Rapid Glycolysis Research Articles

ANALYSIS OF QUALITY INDICATORS OF CHILLED AND FROZEN BEEF

ANALYSIS OF QUALITY INDICATORS OF CHILLED AND FROZEN BEEF

Dietary creatine nitrate enhances muscle creatine loading and delays postmortem glycolysis of broilers that experienced preslaughter transport.

This study investigated the attenuating effects of dietary creatine nitrate (CrN), a novel form of creatine, on energy expenditure and rapid glycolysis in pectoralis major (PM) muscle of broiler induced by preslaughter transport. A total of 288 Arbor Acres broilers (28 day old) were randomly assigned into five dietary treatments, including a basal diet or the basal diet supplemented with 600 mg/kg guanidinoacetic acid (GAA), 300, 600, or 900 mg/kg CrN for 14 d, respectively. On the day of transportation, the broilers from basal diet group were divided into two equal groups: one group was transported for 0.5 h (Control group) and the other group was transported for 3 h (T3h group). Meanwhile, the birds from GAA and CrN supplementation groups were transported for 3 h (identified as GAA600, CrN300, CrN600, and CrN900 group, respectively). The results demonstrated that dietary supplementation of GAA or CrN from 28 to 42 d of age did not significantly affect the growth performance, carcass traits, and textural characteristics (P > 0.05) in PM muscle of transported broilers. Compared with T3h group, GAA600, CrN600, and CrN900 groups increased the pH45min (P < 0.01), and CrN600, CrN900 groups decreased the cooking loss (P < 0.05) of PM muscle. Meanwhile, the muscle of GAA600, CrN600, and CrN900 groups showed a higher glycogen content (P < 0.01) and a lower lactic acid content (P < 0.01). GAA600 and all CrN treatments enhanced muscle Cr content and reduced AMP/ATP ratio (P < 0.01). In addition, GAA600 and all CrN treatments downregulated the relative mRNA expression levels of LKB1 and AMPKα2 (P < 0.001) and the protein expression of p-AMPKαThr172 compared with the T3h group (P < 0.01). All CrN treatments showed lower protein expression levels of LKB1 and p-LKB1Thr189 than those of the T3h group (P < 0.05). In summary, dietary supplementation with GAA and CrN enhanced the content of muscle creatine, and inhibited transport-induced activation of LKB1/AMPK pathway, which is beneficial for delaying rapid muscle glycolysis and improving meat quality.

Designing Lactate Dehydrogenase-Mimicking SnSe Nanosheets To Reprogram Tumor-Associated Macrophages for Potentiation of Photothermal Immunotherapy.

Rapid glycolysis of tumor cells produces excessive lactate to trigger acidification of the tumor microenvironment (TME), leading to the formation of immunosuppressive TME and tumor-associated macrophage (TAM) dysfunction. Therefore, reprogramming TAMs by depleting lactate with nanodrugs is expected to serve as an effective means of tumor-targeted immunotherapy. Herein, we report the use of lactic acid dehydrogenase (LDH)-mimicking SnSe nanosheets (SnSe NSs) loaded with a carbonic anhydrase IX (CAIX) inhibitor to reconstruct an acidic and immunosuppressive TME. As expected, this nanosystem could reprogram the TAM to achieve M1 macrophage activation and could also restore the potent tumor-killing activity of macrophages while switching their metabolic mode from mitochondrial oxidative phosphorylation to glycolysis. In addition, the repolarizing effect of SnSe NSs on macrophages was validated in a coculture model of bone marrow-derived macrophages, in three patient-derived malignant pleural effusion and in vivo mouse model. This study proposes a feasible therapeutic strategy for depleting lactate and thus ameliorating acidic TME employing Se-containing nanosheets, which could further amply the effects of TAM-based antitumor immunotherapy.

The histone deacetylase Hdac7 supports LPS-inducible glycolysis and Il-1β production in murine macrophages via distinct mechanisms.

