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M T Silva

Publications and source records attributed to M T Silva.

At least 73 records · Page 4Linked to original sources

In vivo killing and degradation of Mycobacterium aurum within mouse peritoneal macrophages.

We studied the in vivo killing and degradation of Mycobacterium aurum, a nonpathogenic, acid-fast bacillus, within macrophages after inoculation into the peritoneal cavity of CD-1 mice. The degradative process could be divided in five successive steps that were characterized on ultrastructural and cytochemical grounds and the relative contributions of which were determined by quantitative electron microscopy of samples taken at different times. The main ultrastructural alterations observed during the degradative process were ribosome disaggregation, coagulation of the cytoplasmic matrix, and change in the membrane profile from asymmetric to symmetric, with loss of the polysaccharide components from the outer layer, followed by membrane solubilization and intracellular clearing, followed by digestion of the innermost (peptidoglycan) layer of the cell wall, and at the end of the process, disorganization and collapse of the remaining layers of the cell wall. The correlation between viability and morphology indicated that the first ultrastructural signs of viability loss are cytoplasmic coagulation, change in the membrane geometry, and disappearance of ribosomes. The labeling of lysosomes of peritoneal macrophages with ferritin or by the cytochemical demonstration of inorganic trimetaphosphatase showed that fusion of lysosomes with phagosomes containing mycobacteria occurs in the phagocytes in the mouse peritoneal cavity and is already extensive as soon as 1 h after the inoculation of the bacilli.

Animals↗

Improved Thiéry staining for the ultrastructural detection of polysaccharides.

The use of silver vitellinate instead of silver proteinate in the Thiéry's staining for the ultrastructural detection of polysaccharides has advantages which derive from the lesser reactivity and higher stability of silver vitellinate solutions, allowing the obtention of a finer staining of periodic acid-Schiff (PAS)-positive materials with a cleaner background. The proposed method is particularly useful for the staining of PAS-positive cellular components at high resolution.

Animals↗

Ultrastructure of bacilli and the bacillary origin of the macrophagic inclusions in Whipple's disease.

An electron microscopic and cytochemical study of the Whipple bacillus in jejunal biopsies from three untreated patients was made using fixation procedures developed for the satisfactory preservation of bacterial ultrastructure. The envelopes of the normal-looking bacilli present free in the lamina propria consisted of the following layers. (i) A cytoplasmic membrane with a triple-layered profile and a mean thickness (peak-to-peak distance) of 6.08 nm. (ii) A thick (20 nm) cell wall containing peptidoglycan; the wall had a hitherto undescribed inner layer that contained polysaccharides, possibly teichoic acids. (iii) Surrounding the cell wall, a surface membrane with a symmetric profile and a mean peak-to-peak distance of 4.74 nm. The ultrastructural pattern of the Whipple bacillus wall corresponds to that of Gram-positive bacteria, but with an additional surface membrane. This membrane is different from the outer membrane of Gram-negative bacteria because it has a symmetric profile, is thinner and has no periodic acid-Schiff (PAS)-positive components. Normal-looking bacilli were seen very rarely inside jejunal macrophages, but degenerating bacteria were abundant in these phagocytes. Electron microscopy and ultrastructural cytochemistry of Whipple bacilli inside jejunal macrophages of the three untreated patients showed that the degenerative process is a sequence that leads to the loss of bacillary forms and to the accumulation of bacterial remnants resistant to degradation by the macrophage. These remnants correspond to the innermost, polysaccharide-containing portion of the bacillus wall. The progressive accumulation of these PAS-positive wall remnants is the origin of the intramacrophagic inclusions that are important in the histological diagnosis of Whipple's disease. The reported results indicate that in the three patients studied, the Whipple bacillus multiplies extracellularly, the bacteria that are phagocytosed by macrophages being degraded.

Bacillus↗

Ultrastructural characterization of normal and damaged membranes of Mycobacterium leprae and of cultivable mycobacteria.

Microdensitometry showed that the membrane profiles of normal cultivable mycobacteria were very asymmetric (outer layer denser and thicker than the inner layer), while the profiles of normal-looking M. leprae in lepromatous patients, in experimentally infected armadillos and in nude mice were approximately symmetric; moreover, the membrane of M. leprae was thicker than that of cultivable species. Using two cytochemical methods for the ultrastructural detection of periodic acid-Schiff (PAS)-positive molecules (the Thiéry procedure, and staining with phosphotungstic acid at low pH) we found that the membrane of cultivable mycobacteria, growing in vitro or in vivo, had PAS-positive components exclusively in the outer layer, while the normal-looking M. leprae in patients and in armadillos had membranes with PAS-positive components in both layers. The membranes of damaged cultivable mycobacteria, in vivo or in vitro, and of damaged M. leprae, in patients or armadillos, were PAS-negative.

