The impact of postharvest interventions on the color stability and, subsequently, the palatability of beef from cattle fed wet distillers grain.

Two hundred forty heifers were fed at Oklahoma State University in Stillwater, OK, in 1 of 2 treatment groups: a dry rolled corn (CON) diet or a diet including 30% wet distillers grains plus solubles (WDGS). Chuck rolls (n = 60) and paired strip loins (n = 75 pairs; 38 CON and 37 WDGS) were collected from each treatment group and processed at 3 d and 14 d, respectively. After grinding, each chuck was separated into 8 polyvinyl chloride (PVC) film overwrapped packages and 8 high oxygen modified atmosphere packaging (MAP), each containing approximately 0.23 kg of ground beef, for evaluation by a trained color panel, a trained color panel and a trained sensory panel and for thiobarbituric acid reactive substance (TBARS) analysis. After 14 d, 1 strip loin from each pair was injected with an enhancement solution. Steaks from each strip loin were fabricated and packaged, one-half in PVC and one-half in MAP. In addition to the evaluation by trained color and sensory panels and TBARS analysis, steaks were subjected to instrumental color evaluation using a HunterLab Miniscan XE and Warner-Bratzler Shear Force analysis using an Instron Universal Testing Machine. Ground beef exhibited no significant differences in color between dietary treatments; however, sensory panelists did find MAP WDGS had less beefy flavor (P = 0.05) and more painty flavor (P = 0.01) intensities than the MAP CON ground beef. Cattle fed WDGS discolored more (P = 0.01) and had less bright steaks than cattle fed the CON when MAP and enhanced. Distillers fed, nonenhanced (nonE) MAP steaks were redder and yellower than control steaks (P < 0.05) on removal from simulated retail display. There were no other significant (P > 0.05) color differences between dietary treatments using any other combination of postharvest interventions. Sensory panel results indicated WDGS NE PVC products were juicier and more tender (P < 0.05), initially, and contained less connective tissue (5.3 ± 0.1, 5.5 ± 0.1, and 5.9 ± 0.4, respectively) than the steaks from CON carcasses (5.1 ± 0.1, 5.4 ± 0.1, and 5.8 ± 0.4, respectively). Although WDGS NE MAP steaks showed more oxidation than CON NE MAP steaks on removal from retail case, all TBAR values were well below a threshold of 1 mg malonaldehyde/kg. Essentially, MAP but not enhancing products from cattle fed WDGS may be the best way to maintain a visually appealing appearance in the retail case but at possible risk to product juiciness.

Effects of postmortem calcium chloride injection on meat palatability traits of strip loin steaks from cattle supplemented with or without zilpaterol hydrochloride.

An experiment was conducted to determine the effects of zilpaterol hydrochloride mM supplementation (ZH; 8.3 mg/kg on a DM basis for 20 d) and calcium chloride injection [CaCl(2), 200 at 5% (wt/wt) at 72 h postmortem] on palatability traits of beef (Bos taurus) strip loin steaks. Select (USDA) strip loins were obtained from control (no ZH = 19) and ZH-supplemented carcasses (n = 20). Right and left sides were selected alternatively to serve as a control (no INJ) or CaCl(2)-injected (INJ) and stored at 4°C. Before injecting the subprimals (72 h postmortem), 2 steaks were cut for proximate, sarcomere length, and myofibrillar fragmentation index (MFI) analyses. At 7 d postmortem each strip loin was portioned into steaks, vacuum packaged, and aged for the appropriate period for Warner-Bratzler shear force (WBSF; 7, 14, 21, and 28 d postmortem), trained sensory analysis (14 and 21 d postmortem), purge loss (7 d), and MFI (3, 7, 14, 21, and 28 d postmortem). Results indicated steaks from both ZH supplementation and INJ had reduced WBSF values as days of postmortem aging increased. The WBSF values of ZH steaks were greater (P < 0.05) than no ZH steaks at each postmortem aging period. The INJ steaks had lower WBSF values (P < 0.05) than non-injected steaks. A greater percentage (91 vs. 71%) of steaks had WBSF values < 4.6 kg from steers with no ZH supplementation at 7 d postmortem, but the percentage did not differ (P > 0.05) due to ZH at 14, 21, or 28 d or due to INJ at any aging period. Trained panelists rated tenderness less in ZH steaks than steaks with no ZH at 14 d and 21 d. However, INJ improved (P < 0.05) the tenderness ratings and flavor intensity of the trained panelists, compared with their non-injected cohorts at 21 d. Zilpaterol hydrochloride supplementation reduced (P < 0.05) MFI values, but INJ resulted in greater (P < 0.05) MFI values compared with no INJ. Subprimals from ZH and INJ showed greater purge loss (P < 0.05). Although no interactions were found with ZH and CaCl(2), injecting USDA Select strip loins from ZH-fed cattle can help reduce the normal WBSF variation as it does in steaks from non-ZH-fed cattle.

