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A contemporary review of the studies on the trophic spectrum and feeding ecology of the Bulgarian frogs and toads is presented. The analysis of the Bulgarian herpetological literature showed that currently, there are 18 specialized studies on the diet of the Bulgarian anuran species. Of all 16 anuran species, occurring in Bulgaria, 9 (56.25%) have been studied in connection with their trophic spectrum. For four species (25.00%), there is very little data, and for 7 (43.75%), such studies have not yet been done. All Bulgarian species of amphibians are zoophagous, mainly insectivorous. All studied species are general feeders (polyphages), except Bombina variegata, which shows a slight preference to Coleoptera and Bufo bufo and Bufotes viridis, which show slight preference to Formicidae and Coleoptera. Cannibalism was recorded only in Pelophylax ridibundus. The highest values of the trophic niches breadths are recorded in P. ridibundus, R. graeca, R. temporaria, and R. dalmatina. The trophic niches breadths of the other anuran species found in Bulgaria have significantly lower values, the lowest being in Hyla orientalis.

The aim of the study was to present our own experimental model of blunt chest trauma in small laboratory animals. Fifteen Wistar albino rats (body weight 290.0-320.0 g) were used in the experiment. An originally designed platform was used to inflict blunt thoracic trauma in the rats. Gross examination and histological evaluation of the heart and lung were performed. It was established that blunt chest trauma caused three thoracic injuries: bilateral lung contusion, myocardial contusion and hemopericardium. These thoracic injuries occurred as a result from direct compression of the chest wall towards the lung and the heart. The severity of lung parenchymal and myocardial pathological findings depended on the energy of blunt chest trauma. The study presents a reproducible small-animal experimental model of blunt chest trauma. The model gives opportunities for further studying different aspects of lung and myocardial contusion.

Summary. Coagulation factor V (FV) plays an important role in the blood coagulation cascade as part of the prothrombinase complex. FV deficiency is a rare autosomal recessive bleeding disorder with variable phenotypic expression. Thus, our study reports 39 patients with FV deficiency. In 36 cases, we were able to identify a causative mutation. Of these, 20 patients were heterozygous for the identified mutation, nine were homozygous, six were compound heterozygous and one proband was pseudohomozygous. In the remaining patients, no mutation was found. A total of 42 genetic alterations (of which 33 were uniquely different mutations), comprising 19 missense mutations, eight nonsense mutations, four small deletions and two splice site mutations, were identified by this study. Twenty-three of these were novel sequence variations not previously described in the literature. Interestingly, all changes found in exon 13 resulted in null alleles as either nonsense mutations or small deletions. The overall profile of these new mutations corresponds well with the data published in the F5 database. In those cases, where data were available, information on FV activity levels and/or bleeding history is given. Interestingly, some patients with mild FV deficiency (FV:C about 50% of normal) also exhibited bleeding episodes. Our data substantially contribute to the broadening and better understanding of the FV deficiency mutational spectrum. Identifying the molecular basis of mutations underlying this rare coagulation disorder will allow more insight into the mechanisms involved in the variable clinical phenotypes of patients with FV deficiency.

Molecular genetic background of haemophilia A patients with discrepancy between one-stage and two-stage factor VIII assays

INTRODUCTION: Androgen deficiency anemia occurs most frequently in pharmacogenic suppression of androgen synthesis or with advancing age in men. Bilateral orchiectomy is a surgical modality used in the treatment of metastatic prostate carcinoma. It is accompanied by marked decrease in circulating serum levels of androgens. AIM: The aim of the experimental study was to determine the effect of substitution therapy with testosterone propionate (TP) on some haematological parameters of erythropoiesis in male rats after orchiectomy. MATERIAL AND METHODS: Eighty Wistar male rats with mean weight of 252.3 g were used in the study. The animals were allocated into 2 control orchidectomized groups, 2 shamoperated groups and 4 experimental orchidectomized groups. Testosterone propionate was administered intramuscularly, once a week at a dose of 4 mg and 8 mg per kilogram of body weight for 15 days and for 15 weeks. Erythrocyte count was performed and hemoglobin and hematocrit levels were measured. RESULTS: In the chronic experiment there was a significant decrease in red blood cells and hemoglobin, and a tendency of decrease in hematocrit after orchiectomy. The effect of TP on erythropoiesis in orchiectomised rats is dose-dependent. CONCLUSION: TP replacement therapy in doses of 4 mg/kg and 8 mg/kg has a stimulating effect on erythropoiesis only in chronic administration.

