Time course in calpain activity and autolysis in slow and fast skeletal muscle during clenbuterol treatment.

Calpains are Ca2+ cysteine proteases that have been proposed to be involved in the cytoskeletal remodeling and wasting of skeletal muscle. Cumulative evidence also suggests that ß2-agonists can lead to skeletal muscle hypertrophy through a mechanism probably related to calcium-dependent proteolytic enzyme. The aim of our study was to monitor calpain activity as a function of clenbuterol treatment in both slow and fast phenotype rat muscles. For this purpose, for 21 days we followed the time course of the calpain activity and of the ubiquitous calpain 1 and 2 autolysis, as well as muscle remodeling in the extensor digitorum longus (EDL) and soleus muscles of male Wistar rats treated daily with clenbuterol (4 mg·kg-1). A slow to fast fiber shift was observed in both the EDL and soleus muscles after 9 days of treatment, while hypertrophy was observed only in EDL after 9 days of treatment. Soleus muscle but not EDL muscle underwent an early apoptonecrosis phase characterized by hematoxylin and eosin staining. Total calpain activity was increased in both the EDL and soleus muscles of rats treated with clenbuterol. Moreover, calpain 1 autolysis increased significantly after 14 days in the EDL, but not in the soleus. Calpain 2 autolysis increased significantly in both muscles 6 hours after the first clenbuterol injection, indicating that clenbuterol-induced calpain 2 autolysis occurred earlier than calpain 1 autolysis. Together, these data suggest a preferential involvement of calpain 2 autolysis compared with calpain 1 autolysis in the mechanisms underlying the clenbuterol-induced skeletal muscle remodeling.

The extracellular ß-1,3-endoglucanase EngA is involved in autolysis of Aspergillus nidulans.

AIMS: To elucidate the roles of the ß-1,3-endoglucanase EngA in autolysis of the filamentous fungus Aspergillus nidulans and to identify the common regulatory elements of autolytic hydrolases. METHODS AND RESULTS: A ß-1,3-endoglucanase was purified from carbon-starving cultures of A. nidulans. This enzyme is found to be encoded by the engA gene (locus ID: AN0472.3). Functional and gene-expression studies demonstrated that EngA is involved in the autolytic cell wall degradation resulting from carbon starvation of the fungus. Moreover, regulation of engA is found to be dependent on the FluG/BrlA asexual sporulation signalling pathway in submerged culture. The deletion of either engA or chiB (encoding an endochitinase) caused highly reduced production of hydrolases in general. CONCLUSIONS: The ß-1,3-endoglucanase EngA plays a pivotal role in fungal autolysis, and activities of both EngA and ChiB are necessary to orchestrate the expression of autolytic hydrolases. The production of cell wall-degrading enzymes was coordinately controlled in a highly sophisticated and complex manner. SIGNIFICANCE AND IMPACT OF THE STUDY: No information was available on the autolytic glucanase(s) of the euascomycete A. nidulans. This study demonstrates that EngA is a key element in fungal autolysis, and normal activities of both EngA and ChiB are crucial for balanced production of hydrolases.

The role of autolysis loop in determining the specificity of coagulation proteases.

We recently demonstrated that the substitution of the autolysis loop (residues 143 to 154 in the chymotrypsin numbering system) of activated protein C (APC) with the corresponding loop of factor Xa (fXa) renders the APC mutant (APC/fX143-154) susceptible to inhibition by antithrombin (AT) in the presence of pentasaccharide. Our recent results further indicated, that in addition to an improvement in the reactivity of APC/fX143-154 with AT, both the amidolytic and anti-factor Va activities of the mutant APC have also been significantly increased. Since the autolysis loop of APC is five residues longer than the autolysis loop of fXa, it could not be ascertained whether this loop in the mutant APC specifically interacts with the activated conformation of AT or if a shorter autolysis loop is responsible for a global improvement in the catalytic activity of the mutant protease. To answer this question, we prepared another APC mutant in which the autolysis loop of the protease was replaced with the corresponding loop of trypsin (APC/Tryp143-154). Unlike an approximately 500-fold improvement in the reactivity of APC/fX143-154 with AT in the presence of pentasaccharide, the reactivity of APC/Tryp143-154 with the serpin was improved approximately 10-fold. These results suggest that both the length and structure of residues of the autolysis loop are critical for the specificity of the coagulation protease interaction with AT. Further factor Va inactivation studies with the APC mutants revealed a similar role for the autolysis loop of APC in the interaction with its natural substrate.

