Comb-like oligoaminoethane carriers: change in topology improves pDNA delivery.

Establishing precise structure-activity relationships is important for the optimization of synthetic carriers for gene delivery. Sequence-defined oligomers with branched or linear shapes were synthesized to investigate the influence of topology on their biophysical properties and biological performance. Comb-like structures were synthesized consisting of an oligolysine peptide backbone modified at the ε-amino groups with four different artificial oligoamino acids, succinyl-diethylene triamine (Sdt), succinyl-triethylene tetramine (Stt), succinyl-tetraethylene pentamine (Stp), and succinyl-pentaethylene hexamine (Sph). Optionally the amino acids histidine and alanine were inserted into the oligolysine backbone to assess a possible buffer or spacer effect. After the evaluation of biophysical properties, the best performing oligomers, containing the Stp or Sph building blocks, were compared to corresponding linear oligomers where Stp or Sph are directly integrated into the linear oligolysine row. Clear differences between the comb and linear carriers were observed in the comparison of properties such as DNA complexation ability, buffer capacity, cellular association and internalization, and gene transfer. For the Stp containing structures, the comb topology mediated an increased buffer capacity at endosomal pH. For the Sph containing structures, in sharp contrast, the linear topology displayed advantageous endosomal buffering. Interestingly, for both Stp and Sph carriers, the comb in comparison to the linear topologies mediated a higher overall cellular uptake despite a lower cell association. For Stp combs, the combined advantage in both buffering and cellular uptake resulted in a strong (10- to >100-fold) increase in DNA transfection efficiency. In the case of Sph carriers, comb topology mediated only moderately (maximum 4-fold) enhanced gene transfer over the linear topology.

Zoonotic bacterial populations, gut fermentation characteristics and methane production in feedlot steers during oral nitroethane treatment and after the feeding of an experimental chlorate product.

Nitroethane inhibits the growth of certain zoonotic pathogens such as Campylobacter and Salmonella spp., foodborne pathogens estimated to cause millions of human infections each year, and enhances the Salmonella- and Escherichia coli-killing effect of an experimental chlorate product being developed as a feed additive to kill these bacteria immediately pre-harvest. Limited studies have shown that nitroethane inhibits ruminal methane production, which represents a loss of 2-12% of the host's gross energy intake and contributes to global warming and destruction of the ozone layer. The present study was conducted to assess the effects of 14-day oral nitroethane administration, 0 (0X), 80 (1X) or 160 (2X)mg nitroethane/kg body weight per day on ruminal and fecal E. coli and Campylobacter, ruminal and fecal methane-producing and nitroethane-reducing activity, whole animal methane emissions, and ruminal and fecal fermentation balance in Holstein steers (n=6 per treatment) averaging 403+/-26 (SD) kg BW. An experimental chlorate product was fed the day following the last nitroethane administration to determine effects on E. coli and Campylobacter. The experimental chlorate product decreased (P<0.001) fecal, but not ruminal (P>0.05) E. coli concentrations by 1000- and 10-fold by 24 and 48 h, respectively, after chlorate feeding when compared to pre-treatment concentrations (>5.7 log(10) colony forming units/g). No effects (P>0.05) of nitroethane or the experimental chlorate product were observed on fecal Campylobacter concentrations; Campylobacter were not recovered from ruminal contents. Nitroethane treatment decreased (P<0.01) ruminal (8.46, 7.91 and 4.74+/-0.78 micromol/g/h) and fecal (3.90, 1.36 and 1.38+/-0.50 micromol/g/h) methane-producing activity for treatments 0X, 1X and 2X, respectively. Administration of nitroethane increased (P<0.001) nitroethane-reducing activity in ruminal, but not fecal samples. Day of study affected ruminal (P<0.0001) but not fecal (P>0.05) methane-producing and nitroethane-reducing activities (P<0.01); treatment by day interactions were not observed (P>0.05). Ruminal accumulations of acetate decreased (P<0.05) in 2X-treated steers when compared with 0X- and 1X-treated steers, but no effect (P>0.05) of nitroethane was observed on propionate, butyrate or the acetate to propionate ratio. Whole animal methane emissions, expressed as L/day or as a proportion of gross energy intake (%GEI), were unaffected by nitroethane treatment (P>0.05), and were not correlated (P>0.05) with ruminal methane-producing activity. These results demonstrate that oral nitroethane administration reduces ruminal methane-producing activity but suggest that a microbial adaptation, likely due to an in situ enrichment of ruminal nitroethane-reducing bacteria, may cause depletion of nitroethane, at least at the 1X administration dose, to concentrations too low to be effective. Further research is warranted to determine if the optimization of dosage of nitroethane or related nitrocompouds can maintain the enteropathogen control and anti-methanogen effect in fed steers.

