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PubMed1999

Characterization by gas chromatography/mass spectrometry of sterols in saccharomyces cerevisiae during autolysis.

Yeast autolysis affects membrane stability and induces a release of vacuolar enzymes into the cell cytoplasm. Consecutively, it was important to study the evolution of sterol content in Saccharomycescerevisiae for a fourteen day period of accelerated autolysis. Unesterified and esterified sterols were analyzed both in the biomass and in the autolysis medium. Ten sterols were identified by gas chromatography/mass spectrometry. A second group of six sterols was separated and partially characterized. Among the first group of 10 sterols, a dehydroergosterol was identified as ergosta-5, 7,9(11),22-tetraen-3beta-ol, not yet charaterized in S. cerevisiae. Yeast autolysis induced a decrease of esterified sterol content, especially first intermediates in the sequence of the ergosterol biosynthesis, as zymosterol. In contrast, the yeast autolysis resulted in the release of a low quantity of sterols into the medium. At the end of the fourteenth day of autolysis, 0.015% of the total sterol content of the initial biomass was found in the medium.

Autolysis↗
PubMed1996

Autolysis parallels activation of mu-calpain.

The kinetics of autolysis and activation of mu-calpain were measured with microtubule-associated protein 2 (MAP2) as a very sensitive substrate. The initial rate of MAP2 hydrolysis was found to be a linear function of the autolysed 76 kDa form of mu-calpain large subunit at both 10 and 300 microM Ca2+, and both straight lines intersected the origin. This finding supports the view that native mu-calpain is an inactive proenzyme and that activation is accompanied by autolysis. The first-order rate constant of autolysis, K1(aut), was determined at different Ca2+ concentrations: the half-maximal value was at pCa2+ = 3.7 (197 microM Ca2+), whereas the maximal value was 1.52 s-1, at 30 degrees C. The Ca(2+)-induced activation process was then monitored by using our novel, continuous fluorimetric assay with labelled MAP2 as substrate. The first-order rate constant of activation, k1(act), was derived as the reciprocal of the lag phase ('transit time') at the initial part of the progress curve: half-maximum was at pCa2+ = 3.8 (158 microM Ca2+) and the maximum value was 2.15 s-1. The good agreement between the kinetic parameters of mu-calpain autolysis and activation is remarkable. We claim that this is the first kinetically correct determination of the rate constant of autolysis of mu-calpain. Pre-activated mu-calpain has a Ca2+ requirement that is almost three orders of magnitude smaller [half-maximal activation at pCa2+ = 6.22 (0.6 microM Ca2+)]. We cannot exclude the possibility that the activation process involves other mechanistic steps, e.g. the rapid dissociation of the mu-calpain heterodimer, but we state that in our conditions in vitro autolysis and activation run in close parallel.

Autolysis↗
PubMed1972

Influence of macromolecular biosynthesis on cellular autolysis in Streptococcus faecalis.

The addition of several different antibiotics to growing cultures of Streptococcus faecalis, ATCC 9790, was found to inhibit autolysis of cells in sodium phosphate buffer. When added to exponential-phase cultures, mitomycin C (0.4 mug/ml) or phenethyl alcohol (3 mg/ml) inhibited deoxyribonucleic acid synthesis, but did not appreciably affect the rate of cellular autolysis. Addition of chloramphenicol (10 mug/ml), tetracycline (0.5 mug/ml), puromycin (25 mug/ml), or 5-azacytidine (5 mug/ml) to exponential-phase cultures inhibited protein synthesis and profoundly decreased the rate of cellular autolysis. Actinomycin D (0.075 mug/ml) and rifampin (0.01 mug/ml), both inhibitors of ribonucleic acid (RNA) synthesis, also reduced the rate of cellular autolysis. However, the inhibitory effect of actinomycin D and rifampin on cellular autolysis was more closely correlated with their concomitant secondary inhibition of protein synthesis than with the more severe inhibition of RNA synthesis. The dose-dependent inhibition of protein synthesis by 5-azacytidine was quickly diluted out of a growing culture. Reversal of inhibition was accompanied by a disproportionately rapid increase in the ability of cells to autolyze. Thus, inhibition of the ability of cells to autolyze can be most closely related to inhibition of protein synthesis. Furthermore, the rapidity of the response of cellular autolysis to inhibitors of protein synthesis suggests that regulation is exerted at the level of autolytic enzyme activity and not enzyme synthesis.

