[Molecular Polymorphism of ß-Galactosidase LAC4 Genes in Dairy and Natural Strains of Kluyveromyces Yeasts].

The ability to ferment lactose is a characteristic peculiarity of dairy Kluyveromyces lactis yeasts; the vast majority of other yeast species are not able to assimilate this disaccharide. Molecular polymorphism of LAC4 genes encoding ß-galactosidase controlling lactose fermentation is not well studied, and the published data concern only a single strain (K. lactis var. lactis NRRL Y-1140) isolated from cream in the United States. We studied ß-galactosidase genes in lactose-fermenting К lactis strains isolated from dairy products and natural sources in different regions of the world using molecular karyotyping, Southern hybridization, and sequencing. It was established that the ability to ferment lactose in К. lactis var. lactis dairy yeasts is controlled by at least three polymeric LAC loci with different chromosomal localization: LAC1 (chromosome III), LAC2 (II), and LAC3 (IV). Most of the strains we studied had the LAC2 locus. A comparative analysis of ß-galactosidases of the Kluyveromyces genus yeasts and these enzymes from other yeasts was conducted for the first time. Phylogenetic analysis detected significant differences between the LAC4 proteins of yeasts of the Kluyveromyces genus (K. lactis, К. marxianus, К. aestuarii, К. nonfermentans, К. wickerhamii), Scheffersomyces stipitis, Sugiyamaella lignohabitans, and Debaryomyces hansenii. A correlation between ß-galactosidase sequences and ecological origin (dairy products and natural sources) of Kluyveromyces strains was found. The group of dairy strains is heterogeneous and includes К. lactis var. lactis and К. marxianus yeasts (99.80-100% similarity), which indicates a common origin of their LAC4 genes. Phylogenetic analysis of ß-galactosidases indicates a close genetic relationship of dairy and hospital strains of К. lactis var. lactis and К. marxianus. Clinical isolates are able to ferment lactose and appear to originate from the dairy yeasts.

Isolation and properties of collagenolytic serine proteinase isoenzyme from King Crab Paralithodes camtschatica.

An electrophoretically homogeneous isoenzyme CSP-2 of collagenolytic serine proteinase has been isolated from the total preparation of king crab digestive enzymes. The molecular mass of the proteinase is 24.8 +/- 0.3 kD, pH optimum for activity is 8.5, the temperature optimum for activity is 38-40 degrees C, and the pH range of stability is 7-10. The enzyme has dual substrate specificity, but preference for positively charged amino acid residues in P(1)-position is more pronounced than in the case of the major isoenzyme. The temperature dependence of kinetic constants for synthetic substrate hydrolysis by CSP-2 has been investigated. Inhibition specificity of the enzyme is characteristic of serine proteinases but more like that of crab trypsin than that of the major CSP isoenzyme. The isolated collagenolytic proteinase also cleaves fibrinogen and fibrin and activates plasminogen. The amino acid sequence of the CSP-2 proteinase, which has been partially determined by tandem mass spectrometry, displays some similarity to the sequence of the major CSP isoenzyme.

Coagulation and fibrinolysis in rats after surgery with monopolar electrical scalpel.

Blood coagulation, fibrinolysis in plasma and peritoneal fluid, and activity of tissue plasminogen activator in the peritoneum and uterine horns were studied in albino rats after surgery on the uterine horns with a monopolar electrical scalpel. This instrument induced severe inflammatory reaction and disturbances in the fibrinolysis and coagulation systems.

Depletion of fibrinolysis activators as a result of continuous stress.

The dynamics of disorders in haemostasis and fibrinolysis upon continuous stress was studied in model experiments with rats exposed to emotional-pain-stress. It was shown that the plasminogen activator (PA) is released into the blood within the first few minutes of exposure to stress. Four hours after cessation of continuous stress (6 hours) the enzyme depletion and fibrinolysis depression occurred. Such stress-induced changes in the blood system function can be regarded as a risk factor in the development of thrombosis.

Plasminogen activator content in heart tissue and perfusate under various experimental conditions.

In order to elucidate the mechanisms of plasminogen activator release into the circulation during the first minutes of experimental myocardial ischaemia in vitro, isolated rat hearts were perfused under optimal conditions and under conditions inducing myocardial ischaemia. In ventricular tissues the content of plasminogen a activator was measured; in the perfusate the plasminogen activator and anticoagulating activities were determined. Under optimal conditions the plasminogen activator content in contracting heart was found to be essentially the same as under in vivo conditions; in the perfusates the release of plasminogen activator and of substances possessing the anticoagulating activity was insignificant. Under myocardial ischaemia the release of these substances into the circulation increased, while plasminogen activator content decreased.

Changes of the fibrinolytic system of animals induced by injection of tissue plasminogen activator.

The changes in the fibrinolytic system of rats induced by repeated injections of tissue plasminogen activator (TPA) have been studied. The euglobulin fraction fibrinolytic activity increases 10 min after the first injection of TPA. Repeated injections of TPA for one or more days do not stimulate the fibrinolytic activity or TPA accumulation in the blood. Apparently in the absence of fibrin clots in the blood flow of healthy animals TPA is incapable of activating plasminogen conversion into plasmin. TPA, in its turn, is rapidly bound to the antiactivator and is excreted from the organism.