An indirect screen for brain uptake of 1,2-diarylethane melanocortin 4 receptor antagonists in rats.

Antagonism of the melanocortin 4 receptor (MC4R) has been proposed as a therapeutic intervention for the prevention of lean body mass waste, as in cachexia. Pharmacokinetic profiles of substituted 1,2-diarylethane MC4R antagonists were determined in rats after a single intravenous (IV) administration at 1 mg/kg. Brain and plasma concentrations of these compounds were determined at 1 and 4 hours after an oral dose at 10 mg/kg, since oral administration is the intended clinical dosing route and the pharmacological target is the central nervous system. The brain to plasma concentration ratios (0.10 - 50) after oral dosing correlated well with Vd(ss) (2.21 to 81.4 L/kg; R(2)=0.810) determined after IV administration. A good correlation was also observed between the brain AUC(0-4 hr) (119 - 18400 nM*hr) and Vd(ss) (R(2)=0.981). Thus, further screening and ranking of substituted 1,2-diarylethanes for their brain uptakes could be carried out more efficiently via the simple and indirect Vd(ss) screen after intravenous administration in rats.

Low level nitrate or nitroethane preconditioning enhances the bactericidal effect of suboptimal experimental chlorate treatment against Escherichia coli and Salmonella Typhimurium but not Campylobacter in swine.

An experimental chlorate product that targets the respiratory nitrate reductase enzyme of bacteria such as Salmonella and Escherichia coli has shown promising results in reducing concentrations of these bacteria in the gut of food animals. Because expression of the target enzyme is induced by nitrate, we administered short-duration, low level nitrate or nitroethane preconditioning treatments to finishing swine to see if these would enhance the ability of an experimental chlorate product to kill these bacteria. Results from these studies showed that preconditioning the gut microflora of swine with low levels of nitrate or nitrocompounds enhanced (more than tenfold) the ability of the chlorate product to kill Salmonella and E. coli, but not Campylobacter. Further studies are needed before these compounds can be fed as feed additives to animals, although it is likely that nitrate preconditioning may be more near to market than the nitrocompounds, which may require more comprehensive review by regulatory authorities.

NTP technical report on the toxicity studies of 1,1,2,2-tetrachloroethane (CAS No. 79-34-5) administered in microcapsules in feed to F344/N rats and B6C3F1 mice.

BACKGROUND: 1,1 ,2,2-Tetrachloroethane was widely used in the production of solvents and pesticides. Its production ended in the 1990s, but it is a major component of waste sites. We studied the effects of 1,1 ,2,2-tetrachloroethane on male and female rats and mice to identify potential toxic hazards to humans. METHODS: Because 1,1,2,2-tetrachloroethane can evaporate easily, we enclosed it in starch microcapsules and placed them in the feed of rats and mice for 14 weeks. Male and female rats received up to 4,600 parts per million (ppm) 1,1 ,2,2-tetrachloroethane (equivalent to 0.46%) and mice received up to 9,100 ppm (0.91%). Control animals received empty starch microcapsules in their feed. Tissues from more than 40 sites were examined in all control and high-dose animals; tissues with lesions were examined in the lower exposure groups until no lesions were observed. RESULTS: Rats receiving 1,180 ppm or more 1,1,2,2-tetrachloroethane and mice receiving 2,300 ppm or more weighed less than the control animals. Male and female rats given 1,1 ,2,2-tetrachloroethane had pale and diseased livers and also had atrophy of the bone marrow and of the genital systems. Male and female mice given 1,1,2,2-tetrachloroethane had lesions of the liver and the bile duct. CONCLUSION: We conclude that 1,1,2,2-tetrachloroethane at doses greater than 590 ppm in the feed was toxic to the liver of male and female rats. In mice, 1,1 ,2,2-tetrachloroethane was already known to cause cancer after long-term exposure. In these 14-week studies, 1,1 ,2,2-tetrachloroethane was toxic to the livers of male and female mice.

