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G protein-coupled receptors (GPCRs) have historically been associated with signalling events driven from the plasma membrane. More recently, signalling from endosomes has been recognized as a feature of internalizing receptors. However, there was little consideration given to the notion that GPCRs can be targeted to distinct subcellular locations that did not involve an initial trafficking to the cell surface. Here, we focus on the evidence for and the potential impact of GPCR signalling specifically initiated from the nuclear membrane. We also discuss the possibilities for selectively targeting this and other internal pools of receptors as novel venues for drug discovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Amino acids are crucial nutrients involved in several cellular and physiological processes, including fertilization and early embryo development. In particular, Leucine and Arginine have been shown to stimulate implantation, as lack of both in a blastocyst culture system is able to induce a dormant state in embryos. The aim of this work was to evaluate the effects of Leucine and Arginine withdrawal on pluripotent mouse embryonic stem cell status, notably, their growth, self-renewal, as well as glycolytic and oxidative metabolism. Our results show that the absence of both Leucine and Arginine does not affect mouse embryonic stem cell pluripotency, while reducing cell proliferation through cell-cycle arrest. Importantly, these effects are not related to Leukemia Inhibitory Factor (LIF) and are reversible when both amino acids are reconstituted in the culture media. Moreover, a lack of these amino acids is related to a reduction in glycolytic and oxidative metabolism and decreased protein translation in mouse embryonic stem cells (mESCs), while maintaining their pluripotent status.

Mesenchymal stem cells reside under precise hypoxic conditions that are paramount in determining cell fate and behavior (metabolism, proliferation, differentiation, etc.). In this work, we show that different oxygen tensions promote a distinct proliferative response and affect the biosynthetic demand and global metabolic profile of umbilical cord-mesenchymal stem cells (UC-MSCs). Using both gas-based strategies and CoCl2 as a substitute for the costly hypoxic chambers, we found that specific oxygen tensions influence the fate of UC-MSCs differently. While 5% O2 potentiates proliferation, stimulates biosynthetic pathways, and promotes a global hypermetabolic profile, exposure to <1% O2 contributes to a quiescent-like cell state that relies heavily on anaerobic glycolysis. We show that using CoCl2 as a hypoxia substitute of moderate hypoxia has distinct metabolic effects, when compared with gas-based strategies. The present study also highlights that, while severe hypoxia regulates global translation via mTORC1 modulation, its effects on survival-related mechanisms are mainly modulated through mTORC2. Therefore, the experimental conditions used in this study establish a robust and reliable hypoxia model for UC-MSCs, providing relevant insights into how stem cells are influenced by their physiological environment, and how different strategies of modulating hypoxia may influence experimental outcomes.

Mouse embryonic stem cells (mESCs) can be grown in culture, recapitulating the different states of pluripotency of their in vivo counterparts, with notably different metabolic profiles. mESCs in a naïve pluripotent state present an ambivalent metabolism, using both glycolysis and oxidative phosphorylation as energy sources. Here, we describe a method to evaluate the oxidative function of naïve mESCs using the Seahorse Extracellular Flux Analyzer coupled to flow cytometry analysis of mitochondrial transmembrane potential using the TMRM fluorescence probe, thus assessing both oxygen consumption and mitochondrial membrane potential. This may be a useful protocol for understanding how mitochondrial oxidative function and potential of mESCs change in certain circumstances, and how is it related with various pluripotency/differentiation phenotypes.

DMSO is a commonly used solvent in biological studies, as it is an amphipathic molecule soluble in both aqueous and organic media. For that reason, it is the vehicle of choice for several water-insoluble substances used in research. At the molecular and cellular level, DMSO is a hydrogen-bound disrupter, an intercellular electrical uncoupler, and a cryoprotectant, among other properties. Importantly, DMSO often has overlooked side effects. In stem cell research, the literature is scarce, but there are reports on the effect of DMSO in human embryoid body differentiation and on human pluripotent stem cell priming towards differentiation, via modulation of cell cycle. However, in mouse embryonic stem cell (mESC) culture, there is almost no available information. Taking into consideration the almost ubiquitous use of DMSO in experiments involving mESCs, we aimed to understand the effect of very low doses of DMSO (0.0001%-0.2%), usually used to introduce pharmacological inhibitors/modulators, in mESCs cultured in two different media (2i and FBS-based media). Our results show that in the E14Tg2a mESC line used in this study, even the smallest concentration of DMSO had minor effects on the total number of cells in serum-cultured mESCs. However, these effects could not be explained by alterations in cell cycle or apoptosis. Furthermore, DMSO did not affect pluripotency or differentiation potential. All things considered, and although control experiments should be carried out in each cell line that is used, it is reasonable to conclude that DMSO at the concentrations used here has a minimal effect on this particular mESC line., Competing Interests: The authors declare that no conflict of interest exists., (Copyright © 2020 Maria Inês Sousa et al.)

