Dominant and sensitive control of oxidative flux by the ATP-ADP carrier in human skeletal muscle mitochondria: Effect of lysine acetylation.

The adenine nucleotide translocase (ANT) of the mitochondrial inner membrane exchanges ADP for ATP. Mitochondria were isolated from human vastus lateralis muscle (n = 9). Carboxyatractyloside titration of O2 consumption rate (Jo) at clamped [ADP] of 21 µM gave ANT abundance of 0.97 ±â€¯0.14 nmol ANT/mg and a flux control coefficient of 82% ±â€¯6%. Flux control fell to 1% ±â€¯1% at saturating (2 mM) [ADP]. The KmADP for Jo was 32.4 ±â€¯1.8 µM. In terms of the free (-3) ADP anion this KmADP was 12.0 ±â€¯0.7 µM. A novel luciferase-based assay for ATP production gave KmADP of 13.1 ±â€¯1.9 µM in the absence of ATP competition. The free anion KmADP in this case was 2.0 ±â€¯0.3 µM. Targeted proteomic analyses showed significant acetylation of ANT Lysine23 and that ANT1 was the most abundant isoform. Acetylation of Lysine23 correlated positively with KmADP, r = 0.74, P = 0.022. The findings underscore the central role played by ANT in the control of oxidative phosphorylation, particularly at the energy phosphate levels associated with low ATP demand. As predicted by molecular dynamic modeling, ANT Lysine23 acetylation decreased the apparent affinity of ADP for ANT binding.

Role of adipocyte mitochondria in inflammation, lipemia and insulin sensitivity in humans: effects of pioglitazone treatment.

BACKGROUND/OBJECTIVES: To gain further insight into the role of adipocyte mitochondria in systemic lipid metabolism, inflammation and insulin sensitivity in humans and to provide a better understanding of the mechanisms of action of the peroxisome proliferator-activated receptor gamma agonist pioglitazone. SUBJECTS/METHODS: Mitochondrial DNA (mtDNA) copy number, mitochondrial distribution, mitochondrial and overall cellular protein abundances as well as intrinsic mitochondrial function of subcutaneous adipocytes were assessed by real-time quantitative PCR, MitoTracker staining, global proteomics analyses and NADH cytochrome c reductase activity in insulin-sensitive, normal-glucose-tolerant (NGT) individuals and age, gender, adiposity-matched insulin-resistant individuals with abnormal glucose tolerant (AGT) before and after 3 months of pioglitazone treatment. RESULTS: mtDNA copy number/adipocyte and mtDNA copy number/adipocyte volume were ~55% and ~4-fold lower in AGT than in NGT, respectively, and correlated positively with the M-value of euglycemic clamps and high-density lipoprotein, and negatively with fasting plasma triglyceride, tumor necrosis factor-α and interleukin-6 levels in the entire cohort. mtDNA copy number/adipocyte volume also correlated positively with plasma adiponectin. Pioglitazone, which improved insulin sensitivity, plasma lipids and inflammation, increased the mitochondrial copy number, and led to a redistribution of mitochondria from a punctate to a more reticular pattern as observed in NGT. This was accompanied by disproportionately increased abundances of mitochondrial proteins, including those involved in fat oxidation and triglyceride synthesis. Pioglitazone also increased the abundance of collagen VI and decreased the abundance of cytoskeletal proteins. NADH cytochrome c reductase activity of isolated adipocyte mitochondria was similar in AGT and NGT and unaltered by pioglitazone. CONCLUSIONS: Adipocyte mitochondria are deficient in insulin-resistant individuals and correlate with systemic lipid metabolism, inflammation and insulin sensitivity. Pioglitazone induces mitochondrial biogenesis and reorganization as well as the synthesis of mitochondrial proteins including those critical for lipid metabolism. It also alters extracellular matrix and cytoskeletal proteins. The intrinsic function of adipocyte mitochondria appears unaffected in insulin resistance and by pioglitazone.International Journal of Obesity advance online publication, 31 October 2017; doi:10.1038/ijo.2017.192.

