FcUni-RLuc: an engineered Renilla luciferase with Fc binding ability and light emission activity.

A novel and advanced Fc-binding probe ­ FcUni-RLuc namely ­ has been produced and functionally assayed for labelling IgGs. The Fc antibody binding sequence ­ HWRGWV ­ was fused to Renilla luciferase, and the purified probe was employed for bioluminescence enzyme-linked immunoabsorbance assay of Her2 positive cells.

In vitro antitumor activity of parent and nano-encapsulated mono cobalt-substituted Keggin polyoxotungstate and its ctDNA binding properties.

The parent and nanosized starch, and lipid encapsulated K6[SiW11O39Co(H2O)]·nH2O (abbreviated as SEP, LEP and SiW11Co, respectively), as potent antitumor candidates, were synthesized and characterized by FT-IR spectroscopy, ICP, TG analysis, SEM and TEM images. The results show that the SiW11Co retains its parent structure after encapsulation by starch and lipid nanoparticles. Antitumor activity tests of SiW11Co and its encapsulated forms were carried out on two types of human cancer cells, MCF-7 and HEK-293 by MTT method. The encapsulated forms exhibited the higher antitumor activity compared to the parent SiW11Co. However, this observed enhancement for the lipid encapsulated form is more than the starch counterpart, which can be related to its smaller size. These results showed that these compounds can be novel antitumor candidates. The calf thymus DNA (abbreviated as ctDNA) binding ability of SiW11Co was also investigated, using UV-Vis absorption spectroscopy, fluorescence quenching and fluorescence Scatchard plots. Absorption spectra tracing reveal 10% hyperchromism for SiW11Co. The values of 1.8×10(4) and 1.2×10(4)M(-1) were obtained for association binding constant of SiW11Co to ctDNA at R⩾1 and R<1, respectively (R is defined as the mole ratio of SiW11Co to ctDNA). It was shown that the interaction of SiW11Co with ctDNA depended on the R values. The obtained results of absorption titration rejected the intercalating binding mode and suggest the groove or outside stacking binding for SiW11Co. These results were authenticated by fluorescence quenching experiments and scatchard plots.

ctDNA binding affinity and in vitro antitumor activity of three Keggin type polyoxotungestates.

The ctDNA-binding properties and in vitro antitumor activity of three water soluble Keggin type polyoxometalates (POMs): K6H[CoW11O39CpZr]·nH2O, K6H[CoW11O39CpTi·nH2O and K7H2[CoW11O39CpFe]·nH2O (abbreviated as CoWCpZr, CoWCpTi and CoWCpFe, respectively) were investigated using UV-Vis absorption spectroscopy, fluorescence spectrophotometry, cyclic voltammetry and MTT assay. The results of UV-Vis, fluorescence and cyclic voltammetry rule out intercalating binding mode and propose the groove or outside stacking binding of these POMs with ctDNA. The values of 1.30×10(4) M(-1), 1.15×10(4) M(-1) and 3.10×10(3) M(-1) were obtained for binding constant of CoWCpZr, CoWCpTi and CoWCpFe to ctDNA, respectively. The redox potential of POMs shift to more negative values in the presence of ctDNA which can be related to domination of electrostatic interaction in this system. The antitumor activity tests of these polyoxometalates (POMs) were carried out on two types of human cancer cells, MCF-7 and HEK-293 by MTT method. The results show the higher antitumor activity of CoWCpFe respect to two other that is related to its highest penetrating effectiveness for MCF-7 cells. Therefore, the antitumor activity of these POMs depends not only on their affinity to ctDNA but also strongly on their penetration ability to the cell membrane.

Differential expression of suppressors of cytokine signalling genes in response to nutrition and growth hormone in the septic rat.

