Structural damages in adsorbed vaccines affected by freezing

This study was planned to evaluate structural damages in adsorbed vaccines affected by freezing using scanning electron microscopy and X-ray analysis of the elements. Randomly selected 42 vials of eight different types of WHO pre-qualified adsorbed freeze-sensitive vaccines from 10 manufacturers were included in the study. Vaccines were kept at 5 °C. Selected numbers of vials from each type were then exposed to ?25 °C for 24 h periods. All samples were evaluated for their structure using scanning electron microscopy, X-ray analysis of the elements and precipitation time. Scanning electron microscopy of vaccines affected by freezing showed either smooth or rough surfaced conglomerates associated with phosphate content of the precipitate. These vaccines precipitated 2–15 times faster compared to non-frozen samples. Non-frozen samples showed uniform flocculent structure either dense or dispersed. X-ray analysis of precipitates in frozen samples confirmed that the precipitate is mainly aluminium clutters. Scanning electron microscopy confirmed that the lattice structure of bonds between adsorbent and the antigen is broken and aluminium forms conglomerates that grow in size and weight. The precipitation time of vaccines affected by freezing is 4.5 times faster on average compared to non-frozen samples. These facts form the basis of the "shake test".     

Oxidative DNA Base Modifications and Polycyclic Aromatic Hydrocarbon DNA Adducts in Squamous Cell Carcinoma of Larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.     

Inhibition of jack bean urease by tetrachloro-o-benzoquinone and tetrachloro-p-benzoquinone

Tetrachloro-o-benzoquinone (TCoBQ) and tetrachloro-p-benzoquinone (TCpBQ) were studied as inhibitors of jack bean urease in 20 mM phosphate buffer, pH 7.0, 1 mM EDTA, 25°C. The mechanisms of inhibition were evaluated by analysis of the progress curves obtained with two procedures: the reaction initiated by addition of the enzyme and the reaction initiated by addition of the substrate after preincubation of the enzyme with the inhibitor. The obtained results were characteristic of slow-binding inhibition. The effects of different inhibitor concentrations on the initial and steady-state velocities obeyed the relationships of two-step enzyme-inhibitor interaction, qualified as mechanism B. It was found that TCoBQ and TCpBQ are strong urease inhibitors. TCpBQ is more effective than TCoBQ with the overall inhibition constant of K_i* = 4.5 × 10^{? 7} mM. The respective inhibition constant of TCoBQ was equal to: K_i* = 2.4 × 10^{? 6} mM. The protective experiment proved that the urease active site is involved in the tetrachlorobenzoquinone inhibition process. High effectiveness of thiol protectors against inhibition by TCoBQ and TCpBQ indicates the strategic role of the active site sulfhydryl group in the blocking process. The stability of the complexes: urease-TCoBQ and urease-TCpBQ was tested in two ways: by dilution or addition of dithiothreitol. No recovery of urease activity bound in the urease-inhibitor complexes proves that the complexes are stable and strong.     

Transgenic pigs designed to express human α-galactosidase to avoid humoral xenograft rejection

The use of animals as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human organ donors. However, the presence of xenoreactive antibodies in humans directed against swine Gal antigen present on the surface of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a consequence of hyperacute reaction. To prevent hyperacute rejection, it is possible to change the swine genome by a human gene modifying the set of donor’s cell surface proteins. The gene construct pGal-GFPBsd containing the human gene encoding α-galactosidase enzyme under the promoter of EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a male pronucleus of the fertilised porcine oocyte. As a result, the founder male pig was obtained with the transgene mapping to chromosome 11p12. The polymerase chain reaction (PCR) analysis revealed and the Southern analysis confirmed transgene integration estimating the approximate number of transgene copies as 16. Flow cytometry analysis revealed a reduction in the level of epitope Gal on the cell surface of cells isolated from F0 and F1 transgenic animals. The complement-mediated cytotoxicity assay showed increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of α-galactosidase expression.     

