Cell-Penetrating D-Peptides Retain Antisense Morpholino Oligomer Delivery Activity

Cell-penetrating peptides (CPPs) can cross the cell membrane to enter the cytosol and deliver otherwise non-penetrant macromolecules such as proteins and oligonucleotides. For example, recent clinical trials have shown that a CPP attached to phosphorodiamidate morpholino oligomers (PMO) resulted in higher muscle concentration, increased exon-skipping and dystrophin production relative to another study of the PMO alone in patients of Duchenne muscular dystrophy. Therefore, effective design and study of CPPs could help enhance therapies for difficult-to-treat diseases. So far, the study of CPPs for PMO delivery has been restricted to predominantly canonical L-peptides. We hypothesized that mirror-image D-peptides could have similar PMO delivery activity as well as enhanced proteolytic stability, facilitating their characterization and quantification from biological milieu. We found that several enantiomeric peptide sequences could deliver a PMO-biotin cargo with similar activities, while remaining stable against serum proteolysis. The biotin label allowed for affinity capture of fully intact PMO-peptide conjugates from whole cell and cytosolic lysates. By profiling a mixture of these constructs in cells, we determined their relative intracellular concentrations. When combined with PMO activity, these concentrations provide a new metric for delivery efficiency which may be useful for determining which peptide sequence to pursue in further pre-clinical studies.Abstract Figure

Antioxidant Status in the Soleus Muscle of Sprague-Dawley Rats in Relation to Duodenal-Jejunal Omega Switch and Different Dietary Patterns

Background. Obesity and chronic ingestion of lipid-rich meals are related to an enhanced oxidative stress (OS). Aims. To examine the influence of duodenal-jejunal omega switch surgery in combination with different diets on the antioxidative status in the soleus muscle of rats. Methods. After 8 weeks on a high-fat diet (HF) or control diet (CD), rats underwent duodenal-jejunal omega switch (DJOS) or SHAM (control) surgery. After surgery, for the next 8 weeks, half of DJOS/SHAM animals were kept on the same diet as before, and half had a changed diet. The total superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR) activity as well as malondialdehyde (MDA) concentration were measured in the soleus of rats. Results. CAT and GPx activity were significantly lower after DJOS surgery versus SHAM, regardless of the type of diet. The activity of CAT, SOD, GR, CuZnSOD, and GPx was altered in the CD/HF or HF/CD groups. After DJOS, the lowest muscle concentration of MDA was observed in the CD/CD group and the highest in CD/HF. Conclusions. DJOS surgery significantly decreases the antioxidative system in soleus muscles of rats. CD/HF and HF/CD dietary patterns lead to an increase in antioxidative activity, while remaining on unchanged diet (CD or HF) is associated with a reduced oxidative stress.

Residue Depletion Study of Danofloxacin in Cultured Tilapia (Oreochromis mossambicus)

Danofloxacin is an antibacterial drug of the fluoroquinolone group developed for therapeutic purposes in veterinary medicine. The studies described here include investigations of the residues following a single dose or multiple doses of danofloxacin. Residue depletion studies were performed to determine residues in plasma and tissues of saltwater tilapia fish (Oreochromis mossambicus) after a single oral administration of danofloxacin at the dose of 10 mg/kg body weight and also after daily dose of 10 mg/kg body weight for five consecutive days. Danofloxacin residues were analyzed by HPLC with fluorescence detection. Following a single oral dose, danofloxacin residues in 6 h postdosing tilapia were at a maximum of 1.44, 12.48, and 13.18 μg/g in serum, liver, and kidney samples, respectively, while a peak muscle concentration of 2.15 μg/g was reached at 12 h. From single-dose data, the concentration of danofloxacin in serum, muscle, liver, and kidney samples declined with half-lives of 29, 34, 49, and 44 h, respectively. Based on the maximum residue level (MRL) of 0.1 μg/g in edible tissue for fin fish, the withdrawal times of danofloxacin in muscle were estimated to fall below the MRL after a withdrawal period of 21 days following the multiple-dose administration. These results may be helpful to regulatory agencies as they determine what tissues should be monitored to ensure that the established residue safety tolerance levels are not exceeded.

