Toxicity and proteolysis in gnotobiotic rats with thermal burns]

Experiments were conducted on germ-free (gnotobiotic) rats. A study was made of the toxic properties of the organs and the activity of proteolytic enzymes in them after thermal burns. Despite the absence of pathogenic microbial flora the germ-free rats developed the state of toxemia with an increase of the level of proteolytic enzymes. The extent of the toxic and catabolic reaction depended on the severity of the burn. Intoxication and an increase in proteolysis in the gnotobiotic rats were similar, and, in a number of cases greater than in usual animals. A conclusion was drawn on the important role of the tissue sources of intoxication in burns. A correlation between the toxemia and proteolysis was established.     

Spontaneous variability in a population of Candida albicans strains]

The spontaneous variability of the populations of C. albicans strains of different genesis in the morphological properties of their colonies and in the potential of the activity of their extracellular proteolytic and phospholipid enzymes has been studied. The isolated types of colonies, differing in their morphology, have the phenotypic character of variability. Different populations of strains exhibited variability in the activity of enzymes, depending on morphological variants isolated from these populations. Selected morphological variants with high potential of their proteolytic enzymes retained stability in this property for 5 generations and can be used in medical practice for the isolation of C. albicans antigens.     

Inhibitors of trypsin-like proteolytic enzymes as means of preventing the development of secondary necrosis in burn wounds]

The effect of proteolytic inhibitor contrical on the experimental burn wound healing was studied in rats using biochemical, histological and histochemical methods. In control untreated animals with flame burn of 20% of body surface wound healing was associated with development of secondary necrosis, marked inflammatory reaction, augmented activity of proteases and peptidases. The use of contrical prevented secondary necrosis; this effect was apparently related to reduction of tissue proteolytic enzymes activity.     

放射损伤、烧伤与放烧复合伤血清成份对心肌细胞钙离子通道活动的影响

本实验研究了放射损伤、烧伤与放烧复合伤后血清成份对培养心肌细胞Ｌ－型钙离子通道活动的影响,结果表明：伤后血清对上述通道均有激活作用,从而改变细胞内钙离子水平,此可能为伤后心脏功能抑制的一个重要机理。在作用强度上,复合伤血清重于单一伤、烧伤重于放射损伤,这是导致不同伤后血清对心功能抑制程度不一的重要因素。对伤后血清成分作用的进一步研究表明：伤后血清大分子的作用不明显,主要是血清低分子与血清脂质起作用。其中,放烧复合伤血清低分子不仅使通道开放增加,还使膜片噪声增加,提示其作用包括改变其Ｌ－型钙离子通道活动与改变膜的物理特性二个方面,它们共同导致细胞的功能变化;血清脂质对钙通道的作用可被ＳＯＤ所抑制,表明其作用与氧自由基反应有关。至于伤后血清低分子与血清脂质中的具体毒性成分以及它们对Ｌ－型钙离子通道影响的调控机制,有待于进一步研究     

Study of the nature of toxic factors in burns on sterile guinea pigs]

Experiments were conducted on sterile and contaminated guinea pigs. The toxicity of the serum and the water-salt extracts of the internal organs after the burn was studied by the action on the leukocyte migration in a culture. Despite the complete absence of the microbial flora, the toxic properties of the serum and of the extracts of the internal organs of the sterile animals were the same as in the contaminated organs. The data obtained pointed to the important role of the histiogenic factors in the development of burn intoxication.     

Action de la chlorhexidine sur l'expression de la virulence de Candida albicans

Candida albicans is an opportunistic yeast. Its pathogenicity is linked to the susceptibility of the host surface as well as to particular factors of the strain: adhesion, filamentous growth and secretion of proteolytic enzymes. Chlorhexidine digluconate is an antiseptic with fungicidal properties. The action of the antiseptic on the growth of the yeast shows a minimal inhibitory concentration (MIC) at 50 μg·mL⁻¹ and a minimal fungicidal concentration (MFC) at 100 μg·mL⁻¹. The consequences of antiseptic treatment are studied using two indicators of pathogenicity: filamentation and the secretion of acid proteinase. Concerning the morphological indicator, a complete inhibition of filamentation for antiseptic concentration far below the MIC is observed; concerning the biochemical indicator, the results show a maintenance of proteolytic activity for concentration equal to the MIC and a disappearance of this activity between the MIC and the MFC. Concerning the proteolytic activity, Skim Milk (Difco) was substituted for bovine serum albumin with success. This mixture of casein allows to use a cheaper and more easily available substrate.     

