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PubMed1978

[Proteolytic processes in rat brain and liver during adaptation to cold].

The autolysis and the neutral proteinase activity with respect to protamine in the brain and liver of rats were studied during cold adaptation, on the 1st, 3rd, 7th, 15th, and 45th days in the cold chamber at 2--4 degrees C. A short cold effect (1--3 days) decreases the brain autolysis by 33%, the liver one by 18%. The activity of protamine-lytic peptide-hydrolase diminishes nearly by half both in the brain and in the liver. In cold-adapted rats (45 days) the autolysis increases by 50% in the brain and by 24% in the liver. The activity of neutral peptide-hydrolase remains lowered by 43% in the brain and by 36% in the liver. Thus, only autolytic processes are intensified in cold-adapted animals while the neutral peptide-hydrolase does not participate in the increasing protein catabolism in the brain and liver.

Acclimatization↗
PubMed1994

Crystallographic structure of human gamma-thrombin.

In an effort to prepare crystals and determine the structure of alpha-thrombin complexed to a synthetic peptide inhibitor (MDL-28050) of the hirudin 54-65 COOH-terminal region, it was discovered that the crystals were not those of the complex but of gamma-thrombin. Gel electrophoresis studies revealed that autolytic degradation had occurred prior to crystallization. NH2-terminal sequence analysis of these autolytic fragments confirmed the gamma-thrombin product (cleavages at Arg75-Tyr76 and/or Arg77A-Asn78, and Lys149E-Gly150; chymotrypsinogen numbering) with a minor amount of another autolysis product, beta-thrombin (first two cleavages only). The final structure has an R-factor of 0.156 for 7.0-2.5-A data, and includes 186 water molecules. A comparison of gamma-thrombin with the thrombin structure in the alpha-thrombin-hirugen complex revealed that the two structures agreed well (r.m.s. delta = 0.39 A for main chain atoms). These structures possess uninhibited active sites where the disposition of the catalytic triad residues is nearly identical. The electron density in the vicinity of the gamma-thrombin cleavage regions is poor, and only becomes well-defined several residues prior to and after the actual cleavage sites. The extensive disorder evoked by beta-cleavage(s) in the Lys70-Glu80 loop region indicates that this part of the molecule is severely disrupted by autolysis and is the reason exosite functions are dramatically impaired in beta-and gamma-thrombin. Since autolysis did not lead to a major reorganization of the folded structure of alpha-thrombin, the likely structural features of the interaction of thrombin substrate with thrombin enzyme during beta-cleavage have been modeled by docking the exosite region of one molecule at the active site of another.

Amino Acid Sequence↗
PubMedDate not supplied

[Enzymatic profile of autolysing Saccharomyces yeasts].

The influence of pH on the efficiency of autolysis of brewer's yeast was studied. It is shown that some kinds of peptidase activities in "solid" and "liquid" fractions of autolysates depend on the time of autolysis. The inactivation constants and activation energy of inactivation of yeast proteases were determined. It was found that heat stability of yeast proteases contained in the "solid" fraction of autolysates is significantly higher as compared to the enzyme present in the "liquid" fraction. From this results the conclusion that some of yeast proteases are immobilized on the cell wall fragments. A technique for activation of yeast enzyme systems in the course of autolysis is proposed.

Endopeptidases↗
PubMed1998

[Structural characteristics providing for high specificity of enteropeptidase].

The effects of structural modification upon the specificity of enteropeptidase were studied. A variation in the unique specificity of the enzyme was shown to be the result of an autolysis caused by the enzyme's loss of calcium ions. The cleavage sites of the autolysis were determined. A truncated enzyme containing the C-terminal fragment of its heavy chain (466-800 residues) and the intact light chain were shown to be the products of autolysis. The kinetic parameters of the hydrolysis of trypsinogen, a recombinant protein, and a peptide substrate with both forms of enteropeptidase were determined. Conditions were found that can help regulate the transition of the native enzyme into the truncated form. A hypothesis was proposed concerning the autoactivational character of proenteropeptidase processing.

