Cyclic nigerosyl-1,6-nigerose-based nanosponges: An innovative pH and time-controlled nanocarrier for improving cancer treatment.

The design and structural optimisation of a novel polysaccharide-based nanomaterial for the controlled and sustained release of doxorubicin are here reported. A cross-linked polymer was obtained by reacting a tetraglucose, named cyclic nigerosyl-1-6-nigerose (CNN), with pyromellitic dianhydride. The cross-linking reaction formed solid nanoparticles, named nanosponges, able to swell as a function of the pH. Nanoparticle sizes were reduced using High Pressure Homogenization, to obtain uniform nanosuspensions. Doxorubicin was incorporated into the CNN-nanosponges in a good extent. DSC and solid state NMR analyses proved the drug interaction with the polymer matrix. In vitro studies demonstrated pH-dependent slow and prolonged release kinetics of the drug from the nanoformulation. Doxorubicin-loaded CNN-nanosponges were easily internalized in A2780 cell line. They might considered an intracellular doxorubicin reservoir, able to slowly release the drug over time. CNN-nanosponges may be promising biocompatible nanocarriers for the sustained delivery of doxorubicin with potential localised application in cancer treatments.

Forced expression of OCT4 influences the expression of pluripotent genes in human mesenchymal stem cells and fibroblasts.

Genetic reprogramming of adult cells to generate induced pluripotent stem (iPS) cells is a new and important step in sidestepping some of the ethical issues and risks involved in the use of embryonic stem cells. iPS cells can be generated by introduction of transcription factors, such as OCT4, SOX2, KLF4, and CMYC. iPS cells resemble embryonic stem cells in their properties and differentiation potential. The mechanisms that lead to induced pluripotency and the effect of each transcription factor are not completely understood. We performed a critical evaluation of the effect of overexpressing OCT4 in mesenchymal stem cells and fibroblasts and found that OCT4 can activate the expression of other stemness genes, such as SOX2, NANOG, CMYC, FOXD3, KLF4, and ßCATENIN, which are not normally or are very weakly expressed in mesenchymal stem cells. Transient expression of OCT4 was also performed to evaluate whether these genes are affected by its overexpression in the first 48 h. Transfected fibroblast cells expressed around 275-fold more OCT4 than non-transfected cells. In transient expression, in which cells were analyzed after 48 h, we detected only the up-regulation of FOXD3, SOX2, and KLF4 genes, suggesting that these genes are the earlier targets of OCT4 in this cellular type. We conclude that forced expression of OCT4 can alter cell status and activate the pluripotent network. Knowledge gained through study of these systems may help us to understand the kinetics and mechanism of cell reprogramming.

Functional inactivation by interleukin-1beta of glyceraldehyde-3-phosphate dehydrogenase in insulin-secreting cells.

AIMS/HYPOTHESIS: It is well established that long-term exposure of isolated beta cells to cytokines [e.g., IL-1beta] results in increased expression of inducible nitric oxide synthase and subsequent release of nitric oxide, which in turn, has been shown to mediate a wide array of effects, including alterations in cellular high-energy metabolism. In this context, several extant studies have demonstrated significant reduction in adenine and guanine nucleotide triphosphate levels in beta cells exposed to IL-1beta. Herein, we examined the functional status of glyceraldehyde-3-phosphate dehydrogenase [GAPDH] in insulin-secreting cells exposed to IL-1beta, since it represents the first enzyme in the glycolytic pathway that is involved in the generation of ATP. METHODS: GAPDH was assayed spectrophotometrically in the cytosolic fraction derived from control and IL-1beta -treated [300 pM for 24 hrs] insulin-secreting cell lines [HIT-T15 and RINm5F]. RESULTS: IL-treatment resulted in marked attenuation of GAPDH activity in HIT and RIN cells; such a reduction in this activity was not due to inhibition of its expression by IL-1. Instead, we observed that incubation of HIT and RIN lysates with peroxynitrite, a reactive intermediate of nitric oxide with superoxide anion, resulted in significant reduction in the GAPDH activity. CONCLUSION/INTERPRETATION: These results identify a GAPDH as one of the biochemical loci for the effects of IL-derived peroxynitrite in the islet beta cell. The previously reported reduction in high-energy phosphate levels in an IL-treated beta cell may, in part, be due to inhibition of GAPDH activity, and subsequent reduction in the glycolytic efficiency of the beta cell.

