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PubMed1987

Myofibrillar degeneration and necrosis of the visceral smooth musculature: an ischemic visceral myopathy.

Pathologic studies of the visceral smooth musculature in humans are scant despite the relatively frequent occurrence of alterations in these muscles in autopsy material. We investigated the different types of lesions of this musculature observed in various conditions associated with ischemia--acute tubular necrosis, congenital heart disease (low output syndrome due to open heart surgery), and necrotizing enterocolitis in premature babies. Control cases included normal rat tissue undergoing autolysis and rigor mortis and bowel resected from patients with ulcerative colitis and Hirschsprung's disease. Four histologically distinct lesions were present on hematoxylin--eosin staining in the ischemic group: contraction bands, wavy fibers, thick waves, and coagulation necrosis. These lesions were absent in the control groups. We conclude that myofibrillar degeneration and necrosis of the visceral musculature are common in disorders associated with visceral ischemia. These changes are not artifacts produced by autolysis, rigor mortis, or technical handling, nor are they induced by nonischemic inflammatory conditions. Catecholamines may play a role in their genesis.

Adolescent↗
PubMed2002

Thioester function is conserved in SpC3, the sea urchin homologue of the complement component C3.

The amino acid sequence of the thioester site in the alpha chain of SpC3, the sea urchin homologue of C3, is conserved. This implies a conserved function of covalent bond formation with amine or hydroxyl groups on target molecules. When coelomic fluid (CF) was incubated with 14C-methylamine, a classic assay for thioester binding function, the alpha chain became labeled. When CF was treated to induce autolysis, peptide bond cleavage occurred at the thioester site. Autolysis could be blocked or reduced by pre-treating CF with either methylamine or yeast, both of which are known to bind to thioester sites C3 proteins from other organisms. The data suggest that SpC3 can bind to target cell surfaces, constituting indirect evidence that it can covalently bind to pathogen surfaces and function as an opsonin in vivo. This activity may be an important aspect of host defense in the sea urchin.

Amino Acid Sequence↗
PubMed1999

Potential definition of the time of death from autolytic myocardial cells: a morphometric study.

Morphometric methods and transmission electron microscopy were used to quantify modifications occurring in the mitochondria of dog myocardium during the first four hours of autolysis. Myocardial fragments were obtained from the outer free wall of the left ventricle, during anesthesia (control-zero) and at 15, 45, 120, and 240 min after cardiac arrest, maintaining the heart "in situ" at 22 degrees C. During the 240 min of autolysis, the main parameters evaluated showed: (a) a decrease in the number of mitochondria from 0.31 to 0.12 per micron 3 of cytoplasm. The decrease over the first 45 min reached 50% of the initial value; (b) an increase in mitochondrial volume, three times greater after the first 45 min (from 0.92 to 2.68 micron 3) and four times greater after 240 min (from 0.92 to 3.79 micron 3); (c) an increase in mitochondrial outer membrane surface area from 5.51 to 12.54 micron 2; (d) an increase in the surface area of individual mitochondria inner membrane and cristae from 27.60 to 56.96 micron 2. The progressive nature of the alterations and the difference in the numerically expressed values allow correlation with the time of somatic death. The authors emphasize the need for further studies in order to complement the present study.

Animals↗
PubMed2001

Increasing the thermal stability of euphauserase. A cold-active and multifunctional serine protease from Antarctic krill.

A molecular model of Antarctic krill euphauserase based on the known crystal structure of its fiddler crab analog, collagenase I, indicates that the core structure of these enzymes is almost identical. Euphauserase is a cold-active and thermally sensitive enzyme with a high affinity for Lys, Arg and large hydrophobic amino acids. Residue Phe137 in euphauserase, localized in loop D (autolysis loop), is highly exposed on the surface of the molecule. Therefore, it appeared to be an easy target for autolysis. The broadly specific euphauserase has a low affinity for negatively charged residues. In order to increase the stability of the enzyme, two mutants were created in which residue Phe137 was replaced by a Glu and an Asp residue. Both mutations resulted in increased stability of the recombinant euphauserase towards thermal inactivation.