Amino Acids↗
PubMed1981

Diagnostic significance of nerve cells in human CSF with particular reference to CSF cytology in the brain death syndrome.

Cytological examination of the cerebrospinal fluid (CSF) in 5 cases of the brain death syndrome treated on a respirator revealed the presence of nerve cells (mostly Purkinje cells), massive pleocytosis with many polymorphonuclear leukocytes, and numerous macrophages containing erythrocytes, leukocytes, lipid droplets, and hemosiderin. Portions of connective tissue with well preserved capillaries, and debris which could not be more closely identified, were also found. Neuropathological examination in 4 cases revealed progressive autolysis of brain tissue and displacement of the autolyzed tissue into the subarachnoid space of the spinal canal, partly due to tonsillar herniation. All five patients exhibited clinical signs of brain death and had been placed on respirators for various periods of time. The cytological findings in the CSF, together with the clinical signs of brain death, constitute an intravital morphological indication of brain tissue autolysis.

Autolysis↗
PubMed1986

The kinetics of the autolytic phase of growth in cultures of Aspergillus niger.

The kinetics of the autolytic phase of growth in cultures of Aspergillus niger has been studied. Two different autolytic periods could be distinguished. One, consisting of a rapid (exponential) loss (62%) of mycelial weight, occurred between 36 and 117 hours of incubation. A second, consisting of a slow autolysis, occurred between the 117th and the 190th hour of incubation; the mycelial loss here being 5%. Based on the degree of autolysis (alpha = 67.0%), 92.5% and 7.5% are lost during the first and the second autolytic periods, respectively.

Aspergillus niger↗
PubMed1977

Ultrastructure of striated muscle of the rat after temporary ischemia.

Twenty-six anterior tibial muscle specimens were analyzed with the electron microscope 2 h, 1, 4, 18, 45, 90 and 226 days after temporary ischaemia of the rat hind leg produced by pneumatic tourniquet for 1, 2, 3, 4 and 6 h. No ultrastructural alterations were seen after 1 h ischaemia, but reversible disappearance of muscle glycogen and swelling of muscle and nerve mitochondria occurred after 2 h ischaemia. Irreversible alterations such as disappearance of the Z-line, disruption of cell membrane and vacuolation of muscle mitochondria were observed after ischaemia of 3 h and longer in a part of the muscle fibres. Ischaemia for 6 h was followed by autolysis of all the muscle fibres within the specimens. Autolysis and phagocytosis of the remnants of the affected fibres was present on the 4th day. On that day regenerative phenomena were also observed, such as satellite cells and young myotubes. The fine structure of the muscle fibres was normal on the 18th day after the temporary ishcaemia, although some size variation of the muscle fibres was still observed. Degeneration of a part of the presynaptic nerve endings of motor end-plates with or without simultaneous degeneration of the postsynaptic muscle fibre was seen after ischaemia lasting 3 h or longer. Motor end-plates were first seen on the 45th day in the muscle subjected to 6 h ischaemia and were most likely regenerated ones.

Animals↗
PubMed2004

Physiological and morphological changes in autolyzing Aspergillus nidulans cultures.

Physiological and morphological changes in carbon-limited autolyzing cultures of Aspergillus nidulans were described. The carbon starvation arrested conidiation while the formation of filamentous and "yeast-like" hyphal fragments with profoundly altered metabolism enabled the fungus to survive the nutritional stress. The morphological and physiological stress responses, which maintained the cellular integrity of surviving hyphal fragments at the expense of autolyzing cells, were highly concerted and regulated. Moreover, sublethal concentrations of the protein synthesis inhibitor cycloheximide or the mitochondrial uncoupler 2,4-dinitrophenol completely blocked the autolysis. In accordance with the propositions of the free-radical theory of ageing reactive oxygen species accumulated in the surviving fragments with a concomitant increase in the specific superoxide dismutase activity and a continuous decrease in cell viability. Glutathione was degraded extensively in carbon-starving cells due to the action of gamma-glutamyltranspeptidase, which resulted in a glutathione-glutathione disulfide redox imbalance during autolysis.

