Implications of white striping and spaghetti meat abnormalities on meat quality and histological features in broilers.

During the past few years, there has been an increasing prevalence of broiler breast muscle abnormalities, such as white striping (WS) and wooden breast conditions. More recently, a new muscular abnormality termed as spaghetti meat (SM) because of the altered structural integrity of the Pectoralis major muscle often associated with WS has emerged. Thus, this study aimed at evaluating the effects of WS and SM conditions, occurring alone or combined within the same P. major muscle, on meat quality traits and muscle histology. In two replications, 96 P. major muscles were classified into four classes: normal (N), WS, SM and WS/SM. The whole fillet was used for weight assessment and morphometric measurements, then each sample was cut in order to separate the superficial layer from the deep one and used to evaluate proximate composition, histological features, nuclear magnetic resonance relaxation times, functional properties and both myofibrillar and sarcoplasmic proteins profile. Fillets affected by WS and SM abnormalities exhibited higher weights and increased thickness and length. SM condition was associated with a relevant decrease in protein content coupled with a significant increase in moisture level, whereas fat content was affected only by the simultaneous presence of WS. Histological evaluations revealed that abnormal samples were characterized by several degenerative aspects that almost completely concerned the superficial layer of the fillets. White striped fillets exhibited necrosis and lysis of fibers, fibrosis, lipidosis, loss of cross striation and vacuolar degeneration. Moreover, SM samples were characterized by poor fiber uniformity and a progressive rarefaction of the endo- and peri-mysial connective tissue, whereas WS/SM fillets showed intermediate histological features. Nuclear magnetic resonance relaxation analysis revealed a higher proportion of extra-myofibrillar water in the superficial section of all the abnormal fillets, especially in SM samples, which consequently led to a reduction of the water holding capacity of meat. As for functional properties, abnormal fillets exhibited a lower protein solubility and higher ultimate pH values on both the superficial and deep sections. Although abnormal fillets exhibited higher yellowness values, no relevant effect on meat color was observed. The occurrence of WS and SM abnormalities led to increased carbonylation levels and more intense proteolytic processes. Overall, muscle abnormalities mainly affect the superficial layer of P. major muscle and particularly the occurrence of SM myopathy seems to implicate a more pronounced modification of meat quality traits than the mere presence of WS.

Histology, composition, and quality traits of chicken Pectoralis major muscle affected by wooden breast abnormality.

Only a few years ago, the poultry industry began to face a recent abnormality in breast meat, known as wooden breast, which frequently overlaps with white striping. This study aimed to assess the impact of wooden breast abnormality on quality traits of meat. For this purpose, 32 normal (NRM), 32 wooden (WB), and 32 wooden and white-striped (WB/WS) Pectoralis major muscles were selected from the same flock of heavy broilers (males, Ross 708, weighing around 3.7 kg) in the deboning area of a commercial processing plant at 3 h postmortem and used to assess histology, proximate (moisture, protein, fat, ash, and collagen) and mineral composition (Mg, K, P, Na and Ca), sarcoplasmic and myofibrillar protein patterns, and technological traits of breast meat. Compared to the normal group, WB/WS fillets showed more severe histological lesions characterized by fiber degeneration, fibrosis, and lipidosis, coupled with a significantly harder texture. With regard to proximate and mineral composition, abnormal samples exhibited significantly (P < 0.001) higher moisture, fat, and collagen contents coupled with lower (P < 0.001) amounts of protein and ash. Furthermore, increased calcium (131 vs. 84 mg kg(-1); P < 0.05) and sodium (741 vs. 393 mg kg(-1); P < 0.001) levels were found in WB/WS meat samples. The SDS-PAGE analysis revealed a significantly lower amount of calcium-ATPase (SERCA, 114 kDa), responsible for the translocation of Ca ions across the membrane, in normal breasts compared to abnormal ones. As for meat quality traits, fillets affected by wooden abnormality exhibited significantly (P < 0.001) higher ultimate pH and lower water-holding/water-binding capacity. In particular, compared to normal, abnormal samples showed reduced marinade uptake coupled with increased drip loss and cooking losses as well. In conclusion, this study revealed that meat affected by wooden breast or both wooden breast and white striping abnormalities exhibit poorer nutritional value, harder texture, and impaired water-holding capacity.