Cooling efficiency of cryogen spray during laser therapy of skin.

BACKGROUND AND OBJECTIVES: Cryogen spray cooling (CSC) is used extensively for epidermal protection during laser-induced photothermolysis of port wine stains and other vascular skin lesions. The efficacy of CSC depends critically on the heat transfer coefficient (H) at the skin surface for which, however, no reliable values exist. Reported values for H, based on tissue phantoms, vary from 1,600 to 60,000 W/m(2) K. STUDY DESIGN/MATERIALS AND METHODS: A simple experimental model was designed and constructed, consisting of a pure silver-measuring disk (diameter 10 mm, thickness approximately 1 mm), embedded in a thermal insulator. The disk was covered with a 10 microm thick stratum corneum layer, detached from in vivo human skin. The heat transfer coefficient of the stratum corneum/cryogen interface was measured during CSC with short spurts of atomized tetrafluoroethane. RESULTS: H was found to be dependent on the specific design of the cryogen valve and nozzle. With nozzles used in typical clinical settings, H was 11,500 W/m(2) K, when averaged over a 100 ms spurt, and 8,000 W/m(2) K when averaged over a 200 ms spurt. CONCLUSIONS: The presented model enables accurate prediction of H and thus improve control over temperature depth profile and cooling efficiency during laser therapy. Thereby, it may contribute to improvement of therapeutic outcome.

Dose-dependent protein adduct formation in kidney, liver, and blood of rats and in human blood after perchloroethene inhalation.

Perchloroethene (PER) was a widely used solvent and is an environmental contaminant. In bioassays for carcinogenicity, PER was found to increase the incidence of liver tumors in mice and of renal tumors in male rats. Toxic effects of PER after repeated administration are likely caused by bioactivation. PER bioactivation occurs by two pathways. Oxidation by cytochrome P450 results in trichloroacetyl chloride, which binds to lipids and proteins. Glutathione S-conjugate formation from PER and further processing of the formed S-(trichlorovinyl)glutathione to S-(trichlorovinyl)-L-cysteine, followed by cysteine conjugate beta-lyase catalyzed cleavage, resulted in the reactive dichlorothioketene, which binds to proteins under formation of N epsilon-(dichloroacetyl)-L-lysine in proteins. The objective of this study was to comparatively quantify the dose-dependent formation of protein adducts from PER in rats and humans using antibodies with high specificity for either N epsilon-(trichloroacetyl)-L-lysine or N epsilon-(dichloroacetyl)-L-lysine in proteins. Male and female rats (n = 2, per concentration and time point) were exposed to 400, 40, and 10 ppm PER for 6 h and killed at various time points. Formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was comparatively quantified in subcellular fractions from liver and kidney and in blood. In addition, three male and three female human volunteers were exposed to 10 and 40 ppm PER, and formation of protein adducts in blood was analyzed using the antibodies and GC/MS after immunoaffinity enrichment of modified proteins. In liver and kidney subcellular fractions and blood of PER-exposed rats, dose-dependent formation of N epsilon-(dichloroacetyl)-L-lysine and N epsilon-(trichloroacetyl)-L-lysine in proteins was observed. Highest concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were formed in kidney mitochondria, followed by kidney cytosol. Only low concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were present in liver proteins; blood concentrations of N epsilon-(dichloroacetyl)-L-lysine in proteins were 5 to 10 fold lower than in kidney mitochondria. Highest concentrations of N epsilon-(trichloroacetyl)-L-lysine were found in microsomal and cytosolic proteins from the liver of rats exposed to PER. A higher protein adduct formation was seen in PER-exposed-male than -female rats for N epsilon-(dichloroacetyl)-L-lysine in renal mitochondrial proteins, after exposure to 400 ppm PER. In human blood samples taken 0 and 24 h after the 6 h exposures to PER, N epsilon-(trichloroacetyl)-L-lysine-containing proteins were present in low concentrations. N epsilon-(Dichloroacetyl)-L-lysine-containing proteins were not detected either by Western blotting or GC/MS after immunoaffinity chromatography. The obtained results indicate a dose-dependent covalent binding of PER metabolites to proteins in rat liver, kidney, and blood and suggest that the concentration of covalent protein adducts is much lower in blood of humans as compared to the blood of rats exposed under identical conditions.

