Macdonald Wick

Functional Proteomic Analysis Predicts Beef Tenderness and the Tenderness Differential

Inconsistent tenderness is one of the most detrimental factors of meat quality. Functional proteomics was used to associate electrophoretic bands from the myofibrillar muscle fraction to meat tenderness in an effort to gain understanding of the mechanisms controlling tenderness. The myofibrillar muscle fraction of the Longissimus dorsi from 22 Angus cross steers was analyzed by SDS-PAGE and linearly regressed to Warner-Bratzler shear values. Six significant electrophoretic bands were characterized by electrophoretic and statistical analysis and sequenced by nano-LC-MS/MS. The protein(s)/peptide(s) identified in these bands encompass a wide array of cellular pathways related to structural, metabolic, chaperone, and developmental functions. This study begins to assemble information that has been reported separately into a more complete picture that will lead to the establishment of a coherent mechanism accounting for meat tenderness.

Myofibrillar 1-D fingerprints and myosin heavy chain MS analyses of beef loin at 36 h postmortem correlate with tenderness at 7 days

This study investigated the potential for relating changes in electrophoretic protein patterns derived from the longissimus dorsi of beef cattle 36 h postmortem with tenderness at 7 days. We report finding a significant correlation (R(2)=0.82) between electrophoretic l. dorsi myofibrillar fingerprints at 36 h postmortem and tenderness at 7 days, as determined by Warner-Bratzler shear analysis. In addition, we have used mass spectrometric analyses to identify fragments of bovine myosin heavy chain that are significantly correlated with tenderness. Furthermore, this method offers the potential to increase our understanding of the fundamental cellular mechanisms underlying the proteolytic breakdown of muscle proteins during the aging process.

Proteomic analysis of proteins associated with body mass and length in yellow perch, Perca flavescens.

The goal of commercial yellow perch aquaculture is to increase muscle mass which leads to increased profitability. The accumulation and degradation of muscle‐specific gene products underlies the variability in body mass (BM) and length observed in pond‐cultured yellow perch. Our objective was to apply a combination of statistical and proteomic technologies to identify intact and/or proteolytic fragments of muscle specific gene products involved in muscle growth in yellow perch. Seventy yellow perch randomly selected at 10, 12, 16, 20, and 26 wk of age were euthanized; BM and length were measured and a muscle sample taken. Muscle proteins were resolved using 5–20% gradient SDS‐PAGE, stained with SYPRO® Ruby and analyzed using TotalLab™ software. Data were analyzed using stepwise multiple regression with the dependent variables, BM and length and proportional OD of each band in a sample as a potential regressor. Eight bands associated with BM (R2 = 0.84) and nine bands with length (R2 = 0.85) were detected. Protein sequencing by nano‐LC/MS/MS identified 20 proteins/peptides associated with BM and length. These results contribute the identification of gene products and/or proteolytic fragments associated with muscle growth in yellow perch.

Improvement of the physicochemical properties of pale soft and exudative (PSE) pork meat products with an extract from mechanically deboned turkey meat (MDTM).

Loss of protein functionality of pale soft and exudative (PSE) meat and a surplus of mechanically deboned turkey meat (MDTM) has negative economic impacts on the meat industry. The objective of this research was to add functional proteins from an extract of MDTM to PSE meat to create a value-added pork sausage product. Sausages manufactured from PSE incorporating an MDTM extract exhibited a 30% increase in rigidity (P<0.05) compared to those manufactured using brine alone. Sausages made from PSE and normal pork with the MDTM extract demonstrated little difference in water holding capacity (P>0.05), and reductions of 4.1 and 3.1% in cooking loss (P<0.05), respectively, compared to sausages made employing brine alone. Introducing functional proteins derived from MDTM to processed meat made with PSE pork has the potential to enhance the economic value to both of these low value raw materials.

Identification of a genomic locus containing three slow myosin heavy chain genes in the chicken

Two unique cDNA clones containing chicken slow myosin heavy chain (MyHC) inserts have been isolated from an expression library. Immunochemical analyses of the expressed proteins using different slow MyHC specific monoclonal antibodies were consistent with the two clones encoding slow MyHC 1 (SM1) and slow MyHC 2 (SM2) protein sequences. Northern blot analyses showed that the clones hybridized with 6-kb mRNAs that are differentially expressed in developing and adult slow muscles, further supporting the conclusion that these two clones represent SM1 and SM2 cDNAs. Sequence analyses show that both clones encode the highly conserved light meromyosin portion of the sarcomeric myosin rod and are 78-81% homologous to a mammalian slow/cardiac beta-MyHC cDNA. Hybridization using PCR generated probes specific for SM1 and SM2 sequences demonstrated that the genes encoding these two slow MyHCs colocalized to an 80-kb BssHII genomic fragment. We further show that a probe specific to a third slow MyHC gene also hybridized with the same 80-kb genomic fragment. We conclude that in the chicken genome there is a slow MyHC locus containing at least three distinct slow MyHC genes.