Quantity and functionality of protein fractions in chicken breast fillets affected by white striping.

Recently, white striations parallel to muscle fibers direction have been observed on the surface of chicken breast, which could be ascribed to intensive growth selection. The aim of this study was to evaluate the effect of white striping on chemical composition with special emphasis on myofibrillar and sarcoplasmic protein fractions that are relevant to the processing features of chicken breast meat. During this study, a total of 12 pectoralis major muscles from both normal and white striped fillets were used to evaluate chemical composition, protein solubility (sarcoplasmic, myofibrillar, and total protein solubility), protein quantity (sarcoplasmic, myofibrillar, and stromal proteins), water holding capacity, and protein profile by SDS-PAGE analysis. White-striped fillets exhibited a higher percentage of moisture (75.4 vs. 73.8%; P < 0.01), intramuscular fat (2.15 vs. 0.98%; P < 0.01), and collagen (1.36 vs. 1.22%; P < 0.01), and lower content of protein (18.7 vs. 22.8%; P < 0.01) and ash (1.14 vs. 1.34%; P < 0.01), in comparison with normal fillets. There was a great decline in myofibrillar (14.0 vs. 8.7%; P < 0.01) and sarcoplasmic (3.2 vs. 2.6%; P < 0.01) content and solubility as well as an increase in cooking loss (33.7 vs. 27.4%; P < 0.05) due to white striping defects. Moreover, gel electrophoresis showed that the concentration of 3 myofibrillar proteins corresponding to actin (42 kDa); LC1, slow-twitch light chain myosin (27.5 kDa); and LC3, fast-twitch light chain myosin (16 kDa), and almost all sarcoplasmic proteins were lower than normal. In conclusion, the findings of this study revealed that chicken breast meat with white striping defect had different chemical composition (more fat and less protein) and protein quality and quantity (low content of myofibrillar proteins and high content of stromal proteins) with respect to normal meat. Furthermore, white striped fillets had lower protein functionality (higher cooking loss). All the former changes indicate that white striping has great impact on quality characteristics of chicken breast meat.

Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels.

BACKGROUND AND PURPOSE: ClC-K kidney Cl(-) channels are important for renal and inner ear transepithelial Cl(-) transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above approximately 1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA. EXPERIMENTAL APPROACH: We used site-directed mutagenesis and two-electrode voltage clamp analysis of wild-type and mutant channels expressed in Xenopus oocytes. Guided by the crystal structure of a bacterial CLC homolog, we screened 97 ClC-Ka mutations for alterations of NFA effects. KEY RESULTS: Mutations of five residues significantly reduced the potentiating effect of NFA. Two of these (G167A and F213A) drastically altered general gating properties and are unlikely to be involved in NFA binding. The three remaining mutants (L155A, G345S and A349E) severely impaired or abolished NFA potentiation. CONCLUSIONS AND IMPLICATIONS: The three key residues identified (L155, G345, A349) are localized in two different protein regions that, based on the crystal structure of bacterial CLC homologs, are expected to be exposed to the extracellular side of the channel, relatively close to each other, and are thus good candidates for being part of the potentiating NFA binding site. Alternatively, the protein region identified mediates conformational changes following NFA binding. Our results are an important step towards the development of ClC-Ka activators for treating Bartter syndrome types III and IV with residual channel activity.

Systematic analysis of three FHM genes in 39 sporadic patients with hemiplegic migraine.