Background: Expression of TRAP1, a member of the HSP90 chaperone family, has been implicated in tumour protective effects, based on its differential mitochondrial localization and function., Design: This work was designed to provide new insights into the pathways involved in TRAP1-provided cytoprotection on NSCLC. For this, TRAP1-depleted A549 human NSCLC cells and MRC-5 normal lung fibroblasts were produced using a siRNA approach and main cellular quality control mechanisms were investigated., Results: TRAP1-depleted A549 cells displayed decreased cell viability likely due to impaired mitochondrial function including decreased ATP/AMP ratio, oxygen consumption and membrane potential, as well as increased apoptotic indicators. Furthermore, the negative impact of TRAP1 depletion on mitochondrial function was not observed in normal MRC-5 lung cells, which might be due to the differential intracellular localization of the chaperone in tumour versus normal cells. Additionally, A549 TRAP1-depleted cells showed increased autophagic flux. Functionally, autophagy inhibition resulted in decreased cell viability in both TRAP1-expressing and TRAP1-depleted tumour cells with minor effects on MRC-5 cells. Conversely, autophagy stimulation decreased cell viability of both A549 and MRC-5 TRAP1-expressing cells while in A549 TRAP1-depleted cells, increased autophagy augmented viability., Conclusions: Our results show that even though TRAP1 depletion affects both normal MRC-5 and tumour A549 cell proliferation, inhibition of autophagy per se led to a decrease in tumour cell mass, while having a reduced effect on the normal cell line. The strategy of targeting TRAP1 in NSCLC shows future potential therapeutic applications., (© 2018 Stichting European Society for Clinical Investigation Journal Foundation.)

The objective of this study was to compare 2 commonly used techniques for measuring methane emissions from ruminant animals: the GreenFeed (GF) system and the sulfur hexafluoride (SF6) technique. The study was part of a larger experiment in which a methane inhibitor, 3-nitrooxypropanol, fed at 4 application rates (0, 40, 60, and 80 mg/kg of feed dry matter) decreased enteric methane emission by an average of 30% (measured by both GF and SF6) in a 12-wk experiment with 48 lactating Holstein cows fed a total mixed ration. The larger experiment used a randomized block design and was conducted in 2 phases (February to May, phase 1, and June to August, phase 2), with 2 sets of 24 cows in each phase. Using both GF and SF6 techniques, methane emission data were collected simultaneously during experimental wk 2, 6, and 12 (phase 1) and 2, 9, and 12 (phase 2), which corresponded to a total of 6 sampling periods. During each sampling period, 8 spot samples of gas emissions (staggered over a 3-d period) were collected from each cow using GF, as well as 3×24-h collections using the SF6 technique. Methane emission data were averaged per cow for the statistical analysis. The mean methane emission was 373 (standard deviation=96.3) and 405 (standard deviation=156) g/cow per day for GF and SF6, respectively. Coefficients of variation for the 2 methods were 25.8 and 38.6%, respectively; correlation and concordance between the 2 methods were 0.40 and 0.34, respectively. The difference in methane emission between the 2 methods (SF6 - GF) within treatment was from 46 to 144 and 24 to 27 g/d for phases 1 and 2, respectively. In the conditions of this experiment, the SF6 technique produced larger variability in methane emissions than the GF method. The overall difference between the 2 methods was on average about 8%, but was not consistent over time, likely influenced by barn ventilation and background methane and SF6 concentrations., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The adaptor protein p66Shc modulates cellular redox status integrating oxidative stress with mitochondrial stress responses. Upon oxidative stress, p66Shc is translocated to mitochondria or mitochondria-associated membranes in a multi-step process, resulting in locally increased reactive oxygen species production. This signaling pathway is believed to be important in the context of drug-induced organ toxicity. The use of anthracyclines as anticancer agents is limited due to a dose-dependent and cumulative toxicity resulting in cardiomyopathy. Treatment with the anthracycline doxorubicin (DOX) results in a dose-dependent and cumulative cardiotoxicity which is mediated, at least in part, by increased oxidative stress. In the present study, we investigated for the first time whether p66Shc signaling is activated during DOX treatment of the rat cardiomyoblast H9c2 cell line. We further tested whether the transcriptional factor FoxO3a, which activates target genes responsible for apoptosis and cell cycle arrest, is also involved in p66Shc-dependent redox signaling pathway. Our results suggest that DOX treatment induces p66Shc protein up-regulation specifically in nuclear fractions. Increased nuclear expression of FoxO3a was also detected in H9c2 cells after DOX treatment. Treatment with the antioxidant and protein kinase C (PKC-β) inhibitor hispidin decreased DOX-induced activation of caspase 9 and p66Shc alterations. Taking together, we hypothesize that p66Shc signaling is involved in the activation of stress/toxicity responses elicited by the treatment of H9c2 cells with DOX. Hence, the selective inhibition of this redox pathway may be a promising therapeutic approach to circumvent DOX cardiotoxicity.

Background: The employment of dietary strategies such as ketogenic diets, which force cells to alter their energy source, has shown efficacy in the treatment of several diseases. Ketogenic diets are composed of high fat, moderate protein and low carbohydrates, which favour mitochondrial respiration rather than glycolysis for energy metabolism., Design: This review focuses on how oncological, neurological and mitochondrial disorders have been targeted by ketogenic diets, their metabolic effects, and the possible mechanisms of action on mitochondrial energy homeostasis. The beneficial and adverse effects of the ketogenic diets are also highlighted., Results and Conclusions: Although the full mechanism by which ketogenic diets improve oncological and neurological conditions still remains to be elucidated, their clinical efficacy has attracted many new followers, and ketogenic diets can be a good option as a co-adjuvant therapy, depending on the situation and the extent of the disease., (© 2016 Stichting European Society for Clinical Investigation Journal Foundation.)

