Changes of ultrastructure in spore coat of Bacillus thiaminolyticus during germination and outgrowth.

Electron microscopic observation showed that the spore coat of Bacillus thiaminolyticus consisted of at least four layers; a high electron dense outer spore coat layer with five prominent ridges, a middle spore coat layer including two layers of a high and a low electron density, and an inner spore coat layer composing six to seven laminated layers. Rapid breakdown of the cortex and swelling of the core occurred in spores which were allowed to germinate by L-alanine for 45 min, whereas no change of surface feature was observed by scanning electron microscopy. Germination and outgrowth of spores in nutrient broth proceeded, being accompanied by morphological changes, in three steps; the first is a rapid breakdown of the cortex and swelling of the core, the second degradation of the inner layer at prominent region of the spore coat, and the last rupture of the spore coat and emergence of a young vegetative cell.     

Assessment of mucosal immune response in genitourinary tract using urine.

A novel method to assess mucosal immune response in the genitourinary mucosa after immunization with a mucosal vaccine has been developed. In this method, secretory IgA antibody is measured by a highly sensitive enzyme immunoassay (immune-complex transfer enzyme immunoassay) using urine as a specimen. The urinary IgA antibody response could be detected by the immune-complex transfer enzyme immunoassay. In contrast, a conventional enzyme immunoassay (enzyme-linked immunosorbent assay (ELISA)) could not detect this response because of its low sensitivity. Because urine samples can be collected easily and nontraumatically, not only from experimental animals but also from humans, both males and females, the present method may be applicable for assessing the protective efficacy of candidates for mucosal vaccines against sexually transmitted microorganisms, such as human immunodeficiency virus. Furthermore, the usefulness of this method for novel mucosal vaccine formulae was shown for a model in which vaccine antigen and Bordetella pertussis adjuvant were adsorbed onto CaCO, and enclosed in enteric coated capsules.     

Paraquat Resistant Tobacco Calluses with Enhanced Superoxide Dismutase Activity

Seven paraquat resistant calluses of tobacco (Nicotiana tabacumL. cv. Samsun) were obtained by three successive screeningsof protoplast-derived calluses on a paraquat containing medium.Superoxide dismutase (SOD) activity of the resistant calluseswas 14- to 159-fold that of the leaf cells on protein basis.Paraquat-resistant calluses, however, showed little increasein catalase and peroxidase activities. More than 90% of SODactivity in the resistant calluses was inhibited by KCN, aswas the SOD activity in leaves, indicating that the major SODin the callus appears to be the Cu, Zn containing enzyme. Thecallus cells, however, expressed the immunologically distinguishedSOD isozyme from the enzyme in the leaves. (Received April 23, 1984; Accepted August 6, 1984)     

Chloroplast Ribonucleoproteins (RNPs) as Phosphate Acceptors for Casein Kinase II: Purification by ssDNA-Cellulose Column Chromatography

Using ssDNA-cellulose column chromatography, a 34 kDa ribonucleoprotein(p34) has been purified from a 0.4 M KCl crude extract of spinachchloroplasts as an effective phosphate acceptor for casein kinaseII (CK-II) in vitro. Monomeric and oligomeric CK-IIs were copurifiedwith p34 by the column chromatography and the kinases were separatedfrom p34 by means of Mono Q column chromatography. It was foundthat (i) the purified p34 (pi 4.9) was phosphorylated specificallyby CK-II in vitro; and (ii) similar polypeptides, such as p35(pI 4.7) and p39 (pI 4.9) in maize and p33 (pI 4.7) in liverwort,were detected as ssDNA-binding chloroplast proteins phosphorylatedby CK-II in vitro. The findings suggest that (i) RNPs that functionas phosphate acceptors for CK-II exist commonly in chloroplastsamong plant cells; and (ii) the physiological activity of RNPsis regulated by their specific phosphorylation by CK-II in chloroplasts. (Received July 3, 1995; Accepted October 5, 1995)     

Studies on relationship between cysts and granulomas in murine cryptococcosis

The histopathology of murine cryptococcosis was observed until the 55th day and particular attention was paid to whether or not cysts, which had been formed in the brain, could change to granulomas. Cryptococcus neoformans RIB-12M was used in this experiment. As experimental animals, five-week-old male BALB/c mice, weighing 20–22 g, were used. An infective inoculum was prepared by adjusting the number of cryptococci to 10⁶ or 5 × 10⁶/0.2 ml. Each mouse was inoculated intravenously with 0.2 ml of the cell suspension, and the colony forming unit of the brain and liver, and the histopathological findings in various visceral organs were investigated.40 × 10⁴ colonies grew from 100 mg of the brain tissue of the eighth day. Thereafter, the number increased gradually. It reached 500 × 10⁴ on the 20th day. The colony forming unit from the liver reached a peak on the 12th day (250 × 10⁴) and thereafter the number decreased gradually.Histopathologically, the brain and liver were severely affected with the fungus. In the brain cysts with cryptococci continued to increase until the end of the experiment. On the other hand, in the liver several purulent foci appeared on the second day. On the eighth day numerous mononuclear cells accumulated at the foci and their lesions changed to granulomatous ones with cryptococci. The number of granulomatous lesions reached a peak on the 16th day in the mice inoculated with 5 × 10⁶ cryptococci, and thereafter showed a tendency to decrease gradually.     

Cross-reaction of synthetic alpha-(1----3)-branched glucans with rabbit anti-N4 dextran

Cross-reactions of four synthetic branched glucans (3-O-alpha-D-glucopyranosyl-(1----6)-alpha-D-glucopyranans: V39, V17, V37, and V32, each containing one unit glucose branches amounting to 11-12%, 33-43%, 50-54%, and 71-100%, respectively) with rabbit anti-N4 dextran were examined. All four samples precipitated antibodies raised in rabbits by injecting N4 dextran-concanavalin A conjugate. The ability of glucans to precipitate antibody depended on the quantity of branches, samples with more branches precipitating less antibody nitrogen under the same conditions. This may indicate an inhibitory effect of the branches on precipitation. Oligosaccharide inhibition assay showed that the precipitation reactions were specific for (1----6)-alpha-D-glucopyranosyl linkages, and the maximum size of the alpha-(1----6)-specific antibody combining site corresponded to isomaltopentaose. Determination of antibody nitrogen and glucan in the precipitates indicated that the ratios of one combining site of antibody to numbers of glucose residues were 1:9 (V39), 1:11 (V17), and 1:16 (V37) in the extreme antibody excess region. A synthetic sample of manno-glucan ((1----6)-alpha-D-glucopyranan containing about 27% of randomly linked 3-O-alpha-D-mannopyranosyl side chains) also reacted with the same antibody.     

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.