Animals↗
PubMed1996

mu-calpain activation and calpain-mediated cytoskeletal proteolysis following traumatic brain injury.

Increasing evidence suggests that excessive activation of the calcium-activated neutral protease mu-calpain could play a major role in calcium-mediated neuronal degeneration after acute brain injuries. To further investigate the changes of the in vivo activity of mu-calpain after unilateral cortical impact injury in vivo, the ratio of the 76-kDa activated isoform of mu-calpain to its 80-kDa precursor was measured by western blotting. This mu-calpain activation ratio increased to threefold in the pellet of cortical samples ipsilateral to the injury site at 15 min, 1 h, 3 h, and 6 h after injury and returned to control levels at 24-48 h after injury. We also investigated the effect of mu-calpain activation on proteolysis of the neuronal cytoskeletal protein alpha-spectrin. Immunoreactivity for alpha-spectrin breakdown products was detectable within 15 min after injury in cortical samples ipsilateral to the injury site. The levels of alpha-spectrin breakdown products increased in a biphasic manner, with a large increase between 15 min and 6 h after injury, followed by a smaller increase between 6 and 24 h after the insult. No further accumulation of alpha-spectrin breakdown products was observed between 24 and 48 h after injury. Histopathological examinations using hematoxylin and eosin staining demonstrated dark, shrunken neurons within 15 min after traumatic brain injury. No evidence of mu-calpain autolysis, calpain-mediated alpha-spectrin degradation, or hematoxylin and eosin neuronal pathology was detected in the contralateral cortex. Although mu-calpain autolysis and cytoskeletal proteolysis occurred concurrently with early morphological alterations, evidence of calpain-mediated proteolysis preceded the full expression of evolutionary histopathological changes. Our results indicate that rapid and persistent mu-calpain activation plays an important role in cortical neuronal degeneration after traumatic brain injury. Our data also suggest that specific inhibitors of calpain could be potential therapeutic agents for the treatment of traumatic brain injury in vivo.

Analysis of Variance↗
PubMed1997

Selective loss of fibrinogen clotting in a loop-less thrombin.

The autolysis loop of thrombin comprises nine residues, from Glu146 to Lys149e, five of which (Ala149a-Lys149e) are inserted relative to trypsin and chymotrypsin. Deletion of the insertion Ala149a-Lys149e causes no significant change in the properties of the enzyme, except for a slight enhancement of protein C activation. Deletion of the entire Glu146-Lys149e loop, however, reduces fibrinogen clotting 240-fold, but decreases protein C activation only 2-fold. This loop-less mutant is de facto an exclusive activator of protein C, having lost the primary procoagulant function of thrombin. Because the autolysis loop affects fibrinogen binding, but not protein C activation, it provides a target for new drugs designed to suppress exclusively the procoagulant activity of thrombin.

Autolysis↗
PubMed1986

The amino-terminal hydrophobic region of the small subunit of calcium-activated neutral protease (CANP) is essential for its activation by phosphatidylinositol.

Ca2+-Activated neutral protease (CANP), that consists of 80K and 30K subunits, is converted to a low-Ca2+-requiring form by autolysis in the presence of Ca2+. Phosphatidylinositol greatly reduces the Ca2+-requirement for the autolysis of native CANP. However, this effect was not observed for CANP with a trimmed 30K subunit lacking the NH2-terminal hydrophobic and glycine-rich region. This suggests that the NH2-terminal hydrophobic region of the 30K subunit is important for the interaction of CANP with the cell membrane and that the calcium sensitivity of CANP is increased at the cell membrane through the effect of phosphatidylinositol.

Amino Acids↗
PubMed1986

Independent folding of autolytic fragments of thermolysin and their domain-like properties.

High molecular weight autolysis products from thermolysin have been isolated and identified. The primary fragments correspond to residues 1 to 187-204 (21kD) and residues 187-204 to 316 (12kD), respectively. The fragments are both capable of independent refolding upon removal of denaturant. On the basis of these results, we suggest that the first step in the unfolding pathway of thermolysin involves unfolding of an interdomain region and domain separation. Bound calcium ions at sites 1, 2 and 4 play a major role in protecting the protein against both autolysis and unfolding, probably by stabilizing the interdomain region and enhancing domain-domain interactions.