TLRs reprogram macrophage metabolism, enhancing glycolysis and promoting flux through the tricarboxylic acid cycle to enable histone acetylation and inflammatory gene expression. The histone deacetylase (HDAC) family of lysine deacetylases regulates both TLR-inducible glycolysis and inflammatory responses. Here, we show that the TLR4 agonist LPS, as well as agonists of other TLRs, rapidly increase enzymatic activity of the class IIa HDAC family (HDAC4, 5, 7, 9) in both primary human and murine macrophages. This response was abrogated in murine macrophages deficient in histone deacetylase 7 (Hdac7), highlighting a selective role for this specific lysine deacetylase during immediate macrophage activation. With the exception of the TLR3 agonist polyI:C, TLR-inducible activation of Hdac7 enzymatic activity required the MyD88 adaptor protein. The rapid glycolysis response, as assessed by extracellular acidification rate, was attenuated in Hdac7-deficient mouse macrophages responding to submaximal LPS concentrations. Surprisingly however, reconstitution of these cells with either wild-type or an enzyme-dead mutant of Hdac7 enhanced LPS-inducible glycolysis, whereas only the former promoted production of the inflammatory mediators Il-1β and Ccl2. Thus, Hdac7 enzymatic activity is required for TLR-inducible production of specific inflammatory mediators, whereas it acts in an enzyme-independent fashion to reprogram metabolism in macrophages responding to submaximal LPS concentrations. Hdac7 is thus a bifurcation point for regulated metabolism and inflammatory responses in macrophages. Taken together with existing literature, our findings support a model in which submaximal and maximal activation of macrophages via TLR4 instruct glycolysis through distinct mechanisms, leading to divergent biological responses.

Transcriptional and Metabolic Profiling of Nicotinamide-Enhanced Natural Killer (NAM-NK) Cells (GDA-201)

Adoptive transfer of NK cells is a promising immunotherapeutic modality, however limited NK cell persistence and proliferation in vivo have historically been barriers to clinical success. Nicotinamide (NAM), an allosteric inhibitor of NAD-dependent enzymes, has been shown to preserve cell function and prevent differentiation in ex vivo culture of NK (NAM-NK) and other cells. Clinical responses were observed in a Phase 1 trial of NAM-NK (GDA-201) in patients with refractory non-Hodgkin lymphoma (Bachanova, et. al., Blood 134:777, 2019). We now use transcriptional and metabolic profiling to characterize the mechanisms underlying the activity of NAM-NK.CD3 negative lymphocytes obtained from healthy donors were cultured for 14 days with IL-15 in the presence or absence of NAM (7 mM). Next generation sequencing (NGS), liquid chromatography-mass spectrometry (LC-MS)-based metabolite quantification, and glycolytic/mitochondrial respiration measurements were performed. Transcriptome and pathway enrichment analyses were performed with Ingenuity Pathway Analysis software. Extracted cellular and medium metabolites were analyzed on a Thermo Q-Exactive Plus mass spectrometer coupled with a Vanquish UHPLC system. Extracellular acidification (ECAR) and oxygen consumption rates (OCR) were quantified using a Seahorse Extracellular Flux Analyzer. Glycolysis/citric acid cycle (TCA) rates were measured using isotope-labelled glucose incorporation assays.Transcriptome analyses defined 1,204 differentially expressed (DE) genes in NAM-NK vs. control NK. Biological/functional enrichment and pathway analyses of DE-genes predicted upregulation of cell cycle, DNA replication (CDK4/CDKN2D, CyclinD/E, MAD2L), RNA transcription, translation (SMN1/2, ABCF1, EIF4B, RPL13, RPS6), protein synthesis (EIF2, PABPC1, SOS, 60S complex) mitochondrial energy metabolism (NDUFB8, ATP5G2/E, COX7B/C) migration, homing (CD62L, CD44, DNAM1), and cytokine/chemokine response (IL18R, CXCR3, CCR5, XCL1, SOCS3, LFA1) pathways, with concomitant downregulation of cell exhaustion, senescence (BATF1, FOXP1, STAT1, CD86, LGALS9, LAG3), apoptosis, necrosis (CASP1, MDM2, IKK3), stress response (CALR, HSP90, HSPH1), and lymphoid cellular maturation (IL-2Ra, CD40L, GATA3) pathways in NAM-NK. Metabolomic analyses showed a significant increase of intracellular NAD, NADH, NADP, NADPH, high-energy triphosphates (ATP, UTP, GTP) and overall energy charge ([ATP+0.5*ADP]/[ATP+ADP+AMP]) in NAM-NK. Cellular metabolic fitness analyses revealed increased basal and ATP-linked respiration, mitochondrial maximal respiratory capacity, and glycolytic capacity in NAM-NK compared to control NK. In addition, NAM increased the rate of glucose incorporation into TCA cycle intermediates (acetyl-CoA, succinyl-CoA), consistent with a more rapid glycolysis rate, increased TCA cycling, and improved glucose consumption efficiency.Taken together, results of transcriptome, metabolomic, mitochondrial respiration, and glycolytic rate analyses suggest that NAM pleiotropically modulates key cellular metabolic functions in ex vivo-expanded NK cells, resulting in increased response to cytokine stimulation and enhanced potency. NAM inhibits differentiation, cellular stress, and exhaustion pathways that are typically activated in culture. Moreover, NAM increases cellular metabolic fitness, energy charge, and efficiency of glucose consumption, potentially imparting a protective effect against oxidative stress in the tumor microenvironment. These data offer insight into the mechanism of improved persistence, proliferation, and cytotoxicity observed in in vivo and clinical studies of GDA-201. DisclosuresYackoubov: Gamida Cell: Current Employment. Pato: Gamida Cell: Current Employment. Rifman: Gamida Cell: Current Employment. Cohen: Gamida Cell: Current Employment. Hailu: Gamida Cell: Current Employment. Persi: Gamida Cell: Current Employment. Berhani-Zipori: Gamida Cell: Current Employment. Edri: Gamida Cell: Current Employment. Peled: Biokine Therapeutics Ltd: Current Employment; Gamida Cell: Research Funding. Cichocki: Gamida Cell: Research Funding; Fate Therapeutics, Inc: Patents & Royalties, Research Funding. Rabinowitz: Gamida Cell: Research Funding. Lodie: Gamida Cell: Current holder of stock options in a privately-held company, Ended employment in the past 24 months. Adams: Gamida Cell: Current Employment. Simantov: Gamida Cell: Current Employment. Geffen: Gamida Cell: Current Employment.

