Immunohistochemical analysis of secretoglobin SCGB 2A1 expression in human ocular glands and tissues

Human secretoglobin (SCGB) 2A1 (or lipophilin C, lacryglobin, mammaglobin B) is a small protein of unknown function that forms heterodimers with secretoglobin 1D1 (lipophilin A) in tears. SCGB 2A1 is homologous to mammaglobin (mammaglobin A) and the C3 component of prostatein, the major secretory protein of the rat ventral prostate. Androgen-dependent expression of SCGB 2A1 has been observed in the prostate. Besides identification of SCGB 2A1 in the tear proteome only its mRNA had been detected in the lacrimal gland. Here, we report expression of SCGB 2A1 in all ocular glands and in the keratinized stratified squamous epithelium of the eyelid as well as in the stratified epithelium of the conjunctiva and in the orbicularis oculi muscle. Almost all of these tissues are also known to express the androgen receptor. Therefore, we conclude that presence of the androgen signalling machinery could be the main general determinant of SCGB 2A1 expression. Implications of the presence in tear fluid of an androgen-regulated secretoglobin, which most likely binds hydrophobic ligands, for tear film lipid layer formation and function is discussed.     

High affinity copper binding by stefin B (cystatin B) and its role in the inhibition of amyloid fibrillation

We show that human stefin B, a protease inhibitor from the family of cystatins, is a copper binding protein, unlike stefin A. We have used isothermal titration calorimetry to directly monitor the binding event at pH 7 and pH 5. At pH 7 stefin B shows a picomolar affinity for copper but at pH 5 the affinity is in the nanomolar range. There is no difference in the affinity of copper between the wildtype stefin B (E31 isoform) and a variant (Y31 isoform), whereas the mutant (P79S), which is tetrameric, does not bind copper. The conformation of stefin B remains unaltered by copper binding. It is known that below pH 5 stefin B undergoes a conformational change and amyloid fibril formation. We show that copper binding inhibits the amyloid fibril formation and, to a lesser degree, the initial aggregation. Similarities to and differences from other copper binding amyloidogenic proteins are discussed.     

Antiproliferative effect and genotoxicity of novel synthesized palladium complexes with organoarsenic ligands

Three new palladium complexes with general formula [PdCl₂L₂], where L = heterofunctional organoarsenic ligand: (2-isopropoxyphenyl)diphenylarsine (1), (2-methoxyphenyl)-diphenylarsine (2) and (2-hydroxyphenyl)diphenylarsine (3) have been synthesized and fully characterized, including X-ray crystallographic data. Their potential antitumor effect and genotoxicity have been studied as well. The viability test performed on human tumor (MLS) and normal (Hfl-1) cell lines indicates significant cytotoxicity of complexes, which is higher in tumor cells than in normal cells. The lethal doses are comparable with those of standard metal-based chemotherapeutical drugs (carboplatin and oxaliplatin). These palladium complexes exhibit a higher cytotoxicity against tumor cells as against normal cells in vitro. A new static cytometric method was developed and simultaneously the classic AnnexinV test was performed. Complex 2 has an important capacity to induce apoptosis in tumor cells. The apoptotic process is triggered due to the interaction of these complexes with secondary structure of DNA in treated cells. The alkaline single-cell gel assay shows that the level of DNA damages induced by compounds 2 and 3 are significantly higher in tumor cells as in normal cells. These studies shown that complexes 1, 2 and 3 have biologic activity, the effect of complex 2 being superior to its platinum analogues, attributable to its structure.     

Probing the membrane potential of living cells by dielectric spectroscopy

In this paper we demonstrate a quantitative way to measure the membrane potential of live cells by dielectric spectroscopy. We also show that the values of the membrane potential obtained using our technique are in good agreement with those obtained using traditional methods—voltage-sensitive dyes. The membrane potential is determined by fitting the experimental dielectric dispersion curves with the dispersion curves obtained from a theoretical model. Variations in the membrane potential were induced by modifying the concentration of potassium chloride in the solution of the cell suspension in the presence of valinomycin. For exemplification of the method, E. coli was chosen for our experiments.     

Preparation and Ultraviolet Light-Induced Transformation of an Antifungal Mixture of Heptaene Antibiotics of Streptomyces surinam

Conditions for the production and isolation of an antifungal antibiotic mixture (DJ400) were investigated. Different preparations of DJ400 may contain at least 12 different heptaenes, which were characterized by partition chromatography (peak number) and ultraviolet (UV) spectra (types A, B, C). Irradiation with UV light transformed the predominant UV spectrum of type B into A. Comparison of untreated and UV-irradiated products indicated that the main components may exist in two forms with identical structures except for an all trans-heptaene system in A compounds and one internal cis double bond in B compounds. The ratio of the major components 6B and 8B depended on the media composition. Component 4B is probably a precursor of 6B; 10B and 12 may be precursors of 8B.     

Staphylococci Bind Heparin-Binding Host Growth Factors

Staphylococcus aureus, which mediated binding to heparan sulfate, and also strains of coagulase-negative staphylococci (CNS) adhered in high numbers to polymers with end-point attached heparin. A characteristic feature of several cell growth factors is strong affinity for heparin. In the present study, binding of the ¹²⁵I-labeled heparin-binding growth factors (HBGF), acidic and basic fibroblast growth factor (aFGF, bFGF), and platelet-derived growth factor (PDGF) by S. aureus and CNS strains was examined. Staphylococcal strains used in this study bind bFGF and PDGF, but not aFGF. The binding of bFGF and PDGF was time dependent, influenced by pH and ionic strength for S. aureus Cowan 1. Preincubation of staphylococcal cells with unlabeled bFGF enhanced bFGF binding, but heparin, protamine sulfate, poly-L-lysine, and suramin were potent inhibitors of ¹²⁵I-bFGF binding to cells of S. aureus Cowan 1. Glycosaminoglycans of comparable size (chondroitin sulfate), other polysulfated polymers (λ-carrageenan, fucoidan), and some polysulfated polysaccharides (dextran sulfate, pentosan polysulfate) inhibited binding of both GFs to various extents. The partial inhibition of binding of both GFs after protease and periodate treatments indicates that both proteinaceous and other carbohydrate moieties participate in the binding. A lysozyme cell surface extract and bacterial lysates of S. aureus Cowan 1 competitively inhibited binding of ¹²⁵I-bFGF and ¹²⁵I-PDGF. These results suggest that staphylococci have the ability to bind two of the HBGFs, bFGF and PDGF, but not aFGF, via more than one cell structure. These binding structures seem to be exposed on the cell surface and deeply anchored in the cytoplasmic membrane as well.     

The mechanism of the C-13(3) esterification step in the biosynthesis of bacteriochlorophyll a

5-Aminolaevulinate labelled with 18O at its C-1 carboxy oxygen atoms was prepared and incorporated into bacteriochlorophyll aphytyl of Rhodopseudomonas sphaeroides and bacteriochlorophyll ageranylgeranyl of Rhodospirillum rubrum. The biosynthetic samples of the bacteriochlorophylls were separately processed to obtain their isoprenyl alcohol components from the C-17(3) ester linkages and methanol from the C-13(3) methoxycarbonyl group. Methods were developed for the quantification of the isotopic composition of the various alcohols (methanol, phytol, geranylgeraniol). It was shown that the hydroxyl oxygen atoms of all the three alcohols originated from one of the C-1 oxygen atoms of the precursor 5-aminolaevulinate. In the light of these results the in vivo mechanism for the O-methylation reaction at C-13(3) during the biosynthesis of the two species of bacteriochlorophylls is discussed.