Amino Acid Sequence↗
PubMed1986

Bony fish myelin: evidence for common major structural glycoproteins in central and peripheral myelin of trout.

Peripheral nervous system (PNS) myelin from the rainbow trout (Salmo gairdneri) banded at a density of 0.38 M sucrose. The main myelin proteins consisted of (1) two basic proteins, BPa and BPb (11,500 and 13,000 MW, similar to those of trout central nervous system (CNS) myelin proteins BP1 and BP2), and (2) two glycosylated components, IPb (24,400 MW) and IPc (26,200 MW). IPc comigrated with trout CNS myelin protein IP2 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whereas trout CNS myelin protein IP1 had a lower molecular weight (23,000). Following two-dimensional separation, however, both IPb and IPc from PNS showed two components; the more acidic component of IPc comigrated with IP2 from CNS. PNS tissue autolysis led to the formation of IPa (20,000 MW), consisting of two components in isoelectric focusing of which again the more acidic one comigrated with the CNS autolysis product IP0. Limited enzymatic digestion of isolated IP proteins from PNS and CNS led to closely similar degradation patterns, being most pronounced in the case of IP2 and IPc. Immunoblotting revealed that all IP components from trout PNS and CNS myelins reacted with antibodies to trout IP1 (CNS) and bovine P0 protein (PNS) whereas antibodies to rat PLP (CNS) were entirely unreactive. All BP components from trout PNS and CNS myelins bound to antibodies against human myelin basic protein. On the basis of these studies trout PNS and CNS myelins contain at least one common IP glycoprotein, whereas other members of the IP myelin protein family appear closely related. In the CNS myelin of trout the IP components appear to replace PLP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Role of autolysins in the activities of imipenem and CGP 31608, a novel penem, against slowly growing bacteria.

The carbapenem imipenem and the penem CGP 31608 demonstrated unusually good bactericidal activity against slowly growing bacteria. In contrast to that of penicillin, the rate of killing was independent of growth rate. In logarithmically growing cells, a decrease in growth rate was paralleled by a decrease in the relative activity of only one of four autolysins measured (membrane-bound endopeptidase), suggesting that autolysis induced by penicillin G may be rate limited by this enzyme. Imipenem, on the other hand, appeared to trigger different autolysins in Escherichia coli, as evidenced by differences in the structure of peptidoglycan after imipenem- versus penicillin-induced autolysis.

Anti-Bacterial Agents↗
PubMed1990

Lysis and aberrant morphology of Bacillus subtilis cells caused by surfactants and their relation to autolysin activity.

The surfactants tested in this study lysed Bacillus subtilis 168 cells at the logarithmic growth phase. Results obtained with inhibitors and a mutant that had defective autolytic enzymes suggested that cell lysis resulted from the deregulation of autolysin activity. The addition of surfactants at sublytic concentrations produced twisted cells, filamented cells, or both. Autolysins extracted with 5 M LiCl from the cell wall fraction and lysozyme added to cells that were treated with surfactants restored the apparently normal cell rod morphology, suggesting that surfactants interfere with the role of autolysins in normal construction of the cell envelope. The rates of cellular autolysis and autolysin activity remaining in growing cells after exposure to a surfactant at a sublytic concentration decreased, although the rate of turnover of cell wall peptidoglycan was the same as that of control cells. Surfactants were suggested to interact with the regulatory system of autolysins and, thus, to affect the activities of autolysins in B. subtilis cells and to cause either morphological changes or cell autolysis, depending on the concentration of surfactants.

Autolysis↗
PubMed1972

Factors influencing the production of H and M antigens by Histoplasma capsulatum: effect of physical factors and composition of medium.

