Antimicrobial and anti-biofilm properties of new taxodione derivative from hairy roots of Salvia austriaca.

The aim of the present report was to evaluate antimicrobial/anti-biofilm activity of 7-(2-oxohexyl)-taxodione, a novel taxodione derivative isolated from n-hexane extract of Salvia austriaca hairy roots. Antimicrobial assays showed that 7-(2-oxohexyl)-taxodione was at least 4 times more active than taxodione against methicillin-susceptible as well against methicillin-resistant staphylococci with MIC of 1.25-2.5 µgml(-1). This compound was less active against vancomycin-resistant enterococci (VRE), on the same level as taxodione (MIC ranged 10.0-20.0 µgml(-1)). The presence of 7-(2-oxohexyl)-taxodione in the culture medium (at MIC, ½ MIC or » MIC) decreased adhesion of staphylococci to abiotic surfaces, which in turn caused a reduction in biofilm formation during 24h, by approximately 25-30%. Also, the extent of established biofilm eradication was found to be significant, although it required an increased concentration of the compound. This is the first report on the antimicrobial activity of this, up to now not known compound, isolated from transformed roots of S. austriaca.

Synthesis, structural characterization, antimicrobial and cytotoxic effects of aziridine, 2-aminoethylaziridine and azirine complexes of copper(II) and palladium(II).

The synthesis, spectroscopic and X-ray structural characterization of copper(II) and palladium(II) complexes with aziridine ligands as 2-dimethylaziridine HNCH(2)CMe(2) (a), the bidentate N-(2-aminoethyl)aziridines C(2)H(4)NC(2)H(4)NH(2) (b) or CH(2)CMe(2)NCH(2)CMe(2)NH(2) (c) as well as the unsaturated azirine NCH(2)CPh (d) are reported. Cleavage of the cyclometallated Pd(II) dimer [µ-Cl(C(6)H(4)CHMeNMe(2)-C,N)Pd](2) with ligand a yielded compound [Cl(NHCH(2)CMe(2))(C(6)H(4)CHMe(2)NMe(2)-C,N)Pd] (1a). The reaction of the aziridine complex trans-[Cl(2)Pd(HNC(2)H(4))(2)] with an excess of aziridine in the presence of AgOTf gave the ionic chelate complex trans-[(C(2)H(4)NC(2)H(4)NH(2)-N,N')(2)Pd](OTf)(2) (2b) which contains the new ligand b formed by an unexpected insertion and ring opening reaction of two aziridines ("aziridine dimerization"). CuCl(2) reacted in pure HNC(2)H(4) or HNCH(2)CMe(2) (b) again by "dimerization" to give the tris-chelated ionic complex [Cu(C(2)H(4)NC(2)H(4)NH(2)-N,N')(3)]Cl(2) (3b) or the bis-chelated complex [CuCl(C(2)H(2)Me(2)NC(2)H(2)Me(2)NH(2)-N,N')(2)]Cl (4c). By addition of 2H-3-phenylazirine (d) to PdCl(2), trans-[Cl(2)Pd(NCH(2)CPh)(2)] (5d) was formed. All new compounds were characterized by NMR, IR and mass spectra and also by X-ray structure analyses (except 3b). Additionally the cytotoxic effects of these complexes were examined on HL-60 and NALM-6 human leukemia cells and melanoma WM-115 cells. The antimicrobial activity was also determined. The growth of Gram-positive bacterial strains (S. aureus, S. epidermidis, E. faecalis) was inhibited by almost all tested complexes at the concentrations of 37.5-300.0 µg mL(-1). However, MIC values of complexes obtained for Gram-negative E. coli and P. aeruginosa, as well as for C. albicans yeast, mostly exceeded 300 µg mL(-1). The highest antibacterial activity was achieved by complexes 1a and 2b. Complex 2b also inhibited the growth of Gram-negative bacteria.

Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L.: salvipisone as a potential anti-biofilm agent active against antibiotic resistant Staphylococci.

