Isolation and partial characterization of exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A.

Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 190L. The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained. The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin. The hexagonal array was partially disintegrated with 5 M guanidine-HCl and almost completely disrupted with 8 M urea in combination with 1% mercaptoethanol under alkaline conditions. The purified exosporium fraction was composed mainly of protein (69.1%) and lipids (13.8%). A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected. The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine.     

S layer protein of Clostridium tetani: purification and properties.

S layer protein of Clostridium tetani strain AO 174, a nontoxigenic derivative of strain Harvard A 47, was prepared from the cell walls by 4 M urea extraction and purified by DEAE-Sepharose CL-6B chromatography followed by a combination of anion-exchange chromatography and reverse-phase chromatography using an HPLC system. The molecular weight of the S layer protein was estimated to be 140 kilodaltons (kDa) by SDS-PAGE. The amino acid composition of the 140 kDa protein was very similar to those of S layer proteins from the other bacterial species: it was rich in acidic amino acid and lacked cysteine. Also, the protein was unique in its extremely low content of proline (0.02 to 0.03 mol%). Multiple isoelectric forms ranging from pH 4.0 to 4.5 were observed in the purified preparation. Immunodiffusion analysis showed that the 140 kDa protein was a common antigen to the three strains of C. tetani tested.     

Effect of the orthoquinone moiety in 9,10-phenanthrenequinone on its ability to induce apoptosis in HCT-116 and HL-60 cells

9,10-Phenanthrenequinone (9,10-PQ) is one of the most abundant quinones among diesel exhaust particulates. Recent data have suggested that quinones induce apoptosis in immune, epithelial and tumor cells, leading to respirator illness; however, the mechanisms by which quinones induce apoptosis and the structure required for this remain unknown. We studied the antitumor activity of 9,10-PQ analogs against two human tumor cell lines, HCT-116 colon tumor cells and HL-60 promyelocytic leukemia cells. The loss of the cis-orthoquinone unit in 9,10-PQ abrogated its ability to induce apoptosis in the two tumor cell lines, and the LC₅₀ values of these analogs were indicated over 10 μM. An analog of 9,10-PQ in which the biaryl unit had been deleted displayed a reduced ability to induce tumor cell apoptosis, while the analogs 1,10-phenanthroline-5,6-dione (9) and pyrene-4,5-dione (10), which also had modified biaryl units, exhibited increased tumor cell apoptotic activity. The cis-orthoquinone unit in 9,10-PQ was identified as essential for its ability to induce apoptosis in tumor cells, and its biaryl unit is also considered to influence orthoquinone-mediated apoptotic activity.     

Redox properties and regulatory mechanism of the iron-quinone electron acceptor in photosystem II as revealed by FTIR spectroelectrochemistry

Photosynthesis Research - Photosystem II (PSII) performs oxidation of water and reduction of plastoquinone through light-induced electron transfer. Electron transfer reactions at individual redox...     

Focus: Allergic Diseases and Type II Immunity: Expression of Leucine-rich Repeat-containing Protein 32 Following Lymphocyte Stimulation in Patients with Non-IgE-mediated Gastrointestinal Food Allergies

The lymphocyte stimulation test (LST) facilitates the diagnosis of non-IgE-mediated gastrointestinal food allergies (non-IgE-GI-FAs). However, LSTs require large volumes of blood and prolonged culture durations. Recently, we found that IL2RA mRNA expression in peripheral blood mononuclear cells (PBMCs) of patients with non-IgE-GI-FAs increased after a 24 h stimulation with milk proteins. We designated this gene expression test as the instant peripheral blood allergen stimulation test (iPAST). In this study, we investigated whether other activated T cell-associated genes are superior to IL2RA in the iPAST for the supplementary diagnosis of non-IgE-GI-FAs. After incubating PBMCs with milk proteins for 24 h, the mRNA levels of three genes, LRRC32, TNFRSF4, and CD69, were assessed using quantitative RT-PCR. The diagnostic significance of the mRNA expression was evaluated by analyzing the receiver operating characteristic (ROC) curve. Upon stimulation with α-casein, κ-casein, α-lactalbumin, or a mixture of four milk protein components (Pmix), LRRC32 expression in the PBMCs of 16 patients with non-IgE-GI-FAs was found to be higher than that in their 17 control counterparts, whereas TNFRSF4 and CD69 levels remained unaltered. Except for β-lactoglobulin and cow’s milk (CM), the area under the ROC curve (AUC) for LRRC32 mRNA expression upon stimulation was >0.7, which validated the diagnostic ability of this test. Notably, α-casein and Pmix had higher AUC scores of 0.820 and 0.842, respectively, than other antigens. iPAST assessed by LRRC32 as well as IL2RA may be useful for the supplementary diagnosis of non-IgE-GI-FAs as an alternative to LSTs and provide insight into the pathogenesis of non-IgE-GI-FAs.     