Alcohols↗
PubMed2004

A simple method for the prevention of endometrial autolysis in hysterectomy specimens.

AIMS: Uteri are among the most common surgical pathology specimens. Assessment of the endometrium is often difficult because of pronounced tissue autolysis. This study describes a simple method to prevent endometrial autolysis and aid in interpretation of the endometrium. METHODS: Sixty uteri were injected with formalin using a needle and syringe directed alongside a probe, which was inserted through the external cervical os into the endometrial cavity. Injection was performed on the same day as removal of the uterus. As controls, 60 uteri that were not injected with formalin were examined. The degree of endometrial autolysis was assessed on a four point scale (0-3), with a score of 0 representing no or minimal autolysis and a score of 3 representing extensive autolysis, such that histological interpretation of the endometrium was impossible. RESULTS: In the injected group, the number of cases with scores of 0, 1, 2, and 3 was 42, 13, four, and one, respectively. The corresponding values for the control group were 17, 23, eight, and 12, respectively. This was highly significant (p < 0.001) CONCLUSIONS: There was significantly less endometrial autolysis in uteri injected with formalin. The use of this simple procedure should be encouraged in hysterectomy specimens.

Adult↗
PubMed2001

Immunohistochemistry of PrPsc within bovine spongiform encephalopathy brain samples with graded autolysis.

Bovine spongiform encephalopathy (BSE) is a transmissible neurodegenerative disease of cattle. Clinical diagnosis can be confirmed by investigation of both spongiform changes and abnormal prion protein (PrPsc), a marker considered specific for the disease. Tissue autolysis, often unavoidable in routine field cases, is not compatible with histological examination of the brain even though PrPsc is still detectable by immunoblotting. To determine how autolysis might affect accurate diagnosis using PrPsc immunohistochemistry, we studied 50 field samples of BSE brainstem (obex) with various degrees of autolysis. We demonstrated that the antigen-unmasking pretreatments necessary for PrPsc immunohistochemistry were compatible with the preservation of autolyzed brain sections and that PrPsc detection was unaffected by autolysis, even though anatomic markers were sometimes lost. In tissue samples in which anatomic sites were still recognizable, PrPsc accumulation was detected in specific gray matter nuclei. In samples with advanced autolysis, PrPsc deposits were still observed, at least at the cellular level, as an intraneuronal pattern. We found that the sensitivity of PrPsc immunohistochemistry as a diagnostic method for BSE was undiminished even by severe tissue autolysis.

Animals↗
PubMed1986

Catecholamines in the vitreous fluid and urine of guinea pigs dying of cold and the effect of postmortem freezing and autolysis.