Upregulating sirtuin 6 ameliorates glycolysis, EMT and distant metastasis of pancreatic adenocarcinoma with kr\xfcppel-like factor 10 deficiency

Krüppel-like factor 10 (KLF10) is a tumor suppressor in multiple cancers. In a murine model of spontaneous pancreatic adenocarcinoma (PDAC), additional KLF10 depletion accelerated distant metastasis. However, Klf10 knockout mice, which suffer from metabolic disorders, do not develop malignancy. The mechanisms of KLF10 in PDAC progression deserve further exploration. KLF10-depleted and KLF10-overexpressing PDAC cells were established to measure epithelial-mesenchymal transition (EMT), glycolysis, and migration ability. A murine model was established to evaluate the benefit of genetic or pharmacological manipulation in KLF10-depleted PDAC cells (PDACshKLF10). Correlations of KLF10 deficiency with rapid metastasis, elevated EMT, and glycolysis were demonstrated in resected PDAC tissues, in vitro assays, and murine models. We identified sirtuin 6 (SIRT6) as an essential mediator of KLF10 that modulates EMT and glucose homeostasis. Overexpressing SIRT6 reversed the migratory and glycolytic phenotypes of PDACshKLF10 cells. Linoleic acid, a polyunsaturated essential fatty acid, upregulated SIRT6 and prolonged the survival of mice injected with PDACshKLF10. Modulating HIF1α and NFκB revealed that EMT and glycolysis in PDAC cells were coordinately regulated upstream by KLF10/SIRT6 signaling. Our study demonstrated a novel KLF10/SIRT6 pathway that modulated EMT and glycolysis coordinately via NFκB and HIF1α. Activation of KLF10/SIRT6 signaling ameliorated the distant progression of PDAC.Clinical Trial Registration: ClinicalTrials.gov. identifier: NCT01666184.