Aspergillus nidulans↗
PubMed2000

Degradation of myofibrillar proteins by a calpain-like proteinase in the arm muscle of Octopus vulgaris.

The effects of a calpain-like proteinase (CaDP) isolated from the arm muscle of Octopus vulgaris on the myofibrils and myofibrillar proteins isolated from the same tissue were examined. Our studies clearly showed that treatment of intact myofibrils with CaDP in the presence of 5 mM Ca2+ results in the degradation of the major myofibrillar proteins myosin, paramyosin, and actin. From the isolated alpha- and beta-paramyosins only beta-paramyosin is degraded by CaDP in the presence of 5 mM Ca2+ producing three groups of polypeptides of 80, 75, and 60 kDa, respectively. The degradation rate depends on the proteinase to substrate ratio, temperature, and time of proteolysis and is inhibited by the endogenous CaDP inhibitory factor (CIF), as well as by various known cysteine proteinase inhibitors (E-64, leupeptin, and antipain). From the other myofibrillar proteins examined myosin, but not actin, is degraded by CaDP; myosin heavy chain (MHC, 200 kDa) is degraded by CaDP producing four groups of polypeptides of lower molecular masses (155, 125, 115, and 102 kDa, respectively); the degradation rate depends on the incubation time and the proteinase to substrate ratio. Furthermore, CaDP undergoes limited autolysis in the presence of both the exogenous casein and the endogenous beta-paramyosin producing two large active fragments of 52 and 50.6 kDa, respectively; CIF reversibly inhibits this CaDP autolysis.

Actins↗
PubMed1980

The (iso)enzyme activities of lactate dehydrogenase, alpha-hydroxybutyrate dehydrogenase, creatine kinase and aspartate aminotransferase in human myocardial biopsies and autopsies.

In a study on enzyme activities in normal human myocardial tissue the activities of lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH), creatine kinase (CK), and aspartate aminotransferase (AST), and the activities of the isoenzymes of LDH (1--5) and AST (c and m) were measured in myocardial specimens obtained from live patients (18 biopsies) and postmortem (14 autopsies). Comparison of measurements in 4 right ventricular biopsies from 1 patient with those in 9 right ventricular biopsies from 9 patients shows that (1) the enzymes studied are homogeneously distributed throughout the right ventricular myocardium, (2) errors in the measurement of the activities of the enzymes studied are within 5%, and (3) inter-individual differences in myocardial enzyme activities are quite considerable (10--17%). No significant differences in the activities of the enzymes studied were found between left and right ventricular myocardium. The effect of autolysis on the activities of the enzymes studied in myocardial specimens obtained postmortem is small, amounting to -1% to -6% in the first 10 h, irrespective of whether a linear or an exponential decay of enzyme activity is assumed. Comparison of myocardial enzyme activities in biopsies with those in autopsies, the latter being corrected for autolysis-induced inactivation, reveals significant differences with respect to CK, AST and mAST. At present it is impossible to conclude whether these differences were due to the different conditions existing before the myocardial specimens were frozen at -20 degrees C or to the different types of pathology present in the hearts from which the specimens were taken.

Adult↗
PubMed1983

Exercise myopathy: selectively enhanced proteolytic capacity in rat skeletal muscle after prolonged running.

The proteolytic capacity of rat skeletal muscle was analyzed during the repair of fiber injuries after strenuous exercise. A single bout of prolonged exercise (8 hr running at a speed of 17 m X min-1) caused a slight fiber necrosis and a selective response in the proteolytic activity of rat skeletal muscle. Acid proteolytic capacity (cathepsin D and acid autolysis) was considerably increased on the 4th day after exertion and partially decreased by the 10th day. The acid hydrolytic response was more prominent in red than in white skeletal muscle. Alkaline proteolytic capacity (alkaline and myofibrillar proteases), increased in several atrophic myopathies, was not affected in exercise myopathy. The rate of neutral autolysis slightly increased after exertion. The protein content of skeletal muscle was decreased on the 4th day after exertion. We suggest that the proteolytic responses to acute injuries, as well as to chronic atrophies, are highly selective in skeletal muscle fibers.