Ruminant animals (domesticated or wild) emit methane (CH4) through enteric fermentation in their digestive tract and from decomposition of manure during storage. These processes are the major sources of greenhouse gas (GHG) emissions from animal production systems. Techniques for measuring enteric CH4 vary from direct measurements (respiration chambers, which are highly accurate, but with limited applicability) to various indirect methods (sniffers, laser technology, which are practical, but with variable accuracy). The sulfur hexafluoride (SF6) tracer gas method is commonly used to measure enteric CH4 production by animal scientists and more recently, application of an Automated Head-Chamber System (AHCS) (GreenFeed, C-Lock, Inc., Rapid City, SD), which is the focus of this experiment, has been growing. AHCS is an automated system to monitor CH4 and carbon dioxide (CO2) mass fluxes from the breath of ruminant animals. In a typical AHCS operation, small quantities of baiting feed are dispensed to individual animals to lure them to AHCS multiple times daily. As the animal visits AHCS, a fan system pulls air past the animal's muzzle into an intake manifold, and through an air collection pipe where continuous airflow rates are measured. A sub-sample of air is pumped out of the pipe into non-dispersive infra-red sensors for continuous measurement of CH4 and CO2 concentrations. Field comparisons of AHCS to respiration chambers or SF6 have demonstrated that AHCS produces repeatable and accurate CH4 emission results, provided that animal visits to AHCS are sufficient so emission estimates are representative of the diurnal rhythm of rumen gas production. Here, we demonstrate the use of AHCS to measure CO2 and CH4 fluxes from dairy cows given a control diet or a diet supplemented with technical-grade cashew nut shell liquid.

A quarter of all anthropogenic methane emissions in the United States are from enteric fermentation, primarily from ruminant livestock. This study was undertaken to test the effect of a methane inhibitor, 3-nitrooxypropanol (3NOP), on enteric methane emission in lactating Holstein cows. An experiment was conducted using 48 cows in a randomized block design with a 2-wk covariate period and a 12-wk data collection period. Feed intake, milk production, and fiber digestibility were not affected by the inhibitor. Milk protein and lactose yields were increased by 3NOP. Rumen methane emission was linearly decreased by 3NOP, averaging about 30% lower than the control. Methane emission per unit of feed dry matter intake or per unit of energy-corrected milk were also about 30% less for the 3NOP-treated cows. On average, the body weight gain of 3NOP-treated cows was 80% greater than control cows during the 12-wk experiment. The experiment demonstrated that the methane inhibitor 3NOP, applied at 40 to 80 mg/kg feed dry matter, decreased methane emissions from high-producing dairy cows by 30% and increased body weight gain without negatively affecting feed intake or milk production and composition. The inhibitory effect persisted over 12 wk of treatment, thus offering an effective methane mitigation practice for the livestock industries.

H9c2 myoblasts are a cell model used as an alternative for cardiomyocytes. H9c2 cells have the ability to differentiate towards a cardiac phenotype when the media serum is reduced in the presence of all-trans-retinoic acid (RA), creating multinucleated cells with low proliferative capacity. In the present study, we performed for the first time a transcriptional analysis of the H9c2 cell line in two differentiation states, i.e. embryonic cells and differentiated cardiac-like cells. The results show that RA-induced H9c2 differentiation increased the expression of genes encoding for cardiac sarcomeric proteins such as troponin T, or calcium transporters and associated machinery, including SERCA2, ryanodine receptor and phospholamban as well as genes associated with mitochondrial energy production including respiratory chain complexes subunits, mitochondrial creatine kinase, carnitine palmitoyltransferase I and uncoupling proteins. Undifferentiated myoblasts showed increased gene expression of pro-survival proteins such as Bcl-2 as well as cell cycle-regulating proteins. The results indicate that the differentiation of H9c2 cells lead to an increase of transcripts and protein levels involved in calcium handling, glycolytic and mitochondrial metabolism, confirming that H9c2 cell differentiation induced by RA towards a more cardiac-like phenotype involves remodeled mitochondrial function. PI3K, PDK1 and p-CREB also appear to be involved on H9c2 differentiation. Furthermore, complex analysis of differently expressed transcripts revealed significant up-regulation of gene expression related to cardiac muscle contraction, dilated cardiomyopathy and other pathways specific for the cardiac tissue. Metabolic and gene expression remodeling impacts cell responses to different stimuli and determine how these cells are used for biochemical assays.

Technical-grade cashew nut shell liquid (TCNSL) is a by-product of the cashew nut industry in tropical countries, and is known to exhibit a wide range of biological activities, including inhibitory effect against gram-positive bacteria. This study was conducted to investigate the effects of TCNSL (73.3% cardanol, 16.4% cardol, and 3.0% methylcardol) on rumen methane emission, nutrient digestibility, dry matter intake, and milk yield and composition in dairy cows. Eight multiparous Holstein cows were used in a crossover design trial with two 21-d experimental periods. The diet was based on corn silage and alfalfa haylage and was formulated to meet or exceed the energy and metabolizable protein requirements of the cows. Treatments were control (no TCNSL supplementation) or 30 g/cow per day of TCNSL. Rumen carbon dioxide emission was not affected by TCNSL. Treatment had no effect on methane emission (542 vs. 511±35.3 g/cow per day, respectively) and methane emission intensity (15.0 vs. 13.9±0.58 g/kg of energy-corrected milk, respectively) and tended to decrease methane emission per kilogram of dry matter intake (20.2 vs. 18.6±1.04 g/kg, respectively). Dry matter intake (average 26.9±1.00 kg/d), milk yield (40.0±1.73 kg/d), and milk composition were not different between treatments. The TCNSL had no effect on N losses in urine and feces and total-tract apparent digestibility of nutrients, except digestibility of neutral detergent fiber tended to be increased compared with the control. Plasma urea and glucose concentrations were not affected by TCNSL. Concentration of milk C18:0 tended to be decreased (17%) by TCNSL compared with the control. In this study, TCNSL did not alter absolute methane emission in the rumen, but tended to decrease it by 8% per kilogram of dry matter intake. The TCNSL had no effect on milk yield and composition in dairy cows., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

