Reactive oxygen species and sperm cells

There is a dynamic interplay between pro- and anti-oxidant substances in human ejaculate. Excessive reactive oxygen species (ROS) generation can overwhelm protective mechanism and initiate changes in lipid and/or protein layers of sperm plasma membranes. Additionally, changes in DNA can be induced. The essential steps of lipid peroxidation have been listed as well as antioxidant substances of semen. A variety of detection techniques of lipid peroxidation have been summarized together with the lipid components of sperm membranes that can be subjected to stress. It is unsolved, a threshold for ROS levels that may induce functional sperm ability or may lead to male infertility.     

Immunolocalization of PTHrP in the sublingual glands of three mammals

The present study deals with immunohistochemical localization of PTHrP in sublingual glands of white mouse, bank vole, and common vole. PTHrP immunoreactivity was observed in epithelial cells of striated, interlobular and main excretory ducts of the salivary glands in all the three animal species tested. However, we found no positive reaction for PTHrP in epithelial cells of the intercalated ducts. In striated duct cells, the reaction intensity was species-dependent. In bank vole and common vole, the reaction was very strong, while in white mouse very weak. In the remaining segments of excretory ducts (interlobular and main excretory duct) we found no species-related differences in the reaction intensity or character. Myoepithelial cells surrounding ducts and mucous tubules with serous demilunes in sublingual glands were also PTHrP-negative in all the three animal species tested.     

Comparison of PTHrP expression in the epididymis of juvenile and adult European bisons

The aim of the present study was to compare the expression of PTHrP in the epididymes of adult European bisons, and 12- and 5-month-old calves. The highest PTHrP expression was observed in adult animals in muscle cells and endothelium of large vessels, and in muscle cells of the epididymal duct. In one-year-old calves, the reaction was weaker than in adult bulls, being the weakest in 5-month-old calves. However, in small vessels of adult animals, in vascular cells and smooth muscle cells the reaction for PTHrP was considerably weak, being weaker in one-year-old calves, and negative in 5-month-old calves. A similar trace reaction was observed in muscle cells of the epididymal duct in 5- and 12-month-old calves. The present study has revealed that PTHrP expression in vascular and extravascular smooth muscle cells and endothelial cells in European bison is correlated with the animal age and size of the organ.     

Crystal structure of a covalent intermediate of endogenous human arylsulfatase A

The structures of human arylsulfatase A crystals soaked in solutions containing 4-methylumbelliferyl phosphate and O-phospho-DL-tyrosine have been determined at 2.7- and 3.2-A resolution, respectively. The formylglycine in position 69, a residue crucial for catalytic activity, was unambiguously identified in both structures as forming a covalent bond to the phosphate moiety. A hydroxyl group is present at the Cbeta of residue 69 and the formation of one out of two possible stereomeric forms is strongly favoured. The structures confirm the importance of the gem-diol intermediate in the arylsulfatase's catalytic mechanism. The presence of an apparently stable covalent bond is consistent with the weak phosphatase activity observed for human arylsulfatase A. The structures of the complexes suggest that phosphate ions and phosphate esters inhibit arylsulfatase in non-covalent and covalent modes, respectively. The metal ion present in the active site of arylsulfatase A isolated from human placenta is Ca(2+) and not Mg(2+) as was found in the structure of the recombinant enzyme.     

Expression of high molecular weight cysteine proteinase inhibitor in ovarian cancer tissues: regulation of cathepsin B expression by placental CPI

Mouse polyclonal antibodies against placental cysteine proteinase inhibitor (CPI) react with the placental 67 kDa CPI on Western blots, and CPI present in ovarian cancer homogenate and serum was shown by double immunodiffusion to react with the same antiserum. By immunohistochemical staining, positive expression of high molecular weight CPI was observed on the tumour cell surface in serous and endometrioid ovarian carcinomas with metastasis. Normal endometrioid tissue was not stained with anti-placental CPI antibodies. Cathepsin B and pro-cathepsin B median levels in ovarian cancer tissue homogenates increased progressively with FIGO stage of the disease. The enzyme level decreased 22-fold after treatment of tissue homogenates with 5 nM purified CPI. These results provide evidence that addition of CPI reduces the levels of cysteine-type cathepsins to those of normal non-cancerous values.     

