Morphological study on Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105).

Mycobacterial cell wall consists of rigid cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidiglycan complex, in which mycoloyl structure varies by the mycobacterial species diversely, whereas the arabinogalactan peptidoglycan structure is consistent comparatively. The CWS of Mycobacterium bovis BCG has long been expected as a potent adjuvant for immunotherapy of malignant tumor. Although the chemical structure of CWS has been established in the last few decades, the physicochemical properties of CWS having highly amphipathic micelle structure with very long mycoloyl and carbohydrate chains are not unveiled. In this study, the ultrastructure of CWS of M. bovis BCG Tokyo 172 (SMP-105), suspended in several solvents with different polarity, was investigated with a particle size analyzer, a transmission electron microscope (TEM) and other techniques. As a result, the particle size was about 4.7 to 67.8 microm in physiological saline, but it became smaller and more compact when suspended in hydrophobic solvents. TEM images showed two different morphological forms distinctively: double folded sheet structure in hydrophilic conditions and multilayered rolled sheet structure in hydrophobic conditions. These studies have revealed characteristic surface features of SMP-105, the hydrophobic moiety occupying dominant space and the hydrophilic moiety smaller space, respectively, which may lead to the acceleration of immunological studies on this product.

Preliminary characterization of chemically generated Mycobacterium avium subsp. paratuberculosis cell wall deficient forms (spheroplasts).

Cell wall deficient forms (CWD, spheroplasts) genetically indistinguishable from M. avium subsp. paratuberculosis (MAP) have been isolated from patients with Crohn's disease and sarcoidosis. These MAP CWD may be important in the pathogenesis of these diseases and in Johne's Disease in other animal species. CWD forms are extremely difficult to isolate and generally revert to cell wall competent forms (CWC) when cultured in vitro. Cultured MAP strain 19698 were chemically treated to generate sufficient CWD to compare to CWC organisms by electron microscopy, chemotype profile (matrix solid-phase dispersion and thin layer chromatography), silver-stained SDS-PAGE gels with and without periodic acid treatment and Western blots with antigen recognition by sera from confirmed Johne's positive and Johne's negative cattle. On electron microscopy, CWD organisms were larger and rounder than cell wall competent forms and had lost the majority of their cell walls, being bounded only by a plasma membrane. Chemotype profiles of CWD lacked bands generally associated with cell wall glycolipids. Silver-stained SDS-PAGE gels of CWD demonstrated loss of bands that migrate in the same region as lipoarabinomannan (LAM) and some bands likely representing proteins and weakening of bands that migrate similarly to phosphatidylinositol mannosides (PIM). Western blots of CWD demonstrated bands with loss or attenuation of signal that migrate similarly to LAM and other constituents. In summary, CWD and CWC forms of MAP 19698 had marked differences in morphology, chemotype profile, cell wall constituents, and antigens recognized by Johne's disease positive and negative bovine sera.

Electrophoretic mobility of external invertase from free and gel-immobilized yeast cells.

Electrophoretic mobility of secreted invertase (E.C. 3.2.1.26) from gelatin-immobilized yeast cells was analysed and compared with that of secreted invertase from freely suspended batch-grown cells. Invertase from immobilized cells showed a lower mobility after 24 h of incubation, in medium containing either glucose or raffinose as carbon source. Changes in invertase mobility were also followed in a time course both for immobilized and for freely suspended batch-grown cells. Mobility of invertase from free cells increased after approximately 15 h of incubation, independently of the carbon source, whilst that of invertase from immobilized cells remained constant. The differences observed were attributed to a different level of glycosylation of the protein moiety in free and immobilized cells. The amount of mannoproteins in the cell walls of immobilized cells was also investigated by ConA-ferritin labelling and quantification of ferritin particle density in ultrathin sections; the results of this experiment showed a higher content of mannoproteins in the walls of immobilized cells when compared with free cells. As a whole, these results are indicative of physiological changes that can be ascribed to the peculiar microenvironment of gel-immobilized cells.

Reversible rearrangement of vimentin-type intermediate filaments in cultured human skin fibroblasts from patients with lysosomal storage diseases.

Immunofluorescence microscopy shows that unlike cytoplasmic microtubules (MT), vimentin-type intermediate filaments (IF) are collected into ring-shaped structures in affected fibroblasts. The altered IF organization could be observed in monolayers of polarized fibroblasts in the prolonged stationary growth phase and-after replating-upon their initial spreading. Transition from a discoid to an extended cellular form is accompanied by centrifugal dislocation of ring-shaped IF structures towards the cell's active edge with gradual restoration of the radial fibrillar vimentin network. Spreading of affected cells occurred more slowly than that of control fibroblasts.

Induction of inflammatory cytokines by Nocardia fractions.

NDCM and NLD fractions of Nocardia opaca cell walls were used for in vitro stimulation of human and porcine peripheral blood mononuclear cells. Inflammatory cytokines IL-1, IL-6, tumour necrosis factor (TNF) alpha and interferon (IFN) gamma were detected at the single-cell level using paraformaldehyde-fixed and saponin-permeabilized mononuclears stained with cytokine-specific antibodies and indirect immunofluorescence method. IL-1, IL-6 and TNF were produced by human monocytes stimulated for 2 h, IFN gamma-positive lymphocytes were detected later. IFN gamma was produced also by activated porcine lymphocytes. Cells expressing apoptotic features were found among blood mononuclears treated with Nocardia fractions.