Structural and functional analysis of glycosylated Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103

The fungal strain Humicola lutea 103 produces a naturally glycosylated Cu/Zn-superoxide dismutase (Cu/ZnSOD) (HLSOD). To improve its yield, the effect of increased concentration of Cu2+ (from 1 to 750 microg/ml) on growth and enzyme biosynthesis was studied. The primary structure of this fungal enzyme has been determined by Edman degradation of peptide fragments derived from proteolytic digest. A single chain of the protein, consisting of 152 amino acid residues, reveals a very high degree (74-85%) of structural homology in comparison to the amino acid sequences of other fungal Cu/ZnSODs. The difference of the molecular masses of H. lutea Cu/ZnSOD, measured by MALDI-MS (15,935 Da) and calculated by its amino acid sequence (15,716 Da), is attributed to the carbohydrate chain of one mole of N-acetylglucosamine, attached to the N-glycosylation site Asn23-Glu-Ser. HLSOD protected mice from mortality after experimental influenza A/Aichi/2/68 (H3N2) virus infection. Using the glycosylated HLSOD, the survival rate is increased by 66% (protective index=86.1%) and the survival time prolonged by 5.2 days, similar to the application of ribavarin, while non-glycosylated bovine SOD conferred lower protection.     

Effect of a fungal Cu/Zn superoxide dismutase on the cell-mediated immune response in Graffi tumor bearing hamsters

The antibody-dependent cell cytotoxicity (ADCC) of spleen lymphocytes, isolated from hamsters with progressing myeloid Graffi tumor, was studied. The effect of the application of Cu/Zn superoxide dismutase, isolated from the fungal strain Humicola lutea (HL SOD), before and during tumor transplantation on the lymphocyte ADCC was examined. Myeloid Graffi tumor cells as target cells were used. Antibodies from a rabbit hyper-immune anti-tumor Graffi cells serum, or from tumor-bearing hamsters serum were used in the test. The leukocyte adherence inhibition (LAI) in the presence of tumor antigen was examined also during tumor progression. ADCC of the spleen lymphocytes, determined by both, rabbit and hamster anti-tumor antibodies, decreased during tumor progression. The optimum treatment of the animals by HL SOD induced a 20-30% increase of lymphocyte cytotoxicity against myeloid Graffi tumor cells. Cytotoxicity in presence of tumor bearing hamsters serum was twofold lower as compared to that one determined in the presence of rabbit hyper-immune anti-myeloid Graffi tumor cells serum. Leukocyte adherence inhibition (LAI) index in the presence of tumor antigen increased during tumor development in the groups of treated and untreated animals. The LAI indices of HL SOD-treated tumor-bearing hamsters were lower than that of untreated animals with tumors, what can be explained by a higher adherence ability of leukocytes induced by HL SOD treatment (in formula for calculation of LAI index the adherence value is in the denominator). The results show the beneficial effect of HL SOD on the cell-mediated immune response of myeloid Graffi tumor bearing hamsters, what is probably due to the participation of the enzyme in the host's oxidant-antioxidant balance.     

Studies of Antarctic yeast for β-glucosidase production

Moss and lichen samples from the region of the Bulgarian base on Livingston Island, Antarctica were examined for the presence of yeasts. Six pure cultures were obtained. They were screened for -glucosidase production and two of them were selected. These were identified as Cryptococcus albidus AL₂ and C. albidus AL₃, according to their morphology, reproductive behaviour, and growth at different temperatures, salt concentrations, nutritional characteristics and various biochemical tests. These strains were examined for biosynthesis of -glucosidase on different carbon sources under aerobic conditions. High exocellular and endocellular activities were obtained when they were grown on cellobiose, methyl--D-glucopyranoside and salicin. The time course of growth and -glucosidase production of the yeast was examined by cultivation in a medium with cellobiose under aerobic conditions at temperatures 18 and 24 °C for 96 h. Cryptococcus albidus AL₂ and C. albidus AL₃ synthesized exocellular enzyme, respectively 58.33 and 55.83 U/ml and endocellular enzyme 137.75 and 205.34 U/ml at 24 °C for 72 h of the cultivation.     

Metabolic stability of nonhistone chromosomal proteins in two different classes of developing rat brain nuclei.

