Traditionally, the taxonomy of the genus Nostoc is based on morphological and physiological characters. The extreme morphological variability of the Nostoc species, due to their life cycle and environmental conditions, hampers the correct identification of the individual species. This is also one of the reasons for the disputed taxonomic positions and relationships between the genera Anabaena–Aphanizomenon as well as between Anabaena–Nostoc. Therefore, it is necessary to use additional markers for development of a polyphasic classification system of order Nostocales. In light of this, we here present the first molecular and phy-logenetic characterization of two species of the genus Nostoc (Nostoc linckia and Nostoc punctiforme) based on the cpcB-IGS-cpcA locus of the phycocyanin oper-on. The phylogenetic position of these two species within order Nostocales as well as within division Cyanobacteria has been determined. Our results indicate that genus Nostoc is heterogeneous. Analysis of the IGS region between cpcB and cpcA showed that Nostoc and Anabaena are distinct genera. Reported molecular and phylogenetic data will be useful to solve other problematic points in the tax-onomy of genera Aphanizomenon, Anabaena and Nostoc.

Nonspecific esterases of silkworm (Bombyx mori L.) gut were investigated by means of polyacrilamide gel electrophoresis (PAGE). Stage-specific expression of eleven esterase bands was detected during larval development of breeds and inter-breed hybrids kept in Bulgaria. In two esterase zones, intra- and inter-breed polymorphism was found. The polymorphism in fraction GES I1 specific for the gut may be used for testing of the breeds raised in our country with reference to determining the degree of genetic heterogeneity. The specific expression in zone GES L1 observed at present study confirmed gene determinate polymorphism in zone BES E1, observed earlier.

Thirteen new isolated thermotolerant Bacillus strains and four known Bacillus species were used to evaluate the effect of growth temperature, pH-values and NaCl concentrations on the intracellular glucose-6-phosphate dehydrogenase (G6PDH) activity. Results had shown a significant difference in G6PDH production among all species at all used temperatures (p<0.05). The response of individual new isolates and controls for production of G6PDH under growth conditions was variable. The optimal growth conditions did not correspond to the optimal cultivation conditions for maximum G6PDH production. The growth temperature showed the most significant effect on G6PDH activity. The combined effect of temperature and NaCl on the G6PDH activity was strongly pronounced in comparison with the combined effect of temperature and pH or pH and NaCl. Thermal stability at 53ºC and electrophoretic mobility were also investigated. G6PDH from HUTB41 was the most thermostable G6PDH enzyme with T50% of more than 360 minutes. Electrophoretic study demonstrated that G6PDH was composed of two isoenzymes for all strains except B. marinus and B. schlegelii that had three isoenzymes.

The biological role of blood group antigens (BGA) A and B in tissues of different vertebrates is still controversial. There are few investigations on vertebrate pancreas and no obvious explanation of their tissue expression. The aim of the present study is to follow and compare the pancreatic expression of BGA A and B in representatives of five vertebrate classes. The biotin-streptavidin-proxidase labeling system was used for immunohistochemical detection of BGA by monoclonal antibodies to human A and B antigens. The present study reveals specific immunoreactivity in acinar and epithelial cells of pancreatic efferent ducts in species free-living vertebrates. The immunoperoxidase staining shows antigenic heterogeneity in the cellular localization. The number of positive cells and the intensity of expression vary in different species. Endothelial cells are positive only in the pancreas of Emys orbicularis. The lack of BGA A and B in some species suggests that the expression of these antigens is dependent not only on the evolutionary level of the species, but mainly on some genetic control mechanisms. The production of BGA A and B and the variability in their cellular localization probably reflect the stage of cell differentiation and the mechanisms of pancreatic secretor function. The presence of histo BGA in endodermal acinar pancreatic cells confirms the assumption for the high antigenic stability and conservatism of these molecules in vertebrate histogenesis and evolution.

An in vivo micronucleus (MN) test in peripheral erythrocytes and frequency of bone marrow cells with chromosome aberrations in free-living small rodents, chronically exposed to heavy metal pollution were used for detection the genome response to genotoxic agents in the environment. Yellow-necked mice (Apodemus flavicollis), common vole (Microtus arvalis) and East-Mediterranean (Macedonian) mice (Mus macedonicus) were collected in a polluted region near lead-zinc smelting factory – Asenovgrad (South Bulgaria, near Plovdiv) and in the background region of the Strandzha National Park (Southeastern Bulgaria). Mean frequencies of MN and aberrant cells in the individuals from the impact region were significantly higher compared to the mean frequencies from the same species in the background region. The comparative analysis of results confirmed that the species Apodemus flavicollis and Microtus arvalis may be suitable bioindicators for biomonitoring studies using MN test and chromosome aberrations. Obtained results demonstrated that the in vivo MN test may be a sensitive end-point for the detection of genotoxity that may result from the simultaneous action of several metals and may be useful as a biomarker of environmental stress in situ.