Concentrations of catecholamines in vitreous fluid and urine in guinea pigs dying of cold and the effects of freezing and autolysis on these parameters were studied. The analysis was performed by high performance liquid chromatography with electrochemical detection. Noradrenaline (NA) concentration in vitreous fluid was more than 20 times higher in the cold exposed animals than in controls (44.2 +/- 9.2 versus 2.0 +/- 1.0 ng/mL). Autolysis alone caused an increase to 33.5 +/- 7.7 ng/mL, and freezing alone to 13.4 +/- 5.3 ng/mL. The highest values were in the group with exposure, freezing, and autolysis. Adrenaline (A) concentration in the vitreous fluid increased fourfold (3.9 +/- 1.5 versus 0.7 +/- 0.5 ng/mL) in cold exposure and twofold as a result of autolysis. Dopamine (DA) concentration in vitreous fluid was elevated only in the group with exposure, freezing, and autolysis. The increase of NA concentration in urine was fivefold during the whole exposure (from 19.4 +/- 6.9 to 109 +/- 57.3 ng/mL), but A was increased by twentyfold (from 10 +/- 5.1 to 213.2 +/- 168.7 ng/mL), whereas DA concentration did not change. The increase of average excretion of NA to urine was eightfold during the first 6 h of exposure, and that of A tenfold. According to the present results, elevated concentrations of catecholamines in the vitreous fluid and urine can be used as a diagnostic aid for hypothermia death. Concerning the values of noradrenaline in the vitreous, the increase as a result of autolysis must be taken in account when interpreting the results.

Animals↗
PubMed2006

Role of hydrolytic enzymes and oxidative stress in autolysis and morphology of Blakeslea trispora during beta-carotene production in submerged fermentation.

The role of hydrolytic enzymes (proteases and chitinase) and oxidative stress in the autolysis and morphology of Blakeslea trispora during beta-carotene production from a chemically defined medium in shake flask culture was investigated. The process of cellular autolysis was studied by measuring the changes in biomass dry weight, pH, concentration of beta-carotene, specific activity of the hydrolytic enzymes and micromorphology of the fungus using a computerized image analysis system. In addition, the phenomenon of autolysis was associated with high concentrations of reactive oxygen species (ROS). The accumulation of ROS produced during fermentation causes oxidative stress in B. trispora. Oxidative stress was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). The profile of the specific activities of the above enzymes appeared to correlate with the oxidative stress of the fungus. The high activities of CAT and SOD showed that B. trispora is found under oxidative stress during beta-carotene production. The culture began to show signs of autolysis nearly in the growth phase and autolysis increased significantly during the production phase. The morphological differentiation of the fungus was a result of the degradation of the cell membrane by hydrolytic enzymes and oxidative stress. Increased beta-carotene production is correlated with intense autolysis of clumps, which has as a consequence the increase of the freely dispersed mycelia.

Biotechnology↗
PubMed2000

Role of proteases in autolysis of Penicillium chrysogenum chemostat cultures in response to nutrient depletion.

An industrial strain of Penicillium chrysogenum was subjected to carbon or nitrogen limitation in a chemostat and the response monitored in terms of the "classical" indicators of autolysis (biomass decline and ammonia release), culture degradation (as measured by image analysis) and by obtaining profiles for three classes of proteases implicated in autolysis. Under both sets of conditions (carbon or nitrogen limitation), once started, autolysis involved a succession of different protease activities. The first stages of the process of autolysis in starved chemostat cultures was associated with peaks in the activities of both serine and aspartyl proteases, coinciding with the mobilisation of endogenous energy reserves. Conversely, a peak in the activity of metalloproteases was associated with the later stages of autolysis, perhaps occurring in response to depletion of endogenous energy reserves; the activity of these enzymes led to gross culture degradation, disintegration of ordered mycelial structures and signalled the end of metabolic activity (respiration) within the culture. These findings indicate that strategies intended to control/regulate autolysis in large-scale industrial fungal cultures might profitably be focused on regulation of the activity of key classes of proteases involved in the series of events leading to culture degradation.

Ammonia↗
PubMed2001

Structural and ultrastructural changes in yeast cells during autolysis in a model wine system and in sparkling wines.

This study shows the changes that occur during the autolysis of yeast in a model wine medium and in a sparkling wine after 12 months of aging, using Nomarsky Light Microscopy and Low Temperature Scanning Electron Microscopy (LTSEM). The size of the yeasts after 24 h of autolysis in a model medium is much smaller than when they are in the growth stage. With LTSEM. a large number of folds can be observed on the surface of the yeast and practically empty cells. Greater morphological changes, both structural and ultrastructural, can be observed in the yeast after 12 months of aging in wine than in the yeast after 24 h of induced autolysis. However, less of the cytoplasmic content of the yeast that has undergone autolysis in the wine was solubilized than that of the yeast after 24 h of autolysis in the model wine system. These findings indicate that autolysis of yeast in wine is a long-lasting process, which continues for at least 12 months.

