Ultrastructure of a Hexagonal Array in Exosporium of a Highly Sporogenic Mutant of Clostridium botulinum Type A Revealed by Electron Microscopy Using Optical Diffraction and Filtration

The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing an equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. The adjacent globular subunits appeared to be interconnected by delicate linkers.     

Isolation of Nontoxigenic Variants Associated with Enhanced Sporulation and Alteration in the Cell Wall from Clostridium botulinum Type A 190L by Treatment with Detergents

Nontoxigenic variants were isolated from Clostridium botulinum type A strain 190L after treatment with detergents such as deoxycholate, sodium dodecyl sulfate, Tween 80 and Brij-58. Deoxycholate was most effective for obtaining the variants. The variants exhibited a markedly increased frequency of sporulation compared with the oligosporogenic parent strain. The cell wall of the parent strain was composed of an outer layer and an inner layer, whereas that of the variants lost the outer layer. After treatment with mitomycin C the parent strain was subjected to lysis and produced bacteriophages with a hexagonal head and a contractible tail, while the nontoxigenic variants did not yield bacteriophages or phage-like structures. There appears to be a close relationship among the toxigenic and sporogenic properties, formation of the outer cell wall layer and lysogeny.     

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin‐dependent neutrophil lysis

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA‐treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA‐treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA‐induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.     

Transport of an Mr approximately 300,000 Plasmodium falciparum protein (Pf EMP 2) from the intraerythrocytic asexual parasite to the cytoplasmic face of the host cell membrane

The profound changes in the morphology, antigenicity, and functional properties of the host erythrocyte membrane induced by intraerythrocytic parasites of the human malaria Plasmodium falciparum are poorly understood at the molecular level. We have used mouse mAbs to identify a very large malarial protein (Mr approximately 300,000) that is exported from the parasite and deposited on the cytoplasmic face of the erythrocyte membrane. This protein is denoted P. falciparum erythrocyte membrane protein 2 (Pf EMP 2). The mAbs did not react with the surface of intact infected erythrocytes, nor was Pf EMP 2 accessible to exogenous proteases or lactoperoxidase-catalyzed radioiodination of intact cells. The mAbs also had no effect on in vitro cytoadherence of infected cells to the C32 amelanotic melanoma cell line. These properties distinguish Pf EMP 2 from Pf EMP 1, the cell surface malarial protein of similar size that is associated with the cytoadherent property of P. falciparum-infected erythrocytes. The mAbs did not react with Pf EMP 1. In one strain of parasite there was a significant difference in relative mobility of the 125I-surface-labeled Pf EMP 1 and the biosynthetically labeled Pf EMP 2, further distinguishing these proteins. By cryo-thin-section immunoelectron microscopy we identified organelles involved in the transit of Pf EMP through the erythrocyte cytoplasm to the internal face of the erythrocyte membrane where the protein is associated with electron-dense material under knobs. These results show that the intraerythrocytic malaria parasite has evolved a novel system for transporting malarial proteins beyond its own plasma membrane, through a vacuolar membrane and the host erythrocyte cytoplasm to the erythrocyte membrane, where they become membrane bound and presumably alter the properties of this membrane to the parasite's advantage.     

Ultrastructure of malaria-infected erythrocytes

Knobs, caveolae, caveola-vesicle complexes, cytoplasmic clefts, and electron-dense material are five major ultrastructural changes found in the membrane skeleton and cytoplasm of erythrocytes infected with species of primate malaria. Knobs are electron-dense, conical evaginations of the erythrocyte surface, which are believed to mediate cytoadherence and sequestration of Plasmodium falciparum-infected erythrocytes. Caveolae and caveola-vesicle complexes are flask-shaped invaginations of the membrane skeleton, which may be involved in the uptake or export of host- or parasite-derived substances. Cytoplasmic clefts are flattened or circular membranous structures found in the erythrocyte cytoplasm between the intracellular parasite and the host cell surface. The clefts are variable in length and bounded by two or more membranes. Fine, granular electron-dense material is often found on the cytoplasmic face of clefts or in amorphous packets in the erythrocyte cytoplasm. Immunocytochemistry has demonstrated that all of these ultrastructural changes are associated with the trafficking and interaction of specific malarial antigens with the host erythrocyte.     

