Influence of structure of human,rat, and bovine serum albumins on binding properties of photoactive drug hypericin

Fluorescence binding measurements and molecular modeling were employed to study the interaction of hypericin (Hyp) with human (HSA), rat (RSA), and bovine (BSA) serum albumins. Fluorescence emission data show the solubility of Hyp increasing in the order BSA, HSA, and RSA. Molecular modeling was used to construct the detailed structural models of the complexes and to explain the differences in the binding properties of Hyp. It was shown that the structures of Hyp/HSA and Hyp/RSA complexes are more similar and in some aspects different from those found for the Hyp/BSA complex. The role of the amino acid sequence in the IIA subdomains of HSA, RSA, and BSA is discussed to explain the observed differences.     

Identification and quantification of hypericin and pseudohypericin in different Hypericum perforatum L. in vitro cultures.

Investigations have been made to develop an efficient protocol for micropropagation allowing to improve hypericin and pseudohypericin productions in Hypericum perforatum L. in vitro cultures. The role of growth regulator treatments has been particularly studied. Three in vitro culture lines with different morphological characteristics were obtained during H. perforatum micropropagation and referred to shoots, calli and plantlets according to their appearance. Multiplication and callogenesis from apical segments from sterile germinated seedlings were obtained on solid MS/B5 culture medium in the presence of N6-benzyladenine (BA) (0.1-5.0 mg/l BA). Regenerative potential of shoots was assessed on medium supplemented with auxins (0.05-1.0 mg/l), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). The main goal of the research was to summarize the influence of plant growth regulators on hypericin and pseudohypericin productions in in vitro cultures of Hypericum. A rapid method for naphtodianthrone quantification was developed. The use of a reversed-phase high performance liquid chromatography (HPLC) method with fluorescence detection was used. Identification of the compounds was confirmed by electrospray ionization-mass spectrometry (ESI-MS) with electrospray in negative ion mode [M-H] . Calli, shoots and plantlets of H. perforatum produced hypericin and pseudohypericin. The concentration range of BA from 0.1 to 2.0 mg/l improved the production of hypericin (25-50 microg/g dry mass (DM)) and pseudohypericin (170-350 microg/g DM) in shoots. In callus cultures, BA (4.0-5.0 mg/l) did not changed hypericin contents (15-20 microg/g DM) but influenced pseudohypericin productions (120-180 microg/g DM). In the presence of auxins (IAA and IBA), Hypericum plantlets produced hypericin (30-100 microg/g DM) and pseudohypericin (120-400 microg/g DM). The presence of IAA did not influence naphtodianthrone productions in plantlets, but IBA decreased hypericin and pseudohypericin amounts in plantlets. The specific accumulation of the naphtodianthrones in in vitro cultures was influenced by phytohormonal supplementation of the medium. Results indicated that the production of hypericin and pseudohypericin could be increased by carefully adapted in vitro cultures. Hypericum in vitro cultures represent promising systems for hypericin and pseudohypericin productions.     

Upregulation of CaMKIIδ during ischaemia-reperfusion is associated with reperfusion-induced arrhythmias and mechanical dysfunction of the rat heart: involvement of sarcolemmal Ca2+-cycling proteins

Although Ca(2+)/calmodulin-dependent protein kinase II delta (CaMKIIδ) has been implicated in development of different phenotypes of myocardial ischaemia-reperfusion injury, its involvement in arrhythmogenesis and cardiac stunning is not sufficiently elucidated. Moreover, the mechanisms by which CaMKIIδ mediates disturbances in excitation-contraction coupling, are not exactly known. To investigate this, KN-93 (0.5 μmol/L), a CaMKII inhibitor, was administered before induction of global ischaemia and reperfusion in isolated Langendorff-perfused rat hearts. Expression of CaMKIIδ and the sarcollemal Ca(2+)-cycling proteins, known to be activated during reperfusion, was analyzed using immunoblotting. KN-93 reduced reperfusion-induced ectopic activity and the incidence of ventricular fibrillation. Likewise, the severity of arrhythmias was lower in KN-treated hearts. During the pre-ischaemia phase, neither inotropic nor chronotropic effects were elicited by KN-93, whereas post-ischaemic contractile recovery was significantly improved. Ischaemia-reperfusion increased the expression of CaMKIIδ and sodium-calcium exchanger (NCX1) proteins without any influence on the protein content of alpha 1c, a pore-forming subunit of L-type calcium channels (LTCCs). On the other hand, inhibition of CaMKII normalized changes in the expression of CaMKIIδ and NCX1. Taken together, CaMKIIδ seems to regulate its own turnover and to be an important component of cascade integrating NCX1, rather than LTCCs that promote ischaemia-reperfusion-induced contractile dysfunction and arrhythmias.     

