Aerosol generation by metered-dose inhalers containing dimethyl ether/propane inverse microemulsions

Water soluble compounds were incorporated into metered-dose inhalers (MDIs) by using water-in-propellant lecithin microemulsions, in which dimethyl ether (DME) and propane acted as both continuous phase and propellant. Lecithin, water, and water soluble compounds were added to glass MDI containers, valves were crimped on, and propellants were added using a pressure burette. Aerosols were produced using commercially available actuators, and inertial impaction was used to determine the mass median aerodynamic diameter (MMAD), geometric standard deviation (GSD), and fine particle fraction (FPF) of the resulting aerosols. The DME/propane/lecithin, microemulsion MDIs generated aerosols with particle size distributions suitable for pulmonary delivery (eg, MMAD 3.1 μm, FPF 59% for DME with lecithin content 3%, water content 2.5% [wt/wt]). Increasing water concentration (up to 8% wt/wt) was correlated with a reduction in FPF. Freezing and rewarming had no adverse effect on MMAD, GSD, or FPF. Storage of microemulsion samples for up to 3 weeks did not adversely affect the MMAD, GSD, or FPF. This approach may enable the pulmonary delivery of water soluble therapeutic agents via MDIs.     

p-Nitrophenol and glutathione response in medaka (Oryzias latipes) exposed to MX,a drinking water carcinogen

When chlorine is introduced into public drinking water for disinfection, it can react with organic compounds in surface waters to form toxic by-products such as 3-chloro-4-(dichloromethyl)-5-hydroxy-2[5H]-furanone (MX). We investigated the effect of exposure to MX on cytochrome P450 2E1 (CYP2E1)-like activity and total glutathione (GSH) in the liver of the small fish model, medaka (Oryzias latipes). The multi-site carcinogen methylazoxymethanol acetate (MAMAc) was the positive control compound. Both medaka liver microsome preparations and S-9 fractions catalyzed the hydroxylation of p-nitrophenol (PNP), suggesting CYP2E1-like activity in the medaka. Male medaka exposed for 96 h to the CYP2E1 inducers ethanol and acetone under fasted conditions showed significant increases in PNP-hydroxylation activity. Furthermore, total reduced hepatic GSH was reduced in fish fasted for 96 h, indicating that normal feeding is a factor in maintaining xenobiotic defenses. Exposure to MX and MAMAc induced significant increases in hepatic CYP2E1-like activity, however MX exposure did not alter hepatic GSH levels. These data strengthen the role of the medaka as a suitable species for examining cytochrome P450 and GSH detoxification processes and the role these systems play in chemical carcinogenesis.     

PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum

We have previously shown that PGE(2) and PGI(2) induce recovery of transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in Cl(-) secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight junction assembly, we postulated that this signaling pathway is involved in PG-induced mucosal recovery. Porcine ileum was subjected to 45 min of ischemia, after which TER was monitored for a 180-min recovery period. Endogenous PG production was inhibited with indomethacin (5 microM). PGE(2) (1 microM) and PGI(2) (1 microM) stimulated recovery of TER, which was inhibited by serosal application of the osmotic agent urea (300 mosmol/kgH(2)O). The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in response to PGs or mucosal urea. Immunofluorescence imaging of recovering epithelium revealed that PGs restored occludin and zonula occludens-1 distribution to interepithelial junctions, and this pattern was disrupted by pretreatment with wortmannin. These experiments suggest that PGs stimulate recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of tight junction protein distribution.     

Bryostatin-1 attenuates TNF-induced epithelial barrier dysfunction: role of novel PKC isozymes

Tumor necrosis factor (TNF) increases epithelial permeability in many model systems. Protein kinase C (PKC) isozymes regulate epithelial barrier function and alter ligand-receptor interactions. We sought to define the impact of PKC on TNF-induced barrier dysfunction in T84 intestinal epithelia. TNF induced a dose- and time-dependent fall in transepithelial electrical resistance (TER) and an increase in [(3)H]mannitol flux. The TNF-induced fall in TER was not PKC mediated but was prevented by pretreatment with bryostatin-1, a PKC agonist. As demonstrated by a pattern of sensitivity to pharmacological inhibitors of PKC, this epithelial barrier preservation was mediated by novel PKC isozymes. Bryostatin-1 reduced TNF receptor (TNF-R1) surface availability, as demonstrated by radiolabeled TNF binding and cell surface biotinylation assays, and increased TNF-R1 receptor shedding. The pattern of sensitivity to isozyme-selective PKC inhibitors suggested that these effects were mediated by activation of PKC-epsilon. In addition, after bryostatin-1 treatment, PKC-delta and TNF-R1 became associated, as determined by mutual coimmunoprecipitation assay, which has been shown to lead to receptor desensitization in neutrophils. TNF-induced barrier dysfunction occurs independently of PKC, but selective modulation of novel PKC isozymes may regulate TNF-R1 signaling.     

