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.     

Natural killer (NK) activity of cultured S100β-positive T-leukemia cells

In order to clarify the function of human S100β- positive T-cells, S100β-positive T-leukemia cells (S100β TLC) were examined in vitro. S100β TLC were obtained from the peripheral blood of a patient with S100β-positive T-cell leukemia and enriched by an E-rosetting method. Two dimensional flow cytometric analysis indicated that the vast majority of the E-positive fraction were S100β TLC expressing CD3 and CD8 antigens. Although S100β TLC expressed CD3 antigen, they were negative for the α/β and γ/δ T-cell antigen receptor (TCR) defined by monoclonal antibodies (mabs) WT-31 and δ TCS-1, respectively. It was speculated that S100β TLC initially expressed α/β TCR but lost it during malignant transformation. When S100β TLC were cultured for 24 h, they acquired cytotoxic activity towards various NK-sensitive cell lines including K-562, Molt-3 and CEM-CCLF, but did not exhibit lysing activity towards NK-resistant cell lines including Raji, Daudi and MT-1. Despite the NK-activity of cultured S100β TLC, they lacked the morphological features of large granular lymphocytes (LGL). S100β TLC did not exhibit lymphokine-activated killer (LAK) activity. When S100β TLC were cocultivated with NK-sensitive cells or NK-resistant cells, they selectively bound to NK-sensitive cells, indicating that they lysed target cells by cell-to-cell contact. The finding that S100 β TLC lacked TCR molecules and their NK activity was not inhibited by mabs reactive with the CD3-TCR complex indicated that the CD3-TCR complex was not involved in their target recognition. These findings suggest that S100 β-positive T-cells are functionally similar to NK cells. We discuss the roles of S100 β-positive T-cells in the human immune system.     

Induction of interdigitating reticulum cell-like differentiation in human monocytic leukemia cells by conditioned medium from IL-2-stimulated helper T-cells

Monocytic leukemia (MoL) cells were obtained from the peripheral blood of a patient in whom the leukemic cells infiltrating various lymphoreticular organs exhibited features intermediate between interdigitating reticulum cells (IDC) and ordinary phagocytic macrophages, whereas the leukemic cells in the peripheral blood were essentially monocytic and lacked such features. Peripheral blood CD4+ T-cells were established as an interleukin-2-dependent T-cell line. When the MoL cells were exposed for a few days to conditioned medium from the T-cell line, they extended several dendritic cytoplasmic projections and became intensely positive for HLA-DR antigen, cytoplasmic S-100β protein, and CD1 antigen. Functionally, the conditioned medium significantly down-regulated Fc-mediated and Fc-independent phagocytic activities, and the levels of lysosomal enzymes such as lysozyme and nonspecific esterase in the MoL cells. Moreover, the conditioned medium significantly up-regulated the accessory cell function of the MoL cells as measured by the primary allogenic mixed leukocyte reaction (MLR). Furthermore, the conditioned medium significantly down-regulated the expression of CD14 antigen. Biochemical analysis indicated that the factor responsible for these changes is a protein which is distinct from known human cytokines and whose molecular weight is approximately 31 kDa. These findings suggest that IDC are closely related the monocytic lineage and that helper T-cells play an important role in constructing the microenvironment of T-lymphoid tissues which is necessary for the differentiation and maturation of IDC.     

Microsatellite DNA markers for rice chromosomes

We found 369 complete microsatellites, of which (CGG/GCC)_n was the most frequent, in 11 798 rice sequences in the database. Of these microsatellites, 35 out of 45 could be successfully converted into microsatellite DNA markers using sequence information in their flanking regions. Thus, the time and labor used to develop new microsatellite DNA markers could be saved by using these published sequences. Twenty eight polymorphic markers between Asominori (japonica) and IR24 (indica) have been correctly mapped on the rice genome and microsatellites appear to be randomly distributed in the rice chromosomes. Integration of these markers with the published microsatellite DNA markers showed that about 35% of the rice chromosomes were covered by the 56 microsatellite DNA markers. These microsatellites were hypervariable and were easily to assay by PCR; they were distributed to all chromosomes and therefore, one can easily select plants carrying desired chromosome regions using these microsatellite DNA markers. Thus, microsatellite maps should aid the development of new breeds of rice saving time, labor, and money.     

Effects of the Degree of Polymerization on the Binding of Xyloglucans to Cellulose

Xyloglucan oligosaccharides were isolated with various degreesof polymerization (DP) and reduced with tritiated sodium borohydride.The ³H-oligosaccharides were tested for their ability to bindto amorphous and microcrystalline celluloses and to cellulosefilter paper. The time course of binding indicated that theradiolabeled oligosaccharides continued to be bound for at least1 h after heating at 120°C. The binding probably requiredthe organization of the oligosaccharides and celluloses by gradualannealing after heating. Although neither pentasaccharide (glucose:xylose, 3 : 2), heptasaccharide (glucose: xylose, 4 : 3) andnonasaccharide (glucose : xylose : galactose : fucose, 4 : 3: 1 : 1) failed to bind to the celluloses, binding occurredwith oligosaccharides with DP equivalent to more than four consecutive1,4-ß-glucosyl residues. The extent of binding tothe celluloses increased gradually from octasaccharide (glucose:xylose, 5 : 3) to hendecosanosaccharide (glucose/xylose, 12: 9), with the increase in the DP of 1,4-ß-glucosylresidues. The binding of reduced cello-dextrins to celluloserequired at least 4 consecutive 1,4-ß-glucosyl residues.The extent of binding of cellopentitol or cellohexitol to cellulosewas similar to that of hendecosanosaccharide, showing lowerbinding for xyloglucan oligosaccharides in spite of longer chainsof 1,4-ß-glucosyl residues. These findings suggestthat the mode of binding to cellulose of xyloglucan oligosaccharidesis different from that of cello-oligosaccharides. (Received February 18, 1994; Accepted June 1, 1994)