SummaryAntithrombin (AT) is the most important physiological inhibitor of coagulation proteases. It is activated by glycosaminoglycans such as heparin. Hereditary antithrombin deficiency is a rare disease that is mainly associated with venous thromboembolism. So far, more than 200 different mutations in the antithrombin gene (SERPINC1) have been described. The aim of our study was to characterise the molecular background in a large cohort of patients with AT deficiency. Mutation analysis was performed by direct sequencing of SERPINC1 in 272 AT-deficient patients. Large deletions were identified by multiplex PCR coupled with liquid chromatography or multiplex ligation-dependent probe amplification (MLPA) analysis. To predict the effect of SERPINC1 sequence variations on the pathogenesis of AT deficiency, in silico assessments, multiple sequence alignment, and molecular graphic imaging were performed. The mutation profile consisted of 59% missense, 10% non-sense, 8% splice site mutations, 15% small deletions/insertions/duplications, and 8% large deletions. Altogether 87 different mutations, including 42 novel mutations (22 missense and 20 null mutations), were identified. Of the novel missense mutations, nine are suspected to impair the conformational changes that are needed for AT activation, two to affect the central reactive loop or the heparin binding site, and six to impair the structural integrity of the molecule. Despite the heterogeneous background of AT deficiency, 10 AT variants occurred in multiple index patients. Characterisation of the SERPINC1 mutation profile in large cohorts of patients may help to further elucidate the pathogenesis of AT deficiency and to establish genotype-phenotype associations.

Background: Approximately 25% of severe hemophilia A (HA) patients develop antibodies to factor VIII protein. Patients: In the present case-controlled cohort study, 260 severely affected, mutation-type-matched HA patients were studied for association of human leukocyte antigen (HLA) class II molecules and polymorphisms in the genes encoding interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) and development of inhibitors. Results: Our results demonstrate a higher frequency of DRB1*15 and DQB1*0602 alleles as well as of the haplotype DRB1*15/DQB1*0602 in inhibitor patients [odds ratio (OR) 1.9; P A polymorphism was found with higher frequency in the inhibitor cohort (0.22 vs. 0.13, OR 1.80). This finding was more pronounced for the homozygous A/A genotype (OR 4.7). For IL-10, the -1082G allele was observed more frequently in patients with inhibitors (0.55 vs. 0.43; P = 0.008). The functional cytokine phenotype was determined for the first time, on the basis of the genetic background, and this showed that 12% of patients with inhibitors were high-TNF-alpha/high-IL-10 producers, as compared with 3% of non-inhibitor patients (OR 4.4). A trend for a lower frequency of the A allele of the CT60 polymorphism in CTLA-4 was found in inhibitor patients (0.42 vs. 0.50). Conclusions: In conclusion, the reported data clearly highlighted the participation of HLA molecules in inhibitor formation in a large cohort of patients. The higher frequencies of the -308G > A polymorphism in TNF-alpha and -1082A > G in IL-10 in inhibitor patients confirmed the earlier published data. The CT60 single-nucleotide polymorphism in CTLA-4 is of apparently less importance.

SummaryHuman coagulation factor V (FV), a non-enzymatic cofactor of the prothrombinase complex, is required for the rapid generation of thrombin. FV deficiency is a rare autosomal recessive bleeding disorder. We describe two novel mutations,Tyr91Asn and Asp2098Tyr,found in two probands with a residual FV activity of 51% and 4%, respectively. Modelling and structural analysis of these mutations were performed following short-duration molecular dynamics (MD) simulation.Asp2098Tyr lead to abolishment of the highly conserved salt bridge Asp2098-Arg2171 presumably required for structural integrity of the C2 domain. MD studies suggest that additional conformational changes resulting from this mutation involve local rearrangements at Tyr2063 and Tyr2064 and so affect the phospholipid-membrane binding. MD modelling of the Try91Asn mutant revealed a conformational change nearby the Cu2+ binding site that could affect overall stabilization of the heavy and light chains. These findings suggest that both mutations influence the structural integrity of FV protein. Transient expression data of wild-type and mutant FV variants in 293T human embryonic kidney cells showed FV-specific activity reduced to 26% for Asp2098Tyr and 56% for Tyr91Asn compared to that of wild-type. Thus, both the data from the short duration molecular dynamic simulation and from expression analysis indicate alterations of the FV protein variants that explain the clinical phenotype.