Stagnant culture methods have permitted only limited physiological studies of the production of H and M antigens by Histoplasma capsulatum because, with such methods, antigen production is uncontrolled. In this investigation, a shake culture method was used to convert yeast-phase inoculum to mycelialphase growth at 25 C. Results strongly suggest that the release of H and M antigens relates to autolysis of the cells. Among the factors influencing production of H and M antigens under shaking conditions, choice of strain was the most important. Alterations of carbon or nitrogen source or variations in amino acid to carbohydrate ratios had limited influence on antigen production. With a strain that produced both H and M antigens, however, proportions of titers of M to H antigens could be made to vary considerably by changes in the medium, the pH, and the temperature. Results suggest that the source of M antigen during autolysis is enzymatic dissolution of the cell wall. The source of H antigen is more obscure. Production of both antigens may be differentially controlled under conditions of good reproducibility by a correct choice of strain and manipulation of culture medium.

Antigens↗
PubMed1973

Rapid methods for extracting autolysins from Bacillus subtilis.

Two procedures are described for the extraction of autolysins from whole cells. One method uses 5 M LiCl at 4 C. The amount of enzyme obtained by this method is six times more than that obtained by autolysis of cell walls and fourteen times more than that obtained by extracting cell walls with LiCl. With the other method, cells are extracted with 2% Triton X-100. This is less efficient than the LiCl method but yields about one-half the amount of enzyme obtained by cell wall autolysis and about the same amount as obtained by extracting cell walls with salt. Both procedures yield autolysin with multiple pH optima. Autolysins can be extracted from several bacterial species by either the LiCl or the detergent method. The data suggest that these techniques have sufficient sensitivity to detect small differences in autolytic activity among mutants and various organisms and are also suitable for large-scale isolation of autolysin for purification and characterization studies.

Autolysis↗
PubMed1973

Autolytic activity and its association with the development of competence in group H streptococci.

Seventeen strains of group H streptococci were tested for their ability to develop competence for genetic transformation, either spontaneously or with the addition of competence factor derived from strain Challis supernatant fluids, and for their ability to autolyze. Autolysis was measured as a decline in optical density after washed cells were placed in a buffer at pH 9. Kinetic experiments showed that, in strains Challis, SBE I/II, WE4, SR 30, and a strain (FW 227) cured of its bacteriophage, competence and the ability to autolyze occurred simultaneously. Since autolysis was observed only in (i) competent cells, (ii) cells that passed their peak of competence, and (iii) those cells that exhibit a potential for developing competence but never go on to transform (i.e., lysogenized Challis cells), it is concluded that, in the group H streptococci, autolytic events are associated with the competent state. Strains that transformed but did not autolyze were not found.

Autolysis↗
PubMed2004

Revising the role of the pneumococcal vex-vncRS locus in vancomycin tolerance.

Vancomycin is used increasingly to treat invasive infections caused by multidrug-resistant Streptococcus pneumoniae. Although no vancomycin-resistant strains have been isolated to date, tolerant strains that fail to die rapidly and that cause relapsing disease have been described. The vex123-pep27-vncRS locus, consisting of an ABC transporter, a presumed signaling peptide, and a two-component system, respectively, has been implicated in vancomycin tolerance. Recent findings, however, challenged this model. The data presented here indicate that erythromycin in the growth medium induces a vancomycin-tolerant phenotype and that loss of function of Pep27 or VncRS does not alter autolysis. However, a role for the ABC transporter encoded by the vex123 genes in tolerance was confirmed. A vex3 mutant was considerably more tolerant to vancomycin treatment than the wild-type strain T4, and the strength of the phenotype depended on the orientation of the resistance cassette used to construct the mutant. Microarray results suggested a number of genes that might be involved in tolerance in the vex3 mutant. Although the exact function and regulation of the vex123-pep27-vncRS locus remains to be determined, several factors influence the autolysis behavior of S. pneumoniae, including the bacterial capsule, erythromycin, and the lytA and vex3 gene products.

ATP-Binding Cassette Transporters↗
PubMed1970

Postmortem detection of inapparent myocardial infarction.