Eukaryotic inhibitors or activators elicit responses to chemosensory compounds by ruminal isotrichid and entodiniomorphid protozoa

Our objectives were to evaluate potential signaling pathways regulating rumen protozoal chemotaxis using eukaryotic inhibitors potentially coordinated with phagocytosis as assessed by fluorescent bead uptake kinetics. Wortmannin (inhibitor of phosphoinositide 3-kinase), insulin, genistein (purported inhibitor of a receptor tyrosine kinase), U73122 (inhibitor of phospholipase C), and sodium nitroprusside (Snp, nitric oxide generator, activating protein kinase G) were preincubated with mixed ruminal protozoa for 3h before assessing uptake of fluorescent beads and chemosensory behavior to glucose, peptides, and their combination; peptides were also combined with guanosine triphosphate (GTP; a chemorepellent). Entodiniomorphids were chemoattracted to both glucose and peptides, but chemoattraction to glucose was increased by Snp and wortmannin without effect on chemoattraction to peptides. Rate of fluorescent bead uptake by an Entodinium caudatum culture decreased when beads were added simultaneously with feeding and incubated with wortmannin (statistical interaction). Wortmannin also decreased the proportion of mixed entodiniomorphids consuming beads. Isotrichid protozoa exhibited greater chemotaxis to glucose but, compared with entodiniomorphids, were chemorepelled to peptides. Wortmannin increased chemotaxis by entodiniomorphids but decreased chemotaxis to glucose by isotrichids. Motility assays documented that Snp and wortmannin decreased net swimming speed (distance among 2 points per second) but not total swimming speed (including turns) by entodiniomorphids. Wortmannin decreased both net and total swimming behavior in isotrichids. Results mechanistically explain the isotrichid migratory ecology to rapidly take up newly ingested sugars and subsequent sedimentation back to the ventral reticulorumen. In contrast, entodiniomorphids apparently integrate cellular motility with feeding behavior to consume small particulates and thereby stay associated and pass with the degradable fraction of rumen particulates. These results extend findings from aerobic ciliate models to explain how rumen protozoa have adapted physiology for their specific ecological niches.

Comparative proteomic characterization of the sarcoplasmic proteins in the pectoralis major and supracoracoideus breast muscles in 2 chicken genotypes

The selection processes that have resulted in broiler (meat) and leghorn (eggs) chickens have had very different effects on the pectoralis major and supracoracoideus muscles. The objective of this study, therefore, was to analyze the one-dimensional proteomic profiles of sarcoplasmic protein fractions isolated from the p. major and supracoracoideus muscles collected from 10 chicks from each genotype to compare developmental differences. The sarcoplasmic protein fraction was analyzed by SDS-PAGE. The mean band percentages were analyzed using a mixed model, with strain and muscle type as main effects. Six bands were found to be significantly different across the 2 strains. Strain differences in glycogen phosphorylase, enolase, elongation factor 1, creatine kinase, fructose-bisphosphate aldolase, and glyceraldehyde 3-phosphate-dehydrogenase suggest a genotype-specific shift in energy metabolism during breast muscle growth and development.

The ontogeny of delta-like protein 1 messenger ribonucleic acid expression during muscle development and regeneration: Comparison of broiler and Leghorn chickens

Delta-like protein 1 (DLK1) has been implicated in the muscle hypertrophy observed in DLK1 transgenic mice, callipyge sheep, and mouse paternal uniparental disomy 12 and human paternal uniparental disomy 14 syndromes. The current study was aimed to determine chicken DLK1 (gDLK1) mRNA expression during primary muscle cell differentiation and during muscle regeneration after cold injury and to compare gDLK1 mRNA expression during skeletal muscle development in layers and broilers. In chicken primary muscle cell culture, gDLK1 mRNA expression was significantly increased from 12 to 48 h (P < or = 0.05) when the nascent myotubes were actively formed at d 2 to 3. Myogenin, a late myogenic marker gene, mRNA expression peaked at 36 to 48 h. Myogenic differentiation 1 (MyoD) and paired box gene 7 (Pax7), early myogenic marker genes, mRNA expression gradually decreased during myogenic differentiation. During muscle regeneration, the expression of MyoD and Pax7 peaked at d 2 (P < or = 0.05), and myogenin mRNA expression peaked at d 4 (P < or = 0.05). The induction of gDLK1 gene appeared between d 7 to 10 postinjury (P < or = 0.05) when myotubes were actively formed as also demonstrated in histological sections. The expression of gDLK1 was slowly downregulated to the control levels at d 14 when the damaged muscle appeared nearly healed. These data suggest that gDLK1 may be involved in the late myogenic stages of primary muscle cell differentiation and muscle regeneration. The gDLK1 mRNA in the muscle tissues was very abundant at embryonic ages but decreased after hatching in both broiler and layer chickens. Compared with layers, broiler muscle at embryonic d 13 had a 3-fold greater expression of DLK1 (P < or = 0.01). In addition, the gDLK1 mRNA expression at d 1, 11, and 33 post-hatch was significantly higher in broilers than layers (P < or = 0.05). Therefore, the relatively greater expression of the gDLK1 gene in muscles of broilers compared with layers suggests that gDLK1 may serve as a new selection marker for high muscle growth in chickens. These findings may provide new insight into chicken muscle development and regeneration.