G protein-coupled receptors (GPCRs) play key physiological roles and represent a significant target for drug development. However, historically, drugs were developed with the understanding that GPCRs as a therapeutic target exist solely on cell surface membranes. More recently, GPCRs have been detected on intracellular membranes, including the nuclear membrane, and the concept that intracellular GPCRs are functional is become more widely accepted. Nuclear GPCRs couple to effectors and regulate signaling pathways, analogous to their counterparts at the cell surface, but may serve distinct biological roles. Hence, the physiological responses mediated by GPCR ligands, or pharmacological agents, result from the integration of their actions at extracellular and intracellular receptors. The net effect depends on the ability of a given ligand or drug to access intracellular receptors, as dictated by its structure, lipophilic properties, and affinity for nuclear receptors. This review will discuss angiotensin II, endothelin, and β-adrenergic receptors located on the nuclear envelope in cardiac cells in terms of their origin, activation, and role in cardiovascular function and pathology.

The cardiotoxicity induced by the anti-cancer doxorubicin involves increased oxidative stress, disruption of calcium homeostasis and activation of cardiomyocyte death. Nevertheless, antioxidants and caspase inhibitors often show little efficacy in preventing cell death. We hypothesize that a caspase-independent cell death mechanism with the release of the apoptosis-inducing factor from mitochondria is involved in doxorubicin toxicity. To test the hypothesis, H9c2 cardiomyoblasts were used as model for cardiac cells. Our results demonstrate that z-VAD-fmk, a pan-caspase inhibitor, does not prevent doxorubicin toxicity in this cell line. Doxorubicin treatment results in AIF translocation to the nuclei, as confirmed by Western Blotting of cell fractions and confocal microscopy. Also, doxorubicin treatment of H9c2 cardiomyoblasts resulted in the appearance of 50kbp DNA fragments, a hallmark of apoptosis-inducing factor nuclear effects. Apoptosis-inducing factor knockdown using a small-interfering RNA approach in H9c2 cells resulted in a reduction of doxorubicin toxicity, including decreased p53 activation and poly-ADP-ribose-polymerase cleavage. Among the proteases that could be responsible for apoptosis-inducing factor cleavage, doxorubicin decreased calpain activity but increased cathepsin B activation, with inhibition of the latter partly decreasing doxorubicin toxicity. Altogether, the results support that apoptosis-inducing factor release is involved in doxorubicin-induced H9c2 cell death, which explains the limited ability of caspase inhibitors to prevent toxicity., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The relationship between mitochondrial metabolism and cell viability and differentiation in stem cells (SCs) remains poorly understood. In the present study, we compared mitochondrial physiology and metabolism between P19SCs before/after differentiation and present a unique fingerprint of the association between mitochondrial activity, cell differentiation and stemness. In comparison with their differentiated counterparts, pluripotency of P19SCs was correlated with a strong glycolytic profile and decreased mitochondrial biogenesis and complexity: round, low-polarized and inactive mitochondria with a closed permeability transition pore. This decreased mitochondrial capacity increased their resistance against dichloroacetate. Thus, stimulation of mitochondrial function by growing P19SCs in glutamine/pyruvate-containing medium reduced their glycolytic phenotype, induced loss of pluripotent potential, compromised differentiation and became P19SCs sensitive to dichloroacetate. Because of the central role of this type of SCs in teratocarcinoma development, our findings highlight the importance of mitochondrial metabolism in stemness, proliferation, differentiation and chemoresistance. In addition, the present work suggests the regulation of mitochondrial metabolism as a tool for inducing cell differentiation in stem line therapies.

The ergot alkaloid ergovaline has demonstrated a persistent and sustained contractile response in several different vascular models. It was hypothesized that different alkaloids isolated from tall fescue (Lolium arundinaceum) will contribute to this contractile response differently. The objective was to compare contractile-response patterns of single additions of the ergoline alkaloids lysergic acid, lysergol, and ergonovine and the ergopeptine alkaloids ergotamine, ergocristine, ergocryptine, ergocornine, and ergovaline (provided as tall fescue seed extract). Lateral saphenous veins were collected from 6 Holstein steers (BW = 397 ± 28 kg) immediately after slaughter, sliced into cross-sections, and suspended in myograph chambers containing oxygenated Krebs-Henseleit buffer (95% O2/5% CO2; pH = 7.4; 37°C). Treatments were added at 0 min and buffer was replaced in 15-min intervals for a 120-min incubation. In addition to maximum tension and time to reach maximum tension, percent relaxation and rate of relaxation were determined following maximum tension for each treatment. All compounds tested produced significant contractile responses (P < 0.05). All ergoline alkaloids reached maximum response in less time (P < 0.05) than the remaining compounds and began to relax immediately after first buffer change. Lysergic acid had the greatest (P < 0.05) percent relaxation and ergonovine had the greatest (P < 0.05) rate of relaxation. The ergopeptine alkaloids ergovaline, ergotamine, ergocristine, ergocryptine, and ergocornine had slower developing contractile responses with a longer (P < 0.05) interval until maximum tension was achieved compared to the ergoline alkaloids. Maximal responses to all the ergopeptine alkaloids, however, all persisted for the 120-min duration with negligible relaxation occurring. The different classes of alkaloids differed greatly in the type of contractile response generated in the lateral saphenous vein. Persistence of contractile response is thought to be the primary contributing factor to the vasoconstriction observed in animals demonstrating signs of fescue toxicosis, where different ergot alkaloids can contribute differently.

