Use of electromyography to detect muscle exhaustion in finishing barrows fed ractopamine HCl

Citation: Noel, J. A., Broxterman, R. M., McCoy, G. M., Craig, J. C., Phelps, K. J., Burnett, D. D., . . . Gonzalez, J. M. (2016). Use of electromyography to detect muscle exhaustion in finishing barrows fed ractopamine HCl. Journal of Animal Science, 94(6), 2344-2356. doi:10.2527/jas2016-0398The objectives of this study were to determine the effects of dietary ractopamine HCl (RAC) on muscle fiber characteristics and electromyography (EMG) measures of finishing barrow exhaustion when barrows were subjected to increased levels of activity. Barrows (n = 34; 92 +/- 2 kg initial BW) were assigned to 1 of 2 treatments: a conventional swine finishing diet containing 0 mg/kg ractopamine HCl (CON) or a diet formulated to meet the requirements of finishing barrows fed 10 mg/kg RAC (RAC+). After 32 d on feed, barrows were individually moved around a track at 0.79 m/s until subjectively exhausted. Wireless EMG sensors were affixed to the deltoideus (DT), triceps brachii lateral head (TLH), tensor fasciae latae (TFL), and semitendinosus (ST) muscles to measure median power frequency (MdPF) and root mean square (RMS) as indicators of action potential conduction velocity and muscle fiber recruitment, respectively. After harvest, samples of each muscle were collected for fiber type, succinate dehydrogenase (SDH), and capillary density analysis. Speed was not different (P = 0.82) between treatments, but RAC+ barrows reached subjective exhaustion earlier and covered less distance than CON barrows (P 0.29). There was a treatment x muscle interaction (P = 0.04) for end-point RMS values. The RAC diet did not change end-point RMS values in the DT or TLH (P > 0.37); however, the diet tended to decrease and increase end-point RMS in the ST and TFL, respectively (P 0.10). Muscles of RAC+ barrows tended to have less type I fibers and more capillaries per fiber (P < 0.07). Type I and IIA fibers of RAC+ barrows were larger (P < 0.07). Compared with all other muscles, the ST had more (P < 0.01) type IIB fibers and larger type I, IIA, and IIX fibers (P < 0.01). Type I, IIA, and IIX fibers of the ST also contained less SDH compared with the other muscles (P < 0.01). Barrows fed a RAC diet had increased time to subjective exhaustion due to loss of active muscle fibers in the ST, possibly due to fibers being larger and less oxidative in metabolism. Size increases in type I and IIA fibers with no change in oxidative capacity could also contribute to early exhaustion of RAC+ barrows. Overall, EMG technology can measure real-time muscle fiber loss to help explain subjective exhaustion in barrows

(αMe)Nva: stereoselective syntheses and preferred conformations of selected model peptides

Using different stereoselective chemical and chemoenzymatic approaches we synthesized the chiral, Cα-methylated α-amino acid l-(αMe)Nva with a short, linear side-chain. A set of terminally protected model peptides to the pentamer level containing either (αMe)Nva or Nva in combination with Ala and/or Aib was prepared using solution methods and characterized fully. Two (αMe)Nva peptides were also synthesized using side-chain hydrogenation of the corresponding Cα-methyl, Cα-allylglycine (Mag) peptides. A detailed solution and crystal-state conformational analysis based on FT-IR absorption, 1H NMR and X-ray diffraction techniques allowed us to define that: (i) (αMe)Nva is an effective β-turn and 310-helix former; and (ii) the relationship between (αMe)Nva chirality and the screw sense of the turn/helix formed is that typical of protein amino acids, i.e. l-(αMe)Nva induces the preferential formation of right-handed folded structures. In more general terms, this study reinforced previous conclusions that peptides based on α-amino acids with a Cα-methyl substituent and a Cα-linear alkyl substituent are characterized by a strong tendency to fold into turn and helical structures.The Padova authors gratefully acknowledge M.U.R.S.T., the Ministry of University and Scientific and Technological Research, and C.N.R., the National Council of Research of Italy for their continuous support. The Zaragoza authors acknowledge the Direccion General de Investigacion Cientifica y Tecnica (PB94±0998) for financial support. The Zaragoza and Padova authors are also grateful to the Spain-Italy exchange program Accion Integrada HI 97-20/AzioniIntegrate 1997/99.Peer reviewe

