Fluorescence resonance energy transfer analysis of cytochromes P450 2C2 and 2E1 molecular interactions in living cells

The molecular organization of microsomal cytochromes P450 (P450s) and formation of complexes with P450 reductase have been studied previously with isolated proteins and in reconstituted systems. Although these studies demonstrated that some P450s oligomerize in vitro, neither oligomerization nor interactions of P450 with P450 reductase have been studied in living cells. Here we have used fluorescence resonance energy transfer (FRET) to study P450 oligomerization and binding to P450 reductase in live transfected cells. Cytochrome P450 2C2, but not P450 2E1, forms homo-oligomeric structures, and this self-association is mediated by the signal-anchor sequence. Because P450 2C2, in contrast to P450 2E1, is directly retained in the endoplasmic reticulum (ER), these results could suggest that oligomerization may prevent transport from the ER. However, P450 2C1 signal-anchor sequence chimera defective in ER retention also formed oligomers, and chimera containing the cytoplasmic domain of P450 2C2, which is directly retained in the ER, did not exhibit self-oligomerization, which indicates that oligomerization is not correlated with direct retention. By using FRET, we have also detected binding of P450 2C2 and P450 2E1 to P450 reductase. In contrast to self-oligomerization, the catalytic domain can mediate an interaction of P450 2C2 with P450 reductase. These results suggest that microsomal P450s may differ in their quaternary structure but that these differences do not detectably affect interaction with the reductase or transport from the ER.     

Induction of apoptosis and necrosis in lymphocytes by the cis-Pt(II) complex of 3-aminoflavone in comparison with cis-DDP

cis-Diamminedichloroplatinum(II) (cis-DDP) is one of the most widely administrated antitumor drugs. However, the use of cis-DDP is severely limited because of its toxic side effects. Therefore, efforts are concentrated on the development of improved platinum compounds with a broader activity spectrum and effectiveness in chemotherapy, but lower toxicity. Beneficial properties of flavonoids, e.g. their antitumor activity, encouraged scientists to synthesize cis-bis(3-aminoflavone)dichloroplatinum(II). Abilities of these compounds to induce apoptosis and necrosis were compared by use of trypan blue, fluorochrome staining (Hoechst 33258/propidium iodide double staining) and TUNEL assays. The cytotoxicity was evaluated by MTT. The results obtained show that the cis-Pt(II) complex of 3-aminoflavone is less toxic than cis-DDP. However, the former compound has a faster rate of apoptosis induction in lymphocytes than the latter. The cis-Pt(II) complex of 3-aminoflavone induces apoptosis in normal lymphocytes to a lesser degree and could be a potential antitumor drug.     

Novel enamine derivatives of 5,6-dihydro-2'-deoxyuridine formed in reductive amination of 5-formyl-2'-deoxyuridine

Reductive amination of 5-formyl-3',5'-di-O-acetyl-2'-deoxyuridine with primary amines and sodium triacetoxyborohydride (NaBH(OAc)(3)) afforded novel enamine derivatives of 5,6-dihydro-2'-deoxyuridine as a result of unexpected 1,4-conjugate reduction of intermediate Schiff bases in addition to the secondary amine derivatives of 2'-deoxyuridine, typical 1,2-reduction products.     

Under-age girl as a patient of pediatric and adolescent ginecology outpatient clinic

The research carried out in Poland reflected that sexual initiation before 18 years of age is a common phenomenon and refers to roughly 80% of teenagers. In Poland there is no uniform standing of medical and legal environments with regard to dealing with a juvenile patient who has become sexually active and expects the advice of a gynaecologist, gynaecologic examination and often asks for prescribing contraceptives. The procedures must take into account the fact that in Poland, until 18 years of age, a juvenile functions under the parental or tutelary authority, while a consent for medical service requires beside of the consent of legal guardian also the consent of a juvenile who is 16 years of age and becomes a full-right patient. According to the Act on Health Care Institutions, a patient has the right to self-decisions, the respect of physical and mental integrity and the respect of privacy, while the participation of a statutory representative post 16th year of age refers practically to co-deciding on a medical service provision. Therefore, the information received from such juvenile patient in subjective and objective examination does not have to be passed to the statutory representative, if the juvenile patient requires confidentiality and if this does not affect the patient's health and the planned medical procedures (e.g. the necessity of making an operation). The knowledge of conduct procedures with regard to a juvenile patient as a carrier of rights shall enable doctors to make aware choices of conduct and provide services or, in most cases, only advice, without the necessity to breach the laws of Poland.     