A polymerase chain reaction based assay was developed for authentication of cell lines or tissues from human, pig and chicken origin. Specificity was achieved by species specific primer design targeting the mitochondrial D-loop sequence. Amplicon sizes were 114 bp, 169 bp and 645-648 bp for chicken, human and pig derived cell lines, respectively. Primers were tested for species specificity and non-specificity between haplogroups of the same organisms using BLAST tool and subsequently for cross amplification DNA extracted from human, chicken and pig venous blood as a positive control. Primers were also amplifying specific products in DNA extracted from individual cell line in both functional cell models and intentionally mixed cell lines consisting functional cell models. The PCR assay developed in this study represents a low-cost species specific end-point PCR based assay of the mitochondrial D-loop for the authentication of the cell line origin.

Dextransucrases are enzymes that transfer the glucosyl moiety from sucrose to other acceptor molecules. In the present study, we have investigated the biosynthesis of dextransucrases by two strains of Leuconostoc mesenteroides isolated from Bulgarian fermented products. Extracellular dextransucrase activities of 10.80 U/ml for strain Lm 28 and 9.6 U/ml for strain Lm 22 were measured in a batch culture. The SDS-PAGE analysis showed that the molecular weight of the dextransucrases isolated from both strains was 180 kDa. Enzymes of the studied strains were found to efficiently transfer the glucosyl moiety of sucrose onto maltose acceptor. By increasing the sucrose/maltose ratio, it was possible to catalyze the synthesis of oligosaccharides of increasing degree of polymerization.

Cyanobacteria are among the oldest autotrophic organisms with cosmopolitan distribution and known as producers of secondary metabolites with toxic properties named "cyanotoxins". Studies with respect to toxin production of genus Nostoc are yet limited. In the present study we have investigated two Nostoc species (Nostoc linckia and Nostoc punctiforme) for production of intracellular and/or extracellular compounds with cytotoxic potential. Extracts and algal growth media were assessed by different in vitro tests using freshly established mouse primary cultures from different tissues and one fish cell line. Our data showed that the mouse cells are more sensitive to toxic compounds than the fish cells. Both Nostoc species produced intracellular and extracellular bioactive compounds with different effects on mouse and fish cells. The presence of cyanotoxins as anatoxin-a and microcystins/nodularin was confirmed by HPLC and ELISA analyses. Therefore, Nostoc species are not only sources of bioactive compounds with therapeutic action, but they can be a potential hazard to aquatic systems as well as to animal and human health.

Allozyme genetic polymorphism in Bulgarian honey bee populations from four different locations in the south-eastern part of the Rhodopes Mountain was studied on six enzymic systems (MDH, ME, EST, ALP, PGM and HK) corresponding to six genetic loci. Allozyme analysis revealed that all studied loci were polymorphic in almost all investigated populations. The observed heterozygosity was found to range from 0.110 to 0.208 and Nei's genetic distance – between 0.016 and 0.061 among the studied populations. These honey bee populations were clustered in two groups in the UPGMA dendrogram. The Tihomir population was in a separate clade while other three populations (Kardzhali, Krumovgrad and Dolni Yurutci) were grouped together.

The present investigation focuses on screening and optimization of media components to enhance glucansucrase and glucan production by Pediococcus pentosaceus CRAG3 at shake-flask and bioreactor level using Taguchi orthogonal array design. A three-level Taguchi orthogonal array layout of L27 (33) was employed, in which six variables were studied for their influence on glucansucrase and glucan production. The results showed that sucrose, K2HPO4 and Tween-80 were the most significant factors to improve glucansucrase production while the glucan production was mostly affected by sucrose, peptone and K2HPO4. The optimized medium composition for maximum glucansucrase and glucan production were: sucrose 3.5% and 5%; yeast extract 0.2% and 2.0%; beef extract 0.5% and 0.5%; peptone 3.0% and 1.0%; K2HPO4 0.2% and 0.2%, and Tween-80 1.0 and 0.1%, respectively. The optimized medium gave 10.1 U/ml and 10.2 U/ml glucansucrase activity while glucan concentrations were 56 mg/ml and 80 mg/ml in shake flask and bioreactor level, respectively which were in good agreement with predicted values (10.1 U/ml and 54.5 mg/ml). The optimized medium gave 2 fold enhancement in enzyme activity and 4 fold increase in glucan concentration as compared to non-optimized medium (4.5 U/ml and 15 mg/ml, respectively) at shake flask level.