Neuro-hormonal regulation of cardiac function via cathecol-amines results in increased heart rate and contractility. A persistent adrenergic tone, however, is an insult to the heart, affecting its regular homeostasis, altering morphology and gene expression patterns, as well as inducing apoptosis of cardio-myocytes. At the same time as being the main oxygen consumers, mitochondria are also key to the energy production required for the heart to maintain its vital functions and to integrate a series of signaling pathways that define the life and death of the cell. As α-adrenergic receptors (α-AR) orchestrate multiple biochemical events that can either trigger or inhibit cell death, mitochondria can act as a referee in the entire process. In fact, α-AR subtypes α1 and α2 activate various down-stream pathways which differently modulate intracellular calcium levels and production of mitochondrial reactive oxygen species (ROS). The delicate balance between an adaptive (cardio-protective) response resulting in increased contractility and activation of survival pathways, vs. cell death caused by calcium and ROS-induced mitochondrial disruption, along with evidence of their clinical and potential therapeutic translations, are reviewed in this communication.

β-Adrenergic receptor stimulation plays an important role in cardiomyocyte stress responses, which may result in apoptosis and cardiovascular degeneration. We previously demonstrated that toxicity of the β-adrenergic agonist isoproterenol on H9c2 cardiomyoblasts depends on the stage of cell differentiation. We now investigate β-adrenergic receptor downstream signaling pathways and stress responses that explain the impact of muscle cell differentiation on hyper-β-adrenergic stimulation-induced cytotoxicity. When incubated with isoproterenol, differentiated H9c2 muscle cells have increased cytosolic calcium, cyclic-adenosine monophosphate content and oxidative stress, as well as mitochondrial depolarization, increased superoxide anion, loss of subunits from the mitochondrial respiratory chain, decreased Bcl-xL content, increased p53 and phosphorylated-p66Shc as well as activated caspase-3. Undifferentiated H9c2 cells incubated with isoproterenol showed increased Bcl-xL protein and increased superoxide dismutase 2 which may act as protective mechanisms. We conclude that the differentiation of H9c2 is associated with differential regulation of stress responses, which impact the toxicity of several agents, namely those acting through β-adrenergic receptors and resulting in mitochondrial disruption in differentiated cells only., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A characteristic component of the anti-neoplastic doxorubicin (DOX)-induced cardiac toxicity is the delayed and persistent toxicity, with cancer childhood survivors developing cardiac failure later in life. The mechanisms behind this persistent toxicity are unknown, although one of the consequences of early childhood treatment with DOX is a specific removal of cardiac progenitor cells. DOX treatment may be more toxic to undifferentiated muscle cells, contributing to impaired cardiac development and toxicity persistence. H9c2 myoblasts, a rat embryonic cell line, which has the ability to differentiate into a skeletal or cardiac muscle phenotype, can be instrumental in understanding DOX cytotoxicity in different differentiation stages. H9c2 cell differentiation results in decreased cell proliferation and increased expression of a differentiated muscle marker. Differentiated H9c2 cells accumulated more DOX and were more susceptible to DOX-induced cytotoxicity. Differentiated cells had increased levels of mitochondrial superoxide dismutase and Bcl-xL, an anti-apoptotic protein. Of critical importance for the mechanisms of DOX toxicity, p53 appeared to be equally activated regardless of the differentiation state. We suggest that although more differentiated H9c2 muscle cells appear to have more basal mechanisms that would predict higher protection, DOX toxicity is higher in the differentiated population. The results are instrumental in the understanding of stress responses of this specific cell line in different differentiation stages to the cardiotoxicity caused by anthracyclines.

H9c2 cells are used as a surrogate for cardiac cells in several toxicological studies, which are usually performed with cells in their undifferentiated state, raising questions on the applicability of the results to adult cardiomyocytes. Since H9c2 myoblasts have the capacity to differentiate into skeletal and cardiac muscle cells under different conditions, the hypothesis of the present work was that cells in different differentiation states differ in their susceptibility to toxicants. In order to test the hypothesis, the effects of the cardiotoxicant isoproterenol (ISO) were investigated. The present work demonstrates that differentiated H9c2 cells are more susceptible to ISO toxicity. Cellular content of beta(1)-adrenergic receptors (AR), beta(3)-AR, and calcineurin is decreased as cells differentiate, as opposed to the content on the mitochondrial voltage-dependent anion channel (VDAC) and phosphorylated p38-MAPK, which increase. After ISO treatment, the pro-apoptotic protein Bax increases in all experimental groups, although only undifferentiated myoblasts up-regulate the anti-apoptotic Bcl-2. Calcineurin is decreased in differentiated H9c2 cells, which suggests an important role against ISO-induced cell death. The results indicate that the differentiation state of H9c2 myoblasts influence ISO toxicity, which may involve calcineurin, p38-MAPK, and Bax/Bcl-2 alterations. The data also provide new insights into cardiovascular toxicology during early development.

H9c2 cells, derived from the ventricular part of an E13 BDIX rat heart, possess a proliferative and relatively undifferentiated phenotype but can be readily directed to differentiate under reduced serum conditions originating cells presenting muscle features. Skeletal or cardiac phenotypes can be originated depending on whether or not serum reduction is accompanied by a daily treatment with all-trans-retinoic acid. In the present study, we aimed to characterize and compare the metabolic profile of H9c2 cells at various differentiation states, correlating the differences between different populations with muscle-specific development. We determined that H9c2 myoblasts remodel their metabolism upon differentiation, with undifferentiated cells more reliant on glycolysis, as demonstrated by higher lactate production rates. Differentiated cells adopted a more oxidative metabolism with better coupling between the glycolytic and oxidative pathways, which is indicative of a metabolic evolvement toward a higher energetic efficiency state. Our findings emphasize the metabolic differences between differentiated and undifferentiated H9c2 cells and raise caution on how to adequately select the H9c2 differentiation state that will act as the better model for the design of experimental studies.