Metastatic bone cancer of the foot: a case report.

Metastasis to the foot is rarely presented in the literature. We describe a 56-year-old woman with nonspecific pain in the left foot, ankle, and knee for 3 months. The patient was diagnosed as having urothelial carcinoma with metastasis to the left lower extremity, including the foot. This diagnosis was reached through the combined efforts of radiologic techniques and biopsies. This case discusses the importance of paying close attention to the details of the history and physical examination, performing appropriate tests, and obtaining suitable referrals.

Utilizing fungus myceliated grain for molt induction and performance in commercial laying hens.

Molting in poultry is used to rejuvenate hens for a second or third laying cycle. Feed withdrawal was once the most effective method used for molt induction; however, it has being phased out due to food safety and animal welfare concerns. This study evaluated the utilization of fungus myceliated grain as a safe and effective alternative for inducing molt, enhancing immunity, reducing Salmonella growth, and returning to egg production. Laying hens were subjected to 1 of 5 treatments: 1) nonfed (NF), 2) full-fed (FF), 3) fungus myceliated meal (FM), 4) 90% fungus myceliated meal+10% standard layer ration (FM-90), and 5) 90% alfalfa meal+10% fungus myceliated meal (AF-90). Each treatment condition was replicated 9 times during a 9-d molt period. The results revealed that egg production for treatments 1 and 3 ceased completely by d 5, whereas hens in treatments 4 and 5 ceased egg production by d 6. The percentage of BW loss decreased significantly (P<0.05) in treatments 1 (57%), 2 (8%), 3 (35%), 4 (37%), and 5 (44%). Ovary weights of hens fed all molting diets decreased significantly from the full-fed control but did not differ significantly (P<0.05) from each other. Salmonella population in the crop, ovary, and ceca from hens differed significantly (P<0.05) among treatments. Return to egg production differed between treatments with higher production beginning in treatment 3 and ending in treatment 5. Antibody titers did differ (P<0.05) among treatments. From these results, fungus myceliated meal appears to be a viable alternative to conventional feed withdrawal and other methods for the successful induction of molt and retention of postmolt performance.

MI-63: a novel small-molecule inhibitor targets MDM2 and induces apoptosis in embryonal and alveolar rhabdomyosarcoma cells with wild-type p53.

BACKGROUND: Interruption of the role of p53s as a tumour suppressor by MDM2 may be one of the mechanisms by which cancer cells evade current therapy. Blocking the inhibition of wild-type p53 by MDM2 in cancer cells should reactivate p53's tumour suppressor functions and enhance current cancer treatments. MI-63 is a novel non-peptide small molecule that has shown strong binding affinity (K(i)=3 nM) for MDM2; however, its effects on paediatric cancer cells and the specific mechanism of tumour suppressor reactivation have not been evaluated. METHODS: Rhabdomyosarcoma (RMS), the most common childhood soft tissue sarcoma, expresses either wild-type or mutant p53 protein. We examined the inhibitory effects of MI-63 in embryonal RMS (ERMS) and alveolar RMS (ARMS) cell lines expressing wild-type or mutated p53. RESULTS: Treatment with MI-63 reduced cell viability by 13.4% and by <1%, respectively, at 72 h in both RH36 and RH18 cell lines expressing wild-type p53. In contrast, RH30 and RD2 cells expressing p53 mutants are resistant to MI-63 treatment. An increased expression of p53, p21(WAF1), and Bax protein was observed after treatment with MI-63 in RMS cells with wild-type p53, and apoptosis was confirmed by cleaved PARP and caspase-3 expression. However, RD2 and RH30 RMS cells, as well as human normal skeletal muscle cells, showed a minimal increase in p53 signalling and no induction of cleaved PARP and caspase-3. MI-63 was compared with Nutlin-3, a known MDM2 inhibitor, and was found to be more potent in the inhibition of cell proliferation/viability. Further, synergy was observed when MI-63 was used in combination with doxorubicin. CONCLUSION: These results indicate that MI-63 is a potent therapeutic agent for RMS cells expressing wild-type p53 protein.