Molecular phylogeny of glacial relict species: a case of freshwater Valvatidae molluscs (Mollusca: Gastropoda) in North and East Asia

The study of glacial relict species has been focused on understanding how the biogeographic patterns of species have developed. A number of studies using phylogenetic and population genetics approaches have been conducted for terrestrial glacial relict species, and the mechanisms of their formation have been elucidated. On the other hand, less focus has been placed on glacial relict species inhabiting freshwater systems. In particular, stable lakes can serve as refugia during a glacial period, but research studies on freshwater relict species inhabiting lakes have not been well conducted. In order to clarify the mechanism of the glacial relict species in freshwater, we conducted a molecular phylogeny analysis, divergence time estimation, and a biogeographic reconstruction on freshwater Valvatidae molluscs, which have been considered as a glacial relict in the Japanese Archipelago. Our study shows that the valvatid fauna in the Japanese Archipelago was produced by multiple dispersal events from the Asian continent and by vicariance events during the period of the Pliocene–Quaternary glaciation. It includes multiple relict species that survived interglacial periods in different lakes. These findings suggest that the lakes can serve as refugia not only during glacial periods, but also during interglacial periods.     

Solubilization and partial properties of receptor substance for bacteriophage alpha 2 induced from Clostridium botulinum type A 190L

Bacteriophage alpha 2, one of the two inducible phages from Clostridium botulinum type A 190L, had a latent period of 55 min and an average burst size of 75 in C. botulinum type A Hall used as the host bacterium. The phage particles were adsorbed on the cell walls extracted with hot trichloroacetic acid (TCA-walls). The receptor substance for the phage was solubilized from the TCA-walls with Achromopeptidase and fractionated by gel filtration on Sephadex G-150. The fraction having the highest level of receptor activity for the phage contained large amounts of muramic acid and glucosamine. Both authentic muramic acid and glucosamine significantly inactivated the phage, whereas glucose, galactose, L-and D-alanine, diaminopimeric acid, or D-glutamic acid did not exhibit similar activity. There results strongly suggest that the receptor site for phage alpha 2 is closely associated with glycan moieties of the cell wall peptidoglycan.     

Aluminum-tolerant Protoplasts from Roots Can be Collected with Positively Charged Silica Microbeads: a Method Based on Differences in Surface Negativity

A new technique was developed for the collection of Al-tolerantplant cells. The procedure takes advantage of the differencein surface charge between Al-tolerant and Al-sensitive protoplasts.Equal volumes of freshly prepared, positively charged silicamicrobeads (0.05% w/v) and purified protoplasts derived fromroot tips (2x10⁵ ml^-1) of rice, maize or pea were mixed andthen centrifuged on a discontinuous Ficoll gradient. Intactprotoplasts from the Al-tolerant plant were recovered mostlyin the bottom fraction, those from the Al-sensitive plant wererecoverd at the uppermost interface, and those from the plantwith intermediate tolerance to Al were collected at the middleinterface. Moreover, the pattern of fractionation was unrelatedto the diameter of the protoplasts. The technique should beuseful in future investigations of the physiological and biochemicalproperties of Al-tolerant cells or their plasma membranes. (Received April 19, 1995; Accepted September 1, 1995)     

MPSS profiling of human embryonic stem cells

Background  

Pooled human embryonic stem cells (hESC) cell lines were profiled to obtain a comprehensive list of genes common to undifferentiated human embryonic stem cells.