Effects of preslaughter shackling on postmortem glycolysis, meat quality, changes of water distribution, and protein structures of broiler breast meat

The objective of this study was to investigate the effects of preslaughter shackling on stress, postmortem glycolysis, meat quality, water distribution, and protein structures of pectoralis majors. Before slaughter, Arbor Acres broilers (n = 105, 42 days old, 2.0 to 2.5kg) were randomly categorized into 3 treatment groups: (I) control group without shackling (NS); (II) 2.5min shackling (SS); (III) 4.5min shackling (LS). Each treatment group consisted of 5 replicates with 7 broilers each. Results indicated that preslaughter shackling increased (P < 0.05) plasma corticosterone and adrenocorticotropic hormone concentrations in comparison with the control group. Antemortem shackling increased (P < 0.05) activity of glycogen phosphorylase and phosphofructokinase-1 (PFK-1) accompanying with rapid glycolysis and pH decline at early postmortem. LS treatment led to myosin denaturation, decreased (P < 0.05) α-helix content, and increased (P < 0.05) β-sheet structures proportion in the myofibrillar proteins. Furthermore, meat from LS treatment had higher (P < 0.05) lightness, redness, and poorer water-holding capacity. These results indicated that the longer shackling duration (4.5min) increased stress and the rate of glycolysis, causing myosin denaturation and changes of the secondary structure in the myofibrillar proteins, which aggravated the deterioration of meat quality.

Attenuating effects of guanidinoacetic acid on preslaughter transport-induced muscle energy expenditure and rapid glycolysis of broilers

This study evaluated the effects of dietary guanidinoacetic acid (GAA) supplementation on growth performance, plasma variables, muscle energy status, glycolytic potential, and meat quality of broilers experiencing transport stress during the summer. A total of 320 28-day-old male Arbor Acres broilers were randomly allotted to 3 dietary treatments, including a GAA-free basal control diet (160 birds) and basal diet supplementation with 600 (80 birds) or 1,200mg/kg (80 birds) GAA for 14 D. On the morning of day 42, after an 8-h fast, the birds fed basal diets were divided into 2 equal groups, and all birds in the 4 groups of 80 birds were transported according to the following protocols: 1) a 0.5-h transport of birds on basal diets (as a lower-stress control group), 2) a 3-h transport of birds on basal diets, and a 3-h transport of birds on basal diets supplemented with either 3) 600 or 4) 1,200mg/kg GAA. The results revealed that dietary supplementation with GAA at 600 and 1,200mg/kg for 14 D prior to slaughter did not affect growth performance, carcass traits, and most textural characteristics and chemical composition of the pectoralis major (PM) muscle (P > 0.05). In the GAA-free group, a 3-h transport increased the broiler live weight loss, elevated the plasma corticosterone concentration, decreased the plasma glucose concentration, muscle concentrations of ATP, creatine and energy charge value, increased the muscle AMP concentration and AMP/ATP ratio, and accelerated glycolysis metabolism, which resulted in inferior meat quality (lower pH and higher drip loss, P < 0.05). However, dietary addition of GAA at 1,200mg/kg increased the mRNA expression of S-adenosyl-l-methionine: N-guanidino-acetate methyltransferase in the liver and creatine transporter in both the liver and PM muscle. It also elevated muscle concentrations of creatine and phosphocreatine (P < 0.05), which helps improve meat quality by ameliorating the 3-h transport-induced muscle energy expenditure and delaying anaerobic glycolysis of broilers.