The metabolic behaviour of NHCP was studied in rat brain nuclei from fully differetiated cells and in nuclei from still differentiating cells. Eight-day-old rats were injected intracisternally with [14C]thymidine and [3H]tryptophan and the 3H/14C ratio of the chromatin was followed for a period of 33 days. It was found that in the fraction of differentiated cells this ratio remained unchanged, showing the presence of metabolically stable NHCP. At the same time in the fraction containing nondifferentiated cells a substantial part of the NHCP was turned over, which was indicated by a sharp decrease in their 3H/14C ratio with time.     

Molecular modeling of cobalt(II) hyaluronate

Structural data for complexes of hyaluronic acid and 3d metals(II) of the fourth group of the periodic table are lacking. A combined QM/MM method was used to solve the structure of the first coordination sphere around the cobalt(II) ion. Some available experimental data were compared with the results obtained via computation and were found to be in good agreement. Our results open the way for using molecular modeling to solve the structure of other metal(II) hyaluronates.     

Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer.     

In vitro antioxidant activity of polyphenol extracts with antiviral properties from Geranium sanguineum L

Recent evidence shows that plant polyphenols exhibit antioxidant and radical scavenging properties. By three separate and complementary methods--DPPH assay, beta-carotene-linoleic acid assay and NBT-reduction assay it was established that a polyphenol-rich extract from the medicinal plant Geranium sanguineum L. with strong anti-influenza virus activity, possessed antioxidant and radical scavenging capacities. For comparative reasons caffeic acid and the synthetic antioxidant BHT were used. Total soluble phenolic constituents of the MeOH extract measured by Folin-Ciocalteu reagent were found as 34.60% (w/w). Further it was demonstrated that the EtOAc fraction, retaining the majority of the in vivo protective effect exhibited a strong O2-scavenging activity while the n-BuOH fraction, containing the majority of the in vitro antiviral activity provoked generation of O2-. The O2- scavenging activity of all three preparations correlated with the rate of the protective effect shown in the murine model of experimental influenza virus infection. The present results are in accordance with our intensive studies on the mode of the protective effect of the plant extract which showed positively that the protection may possibly be attributed to the combination of more than one biological activities and that the use of antioxidants might be an useful approach in the treatment of influenza infection.     

Effect of Cu/Zn-superoxide dismutase from the fungal strain Humicola lutea 103 on antioxidant defense of Graffi tumor-bearing hamsters

A novel Cu/Zn-containing superoxide dismutase (SOD) was isolated from the fungal strain Humicola lutea 103. Previously, a protective effect of this enzyme (HLSOD) against tumor growth and also superoxide production in Graffi tumor-bearing hamsters (TBH) were established. The aim of the present study was to investigate the effect of HLSOD on the activity of endogenous SOD and catalase in the cells from TBH during tumor progression. Our results point out that transplantation of Graffi tumor causes a significant decrease in SOD activity in the cells from liver of the hosts (from 35 to 59% compared to the control). In the tumor cells relatively low levels of SOD (about 7 U mg protein(-1)) were found, and Cu/ZnSOD was the main isoenzyme in total SOD activity. Tumor growth resulted in a reduction of catalase activity, which correlated with the process of tumor progression. A single dose (65 U) treatment with HLSOD caused an increase in endogenous SOD and catalase activity in healthy animals and resulted in restoration of the antioxidant ability in liver cells of the hosts at the early stage of tumor progression. The results show the possible participation of HLSOD in the host oxidant-antioxidant balance, which is probably one of the factors of its immunoprotective action established earlier.     

Adaptation of fungi,including yeasts,to cold environments

A wide range of cold environments exist, with an equally broad variety of fungi and yeasts that have adapted to such environments. These adaptations, which affect membranes, enzymes and other cellular components, such as radical scavenging molecules, display a great potential for exploitation in biotechnology. Alterations have been detected in membrane lipids, with an increase in fatty acid unsaturated bonds that enhance their fluidity. We report new data on the different phospholipid composition in membrane lipids in the same fungal species from both Antarctic and temperate regions. The decrease in temperature causes intracellular oxidative stress by inducing the generation of reactive oxygen species. We report the results of the first analysis of the non-enzymatic antioxidant response and phenolic compound production by an Antarctic strain of Geomyces pannorum. A survey on yeasts from the cryosphere is reported with a focus on their adaptation to a cold environment. Some studies have shown that the number of macrofungi in glacier forefronts rises as deglaciation increases. The survival success of many plants in such areas may be attributed to their mycorrhizal associations. We highlighted the macrofungal biodiversity of some Italian alpine habitats, in which we Inocybe microfastigiata, Laccaria montana and Lactarius salicis-herbaceae were recorded for the first time in Lombardy (Italy).