Bacteriolysis↗
PubMed1998

Purification of native p94, a muscle-specific calpain, and characterization of its autolysis.

p94, a skeletal muscle-specific calpain, has attracted much attention because its gene is responsible for limb-girdle muscular dystrophy type 2A. p94, however, has not been characterized at the protein and enzyme levels, owing to its very rapid autolysis. In the present study, a purification procedure for p94 was first established by using a recombinant inactive p94 expressed in COS cells in which the active site cysteine residue was changed to serine [p94(C129S)]. The isolation of native p94 from rabbit skeletal muscle by the established method with conventional procedures was extremely difficult because p94 became highly unstable in a crude extract on the addition of NaCl for separation. Purification of native p94 was possible with an antibody-affinity column but only as an inactive enzyme; p94(C129S) was purified as a homodimer. Characterization of p94, especially autolysis, was performed with partly purified native p94 and p94(C129S). The autolysis of p94, which consisted at least partly of an intermolecular reaction, proceeded in three consecutive steps; 60 and 58 kDa fragments were produced as intermediates before a stable 55 kDa fragment appeared. Autolysis of p94 was regarded as a degradative step rather than for the activation of the enzyme. All the autolysis cleavage sites were located in the p94-specific insertion sequence 1 region, which explains why p94 is unstable compared with the other calpains. The autolysis sites in p94 clearly showed a different specificity relative to the autolytic and proteolytic cleavage sites of the ubiquitous mu- and m-calpains, in its preference for residues at the P3 to P1' sites, indicating a distinct substrate specificity and function for the muscle enzyme.

Amino Acid Sequence↗
PubMed1976

Autolysis in strains of viridans streptococci.

Seven strains of viridans streptococci of the species Streptococcus sanguis, S. mutans and S. mitis were investigated for autolysis. The effect of pH, salt concentration and temperature on the autolytic process was studied in Na2HPO4/NaH2PO4 buffer. Whole cells and walls of all strains autolysed most rapidly at pH values above 7. Autolysis of whole cells of S. sanguis and one strain of S. mitis (ATCC15909) was maximal in 0-05 TO 0-2 M buffer, while the two S. mutans strains and S. mitis ATCC15912 showed maximal autolysis in 0-5 and 1-0 M buffers. Cultures harvested in the stationary phase of growth possessed only slightly decreased autolytic activity compared with those from the exponential phase. Whole cells autolysed more rapidly at 37 degrees C Than at 45 degrees C and 10 degrees C. Autolysis of isolated walls of three strains of S. mitis (ATCC903, ATCC15909 and ATCC15912) was maximal at pH 7-0 AND 7-5 and in 1-0 M buffers. Streptococcus mitis ATCC15909 also showed maximal lysis in 0-01 M and 0-5 M buffers. An endopeptidase action of the autolytic system of S. mitis ATCC15912 was indicated by the progressive release of soluble amino groups during autolysis of the walls. No release of reducing groups was observed. Several free amino acids were released during autolysis of these walls, alanine, lysine and glutamic acid being in greatest quanitity.

Amino Acids↗
PubMed2006

Transcriptomic and functional analysis of an autolysis-deficient, teicoplanin-resistant derivative of methicillin-resistant Staphylococcus aureus.