Sanguinarine (SANG) is an alkaloid recognized to have anti-proliferative activity against various human tumour cell lines. No data is available on the susceptibility of advanced malignant melanoma to SANG, although this disease has a very poor prognosis if not detected in time due to the resistance to conventional chemotherapy. The present work was designed to study the nuclear and mitochondrial involvement in the pro-apoptotic effect of SANG in an invasive mouse melanoma cell line. The results obtained show that SANG is primarily accumulated by the cell nuclei, causing inhibition of cell proliferation and inducing cell death, as confirmed by an increase in sub-G1 peaks. At low concentrations, SANG induces mitochondrial depolarization in a sub-population of melanoma cells, which also generally displayed strong nuclear labelling of phosphorylated histone H2AX. Western blotting revealed an increase in p53, but not Bax protein, in both whole-cell extracts and in mitochondrial fractions. Isolated hepatic mitochondrial fractions revealed that SANG affects the mitochondrial respiratory chain, and has dual effects on mitochondrial calcium loading capacity. We suggest that SANG is able to induce apoptosis in metastatic melanoma cells. The knowledge of mitochondrial vs. nuclear effects of SANG is important in the development of this promising compound for clinical use against aggressive melanoma.

Objectives were to determine effects of feeding a culture of Saccharomyces cerevisiae on performance, health, and immunocompetence of calves in the first 70 d of age. Holstein calves (n = 512) at 2 +/- 1 d of age were randomly assigned to yeast culture (YC, 218 females and 37 males) or control (223 females and 34 males). Yeast culture was fed at 2% of the grain dry matter. All calves received colostrum during the first 24 h, pasteurized milk thereafter until 60 d of age, and grain was fed ad libitum for the first 70 d of age. Calves were housed in individual hutches, and grain intake was measured 5 d/wk. Body weight was measured at 5, 30, and 68 d of age, and attitude and fecal consistency were scored daily. Incidence and duration of health disorders and treatments were recorded. Neutrophil phagocytic and killing activities and antibody response to immunization with ovalbumin were measured. Concentrations of glucose and 3-hydroxybutyrate were measured in plasma. Grain intake did not differ between treatments and averaged 908 g/d throughout the study. Body weight change, concentrations of glucose, and 3-hydroxybutyrate did not differ between YC and control. Minor effects on neutrophil function were observed, and YC tended to increase the number of phagocytized bacteria and killing of phagocytized bacteria but did not influence humoral immune response. Attitude scores were similar between treatments throughout the study. Almost all calves experienced mild diarrhea during the study, but feeding YC improved fecal scores, reduced days with watery feces, incidence of fever and diarrhea, and risk of health disorders. Because of the high incidence of diarrhea, mortality preweaning was also high, but YC improved survival of calves by decreasing mortality rate past 13 d of age. Income at the end of the study was improved by $48/calf with YC. Feeding yeast culture in grain improved health, minimized frequency of health treatments, and reduced risk of morbidity and mortality in dairy calves.

Berberine [Natural Yellow 18, 5,6-dihydro-9,10-dimethoxybenzo(g)-1,3-benzodioxolo(5,6-a)quinolizinium] is an alkaloid present in plant extracts and has a history of use in traditional Chinese and Native American medicine. Because of its ability to arrest the cell cycle and cause apoptosis of several malignant cell lines, it has received attention as a potential anticancer therapeutic agent. Previous studies suggest that mitochondria may be an important target of berberine, but relatively little is known about the extent or molecular mechanisms of berberine-mitochondrial interactions. The objective of the present work was to investigate the interaction of berberine with mitochondria, both in situ and in isolated mitochondrial fractions. The data show that berberine is selectively accumulated by mitochondria, which is accompanied by arrest of cell proliferation, mitochondrial fragmentation and depolarization, oxidative stress, and a decrease in ATP levels. Electron microscopy of berberine-treated cells shows a reduction in mitochondria-like structures, accompanied by a decrease in mitochondrial DNA copy number. Isolated mitochondrial fractions treated with berberine had slower mitochondrial respiration, especially when complex I substrates were used, and increased complex I-dependent oxidative stress. It is also demonstrated for the first time that berberine stimulates the mitochondrial permeability transition. Direct effects on ATPase activity were not detected. The present work demonstrates a number of previously unknown alterations of mitochondrial physiology induced by berberine, a potential chemotherapeutic agent, although it also suggests that high doses of berberine should not be used without a proper toxicology assessment.

