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.     

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.     

Enantioselectivity in glutathione conjugation of 1,2-epoxy-1,2,3,4-tetrahydronaphthalene by hepatic glutathione S-transferase

Enantiomers of 1,2-epoxy-1,2,3,4-tetrahydronaphthalene (ETN) were conjugated with glutathione (GSH) specifically at their benzylic oxiran carbons, with a marked difference in rate [(1R,2S)-(+)- less than (1S,2R)-(-)-ETNs] as well as in affinity for GSH S-transferase [Km: (1S,2R)-(-)- less than (1R, 2S)-(+)-ETNs], in rat liver cytosol to yield two diastereomeric S-(2-hydroxy-1,2,3,4-tetrahydronaphth-1-yl)glutathiones which were separable by reverse partition hplc. Enzymatic GSH conjugation of racemic ETN occurred preferentially with the (1S,2R)-(-)-component as a result of its retarding effect on the conjugation of the (1R,2S)-(+)-counterpart, one half of which remained in enantiomerically pure form in the incubation medium when the (1S,2R)-(-)-component had been completely conjugated.     

An autoradiographic study of calcium transport in spicule formation in the gorgonian Leptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary The route of calcium transport to the sites of spicule formation in the gorgonian Leptogorgia virgulata has been examined by the use of ⁴⁵Ca as a tracer in light- and electron-microscopic autoradiography. From 1 to 15 min after the 15-min incubation the tracer accumulates in the axis. After 15 min there is a movement of label out of the axis largely to the peripheral region of the axis, the axial epithelium, and the mesoglea. By 60 min much of the label is in the spicules reaching its maximum level at 120 min. When calcium enters the scleroblast from the mesoglea, it appears to be transported to the spicule by electron-dense bodies. There does not appear to be a simultaneous release of all ionic calcium from the axis, but rather a continuously increasing efflux which levels off at 60–120 min. Not all of the calcium reaching the axis will traverse it en route to spicules; instead, a portion of it apparently precipitates as an amorphous compound.Contribution No. 550; Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina, Columbia, South Carolina 29208 USA     

Isolation and characterization of forespores from Bacillus megaterium

A procedure for isolation of intact forespores from sporulating Bacillus megaterium cells was developed. The cells were digested with lysozyme and made to release free forespores from the protoplasts by disruption of the cytoplasmic membrane with sonication in phosphate buffer containing 10% glycerol. The suitability of the procedure was confirmed by recovery of dipicolinic acid in the isolated forespores and an electron microscopic observation. The fine structure of the forespores prepared at 6 hr (t6) after initiation of sporulation was similar to that of mature spores, except that the cortex layer and primordial cell wall were thinner and the core was larger. The density, determined by density gradient centrifugation, of the forespores isolated at t6, t10, t12, and mature spores was estimated to be 1.2783, 1.2875, 1.2861, and 1.2858, respectively. The isolated forespores at t6 and t8 were extremely heat labile (D80 of 9.5 and 21.5 min, respectively) relative to mature spores (D80 of 277.8 min). These forespores were also less resistant to organic solvents. Germination of the forespores as well as mature spores was induced by KNO3, D-glucose, and L-leucine. Forespores at t6 were more sensitive to KNO3-induced germination than those at t10, t12, and mature spores when measured by reduction in the optical density of cell suspension.