IL-1beta-induced nitric oxide release from insulin-secreting beta-cells: further evidence for the involvement of GTP-binding proteins.

Recently, we have demonstrated regulatory roles for G-proteins (e.g., H-Ras) in IL-1beta induced NO release from HIT-T15 cells. Herein, we report a similar regulatory mechanism for IL-1beta induced NO release from RIN5F and INS-1 cells. Our data indicate that functional inactivation of Ras, either by Clostridial toxins or by specific inhibitors of Ras function, results in a significant inhibition in IL-1beta induced NO release, suggesting that activation of specific G-proteins is essential for IL-1beta induced NO release. In the present study, we report possible loci where IL-1beta treatment might result in functional activation of these G-proteins. For example, IL-1beta treatment resulted in significant reduction in (high-and low-affinity) GTPase activities in lysates derived from normal rat islets; such a scenario might lead to retention of candidate G-proteins in GTP-bound, active conformation. Further, IL-1beta treatment increased the G-protein carboxyl methyl transferase activity as well as carboxyl methylation of endogenous beta-cell proteins; such a modification has been shown to increase the membrane association and interaction of these G-proteins with their respective effector proteins. Also, we report immunologic localization of H-Ras regulatory proteins including its nucleotide exchange factor (GRF-1) and its effector protein (eg., Raf-1) in isolated beta-cells. Together, our data indicate localization, and regulation by IL-1beta, of specific enzymes that are critical to activation of G-proteins. Based on these preliminary findings, we propose a model for the involvement of G-proteins in IL-1beta induced NO release and subsequent demise of the pancreatic beta-cell.

Positive modulation by Ras of interleukin-1beta-mediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells.

In the present study, we have shown that exposure of insulin-secreting clonal beta (HIT-T15) cells to interleukin-1beta (IL-1beta) results in a time- and concentration-dependent increase in nitric oxide (NO) release. These effects by IL-1beta on NO release were mediated by induction of inducible nitric oxide synthase (iNOS) from the cells. Preincubation of HIT cells with Clostridium sordellii lethal toxin-82, which irreversibly glucosylates and inactivates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1beta-induced NO release. Pre-exposure of HIT cells to C. sordellii lethal toxin-9048, which monoglucosylates and inhibits Ras, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1beta-mediated NO release. These data indicate that activation of Ras and/or Rac may be necessary for IL-1beta-mediated NO release. Preincubation of HIT cells with C. difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no demonstrable effects on IL-mediated NO release, ruling out the possibility that Rac may be involved in this signaling step. Further, two structurally dissimilar inhibitors of Ras function, namely manumycin A and damnacanthal, inhibited, in a concentration-dependent manner, the IL-1beta-mediated NO release from these cells. Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1beta-mediated NO release and, presumably, the subsequent dysfunction of the pancreatic beta cell. Our data also provide a basis for future investigations to understand the mechanism of cytokine-induced beta cell death leading to the onset of insulin-dependent diabetes mellitus.

S-nitrosoglutathione photolysis as a novel therapy for antifibrosis in filtration surgery.