Effect of pre-slaughter shackling and wing flapping on plasma parameters, postmortem metabolism, AMPK, and meat quality of broilers

The objective of this study was to determine the effects of shackling and wing flapping on stress, postmortem metabolism, AMP-activated protein kinase (AMPK), and quality of broiler pectoralis major. Before slaughter, a total of 80 Arbor Acres broilers was randomly categorized into 2 replicate pens (40 broilers per pen) and each pen randomly divided into 2 groups (shackling, T; control, C). Corticosterone, creatine kinase, and lactate dehydrogenase were determined on blood plasma parameters. Pectoralis major were removed after evisceration and used for determination of energy metabolism, meat quality, and AMPK phosphorylation. In this study, shackling and wing flapping increased (P < 0.05) plasma corticosterone level, creatine kinase activity, and lactate dehydrogenase activity. Shackling and wing flapping increased (P < 0.05) AMPKα(Thr172) and acetyl-CoA carboxylase (ACC) phosphorylation, followed by rapid glycolysis and accumulation of lactic acid, and leading to a fast pH decline in the initial postmortem meat. Shackling and wing flapping have an adverse effect on final meat quality, which increased (P < 0.05) muscle lightness, drip loss, and cooking loss. The results indicate that antemortem shackling and wing flapping increased stress and AMPKα(Thr172) phosphorylation, which may accelerate glycolysis and lead to a low water-holding capacity of broiler meat.

Creatine Monohydrate Enhances Energy Status and Reduces Glycolysis via Inhibition of AMPK Pathway in Pectoralis Major Muscle of Transport-Stressed Broilers.

Creatine monohydrate (CMH) contributes to reduce transport-induced muscle rapid glycolysis and improve meat quality of broilers, but the underlying mechanism is still unknown. Therefore, this study aimed to investigate the molecular mechanisms underlying the ameliorative effects of CMH on muscle glycolysis metabolism of transported broilers during summer. The results showed that 3 h transport during summer elevated chicken live weight loss and plasma corticosterone concentration; decreased muscle concentrations of ATP, creatine, and energy charge value; increased muscle AMP concentration and AMP/ATP ratio; and upregulated muscle mRNA expression of LKB1 and AMPKα2, as well as protein expression of p-LKB1Thr189 and p-AMPKαThr172, which subsequently resulted in rapid glycolysis in the pectoralis major muscle and consequent reduction of meat quality. Dietary addition of CMH at 1200 mg/kg ameliorated transport-induced rapid muscle glycolysis and reduction of meat quality via enhancement of the energy-buffering capacity of intramuscular phosphocreatine/creatine system and inhibition of AMPK pathway.

Study of blood sugar levels in high risk neonates using glucometer method and laboratory glucose oxidase peroxidase method

Background: The glucose oxidase method used for determining the blood glucose concentration is time consuming, requires more quantity of blood and rapid glycolysis of red blood cells glucose which mandates early estimation using faster techniques. A blood glucose method requiring one drop of blood and giving reliable results within a minute would help greatly in identifying and treating the infants who require extra carbohydrate. Glucometers have the advantage of being simpler, uses less quantity of blood, quick reading and can be handled even by unskilled personnel. Hence, there is a need to know the efficacy and accuracy of glucometer used in our set up.Methods: About 150 infants admitted in NICU and high risk neonates in post-natal ward in department of pediatrics at our Hospital from June 2014 to June 2015 were included in the study. The blood samples were collected immediately after birth or within 5 minutes of admission. Comparison of variables representing categorical data (between two markers) were estimated by Chi Square Test. Sensitivity, Specificity and Predictive Values of Glucometer were calculated by using formulas. All values will be reported based on two-sided and all the statistical tests were interpreted at 5% level of significance level (i.e. p &lt;0.05).Results: After the comparison between glucometer and laboratory method, results show that blood glucose estimation using capillary blood has an excellent pick-up rate of detecting neonatal hypoglycemia. More than 93% of the proven cases of hypoglycemia proved by lab method] were diagnosed when blood glucose estimation with glucometer was done. The overall pick-up rate of hypoglycemia by glucometer is very good compared to the laboratory method.Conclusions: Blood glucose estimation using Glucometer method is an effective method in detecting neonatal hypoglycemia.