Some properties of proteolytic enzymes from Tribolium confusum

The digestive tract of Tribolium confusum Duv. larvae was studied for proteolytic enzymes properties. The pH optima are determined for the enzymes effect on various substrates. Proteases were partially purified by gel chromatography on Sephadex G = 100 and investigated for thyol compound influence on their activity. The activity of the enzymes is shown to increase considerably with addition of cystein, glutathione, 2-mercaptoethanol. dithiotreitol and EDTA. Dithiotreitol produces the strongest restoring effect and in concentration of 10(-6) M it activates the enzyme almost twice. Storage for 48 h at 4 degrees C induced a 2.5-fold decrease in the proteolytic enzyme activity; SH-groups in the catalytic action of enzymic solutions is shown. The maximum proteolytic activity is found in extracts from 14-day insects.     

Trichothecene inactivation by proteolytic enzymes from Trichothecium roseum Lk ex Fr]

Trichothecium roseum Lk ex Fr produces simultaneously trichothecin and proteolytic enzymes possessing fibrinolytic, thrombolytic and esterase activity. In addition to the function of splitting and consuming the substrate, the proteolytic enzymes of T. roseum are probably able to hydrolize the other bond in the molecule of trichothecin, which results in partial inactivation of the antibiotic in both the fungus culture and the model experiment on trichothecin contact with the preparations of the proteolytic enzymes. Presence of proteases of T. roseum may be considered as a protective mechanism for detoxication of the metabolites toxic for the organism.     

Detection of proteolytic enzymes in agar electrophoresis

An agar substrate containing hyaluronic acid and horse serum for the detection of hyaluronidase activity following electrophoresis was found suitable for the detection of proteolysis. The substrate was refined for the detection of only proteolysis by elimination of the hyaluronic acid and substitution of bovine serum albumin in place of the serum. The refined substrate revealed two bands of proteolytic activity in papain. The refined substrate also revealed weak proteolytic activity in a “chromatographically pure” preparation of hyaluronidase. The method should be adaptable for the detection of multiple electrophoretic forms of several proteolytic enzymes.     

Experimental study of protease C--proteolytic enzyme of microbial origin]

The specific activity of protease C, a proteolytic enzyme isolated from Acremonium chrysogenum was studied under experimental conditions. Protease C was shown to lyse necrotic biological substrates (dry crusts of burn wounds) and blood clots. By the nature of the effect protease C was analogous to terrilytin and by the level of the effect it was superior in some experiments. Protease C was low toxic and had no mutagenic action.     

Lysosomal proteolysis: effects of aging and insulin

Age-related characteristics of the effect of insulin on the activity of lysosomal proteolytic enzymes were studied. The relationship between the insulin effect on protein degradation and insulin degradation was analyzed. The effect of insulin on the activities of lysosomal enzymes was opposite in young and old rats (inhibitory in 3-month-old and stimulatory in 24-month-old animals). The activities of proteolytic enzymes were regulated by insulin in a glucose-independent manner: similar hypoglycemic effects of insulin in animals of different ages were accompanied by opposite changes in the activities of lysosomal enzymes. The inhibition of lysosomal enzymes by insulin in 3-month-old rats is consistent with a notion on the inhibitory effect of insulin on protein degradation. An opposite insulin effect in 24-month-old rats (i.e., stimulation of proteolytic activity by insulin) may be partly associated with attenuation of the degradation of insulin, resulting in disturbances in signaling that mediates the regulatory effects of insulin on protein degradation.     