Animals↗
PubMed1987

Autolytic changes of human white matter: an electron microscopic and electrophoretic study.

The effect of autolysis on the electrophoretic pattern of the 3H-labeled membrane glycoproteins derived from human brain white matter (WM) was investigated and correlated with electron microscopic findings. Samples were taken from the WM of outer zones of routine surgical specimens sent for pathologist's examination. The WM samples were incubated at +4 and +25 degrees C for 6 and 24 hr and analyzed by electron microscopy and by galactose oxidase labeling with subsequent SDS-polyacrylamide gradient gel electrophoresis. The effect of freezing and thawing was also studied. In electron microscopic examination myelin was found to have degenerated after an incubation of 24 hr at +4 and +25 degrees C, and it was severely disrupted after 24 hr at +25 degrees C, when a periaxonal network-like change was observed. The major labeled membrane glycoprotein band with an estimated molecular weight (MW) of 138,000 was stable during the first 24 hr of incubations at both +4 and +25 degrees C. No marked changes were noticed in the electrophoretic pattern of all of the WM membrane proteins labeled by [3H]acetic anhydride technique. In frozen and thawed specimens there was an increase in the intensity of the band with a MW of 86,500 and a new band appeared at the MW region of 25,000. The present results suggest that despite the apparent stability of the major glycoprotein band during the first 24 hr of autolysis, there are ultrastructural abnormalities in the myelin sheath, which should be taken into consideration during interpretation of changes in pathologic conditions.

Autolysis↗
PubMed1993

FVIIa derivatives obtained by autolytic and controlled cathepsin G mediated cleavage.

The heavy chain of coagulation factor VII contains a serine esterase entity. A partial cleavage in the heavy chain occurs during purification and activation of the single-chain zymogen, presumably as a result of autolysis. Neutrophil cathepsin G initially generates a Gla-domainless FVIIa without coagulant activity. However, on extended exposure cleavage also occurs in the heavy chain, resulting in a complete loss of enzyme activity. Four cleavage sites on the heavy chain, two susceptible to trypsin-like autolysis and two susceptible to chymotrypsin-like cathepsin G-mediated catalysis have been identified. The hydrolysis of peptide bonds in the heavy chain might contribute to regulation of the coagulation process in vivo.

Amino Acid Sequence↗
PubMed1991

Free calcium and calpain I activity.

Activation of purified calpain I proceeds through a Ca(2+)-induced autolysis from the 80 kDa catalytic subunit to a 76 kDa form via an intermediate 78 kDa form, and from a 30 kDa form to a 18 kDa form as the result of two autocatalytic processes (intra and intermolecular). The minimum Ca2+ requirements for autolysis and proteolysis have been determined by physico-chemical and electrophoretic methods in the presence or absence of a digestible substrate. According to our results the activation process needs less free Ca2+ than the proteolysis of a digestible substrate, which means that proteolysis is really subsequent to activation. For very low Ca2+ levels, a digestible substrate does not initiate the calpain I activation process. In the presence of phospholipid vesicles, such as PI, PS or a mixture of PI (20%), PS (20%) and PC (60%), the apparent kinetic constants of activation are greatly increased without any change in the initial velocity of the substrate proteolysis. Thus, enzyme activation and substrate proteolysis are observed as independent phenomena. These results obtained from experiments using low free Ca2+ concentrations enable us to propose a hypothesis for the mechanism of regulation by which the enzyme could be activated in the living cell.

Autolysis↗
PubMed1990

Control of the activity of the soluble lytic transglycosylase by the stringent response in Escherichia coli.