CD13/Aminopeptidase N overexpression by basic fibroblast growth factor mediates enhanced invasiveness of 1F6 human melanoma cells

CD13/Aminopeptidase N (CD13) is known to play an important role in tumour cell invasion. We examined whether basic fibroblast growth factor (bFGF) is involved in the regulation of CD13 expression in human melanoma cells. 1F6 human melanoma cells were stably transfected with constructs encoding either the 18 kDa (18kD) or all (ALL) bFGF isoform proteins. We observed highly increased CD13 mRNA and protein expression in the 1F6 clones regardless of the overexpression of either the 18kD or all isoform proteins. Neutral aminopeptidase activity was increased five-fold and could be inhibited by bestatin and the CD13-neutralising antibody WM15. The enhanced invasion through Matrigel, but not migration in a wound assay, was efficiently abrogated by both bestatin and WM15. Upregulation of CD13 expression was the result of increased epithelial and myeloid promoter activity up to 4.5-fold in 1F6-18kD and 1F6-ALL clones. Interestingly, in a panel of human melanoma cell lines, a significant correlation (r2=0.883, P<0.05) between bFGF and CD13 mRNA and protein expression was detected. High bFGF and CD13 expression were clearly related with an aggressive phenotype. Taken together, our data indicate that high bFGF expression upregulates CD13 expression in human melanoma cells by activating both the myeloid and the epithelial CD13 promoter. In addition, we show that high bFGF and CD13 expression results in enhanced invasive capacity and metastatic behaviour of human melanoma cells

Neuropilin-1 antagonism in human carcinoma cells inhibits migration and enhances chemosensitivity

BACKGROUND: Neuropilin-1 (NRP1) is a non-tyrosine kinase receptor for vascular endothelial growth factor (VEGF) recently implicated in tumour functions.METHODS: In this study we used a specific antagonist of VEGF binding to the NRP1 b1 domain, EG3287, to investigate the functional roles of NRP1 in human carcinoma cell lines, non-small-cell lung A549, kidney ACHN, and prostate DU145 cells expressing NRP1, and the underlying mechanisms involved.RESULTS: EG3287 potently displaced the specific binding of VEGF to NRP1 in carcinoma cell lines and significantly inhibited the migration of A549 and ACHN cells. Neuropilin-1 downregulation by siRNA also decreased cell migration. EG3287 reduced the adhesion of A549 and ACHN cells to extracellular matrix (ECM), and enhanced the anti-adhesive effects of a beta 1-integrin function-blocking antibody. EG3287 increased the cytotoxic effects of the chemotherapeutic agents 5-FU, paclitaxel, or cisplatin on A549 and DU145 cells, through inhibition of integrin-dependent cell interaction with the ECM.CONCLUSIONS: These findings indicate that NRP1 is important for tumour cell migration and adhesion, and that NRP1 antagonism enhances chemosensitivity, at least in part, by interfering with integrin-dependent survival pathways. A major implication of this study is that therapeutic strategies targeting NRP1 in tumour cells may be particularly useful in combination with other drugs for combating tumour survival, growth, and metastatic spread independently of an antiangiogenic effect of blocking NRP1. British Journal of Cancer (2010) 102, 541-552. doi:10.1038/sj.bjc.6605539 www.bjcancer.com Published online 19 January 2010 (C) 2010 Cancer Research U

Cytotoxicity of rhein, the active metabolite of sennoside laxatives, is reduced by multidrug resistance-associated protein 1

Anthranoid laxatives, belonging to the anthraquinones as do anthracyclines, possibly increase colorectal cancer risk. Anthracyclines interfere with topoisomerase II, intercalate DNA and are substrates for P-glycoprotein and multidrug resistance-associated protein 1. P-glycoprotein and multidrug resistance-associated protein 1 protect colonic epithelial cells against xenobiotics. The aim of this study was to analyse the interference of anthranoids with these natural defence mechanisms and the direct cytotoxicity of anthranoids in cancer cell lines expressing these mechanisms in varying combinations. A cytotoxicity profile of rhein, aloe emodin and danthron was established in related cell lines exhibiting different levels of topoisomerases, multidrug resistance-associated protein 1 and P-glycoprotein. Interaction of rhein with multidrug resistance-associated protein 1 was studied by carboxy fluorescein efflux and direct cytotoxicity by apoptosis induction. Rhein was less cytotoxic in the multidrug resistance-associated protein 1 overexpressing GLC4/ADR cell line compared to GLC4. Multidrug resistance-associated protein 1 inhibition with MK571 increased rhein cytotoxicity. Carboxy fluorescein efflux was blocked by rhein. No P-glycoprotein dependent rhein efflux was observed, nor was topoisomerase II responsible for reduced toxicity. Rhein induced apoptosis but did not intercalate DNA. Aloe emodin and danthron were no substrates for MDR mechanisms. Rhein is a substrate for multidrug resistance-associated protein 1 and induces apoptosis. It could therefore render the colonic epithelium sensitive to cytotoxic agents, apart from being toxic in itself