Dose- and time-dependent effects of a novel (−)-hydroxycitric acid extract on body weight,hepatic and testicular lipid peroxidation,DNA fragmentation and histopathological data over a period of 90 days

(–)-Hydroxycitric acid (HCA), a natural extract from the dried fruit rind of Garcinia cambogia (family Guttiferae), is a popular supplement for weight management. The dried fruit rind has been used for centuries as a condiment in Southeastern Asia to make food more filling and satisfying. A significant number of studies highlight the efficacy of Super CitriMax (HCA-SX, a novel 60% calcium-potassium salt of HCA derived from Garcinia cambogia) in weight management. These studies also demonstrate that HCA-SX promotes fat oxidation, inhibits ATP-citrate lyase (a building block for fat synthesis), and lowers the level of leptin in obese subjects. Acute oral, acute dermal, primary dermal irritation and primary eye irritation toxicity studies have demonstrated the safety of HCA-SX. However, no long-term safety of HCA-SX or any other (–)-hydroxycitric acid extract has been previously assessed. In this study, we have evaluated the dose- and time-dependent effects of HCA-SX in Sprague-Dawley rats on body weight, hepatic and testicular lipid peroxidation, DNA fragmentation, liver and testis weight, expressed as such and as a % of body weight and brain weight, and histopathological changes over a period of 90 days. The animals were treated with 0, 0.2, 2.0 and 5.0% HCA-SX as feed intake and the animals were sacrificed on 30, 60 or 90 days of treatment. The feed and water intake were assessed and correlated with the reduction in body weight. HCA-SX supplementation demonstrated a reduction in body weight in both male and female rats over a period of 90 days as compared to the corresponding control animals. An advancing age-induced marginal increase in hepatic lipid peroxidation was observed in both male and female rats as compared to the corresponding control animals. However, no such difference in hepatic DNA fragmentation and testicular lipid peroxidation and DNA fragmentation was observed. Furthermore, liver and testis weight, expressed as such and as a percentage of body weight and brain weight, at 30, 60 and 90 days of treatment, exhibited no significant difference between the four groups. Taken together, these results indicate that treatment of HCA-SX over a period of 90 days results in a reduction in body weight, but did not cause any changes in hepatic and testicular lipid peroxidation, DNA fragmentation, or histopathological changes.     

Dissociation constants of protonated methionine species in NaCl media

The sulfur-containing amino acid, methionine, has a role in the physiological environment because of its strong interactions with metals. To understand these interactions of metals with methionine, one needs reliable dissociation constants for the protonated methionine species (NH(3)(+)CH(CH(2)CH(2)SCH(3))COOH; H(2)B(+)). The values of stoichiometric dissociation constants, pK(i)*, for protonated methionine species (H(2)B(+) if H(+)+HB, K(1); HB if H(+)+B(-), K(2)) were determined from potentiometric measurements in NaCl solutions as a function of ionic strength, 0.25-6.0 mol (kg H(2)O)(-1) and temperature (5-45 degrees C). The results were extrapolated to pure water using the Pitzer equations to estimate the activity of H(+), H(2)B(+), HB and B(-) as a function of ionic strength and temperature. The resulting thermodynamic values of K(1) and K(2) were fit to the equations (T/K): ln K(1)=69.0013-3496.58/(T/K)-10.9153 ln (T/K); ln K(2)=116.4162-10638.02/(T/K)-18.0553 ln (T/K) with standard errors of 0.003 and 0.033, respectively, for ln K(1)* and ln K(2)*. Pitzer interaction parameters (lambda(HB-Na) and zeta(HB-Na-Cl)) for the neutral HB were determined from literature data. The Pitzer parameters (beta(0)(H(2)BCl), beta(1)(H(2)BCl) and C(phi)(H(2)BCl)) for the interactions of H(2)B(+) with Cl(-) and Na(+) with and B(2-) (beta(0)(NaB), beta(1)(NaB) and C(phi)(NaB)) were also determined. These coefficients can be used to make reasonable estimates of the activity coefficients of methionine species and the pK(i)(*) for the dissociation of methionine in physiological solutions, composed mostly of NaCl over a wide range of temperature and ionic strength.     

Determination of nuclear receptor corepressor interactions with the thyroid hormone receptor

The thyroid hormone receptor (TR) recruits the nuclear corepressors, nuclear receptor corepressor (NCoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT), to target DNA elements in the absence of ligand. While the TR preferentially recruits NCoR, the mechanism remains unclear. The corepressors interact with the TR via interacting domains (IDs) present in their C terminus which contain a conserved motif termed a CoRNR box. Despite their similarity, the corepressor IDs allow for nuclear receptor specificity. Here we demonstrate that NCoR stabilizes the TR homodimer when bound to DNA by preventing its dissociation from thyroid hormone response elements. This suggests that NCoR acts to hold the repression complex in place on target elements. The TR homodimer recruits NCoR through two of its three IDs, one of which is not present in SMRT. This unique ID, N3, contains a CoRNR box but lacks the extended helical motif present in each of the other IDs. Instead, N3 contains an isoleucine just proximal to this motif. This isoleucine is also conserved in N2 but not in the corresponding S2 domain in SMRT. On thyroid hormone response elements and in mammalian cells this residue is critical in both N3 and N2 for high-affinity TR binding. In addition, this residue also controls specificity for the interactions of TR with NCoR. Together these data suggest that the specific recruitment of NCoR by the TR through a unique motif allows for stabilization of the repression complex on target elements.