Animals↗

A turbidimetric and electron microscopic study of the effects of several parameters on the lysis of Streptococcus faecalis by lysozyme.

The lytic effect of lysozyme on Streptococcus faecalis ATCC 9790 was studied by spectrophotometry and electron microscopy and it was found to be highly dependent on the ionic strength of the suspending media and on the ratio lysozyme to bacterial cell mass. When 7.2 X 10(8) bacteria/mL are exposed to 0.4 mg/mL of lysozyme in media with low ionic strength, the enzyme is bound in great amounts, as deduced from protein determinations and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS--PAGE); the binding prevents bacteriolysis in spite of the removal of the cell wall. Extensive lysis of S. faecalis could be obtained by reducing the ratio of lysozyme to bacterial cell mass. Stabilization of S. faecalis by lysozyme was also observed when exponential phase cells incubated under conditions that promote spontaneous autolysis (incubation in 0.05 M tris(hydroxymethyl)aminomethane buffer, pH 8.0, ionic strength = 0.01675) do not lyse and do not leak material which absorbs at 260 nm when lysozyme was present at the highest concentration.

Enterococcus faecalis↗

Electron microscopic study of Mycobacterium leprae membrane.

We report the results of the study by transmission electron microscopy of normal Mycobacterium leprae in the tissues of experimentally infected armadillos. Several fixation procedures were used and compared to those previously employed in the study of M. leprae in lepromatous leprosy patients. The results show that the ultrastructure of M. leprae is identical in both hosts. The demonstration of a symmetric membrane profile in M. leprae in armadillos confirms our previous results. This characteristic of the M. leprae membrane is peculiar in that it is not shared by any of the easily cultivable species of mycobacteria we have studied so far.

Animals↗

The interpretation of the ultrastructure of mycobacterial cells in transmission electron microscopy of ultrathin sections.

In the present report the authors discuss several aspects of the ultrastructure of mycobacterial cells as seen by transmission electron microscopy of ultrathin sections that are relevant in the characterization of normal versus altered bacteria. The importance of the use of adequate fixation conditions is stressed and illustrated with examples showing that normal, but inadequately fixed, mycobacterial cells may exhibit micromorphological alterations similar to those typical of cells affected in several situations such as autolysis, heterolysis, and antibacterial treatments.

Humans↗

A Bacillus subtilis mutant requiring dipicolinic acid for the development of heat-resistant spores.

A Bacillus subtilis mutant is described which forms heat-resistant spores only in the presence of external dipicolinic acid (DPA). The mutation, dpa-1, is localized in a new sporulation locus, linked to pyrA. The dpa-1 strain is unable to synthesize DPA but can incorporate external DPA. The amount of DPA incorporated, the frequency of heat-resistant spores and their degree of resistance are all dependent on the concentration of external DPA. Spores of dpa- 1 strains exhibit normal resistance to most chemicals, including octanol and chloroform, but not to ethanol, pyridine, phenol and trichloroacetic acid. Complete resistance to the latter group depends on DPA. DPA incorporation is slow and apparently requires an energy supply but not protein synthesis. Direct involvement of DPA in the heat-resistance of the spores is suggested. Thin sections of DPA-less spores exhibit clearly visible cytoplasmic membranes and ribosomes. These structures are absent or less visible in the core of spores obtained with added DPA.

Bacillus subtilis↗

Decadent sporulation mutants of Bacillus subtilis.

In decadent sporulation mutants, sporulating populations are heterogeneous: the cells reach successive chemical and physical resistances with progressively decreasing frequencies. Each decadent mutant can be characterized by the shape and slope of the curve describing the frequency of cells resistant to various agents ('the resistance spectrum'). In some mutants the resistance spectrum decreases progressively from xylene resistance to heat resistance; in other mutants it decreases rapidly between octanol resistance and chloroform resistance. Electron microscopy showed that in two mutants the majority of the cells are blocked at stages III and IV; the number of cells that develop further to reach successive morphological stages falls off progressively. In two other mutants most cells reach stage V. Cortexless spores are also frequent. One of the decadent mutations, SpoL1, was localized between aroD and acf. The phenotype of decadent mutants is discussed in terms of sequential gene activation.

Bacillus subtilis↗

Genetic instability of sporulation-associated characters in a Bacillus subtilis mutant: relationship between sporulation, segregation and the synthesis of extracellular enzymes (kinetic studies).

