight glycoprotein detectable in human milk and breast carcinomas

The murine monoclonal antibody (MAb) designated DF3 has defined a high m.w. antigen detectable in human breast carcinomas and in human milk. DF3 antigen is detectable on apical borders of secretory mammary epithelial cells and in the cytosol of less differentiated malignant cells. DF3 antigen expression has been shown to correlate with the degree of human breast tumor differentiation, and the detection of a cross-reactive species in human milk has suggested that DF3 antigen might be useful as a biochemical marker of differentiated mammary epithelial cells. To further characterize DF3 antigen, we have developed an approach to purify the cross-reactive species by using gel filtration and antibody affinity chromatography. The affinity column-purified DF3 antigen was absorbed by wheat germ agglutinin and peanut agglutinin, but not by concanavalin A or lentil lectin. In contrast, wheat germ agglutinin inhibited MAb DF3 reactivity with the purified antigen, whereas there was little, if any, inhibition when using peanut agglutinin. These findings are thus consistent with the involvement of terminal N-acetyl-D-neuraminic acid and/or N-acetylglucosamine residues in the antigenic site. DF3 antigenicity was also sensitive to neuraminidase, but not chondroitinase ABC, chondroitinase AC, chondroitin-4-sulfatase, or hyaluronidase. Furthermore, DF3 antigen was sensitive to Pronase, subtilisin BPN', and alpha-chymotrypsin. The presence of O-glycosidic linkages between carbohydrate and protein in the DF3 antigenic site was further supported by the presence of NaBH4-sensitive sites. Together, these results suggest that sialyl oligosaccharides present on a peptide backbone are required for maintaining DF3 antigenicity. Similar findings have been demonstrated for DF3 antigen purified from both human milk and breast cancer effusions. However, the DF3 antigen in human milk consisted of a single high m.w. species, whereas the tumor-associated antigen consisted of two distinct glycoproteins with m.w. of 330,000 and 450,000. These findings may be relevant to the recent demonstration that distinct high m.w. DF3 antigens are elevated in the circulation of patients with breast carcinoma.     

Vascular permeability-increasing activity possessed byTreponema phagedenis (Reiter strain)

Treponema phagedenis possessed the ability to induce an increase in vascular permeability (blueing) in guinea pig skin, which was exerted not only by cell-free preparations but also directly by intact cells. Cell-free preparations induced maximum blueing 0 h after sample injection, while whole cells did so after 1 h. Log dose-response curves for cell-free preparations were linear within the range of blue spots with diameters of 10–24 mm, with slopes ( ) of 8.5–8.7. The vascular permeability-increasing activity was not ascribable to hyaluronidase and probably to -galactosidase; this organism did not produce any detectable hyaluronidase activity.     

The role of Gαq/Gα11 signaling in intestinal epithelial cells

Intestinal homeostasis and the coordinated actions of digestion, absorption and excretion are tightly regulated by a number of gastrointestinal hormones. Most of them exert their actions through G-protein-coupled receptors. Recently, we showed that the absence of Gα_q/Gα₁₁ signaling impaired the maturation of Paneth cells, induced their differentiation toward goblet cells, and affected the regeneration of the colonic mucosa in an experimental model of colitis. Although an immunohistochemical study showed that Gα_q/Gα₁₁ were highly expressed in enterocytes, it seemed that enterocytes were not affected in Int-G_q/G₁₁ double knock-out intestine. Thus, we used an intestinal epithelial cell line to examine the role of signaling through Gα_q/Gα₁₁ in enterocytes and manipulated the expression level of Gα_q and/or Gα₁₁. The proliferation was inhibited in IEC-6 cells that overexpressed Gα_q/Gα₁₁ and enhanced in IEC-6 cells in which Gα_q/Gα₁₁ was downregulated. The expression of T-cell factor 1 was increased according to the overexpression of Gα_q/Gα₁₁. The expression of Notch1 intracellular cytoplasmic domain was decreased by the overexpression of Gα_q/Gα₁₁ and increased by the downregulation of Gα_q/Gα₁₁. The relative mRNA expression of Muc2, a goblet cell marker, was elevated in a Gα_q/Gα₁₁ knock-down experiment. Our findings suggest that Gα_q/Gα₁₁-mediated signaling inhibits proliferation and may support a physiological function, such as absorption or secretion, in terminally differentiated enterocytes.     

The Regulatory Functions of the rolB and rolC Genes of Agrobacterium rhizogenes are Conserved in the Homologous Genes (Ngrol) of Nicotiana glauca in Tobacco Genetic Tumors