Photosensitizer effects on cancerous cells: a combined study using synchrotron infrared and fluorescence microscopies

Hypocrellin A (HA), a lipid-soluble peryloquinone derivative, isolated from natural fungus sacs of Hypocrella bambusae, has been reported to be a highly potential photosensitizer in photodynamic therapy (PDT). It has been studied increasingly because of its anticancer activities when irradiated with light. We have studied the interaction mechanisms of HA with HeLa cells as a function of incubation time. Fluorescence microscopy confirmed that HA localisation is limited in the cytoplasm before eventually concentrating in clusters around the nucleus. The IR spectra of HA-treated, PDT-treated and control HeLa cells were recorded at the ESRF Infrared beamline (ID21). Principal component analysis has been used to assess the IR spectral changes between the various HeLa cells spectral data sets (The Unscrambler software, CAMO). PCA revealed that there is a frequency shift of protein amide I and amide II vibrational bands, indicating changes in the protein secondary structures of the HA-treated and PDT-treated cancer cells compared to the control cells. In addition, the relative DNA intensity in HA-treated cells decreases gradually along the incubation time. The use of synchrotron infrared microscopy is shown to be of paramount importance for targeting specifically the biochemical modification induced in the cell nucleus.     

Mitigation of postischemic cardiac contractile dysfunction by CaMKII inhibition: effects on programmed necrotic and apoptotic cell death

While Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) has been suggested to be an important protein regulating heart function upon ischemia/reperfusion (I/R), the mechanisms responsible are not fully known. Furthermore, it is not known whether CaMKII activation can modulate necroptosis, a recently described form of programmed cell death. In order to investigate these issues, Langendroff-perfused rat hearts were subjected to global ischemia and reperfusion, and CaMKII inhibition was achieved by adding the CaMKII inhibitor KN-93 (0.5 μmol/dm³) to the perfusion solution before the induction of ischemia. Immunoblotting was used to detect changes in expression of proteins modulating both necroptotic and apoptotic cell death. CaMKII inhibition normalized I/R induced increases in expression of necroptotic RIP1 and caspase-8 along with proteins of the intrinsic apoptotic pathway, namely cytochrome c and caspase-9. In addition, it increased the Bcl-2/Bax ratio and reduced caspase-3 and cleaved PARP1 content suggesting reduction of cell death. These changes coexisted with improvement of postischemic contractile function. On the other hand, there was no correlation between levels of pT287-CaMKIIδ and LVDP recovery after I/R. These results demonstrate for the first time that CaMKII inhibition may mitigate cardiac contractile dysfunction, at least partially, by limiting the contents of not only apoptotic, but also necroptotic proteins. Phosphorylation of CaMKII seems unlikely to determine the degree of postischemic recovery of contractile function.     

The effects of spirotetramat on life history traits and population growth of Tetranychus urticae (Acari: Tetranychidae)

The effects of spirotetramat, a tetramic acid derivative, on gross fertility, net fertility, female longevity and the instantaneous rate of increase of two-spotted spider mite, Tetranychus urticae (Acari: Tetranychidae) were investigated after treatment of female teleiochrysalises (the first assay) and pre-ovipositional females (the second assay). Spirotetramat was applied to the leaf discs by Potter spray tower and the following series of concentrations was applied: 200, 60, 18, 5.4 and 1.62 mg/l. In both assays after 24 h of exposure, surviving females without symptoms of poisoning were used for further procedure. In the first assay, gross fertility of treated females was reduced by 2.4–64.7% and net fertility by 12.4–88.8%, compared to the control. Gross fertility of the females treated with 1.62 and 5.4 mg/l did not significantly differ from the control, whereas all concentrations, except the lowest, significantly reduced net fertility and female longevity. Treatments with 200, 60, and 18 mg/l significantly reduced the instantaneous rate of increase. In the second assay, gross fertility and net fertility were reduced by 43.7–93.3% and 73.8–98.5%, respectively. All concentrations, except the lowest, significantly reduced gross fertility, whereas net fertility and longevity in all treated females were significantly lower compared to the control. All concentrations, except the lowest, significantly reduced the instantaneous rate of increase, provided that concentrations of 200, 60 and 18 mg/l caused population decline. The effects of spirotetramat and its impact on T. urticae management are discussed.     

The effects of spiromesifen on life history traits and population growth of two-spotted spider mite (Acari: Tetranychidae)

Laboratory bioassays were conducted to evaluate the effects of spiromesifen on gross fecundity, gross fertility, net fertility and population growth of two-spotted spider mite (Tetranychus urticae Koch) after treatments with four acaricide concentrations: 180 mg/l, i.e. maximum recommended concentration for use in glasshouses against spider mites, 18, 1.8, and 0.18 mg/l, i.e. concentration discriminative for eggs and immatures in preliminary studies which produced 100% mortality of these stages. Quiescent female deutonymphs were treated in the first assay, and young pre-ovipositing females in the second and third, in which exposure lasted 6 h and 20 h, respectively. In the first assay, the 180, 18, and 1.8 mg/l concentrations significantly reduced gross fecundity (61–85%), gross fertility (64–87%) and net fertility (85–94%) of the surviving females. In the second one, only the highest concentration achieved a significant statistical reduction in gross fecundity (52%), gross fertility (67%) and net fertility (84%). In the third assay, fecundity and fertility reduction under the two highest concentrations was 98–99% and 93–98%, whereas it was 50–74% under the 1.8 mg/l concentration, and statistically different from control values. In all three trials, treatments with 180, 18, and 1.8 mg/l concentrations significantly reduced the instantaneous rate of increase. In the third assay, treatments with the two highest concentrations caused population decline. Sublethal activity of the 0.18 mg/l concentration was not found in any assay to be statistically significant. Sublethal effects of spiromesifen and its impact on T. urticae management are discussed.