In the genetically unstable, protease-overproducing 'medusa (M) strains of Bacillus subtilis, segregation of stable, wild-type-like B cells occurred mainly during sporulation. After the end of the exponential growth phase, a small fraction of M cells sporulated quickly and formed M spores, while the majority of the cells, after a 'critical period', gave rise to B segregants which sporulated after a delay. Segregation occurred without cell division. Delayed sporulation, segregation and protease overproduction are related. Similar but more complex results were obtained with the highly unstable TD strains. Sporulation and the kinetics of protease overproduction were also followed in several stable segregants. Depending on the strain, either the rate of protease production or both the rate and time course were affected. The results are interpreted in terms of sequential activation and de-activation of sporulation genes. The production of the alkaline and the neutral proteases was, in general, under common genetic control. In some strains alpha-amylase was also overproduced.

Alleles↗

Effects of local anesthetics on bacterial cells.

The membrane effects of chlorpromazine, nupercain, tetracain, and procain were studied using Bacillus cereus, B. megaterium, B. subtilis, and Streptococcus faecalis, protoplasts from S. faecalis, and isolated membranes from B. subtilis. Chlorpromazin, nupercain, and tetracain produced characteristic micromorphological alterations after treatment for 5 to 30 min at pH 7.0 and 20 degrees C; the membrane staining pattern changed from asymmetric to symmetric, complex mesosome-like structures appeared, and membrane fractures and solubilization occurred. Procain at concentrations up to 100 mM did not induce detectable alterations. Protoplasts were quickly lysed by 10 mM tetracain. A rapid and extensive leakage of K+ was induced by chlorpromazin, nupercain, and tetracain. Procain (100 mM) induced a slight K+ leakage. The membrane respiratory activity of intact B. cereus cells (as measured by the triphenyl tetrazolium reduction) and the succinic dehydrogenase activity of B. subtilis isolated membranes were found to be inhibited by the four local anesthetics. The concentrations that produced 50% inhibition of those activities are correlated with the hydrophobicities of the anesthetic molecules.

Anesthetics, Local↗

Ultrastructure and development of an exosporium-like outer spore envelope in Bacillus subtilis.

An exosporium-like outermost envelope is occasionally observed in thin sections of Bacillus subtilis spores. Treatment of the mature spores with urea and mercaptoethanol (sometimes completed by sodium dodecyl sulfate) or with NaOH, disorganizes and partially solubilizes the outer spore coat. This treatment permits a clear visualization of the exosporium in all spores of several B. subtilis strains. Exosporium appears either as a single sheet, 8-9 nm thick, or with a triple-layered unit membrane-like profile. Frequently it exhibits a crystal-like periodic pattern. The exosporium primordium appears first at stage IV, and its development is apparently independent of the formation of the cortex and of the spore coats. No morphological relationship was found between the outer forespore membrane and the exosporium.

Bacillus subtilis↗

Ultrastructural effects of chemical agents and moist heat on Bacillus subtilis. I--Effects on vegetative cells.

The ultrastructural alterations induced by treatment of vegetative Bacillus subtilis cells with organic solvents, trichloroacetic acid (TCA) and moist heat were examined by electron microscopy. Organic solvents disorganize the membrane and change the asymmetric unit membrane profile to a symmetric profile. They also lead to partial solubilization of the membrane and produce small or extensive gaps. Membrane damaging activity increases in the following order: xylene = toluene less than octanol less than choroform. TCA coagulates the cytoplasm which shows large, electron-dense, pronase-sensitive blocks. Moist heat alters both the membrane and the cytoplasm.

Bacillus subtilis↗

Ultrastructural effects of the chemical agents and moist heat on bacillus subtilis. II.--Effects on sporulating cells.

When sporulating cells are treated with the organic solvents (xylene, toluene, octanol and chloroform), with TCA or with moist heat (10 min at 80 degree C), the sporangial cells exhibit the same ultrastructural changes as do the vegetative cells. The forespores undergo similar changes after early but not after late treatment. Resistance toward the killing effect and toward ultrastructural alterations appear in the same order. One could correlate the appearance of resistances with precise ultrastructural events as follows:--xylene resistance: cortex formation (stage IV);--resistance to toluene, octanol and chloroform: coat development and cortex maturation (respectively early, middle and late stage V);--TCA and heat resistance: spore maturation (stage VI). The possible mechanisms of the chemical resistances are discussed.

Bacillus subtilis↗

Saccharin aversion in the rat following adrenalectomy.

Changes in saccharin preference as a function of concentration were studied in rats after either adrenalectomy of sham surgery. A 48 hr two-bottle preference test was used. Under two different sets of testing conditions, adrenalectomized animals rejected saccharin solutions at concentrations that were highly acceptible to controls and did not show an appetitive behavior towards andy of the concentrations tested. This aversion pattern persisted even when preference tests were conducted for as long as 10 days. However, it was abolished when the adrenalectomized rats wre exposed to saccharin for 6 days prior to surgery, or when they were administered the glucocorticoid dexamethasone. Results are discussed with respect to the effects of adrenal hormones on sensory systems and to taste-aversion learning.

Adrenalectomy↗