The antimicrobial activities of crude dichloromethane fractions from acetone extracts of Agrobacterium rhizogenes transformed roots and roots of field-grown plants of Salvia sclarea as well as four pure abietane diterpenoids isolated from the hairy root cultures were determined. The growth of Gram-positive bacteria (Staphylococcus aureus, S. epidermidis, Enterococcus faecalis) but not Gram-negative ones (Escherichia coli, Pseudomonas aeruginosa) or pathogenic fungi (Candida albicans) was inhibited by fractions tested at concentrations of 37.5-75.0 microgml(-1). Abietane diterpenoids: salvipisone, aethiopinone, 1-oxoaethiopinone and ferruginol were shown to be bacteriostatic as well as bacteriocidal for the cultures of S. aureus and S. epidermidis strains, regardless of their antibiotic susceptibility profile. This was demonstrated by using simultaneously the optical density measuring method and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide-reduction assay. The highest activity was shown by salvipisone which demonstrated also a very interesting activity when its effect on 24-h-old staphylococcal biofilm cells viability was examined. It limited the survival of biofilms formed by S. aureus as well as by S. epidermidis, putting this compound to the list of potential anti-biofilm agents, better than most of known antibiotics.

Effects of fibrinogen receptor antagonist GR144053F and aurintricarboxylic acid on platelet activation and degranulation.

Activated blood platelets play crucial role in restenosis due to their fundamental significance in thrombus formation. Therefore, platelets are attractive targets for the inhibition with a variety of antagonists. In this study, we present direct evidence that GR144053F [non-peptide antagonist of glycoprotein IIb-IIIa complex (GPIIb-IIIa)] inhibits activation and degranulation of human platelets, and opposes the action of aurintricarboxylic acid (ATA), the antagonist of von Willebrand factor, which augments platelet secretion. The effects of both drugs on platelet function were monitored by using various instrumental methods. Platelet-rich plasma and whole-blood aggregation was measured by using ADP and collagen as agonists. Platelet degranulation was assessed based on the expression of surface membrane activation markers: P-selectin, glycoprotein Ib, and activated GPIIb-IIIa complex. Measurements of closure time with platelet function analyzer PFA-100 enabled us to reason on primary hemostatic capacity and reflected both aggregability and adhesiveness. GR144053F markedly reduced primary hemostatic platelet response (IC(50) = 114.0 +/- 9.6 nM) under conditions that closely mimicked natural blood flow in circulation, and inhibited aggregation in platelet-rich plasma (IC(50) = 17.7 +/- 7.0 nM). It was equally potent inhibitor of platelet activation, degranulation, fibrinogen binding, platelet consumption, and aggregate formation. Also, ATA was efficient in inhibition of platelet aggregation and adhesion (by up to 50% at 100 microM), but the combined action of both drugs on primary haemostatic capacity was not additive. GR144053F suppressed the activating effects of ATA on platelet degranulation and secretion. Overall, our data indicate that GR144053F is not only the efficient blocker of fibrinogen binding to GPIIb-IIIa, but also hampers platelet degranulation and may attenuate the activating effects of ATA.

Modulators of intraplatelet calcium concentration affect the binding of thrombospondin to blood platelets in healthy donors and patients with type 2 diabetes mellitus.

UNLABELLED: Thrombospondin (TSP), which is secreted from alpha-granules of activated platelets, binds to its surface receptor (CD36) in the presence of Ca2+. OBJECTIVES: We monitored how the modulation of intraplatelet Ca2+ affects TSP binding to CD36 on platelets from healthy donors and patients with type 2 diabetes mellitus. We also aimed to verify whether the impaired Ca2+ mobilisation in diabetes influences TSP binding upon the pharmacological modulation of calcium transport. METHODS: Whole blood cytometry was used to monitor TSP release/binding and CD36 presentation in platelets from 28 type 2 patients and 33 healthy donors. RESULTS: No significant changes in TSP and CD36 levels were revealed between the groups in circulating platelets and TRAP-, collagen- or thrombin-activated platelets. In healthy donors, 1 microM thapsigargin (TG) elevated the TRAP-activated TSP binding (by up to 50%, p<0.001), 5 mM EGTA reversed the effect (by up to 85%, p<0.001), and overcame the effect of TG when used together. Less profoundly expressed effects occurred in the NIDDM group. In both groups TG increased the presentation of CD36 in TRAP-stimulated platelets (p<0.05), whereas EGTA lowered the TRAP-stimulated increase in CD36 (p<0.001). The inhibition of CD36 by EGTA was stronger in healthy volunteers (41% vs. 32%, respectively, p<0.05), whereas the activation by TG was higher in the NIDDM group (11% vs. 27%, p<0.05). When acting together the suppressive effects of EGTA on TG-dependent Ca2+ mobilisation were much attenuated in diabetic subjects (p<0.05). CONCLUSION: Both the release of TSP and CD36 presentation are under the influence of agents modulating intracellular Ca2+. Diabetic platelets seem more vulnerable to the releasers of cytosolic [Ca2+] and more resistant to the blockers of cytosolic [Ca2+] mobilisation.