Warner-Bratzler and slice shear force measurements of 3 beef muscles in response to various aging periods after trenbolone acetate and estradiol implants and zilpaterol hydrochloride supplementation of finishing beef steers.

Our objectives were to determine the effects of zilpaterol hydrochloride (ZH) and the release rate of trenbolone acetate and estradiol-17ß on the Warner-Bratzler shear force (WBSF) and slice shear force (SSF) of longissimus lumborum (LL) and the WBSF of gluteus medius (GM) and psoas major (PM) in response to various aging periods. British × Continental steers (n = 168) were assigned to treatments in a 3 × 2 factorial. The main effects of treatment were implant (no implant, Revalor-S, Revalor-XS, Intervet/Schering Plough Animal Health, De Soto, KS) and ZH (0 or 8.3 mg/kg of DM for 20 d). Slaughter group was included as a random effect to account for the variation in days on feed (153 or 174 d). Loins (n = 96) were fabricated to obtain strip loin, top sirloin butt, and tenderloin subprimals. Five 2.54-cm steaks were cut from each subprimal and assigned to 1 of 5 aging periods (7, 14, 21, 28, or 35 d postmortem). Feeding ZH increased (P ≤ 0.01) LL WBSF and SSF values at each aging period compared with controls. Implanting increased (P < 0.05) LL WBSF values at 14 and 21 d, but did not affect LL SSF values (P > 0.05). Only Revalor-S increased (P ≤ 0.05) WBSF values at 28 and 35 d compared with no implant or Revalor-XS. The percentage of LL steaks with a WBSF value below 4.6 kg did not differ (P > 0.05) between ZH supplementation or implant strategy at any aging period, and by d 28, more than 99% of LL steaks registered WBSF values below 4.6 kg. Feeding ZH increased (P < 0.05) GM WBSF values only on d 21. Implant had no effect (P > 0.05) on GM WBSF values. The percentage of GM steaks with a WBSF value below 4.6 kg did not differ (P > 0.05) between ZH supplementation or implant strategy at any aging period. Neither ZH nor implant strategy affected PM WBSF values (P > 0.05). All PM WBSF values were below 4.6 kg on d 7. The results of this study indicated that feeding ZH increased WBSF and SSF of LL steaks, regardless of the aging period; however, the percentage of steaks with WBSF below 4.6 kg did not differ because of ZH or implant. Implanting increased LL WBSF values, but not SSF values. These results showed that although differences existed between implanting, as well as ZH supplementation of British × Continental steers, 99% of LL steaks were classified as tender based on WBSF values by extending aging to 28 d postmortem. It should be noted that 21.2% of 7-d, 13.8% of 14-d, and 17.3% of 21-d ZH steaks had WBSF values greater than 4.6 kg, but 0% of nonsupplemented steaks were greater than 4.6 kg at these aging periods. However, because ZH and implants can increase retail yield of valuable subprimals, such as the tenderloin, considerable value could be captured through ZH supplementation with anabolic implants because shear force was not affected in PM steaks.

Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps.

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.

Substrate-induced nuclear export and peripheral compartmentalization of hepatic glucokinase correlates with glycogen deposition.

Hepatic glucokinase (GK) is acutely regulated by binding to its nuclear-anchored regulatory protein (GKRP). Although GK release by GKRP is tightly coupled to the rate of glycogen synthesis, the nature of this association is obscure. To gain insight into this coupling mechanism under physiological stimulating conditions in primary rat hepatocytes, we analyzed the subcellular distribution of GK and GKRP with immunofluorescence, and glycogen deposition with glycogen cytochemical fluorescence, using confocal microscopy and quantitative image analysis. Following stimulation, a fraction of the GK signal translocated from the nucleus to the cytoplasm. The reduction in the nuclear to cytoplasmic ratio of GK, an index of nuclear export, correlated with a >50% increase in glycogen cytochemical fluorescence over a 60 min stimulation period. Furthermore, glycogen accumulation was initially deposited in a peripheral pattern in hepatocytes similar to that of GK. These data suggest that a compartmentalization exists of both active GK and the initial sites of glycogen deposition at the hepatocyte surface.

Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet beta cells.