Effect of ginger extract and vinegar on ATP metabolites, IMP‐related enzyme activity, reducing sugars and phosphorylated sugars in silver carp during postslaughter storage

SummaryThis study aimed to investigate the utilisation of ginger extract and vinegar for retarding decomposition of inosine monophosphate (IMP), and IMP‐related enzyme activity (the activity of acid phosphatase (ACP) and alkaline phosphatase (ALP)) in salted silver carp (Hypophthalmichthys molitrix) during early postslaughter and chilled storage. We also quantified the decomposition pathway of ATP‐related compounds, the reducing and phosphorylated sugar, and free amino acids (FAAs) in samples. Rapid glycolysis in control occurred during postmortem storage. Vinegar and ginger extract inhibited the activity of ACP and ALP, thereby inhibiting the decomposition of IMP and the increase in hypoxanthine (Hx). IMP concentrations in treated sample were significantly higher than those of the control. Moreover, vinegar and ginger extract promoted the increase in FAAs. Therefore, vinegar and ginger extract could inhibit ACP and ALP activities and contributed to the accumulation of IMP and FAAs in silver carp during chilled storage.

Effect of transportation and pre-slaughter water shower spray with resting on AMP-activated protein kinase, glycolysis and meat quality of broilers during summer.

The aim of this study was to determine the effects of pre-slaughter transport during summer and subsequent water shower spray on stress, postmortem glycolysis, energy metabolism and adenosine monophosphate-activated protein kinase (AMPK) in Pectoralis major (PM) muscle of broilers. Results indicated that transport during high ambient temperature induced the release of plasma corticosterone, which significantly affected stress conditions. Moreover, we found a lower energy status in the early postmortem period compared to the control group. AMPK was activated in this situation, following by the rapid glycolysis and accumulation of lactic acid, leading to a high incidence of pale, soft, exudative (PSE)-like meat. Water shower spray with resting after transport relieved the stress situation, recovered energy homeostasis and lessened the deterioration of meat quality. As a key molecular target for the control of energy status, AMPK has a similar potential in regulating postmortem muscle glycolysis of broilers as in mammals.

Regulation of Aerobic Glycolysis by microRNAs in Cancer.

One of the most common and profound biochemical phenotypes of animal and human cancer cells is their ability to metabolize glucose at high rates, even under aerobic conditions. Such alterations lead to establishment of tumor-specific metabolic machinery that is sufficient for supporting the biosynthetic and energy requirements of the tumor cells for facilitating rapid tumor growth and adaptation to new metastatic niches. These changes entail rapid glycolysis by the tumor cells, shifting the flux of glucose from tricarboxylic acid (TCA) cycle to glycolysis, resulting in generation of vast amounts of lactate, which is then secreted outside the tumor cells. This phenomenon is also termed as Warburg effect, as originally described by Otto Warburg. Several oncogenes and tumor suppressors have been implicated in altering tumor cell metabolism in order to facilitate tumor growth and metastasis. MicroRNAs mediate fine-tuning of the cancerassociated glycolytic pathways either directly or at the level of oncogenes. This article intends to review the mechanisms and pathways by which miRNAs regulate the aerobic glycolysis in cancer.

High early post-mortem temperature induces activation of AMP-activated protein kinase and development of pale, soft and exudative characteristics in turkey muscles

This study investigated the effect of early post-mortem temperature on development of a turkey muscle's pale, soft and exudative (PSE) characteristics. Muscles obtained at 20min post-mortem were incubated at 0, 20 and 40°C until 4h post-mortem and then stored at 4°C. During incubation, the 40°C group had greater rate of pH decline, lactate accumulation and R-values increase than other groups. Moreover, AMP-activated protein kinase (AMPK) activation at 1h post-mortem was higher in the 40°C group than the other groups. At 24h post-mortem, the 40°C group had higher L* values and drip loss; SDS-PAGE and western blotting indicated that lower protein solubility (sarcoplasmic, myofibrillar) in the 40°C group resulted from phosphorylase denaturation and further adhere to myofibrillar fraction. These results suggest that high temperature early post-mortem could induce AMPK activation, which results in rapid glycolysis, thus affecting protein solubility and generating PSE characteristics.

