Modification of immunocytochemical ZAP-70 assay for potential clinical application in B-cell chronic lymphocytic leukemia

The ZAP-70 protein is a member of the Syk/ZAP protein tyrosine kinase family, normally expressed in T cells and NK cells but not found in normal, mature B cells. The protein plays a critical role in the initiation of T-cell signaling. Leukemic cells from patients with B-cell chronic lymphocytic leukemia (B-CLL) that expressed nonmutated immunoglobulin V genes were found to express levels of ZAP-70 protein that were comparable to those detected in T cells of healthy adults. The ZAP-70 protein expression can be evaluated by flow cytometry and may be used as a prognostic marker in B-CLL patients. We modified the method of immunocytochemical assessment of ZAP-70 expression. The traditional two-step method with monoclonal anti-ZAP-70 antibody in the first step followed by FITC-conjugated goat anti-mouse IgG was changed for one-step method with monoclonal anti-ZAP-70 antibody labeled by Zenon Alexa Fluor 488. The method is simple and fast. The major advantage of Zenon labeling technique is its compatibility with simultaneous staining of surface antigens. The cells may be earlier immunostained for CD3, CD19 and/or CD5 to compare of the ZAP-70 kinase expression in B and T cells.     

Epidermal growth factor in Dupuytren's disease

The aim of this article was to show the participation of epidermal growth factor (EGF) in the pathogenesis of Dupuytren's disease (palmar contracture). The concentration of EGF in specimens obtained from 68 patients with Dupuytren's contracture and 14 controls was examined immunochemically with the use of enzyme-linked immunosorbent assay. The determined EGF concentration in pathologic aponeurosis with symptoms of Dupuytren's disease (median, 6.29 ng/g; range, 1.67 to 63.09 ng/g) showed significantly different values (p = 0.036) in comparison with the control group (median, 10.1 ng/g; range, 5.13 to 39.81 ng/g). The changes in EGF concentration were shown in tested groups of pathologic tissues that were formed according to the clinical stage of disease progression. The significantly lower concentration than that seen in the control group characterizes tissues with first and third degrees of palmar contracture progression (p = 0.025 and p = 0.018, respectively). In the group of patients with second-degree disease progression, the EGF level increased transiently. Nevertheless, in comparison with the other groups, the difference was not significant. The group with the fourth degree of the disease showed EGF concentrations that resembled the control values. The authors conclude that significant differences in levels of EGF concentration between contractured and normal fasciae may suggest the participation of this cytokine in the pathogenesis of Dupuytren's disease.     

Inhibition of zeaxanthin epoxidase activity by cadmium ions in higher plants

The effect of cadmium and zinc ions on violaxanthin cycle enzymes, violaxanthin de-epoxidase and zeaxanthin epoxidase, has been investigated on selected plant species, as well as in vitro. About 50% inhibition of zeaxanthin epoxidase by cadmium ions was found for duckweed (Lemna trisulca) and tomato (Lycopersicon esculentum) leaves but for apricot (Prunus armeniaca) leaves no cadmium inhibition of the epoxidation reaction was observed. The cadmium inhibition of zeaxanthin epoxidase in tomato was abolished by zinc ions. Zinc ions alone did not affect the activity of neither of the enzymes of the violaxanthin cycle. This suggests that mechanism of cadmium inactivation of the enzyme relies on cadmium interaction with a cysteine residue of the protein, important for the enzyme activity. The target cysteine in tomato epoxidase could be the cysteine residue present in the most conservative part of the molecule which is not present in the apricot enzyme sequence. Neither stimulation nor inhibition of violaxanthin de-epoxidase by cadmium ions both in vivo and in vitro studies was detected. It confirms the proposed mechanism of zeaxanthin epoxidation inhibition by cadmium ions because the cysteine residue in the conservative motif of violaxathin de-epoxidase is not present.     

Assimilatory ferric reductases in enterococci

Enterococci produced assimilatory ferric reductases which are surface-associated enzymes. This is the first report of the intracellular enzymic reduction of iron by enterococci. A correlation between ferric reductases activity and species affiliation and origin of strains was found. The expression of ferric reductases has not affected by the presence or absence of iron, hemin and hemoglobin in the growth medium. Enterococcal ferric reductases exhibit a very broad specificity. A number of different ferric organic and inorganic compounds, natural and synthetic iron chelators and iron body sources including lactoferrin, transferin, ferritin, haemoglobin, could be reduced. A surface-associated ferric reductases may be one component of a general iron scavenging mechanism which can be used by enterococci growing in a variety of environments.     

