Response Hunsberger, S.; Rubinstein, L.; Boerner, S. A. ...
JNCI : Journal of the National Cancer Institute,
07/2013, Letnik:
105, Številka:
13
Journal Article
The pyridine nucleotide 6-aminonicotinamide (6AN) was shown recently to sensitize a number of human tumor cell lines to cisplatin
in vitro. The present studies were undertaken to compare the drug ...concentrations and length of exposure required for this sensitization
in vitro with the drug exposure that could be achieved in mice
in vivo. Human K562 leukemia cells and A549 lung cancer cells were incubated with 6AN for various lengths of time, exposed to cisplatin for 1–2 hr, and assayed for Pt-DNA adducts as well as the ability to form colonies. K562 cells displayed progressive increases in Pt-DNA adducts and cisplatin sensitivity during the first 10 hr of 6AN exposure. An 18-hr 6AN exposure was likewise more effective than a 6-hr 6AN exposure in sensitizing A549 cells to cisplatin. HPLC analysis of 6AN and its metabolite, 6-amino-NAD
+, permitted assessment of exposures achieved
in vivo after i.v. administration of 10 mg/kg of 6AN to CD2F
1 mice. 6AN reached peak serum concentrations of 80–90 μM and was cleared rapidly, with T
1/2α and T
1/2β values of 7.4 and 31.3 min, respectively. Bioavailability was 80–100% with identical plasma pharmacokinetics after i.p. administration. At least 25% of the 6AN was excreted unchanged in the urine. The metabolite 6-amino-NAD
+ was detected in perchloric acid extracts of brain, liver, kidney, and spleen, but not in serum. Efforts to prolong systemic 6AN exposure by administering multiple i.p. doses or using osmotic pumps resulted in lethal toxicity. These results demonstrated that 6AN exposures required to sensitize tumor cells to cisplatin
in vitro are difficult to achieve
in vivo.
The beginning of lactation requires huge metabolic adaptations to meet increased energy demands for milk production of dairy cows. One of the adaptations is the mobilization of body reserves mainly ...from adipose tissue as reflected by increased plasma nonesterified fatty acid (NEFA) concentrations. The capacity of the liver for complete oxidation of NEFA is limited, leading to an increased formation of ketone bodies, reesterification, and accumulation of triglycerides in the liver. As the skeletal muscle also may oxidize fatty acids, it may help to decrease the fatty acid load on the liver. To test this hypothesis, 19 German Holstein cows were weekly blood sampled from 7wk before until 5wk after parturition to analyze plasma NEFA concentrations. Liver biopsies were obtained at d 3, 18, and 30 after parturition and, based on the mean liver fat content, cows were grouped to the 10 highest (HI) and 9 lowest (LO). In addition, muscle biopsies were obtained at d −17, 3, and 30 relative to parturition and used to quantify mRNA abundance of genes involved in fatty acid degradation. Plasma NEFA concentrations peaked after parturition and were 1.5-fold higher in HI than LO cows. Muscle carnitine palmitoyltransferase 1α and β mRNA was upregulated in early lactation. The mRNA abundance of muscle peroxisome proliferator-activated receptor γ (PPARG) increased in early lactation and was higher in HI than in LO cows, whereas the abundance of PPARA continuously decreased after parturition. The mRNA abundance of muscle PPARD, uncoupling protein 3, and the β-oxidative enzymes 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase, very long-chain acyl-CoA dehydrogenase, and 3-ketoacyl-CoA was greatest at d 3 after parturition, whereas the abundance of PPARγ coactivator 1α decreased after parturition. Our results indicate that around parturition, oxidation of fatty acids in skeletal muscle is highly activated, which may contribute to diminish the fatty acid load on the liver. The decline in muscle fatty acid oxidation within the first 4wk of lactation accompanied with increased feed intake refer to greater supply of ruminally derived acetate, which as the preferred fuel of the muscle, saves long-chain fatty acids for milk fat production.
