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Biomedical subjects

A Ryter

Publications and source records attributed to A Ryter.

At least 55 records · Page 3Linked to original sources
PubMed1983

Ultrastructure and extreme heat resistance of spores from thermophilic Clostridium species.

The heat resistance and ultrastructural features of spore suspensions prepared from Clostridium thermocellum LQRI, Clostridium thermosulfurogenes 4B, and Clostridium thermohydrosulfuricum 39E were compared as a function of decimal reduction time. The decimal reduction times at 121 degrees C for strains LQRI, 4B, and 39E were 0.5, 2.5, and 11 min. The higher degree of spore heat resistance was associated with a spore architecture displaying a thicker cortex layer. Heat resistance of these spores was proportional to the ratio of spore cortex volume to cytoplasmic volume. These ratios for spores of strains LQRI, 4B, and 39E were 1.4, 1.6, and 6.6, respectively. The extreme heat resistance and autoclavable nature of C. thermohydrosulfuricum spores under routine sterilization procedures is suggested as a common cause of laboratory contamination with pure cultures of thermophilic, saccharide-fermenting anaerobes.

Clostridium↗
PubMed1983

Membrane shuttle between plasma membrane, phagosomes, and pinosomes in Dictyostelium discoideum amoeboid cells.

The intracellular redistribution of membrane internalized during endocytosis was studied quantitatively by a biochemical approach and by a morphometric analysis of autoradiographs in electron microscopy. Plasma membrane glycoconjugates, enzymatically labelled with radioactive galactose, were used as a membrane marker. In cells labelled at their surface either before or after the phagocytotic uptake of latex beads, subsequent endocytosis led to a redistribution of label between the plasma membrane and endosomal membranes until a steady-state was reached after about 1 h with 43% of the label on the plasma membrane. The steady-state resulted when all participating membranes carried the same surface density of label. During phagocytosis or pinocytosis the equivalent of the plasma membrane was internalized and recycled once every 20 min or 40 min, respectively. Compared to this rate a very rapid and complete mixing of membranes was observed between newly formed phagosomes and preexisting digestive vacuoles or between newly formed pinosomes and preexisting phagosomes. Due to this rapid mixing, the membranes enclosing undigestible latex beads remained fully linked to the shuttle of membrane to and from the cell surface.

Animals↗
PubMed1982

Destruction of Leishmania mexicana amazonensis amastigotes within macrophages in culture by phenazine methosulfate and other electron carriers.

Exposure of macrophages infected with Leishmania mexicana amazonensis to phenazine methosulfate (PMS) resulted in rapid damage and disappearance of the intracellular amastigotes without obvious ill effects to the host cells. The reduction of the percent infection was related to the concentration of PMS and to the duration of the pulse. Most Leishmania disappeared within 2 h of a 2-h pulse with 10 muM of the drug. In contrast, pretreatment of the macrophages with PMS followed by removal of the drug before infection did not result in disappearance of the parasites. The pH of the PMS medium markedly influenced the disappearance of Leishmania: maximum effect was observed at pH 8.0, while the effect was negligible at pH 6.3. The pH effect may be related to pseudobase formation by the PMS cation. Dose-response curves for PMS were similar for resident, elicited, or activated macrophages. Observations by time-lapse cinemicrography documented the explosion-like fragmentation of the amastigotes within 1-2 h of exposure of infected macrophages to the drug. Parasite-derived granules and vacuoles were seen to scatter within the parasitophorous vacuoles. This early damage to the parasites was confirmed by transmission electron microscopic observations. Infected macrophages incubated with PMS displayed detectable vacuolar fluorescence, indicating that PMS or a metabolite of PMS had access to the vacuoles. A series of other electron carriers, including phenyl methanes, phenazines, oxazines, a xanthene, and a naphthoquinone, given continuously for 18 h, also induced the disappearance of the Leishmania. The most potent was crystal violet, active at 70 nM. The presence of apolar substituents enhanced activity and this is probably related to increased permeation of the dyes. Finally, PMS, as well as other electron carriers examined, also reduced the growth of Leishmania promastigotes in culture. The results are compatible with a direct effect of the drugs on the intracellular amastigotes, involving only a permissive participation of the macrophages. We propose that the diverse agents destroy the amastigotes by redox-cycling generation of active oxygen metabolites at or near the parasites. Alternatively, the effect of the drugs could be mediated by toxic free radical reduction species of the drugs or by interference with electron flow or with the intermediary metabolism of Leishmania.

Animals↗
PubMed1982

Concanavalin-A-mediated thymocyte agglutination: a model for a quantitative study of cell adhesion.