Study of catalase and proteolytic activities of different variants of Bacillus mesentericus]

Catalase and proteolytic activity of the culures and morphological variants of Bacillus mesentericus fuscus, Bac. mesentericus vulgatus were studied. The variants were obtained as a result of prolonged cultivation of the stock strains in the potato mash under the layer of vaseline oil. The level of catalase activity varies in different morphological variants of the same culture, changes with age and depends on the storage conditions. The catalase activity in the rough, smooth and papillar variants that were freshly isolated from the potato mash was 1.5=2.5 times lower than that in the variants long kept on the agar medium. The quantitative indexes of the proteolytic activity of different variants also varied.     

Anchorage independent growth and plasminogen activator production by bovine endothelial cells

Endothelial cells obtained from the aortae of 1- to 2-d-old calves were cloned at high efficiency using fibrin-coated dishes. Primary cultures as well as clones derived from them produced high fibrinolytic activity when grown on 125I-fibrin-coated dishes which was 90% dependent upon the presence of plasminogen. High plasminogen-dependent proteolytic activity was also demonstrated in endothelial cell lysates and in the culture medium of the cells. The production and secretion of the plasminogen activator(s) were found to increase during the log phase of cell growth and to reach a maximum level at confluence. These endothelial cells exhibited morphological phenotypes comparable to those of transformed cells when grown in the presence of acid-treated fetal calf, dog, or human serum. Furthermore, they demonstrated anchorage independent growth, and large colonies were formed in semisolid media. Spontaneous neoplastic transformation of these cells was excluded by karyotypic analysis, lack of tumorigenicity in athymic nude mice, and limited lifespan in culture. Cell clones isolated from colonies grown in agarose demonstrated the same growth characteristics and proteolytic activity as before plating in agarose. High fibrinolytic activity, morphological changes in the appropriate serum, and growth in semisolid media may therefore be indicative of the migratory and/or invasive capacity of both nontransformed endothelial cells as well as tumor cells.     

The production of toxic protease by the entomopathogenous fungus Metarhizium anisopliae in submerged culture

The production of a toxic complex of proteolytic enzymes by Metarhizium anisopliae was evaluated with 29 nitrogen sources in modified Czapek-Dox medium in submerged cultures. The proteolytic complex is more constitutive than that of Beauveria bassiana and its production is influenced by the quality of complex natural media. The highest activity was attained with Galleria mellonella proteins. The proteolytic complex manifests proteolytic activity of two pH optima, 5.5 and 8.0. The ratio of these two activities differs markedly with the nitrogen source used, but the major proteolytic activity occurs at pH 5.5.     

放射损伤,烧伤与放烧复合伤血清成份对心肌细胞钙离子…

本实验研究了放射损伤,烧伤与放烧复合伤后血清成份对培养心肌细胞Ｌ－型钙离子通道活动的影响。结果表明：伤后血清对上述通道的有激活作用,从而改变细胞内钙离子水平,此可能为伤后心脏功能抑制的一个重要机理。在作用强度上,复合伤血清重于单一伤,烧伤重于放射损伤,这是导致不同伤后血清对心功能抑制程度不一的重要因素。     

Levels of alpha 1-antitrypsin and alpha 2-macroglobulin in blood serum and its antitryptic capacity

Immunochemical analysis in combination with gel filtration and isoelectric focusing made it possible to state that in blood serum of healthy people 81.3 +/- 0.5% of administered trypsin is bound with alpha 1-antitrypsin and 18.7 +/- 0.6%--with alpha 2-macroglobulin. The latter is functionally heterogeneous, only 40% of it is bound with trypsin and in the formed complex the antigenic properties of trypsin and alpha 2-macroglobulin are lost. A great number of blood serum alpha 1-antitrypsin cannot fix trypsin. The content of such alpha 1-antitrypsin rises sharply with pathology available. In the immunochemical estimation of the organism inhibitory potential relative to proteolytic enzymes not only the amount of the inhibitor but also its functional activity should be taken into account. The data of immunochemical research of the blood serum isoelectrophoregrams show that the most considerable changes under conditions of pathology occur in alpha 2-macroglobulin.     