The effect of mushroom and pokeweed extract on salmonella, egg production, and weight loss in molting hens.

An experiment was conducted to evaluate the effects of mushroom and pokeweed extract alone or in combination with alfalfa meal on Salmonella spp. population, egg production, and weight loss in laying hens during a 10-d molting period. The trial used 54 active laying hens approximately 77 wk of age that were naturally infected with Salmonella spp. The layers were subjected to 1 of 9 treatment groups, replicated 3 times with 2 hens per replicate cage. The treatment conditions were as follows: 1) full-fed + H(2)0 (FFW), 2) full-fed + mushroom (FFM), 3) full-fed + pokeweed (FFP), 4) nonfed + H(2)0 (NFW), 5) nonfed + mushroom (NFM), 6) nonfed + pokeweed (NFP), 7) full-fed alfalfa meal + H(2)0 (FFAW), 8) full-fed alfalfa meal + mushroom (FFAM), and 9) full-fed alfalfa meal + poke-weed (FFAP). The results showed that the base-10 logarithm values of Salmonella from the ceca significantly increased (P

Developmental and neuropsychological perspectives on the Wisconsin Card Sorting Test in children.

The purpose of this study was to advance our understanding of the Wisconsin Card Sorting Test (WCST) as a measure of a set-shifting component of neuropsychological executive function among children by investigating the level of difficulty posed by the order of administering number (vs. shape and color) as a sorting criterion in the test. A total of 196 participants at three different ages groups (6, 11-12, and 18-19 yrs.) were administered the standard or a modified version of the WCST. Results revealed several age-related trends: (a) increases in the number of categories completed, (b) increases in test efficiency, and (c) differences in task difficulty as a function of the order in which the number sorting criterion was administered in the test. Implications of these findings are discussed in terms of the construct validity of the WCST for young children.

Investigating the effects of dietary probiotic feeding regimens on broiler chicken production and Campylobacter jejuni presence.

An experiment was conducted to investigate the effect of restricted feeding of a commercially available probiotic diet on production/processing performance, Campylobacter jejuni prevalence, and organ weights in broiler chickens. Five hundred forty 1-d-old broiler chicks were randomly assigned to a control or a direct-fed microbial (PrimaLac, DFM) diet and subjected to ad libitum full-fed (A), restricted 8-h (R), or skip-a-day (S) feeding regimens. Each of the 6 treatments was replicated 3 times with 15 male and 15 female chicks per pen for 49 d. Significant (P<0.05) differences between BW in the control and DFM groups with regard to feed type were found at d 7 (A), female d 21 (R) male and females, and d 49 (A and S) male and females. Body weights of males in the control group were significantly higher than the DFM (A) and differed by regimens (A>R>S) at d 49, whereas weights of females did not differ in regimens A and S. Body weight in the control females of regimen R was significantly higher than those in regimens A and S. Carcass yield was significantly higher for males in the control regimen A, 78.1 vs. 74.6% for the DFM regimen A; however, females did not differ significantly in this regimen, but did so in regimen S with 72.6 vs. 69.0%. The gizzard weights were significantly higher for broilers exposed to S and R regimens when compared with the A regimen. The prevalence of C. jejuni in the DFM-treated broilers regimen R was lower (33 vs. 60% positive) for the control group at 21 d. The weekly BW throughout the study reflected many variations, but broiler chickens receiving the control feed on regimen A performed better than those receiving the DFM feed. From the present results, it was concluded that supplementation of DFM reduced the presence of C. jejuni but had no significant effect on the growth performance of broilers; however, there were some significant trends regarding sex, feed, and feeding methods on the performance results.

Effect of a polymorphism in the ND1 mitochondrial gene on human skeletal muscle mitochondrial function.