Platelet hyperreactivity after coronary artery bypass grafting: the possible relevance to glycoprotein polymorphisms. A preliminary report.

Coronary artery bypass grafting (CABG) surgery impairs platelet function and reactivity to a considerable extent. However, variability in the individual patients' responses makes any generalised statement uncertain. The observed variability is nowadays thought to relate to platelet glycoprotein polymorphisms. Our objective was to investigate the association between platelet reactivity and the restoration of platelet functional response to agonists during the period following cardiosurgical operation and some genetic polymorphisms of selected platelet membrane glycoproteins. Platelet reactivity was monitored in 32 IHD patients (56 +/- 8 years) subjected to CABG surgery by means of whole blood impedance aggregometry and concurrently using the platelet function analyser (PFA-100 at four time intervals: prior to operation (A), 2 h after administration of protamine sulfate (B), 3 days after (C) and 7 days after CABG surgery (D). Three important findings were made. First, in all patients platelet reactivity became decreased 2 h postoperatively (aggregation with 20 microM ADP reduced by up to 49%, P < 0.02) and vastly increased 7 days after CABG surgery (CT(CADP) reduced down to 87% of initial value, P < 0.05, ADP-induced aggregation enhanced up to 167%, P < 0.001, and that with collagen up to 131% of the initial value, P < 0.01). Second, the frequencies of the 'prothrombotic' phenotype variants of platelet membrane glycoproteins were higher in patients referred to as the carriers of more reactive platelets compared to those with less reactive platelets (GPIa (807)T-positive, 50 vs. 28%; GPIIIa Pl(A2)-positive, 27 vs. 21%; GPIb Met(145)-positive and GPIb VNTR B-positive, 13 vs. 0%. Lastly, the restoration in platelet hyperreactivity in CABG surgery patients was recorded more often in patients who underwent postoperative myocardial ischaemic episode(s), and was associated with significantly higher frequency of the 'prothrombotic' allele (807)T of the collagen receptor glycoprotein Ia (GPIa) in these subjects (83 vs. 61%). In conclusion, in patients with ischaemic episodes after CABG, we demonstrated a fast postoperative restoration of haemostatic capacity and evidence of platelet hyperreactivity at 7 days after CABG surgery. The platelet hyperfunction seems to relate to the occurrence of platelet glycoprotein polymorphisms GPIa(807)C/T and GPIIIa PlA(1/A2) and may be important in predicting postoperative vascular complications in CABG patients.

Activation of circulating platelets and platelet response to activating agents in children with cyanotic congenital heart disease: their relevance to palliative systemic-pulmonary shunt.

Abnormal platelet function has been hypothesised to play a role in the haemostatic abnormalities in cyanotic congenital heart disease (CCHD) patients. Using whole blood flow cytometry we found that platelets from cyanotic patients were hyperreactive and we related such hyperreactivity directly to young age, unoperated state, high haematocrit, reduced saturation with oxygen and low platelet count. Circulating platelets from CCHD children showed significantly enhanced P-selectin expression (P<0.004) and remained more reactive to 0.2 IU/ml thrombin, 1-8 microM TRAP and 2-4 microM ADP (P<0.04), especially in younger (0-3-year-olds) patients. Such a platelet 'priming' largely concerned CCHD children who were not subjected to modified Blalock-Taussig shunts in the past (non-MBTS). Only non-MBTS cyanotic children, but not MBTS-operated patients, showed significantly higher platelet reactivity compared to controls in response to ADP or 1 microM TRAP with respect to P-selectin expression (p<0.05) and in response to all examined agonists with respect to GPIb expression (P<0.045). The enhanced P-selection expression in MBTS-operated CCHD children and reduced GPIb expression in non-MBTS patients, especially in younger patients, were positively associated with the occurrence of the polymorphic variant Pl(A2) of platelet membrane glycoprotein IIIa gene. Altered blood morphology parameters (elevated RBC, Hb, Hct and MCHC, for all P<0.0005) in CCHD children correlated with the enhanced degranulation of circulating blood platelets and their hyperreactivity in response to some agonists (P<0.05). Overall, our data encourage the reasoning that circulating platelets are remarkably hyperreactive in non-MBTS cyanotic children, which are at higher risk to often encounter platelets activation in circulation. It seems unlikely that the apparently unchanged platelet reactivity in MBTS-operated children is due to the advantageous effects of the shunt, since these patients showed neither altered haematological parameters nor improved oxygen carrying capacity. Otherwise, it may rather result from more frequent episodes of platelet degranulation and preactivation in the past, and/or post-operative enhanced platelet consumption.