This report describes a quantitative study of the agglutination of rat thymocytes with concanavalin A (ConA). The probability that two ConA-coated cells remain bound after centrifugation was determined over a wide range of lectin concentrations. The minimal force required to separate agglutinated cells and the number of ConA molecules bound per cell were measured in similar experimental conditions. Agglutinated cells were examined by electron microscopy to estimate the area of membrane involved in adhesion. The dependence of agglutination on cell metabolism was studied: cold (4 degrees C), sodium azide (15 mM) and cytochalasin B (10 micrograms/ml) inhibited thymocyte adhesion. The importance of lateral movements of ConA molecules was assayed by measuring the adhesion of ConA-coated glutaraldehyde-fixed thymocytes to untreated cells: substantial binding occurred, but at a reduced level relative to untreated cells. A mathematical analysis of experimental data allowed the following conclusions. (1) At least 10(3) ConA bonds were involved in cross-linking two bound cells, which required the lectin molecules to be concentrated in the binding area, at least when low ConA concentrations (0.5 microgram/ml or less) were used. (2) The dependence of the binding probability on lectin concentration was fairly linear when the latter was small, which implied that the limiting step in cell-cell adhesion was the formation of a bond between a single ConA molecule and a ligand on the other cell. (3) The mean intercellular-contact time for the formation of this first bond was about 10 S for high concentrations of ligand (8 micrograms/ml). It was possible to fit the above data into a physically consistent quantitative model of cell adhesion.

Animals↗
PubMed1981

Multiple drug resistance in Mycobacterium avium: is the wall architecture responsible for exclusion of antimicrobial agents?

Whole cells of Mycobacterium avium, characterized by their negative response in the nine biochemical tests used for mycobacterial identification in our laboratory, turned positive for nitrate reductase, Tween-80 hydrolysis, beta-glucosidase, acid phosphatase, alkaline phosphatase, penicillinase, and trehalase after their wall portion was removed to yield spheroplasts. This suggested that the negative results in most of the biochemical procedures were caused by the exclusion mechanism at the wall level. Preliminary transmission and scanning electron microscopic studies showed differences at wall level between laboratory-maintained opaque, dome-shaped (SmD) and host-recycled smooth, transparent (SmT) colony type variants of M. avium and suggested the presence of an outer regularly structured layer in SmT variants. Comparative ultrastructural studies utilizing different polysaccharide coloration methods confirmed the presence of an outer polysaccharide layer in SmT variants which was probably related to their enhanced pathogenicity for experimental animals and drug resistance as compared to that of SmD variants. These findings are discussed with respect to multiple drug resistance, virulence, and gene expression of M. avium.

Anti-Bacterial Agents↗
PubMed1981

Freeze-fracture study of phagocytosis in Dictyostelium discoideum.

The plasma membrane and its derivative, the phagosome membrane, were studied during and after ingestion of yeast of latex beads in Dictyostelium discoideum. Freeze-fracture electron microscopy, which provides information on the internal architecture of the membranes, and observation of thin sections of cells treated by cytochemical methods were used in parallel. For visualization of membrane sterols in the replicas, the cells were fixed in the presence of digitonin or the antibiotic filipin. No lateral phase separation occurred during yeast engulfment: the intramembranous particles (IMPs), phospholipids and sterols remained distributed at random in the forming phagosome membrane. In contrast architectural modifications of the membrane were observed upon phagosome internalization. Compared to the plasma membrane, the phagosome membrane displayed 2-3 times more IMPs a shift in the IMP size distribution and a higher sterol content. These changes were completed soon after phagosome closure; they were not related either to the nature of the ingested particles (yeast, latex beads) or to the pH in the membrane environment. The membrane changes too place when the phagosomes began to fuse with pre-existing digestive or autophagic vacuoles and lysosomes. Some of the experimental evidence suggests that the restructuring of the membrane may be related to the presence of hydrolases.

Cell Membrane↗
PubMed1980

Bacillus megaterium sporal peptidoglycan synthesis studied by high-resolution autoradiography.

Cells of a Dap- Lys- mutant strain of Bacillus megaterium were pulse labeled with [3H]diaminopimelic acid at different times of growth and sporulation. They were processed for radioactivity measurements and high-resolution autoradiography either just after the pulse or after a chase in a nonradioactive medium until refractile forespores started to appear at time (t)4,5. In the pulse-labeled cells, autoradiographs and radioactivity measurements showed that the radioactivity incorporated during a pulse decreased abruptly after t0 and stayed at a low level until t5, although the forespore wall and cortex were formed between t4 and t5. In the pulse-chased bacteria, the acid-insoluble radioactivity, as well as the number of silver grains on autoradiographs, increased during the chase in cells labeled at t1 to t2, whereas it decreased in those labeled before t0. Furthermore, analysis of silver grain distribution showed that, in stage IV bacteria, grains were distributed at the outside of the forespore, mostly on the sporangium cell wall, when pulse-labeling occurred before or at t0; they were located along the cortex and in the forespore cytoplasm when labeling was made at t1 or t2. These facts show that [3H]diaminopimelic acid necessary for spore envelope synthesis was incorporated before their morphological appearance. Free or small diaminopimelic acid precursors entered the sporangium between t1 and t2. The appearance of silver grains in the forespore cytoplasm suggests that the forespore is implicated in sporal peptidoglycan synthesis.