Identification and functional characterization of AclB, a novel cell-separating enzyme from Lactobacillus casei.

Autolysis of nonstarter lactic acid bacteria (NSLAB) was favorable for the development of flavor compounds during cheese manufacture. Among these bacteria, Lb. casei was regarded as the most important microbiota involved in cheese processes. In this study, a novel autolysin named AclB was identified in the genome of Lb. casei BL23 and its modular structure was predicted through bioinformatic approaches. Subsequently, its transcription profile in the exponential phase, hydrolytic activities against cell walls, enzymatic properties under different conditions, physiological function via gene inactivation and upregulation assays, as well as potential applications to NSLAB's autolysis were fully investigated. According to the results, AclB was recognized as a species-specific cell-separating enzyme, responsible for cell separation after cell division in Lb. casei BL23. The purified AclB showed considerable hydrolyzing activities towards cell walls, indicating its enzymatic nature as peptidoglycan hydrolase, or autolysin. The highest activity of AclB was determined at pH5.0 and 37°C, and the expression vector constructed based on AclB was shown to facilitate the controlled lysis of Lb. casei BL23 hosts. In summary, this study provided insight into the enzymatic properties of a novel autolysin involved in cell separation of Lb. casei BL23, which is promising to accelerate cheese ripening and improve cheese quality.

Biochemical, morphological and cytochemical studies of enhanced autolysis of Saccharomyces cerevisiae. 2. Morphological and cytochemical studies.

Morphological and cytochemical observation of Saccharomyces cerevisiae undergoing of induced autolysis were done in response to various chemical inducers of autolysis (NaCl, ethanol, fresh autolyzate). Changes in the inner structure of yeast cells were monitored by transmission electron microscopy and the surface of the cell wall was observed by scanning electron microscopy during autolysis. Cytochemical characterization of autolyzed cells was performed using four synthetic substrates for determination of proteinase activities but only carboxypeptidase Y could be detected in the vacuolar membranes. The morphological studies supported the data obtained from biochemical studies and confirmed that optimized conditions of autolysis have a significant effect on the structural changes of autolyzed yeast.

[Aminoacyl-tRNA-synthetases and their high molecular weight complexes in experimental myocardial ischemia]. / Aminoatsil-tRNK-sintetazy i ikh vysokomolekuliarnye kompleksy pri éksperimental'noi ishemii miokarda.

A number of aminoacyl-tRNA synthetases from rabbit liver during experimental myocardial infarction and from pig myocardium upon 15-min of autolysis were found to increase their activity in aminoacylation. Direct correlations between the activities of high molecular weight complexes and of the total extracts were not observed. It was shown that the specific activity of endogenous inorganic pyrophosphatase increased markedly during the ischemia of myocardium both in total myocardium extracts and in high molecular weight complexes.

[The effect of autolysis on the conduction system in young rats. An enzyme histochemistry study]. / Vliv autolýzy na prevodní systém mladých krys. Enzym-histochemická studie.

The conducting system of young rats was studied by histochemistry during autolysis at room temperature lasting 1, 3, 6, 24 and 48 hours. The results were compared with working muscle tissue. The most sensitive was the "glycogen-dependent" phosphorylase and the activity decreased after 1 hour. It was slightly detectable as late as after 24 hours when the working muscle was already negative. Other enzyme activities (oxidoreductases and hydrolases) were practically uninfluenced by 6 hour autolysis. A substantial decrease of activities was found in conducting system after 48 hours in comparison to the working muscle. A solitary relatively strong residual activity belonged to acetylcholine-esterase in the conducting system. Detection of acetylcholine-esterase can be useful in identification of the conducting system even later after death.

[The relation between corneal autolysis and the lysosomal enzyme level in aqueous humor and corneal storage medium].

The activity of acid phosphatase, a marker enzyme of lysosomal enzymes, in aqueous humor of wet-chamber stored eyeball and corneal storage medium was determined. The results showed a significant rise in the enzyme activity as the storage time increased, i.e. the degree of autolysis of the cornea progressed.

[Change in the alpha-glucosidase activity of cattle muscle tissue during slow autolysis]. / Izmenenie aktivnosti al'fa-gliukozidazy pri medlennom avtolize mushechnoi tkani krupnogo rogatogo skota

Changes in the alpha-glucosidase activity of the cattle muscular tissue (oxen eye muscle of loin) were evaluated during storage at 2 degrees. Under these conditions both lysosomal and extralysosomal alpha-glucosidase activities underwent no significant changes during a long period of time (12 days). Activity of lysosome-bound alpha-glucosidase was about 10% of total enzyme activity in the homogenate; the remaining part of alpha-glucosidase was contained outside lysosomes.