Do determinants of platelet function co-segregate with genetic markers of type 1 diabetes mellitus?

This study examined the significance of selected parameters of primary haemostasis to discriminate between relatives of children with insulin-dependent diabetes mellitus (IDDM). Platelet function, including markers of spontaneous and agonist-induced platelet activation (CD62), platelet consumption (microparticles) and clumping (aggregates), as well as selected parameters of the fibrinolytic system (t-PA and PAI-1), were studied in IDDM children ( n = 45), their parents ( n = 65), siblings ( n = 17) and unrelated healthy controls ( n = 51). The fraction of activated platelets circulating in whole blood amounted to 4.3 +/- 2.1% in IDDM children, and significantly exceeded the level found in parents (1.3 +/- 0.7%, P < 0.002), siblings (1.2 +/- 1.0%, P < 0.002), and controls (1.2 +/- 0.6%, P < 0.002). Furthermore, an enhanced formation of platelet microparticles was observed in the IDDM group, both in resting platelets and also when platelets were stimulated with thrombin. Significantly decreased total PAI-1 occurred in IDDM children ( P < 0.02 versus parents); also slightly lowered active PAI-1 and t-PA antigen were noticed in IDDM subjects compared to other groups, however, the differences were not statistically significant. To assess dissimilarities between the groups of subjects we applied the forward stepwise model of discriminant function analysis, which included platelet flow cytometry parameters. The best separation and the highest discrepancy (expressed as the so called squared Mahalanobis distances, d ) was M revealed between controls and IDDM patients ( P < < 0.0001) and between controls and parents ( P < < 0.0001). The values of d found between IDDM children and their siblings (P < 0.001), as well as parents ( P < 0.01), were M of much lower significance. The finding that the control group, representing unrelated subjects, remains particularly well separated from the other groups, more or less clustered together, implies the possible involvement of genetic factor(s) which might potentially affect platelet activation and reactivity. In addition, the distinguished distribution of HLA DQAI(52) and HLA DQBI(57) genotypes in the groups further validates the suspicion that the altered platelet function and response in diabetes might be associated with some independent genetic factor(s), and is not likely to result from HLA DQAI(52) and HLA DQBI(57) impact.

Expression of 65-kDa oncofetal protein in experimental hepatoma after anticancer therapy.

We have tested the expression of a 65-kDa oncofetal protein (p65) after combined treatment with menadione and methotrexate in hamsters transplanted with Kirkman-Robins hepatoma. The treatment of tumor-bearing animals with these compounds significantly inhibited both the tumor development and the expression of p65. This inhibition in tumor tissue was calculated from densitograms of Western blots. The inhibition of p65 expression was also confirmed in the serum of hepatoma bearing animals by using solid-phase radioimmunoassay (RIA) to quantify the specificity of polyclonal antibodies to fetal p65 molecules. Additionally, p65 was shown to localize both in cytoplasm and in the nuclear extracts prepared from hepatoma tissue.

Monoclonal antibodies against histone H5. Epitope mapping and binding to chromatin.

Monoclonal antibodies against chicken erythrocyte histone H5 were produced. Nine hybridomas of different clonal origin were selected, and the antibodies were purified by affinity chromatography. Typing of the antibodies indicated that all but one (IgM) belong to the IgG1 class and contain kappa light chains. Indirect immunoprecipitation, solid-phase radioimmunoassay, and competitive inhibition assays using various H5 fragments revealed that the antigen-binding sites were localized on the central region of H5 (GH5, residues 22-100). Results of immunoblots from gels containing different denaturing agents indicate that some of the antibodies recognize related continuous epitopes localized at the junction of the GH5 with the rest of the molecule. Competition experiments between pairs of the eight different IgGs suggest that they recognize at least seven distinct sites on GH5. The epitopes appear to represent different regions of GH5 although some of them overlap. In general, the antibodies recognize epitopes which are not too accessible to the environment in the native conformation of the histone. All of the antibodies examined, except one of them (5H10), react with nuclei and chromatin from the erythroid cells but not from other cell lines. The site recognized by 5H10 is likely to be one of the regions where GH5 interacts with the nucleosome. No cross-reactivity of the antibodies with other histones including H1, H2A, H2B, H3, H4, and rat liver histone H1(0) was observed.