To compare patterns of expression between the Ngrol genes ofN. glauca and the Rirol genes of Agrobacterium rhizogenes, weperformed fluorometric and histochemical analysis of transgenicgenetic tumors on the hybrid of Nicotiana glauca x N. langsdorffü(Fl) that harbored a rß- glucuronidase (GUS) reportergene fused to the promoter of NgrolB, NgrolC, RirolB or RirolC The promoters of NgrolB and NgrolCNgrolC had 2- to 3-fold loweractivity than those of RirolB and RirolC However, the changesin patterns of GUS activity caused by deletion of NgrolB andNgrolCpromoters were similar to those of RirolB and RirolC promoters.This result suggests that the cis-acting sequences that regulatethe level of expression of RirolB and RirolC are conserved inthe NgrolB and NgrolC promoters. Furthermore, an auxin dependent(NAA-dependent) increase in GUS activity was observed in thecase of NgrolB-GUS and RirolB-GUS. Histochemical analysis showedGUS activity encoded by both NgrolB-GUS and RirolB-GUS in normal-typeFl transgenic plants was located in meristematic zones, whilethat encoded by NgrolC-GUS and RirolC-GUS was detected mainlyin vascular systems of various organs. Thus, the patterns ofexpression of the Ngrol genes were the same as those of theRirol genes in terms of promotion by auxin and tissue-specificity,indicating that regulatory mechanisms for both sets of geneshave been conserved during the evolution of the genus Nicotianaafter transfer from a progenitor of Agrobacterium to that ofNicotiana. (Received May 2, 1995; Accepted June 13, 1995)     

Two new species ofConiochaeta with a cephalothecoid peridium wall

Two new species ofConiochaeta, isolated from Japanese soils, are described and illustrated:C. cephalothecoides, which is characterized by dark brown ascomata clothed with short setae, cylindrical asci and ovoid to almond-shaped or pyriform ascospores with a longitudinal germ slit; andC. dumosa, which is characterized by dark brown ascomata clothed with short setae and dense hyphal hairs, cylindrical asci and ellipsoid-fusoid ascospores with a longitudinal germ slit. These species are distinguished from most species of the genus by the unique cephalothecoid peridium of their ascomata. The associated anamorphs of both species are assignable to the form-genusLecythophora.     

Two new species ofHeleococcum withAcremonium anamorphs

Two new species ofHeleococcum (a cleistothecial nectrioid genus in the Hypocreaceae) are described and illustrated.Heleococcum alatosporum, isolated from Indonesian soil, is recognized by the production of salmon-colored ascomata, cylindrical asci, and hyaline, small, bicellular ascospores with walls that are verruculose and ornamented with longitudinal ridges.Heleococcum inapertum, isolated from Philippine soil, is characterized by yellow ascomata, clavate asci, and pale yellow, middle-sized, bicellular, verruculose to weakly striate ascospores surrounded with a hyaline sheath. Anamorphs of the new species are included inAcremonium. A key to the accepted species of the genus is provided.     

Reproducible Alterations of DNA Methylation at a Specific Population of CpG Islands during Blast Formation of Peripheral Blood Lymphocytes

We investigated the changes in the methylation patterns of CpGislands associated with blast formation of human peripheralblood lymphocytes activated by anti-CD3 and interleukin-2 (IL-2),using restriction landmark genomic scanning with a methylation-sensitiverestriction enzyme (RLGS-M) system. Of about 2,100 Not I spot/lociwhich were analyzed, only 10 showed changes, whereas drasticchanges have been observed in cases of malignant and SV40 transformation.These changes were highly reproducible for samples from boththe same and different individuals. Even the timing of the changesafter cultivation was the same. Thus, we concluded that at leastthe genomic DNA methylation state in vivo was essentially retainedin T blast cells activated in vitro by induction with IL-2 andanti-CD3, which are commonly used in biological experimentsas well as clinical diagnosis and therapy.     

Structural Features of Membrane Fusion between Influenza Virus and Liposome as Revealed by Quick-Freezing Electron Microscopy

The structure of membrane fusion intermediates between the A/PR/8(H1N1) strain of influenza virus and a liposome composed of egg phosphatidylcholine, cholesterol, and glycophorin was studied using quick-freezing electron microscopy. Fusion by viral hemagglutinin protein was induced at pH 5.0 and 23°C. After a 19-s incubation under these conditions, small protrusions with a diameter of 10–20 nm were found on the fractured convex faces of the liposomal membranes, and small pits complementary to the protrusions were found on the concave faces. The protrusions and pits corresponded to fractured parts of outward bendings of the lipid bilayer or “microprotrusions of the lipid bilayer.” At the loci of the protrusions and pits, liposomal membranes had local contacts with viral membranes. In many cases both the protrusions and the pits were aligned in regular polygonal arrangements, which were thought to reflect the array of hemagglutinin spikes on the viral surface. These structures were induced only when the medium was acidic with the virus present. Based on these observations, it was concluded that the microprotrusions of the lipid bilayer are induced by hemagglutinin protein. Furthermore, morphological evidence for the formation of the “initial fusion pore” at the microprotrusion was obtained. The protrusion on the convex face sometimes had a tiny hole with a diameter of <4 nm in the center. The pits transformed into narrow membrane connections <10 nm in width, bridging viruses and liposomes. The structures of the fusion pore and fusion neck with larger sizes were also observed, indicating growth of the protrusions and pits to distinct fusion sites. We propose that the microprotrusion of the lipid bilayer is a fusion intermediate induced by hemagglutinin protein, and suggest that the extraordinarily high curvature of this membrane structure is a clue to the onset of fusion. The possible architecture of the fusion intermediate is discussed with regard to the localization of intramembrane particles at the microprotrusion.