Increased numbers of small circulating endothelial cells in renal cell cancer patients treated with sunitinib

Mature circulating endothelial cell (CEC) as well as endothelial progenitor populations may reflect the activity of anti-angiogenic agents on tumor neovasculature or even constitute a target for anti-angiogenic therapy. We investigated the behavior of CECs in parallel with hematopoietic progenitor cells (HPCs) in the blood of renal cell cancer patients during sunitinib treatment. We analyzed the kinetics of a specific population of small VEGFR2-expressing CECs (CD45(neg)/CD34(bright)), HPCs (CD45(dim)/CD34(bright)), and monocytes in the blood of 24 renal cell cancer (RCC) patients receiving 50 mg/day of the multitargeted VEGF inhibitor sunitinib, on a 4-week-on/2-week-off schedule. Blood was taken before treatment (C1D1), on C1D14, C1D28, and on C2D1 before the start of cycle 2. Also plasma VEGF and erythropoietin (EPO) were determined. Remarkably, while CD34(bright) HPCs and monocytes decreased during treatment, CD34(bright) CECs increased from 69 cells/ml (C1D1) to 180 cells/ml (C1D14; P = 0.001) and remained high on C1D28. All cell populations recovered to near pre-treatment levels on C2D1. Plasma VEGF and EPO levels were increased on C1D14 and partly normalized to pre-treatment levels on C2D1. In conclusion, opposite kinetics of two circulating CD34(bright) cell populations, HPCs and small CECs, were observed in sunitinib-treated RCC patients. The increase in CECs is likely caused by sunitinib targeting of immature tumor vessel

Indomethacin-induced activation of the death receptor-mediated apoptosis pathway circumvents acquired doxorubicin resistance in SCLC cells

Small-cell lung cancers (SCLCs) initially respond to chemotherapy but are often resistant at recurrence. A potentially new method to overcome resistance is to combine classical chemotherapeutic drugs with apoptosis induction via tumour necrosis factor (TNF) death receptor family members such as Fas. The doxorubicin-resistant human SCLC cell line GLC(4)-Adr and its parental doxorubicin-sensitive line GLC(4) were used to analyse the potential of the Fas-mediated apoptotic pathway and the mitochondrial apoptotic pathway to modulate doxorubicin resistance in SCLC. Western blotting showed that all proteins necessary for death-inducing signalling complex formation and several inhibitors of apoptosis were expressed in both lines. The proapototic proteins Bid and caspase-8, however, were higher expressed in GLC(4)-Adr. In addition, GLC(4)-Adr expressed more Fas (3.1x) at the cell membrane. Both lines were resistant to anti-Fas antibody, but plus the protein synthesis inhibitor cycloheximide anti-Fas antibody induced 40% apoptosis in GLC(4)-Adr. Indomethacin, which targets the mitochondrial apoptotic pathway, induced apoptosis in GLC(4)-Adr but not in GLC(4) cells. Surprisingly, in GLC(4)-Adr indomethacin induced caspase-8 and caspase-9 activation as well as Bid cleavage, while both caspase-8 and caspase-9 specific inhibitors blocked indomethacin-induced apoptosis. In GLC(4)-Adr, doxorubicin plus indomethacin resulted in elevated caspase activity and a 2.7-fold enhanced sensitivity to doxorubicin. In contrast, no effect of indomethacin on doxorubicin sensitivity was observed in GLC(4). Our findings show that indomethacin increases the cytotoxic activity of doxorubicin in a doxorubicin-resistant SCLC cell line partly via the death receptor apoptosis pathway, independent of Fas