Two methods of detecting early inapparent myocardial infarcts have been studied and their value in diagnostic practice compared. The better method proved to be the determination of the potassium to sodium ratio (ionic ratio) which falls in infarcted tissue within minutes of the onset of anoxia. The second method was nitro blue tetrazolium staining of gross sections of myocardium which revealed any infarct older than three and a half hours. As staining is dependent upon enzyme activity, the latter method is disturbed by autolysis. It was shown, on the other hand, that the ionic ratio (K(+)/Na(+)) was not affected by autolysis and was therefore well suited to forensic practice. Sixteen non-infarcted control hearts, plus the nine from cases of sudden death due to causes other than myocardial infarction, all yielded high ionic ratios (K(+)/Na(+)), average 1.4, and stained normally with tetrazolium (the normal controls). Positive control was provided by 20 histologically proven infarcts of which the ionic ratios (K(+)/Na(+)) were all low (average 0.7). Histochemical staining with tetrazolium delineated infarcted areas in each case. In a series of 29 sudden deaths, a cause of death other than myocardial infarction was found at necropsy in nine, mentioned above as normal controls. The remaining 20 hearts were not infarcted histologically, but were shown to be infarcted by examination of the ionic ratios (K(+)/Na(+)). These ratios were low (average 0.8) including three borderline ratios. Confirmatory evidence of infarction included nitro blue tetrazolium staining which revealed infarcts in 10 of the 20 cases, and clinical and necropsy observations. The ionic ratio (K(+)/Na(+)) decreases as the age of the infarct increases for at least 24 hours. Thereafter as healing proceeds, the ratio gradually reverts to normal. Thus, previous infarction and replacement fibrosis do not significantly alter the ionic ratio (K(+)/Na(+)). Nor is it changed by left ventricular hypertrophy, the presence of congestive cardiac failure, or digitalis therapy. It is suggested that macroscopic tetrazolium staining is a useful screening test for early inapparent myocardial infarcts. In cases where no infarct is delineated with that method estimation of the ionic ratio (K(+)/Na(+)) should be carried out on myocardium removed from standard areas on the anterior and posterior left ventricular walls.

Autolysis↗
PubMed1978

Influence of denervation and reinnervation on autolytic activity and on protein composition of skeletal muscle in rat.

Changes in the proteolytic activity and in the relative content of protein in soluble, myofibrillar and insoluble fractions were investigated following denervation and reinnervation of the soleus and tibialis anterior muscles of the rat. After denervation an increase of autolysis in the acid and neutral pH range, but not in the alkaline one, was found in both muscles. An increased autolysis at the acid and neutral pH range was also observed in both muscles after reinnervation, when the weight of the muscles increased. The results indicate the lack of inverse relationship between the changes of proteolytic activity and the decrease or increase of the amount of muscle protein in the course of muscle atrophy and regeneration.

Animals↗
PubMed2005

A comparison of rapid bovine spongiform encephalopathy testing methods on autolyzed bovine brain tissue.

In 1999, the European Union (EU) approved 3 rapid methods for the testing of bovine brain samples for the presence of bovine spongiform encephalopathy (BSE). The evaluation that led to the approval did not include an analysis of autolyzed material. Member states of the EU have active surveillance programs for BSE, which target fallen stock as well as other categories of cattle. Autolysis is a common feature of fallen stock samples because there can be a considerable delay between death and collection of samples. Therefore, it is important to know whether these tests perform optimally on autolyzed samples. The Veterinary Laboratories Agency (VLA) selected 250 positive fallen stock samples. These had been detected during routine testing using the Prionics-Check Western blot and confirmed as BSE cases by immunohistochemistry or electron microscopy. Samples were graded according to the degree of autolysis and then tested by the 3 methods: Prionics-Check Western blot, Platelia test, and Enfer test. All 3 methods correctly classified the samples as positive BSE cases, therefore alleviating doubt about their ability to do so. Subsequent EU validation exercises, such as those conducted in 2002--2003, have included the testing of autolyzed material. It is important that all new methods be evaluated on autolyzed tissue before approval for official use.

Animals↗