Helicobacter pylori VacA cytotoxin interacts with fibronectin and alters HeLa cell adhesion and cytoskeletal organization in vitro

Helicobacter pylori vacuolating cytotoxin VacA causes multiple effects on epithelial cell function and morphology, but the effects of VacA on signal transduction pathways and the cytoskeleton have not been investigated in detail. In this study, we analyzed the effects of native VacA on HeLa and AGS cell adhesion to fibronectin and laminin under serum-free conditions. Confocal microscopic examination revealed increased number of cells with rounded morphology and inhibition of actin fiber formation, in the presence of VacA. VacA binds to fibronectin in vitro in a dose-dependent manner. This interaction was partly inhibited by a peptide containing an arginine-glycine-aspartic acid motif. The adhesion of HeLa cells to fibronectin, but not to laminin, was decreased in the presence of VacA. Thus, VacA may interact with fibronectin and influence integrin receptor-induced cell signaling and cytoskeleton-dependent cell functions.     

Analysis of the human <Emphasis Type="Italic">Alu</Emphasis> Ye lineage

Background  

Alu elements are short (~300 bp) interspersed elements that amplify in primate genomes through a process termed retroposition. The expansion of these elements has had a significant impact on the structure and function of primate genomes. Approximately 10 % of the mass of the human genome is comprised of Alu elements, making them the most abundant short interspersed element (SINE) in our genome. The majority of Alu amplification occurred early in primate evolution, and the current rate of Alu retroposition is at least 100 fold slower than the peak of amplification that occurred 30–50 million years ago. Alu elements are therefore a rich source of inter- and intra-species primate genomic variation.     

Theoretical model of the aqua-copper [Cu(H2O)5]+cation interactions with guanine

Pentaaqua complexes of Cu(I) with guanine were optimized at the DFT B3PW91/6-31G(d) level. For the most stable structures, vibration frequencies and NBO charges were computed followed by energy analyses. The order of individual conformers was very sensitive to the method and basis sets used for the calculation. Several conformers are practically degenerated in energy. The inclusion of an entropy term changes the order of the conformers stability. Water molecules associated at the N9 position of guanine are favored by the inclusion of the entropy correction. Bonding energies of Cu–O(aqua) interactions were estimated to be about 60 kcal mol^–1 and for Cu–N7 bonding in the range of 75–83 kcal mol^–1. The broad range in Cu–N interaction energies demonstrates the role of induction effects caused by water molecules associated at the various sites of guanine. The charge distribution of the guanine molecule is changed remarkably by the coordination of a Cu(I) cation, which can also change the base-pairing pattern of the guanine.     

Inhibitors of phenylalanine ammonia-lyase (PAL): synthesis and biological evaluation of 5-substituted 2-aminoindane-2-phosphonic acids

A series of 5-substituted derivatives of the potent phenylalanine ammonia-lyase (PAL) inhibitor 2-aminoindane-2-phosphonic acid (AIP; 2) were synthesized. The AIP analogues 3-7, with additional NO2, NH2, Me, Br, and OH groups, respectively, were tested as in vitro inhibitors of buckwheat PAL, and as in vivo inhibitors of anthocyanin biosynthesis. Within this series, the racemic 5-bromo (6) and 5-methyl (7) congeners were biologically most active (Table), although being ca. one order of magnitude less potent than AIP proper.     

Spectroscopic characteristics of the micro-environmentally induced H-bond transformation in anil-type species: experimental and theoretical study

The results of combined experimental and theoretical investigations of the spectral behavior of anil-type systems are presented. Two species: N-triphenylmethylsalicylidene imine (MS1) and N-salicylidene methylamine (SmA) were studied. The electronic (absorption, emission and excitation) spectra of MS1 at room temperature were investigated in pure isooctane as well as in acetonitrile and methanol solutions by the steady-state experiments. A mechanism of molecular processes in the ground and excited states in different microenvironments is also proposed. It includes formation of intra- and intermolecular hydrogen bonds, their role in stabilization of molecular conformations and conformation equilibria. The "solvent assisted" proton transfer reaction and rearrangement were modeled using complexes obtained by attaching methanol molecules to the species studied. The OH-rotamer of SmA was also considered. Infrared and Raman spectra were predicted for MS1 and SmA and compared with the experimental data. An analysis of fundamental vibration frequencies was carried out. Quantum chemical ab initio calculations at the HF/6-31G** level were performed for the species studied and their complexes. Chemical formula of anil-type compound: N-salicylidene methylamine (SmA), N-salicylideneaniline (SA) and N-triphenylmethylsalicylidene imine (MS1). [Structure: see text].