Two-photon excitation microscopy was used to image and quantify NAD(P)H autofluorescence from intact pancreatic islets under glucose stimulation. At maximal glucose stimulation, the rise in whole-cell NAD(P)H levels was estimated to be approximately 30 microM. However, because glucose-stimulated insulin secretion involves both glycolytic and Kreb's cycle metabolism, islets were cultured on extracellular matrix that promotes cell spreading and allows spatial resolution of the NAD(P)H signals from the cytoplasm and mitochondria. The metabolic responses in these two compartments are shown to be differentially stimulated by various nutrient applications. The glucose-stimulated increase of NAD(P)H fluorescence within the cytoplasmic domain is estimated to be approximately 7 microM. Likewise, the NAD(P)H increase of the mitochondrial domain is approximately 60 microM and is delayed with respect to the change in cytoplasmic NAD(P)H by approximately 20 sec. The large mitochondrial change in glucose-stimulated NAD(P)H thus dominates the total signal but may depend on the smaller but more rapid cytoplasmic increase.

Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic coupling events in glucose-stimulated insulin secretion.

The relationship between glucokinase (GK) and glucose-stimulated metabolism, and the potential for metabolic coupling between beta cells, was examined in isolated mouse islets by using a recombinant adenovirus that expresses Cre recombinase (AdenoCre) to inactivate a conditional GK gene allele (gklox). Analysis of AdenoCre-treated islets indicated that the gklox allele in approximately 30% of islet cells was converted to a nonexpressing variant (gkdel). This resulted in a heterogeneous population of beta cells where GK was absent in some cells. Quantitative two-photon excitation imaging of NAD(P)H autofluorescence was then used to measure glucose-stimulated metabolic responses of individual islet beta cells from gklox/lox mice. In AdenoCre-infected islets, approximately one-third of the beta cells showed markedly lower NAD(P)H responses. These cells also exhibited glucose dose responses consistent with the loss of GK. Glucose dose responses of the low-responding cells were not sigmoidal and reached a maximum at approximately 5 mM glucose. In contrast, the normal response cells showed a sigmoidal response with an KcatS0.5 of approximately 8 mM. These data provide direct evidence that GK is essential for glucose-stimulated metabolic responses in beta cells within intact islets and that intercellular coupling within the islet plays little or no role in glucose-stimulated metabolic responses.

Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.

We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.

Molecular cloning, sequencing, chromosomal localization, and tissue distribution of the human Na+/H+ exchanger (SLC9A2).

The Na+/H+ exchangers belong to a family of transport proteins involved in intracellular pH regulation and vectorial sodium transport across various epithelial tissues. We have cloned a unique isoform (NHE-2) by screening a rat intestinal cDNA library utilizing a human NHE-1 fragment. In this report we describe molecular cloning of the human Na+/H+ exchanger (NHE-2, HGMW-approved symbol SLC9A2). The cDNA encodes a protein of 698 amino acid residues. Human NHE-2 is widely distributed in tissues of the gastrointestinal tract, kidney, heart, testes, uterus, and adrenal glands. Hydropathy plot indicates that the translated protein is predicted to have 10 transmembrane domains with three potential N-glycosylation sites and four potential cAMP- and cGMP-dependent protein kinase phosphorylation sites. This cDNA maps the location of the gene encoding NHE-2 to human chromosome 2q11.2.

Time course of adaptation to a high-fat diet in obesity-resistant and obesity-prone rats.

The purpose of the present study was to characterize the time course of adaptation (i.e., circulating metabolites and hormones, fat pad mass, lipoprotein lipase) to a high-fat diet in obesity-prone (OP) and obesity-resistant (OR) male Wistar rats. Delineation of OP and OR was based on body weight gain (upper tertile for OP; lower tertile for OR) after 1 wk on a high-fat diet (60% of kcal from corn oil). Rats were killed after 1, 2, or 5 wk of the dietary period. Increased body weight and percent body fat in OP rats at 1 wk could not be accounted for by increased retroperitoneal or epididymal fat pad weight. Plasma nonesterified fatty acids and triglycerides, as well as blood concentrations of glucose, lactate, and glycerol, were similar throughout the study. Plasma insulin was significantly greater in OP vs. OR rats and low-fat diet (LFD; 20% of kcal from corn oil) controls at 5 wk only, and blood beta-hydroxybutyrate (mM) was significantly higher in OR compared with OP and LFD rats at 1, 2, and 5 wk. Lipoprotein lipase mRNA and activity were significantly greater in epididymal fat pad and significantly lower in gastrocnemius muscle of OP vs. OR rats at 1 wk. Results suggest that early (i.e., 1 wk) differences in body weight and fat weight between OP and OR rats are not due to fat deposition in retroperitoneal or epididymal fat depots, and tissue-specific changes in LPL (increase in epididymal fat pad and decrease in gastrocnemius muscle) that occur in OP compared with OR rats after 1 wk on a high-fat diet provide a metabolic environment favoring fat storage.