BACKGROUND: Familial (FHM) and sporadic (SHM) hemiplegic migraine are severe subtypes of migraine associated with transient hemiparesis. For FHM, three genes have been identified encoding subunits of a calcium channel (CACNA1A), a sodium-potassium pump (ATP1A2), and a sodium channel (SCN1A). Their role in SHM is unknown. Establishing a genetic basis for SHM may further the understanding of its pathophysiology and relationship with common types of migraine. It will also facilitate the often difficult differential diagnosis from other causes of transient hemiparesis. METHODS: We systematically scanned 39 well-characterized patients with SHM without associated neurologic features for mutations in the three FHM genes. Functional assays were performed for all new sequence variants. RESULTS: Sequence variants were identified in seven SHM patients: one CACNA1A mutation, five ATP1A2 mutations, and one SCN1A polymorphism. All six mutations caused functional changes in cellular assays. One SHM patient later changed to FHM because another family member developed FHM attacks. CONCLUSION: We show that FHM genes are involved in at least a proportion of SHM patients without associated neurologic symptoms. Screening of ATP1A2 offers the highest likelihood of success. Because FHM gene mutations were also found in family members with "nonhemiplegic" typical migraine with and without aura, our findings reinforce the hypothesis that FHM, SHM, and "normal" migraine are part of a disease spectrum with shared pathogenetic mechanisms.

Niflumic acid inhibits chloride conductance of rat skeletal muscle by directly inhibiting the CLC-1 channel and by increasing intracellular calcium.

BACKGROUND AND PURPOSE: Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage-gated chloride channel CLC-1, in controlling muscle excitability, the availability of ligands modulating CLC-1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non-steroidal anti-inflammatory drugs (NSAID). EXPERIMENTAL APPROACH: Rat muscle Cl(-) conductance (gCl) and heterologously expressed CLC-1 currents were evaluated by means of current-clamp (using two-microelectrodes) and patch-clamp techniques, respectively. Fura-2 fluorescence was used to determine intracellular calcium concentration, [Ca(2+)](i), in native muscle fibres. KEY RESULTS: NFA inhibited native gCl with an IC(50) of 42 muM and blocked CLC-1 by interacting with an intracellular binding site. Additionally, NFA increased basal [Ca(2+)](i) in myofibres by promoting a mitochondrial calcium efflux that was not dependent on cyclooxygenase or CLC-1. A structure-activity study revealed that the molecular conditions that mediate the two effects are different. Pretreatment with the Ca-dependent protein kinase C (PKC) inhibitor chelerythrine partially inhibited the NFA effect. Therefore, in addition to direct channel block, NFA also inhibits gCl indirectly by promoting PKC activation. CONCLUSIONS AND IMPLICATIONS: These cellular effects of NFA on skeletal muscle demonstrate that it is possible to modify CLC-1 and consequently gCl directly by interacting with channel proteins and indirectly by interfering with the calcium-dependent regulation of the channel. The effect of NFA on mitochondrial calcium stores suggests that NSAIDs, widely used drugs, could have potentially dangerous side-effects.

Channel or transporter? The CLC saga continues.

It was recently shown that the putative bacterial Cl- channel, ClC-ec1, is in reality a Cl--H+ antiporter. Our group has now shown that this is also the case for two human CLCs, ClC-4 and ClC-5. We found that the flux of Cl- in one direction is stoichiometrically coupled to the movement of protons in the opposite direction, unveiling a behaviour that is typical of a transporter rather than a channel. This discovery will surely stimulate further research to elucidate the molecular elements responsible for the behaviour as a transporter. On the physiological level, the antiport activity of ClC-4/ClC-5 must lead to a review of the role of CLC proteins in intracellular compartments. Small organic molecules have been extremely useful tools for studying ion channels and many commercial drugs target specific ion channel proteins. Several blockers have been found to inhibit the plasma membrane-localized CLC channels ClC-0, ClC-1 and ClC-Ka. These compounds include 9-anthracene-carboxylic acid (9-AC), p-chlorophenoxy-propionic acid (CPP) and its derivatives, and 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). Two different binding sites have been identified, one extracellular and one intracellular. However, high-affinity ligands for most CLC proteins are still missing. Apart from being useful biophysical tools, such drugs may provide a way to modulate protein function in vivo. With these tasks to be accomplished, it is definitely an exciting time in the chloride transport field.

Characterization of BphF, a Rieske-type ferredoxin with a low reduction potential.