Role of molecular diffusion in conventional and high frequency ventilation.

The influence of molecular diffusion on gas-mixing during conventional mechanical ventilation (CMV) and high frequency ventilation (HFV) was studied by observing the wash-in of six poorly soluble, inert gases in arterial blood. Anesthetized dogs were ventilated either with CMV or HFV. Following a step change in inspired gas composition, the increase in arterial concentrations of hydrogen, helium, methane, ethane, isobutane, and sulfur hexafluoride was determined by gas chromatography. The relative gas diffusivities encompassed a range of almost one order of magnitude. Propane, present in inspired gas during both the control and wash-in phases, served as an internal reference for calculation of blood tracer concentrations. The wash-in of all six inert gases followed a single exponential time course during both CMV and HFV. The rate of wash-in of each gas decreased with increasing molecular weight (MW). The relationship of rate constants to a measure of relative diffusivity (MW-0.5) was significantly different than zero for both types of ventilation. The slope of this relationship was three times larger for CMV than HFV, indicating that molecular diffusion has a greater role in gas mixing during ventilation with large tidal volumes. Diffusion has a minor role in gas mixing during high frequency ventilation with small tidal volumes. Demonstration of the presence of gas separation secondary to molecular diffusion during HFV is enhanced by measuring wash-in, rather than wash-out, of inert gases because gas separation is likely to be obscured as exhaled gases pass through the well-mixed central airways during gas wash-out.

Enhanced degradation of 3-nitropropanol by ruminal microorganisms.

Ruminal fluid was obtained over a 4-yr period from cattle on various diets and supplements to determine the effects of different inocula on the microbial degradation of 3-nitropropanol (NPOH), a toxic metabolite in certain Astragalus spp. (Leguminosae). Nitrite (NO2-) metabolism was also studied in vitro because rapid NO2- reduction is required for the overall detoxification of NPOH. Intra-ruminal supplements of sulfite were ineffective and produced toxic signs in treated animals. Ruminal fluid from cattle on fresh pasture diets enhanced the in vitro metabolism of NO2-, but rates of NPOH disappearance were not significantly affected. Rates of NPOH degradation increased when orchardgrass pasturage was supplemented with molasses. Enhancement of NPOH degradation was achieved with supplements of nitroethane given intra-ruminally at 6.5 or 10 mg/kg body weight. The effect of nitroethane on NO2- reduction was not always observed, but the NO2- rates of metabolism always exceeded those of NPOH. The rate of NPOH degradation also increased when nitroethane was added to a molasses supplement. However, the volatility of nitroethane under field conditions prompted a search for a more stable inducer and the sodium salt of nitroethane was subsequently evaluated. The salt of nitroethane, given intra-ruminally at 20 mg nitroethane/kg body weight, resulted in the highest rate of NPOH degradation; this was similar to that reported for 3-nitropropionic acid, a nitroalkane that is much less toxic to ruminants than NPOH.

Comparison of acute toxic effects of intraperitoneally injected nitromethane and nitroethane in rats.

Male 3-month-old Wistar rats dosed i.p. with 200 mg/kg of nitromethane or -ethane showed increased acid proteinase activity in the brain 4 h after the injection. The change was accompanied by a marginal increase in the cerebral glutathione concentration. Nitroethane caused enhanced epoxide hydrolase and UDP-glucuronosyltransferase activity in the hepatic microsomal fraction up to 48 h while 7-ethoxycoumarin o-deethylase decreased. These biochemical changes were accompanied by proliferation of smooth endoplasmic reticulum and degranulation and disorganization of the rough endoplasmic reticulum of the nitroethane-exposed liver cells. The hepatic effects of nitromethane were restricted to decreased cytochrome c reductase activity with proliferation of smooth endoplasmic reticulum. The results point at limited peroxidative damage possibly involving reduction of the nitrogroup.