PURPOSE: To determine whether a novel peroxynitrite-based photosensitizer S-nitrosoglutathione (GSNO) can produce specific in vitro light-induced cell death of both standard animal lung and human Tenon's capsule (TC) fibroblasts and to compare this effect with that produced by the established photodynamic porphyrin precursor 5-aminolevulinic acid (ALA). METHODS: V79-4 Chinese hamster lung and human TC fibroblasts were established in tissue culture. GSNO, together with its radioactive tritiated and fluorescent dansylated derivatives, were synthesized. The labeled molecules were prepared to determine the time course of uptake into the fibroblasts. Uptake was monitored by scintillation counting for the tritiated GSNO and confocal fluorescence microscopy for the dansylated GSNO. The uptake of ALA and biosynthesis of its photosensitive product were determined by fluorescence emission spectroscopy of a separate set of fibroblasts. Once uptake was established, both cell lines were incubated with varying concentrations of GSNO or ALA as a function of time (0, 4, or 24 hours) before light exposure (200 msec pulsed visible light, 0.068 W per pulse, for 10 minutes at a distance of 10 cm). After 10 minutes of irradiation, the cells were washed and exposed to fresh tissue culture medium. The effect of the treatment was determined 24 hours later by measuring cell viability. RESULTS: A 2-minute drug treatment time (0 hours incubation) with GSNO, followed by 10 minutes of irradiation, resulted in approximately 78% of fibroblast cell death at the lowest concentration of GSNO used compared with the control, which was exposed to light, but no GSNO. The higher concentrations of GSNO, or longer drug treatment times before irradiation, did not statistically increase cell death. Maximal cell death was thus obtained using the lowest GSNO concentration (50 mM) and drug treatment time (2 minutes). In contrast, the well-established photosensitizer ALA killed only approximately 4% of cells at the lowest concentration and drug treatment time tested. At drug treatment times of 4 hours and less, increased concentrations of ALA did not produce cell death of more statistical significance. It was not until 24 hours of drug treatment that comparable amounts of cell death were produced by ALA and GSNO. In all experiments similar results were obtained with the animal lung and human TC fibroblasts, suggesting that the source of the fibroblast had no effect on the outcome. The differences in treatment effects between GSNO and ALA were statistically significant under all conditions tested. CONCLUSIONS: GSNO is able to cause light-specific cell death of human TC fibroblasts at drug treatment times (2 minutes) and irradiation times (10 minutes) that would be compatible with its use in glaucoma filtering surgery. This in vitro performance was superior to that of the well-established photosensitizer ALA, which required treatment times longer than 4 hours to approach the light-specific cell death produced by only 2 minutes of GSNO treatment.

Atorvastatin increases ecNOS levels in human platelets of hyperlipidemic subjects.

BACKGROUND: The purpose of this study was to probe the pleiotrophic effects of Atorvastatin on intraplatelet-nitric oxide metabolism. METHODS AND RESULTS: Hyperlipidemic subjects (n = 19) were treated for 1 month (following a 3-week washout) with either Atorvastatin or placebo in a double-blinded randomized (n = 2, crossover), placebo-controlled study. Changes in the levels of intraplatelet nitric oxide synthase, nitrotyrosine were correlated with cholesterol, LDL-C, HDL-C and triglyceride levels. These studies indicate that with atrovastatin ecNOS levels increased on average by approximately approximately 1.7-fold (paired t-test p = 0.009). Interestingly, levels of nitrotyrosylated platelet proteins, an indication of peroxynitrite damage, decreased as ecNOS levels increased in presence of the drug (paired t-test p = 0.33). Atorvastatin, at 10 mg per day, lowered cholesterol and LDL-C levels in all patients with the average lowering of approximately 21% and approximately 17% respectively. The effect on HDL was not significant whilst triglyceride levels were lowered by an average of approximately 18%. CONCLUSIONS: This study adds to the volume of evidence that statins have beneficial effects other than lipid lowering. Here, Atorvastatin is shown to significantly elevate intraplatelet ecNOS levels in hyperlipidemic subjects without affecting iNOS expression. The net result of this would be the elevation of NO production which would promote platelet deaggregation and vasodilation.

Effects of therapeutic touch on biochemical and mood indicators in women.

Previous research has shown therapeutic touch (TT) to be effective in reducing anxiety and discomfort and promoting relaxation. The present investigation experimentally evaluated the effects of TT on biochemical indicators and moods in a sample of 41 healthy female volunteers. Participants were randomly assigned to either an experimental group who received TT or to a control group who did not receive TT. Pretest and posttest urine samples were collected, and personality and mood inventories were administered across three consecutive monthly sessions. Results indicated that mood disturbance in the experimental group decreased significantly over the course of the three sessions, while the control group increased in mood disturbance over time. Specifically, experimental group participants showed significant reductions in tension, confusion, and anxiety and a significant increase in vigor across sessions. Analyses of the biochemical data indicated that TT produced a significant decrease in levels of nitric oxide in the experimental group by the third TT session. The results of the present investigation have important implications for reducing symptom distress in cancer patients undergoing chemotherapy.