62 EXPRESSION OF KEY ENZYMES OF THE FRUCTOSE METABOLIC PATHWAY IN NEWBORN IN VITRO-DERIVED CALVES

Newborn calves derived from IVF often have difficulties in adapting to life ex utero. Among physiological deviations, plasma fructose levels have been shown to be higher in in vitro-produced calves than in controls during the immediate neonatal period. Interestingly, as plasma fructose levels decrease, plasma lactate levels increase in the first hours of life, which may indicate a biochemical relationship between these substrates (Bertolini et al. 2004 Reproduction 128, 341–354). Fructokinase, the primary enzyme in the fructose metabolic pathway, bypasses the regulatory step catalyzed by phosphofructokinase, allowing fructose to undergo more rapid glycolysis in the liver than does glucose. However, information about the fructose metabolic pathway and fructokinase activity in the liver of neonates is lacking. This study was designed to verify the presence of gene transcripts for the 3 enzymes of the fructose pathway (fructokinase, aldolase B and triokinase) and the relative abundance of transcripts for fructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the enzyme that links the fructose metabolic pathway with glycolysis, in the liver of in vivo- or in vitro-produced newborn calves within the first hour of life. In vivo- or in vitro-produced newborn Flemish calves derived either by superovulation (n = 5) or by IVF (n = 4), respectively, were subject to liver biopsy procedures within the first hour after birth. Tissue samples were placed in cryotubes containing RNAlater™ (Qiagen Inc., Valencia, CA, USA). The RNA was extracted using Trizol® reagent (Invitrogen Corp., Carlsbad, CA, USA) and 300 ng of total RNA from each sample was used for cDNA synthesis with the SuperScript® III First-Strand Synthesis System (Invitrogen Corp.). Each PCR reaction used 5 μM of each specific primer to bovine fructokinase, aldolase B, triokinase and GAPDH (Table 1), 700 ng of cDNA and the PCR Master Mix (Quatro G Ltd., São Paulo, Brazil). For the quantitative PCR (qPCR), the Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) was used in the iQ5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Data for gene expression were normalized with the β-actin housekeeping gene, with the statistical analysis done by t-test (statistical analysis system). The PCR results indicated high fructokinase, aldolase B and triokinase expression levels in the liver of all newborn calves, irrespective of the group. Our preliminary qPCR data showed no differences in the hepatic expression levels for fructokinase (0.319 ± 0.136 vs 0.320 ± 0.179) and GAPDH (0.421 ± 0.243 vs 0.212 ± 0.147) between in vivo- and in vitro-produced calves, respectively. These data suggest that the fructose metabolic pathway is active in in vivo- and in vitro-produced newborn calves in the first hours of life. Consequently, excessive plasma fructose concentrations, as seen in some in vitro-produced calves, may affect carbohydrate metabolism, the acid-base balance and even survival during the neonatal period. Table 1.PCR primers and Gene Bank accession numbers for genes analysed in newborn calves Funded by the RECODISA Project, FINEP/MCT/Brazil.

High post-mortem temperature combined with rapid glycolysis induces phosphorylase denaturation and produces pale and exudative characteristics in broiler Pectoralis major muscles

This study investigated the effect of early post-mortem temperature on broiler protein characteristics and meat quality. Muscles were kept at different temperatures (0, 20 and 40 °C) until 4 h post-mortem and then stored at 4 °C. Rapid degradation of ATP and glycogen, thus inducing a high rate of lactate formation and pH drop, were found in the 40 °C group during incubation. When extracting proteins, a lower protein content of the sarcoplasmic fraction and a higher protein content of the myofibrillar fraction were found in the 40 °C group at 24 h post-mortem; SDS-PAGE and western-blotting results revealed that phosphorylase was associated with the myofibrillar fraction. Furthermore, the 40 °C group had paler surfaces, higher drip loss and lower processing properties. These data suggest that elevated temperature during early post-mortem period, resulting in rapid glycolysis, induced phosphorylase denaturation and association with myofibrillar proteins thus generating pale and exudative characteristics.