GH treatment during critical illness and sepsis may increase mortality. A family of negative regulators of cytokine signalling, the suppressors of cytokine signalling (SOCS), have been characterised. SOCS provide a mechanism for cross-talk between the cytokine receptors, including GH. Here, we have investigated the impact of nutrition and GH treatment on GH receptor, SOCS1, SOCS-2, SOCS-3 and cytokine-inducible SH2-containing protein (CIS) hepatic mRNA expression in a rat model of sepsis, caecal ligation and puncture (CLP). Four groups of rats were studied: control (food given ad libitum, n=7), CLP only (n=8), CLP and total parenteral nutrition (TPN) (n=9), and CLP, TPN and GH (n=10). CLP rats underwent surgery and 18 h later received saline or TPN or TPN+GH for 6 h before they were killed. Serum IGF-I levels were lower in all CLP groups (P<0.001). The combination of TPN and GH treatment increased IGF-I levels compared with the saline-treated CLP rats (P<0.01), but IGF-I levels remained lower than control animals (P<0.001). GH receptor and GH-binding protein expression in liver was reduced in animals subjected to CLP and was unaffected by nutrition or GH treatment. Hepatic SOCS-1 was detectable in normal rats, induced in all CLP animals but was unaffected by nutrition and GH. Hepatic SOCS-2 expression was difficult to detect in normal and CLP rats but was greatly induced in CLP rats treated with GH. Hepatic SOCS-3 expression was only just detectable in the control group but was elevated in all CLP groups and unaffected by nutrition and GH. Hepatic CIS expression was difficult to detect in normal rats, was not induced by CLP but was induced by both nutrition and GH. In conclusion, CLP induced low IGF-I levels associated with increased expression of SOCS-1 and SOCS-3, both of which are known to inhibit GH receptor signalling. GH induced SOCS-2 and CIS in the CLP rat despite resistance with respect to IGF-I generation, and parenteral feeding induced CIS in the CLP rat. Thus, there is potential for a complex interaction between GH and cytokine signalling at the level of SOCS expression whereby the inflammatory response may alter GH signalling and GH may influence the inflammatory response.

High-dose growth hormone does not affect proinflammatory cytokine (tumor necrosis factor-alpha, interleukin-6, and interferon-gamma) release from activated peripheral blood mononuclear cells or after minimal to moderate surgical stress.

High-dose GH therapy, with GH doses 10-20 times the normal replacement dose for GH-deficient adults, has been used as an anti-catabolic agent in a number of different patient groups. A recent study, however, has shown an increase in mortality in critically ill patients treated with high-dose GH. The increased mortality was associated with multiorgan failure, septic shock, and uncontrolled infection, suggesting that GH may have altered the immune response. The GH receptor and GH are both expressed in peripheral blood mononuclear cells (PBMCs); thus, GH could act as either an endocrine or an autocrine modulator of the immune response. We have examined the hypothesis that high-dose GH therapy may induce proinflammatory cytokines, which are implicated in septic shock. To do this we measured cytokine production by PBMCs incubated in conditions that simulated high-dose GH therapy, and we measured cytokine levels in patients undergoing laparoscopic cholecystectomy who were randomized to receive either high-dose GH therapy (13 IU/m2 x day) or placebo. To confirm the biological activity of GH in our cell culture system we used a Stat5 functional assay. In this assay GH induced a bell-shaped curve, with a maximal response at GH levels between 100-1,000 ng/mL. PBMCs from healthy volunteers were incubated with GH in doses from 1-1,000 ng/mL for 6-72 h under resting conditions and after activation with endotoxin and the mixed lymphocyte reaction. Studies were repeated with PBMCs from six individuals using a GH dose of 100 ng/mL (the level of GH found after high-dose GH therapy) and an endotoxin dose that gave a submaximal response (0.01 ng/mL). GH had no effect on cell proliferation or the production of tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), or interferon-gamma (IFNgamma). In patients undergoing laparoscopic cholecystectomy there was a time-related effect of surgery on cytokine levels. There was a rise in IL-6 and a fall in TNFalpha at 24 h after surgery; however, high-dose GH therapy had no effect on the cytokine response. We considered the possibility that endogenous GH production by PBMCs could influence the cytokine response in activated PBMCs; however, incubation of PBMCs in the presence of the GH receptor antagonist, B2036, had no effect on TNFalpha, IL-6, or IFNgamma production by PBMCs in either the mixed lymphocyte reaction or when activated by endotoxin. These results suggest that high-dose GH therapy does not alter the proinflammatory cytokine response to surgery or endotoxin. The results do not exclude an effect of GH on the immune response, but they suggest that the mortality seen in critically ill patients may be due to factors other than immune modulation.