Eight multiparous Holstein cows averaging 570 +/- 43 kg of body weight and 60 +/- 20 d in milk were used in a double Latin square design with four 21-d experimental periods to determine the effects of feeding ground or whole flaxseed with or without monensin supplementation (0.02% on a dry matter basis) on milk production and composition, feed intake, digestion, blood composition, and fatty acid profile of milk. Intake of dry matter was similar among treatments. Cows fed whole flaxseed had higher digestibility of acid detergent fiber but lower digestibilities of crude protein and ether extract than those fed ground flaxseed; monensin had no effect on digestibility. Milk production tended to be greater for cows fed ground flaxseed (22.8 kg/d) compared with those fed whole flaxseed (21.4 kg/d). Processing of flax-seed had no effect on 4% fat-corrected milk yield and milk protein and lactose concentrations. Monensin supplementation had no effect on milk production but decreased 4% fat-corrected milk yield as a result of a decrease in milk fat concentration. Feeding ground compared with whole flaxseed decreased concentrations of 16:0, 17:0, and cis6-20:4 and increased those of cis6-18:2, cis9, trans11-18:2, and cis3-18:3 in milk fat. As a result, there was a decrease in concentrations of medium-chain and saturated fatty acids and a trend for higher concentrations of long-chain fatty acids in milk fat when feeding ground compared with whole flaxseed. Monensin supplementation increased concentrations of cis9 and trans11-18:2 and decreased concentrations of saturated fatty acids in milk fat. There was an interaction between flaxseed processing and monensin supplementation, with higher milk fat concentration of trans11-18:1 for cows fed ground flaxseed with monensin than for those fed the other diets. Flaxseed processing and monensin supplementation successfully modified the fatty acid composition of milk fat that might favor nutritional value for consumers.

Steers (379 +/- 10 kg) with ruminal, duodenal, and ileal cannulas were used in a 5 x 5 Latin square digestion trial to quantify and evaluate the relationship between intestinal protein supply and intestinal starch disappearance. Treatments were infusions of 0, 50, 100, 150, or 200 g/d of casein along with 1,042 g/d of raw cornstarch. Abomasal infusions were accomplished by passing tubing and a pliable retaining washer through the reticular-omasal orifice into the abomasum. Steers were fed a 93% corn silage, 7% supplement diet that contained 12% crude protein at 1.65% body weight in 12 equal portions/d. Periods lasted 17 d (12 d for adaptation, 2 d of collections, and 3 d of rest). The quantity and percentage of organic matter and protein disappearance from the small intestine increased linearly (P < 0.03) with infused casein. Greater quantities of starch disappeared with increased casein infusion (P < 0.01). The infusion of 200 g/d of casein increased small intestinal starch disappearance by 226 g/d over the control. Casein infusion did not affect the quantity or percent of organic matter, starch, or protein disappearance in the large intestine. Treatments did not change ruminal ammonia N, ruminal pH, or plasma glucose concentrations. Starch disappearance from the small intestine was increased with greater protein flow to the duodenum of steers.

Thirteen steers (378+/-23 kg) were used in a split-plot experimental design to evaluate the effect of small intestinal carbohydrate on sodium-glucose cotransport in brush border membrane vesicles prepared from five equidistant sites along the small intestine. The steers consumed 7.2+/-0.4 kg/d ground fescue hay and soybean meal-based supplement and were infused ruminally or postruminally with a partial alpha-amylase starch hydrolysate (914.5+/-8.3 g/d) for 7 d. On d 7, five equidistant 1-m small intestinal sections were harvested and frozen in liquid N for later preparation of brush-border membrane vesicles. Maltase activity of the homogenate and vesicle preparations changed (P < 0.001; lowest in the duodenum, highest in the jejunum) and alkaline phosphatase decreased (P < 0.001) along the small intestine. With respect to the original homogenates, the vesicle preparations were enriched 9.80+/-0.83- and 7.64+/-0.67-fold for alkaline phosphatase and maltase, respectively; enrichments were not different between treatments (P = 0.76 and 0.39, respectively). However, alkaline phosphatase and maltase enrichment changed (P < 0.001) along the small intestine. Recoveries of alkaline phosphatase and maltase activities (25.0+/-0.2% and 19.5+/-0.2%, respectively) in the vesicle preparation were not affected (P = 0.29 and 0.21, respectively) by treatment but changed (P < 0.001) along the intestine. Recovery of protein in the vesicle preparation was 2.60+/-0.01% and was not affected by treatment or intestinal site. Sodium-glucose cotransport activity (220+/-44 pmol x mg(-1) x s(-1)) was not affected (P = 0.34) by treatment but did change (P < 0.001; lowest in the ileum, highest in the proximal and mid-jejunum) along the small intestine. Apparent Km of the sodium-glucose cotransporter for glucose was 62.8+/-5.8 microM. The specific activity of maltase was highest in the jejunum, and sodium-glucose cotransport was highest in the first two jejunal sites. However, duodenal maltase activity was lowest and ileal sodium-glucose cotransport activity was lowest. Sodium-glucose cotransport activity may limit small intestinal starch assimilation in the distal small intestine. It does not seem that glucose arising from carbohydrate hydrolysis regulates activity of sodium-dependent glucose transport in cattle.

We measured the effects of the addition of tannic acid on chemical composition and crude protein (CP) ruminal degradability of bermudagrass, cv. coast cross (Cynodon dactylon L.) and alfalfa (Medicago sativa L.) silages with nylon bags incubated in the rumen of two fistulated lactating cows. Silage pH was greater for alfalfa than for bermudagrass. The addition of tannic acid had no effect on silage pH. Ammonia N was greater in alfalfa than in bermudagrass silage. Treated and control silages had similar ammonia N concentrations. The percentage of CP that was solubilized at time 0 was lower for alfalfa than for bermudagrass silage. The addition of tannic acid decreased the portion of CP solubilized at time 0 and increased the potentially degradable fraction of CP. The potentially degradable fraction of CP was greater for alfalfa than for bermudagrass. There was an interaction of species and treatment; the disappearance rate of CP increased with tannic acid treatment of alfalfa but decreased with acid treatment of bermudagrass. The effective degradability of CP was similar for control and treated alfalfa but lower for treated than for control bermudagrass. Tannic acid treatment is effective in decreasing the rapidly soluble fraction of alfalfa and bermudagrass silages, which could be beneficial to the animal because it would decrease the excess of N in the rumen after feeding. Tannic acid treatment decreased CP degradability of bermudagrass silage but had no effect on alfalfa silage, suggesting that tannic acid concentration required to effectively decrease CP degradability differs among forages.