Disruption of BCAA metabolism in mice impairs exercise metabolism and endurance

Exercise enhances branched-chain amino acid (BCAA) catabolism, and BCAA supplementation influences exercise metabolism. However, it remains controversial whether BCAA supplementation improves exercise endurance, and unknown whether the exercise endurance effect of BCAA supplementation requires catabolism of these amino acids. Therefore, we examined exercise capacity and intermediary metabolism in skeletal muscle of knockout (KO) mice of mitochondrial branched-chain aminotransferase (BCATm), which catalyzes the first step of BCAA catabolism. We found that BCATm KO mice were exercise intolerant with markedly decreased endurance to exhaustion. Their plasma lactate and lactate-to-pyruvate ratio in skeletal muscle during exercise and lactate release from hindlimb perfused with high concentrations of insulin and glucose were significantly higher in KO than wild-type (WT) mice. Plasma and muscle ammonia concentrations were also markedly higher in KO than WT mice during a brief bout of exercise. BCATm KO mice exhibited 43–79% declines in the muscle concentration of alanine, glutamine, aspartate, and glutamate at rest and during exercise. In response to exercise, the increments in muscle malate and α-ketoglutarate were greater in KO than WT mice. While muscle ATP concentration tended to be lower, muscle IMP concentration was sevenfold higher in KO compared with WT mice after a brief bout of exercise, suggesting elevated ammonia in KO is derived from the purine nucleotide cycle. These data suggest that disruption of BCAA transamination causes impaired malate/aspartate shuttle, thereby resulting in decreased alanine and glutamine formation, as well as increases in lactate-to-pyruvate ratio and ammonia in skeletal muscle. Thus BCAA metabolism may regulate exercise capacity in mice.

Near-infrared spectroscopy and indocyanine green derived blood flow index for noninvasive measurement of muscle perfusion during exercise

Near-infrared spectroscopy (NIRS) with the tracer indocyanine green (ICG) may be used for measuring muscle blood flow (MBF) during exercise, if arterial ICG concentration is measured simultaneously. Although pulse dye densitometry allows for noninvasive measurement of arterial dye concentration, this technique is sensitive to motion and may not be applicable during exercise. The aim of this study was to evaluate a noninvasive blood flow index (BFI), which is derived solely from the muscle ICG concentration curve. In 10 male cyclists 5 mg ICG were injected into an antecubital vein at rest and during cycling at 30, 60, 70, 80, 90, and 100% of previously determined maximal work load. Simultaneously blood was withdrawn through a photodensitometer at 20 ml/min from the radial artery to measure arterial ICG concentration. To measure muscle tissue ICG concentrations, two sets of NIRS optodes were positioned on the skin, one over the left seventh intercostal space and the other over the left vastus lateralis muscle. MBF was calculated from the arterial and muscle concentration data according to Fick's principle. BFI was calculated solely from the muscle concentration curve as ICG concentration difference divided by rise time between 10 and 90% of peak. During exercise mean BFI values changed similarly to MBF in both intercostal and quadriceps muscles and showed excellent correlations with MBF: r = 0.98 and 0.96, respectively. Individual data showed some scattering among BFI and MBF values but still reasonable correlations of BFI with MBF: r = 0.73 and 0.72 for intercostal and quadriceps muscles, respectively. Interobserver variability, as analyzed by Bland-Altman plots, was considerably less for BFI than MBF. These data suggest that BFI can be used for measuring changes in muscle perfusion from rest to maximal exercise. Although absolute blood flow cannot be determined, BFI has the advantages of being essentially noninvasive and having low interobserver variability.

Sodium fluoroacetate residues and carcass degradation of free-ranging feral pigs poisoned with 1080

Sodium fluoroacetate (1080) residues in muscle and liver of free-ranging feral pigs, poisoned with 1080-treated grain in a range of habitats, were determined. The incidence of vomiting, and the degradation of poisoned carcasses were also monitored. The maximum recorded concentrations in muscle (n = 79) and liver (n = 16) were 2.42 and 4.28 µg g–1 tissue, respectively. Mean (±s.d.) concentrations were 0.702 ± 0.535 and 0.635 ± 1.091 µg g–1, respectively. Muscle concentration in pigs sampled within 24 h of death were similar between those pigs poisoned with wheat (0.993 µg g–1, n = 21) and malted barley (1.012 µg g–1, n = 20) (P > 0.05), but muscle residues may have been lower in those pigs poisoned with lupin bait (0.178 µg g–1, n = 3). Muscle concentrations were also lower in those pigs sampled 24–48 h after death (0.481 µg g–1, n = 13) (P = 0.004). There were no differences between the sexes (northern rangeland: mean, females 0.883, males 0.869 µg g–1; agricultural: mean, 0.420 and 0.324 µg g–1) (P > 0.05), but adult pigs had lower muscle concentrations than did non-adult pigs (P < 0.001). There was no evidence of vomiting by any recovered poisoned pigs (n = 85), and all but one stomach contained substantial amounts of bait and other foods. Scavengers (mainly raptors) rapidly consumed poisoned pigs weighing <16 kg, within 2 days with no apparent ill-effects. Poisoned adults (≥25 kg) were scavenged less frequently but, because of microbial action and the activity of invertebrates (e.g. fly larvae), these pigs were degraded within 7–10 days (i.e. no longer represented a potential food source for vertebrates). The levels of residues recorded were such that 1080-poisoned pig carcasses pose little potential risk to the long-term viability of non-target species.