The liver plays a central role in adaptation for energy requirements around calving, and changes in the effects of insulin on hepatic energy metabolism contribute to metabolic adaptation in dairy ...cows. Hepatic insulin effects may depend on body fat mobilization. The objective of this study was to investigate the effects of insulin on the hepatic gene expression of enzymes involved in energy metabolism and factors related to nutrition partitioning in cows with high and low total liver fat concentration (LFC) after calving. Holstein cows were retrospectively grouped according to their LFC after calving as a proxy for body fat mobilization. Cows were classified as low (LLFC; LFC <24% fat/dry matter; n = 9) and high (HLFC; LFC >24.4% fat/dry matter; n = 10) fat-mobilizing after calving. Euglycemic-hyperinsulinemic clamps 6 mU/(kg × min) of insulin for 6 h were performed in wk 5 antepartum (ap) and wk 3 postpartum (pp). Before and at the end of the euglycemic-hyperinsulinemic clamps, liver biopsies were taken to measure the mRNA abundance of enzymes involved in carbohydrate and lipid metabolism, expression related to the somatotropic axis, and adrenergic and glucocorticoid receptors. The mRNA abundance of pyruvate carboxylase, cytosolic phosphoenolpyruvate carboxykinase (PEPCK; PCK1), acyl-CoA-dehydrogenase very long chain (ACADVL), and hydroxyl-methyl-glutaryl-CoA-synthase 1 increased, but the mRNA abundance of solute carrier family 2 (SLC2A2 and SLC2A4), growth hormone receptor 1A (GHR1A), insulin-like growth factor 1 (IGF1), sterol regulatory element binding factor 1, adrenoceptor α 1A, and glucocorticoid receptor decreased from ap to pp. Insulin treatment was associated with decreased PCK1, mitochondrial PEPCK, glucose-6-phosphatase, propionyl-CoA-carboxylase α, carnitine-palmitoyl-transferase 1A, ACADVL, and insulin receptor mRNA, but increased IGF1 and SLC2A4 mRNA ap and pp and GHR1A mRNA pp. The mRNA abundance of SLC2A4 was greater, and the mRNA abundance of GHR1A and IGF1 tended to be lower in LLFC than in HLFC. Administration of insulin, albeit at a supraphysiological dose, was associated with inhibition of gene expression related to glucose production and β-oxidation, but we observed variable effects in the degree of insulin depression of individual genes. Insulin status is important for regulation of nutrient partitioning, but different LFC pp had very little influence on changes in hepatic gene expression following administration of insulin.
High performing dairy cows experience distinct metabolic stress during periods of negative energy balance. Subclinical disorders of the cow’s energy metabolism facilitate failure of adaptational ...responses resulting in health problems and reduced performance. The autonomic nervous system (ANS) with its sympathetic and parasympathetic branches plays a predominant role in adaption to inadequate energy and/or fuel availability and mediation of the stress response. Therefore, we hypothesize that indices of heart rate variability (HRV) that reflect ANS activity and sympatho-vagal balance could be early markers of metabolic stress, and possibly useful to predict cows with compromised regulatory capacity. In this study we analysed the autonomic regulation and stress level of 10 pregnant dried-off German Holstein cows before, during and after a 10-h fasting period by using a wide range of HRV parameters. In addition heat production (HP), energy balance, feed intake, rumen fermentative activity, physical activity, non-esterified fatty acids, β-hydroxybutyric acid, cortisol and total ghrelin plasma concentrations, and body temperature (BT) were measured. In all cows fasting induced immediate regulatory adjustments including increased lipolysis (84%) and total ghrelin levels (179%), reduction of HP (−16%), standing time (−38%) and heart rate (−15%). However, by analysing frequency domain parameters of HRV (high-frequency (HF) and low-frequency (LF) components, ratio LF/HF) cows could be retrospectively assigned to groups reacting to food removal with increased or decreased activity of the parasympathetic branch of the ANS. Regression analysis reveals that under control conditions (feeding ad libitum) group differences were best predicted by the nonlinear domain HRV component Maxline (L
MAX, R
2=0.76, threshold; TS=258). Compared with cows having L
MAX values above TS (>L
MAX: 348±17), those with L
MAX values below TS (<L
MAX: 109±26) had higher basal blood cortisol levels, lower concentrations of insulin, and respond to fasting with a shift of their sympatho-vagal balance towards a much stronger dominance of the sympathetic branch of the ANS and development of stress-induced hyperthermia. The data indicate a higher stress level, reduced well-being and restricted regulatory capacity in <L
MAX cows. This assumption is in accord with the lower dry matter intake and energy corrected milk yield (16.0±0.7 and 42±2 kg/day) in lactating <L
MAX compared with >L
MAX cows (18.5±0.4 and 47.3 kg/day). From the present study, it seems conceivable that L
MAX can be used as a predictive marker to discover alterations in central autonomic regulation that might precede metabolic disturbances.
A number of methods are commonly employed for the determination of protein in biological samples. Unfortunately, several compounds that are constituents of biological buffers interfere with these ...methods, limiting their application. Previous studies have demonstrated that tyrosine rapidly undergoes nitration in nitric acid to yield 3-nitrotyrosine, which has a λmaxof 358 nm. Utilizing this reaction, we have developed a one-step method for the assessment of protein content in biological samples. Common interfering substances, including SDS, urea, glycerol, ammonium sulfate, and β-mercaptoethanol, do not interfere with this method. Because of its simplicity, this reaction might be useful for estimating protein content in a variety of biological samples.
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