BphF is a small, soluble, Rieske-type ferredoxin involved in the microbial degradation of biphenyl. The rapid, anaerobic purification of a heterologously expressed, his-tagged BphF yielded 15 mg of highly homogeneous recombinant protein, rcBphF, per liter of cell culture. The reduction potential of rcBphF, determined using a highly oriented pyrolytic graphite (HOPG) electrode, was -157+/- 2 mV vs the standard hydrogen electrode (SHE) (20 mM MOPS, 80 mM KCl, and 1 mM dithiothreitol, pH 7.0, 22 degrees C). The electron paramagnetic resonance spectrum of the reduced rcBphF is typical of a Rieske cluster while the close similarity of the circular dichroic (CD) spectra of rcBphF and BedB, a homologous protein from the benzene dioxygenase system, indicates that the environment of the cluster is highly conserved in these two proteins. The reduction potential and CD spectra of rcBphF were relatively independent of pH between 5 and 10, indicating that the pK(a)s of the cluster's histidinyl ligands are not within this range. Gel filtration studies demonstrated that rcBphF readily oligomerizes in solution. Crystals of rcBphF were obtained using sodium formate or poly(ethylene glycol) (PEG) as the major precipitant. Analysis of the intermolecular contacts in the crystal revealed a head-to-tail interaction that occludes the cluster, but is very unlikely to be found in solution. Oligomerization of rcBphF in solution was reversed by the addition of dithiothreitol and is unrelated to the noncovalent crystallographic interactions. Moreover, the oligomerization state of rcBphF did not influence the latter's reduction potential. These results indicate that the 450 mV spread in reduction potential of Rieske clusters of dioxygenase-associated ferredoxins and mitochondrial bc(1) complexes is not due to significant differences in their solvent exposure.

Methyl p-hydroxybenzoate (E-218) a preservative for drugs and food is an activator of the ryanodine receptor Ca(2+) release channel.

1. Haloperidol is a drug used in the management of several psychotic disorders and its use has been linked to Neuroleptic Malignant Syndrome. In the present study we have investigated the effect of a commercial preparation of haloperidol, Serenase, on skeletal muscle sarcoplasmic reticulum. 2. Addition of Serenase to isolated terminal cisternae caused a rapid release of calcium. We tested whether the active Ca(2+)-releasing substance was haloperidol or another compound present in the preparation. 3. Our results show that methyl p-hydroxybenzoate, one of the preservatives and a commonly used anti-microbial agent (E-218) is an activator of Ca(2+) release (E.C. 50=2.0 mM), mediated by a ruthenium red-sensitive Ca(2+) release channel present in skeletal muscle terminal cisternae.

Reconstitution of a voltage and calcium dependent potassium channel from rat cerebellum.

Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca2+]i, (2) a calcium and voltage dependent potassium channel (KCa). KCa channels had a conductance of (302+/-15) pS (n=5) and were activated by [Ca2+]i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on KCa channels from rat cerebellum.

Experimental evidence for the role of buried polar groups in determining the reduction potential of metalloproteins: the S79P variant of Chromatium vinosum HiPIP.

The amide group between residues 78 and 79 of Chromatium vinosum high-potential iron-sulfur protein (HiPIP) is in close proximity to the Fe4S4 cluster of this protein and interacts via a hydrogen bond with S gamma of Cys77, one of the cluster ligands. The reduction potential of the S79P variant was 104 +/- 3 mV lower than that of the recombinant wild-type (rcWT) HiPIP (5 mM phosphate, 100 mM NaCl, pH 7, 293 K), principally due to a decrease in the enthalpic term which favors the reduction of the rcWT protein. Analysis of the variant protein by NMR spectroscopy indicated that the substitution has little effect on the structure of the HiPIP or on the electron distribution in the oxidized cluster. Potential energy calculations indicate that the difference in reduction potential between rcWT and S79P variant HiPIPs is due to the different electrostatic properties of amide 79 in these two proteins. These results suggest that the influence of amide group 79 on the reduction potential of C. vinosum HiPIP is a manifestation of a general electrostatic effect rather than a specific interaction. More generally, these results provide experimental evidence for the importance of buried polar groups in determining the reduction potentials of metalloproteins.