[The morphogenesis of pancreonecrosis]. / Morfogenez pankreonekroza.

Pancreas was studied histologically, histochemically and electron microscopically in 117 autopsy cases and in the experiment (25 dogs and 60 white rats). Morphogenesis of pancreonecrosis proceeds through certain stages. The initial stages of an acute pancreatic edema is characterized by degenerative and necrotic changes of pancreocytes with parapedesis of pancreatic enzymes into the interstitium and a reactive stromal edema. The following stages are distinguished in the progressing phase of pancreonecrosis: hemorrhagic pancreonecrosis when the proteolytic enzymes provoke a colliquation necrosis of the acinar tissue, fibrinoid necrosis of vascular walls and disturbances of the intravascular hemorheology resulting in the enhancement of destructive processes and hemorrhagic inhibition of tissues; fat pancreonecrosis in which lipolytic enzymes lead to the coagulation necrosis of the acinar and fat tissue while a non-completed proteolysis of necrotic tissues stipulates the intensity of the reactive inflammation.

[Aminoacyl-tRNA synthetases and their high molecular weight complexes in autolysis of the swine heart]. / Aminoatsil-tRNK-sintetazy i ikh vysokomolekuliarnye kompleksy pri autolize serdtsa svin'i.

In pig myocardial extracts autolyzed within 15 min alanyl-, glycyl-, glutamyl-, leucyl- and seryl-tRNA synthetase activities were increased as compared with controls. The enzymatic activities were decreased after autolysis for 30 min. The 15 min autolysis was shown to decrease the molecular mass of the glycyl-tRNA synthetase complex. Within both 15 min and 30 min autolysis inorganic pyrophosphatase was markedly activated either in myocardium extracts or in high molecular complexes; this phenomenon may be responsible for activation of a number of aminoacyl-tRNA synthetases.

Postmortem radiography of gastromalacia: case reports.

Gastromalacia is a postmortem artifact resulting from autolysis of the gastric walls. Gastromalacia is autolytic rupture of the stomach caused by endogenous enzymes, and it is devoid of any vital reactions. The left leaf of the diaphragm is occasionally perforated by a ragged fenestration, with escape of gastric contents into the pleural cavity. This rupture may lead to pneumoperitoneum or pneumothorax. For diagnostic radiologists, gastromalacia is rarely encountered. Therefore, they should be aware of this entity to avoid misdiagnosis when performing postmortem radiography.

Acute pancreatitis.

Acute pancreatitis (AP) is an important cause of morbidity and mortality worldwide and the annual incidence appears to be increasing. It presents as a mild self-limiting illness in 80% of patients. However, one-fifth of these develop a severe complicated life-threatening disease requiring intensive and prolonged therapeutic intervention. Alcohol and gallstone disease remain the commonest causes of AP but metabolic abnormalities, obesity and genetic susceptibility are thought be increasingly important aetiological factors. The prompt diagnosis of AP and stratification of disease severity is essential in directing rapid delivery of appropriate therapeutic measures. In this review, the range of diagnostic and prognostic assays, severity scoring systems and radiological investigations used in current clinical practice are described, highlighting their strengths and weaknesses. Increased understanding of the complex pathophysiology of AP has generated an array of new potential diagnostic assays and these are discussed. The multidisciplinary approach to management of severe pancreatitis is outlined, including areas of controversy and novel treatments.

Purification and properties of a chitinase from Penicillium oxalicum autolysates.

A chitinase (EC. 3.2.1.14) from autolysed culture filtrate of Penicillium oxalicum was purified by precipitation with ammonium sulphate, gel filtration and ion exchange chromatographies. The purified enzyme showed a single protein band in SDS gel electrophoresis. The enzyme is an acidic protein with a pI of 4.5 and has a molecular weight of 54,900 as estimated from SDS gel electrophoresis and 21,500 from gel filtration. The optimum pH and temperature were 5.0 and 35 degrees C, respectively. The enzyme was stable at temperatures up to 45 degrees C and in a pH range between 4.0 and 6.0. The Km was 2.5 mg ml-1 for colloidal chitin, Hg2+ and Ag+ were effective inhibitors. The viscosimetric study carried out using carboxymethyl chitin as substrate revealed the endotype action of this enzyme.