OBJECTIVE: A non-silent polymorphism in the mitochondrial coding region of the ND1 gene, a subunit of reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase is associated with resting metabolic rate (RMR) in 245 non-diabetic Pima Indians. The purpose of this investigation was to determine the effect of the ND1 gene polymorphism on mitochondrial function in 14 male Pima Indians. METHODS AND PROCEDURES: Seven subjects with an A at site 3547 of the ND1 gene (Ile at amino acid 81), and seven with a G at this site (Val) were studied. Mitochondria were isolated from 0.8 to 1.5 g of skeletal muscle obtained by needle biopsy of the lateral quadriceps muscle. In intact mitochondria, maximal (state-3) and resting (state-4) respiration rates were measured polarographically at 37 degrees C with a variety of single substrates or substrate combinations. Disrupted mitochondria were analyzed for maximal capacities through the entire electron transport chain (ETC) (NADH oxidase (NADHOX)), as well as through a segment of Complex I that is independent of the ND1 component (NADH-ferricyanide (NADH-FeCN) reductase). RESULTS: Mitochondria were well coupled and exhibited higher respiratory control ratios (RCRs) than rodent muscle. There were no differences between the two groups for any of the measured parameters. DISCUSSION: These results indicate that the cause of the observed association between RMR and the ND1 polymorphism is not related to in vitro mitochondrial function.

Performance assessment of broiler chickens given mushroom extract alone or in combination with probiotics.

A study was conducted to evaluate the effect of combined Shiitake mushroom (Lentinus edodes) extract with probiotics (PrimaLac) on the growth and health of broiler chickens. In trial 1, 540 d-of-hatch chicks were randomly assigned to 6 treatment groups, replicated 3 times, with 15 males and 15 females per pen for 3 wk. Dietary probiotics and mushroom treatments were as follows: 1) control feed + ad libitum tap water; 2) control feed + skip-a-day mushroom water; 3) control feed + ad libitum mushroom water; 4) probiotic feed + ad libitum tap water; 5) probiotic feed + skip-a-day mushroom water; 6) probiotic feed + ad libitum mushroom water. Body weight gain, feed consumption and efficiency, mortality, bursa, liver, and spleen relative weights of chicks were taken. In trial 2, the performance of broilers 3 to 7 wk withdrawn from the mushroom extract was evaluated along with the comparative level of fecal biofidobacteria in the control and mushroom extract treatment (trt). Mortality, weight gain, feed consumption and efficiency, carcass yield, fat pads, bursa weights and fecal bifidobacteria were measured in trial 2. In trial 1, significant differences (P < 0.05) in female weight gain (trt 4-0.62 vs. trt 1-0.54 kg) and male spleen weights were observed. In trial 2, significant differences were observed in male weight gain (trt 2-2.40 vs. trt 4-1.12 kg), male and female fat pads, male bursa weights (trt 3-0.15 vs. trt 6-0.39), female carcass yield percentage (trt 1-77.8 vs. trt 4-66.4), and feed consumption and efficiency. Body weights were severely depressed in the male broilers receiving the probiotics feed in treatments 4, 5, and 6, but not in the female broilers. These results indicate that performance differences in gender occur with additives during different grow-out periods, and mushroom extract promotes bifidobacteria growth in broiler chickens after 4 wk of withdrawal. It appears that probiotics and mushroom extract offered no combination potential for weight gain, which was compromised in this study, but possible health-enhanced attributes.

Activation of protein kinase B/Akt in the periphery contributes to pain behavior induced by capsaicin in rats.