Ovaries and phylogeny of dermapterans once more: Ovarian characters support paraphyly of Spongiphoridae

The Dermaptera is an insect order with ca. 2200 described species classified in 11 families. Interestingly, recent morphological and molecular data suggest that at least three dermapteran families (Diplatyidae, Pygidicranidae and Spongiphoridae) are paraphyletic. Here we present results of histological analyses of ovaries and ovarioles in two representatives of Spongiphoridae: Chaetospania borneensis and Irdex chapmani. We show that both the ovaries and ovarioles of studied species are morphologically disparate. The ovaries of C. borneensis consist of shortened ovarioles attached to elongated lateral oviducts and are apparently similar to the ovaries of the Eudermaptera. In contrast, I. chapmani share all the important ovarian characters with more basal taxa, i.e. Anisolabididae and Labiduridae. These findings lend additional support to the paraphyly of Spongiphoridae.     

Uptake of cadmium ions in white-rot fungus Trametes versicolor: effect of cd (II) ions on the activity of laccase

The effects of cadmium Cd (II) ions on the physiology and biological activity of Trametes versicolor, a strain belonging to white-rotting Basidiomycetes, were examined. Cd (II) ions were added to 10-day-old cultures grown on a liquid medium, or at the time of inoculation. Our experiments showed that T. versicolor is a good cadmium biosorbent from aqueous solution, this strain removing almost all the Cd (ll) ions over the first 2h of incubation by what appears to be a rapid, energy-independent surface binding phenomenon, at the rate of approximately 2mg Cd per g mycelial dry weight. An additional slower and energy-dependent transport mechanism was also present, taking in approximately 0.3mg Cd (II) perg dry weight. It is also shown that these Cd (II) ions significantly stimulate the activity of extracellular laccase when added to 10-day-old cultures.     

Poly(L-lactide-co-glycolide) thin films can act as autologous cell carriers for skin tissue engineering

Degradable aliphatic polyesters such as polylactides, polyglycolides and their copolymers are used in several biomedical and pharmaceutical applications. We analyzed the influence of poly(L-lactide-co-glycolide) (PLGA) thin films on the adhesion, proliferation, motility and differentiation of primary human skin keratinocytes and fibroblasts in the context of their potential use as cell carriers for skin tissue engineering. We did not observe visible differences in the morphology, focal contact appearance, or actin cytoskeleton organization of skin cells cultured on PLGA films compared to those cultured under control conditions. Moreover, we did not detect biologically significant differences in proliferative activity, migration parameters, level of differentiation, or expression of vinculin when the cells were cultured on PLGA films and tissue culture polystyrene. Our results indicate that PLGA films do not affect the basic functions of primary human skin keratinocytes and fibroblasts and thus show acceptable biocompatibility in vitro, paving the way for their use as biomaterials for skin tissue engineering.     

Discovery of non-peptide, small molecule antagonists of α9α10 nicotinic acetylcholine receptors as novel analgesics for the treatment of neuropathic and tonic inflammatory pain

A series of azaaromatic quaternary ammonium analogs has been discovered as potent and selective α9α10 nicotinic acetylcholine receptor (nAChR) antagonists. The preliminary structure-activity relationships of these analogs suggest that increased rigidity in the linker units results in higher potency in inhibition of α9α10 nAChRs and greater selectivity over α7 nAChRs. These analogs represent a new class of analgesic for the treatment of neuropathic and tonic inflammatory pain.     

Coordination of thiouridine monophosphates with selected metal ions

Potentiometric and spectroscopic data obtained for the complexes of two thio-substituted uridine-monophosphates with Cu(2+), Ni(2+) and Cd(2+) ions have shown that both thionucleotide are more effective ligands than their nucleoside analogues. The basic binding site for all metal ions is the sulfur atom. The chelation by adjacent N(3) donor is also likely, although unfavorable four-membered chelate ring is formed.