Evidence for iNOS-dependent peroxynitrite production in diabetic platelets.

AIMS/HYPOTHESIS: The aim of the present study was twofold. Firstly, to determine whether diabetic platelets produce more peroxynitrite than normal platelets and secondly to correlate the peroxynitrite production with the intraplatelet induction of the inducible isoform of nitric oxide-synthase. METHODS: Intraplatelet peroxynitrite production was monitored with dichlorofluorescin acetate with a combination of confocal microscopy and steady-state fluorescence. The platelets were probed for the induction of the inducible-nitric oxide-synthase by western immunoblotting. RESULTS: In the presence of extracellular L-arginine (100 micromol/l), platelets from subjects with Type I (insulin-dependent) diabetes displayed about 5 times higher fluorescence than those from control subjects. To determine whether inducible-nitric oxide-synthase was the source of peroxynitrite, dichlorofluorescein production was quantified as a function of L-arginine as well as nitric oxide-synthase inhibitors, in platelets from control subjects, subjects with Type I diabetes and subjects with Type II (non-insulin-dependent) diabetes mellitus. Platelets from subjects with Type I yielded about sevenfold and those from Type II about threefold larger amounts of L-arginine/nitric oxide-synthase-dependent dichlorofluorescein fluorescence than those from control subjects. The platelets were then immunologically probed for inducible-nitric oxide-synthase, which has previously been implicated in peroxynitrite production and detected in megakaryocytes of subjects with coronary heart disease. Western immunoblots of intraplatelet proteins indicated that the inducible-nitric oxide-synthase was absent in control subjects. Platelets from both Type I and Type II diabetic subjects, however, contained inducible-nitric oxide-synthase. CONCLUSION/INTERPRETATION: Inducible-nitric oxide-synthase-derived peroxynitrite is a source of platelet damage in diabetes.

Cytotoxic activities of Mannich bases of chalcones and related compounds.

Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.

A photo-activated, protein-based, NO/H2O2 generating system with tumoricidal activity composed of the nitric oxide derivative of apo-metallothionein (thionein-NO) and glucose oxidase.

The S-nitroso derivative of apo-metallothionein (thionein) was prepared by transnitrosation with S-nitrosoglutathione. The thionein-NO thus formed has an absorption maximum at 334 nm. Light-induced NO release from thionein-NO was demonstrated by flash photolysis. This system produces peroxynitrite at neutral pH as evidenced by nitrotyrosine formation. The cytotoxic potential of this protein-based, light-activated NO/H2O2 generating system was demonstrated by exposing human colon adenocarcinoma cells (SW 948) in culture to thionein-NO and glucose oxidase in the presence and absence of light. The cell density of the samples, 72 h subsequent to receiving 1 h of light exposure, decreased by approximately 98%, relative to controls. In comparison, cell density of the samples that were incubated in the presence of catalase and did not receive light treatment, decreased by only approximately 22% after 72 h.

ELISA for in vivo assessment of nonenzymatically glycated platelet glutathione peroxidase.

Using a combination of boronate affinity chromatography and ELISA methodology, a simple procedure was devised to selectively determine the in vivo glycated state of the platelet glutathione peroxidase (GSH-Px) from normal and diabetic subjects. The mean total GSH-Px levels in the normal (n = 14) and diabetic (n = 18) platelets were 1167 +/- 97 and 1007 +/- 73 ng/mg protein, respectively. The mean percentage glycated GSH-Px in the normal and diabetic platelets were 5.79 +/- 0.72% and 11.68 +/- 0.95%, respectively. When the percentage glycated GSH-Px was compared with the fructosamine values, a correlation coefficient of 0.71 was obtained. This indicates that the glycation status of platelet GSH-Px can be utilized as a sensitive, short-term index of plasma glucose levels.