Autoradiography↗
PubMed1980

Peptidoglycan turnover during growth recovery after chloramphenicol treatment in a Dap-Lys-mutant of Bacillus megaterium.

The study of diaminopimelic acid (DAP) incorporation and turnover during growth recovery in chloramphenicol-treated (CMP-treated) Bacillus megaterium cells showed that two kinds of turnover occurred. A low acid-soluble turnover appeared as soon as growth resumed in bacteria labeled before the CMP treatment and at the middle of the first generation in those labeled during the treatment. The acid-insoluble turnover appeared only at the beginning of the second generation of growth resumption in bacteria labeled before CMP addition and at the beginning of the third generation in those labeled during the CMP treatment. The acid-soluble release observed during the period of cell wall thinning is too low to account for the decrease of the wall thickness and the acid-insoluble loss appears after this period. When bacteria were transferred into partially spent medium instead of fresh culture medium the acid-insoluble release started to appear half a generation sooner. Electron microscopic observations showed that in this case, large scales detached from the cell wall. This activity of wall degradation was not observed when the partially spent medium was previously heated for 10 min at 100 degree C. The persistence of a thick wall on cell ends during the first generation does not reflect an absence of growth sites because their labeling on autoradiographs is high. Rather, it seems to be due to a low lytic activity at the poles.

Bacillus megaterium↗
PubMed1980

Electron-microscope study of Dictyostelium discoideum plasma membrane and its modifications during and after phagocytosis.

The study of plasma membrane and phagosome membrane of Dictyostelium discoideum was performed using 2 lectins: concanavalin A (Con A) and Wheat Germ Agglutinin (WGA), and 2 markers of anionic sites: colloidal iron hydroxide (CIH) and cationized ferritin (CF). These labellings were applied to fixed partially broken cells, which had ingested a large quantity of yeast. They showed that Con A and CF labelled both the outer and inner faces of the plasma membrane, whereas CIH and WGA were deposited on the outer face only. Phagosome membranes displayed the same location as the plasma membrane for Con A, CF and CIH even in very old phagosomes. This suggests that most receptors of these 3 markers were not degraded by hydrolases. In contrast, phagosome membranes of young and old phagosomes did not react with WGA. When labellings were made on yeast phagocytozing cells, the membrane of phagocytic cups were also devoid of WGA, while it was labelled with the 3 other markers. The absence of WGA labelling was not observed during ingestion of bacteria and latex beads, suggesting a specific relationship existed between yeast cells and WGA receptors.

Binding Sites↗
PubMed1980

Relationships between anionic sites and lectin receptors in the plasma membrane of Dictyostelium discoideum and their role in phagocytosis.

The disappearance of Wheat Germ Agglutinin (WGA) receptors from the membrane of yeast-engulfing-phagocytic cups in Dictyostelium suggested that these receptors could play a role in yeast adsorption or ingestion. This problem was approached by comparing the fate of WGA, Concanavalin A (Con A) and cationized ferritin (CF) and their effects on the phagocytosis of yeast, bacteria and latex beads. It can be concluded that CF capped in about 30 min and inhibited phagocytosis of any kind of particles for about 15 min. Con A capped in 20--60 min and inhibited phagocytosis of all particles for 1 h 30 min. The time at which phagocytosis started to occur corresponded approximately to the moment at which large areas of plasma membrane were totally devoid of marker. WGA did not cap but induced the formation of large and tight aggregates. The surface of the peripheral cells progressively released WGA in 1 h 30 min. Afterwards, the cells were able to ingest latex beads and bacteria but did not phagocytoze yeast. The latter started to be adsorbed onto the cells and to be ingested only 1 h later. Double labelling experiments showed that CF and Con A receptors were still absent in the plasma membrane, when phagocytosis of any kind of particles started to occur. WGA-labelled cells ingested latex beads and bacteria when their plasma membrane was still devoid of WGA receptors but were able to ingest yeast only after their regeneration. These observations strongly suggest that WGA receptors may correspond to specific receptors for yeast phagocytosis.