The soluble lytic transglycosylase (Slt) of Escherichia coli is known to be a powerful murein hydrolase in vitro. It is shown here to act as an autolysin in vivo as well. Rapid autolysis of Slt overproducing cells was induced by protein biosynthesis inhibitors, which also block the fomration of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). When amino acid starvation was used to inhibit protein synthesis, autolysis was suppressed in relA+ but not in relA- cells. These findings indicate that the stringent control modulates the enzymatic activity of the soluble lytic transglycosylase in vivo.

Amino Acids↗
PubMedDate not supplied

[Ultrastructure of intravital, postmortem and autolysed fibrin].

Investigations on animal experimental and human tissue material (skin, musculature, liver) with the scanning electron microscope are reported. These studies were carried out to clarify open questions on extravascular fibrin structures after blunt and sharp traumatization of vital and postmortem nature. The network formation which was already characterized earlier as typically vital and the irregular structure of postmortem coagula could be demonstrated even with modification of the traumatization, especially in tissue hematomas. Experimental investigations on autolysis revealed an exceedingly good preservation of the structure of vital coagulum material with massive damage and granular decomposition of fibrin fiber structures produced postmortem. The structural differences in the mode of interlinking of the fibrin was shown up especially clearly by the autolysis, which suggests an application as preparation technique for the question specified. Special modifications of the preparation technique (fixation according to Karnovsky, critical-point drying) did not provide such substantial advantages for routine investigations that they might be generally recommended.

Animals↗
PubMed2006

Chromatin immunoprecipitation in postmortem brain.

Methylation and other covalent modifications of nucleosome core histones are key regulators of chromatin structure and function, including epigenetic control of gene expression. For the human brain, however, very little is known about the regulation of histone modifications at specific genomic loci. Furthermore, chromatin immunoprecipitation protocols applicable to postmortem tissue are lacking, and the impact of potential confounds such as autolysis time or tissue pH is unknown. We treated cerebral cortex from human postmortem brain and mice by micrococcal nuclease digestion or, alternatively, by formaldehyde-crosslinking and sonication. We show that the bulk of nucleosomal DNA remains attached to histones during the first 30 h after death. Immunoprecipitation with antibodies against methylated histones was at least 10-fold more effective in unfixed, micrococcal nuclease-digested samples, in comparison to extracts prepared by fixation and sonication. Histone methylation differences across various genomic sites were maintained within a wide range of autolysis times and tissue pH. Therefore, immunoprecipitation of micrococcal nuclease-digested tissue extracts is a feasible approach to profile histone methylation at defined genomic loci in postmortem brain.

Aged↗
PubMed1979

Autolytic changes in the rat adenohypophysis. A histologic, immunocytologic and electron microscopic study.

Thirty-nine adult female Long Evans rats were decapitated and the heads stored at room temperature. The pituitaries were removed at intervals from 30 minutes to seven days, fixed, embedded and studied by histology, immunocytology and electron microscopy. Histologically, changes were noticeable after two hours postmortem. Immunoperoxidase staining showed postivity for growth hormone, prolactin, FSH, LH and TSH up to seven days after sacrifice, appearing even stronger in the advanced stages of autolysis. Fine structural alterations were evident at 30 minutes and more conspicuous later. Changes included dilation, partial degranulation and whorl formation of RER, swelling of Golgi complexes and mitochondria, chromatin clumping, lysis, rhexis and pyknosis of nuclei, cytosegresome formation and disruption of cell membranes. Secretory granules remained well preserved throughout, although some exhibited fusion or reduced electron density. Dilation of capillaries with accumulation of erythrocytes, platelets and fibrin fibers were prominent findings. The severity of changes varied considerably from cell to cell indicating that the rate of autolysis is not the same among different cell types and is possibly affected by the actual functional state of the cell. It appears that increased membrane permeability and disruption of plasmalemma represent important steps in the autolytic process.

Animals↗