The molecular basis of glycopeptide-intermediate S. aureus (GISA) isolates is not well defined though frequently involves phenotypes such as thickened cell walls and decreased autolysis. We have exploited an isogenic pair of teicoplanin-susceptible (strain MRGR3) and teicoplanin-resistant (strain 14-4) methicillin-resistant S. aureus strains for detailed transcriptomic profiling and analysis of altered autolytic properties. Strain 14-4 displayed markedly deficient Triton X-100-triggered autolysis compared to its teicoplanin-susceptible parent, although microarray analysis paradoxically did not reveal significant reductions in expression levels of major autolytic genes atl, lytM, and lytN, except for sle1, which showed a slight decrease. The most important paradox was a more-than-twofold increase in expression of the cidABC operon in 14-4 compared to MRGR3, which was correlated with decreased expression of autolysis negative regulators lytSR and lrgAB. In contrast, the autolysis-deficient phenotype of 14-4 was correlated with both increased expression of negative autolysis regulators (arlRS, mgrA, and sarA) and decreased expression of positive regulators (agr RNAII and RNAIII). Quantitative bacteriolytic assays and zymographic analysis of concentrated culture supernatants showed a striking reduction in Atl-derived, extracellular bacteriolytic hydrolase activities in 14-4 compared to MRGR3. This observed difference was independent of the source of cell wall substrate (MRGR3 or 14-4) used for analysis. Collectively, our results suggest that altered autolytic properties in 14-4 are apparently not driven by significant changes in the transcription of key autolytic effectors. Instead, our analysis points to alternate regulatory mechanisms that impact autolysis effectors which may include changes in posttranscriptional processing or export.

Bacteriolysis↗
PubMed1975

Autolysis of Neisseria gonorrhoeae.

Physiological conditions that would provide maximal rates of autolysis of Neisseria gonorrhoeae were examined. Autolysis was found to occur over a broad pH range with the optimum at pH 9.0 IN 0.05 M tris(hydroxymethyl)amino-methane-maleate buffer. The temperature optimum was found to be 40 C. Potassium ions greatly stimulated autolysis at a concentration of 0.01 M. Exposure of growing N. gonorrhoeae cells to penicillin, vancomycin, or D-cycloserine influenced the susceptibility to the autolysis, whereas chloramphenicol afforded some protection against autolysis. The primary structure of the peptidoglycan is composed of muramic acid/glutamic acid/alanine/diaminopimelic acid/glucosamine in approximate molar ratios of 1:1:2:1:1, respectively. Exogenous radioactive diaminopimelic acid, D-glucosamine, and D-alanine were incorporated into peptidoglycan. During autolysis these radioactive fragments were released from cells.

Alanine↗
PubMed1985

Correlation between degradation and ultrastructure of peptidoglycan during autolysis of Escherichia coli.

The kinetics of peptidoglycan degradation were examined under different conditions of autolysis of Escherichia coli. With cephaloridine- or moenomycin-induced autolysis, degradation did not exceed 25 to 35%, whereas in EDTA-induced autolysis it rapidly reached 65 to 70%. When nonautolyzing cells were fixed overnight with glutaraldehyde, followed by an osmium fixation, and thin sections were stained by the phosphotungstic acid method, a dark, 15-nm-thick layer of uniform appearance and constant width occupied the whole area between the inner and outer membranes of the envelope. The stained material was tentatively identified with peptidoglycan. Ultrastructural changes in this phosphotungstic acid-stained periplasmic space were investigated at different time intervals after induction of autolysis. In all cases, breakdown proceeded over the whole cell surface. During antibiotic-induced autolysis a progressive thinning down limited to the inner side of the layer was observed. During EDTA-induced autolysis, the rapid decrease in thickness correlated well with the important loss of material labeled with [3H]diaminopimelic acid. Considering these changes and the insufficient amounts of peptidoglycan (1.3 U/nm2) necessary to account for a regularly structured polymer occupying the whole 15-nm layer, it was speculated that peptidoglycan might be unevenly distributed throughout the periplasmic space.

Anti-Bacterial Agents↗
PubMed1999

Autolysis of Listeria monocytogenes.