BCAA intake affects protein metabolism in muscle after but not during exercise in humans

Branched-chain amino acids (BCAA) or a placebo was given to seven subjects during 1 h of ergometer cycle exercise and a 2-h recovery period. Intake of BCAA did not influence the rate of exchange of the aromatic amino acids, tyrosine and phenylalanine, in the legs during exercise or the increase in their concentration in muscle. The increase was ∼30% in both conditions. On the other hand, in the recovery period after exercise, a faster decrease in the muscle concentration of aromatic amino acids was found in the BCAA experiment (46% compared with 25% in the placebo condition). There was also a tendency to a smaller release (an average of 32%) of these amino acids from the legs during the 2-h recovery. The results suggest that BCAA have a protein-sparing effect during the recovery after exercise, either that protein synthesis has been stimulated and/or protein degradation has decreased, but the data during exercise are too variable to make any conclusions about the effects during exercise. The effect in the recovery period does not seem to be mediated by insulin.

Lower calorie intake enhances muscle insulin action and reduces hexosamine levels

Previous studies have demonstrated enhanced insulin sensitivity in calorie-restricted [CR, fed 60% ad libitum (AL) one time daily] compared with AL-fed rats. To evaluate the effects of reduced food intake, independent of temporal differences in consumption, we studied AL (unlimited food access)-fed and CR (fed one time daily) rats along with groups temporally matched for feeding [fed 3 meals (M) daily]: MAL and MCR, eating 100 and 60% of AL intake, respectively. Insulin-stimulated glucose transport by isolated muscle was increased in MCR and CR vs. AL and MAL; there was no significant difference for MCR vs. CR or MAL vs. AL. Intramuscular triglyceride concentration, which is inversely related to insulin sensitivity in some conditions, did not differ among groups. Muscle concentration of UDP- N-acetylhexosamines [end products of the hexosamine biosynthetic pathway (HBP)] was lower in MCR vs. MAL despite unaltered glutamine-fructose-6-phosphate aminotransferase activity (rate-limiting enzyme for HBP). These results indicate that the CR-induced increase in insulin-stimulated glucose transport in muscle is attributable to an altered amount, not timing, of food intake and is independent of lower triglyceride concentration. They further suggest that enhanced insulin action might involve changes in HBP.

Penciclovir Pharmacokinetics and Distribution to the Brain and Muscle of Rats, Studied by Microdialysis

Famciclovir, the oral form of penciclovir, is a potent, highly selective antiherpesvirus agent licenced for the treatment of herpes zoster (shingles). Some herpesviruses are prone to infect the central nervous system. To obtain guidance for the possible treatment of herpes encephalitis it is important to study the extent of transport of antiviral agents into the brain. We have used microdialysis to sample the unbound extracellular concentration of penciclovir in the gastrocnemic muscle (which corresponds directly to plasma free concentrations) and in the brain of rats under halothane anaesthesia. Penciclovir (50 mg kg−1) was given intravenously (i.v.) and samples were taken for 5 h after administration. The AUC (area under the time versus concentration curve) (0–5 h) of penciclovir in the brain was 0.096±0.018 (mean±SEM) of the AUC in muscle while the mean ratio of brain to muscle concentration 5 h post-injection was 0.1 80±0.084. Famciclovir given per os to rat at a dose of 1 20 mg kg−1 resulted in a concentration ratio for penciclovir between brain and muscle of 0.415±0.078 at 5 h after administration, while the AUC ratio (0–5 h) was 0.143±0.012. Both of these are higher than after i.v. injection of penciclovir. Penciclovir and famciclovir were also administrated by i.v. infusion (60 and 80 mg kg−1 h−1 respectively). Famciclovir administration (AUC 0.075±0.025 mmol h L−1) did not increase penciclovir transport to the brain compared with penciclovir administration (AUC 0.163±0.018 mmol h L−1).