Antimicrobial activity of chicken egg white cystatin

The cysteine protease inhibitor cystatin was purified from chicken egg white and its antimicrobial activity determined for a series of pathogenic bacteria. The results indicate that Acinetobacter lwoffii, Escherichia coli, Oligella sp. and Pseudomonas aeruginosa are highly sensitive to low concentrations of cystatin, which possesses bactericidal activity. No inhibition was observed with a Citrobacter freundii strain. Fifty percent growth inhibition (IC₅₀) was observed at cystatin concentrations in the range of 80 and 100g/ml, and the growth was completely inhibited at concentrations in the range of 100 and 200g/ml. Fifty percent growth inhibition (IC₅₀) for Staphylococcus aureus, Staphylococcus gallinarum, and Staphylococcus xylosus strains was observed at 150 and 200 g of cystatin/ml respectively, and growth was completely inhibited at cystatin concentrations in the range of 300 and 1000 g/ml. The activity of cysteine proteases was significantly decreased in the culture supernatant of microorganisms when chicken cystatin was added. In this study, we observed that chicken cystatin may be a candidate for antibacterial drug development aiming at controlling bacterial pathogens including Escherichia coli, Pseudomonas aeruginosa, and another possible application might be as a therapeutic agent for health improvement.     

Determination of transmission patterns in multichannel data

The power of today's laboratory equipment allows more and more data channels to be easily recorded. However, some misunderstandings about processing such multivariate data may still be found in the literature. The typical mistake is to treat a multichannel system as comprising pairs of channels; this approach does not use all the collected information about the investigated system, and may lead to erroneous conclusions. In this paper, the differences between single- and multichannel approaches will be briefly summarized and some examples of problems will be described.     

3D domain-swapped human cystatin C with amyloidlike intermolecular beta-sheets

Oligomerization of human cystatin C (HCC) leads to amyloid deposits in brain arteries, and this process is greatly accelerated with a naturally occurring L68Q variant. The crystal structures of N-truncated and full-length HCC (cubic form) showed dimer formation via three-dimensional (3D) domain swapping, and this observation has led to the suggestion that an analogous domain-swapping mechanism, but propagated in an open-ended fashion, could be the basis of HCC fibril formation. Here we report that full-length HCC, when crystallized in a new, tetragonal form, dimerizes by swapping the same secondary structure elements but with a very different overall structure generated by the flexibility of the hinge linking the moveable elements. The beta-strands of the beta-cores of the two folding units of the present dimer are roughly parallel, while they formed an angle of about 100 degrees in the previous two structures. The dimers pack around a crystallographic dyad by extending their molecular beta-sheets in an intermolecular context. At the other edge of the molecular beta-sheet, side-chain-side-chain hydrogen bonds propagate the beta-structure in the same direction. In consequence, a supramolecular crystal structure is generated, with all the beta-strands of the domain-swapped dimers being perpendicular to one crystallographic direction. This observation is relevant to amyloid aggregation of HCC, as X-ray diffraction studies of amyloid fibrils show them to have ordered, repeating structure, consistent with the so-called cross-beta structure, in which extended polypeptide chains are perpendicular to the fiber axis and form infinite beta-sheets that are parallel to this axis.     

Biodegradation of monochloroacetic acid used as a sole carbon and energy source by <Emphasis Type="Italic">Xanthobacter autotrophicus </Emphasis>GJ10 strain in batch and continuous culture

Batch and continuous mode degradation of monochloroacetic acid used as a sole carbon and energy source in the concentration range of 0.9–48.4 mM by pure culture of Xanthobacter autotrophicusGJ10 was investigated. The substrate was completely degraded in each flask in batch system. Partial substrate inhibition occurred at the concentrations exceeding 25.4mM. Temporary accumulation of glycolic acid in the medium indicated that dehalogenation was undergoing faster than further utilization of glycolate. Three different carbon substrates were used for inoculum preparation – 1,2-dichloroethane, tri-sodium citrate and a nutrient broth. The fastest growth on monochloroacetate occurred for 1,2- dichloroethane-grown inoculum. The assays of haloacid dehalogenase in crude extract indicated that the bacteria grown on 1,2-dichloroethane possessed higher level of the enzyme. The response of the GJ10 culture towards spikes of 20mM monochloroacetate was tested in 2.5-l continuously stirred tank fermentor. The substrate was readily utilized within 7–8h. Continuous degradation of monochloroacetate in the fermentor was demonstrated for monochloroacetate concentration of 20mM and dilution rate 0.016 h^–1. Quantitative agreement between the amount of monochloroacetate introduced and chloride released was found. The results demonstrated that the strain X. autotrophicus GJ10 might be suitable for biodegradation of monochloroacetate contaminated media.     

Direct comparison of the crystal and solution structure of glucose/xylose isomerase from Streptomyces rubiginosus

Glucose isomerase (D-xylose ketol-isomerase, EC 5.3.1.5.) catalyses the isomerization reaction of glucose and xylose. The small angle X-ray scattering (SAXS) data of glucose/xylose isomerase from Streptomyces rubiginosus were recorded for protein solution using synchrotron radiation. The experimental data were compared with theoretical scattering calculated on the basis of the known crystal structure (PDB code: 1OAD). The radius of gyration measured by SAXS (R(G)=3.30 nm) was almost identical and the maximum dimension in the distance distribution function was by about 2.5 % lower than the corresponding values calculated on the basis of the crystal structure.