Physiological conditions that could provide maximal rates of autolysis of Listeria monocytogenes were examined. L. monocytogenes was found to be refractory to most treatments that promote rapid autolysis in other bacteria. Best rates of autolysis were obtained after resuspending the cells in Tris-hydrochloride buffer at 37 degrees C with the pH optimum at 8.0. Autolysis was also efficiently promoted by the surfactant Triton X-100. Antibiotics that interfere with the biosynthesis of the cell wall murein (peptidoglycan) caused death of the cells without autolysis after prolonged incubation in the presence of the drug. Only nisin, which has been shown to bind in vitro to the murein precursors lipid I and lipid II brings about autolysis of L. monocytogenes cells, although with slower kinetics than in the case of Tris-HCl and Triton.

Anti-Bacterial Agents↗
PubMed1979

Investigation of the short-time autolysis of rat hearts by means of SDS. Polyacrylamide gel electrophoresis and electron microscopy.

The short-time autolysis of hearts was regarded as a model of ischaemic heart failure. Therefore, isolated rat hearts were subjected to 30--120 min autolysis in a Locke solution at 37 degrees C. Electron microscopic examinations and myofibrillar preparations were made from the autolysed heart ventricles. The myofibrillar proteins were resolved by SDS-polyacrylamide gel electrophoresis. After 30 min autolysis the amount of a protein of 192,000 daltons greatly increased. At the same time on the electron micrographs the focal destruction of filament destruction on the A filament area and the mitochondrial structure altered too. After 60 min autolysis another protein of 36,400 daltons appeared. On the electron micrographs the focal desintegration of Z membranes and the focal destruction of I filaments can be observed. After 120 min autolysis further proteolytic products could not be detected by gel electrophoresis but on the electron micrographs the destruction of Z membranes and I filaments became more pronounced.

Animals↗
PubMed1998

Pleiotropic mutations alter the kinetics of calcium transport, competence regulation, autolysis and experimental virulence in Streptococcus pneumoniae.

Streptococcus pneumoniae is a pathogen in which the extracellular calcium concentration plays a major physiological role, in growth as well as in the induction of competence for genetic transformation and activation of autolysis. Both responses are under the control of a protein activator exported in the medium. We have checked the impact of mutations which alter the regulation of competence and autolysis on experimental virulence. Isogenic encapsulated derivatives carrying the relevant mutations were serotype 3 smooth clones, obtained by transformation of the relevant rough strains with DNA from a serotype 3 smooth isolate. Survival kinetics and bacterial clearance from the blood were followed after intraperitoneal infection of Swiss mice with the different bacterial cultures. In this model, mutants showing an attenuation of virulence relative to the wild type fell into two classes. In the first, represented by the lytA::ery mutant V1095 defective for calcium-induced autolysis, attenuated virulence could be correlated with rapid bacterial clearance from the blood. In the second, represented by the dmb mutants V2200 and V3300, attenuation was associated with delayed bacterial clearance from the blood, and correlated with altered kinetics of calcium transport and of regulation of competence and autolysis. It appeared unlikely that attenuation of virulence for strains V2200 and V3300 was a direct consequence of their competence phenotype, since the com::ery mutants V1008 and V1019, defective for the production of the competence activator, were as virulent as the wild-type strain. Autolysis involving an N-acetyl-muramyl-alanine amidase encoded by lytA was also regulated by calcium. The inserted allele lytA0::ery further reduced virulence in the dmb1 background (V2200). This additive effect of lytA- to dmb1 points to different routes of virulence regulation by LYT and DMB1 and suggests that the kinetics of calcium traffic controls several pathways involved in the virulence of pneumococcus.

Animals↗
PubMed2003

Rapid monitoring of autolysis process of proteases by capillary electrophoresis.

A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 degrees C-50 degrees C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 degrees C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 degrees C. Similar results were obtained when MCP-01 was incubated at 20 degrees C, 40 degrees C and 50 degrees C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.

Autolysis↗