Antimutagenic effect of volatile decomposition products from thermally oxidized linoleate

The antimutagenic effect of volatile decomposition products from thermally oxidized linoleate on mutagenesis by UV irradiation was investigated in Escherichia coli B/r WP2. When added to an agar medium, these products greatly reduced the number of Trp+ revertants. The same antimutagenic effect was observed by acrolein, 2-hexenal, 2-heptenal, 2-nonenal and 2,4-decadienal; these unsaturated aldehydes were components of volatile products.     

Plasmodium gallinaceum: critical role for microtubules in the transformation of zygotes into Ookinetes

The role of microtubules and microfilaments in the transformation of spherical zygotes of Plasmodium gallinaceum (avian malaria parasite) into vermiform ookinetes has been studied by using specific drugs (taxol, colchicine, and cytochalasin-B). Both taxol and colchicine completely abolished the transformation of zygotes into ookinetes. The inhibitory effect was seen only if the drugs were added during the initial 6 hr of total time (20-24 hr) required for complete transformation; the addition of drugs after 6-8 hr of initiation of transformation had no effect. Electron microscopy revealed that microtubules were depolymerized by colchicine treatment, whereas in taxol-treated cells there was an extensive array of cytoplasmic and nuclear microtubules which appeared to be clumped in bundles. In contrast to the effects of taxol and colchicine, cytochalasin-B, which affects the microfilament system, had no effect on the transformation. Protein synthesis and expression of two ookinete-specific surface proteins were not affected in the drug-inhibited parasites. Zygotes treated with taxol for 4 hr at room temperature failed to develop into oocysts when they were subsequently fed to mosquitoes. These studies demonstrate a critical role for microtubules in the initial stages of transformation of zygotes into ookinetes.     

Isolation and characterization of human placenta fibronectin

Fibronectin was isolated from human placenta tissues and compared with human plasma fibronectin. Placenta and plasma fibronectins had similar amino acid compositions, immunological properties, and cell attachment-promoting activities, but differed in apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which could be accounted for at least partly by the difference in carbohydrate composition. Unlike plasma fibronectin, placenta fibronectin failed to form a precipitin line with concanavalin A in a double diffusion system. The non- or low-reactivity of placenta fibronectin with this lectin was also demonstrated by affinity chromatography with concanavalin A-agarose, in which more than 90% of the radiolabeled glycopeptides derived from placenta fibronectin was not retained on the gel. The two fibronectins also differed in the reactivity with Lens culinaris agglutinin of their glycopeptide fractions. These data indicate that placenta and plasma fibronectins are different in their carbohydrate structures and, therefore, suggest the presence of a tissue- or cell-specific mechanism for processing the carbohydrates of this glycoprotein.     

In vitro immune mechanisms associated with clearance of microfilariae of Dirofilaria immitis

An in vitro system has been developed to elucidate potential immune mechanisms associated with clearance of microfilariae (Mf) from the bloodstream in canine Dirofilaria immitis infection. Granulocytes as well as mononuclear cells adhere to Mf of Dirofilaria immitis in the presence of immune serum. Only granulocytes, however, were capable of killing Mf, whereas PBMC attach to but do not effectively kill Mf. In the presence of granulocytes 1% +/- 1, 10% +/- 2, and 12% +/- 3 of Mf were killed by heated normal (NDS), patent (PS), and occult serum (OS), respectively, after an 18-hr incubation. With the addition of fresh NDS there was an increase in killing to 5% +/- 1 (p less than 0.025) with heat-inactivated NDS, to 12% +/- 3 in the presence of PS and to 77% +/- 12 (p less than 0.005) in the presence of OS. On further purification of granulocyte cell populations with metrizamide gradients, neutrophils were found to be the predominant effector cells with 73% +/- 18 killing with neutrophils and 18% +/- 6 with eosinophils (p less than 0.0005). Only with neutrophils was a significant increase in killing of Mf observed when fresh NDS was added to delta OS. Fractionation of OS by gel filtration suggested that IgM was the opsonizing antibody in the occult serum. In addition, immunofluorescent studies showed only IgM bound to the surface on Mf on incubation in OS. The involvement of complement in the fresh serum enhancement of killing was supported by the finding, by immunofluorescence, of surface C3 on Mf after incubation in fresh OS.