Kinetic characterization of a stably expressed novel Na+/H+ exchanger (NHE-2).

We have recently reported the molecular cloning, sequencing and tissue distribution of a novel Na+/H+ exchanger (NHE-2). The cDNA for NHE-2 was cloned by screening a rat intestinal cDNA library. This clone was unique due to the fact that it lacks the first two transmembrane domains which are present in the other Na+/H+ exchanger isoforms (NHE-1, NHE-3, NHE-4). This structural change in the cDNA offered a unique opportunity to study in detail the properties of this stably expressed cDNA in chinese lung fibroblasts that lack the Na+/H+ exchanger (PS120) cells. Amiloride-sensitive Na+ uptake was linear up to 2 min in PS120 cells transfected with the cDNA. Kinetics of the amiloride-sensitive Na+ uptake showed a Vmax of 24.7 +/- 5 nmol/microliters ICW per min and a Km of 33.1 +/- 2.0 mM. The inhibitory constant (KI) for amiloride and its analogue 5-N-ethyl-N-isopropylamiloride (EIPA) was 0.15 microM and 0.66 microM, respectively. Epidermal growth factor, a known stimulator of NHE-1, also upregulated the expressed NHE-2. These results characterize the kinetic properties of this unique exchanger and suggests that the first two transmembrane domains of the Na+/H+ exchanger isoforms are not essential for the expression of amiloride-sensitive Na+ uptake.

Induction of aP2 gene expression by nonmetabolized long-chain fatty acids.

Long-chain fatty acids (FA) have been shown to regulate expression of the gene for the adipocyte FA-binding protein aP2. We examined whether this effect was exerted by FA themselves or by a FA metabolite. The alpha-bromo derivative of palmitate, an inhibitor of FA oxidation, was synthesized in the radioactive form, and its metabolism was investigated and correlated with its ability to induce aP2 in Ob1771 preadipocytes. alpha-Bromopalmitate was not utilized by preadipocytes. It was not cleared from the medium over a 24-hr period and was not incorporated into cellular lipids. Short incubations indicated that alpha-bromopalmitate exchanged across the preadipocyte membrane but remained in the free form inside the cell. In line with this, preadipocyte homogenates did not activate alpha-bromopalmitate to the acyl form. However, although it was not metabolized, bromopalmitate was much more potent than native FA in inducing aP2 gene expression. Induction exhibited the characteristics previously described for native FA, indicating that a similar if not identical mechanism was involved. The data indicated that induction of aP2 was exerted by unprocessed FA. Finally, in contrast to preadipocytes, adipocytes metabolized bromopalmitate. This reflected increased activity with cell differentiation of a palmitoyl-CoA synthase that could activate palmitate and bromopalmitate at about one-fifth the rate for palmitate. In preadipocytes, the predominant fatty-acyl-CoA synthase, arachidonyl-CoA synthase, had very low affinity for both FA. Increased activity of the palmitoyl-CoA synthase, which has a wider substrate range, is likely to be important for initiation of lipid deposition.

Biphasic changes in phospholipid hydroperoxide levels during renal ischemia/reperfusion.

The involvement of lipid peroxidation in renal ischemia/reperfusion was explored by measuring changes in the cortical content of specific primary lipid hydroperoxides (using chemluminescent detection with HPLC) following ischemia and reperfusion and by correlating the changes in hydroperoxide content with measurements of renal blood flow. Phosphatidylcholine and phosphatidylethanolamine hydroperoxide concentrations were significantly lowered during 30 or 60 min of ischemia (to levels less than 50% of control at 60 min). Following 30 min of renal ischemia, reperfusion resulted in a rebound of phospholipid hydroperoxide tissue content to levels higher than controls. Increased phospholipid hydroperoxide formation was not, however, observed in response to reperfusion following long-term (60 min) ischemia. In separate animals it was demonstrated that following 30 min ischemia and reperfusion, renal blood flow recovers to about 65% of control in 1 h. In contrast, following 60 min ischemia and reperfusion, the renal blood flow remains more highly impaired (less than 25% recovery for periods up to 24 h). These results imply that phospholipid hydroperoxides are produced and accumulate in the kidneys under normal aerobic conditions and that lipid peroxidative activity increases during renal ischemia/reperfusion to an extent dependent on the degree of local blood perfusion.