On the role of carbohydrate-protein highly selective interactions in the biological activity of glycoconjugates: <Emphasis Type="Italic">Grifola frondosa</Emphasis> (Fr.) S.F. Gray lectin binding to specific and non-specific antibodies

Both quantitative and qualitative estimations of the basidiomycete Grifola frondosa lectin binding to the specific and non-specific polyclonal rabbit antibodies were attempted. The lectin complexation with homological antibodies was shown to be characterized by greater binding constants as compared to non-homological antibodies. Therewith the values of changes in standard free energy ∆G ⁰ displaying a strength of both complexes were essentially the same. The data obtained testify to universality of biospecific reactions “antigen–antibody” and “lectin–carbohydrate” at a molecular level, and could give new insight into the phenomenon of these biospecific interactions. The actual specificity in the above bio-recognition processes could differ from the results of in vitro assays using lectins, and, therefore, should be interpreted carefully when concluding on the lectins behaviour in living systems.     

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (³⁵S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.     

Effect of culture medium composition on the activity of extracellular lectins of <Emphasis Type="Italic">Lentinus edodes</Emphasis>

The time course of lectin production in culture liquid of the basidial fungus Lentinus edodes strain F-249 in different media under submerged culture conditions was studied. The activity of agglutinins depended on the ratio between carbon and nitrogen sources and the pH of the culture medium. The lectin activity in culture medium was maximal when the fungus was grown in a medium containing L-arabinose as a source of carbon and L-asparagine as a source of nitrogen (C:N ratio, (9.5–12):1) on day 15–18 of culturing at pH 8.0–9.0.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 200–203.Original Russian Text Copyright © 2005 by Tsivileva, Nikitina, Garibova.     

Isolation and Purification of Enzymes from Ligninolytic Complex of the Basidial Fungus <Emphasis Type="Italic">Trametes pubescens</Emphasis> (Schumach.) Pilat and Study of Their Properties

A method for purification of enzymes from the ligninolityc complex of the basidiomycete Trametes pubescens (Schumach.) Pilat has been elaborated. Two homogeneous isoforms of laccases (laccase 1 and laccase 2) as well as a homogeneous preparation of lignin peroxidase were isolated. Basic biochemical parameters of the enzymes were determined, such as the molecular weights (67, 67, and 45 kD, respectively), isoelectric points (5.3, 5.1, and 4.2, respectively), as well as content and composition of the carbohydrate moiety of the laccases (N-acetylglucosamine, mannose, and xylose). The pH dependences and thermal stabilities of the laccases were investigated. The kinetic parameters of the enzymatic reactions catalyzed by the laccases were determined using different substrates, such as catechol, hydroquinone, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate), and K4Fe(CN)6. The structure of the active sites of both laccases and the lignin peroxidase were studied by EPR, CD, and UV-VIS spectroscopy, as well as using fluorescence analysis. Our studies showed similarity of the spectral characteristics of the two laccases, whereas their kinetic properties were found to be different.     

Maternal cell contamination of cultures of spontaneous abortion fibroblasts: importance for cytogenetic analysis of embryonic lethality

The results of standard cytogenetic analysis of the long-term cultures of embryonic fibroblasts of 478 first-trimester spontaneous abortions were retrospectively reviewed. In 16% of embryos with cytogenetically confirmed karyotype 46,XX, the Y chromosome was found by molecular genetic methods. Prior to obtaining the chromosome preparations, the cell cultures of Y chromosome-carrying embryos were maintained for a longer period than the cultures of embryos without the Y chromosome. Thus, a late entry of a culture into the logarithmic growth phase serves as marker of maternal cell contamination. We developed a mathematic model for assessment of karyotype incidence and the "sex ratio" of spontaneous abortions, taking into account risk of maternal cell contamination in extraembryonic tissue cultures. Thus estimated, the incidence of chromosomal abnormalities in the studied sample increased from 54.6 to 60.3% and the expected sex ratio increased from 0.66 to 1.02 in abortions with normal karyotype. Using molecular analysis of inheritance of polymorphic DNA markers of six autosomes (2, 11, 16, 19, 20, and 21), the proposed model was tested on 60 embryos with karyotype 46,XX and their parents. Numerical chromosome abnormalities were revealed in uncultured tissues of seven abortions (11.7%), including four without the Y chromosome, which is in a good agreement with the expected incidence of karyotype abnormalities (8.3%) predicted by our model. In view of this, estimating risk of maternal cell contamination in embryonic cell cultures seems necessary for correctly assessing the effect of natural selection in humans, for understanding the mechanisms that determine the sex ratio, and for evaluating the precision of prenatal cytogenetic diagnosis of chromosomal abnormalities.     

Collagenolytic activity in several species of deuteromycetes under various storage conditions

The ability of deuteromycetes of the genera Penicillium, Aspergillus, and Botrytis to retain collagenolytic activity was studied after both 2 and 10 years of storage on a Czapek medium under a layer of mineral oil at 4 degrees C, as well as in silica gel granules at 20 and -60 degrees C. The enzymatic activity of several species, including Botrytis terrestris, Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum, was retained under both conditions of storage. Aspergillus repens retained enzymatic activity only if stored under a layer of mineral oil. The viability of conidia and the collagenolytic activity of Botrytis terrestris, P. janthinellum, P. chrysogenum, and Penicillium citrinum, maintained on silica gel for 10 years, depended on the storage temperature. The viability of the test strains improved after storage on a silica gel at -60 degrees C. A strain of Aspergillus repens lost its ability to dissolve collagen at various storage temperatures on the silica gel. The index of lysis for three strains of Penicillium deuteromycetes (Penicillium janthinellum, Penicillium chrysogenum, and Penicillium citrinum) increased after a 10-year storage on silica gel at -60 degrees C.     

Application of in vitro motility assay to studying the calcium-mechanical relationship in skeletal and cardiac muscles

In a set of experiments on regulated contractile systems (i.e., in vitro motility assay with a reconstructed thin filament), the velocity of a thin filament on the surface coated with rabbit skeletal or rat cardiac myosin was estimated at various calcium ion concentrations in solution (pCa 4–8). The velocity versus pCa curve proved to be sigmoid. The velocity of a regulated thin filament at a saturating calcium concentration (pCa 4) exceeded that of a nonregulated thin filament by 65 and 87% for skeletal and cardiac myosin, respectively. The Hill coefficient was 1.95 and 2.5 for skeletal and cardiac muscles, respectively; this difference was discussed in terms of the different contributions of cooperativity mechanisms of contractile and regulatory proteins to the regulation of contraction in these types of muscle.     

The in vitro motility assay to study the calcium-mechanical relationship in skeletal and cardiac muscles

In a series of experiments on regulated contractile systems (i.e., in vitro mobile systems with reconstructed thin filaments), the velocities of the movement of a thin filament on the surface covered by either rabbit skeletal or rat cardiac myosin at various concentrations of calcium ions in solution (in the pCa range from 4 to 8) were assessed. The corresponding "pCa-velocity" relationships were plotted, which proved to be of the sigmoid form. It was found that, at a saturating calcium concentration (pCa 4), the velocity of regulated thin filaments was 65% higher than for unregulated ones in the case of skeletal myosin and 87% higher than for unregulated thin filaments in the case of cardiac myosin. It was also found that the Hill coefficient was 1.95 and 2.5 for skeletal and cardiac myosins, respectively. The difference in the Hill coefficients for skeletal and cardiac myosins is discussed in terms of the difference in contribution of cooperativity mechanisms of contractile and regulatory proteins in the regulation of contraction in these types of muscles.