Never ending story: a lesson in using sampling efficiency methods with ground beetles

Biological inventory is a crucial activity in life sciences field research. However, it is sometimes time-consuming and laborious to take representative samplings of communities, especially in the case of invertebrates. In this paper, we address the issue of sampling efficiency and its influence on obtained results. As a study system, we used data on epigeic carabid beetles (Carabidae) collected in 1999–2001 in the Warta River valley of western Poland. We trapped a total of 17,722 individuals belonging to 108 species. However, due to rarefaction methods, the expected number of species was estimated at 134–140, suggesting that from 26 to 32 species are missing from the material, even expressed as a huge number of collected specimens. The estimated probability that another captured individual will represent a new species (i.e. a species that was not already recorded) is 0.0010. In order to record all the species present in the study area, another 193,338 individuals need to be sampled (abundance-based approach) or another 1,871 samples need to be collected (incidence-based approach). This means that the collected material should be 10.9 times greater (or 7.9 times greater for incidence-based data) than what was actually collected in order to record all the species present in the study area. The results show that, in practice, full inventory is simply nearly impossible to achieve, and this knowledge should be included in inventory planning. Therefore, we argue that species accumulation curves and unseen species estimators need to be carefully examined and threshold probability of detecting a new species should be built into the design of inventory science. The ratio between recorded and estimated species richness and the estimated efficiency of further sampling can be easily computed with available freeware software and should be incorporated when performing biological inventories.     

The choice of seed tracking method influenced fate of beech seeds dispersed by rodents

We tracked seeds of European beech (Fagus sylvatica) dispersed by rodents in Gorzowska Forest (western Poland). We used two seed labeling methods, marking with UV-fluorescent powder and with plastic tags, to test whether using different marking methods influences results of seed tracking. The removal rates did not differ among seeds marked with UV-powder, seeds labeled with tags, and unmanipulated seeds. We found 78 % of removed seeds marked with tags, but only 25 % of UV-marked seeds. The consumption rates of tagged and UV-marked seeds were dramatically different: rodents ate 83 % of the former and 26 % of the later. The average dispersal distance was larger for seeds marked with UV-powder than for tagged seeds. Our findings suggest that the choice of seed tracking method might influence results of seed dispersal studies.     

Quantitative trait loci for leaf chlorophyll fluorescence parameters, chlorophyll and carotenoid contents in relation to biomass and yield in bread wheat and their chromosome deletion bin assignments

Relatively little is known of the genetic control of chlorophyll fluorescence (CF) and pigment traits important in determining efficiency of photosynthesis in wheat and its association with biomass productivity. A doubled haploid population of 94 lines from the wheat cross Chinese Spring × SQ1 was trialled under optimum glasshouse conditions for 4 years to identify quantitative trait loci (QTL) for CF traits including, for the first time in wheat, JIP-test parameters per excited cross section (CS_m): ABS/CS_m, DI_o/CS_m, TR_o/CS_m, RC/CS_m and ET_o/CS_m, key parameters determining efficiency of the photosynthetic apparatus, as well as chlorophyll and carotenoid contents to establish associations with biomass and grain yield. The existing genetic map was extended to 920 loci by adding Diversity Arrays Technology markers. Markers and selected genes for photosynthetic light reactions, pigment metabolism and biomass accumulation were located to chromosome deletion bins. Across all CF traits and years, 116 QTL for CF were located on all chromosomes except 7B, and 39 QTL were identified for pigments on the majority of chromosomes, excluding 1A, 2A, 4A, 3B, 5B, 1D, 2D, 5D, 6D and 7D. Thirty QTL for plant productivity traits were mapped on chromosomes 3A, 5A, 6A, 7A, 1B, 2B, 4B, 6B, 7B, 3D and 4D. A region on chromosome 6B was identified where 14 QTL for CF parameters coincided with QTL for chlorophyll content and grain weight per ear. Thirty-five QTL regions were coincident with candidate genes. The environment was shown to dominate in determining expression of genes for those traits.     

Molecular diagnosis of cystic echinococcosis in humans from central Poland

The identity of the causative agent of cystic echinococcosis (CE) in humans from central Poland receiving treatment between 2000 and 2010 was determined. A total of 47 samples obtained after hepatectomy were examined and protoscoleces were identified in wet preparations in 27 cases. Using DNA extracted from the samples, two mitochondrial regions (nad1 and cox1 genes) were amplified and the nad1 fragment was sequenced. This PCR analysis confirmed the presence of Echinococcus species in 30 cases and nad1 sequence alignments showed identity with the G7 (pig) strain, Echinococcus canadensis. These data demonstrate that the pig strain of this parasite is the most frequent causative agent of human cystic echinococcosis in central Poland.