Protein kinase B (PKB/Akt) is a member of the second-messenger regulated subfamily of protein kinases. It is implicated in signaling downstream of growth factors, insulin receptor tyrosine kinases and phosphoinositide 3-kinase (PI3K). Current studies indicate that nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and PI3K help mediate inflammatory hyperalgesia. However, little is known about the role of PKB/Akt in the nociceptive system. In this study, we investigated whether PKB/Akt in primary sensory neurons is activated after noxious stimulation and contributes to pain behavior induced in rats by capsaicin. We demonstrated that phospho-PKB/Akt (p-PKB/Akt) is increased in dorsal root ganglia (DRG) at 5 min after intradermal injection of capsaicin. p-PKB/Akt is distributed predominantly in small- and medium-sized DRG cells. After capsaicin injection, p-PKB/Akt (473) is colocalized with isotectin-B4 (IB4), tyrosine kinase A (TrkA), and calcitonin gene-related peptide (CGRP). Furthermore, most transient receptor potential vanilloid type 1 (TRPV1) positive DRG neurons double label for p-PKB/Akt. Behavioral experiments show that intradermal injection of a PI3K (upstream of PKB/Akt) inhibitor, wortmannin, dose-dependently inhibits the changes in exploratory behavior evoked by capsaicin injection. The PKB/Akt inhibitor, Akt inhibitor IV, has the same effect. The results suggest that the PKB/Akt signaling pathway in the periphery is activated by noxious stimulation and contributes to pain behavior.

Neural substrates of childhood attention-deficit/hyperactivity disorder: electroencephalographic and magnetic resonance imaging evidence.

Research methods based on electroencephalogram (EEG) and anatomical and functional MRI have been used with increasing frequency in the study of childhood Attention-Deficit/Hyperactivity Disorder (ADHD). Both methods are safe and noninvasive, and their results can complement each other because of the good temporal (but relatively poorer spatial) resolution of EEG and the good spatial (but relatively poorer temporal) resolution of MRI. These methods are described, and associated recent research on childhood ADHD is summarized and critically examined. Results of this research support theories of ADHD that focus on a frontal-striatal neurological circuitry substrate, which has been implicated in neuropsychological executive functioning. A number of issues, however, such as the specificity of this finding for ADHD, remain unresolved. We conclude with an overview of advances and issues to be considered in future research on the neural substrates of childhood ADHD and advocate a developmental-contextual perspective on this disorder that acknowledges the reciprocal relations between neural structures and functions.

The effect of a kainate GluR5 receptor antagonist on responses of spinothalamic tract neurons in a model of peripheral neuropathy in primates.

The responses of antidromically identified spinothalamic tract (STT) neurons to mechanical and thermal stimuli were compared in anesthetized normal and neuropathic monkeys before and after administration of a GluR5 kainate receptor antagonist (LY382884) into the spinal cord dorsal horn through a microdialysis fiber. Peripheral neuropathy was induced by tight ligation of the L7 spinal nerve 13-15 days prior to the experiment. STT neurons recorded in the animals with neuropathy showed increased responsiveness to weak mechanical stimuli and to heating and cooling of the skin compared to STT cells in normal animals. In both normal and the neuropathic monkeys the responses of the STT neurons to mechanical and thermal stimuli were attenuated by LY382884 application in a concentration-dependent manner. Intraspinal application of LY382884 in the neuropathic animals led to a potent reduction of those responses of the STT neurons that were aggravated by the peripheral neuropathy (weak mechanical, heat and innocuous cooling stimuli). These results suggest that kainate receptors are involved in synaptic activation of STT cells in the normal state and may also play an important role in pathological pain states such as peripheral neuropathy in primates. Kainate receptor antagonists could thus be useful for the treatment of certain forms of allodynia and hyperalgesia.

Locomotor activity and behavior of mutant mice deleted for gastrin gene expression.