Summary. Haemophilia A is the most common X-linked recessive bleeding disorder. In 5% of severely affected patients the mutations responsible for the disease are large deletions encompassing from one exon to the complete Factor 8 (F8) gene. Large deletions in a male haemophilic patient are easily detected by the absence of the corresponding PCR product. However, in female carriers, identification of the various heterozygous large deletions is difficult representing a major limitation to accurate carrier diagnosis. The deletion is masked by the presence of the second allele that serves as template for the PCR reaction. Quantitative PCR can differentiate between the presence of one or two alleles. Here we report an assay based on multiplex amplification of several exons of the F8 gene of various length and subsequent quantitative evaluation of the amplicons by liquid chromatogphy (LC). Using this approach we achieved an accurate classification of 16 female carriers and eight non-carriers for deletions in the F8 gene in 19 investigated families. One mother and one grandmother were classified as non-carriers, underlining the high de novo mutation rate of large deletions in female germ cells. The large deletions in three families were confirmed by fluorescent in situ hybridization. In conclusion, the multiplex PCR-LC technique represents a rapid, simple and reliable method for detection of heterozygous large deletions in female carriers.

Resistance to activated protein C (APC resistance) is the most frequent inherited hypercoagulable state that represents a common risk factor for venous thrombosis [1]. This resistance is due to a G > A substitution in position 1691 of factor V (FV) gene which leads to the synthesis of an abnormal FV molecule (FV Leiden) where Arg 506, the first cleavage site of APC is replaced by Gln [2]. As a consequence FV Leiden heterozygotes experience a seven-fold increased risk of venous thromboembolism and homozygotes an 80-fold increased risk when compared with non-carrier [3]. Although there is a good correlation between the APC-ratio laboratory test and genotype for the Leiden mutation, a number of genotype/phenotype discrepancies have been observed. Among these is the so called pseudo-homozygous APC resistance. This condition is characterized by co-inheritance of the FV Leiden and FV null mutation on different alleles [4, 5]. Although partial FV deficiency could compensate for the thombotic defect, all of the very few cases reported so far are thrombophilic patients. Here we report a case of pseudo-homozygous FV Leiden associated with FV deficiency due to a null mutation in the FV gene.

Human coagulation factor V (FV) provides an essential function in the coagulation pathway. As a non-enzymatic cofactor of the prothrombinase complex, it is required for the rapid generation of thrombin [1, 2]. The gene for coagulation FV is located on chromosome 1q24.2 and spans more then 80 kilobases (kb) [3]. It consists of 25 exons encoding a 25 amino acids leader peptide and 2196 amino acids mature protein organized in A1-A2-B-A3-C1-C2 domain structure [4]. The activated factor Va is composed of a heavy chain (domains A1 and A2, Ala1-Arg709) and a light chain (domains A3, C1, and C2, Ser1546-Tyr2196), noncovalently associated in the presence of divalent metal ions.

Identifying the molecular basis of mutations underlying this rare coagulation disorder will aid to obtain more insight into the mechanisms involved in the variable clinical phenotype of patients with FV deficiency. The computer mutation modeling could help to predict the possible alterations of FV protein structure and causality of mutations.

In conclusion MP/DHPLC technique can reliable identify hemophilia A female carriers, harboring FVIII gene copy number aberrations and it appears to be a powerful tool in genetic concealing of hemophilia families.

The detection of large gene deletions and rearrangements in AT gene is essential in order to provide an appropriate genetic counseling and treatment in antithrombin deficient patients. We have developed a DHPLC method combined with a quantitative multiplex PCR as an approach to screen for large heterozygous gene rearrangements in AT gene. This technique turned out to be reliable, easy to perform, cost effective and can be well applied to other genetic disorders with heterozygous large defects.

We describe a simple, new technique for producing of nanosecond pulses with rectangular (or triangular) temporal shape and with tunable wavelength. The technique is based on a pumping of a tunable dye laser by pulses with formed rectangular (or triangular) shape, emitted from a standard Nd:YAG (2 -nd harmonic) or Copper-vapor lasers. The pump pulses are formed in a simple electro-optical system. We have shown theoretically that the main problem to reproduce the pump pulse shape in a nanosecond dye laser -- a 'spiking' phenomenon, can be avoided using an appropriate pre-pulse pumping of the dye before the main, formed pump pulse. The experimental check confirms the theoretical predictions.

We report an original approach for tunable subnanosecond pulse (200 - 500 ps) generation in a ring dye laser. It is based on limitation of generation to a single `spike' from the starting transient process using for excitation a quasi- rectangular pulse with duration less or comparable with the time interval between the spikes. The pumping pulse (3 - 7 ns) is formed both by electrooptical division of a standard nanosecond (15 - 50 ns) pulse emitted by a Q-modulated laser in an original scheme and by overlapping of the obtained partial pulses which are fed bi-directionally into the active medium of the ring laser.

Lonetil is a new tranquilizer with great therapeutic range. With its influence on the pentylenetetrazol seizures, hypothermal effect and depression of provoked fight it is similar to the classic tranquilizers. The drug possesses also marked neuroleptic action: depression of orientation reflex and arousal reaction in case of RF stimulation, great antimorphine effect.