Effect of Harvest Treatment on Biochemical Properties of Farmed Chinook Salmon (Oncorhynchus tshawytscha) Tissue during Frozen and Thawed Storage

Two different harvest procedures were employed to investigate whether the method of harvest has an effect upon deteriorative processes that occur during the frozen storage of Chinook salmon (Oncorhynchus tshawytscha) white muscle tissue. These 2 harvest methods, termed "rested"--involving sedation with the aquatic anesthetic AQUI-S and "exercised"--a simulated conventional harvest not involving sedation, contrasted levels of activity of the animal prior to and upon slaughter. Rested and exercised harvesting protocols produced tissue in significantly different postmortem physiological states prior to freezing. Rested, postharvest tissue maintained high metabolic energy stores of ATP and glycogen within the tissue, with low concentrations of tissue and plasma lactate. Exercised tissue exhibited near depleted concentrations of ATP and glycogen and a marked lactate accumulation. In both treatments, no significant change in metabolite levels was seen over a 6-mo storage period at -19 degrees C when tissue was frozen immediately postharvest. Transfer of tissue from frozen temperatures (-80 and -19 degrees C), to refrigerated (-1 and +4 degrees C, respectively) resulted in rapid glycolysis, depleting tissue ATP and glycogen stores and increasing tissue lactate concentrations. Metabolic activity was more significant in rested tissue owing to the larger concentrations of metabolic energy stores and occurred at temperatures between -3 and -1.5 degrees C. During frozen storage (-19 degrees C), there was an increase in the secondary lipid oxidation product TBARS, but harvest treatment had no effect. However, following transfer from frozen to refrigerated (+4 and -1 degrees C) storage, rested tissue showed a significant ability to retard the development of TBARS products.

Different rates of glycolysis affect glycolytic activities and protein properties in turkey breast muscle

Protein alterations of turkey breast muscles (Pectoralis major) were investigated at 20 min and 24 h post mortem. Specific activities, quantities and kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase A were also determined at 20 min post mortem. Based on the pH values at 20 min post mortem, two groups of samples were classified as rapid glycolysis group (RG; pH20 min = 5.80 ± 0.07, n = 20) and normal glycolysis group (NG; pH20 min = 6.21 ± 0.01, n = 20). RG had lower specific activities of GAPDH and aldolase A than NG while Vm and Km values of both enzymes were not different between groups. RG showed lower high ionic strength (HIS) and pellet protein extractabilities at 20 min post mortem. It also had lower low ionic strength (LIS) and HIS protein extractabilities at 24 h post mortem. Besides pellet protein, muscular protein extractabilities at 24 h post mortem were higher than at 20 min post mortem. From SDS-PAGE of samples at 24 h post mortem, RG exhibited lower band intensities at 45 and 200 kDa, which were further identified as actin and myosin heavy chain (MHC), respectively. Western blots revealed that relative amounts of actin and MHC at 20 min post mortem were not different between groups. However, RG muscles had less relative amount of actin at 24 h post mortem. It also indicated that amounts of actin and MHC increased with regard to post mortem time.

Pre-slaughter transport, AMP-activated protein kinase, glycolysis, and quality of pork loin

Numerous studies have revealed that pre-slaughter stress, like transport, increases the occurrence of pale, soft, and exudative (PSE) pork meat. The molecular mechanism underlying this phenomenon, however, is poorly defined. In this study, we investigated the effects of pre-slaughter transport and subsequent rest on energy metabolism, AMP-activated protein kinase (AMPK) activation, and glycolysis in postmortem pork loin. Results indicated that pre-slaughter transport accelerated ATP depletion, which led to lower energy status in postmortem muscle immediately post-exsanguination when compared with control. The lower energy status led to AMPK activation within 1 h postmortem, subsequently increasing glycolysis, leading to rapid glycolysis and high incidence of PSE meat. Allowing pigs to rest after transport restored energy status in muscle ante-mortem. Higher energy status then prevented premature and rapid AMPK activation in postmortem muscle and lessened the negative effects of pre-slaughter transport on meat quality. AMPK regulated glycolysis in postmortem muscle, at least partially, through phosphorylation and activation of phosphofructose kinase-2, since fructose-2,6-diphosphate content, an allosteric activator of phosphofructose kinase-1, was well correlated with AMPK activation and glycolytic rate. This suggests that AMPK is a potential molecular target for the control of PSE incidence in pork.