Four Angus steers (318 +/- 16 kg) fitted with ruminal, duodenal, and ileal cannulas were used in a 4 x 4 Latin square design to determine carbohydrate disappearance from the small intestine (SI). Steers were fed fescue hay at 1.8% of BW and abomasally infused with starch hydrolysate (SH) at 10, 20, or 40 g/h or glucose (G) at 30 g/h. Starch hydrolysate was raw cornstarch digested by a heat-stable alpha-amylase. Experimental periods were 10 d with 6 d of adaptation, 3 d of digesta and feces collection, and 1 d of rest. Glucose (% of infused) had greater (P < .001) apparent small intestinal and postruminal disappearance (% of infused) compared with 20 and 40 g/h SH. Starch hydrolysate infusion linearly increased (P < .001) apparent SI, large intestinal (LI), and total intestinal starch disappearance (g/d) and quadratically increased (P < .003) apparent SI and total intestinal starch disappearance (% of infused). Ileal starch flow from infusion increased quadratically (P < .03) as SH infusion increased. True SI and total intestinal starch disappearance increased linearly (P < .001; g/d) with SH infusion. However, SH infusion quadratically decreased (P < .02) efficiency of true SI starch disappearance (% of infused). True LI starch disappearance (g/d and % of infused) quadratically increased (P < .03) as SH infusion increased. These data demonstrate that, even in animals fed all-forage diets, there is a significant flow of alpha-glucosides, and these need to be considered when evaluating intestinal carbohydrate digestion.

Six Angus steers (260+/-4 kg initial BW) fitted with ruminal, duodenal, and ileal cannulas were used in a 6 x 6 Latin square design to evaluate the effect of feeding poultry by-product meal (PBM) on small intestinal flow and disappearance of amino acids. The diets were provided at 2% of BW on a DM basis, formulated to contain 11.5% CP, and consisted of 49% corn silage, 36% cottonseed hulls, and 15% supplement on a DM basis. Supplements were formulated to contain 37% CP with sources of supplemental N being soybean meal (100% SBM) and 0, 25, 50, 75, and 100% PBM, with urea used to balance for N. Duodenal flow of all amino acids increased linearly (P < .07) as PBM increased in the diet and, except for His, increased (P < .09) for 100% PBM compared with 100% SBM. Similar results were observed for duodenal flow of nonbacterial amino acids, which linearly increased (P < .05) with PBM and were greater (P < .05) for 100% PBM than for 100% SBM. Soybean meal increased (P < .09) the duodenal flow of nonbacterial Lys compared with 0% PBM, and 0% PBM increased (P < .04) flow of Val, Ala, and Pro compared with 100% SBM. Duodenal bacterial essential, nonessential, and total amino acid flows were not affected (P > .80) by PBM; however, they were greater (P < .02) for 100% SBM than for 100% PBM. In addition, nonessential and total bacterial amino acid flows were increased (P < .06) for 100% SBM compared with 0% PBM. Small intestinal disappearance of Lys and Pro increased linearly (P < .09) as PBM increased, and 100% PBM increased (P < .07) disappearance of Arg and Ala compared with 100% SBM. Supplemental N source had no effect (P > .31) on apparent small intestinal disappearance of essential, nonessential, and total amino acids. These data suggest that when PBM, SBM, and urea were used as sources of supplemental N, the daily disappearance of amino acids from the small intestine of steer calves consuming a corn silage- and cottonseed hull-based diet was similar.

We conducted three studies with steers to evaluate poultry by-product meal (PBM) as a supplemental N source for ruminants. An in situ study compared the solubility, degradation rate, and ruminal escape of PBM N with blood meal (BM), corn gluten meal (CGM), and soybean meal (SBM) N. Additionally, an 84-d growth study (n = 95, 228+/-5 kg BW) and a digestion trial (6 x 6 Latin square) were conducted. The basal diet for the growth and digestion studies consisted of 49% corn silage, 36% cottonseed hulls, and 15% supplement (DM basis). Sources of supplemental N (% of total supplemental N) were 100% SBM and 0, 25, 50, 75, and 100% PBM, with urea used to balance for N. In situ ruminal escape N (25.2, 55.3, 86.7, and 98.9% for SBM, PBM, CGM, and BM, respectively) was greater (P < .05) for PBM than for SBM; however, a greater (P < .05) proportion of BM and CGM N escaped ruminal degradation compared with PBM. Dry matter intake, ADG and gain/ feed increased linearly (P < .003) as PBM increased; however, no differences (P > .48) were observed in these variables for 100% PBM compared with 100% SBM. Duodenal N flow and small intestinal N disappearance increased linearly (P < .05) as PBM increased in the diet. Bacterial N flow to the small intestine was not affected (P > .19) by treatment; however, 100% SBM decreased (P < .04) bacterial CP synthesis (g bacterial N/kg OM disappearance from the stomach) compared with 0 and 100% PBM. In vivo ruminal escape N of PBM and SBM was 40.6 and 13.7%, respectively. Ruminal NH3 N decreased linearly (P < .001) as PBM increased. These data suggest that PBM can replace SBM as a source of supplemental N for steer calves that consume a diet based on corn silage and cottonseed hulls.