The current studies were initiated to investigate the role of brain-gut peptide, gastrin, on locomotor activity and anxiety-like behavior. Young, male mutant mice, lacking gastrin gene expression (GAS-KO mice), were used in the experiments. The locomotor activity of GAS-KO vs wild type (WT) mice was compared by open field test. The anxiety-like behavior was examined using elevated plus maze. The time and entries to the open arms of the elevated plus maze were used as an indicator for the anxiety-like behavior and the data were analyzed using Hindsight program. On the open field test, locomotor activity of GAS-KO mice was similar to that of the WT mice for the first 10 min of the test, but decreased significantly after that. Anxiety-like behavior was more evident in the GAS-KO vs WT mice in the elevated plus maze experiments. The number of entries to and time spent on the open arms of plus-maze were significantly reduced for the GAS-KO vs WT mice suggesting an increased anxiety-like behavior of GAS-KO mice. Our studies suggest that normal circulating levels of gastrins may play a direct or indirect role in the regulation of locomotor activity and anxiety-like behavior.

Involvement of peripheral neuropeptide Y receptors in sympathetic modulation of acute cutaneous flare induced by intradermal capsaicin.

In a recent study, we have demonstrated that the dorsal root reflex (DRR)-mediated acute cutaneous neurogenic inflammation following intradermal injection of capsaicin (CAP) is sympathetically dependent and subject to modulation by peripheral alpha(1)-adrenoceptors. Postganglionic sympathetic neurons contain not only adrenergic neurotransmitters, but also non-adrenergic substances, including neuropeptide Y (NPY). In this study, we examined if peripheral NPY receptors participate in the flare following CAP injection. Different NPY receptor subtypes were studied by using relatively specific agonists and antagonists for the Y(1) and Y(2) subtypes. Changes in cutaneous blood flow on the plantar surface of the foot were measured using a laser Doppler flowmeter. Following CAP injection, cutaneous flare spread more than 20 mm away from the site of CAP injection. Removal of the postganglionic sympathetic nerves by surgical sympathectomy reduced dramatically the CAP-evoked flare. If the foot of sympathectomized rats was pretreated with either NPY or Y(2) receptor agonists by intra-arterial injection, the spread of flare induced by CAP injection could be restored and prolonged. However, if the spinal cord was pretreated with a GABA(A) receptor antagonist, bicuculline, to prevent DRRs, NPY or an Y(2) receptor agonist no longer restored the CAP-evoked flare. A Y(1) receptor agonist did not affect the CAP-evoked flare in sympathectomized rats. In sympathetically intact rats, blockade of either peripheral NPY or Y(2) receptors with [D-Trp(32)]-NPY or BIIE0246 markedly reduced the flare induced by CAP injection, whereas blockade of peripheral Y(1) receptors by BIBP3226 did not obviously affect the flare. It is suggested that NPY is co-released with NE from the postganglionic sympathetic terminals to activate NPY Y(2) and alpha(1) receptors following CAP injection. Both substances are involved, at least in part, in modulation of the responses of CAP sensitive afferents thereby affecting their ability to evoke the release of inflammatory agents from primary afferents.

Increased phosphorylation of the GluR1 subunit of spinal cord alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor in rats following intradermal injection of capsaicin.

Ionotropic glutamate receptors are ligand-gated ion channels that help mediate rapid excitatory neurotransmission in the CNS. alpha-Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are critical for synaptic plasticity in central nociceptive transmission. The current study was designed to investigate the role of the AMPA receptor subunit, GluR1, and its phosphorylated forms (at Ser-831 and Ser-845) in central sensitization in rat spinal cord. Western blots and immunohistochemistry were performed to examine the expression and localization of GluR1 and the phosphorylated forms of GluR1 (phospho-GluR1) at Ser-831 and Ser-845 with specific antibodies. Results showed that immunolabeling of GluR1 protein in rat spinal cord can be detected at 110 kD, and two phospho-GluR1 proteins were found at 106 kD. A significant upregulation of phospho-GluR1 both at Ser-831 and Ser-845 was found by 5 min after capsaicin treatment, and this increase lasted at least 60 min. Immunostaining showed that GluR1 and its phosphorylated forms were localized in the superficial laminae of dorsal horn and quantitative image analysis supported the immunoblotting results. Our findings are consistent with the suggestions that AMPA receptors show increased responsiveness because of their phosphorylation and that this may contribute to central sensitization following intradermal injection of capsaicin.