Binding Sites↗
PubMed1980

Non-specific binding by macrophages: existence of different adhesive mechanisms and modulation by metabolic inhibitors.

Rat peritoneal cells were made to bind test particles of five different species: immunoglobulin-coated sheep red cells (IGSRC), glutaraldehyde-treated sheep red cells (GSRC), leishmania, latex beads and tumour cells. The dependence of binding on various physicochemical parameters was studied: the binding of latex or leishmania resisted cold (4 degrees), azide, cytochalasin B, ethyleneglycol and dimethylsulphoxide (DMSO). The binding of IGSRC resisted cold and azide, but it was inhibited by cytochalasin B, ethyleneglycol and DMSO. The binding of GSRC was inhibited by cold, azide and ethyleneglycol, but not by cytochalasin B and DMSO. The binding of tumour cells was inhibited by azide, cytochalasin B, ethyleneglycol and DMSO. An attempt was made to saturate selectively some adhesive sites of macrophage membranes: glutaraldehyde-treated bovine albumin (GBSA) inhibited the ingestion of latex and GSRC, not leishmania and IGSRC, whereas a crude leishmania extract (CLE) inhibited the ingestion of all tested particles except latex. Antigen-antibody complexes inhibited the ingestion of IGSRC and leishmania, not latex and GSRC. Rat macrophages were made to bind radio-iodinated GBSA (GIBSA). The uptake of glutaraldehyde-treated albumin was proportional to the amount of substrate the cells were incubated with over a wide concentration range. This uptake was not a result of endocytosis, and it was far more efficient than that of peroxidase. Macrophage-particle interaction was studied with electron microscopy. The binding of IGSRC and leishmania to macrophages involved large areas where the interacting membranes were separated by a low density gap of constant width. The interaction of GSRC and latex with macrophages was much more patchy and irregular. Further, no redistribution of the macrophage surface polysaccharides seemed associated with the binding of GSRC. It was concluded that several different mechanisms and different membrane adhesive structures are involved in non-specific recognition by macrophages. Further, non-specific binding sometimes requires an active cell participation. Several testable hypotheses are described, which suggest further experiments in order to obtain a fuller insight into the mechanism of rosette formation.

Animals↗
PubMed1979

Peptidoglycan turnover during growth of a Bacillus megaterium Dap- Lys- mutant.

The aim of this study was to ascertain whether or not the absence of cell wall growth zones, deduced from the analysis of autoradiographs of DL-[3H]mesodiaminopimelic acid pulse-labeled cells of a Dap- Lys- mutant of Bacillus megaterium, was due to a high peptidoglycan turnover. Turnover was determined in very precise experimental conditions because two kinds of turnover occurred: a low, acid-soluble turnover and a high, acid-insoluble one. The latter was detected during a chase in the culture medium when bacteria were centrifuged before treatment with trichloroacetic acid. Otherwise the acid-insoluble released material precipitated with the bacteria. In the electron microscope this material presented a globular structure and contained both peptidoglycan and teichoic acid. The acid-insoluble turnover was mainly produced by a lytic acitivity that was released into the culture medium. This thermolabile activity was not due to cell lysis. It was implicated in septum cleavage and in the detachment of wall fragments from the cell surface, but did not seem indispensable for cell elongation. The acid-soluble turnover was much weaker and seemed to be indispensable for cell elongation.

Bacillus megaterium↗
PubMed1978

Uracil incorporation in the forespore and the mother cell during spore development in Bacillus subtilis. Autoradiographic electron microscopic study.

The transcriptional activity of the two genomes of the sporangium during spore formation was determined by pulse-labeling bacteria with 3H-uracil at different times of sporulation and preparing them for high resolution autoradiography. The quantitative analysis of autoradiographs shows that uracile incorporation in the whole sporangium decreases considerably between stages II and IV. However, the variations of the transcriptional activity are not identical in the mother cell and in the forespore. The one of the mother cell decreases rapidly between stages II and III and then remains stable until the end of stage IV, whereas that of the forespore which is low at stage II increases as the forespore grows ovoid and then quickly diminishes. It is very weak at the beginning of stage IV and negligible at the end of this stage. Pulse-chase experiments made in the presence of rifampine indicate that about 30% of the uracile incorporated is located in stable RNA. This value is found at any stage of sporulation in both cellular compartments whatever their rate of uracile incorporation. A relationship can be made between the nuclear shape and the activity of the genetic material. This confirms observations made by several authors in other bacterial species and other physiological conditions that the condensed shape corresponds to a state of low transcriptional activity whereas the more irregular and dispersed shape corresponds to a state of high activity.

Autoradiography↗