Intraspecific groups of Umbelopsis ramanniana inferred from nucleotide sequences of nuclear rDNA internal transcribed spacer regions and sporangiospore morphology

Umbelopsis ramanniana is a well-known species in this genus. A characteristic morphological feature of this fungus is the remarkable variation in the sporangiospore shape, which implies the genetic variations occur in the nucleotide sequences of the internal transcribed spacer (ITS) regions of the nuclear ribosomal DNA (nrDNA) in the U. ramanniana isolates. The relationship between the variations of the sequences of the nrDNA ITS regions and those of the sporangiospore morphology was investigated for 12 isolates of U. ramanniana collected in Europe. Neighbor-joining and parsimony analyses on the sequences suggested that these isolates split into three groups. Precise examination of the morphology showed that the isolates of those respective groups were different from each other in their sporangiospore shape. The present study implies at least three intraspecific groups exist in U. ramanniana and that the variations in the nucleotide sequences of the nrDNA ITS regions correlate well with those in the sporangiospore shape in these intraspecific groups.     

Growth-promoting effects of lactoferrin on L. acidophilus and Bifidobacterium spp.

We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.     

Local and chemical distribution of phlorotannins in brown algae

The local and chemical distribution of phlorotannins among the Japanese Laminariaceae, Eisenia bicyclis, Ecklonia cava and Ecklonia kurome, was investigated. As a result of light microscopy observations with vanillin-HCl staining, phlorotannins were found to be accumulated within the vegetative cells of the outer cortical layer of the thalli, regardless of the species, stage of growth or organ. Crude phlorotannins comprised about 3.0% of the algal powder for each of the algae. High-performance liquid chromatography (HPLC) showed that the phlorotannins of E. bicyclis were composed of phloroglucinol (0.9%), phloroglucinol tetramer (4.4%), eckol (7.5%), phlorofucofuroeckol A (21.9%), dieckol (23.4%), and 8,8'-bieckol (24.6%), plus some other unknown phenolic compounds (17.3%). The composition of the phlorotannins differed little among the Laminariaceae, except for a significantly larger amount of the tetramer, MW 478, in E. bicyclis.     

Maximum Likelihood Analysis of the Complete Mitochondrial Genomes of Eutherians and a Reevaluation of the Phylogeny of Bats and Insectivores

The complete mitochondrial genomes of two microbats, the horseshoe bat Rhinolophus pumilus, and the Japanese pipistrelle Pipistrellus abramus, and that of an insectivore, the long-clawed shrew Sorex unguiculatus, were sequenced and analyzed phylogenetically by a maximum likelihood method in an effort to enhance our understanding of mammalian evolution. Our analysis suggested that (1) a sister relationship exists between moles and shrews, which form an eulipotyphlan clade; (2) chiropterans have a sister-relationship with eulipotyphlans; and (3) the Eulipotyphla/Chiroptera clade is closely related to fereuungulates (Cetartiodactyla, Perissodactyla and Carnivora). Divergence times on the mammalian tree were estimated from consideration of a relaxed molecular clock, the amino acid sequences of 12 concatenated mitochondrial proteins and multiple reference criteria. Moles and shrews were estimated to have diverged approximately 48 MyrBP, and bats and eulipotyphlans to have diverged 68 MyrBP. Recent phylogenetic controversy over the polyphyly of microbats, the monophyly of rodents, and the position of hedgehogs is also examined. Received: 21 December 2000 / Accepted: 16 February 2001     

Lactoferrin-binding proteins in Bifidobacterium bifidum.

Lactoferrin is an iron-binding glycoprotein and its bacteriostatic and bactericidal effects on gram-positive and gram-negative bacteria are well known. On the other hand, it is known that certain kinds of lactic acid bacteria are resistant to its antibacterial effects. Moreover, it is reported that lactoferrin promotes the growth of bifidobacteria in in vitro and in vivo experiments. In our experiments, lactoferrin-binding protein was found both in the membrane and cytosolic fractions of Bifidobacterium bifidum Bb-11. The bifidobacteria were grown in anaerobic conditions with lactobacilli MRS broth containing cysteine, harvested by centrifugation, and processed by sonication. The lactoferrin-binding proteins on the PVDF-membrane transferred after SDS-PAGE were detected by far-Western (western-Western) method using biotinylated lactoferrin and streptavidin-labelled horse radish peroxidase. The molecular weights of the lactoferrin binding protein detected in the membrane fraction were estimated to be 69 kDa and those in cytosolic fractions were 20, 35, 50, and 66 kDa.     

Two Distinct Populations of Primary Cytotoxic Cells Infiltrating into Allografted Tumor Rejection Sites: Infiltration of Macrophages Cytotoxic against Allografted Tumor Precedes That of Multiple Sets of Cytotoxic T Lymphocytes with Distinct Specificity to Alloantigens

It has been reported that the rejection of tumor allografts is mainly mediated by cytotoxic T lymphocytes (CTLs). Here, we characterized the cytotoxic effector cells of C57BL/6 (B6; H-2^b) mice infiltrating into the rejection site of the i.p. allografted Meth A fibrosarcoma (or P815 mastocytoma) cells of H-2^d origin. Two types of cytotoxic cells (i.e., CD8⁺ CTLs and macrophages (Mφs)) were identified by flow cytometric fractionation of the infiltrates or by specific in vitro elimination of cells either with antibody (Ab)-coated beads or with an Ab-plus complement. Of particular interest, these effector cells showed distinct and unique target specificities. First, the CTLs were inactive against transplanted tumor (e.g., Meth A) cells, whereas they were cytotoxic against donor-related concanavalin A (Con A) blasts as well as CTLL-2 (H-2^b) cells transfected with a class I gene of H-2^d origin. A cold target competition assay suggested that the CTLs were composed of multiple sets of T cells, each of which specifically recognized different allo-antigens. Second, the Mφs lysed the allografted tumor cells but were inert toward the Con A blasts and the CTLL-2 transfectants. Unexpectedly, the infiltration of Mφs preceded the infiltration of CTLs by several days during the course of rejection. These results indicate that two distinct populations of unique cytotoxic cells (i.e., CTLs and Mφs) are induced in the allografted tumor rejection site, and that the infiltration of cytotoxic Mφs responsible for rejection precedes that of the CTLs cytotoxic against cells expressing donor-related allo-antigens.