Fate of egg white trypsin inhibitor and start of proteolysis in developing chick embryo and newly hatched chick

Trypsin inhibitor and proteolytic activities were studied in incubated eggs, embryos, and newly hatched chicks. After rupture of the secondary seroamniotic suture at 11 days, the trypsin inhibitor content of the albumen gradually passes into the amniotic cavity; from there it is taken up orally by the chick embryo. It is supposed that between 11 and 18 days of embryonic development the trypsin inhibitor passes from the gut to the yolk sac through the vitellointestinal duct. The thin yolk contained only traces of trypsin inhibitor, and the allantoic fluid was entirely free from it. The amylase activity demonstrable in the liquid intestinal contents of the chick embryo indicates the presence of pancreatic secretion. The trypsin inhibitor probably suppresses the proteases not only directly, but also through prevention of the activation of zymogens. Enterocytes of chick embryos showed no morphological indication of the absorption of undigested proteins on histological examination. The cloacal membrane of the newly hatched chick ruptures shortly after the bird has dried up, and the trypsin inhibitor is subsequently eliminated along with the intestinal contents. The intestinal proteolytic enzymes appear immediately afterward. The proteolytic activity appeared regardless of whether the birds were or were not fed. Maximum proteolytic activity was measured in the small intestine of chicks that were fasted for 2 days after hatching. The pattern of proteolytic enzymes as well as their sensitivity to protease inhibitors did not notably differ from that of mammals.     

Phytotoxicity of pesticides mancozeb and chlorpyrifos: correlation with the antioxidative defence system in <Emphasis Type="Italic">Allium cepa</Emphasis>

Pesticides are a group of chemical substances which are widely used to improve agricultural production. However, these substances could be persistent in soil and water, accumulative in sediment or bio-accumulative in biota depending on their solubility, leading to different types of environmental pollution. The present study was done to assess the impact of pesticides-mancozeb and chlorpyrifos, via morphological and physiological parameters using Allium cepa test system. Phytotoxic effects of pesticides were examined via germination percentage, survival percentage, root and shoot length, root shoot length ratio, seedling vigor index, percentage of phytotoxicity and tolerance index. Oxidative stress on Allium seedlings caused by pesticides was also assessed by investigating the activity of antioxidative enzymes viz. catalase, peroxidase and superoxide dismutase. Correlation was worked out between morphological parameters and antioxidative enzymes to bring out the alliance between them. Mancozeb and chlorpyrifos concentrations were significantly and positively correlated with the activity of antioxidative enzymes and negatively correlated with morphological parameters. Significant positive correlation between various morphological parameters showed their interdependency. However, negative correlation was obtained between activity of antioxidative enzymes and morphological parameters. The enzymes however, showed positive correlation with each other. Based on our result we can conclude that all morphological parameters were adversely affected by the two pesticides as reflected by phytotoxicity in Allium. Their negative correlation with activity of antioxidative enzymes indicates that upregulation of antioxidative enzymes is not sufficient to overcome the toxic effect, thereby signifying the threat being caused by the regular use of these pesticides.     

Enzymes Produced by a Pseudomonas Species Which Inactivate Inhibitors of Certain Viral Hemagglutinins. I. Identification and Purification of a Proteinase and Phospholipase C

Filtrates from cultures of a psychrophilic Pseudomonas species, which inactivate serum inhibitors of certain viral hemagglutinins, were shown to contain both lecithinase (phospholipase C) and a proteolytic enzyme with elastase activity. The bacterium was cultivated under conditions favoring production of the respective enzymes, and the enzymes were purified by ammonium sulfate precipitation followed by column chromatography or by gel filtration. The elastase was obtained in crystalline form and was recrystallized. It has properties similar to those of a number of other bacterial elastases but is more heat-labile than most. Although a high degree of purification was achieved for the lecithinase, as evidenced by an increase in specific activity, it was not obtained in crystalline form. Partially purified preparations of the lecithinase had extremely high activity compared to that of commercial preparations of phospholipase C from Clostridium welchii.     

Studies on the proteolytic enzymes of invertebrates constituting fish food

Studies were carried out on the proteolytic activity of trypsin and pepsin-like enzymes of invertebrates consituting fish food. Relations between proteolytic activity of enzymes, pH, and temperature were established.