Muscle fiber characteristics of pectoralis major muscle as related to muscle mass in different Japanese quail lines.

The objectives of this study were to investigate the muscle fiber characteristics of the pectoralis major muscle, and its relation to growth performance in the random bred control (RBC) and heavy weight (HW) Japanese quail lines at 42 days of age. The HW line had greater body (232.0 v. 100.2 g, P < 0.001) and pectoralis major muscle (19.0 v. 6.2 g, P < 0.001) weights than the RBC line. Color differences were observed between the superficial and deep regions of the pectoralis major muscle, with the superficial region showing a higher value of lightness than the deep region of the RBC or HW lines (P < 0.001). The percentage of the superficial region in the pectoralis major muscle was higher in the HW line compared with the RBC line (46.2% v. 38.0%, P = 0.017). There were no significant differences in the total fiber number in the superficial and deep regions between the two quail lines (P = 0.718). The HW quail line showed a larger mean fiber cross-sectional area (CSA; 375.5 v. 176.6 μm², P < 0.001) and type IIA fiber CSA (243.7 v. 131.9 μm², P < 0.001) than the RBC quail line. The HW line also had greater CSA percentage (60.2% v. 34.2%, P < 0.001) and number percentage (41.6% v. 14.2%, P < 0.001) of type IIB fibers, although there were no significant differences in type IIB fiber CSA between the RBC and HW lines (P = 0.219). Therefore, greater body and muscle weights of the HW line are caused by differences in muscle fiber characteristics, especially the proportion of type IIB fiber and the CSA of type IIA fiber, compared with the RBC line. The results of this study suggest that muscle fiber hypertrophy has more impact on body and muscle weights of the different quail lines than muscle fiber hyperplasia.

Temporal myosin heavy chain isoform expression transitions faster in broiler chickens compared with Single Comb White Leghorns

Myosin heavy chain (MyHC), one of the major components in the contractile machinery of skeletal muscle fibers, is found in several isoforms during myogenesis. During chicken development, embryonic, neonatal, and adult MyHC isoforms are expressed. Broiler chickens have been selected for fast and large muscle growth, whereas Single Comb White Leghorn (SCWL) chickens have been selected for egg laying capabilities. This has led to an obvious difference in muscle growth and development with broilers being much larger than SCWL. The objective of this study was to determine if differences in muscle growth and development of SCWL and broilers are associated with differences in temporal expression of MyHC isoforms in skeletal muscle between the 2 breeds. Pectoralis major muscle (PM) was collected from SCWL and broilers at embryonic d 15, 17, and 19 and 1, 5, 11, 20, 27, and 33 d posthatch with n = 3 samples per time point and breed. Western blotting using 3 monoclonal antibodies (EB165, 2E9, and AB8) was performed to compare the expression patterns of embryonic/adult, neonatal, and adult isoforms of MyHC, respectively, for all time points in both SCWL and broiler chickens. Both broiler and SCWL chickens began expressing the neonatal MyHC isoform on d 5; however, SCWL chickens expressed the neonatal isoform much longer than broilers. The SCWL chickens had sustained expression of the neonatal MyHC isoform through d 27, whereas in broiler chickens the neonatal isoform was not expressed at d 20. Pectoralis major tissue from broiler chickens expressed the adult MyHC isoform as early as d 20, whereas the SCWL chickens began expressing the adult isoform later. The rate of transition to neonatal and adult MyHC isoforms in broilers and Leghorns is consistent with the faster maturation and growth of broilers relative to Leghorns. This relationship between faster growth of the PM and the rate of transition of MyHC isoforms within the fast skeletal muscle of the PM may indicate a selection marker for improvement of broiler PM growth.