Skeletal Muscle Of Chickens Research Articles

Lactobacillus-derived protoporphyrin IX and SCFAs regulate the fiber size via glucose metabolism in the skeletal muscle of chickens.

The gut microbiota contributes to skeletal muscle energy metabolism and is an indirect factor affecting meat quality. However, the role of specific gut microbes in energy metabolism and fiber size of skeletal muscle in chickens remains largely unknown. In this study, we first performed cecal microbiota transplantation from Chinese indigenous Jingyuan chickens (JY) to Arbor Acres chickens (AA), to determine the effects of microbiota on skeletal muscle fiber and energy metabolism. Then, we used metagenomics, gas chromatography, and metabolomics analysis to identify functional microbes. Finally, we validated the role of these functional microbes in regulating the fiber size via glucose metabolism in the skeletal muscle of chickens through feeding experiments. The results showed that the skeletal muscle characteristics of AA after microbiota transplantation tended to be consistent with that of JY, as the fiber diameter was significantly increased, and glucose metabolism level was significantly enhanced in the pectoralis muscle. L. plantarum, L. ingluviei, L. salivarius, and their mixture could increase the production of the microbial metabolites protoporphyrin IX and short-chain fatty acids, therefore increasing the expression levels of genes related to the oxidative fiber type (MyHC SM and MyHC FRM), mitochondrial function (Tfam and CoxVa), and glucose metabolism (PFK, PK, PDH, IDH, and SDH), thereby increasing the fiber diameter and density. These three Lactobacillus species could be promising probiotics to improve the meat quality of chicken.IMPORTANCEThis study revealed that the L. plantarum, L. ingluviei, and L. salivarius could enhance the production of protoporphyrin IX and short-chain fatty acids in the cecum of chickens, improving glucose metabolism, and finally cause the increase in fiber diameter and density of skeletal muscle. These three microbes could be potential probiotic candidates to regulate glucose metabolism in skeletal muscle to improve the meat quality of chicken in broiler production.

Characterization of lncRNA/circRNA-miRNA-mRNA network to reveal potential functional ceRNAs in the skeletal muscle of chicken.

Skeletal muscle, comprising approximately 40% of body mass, is a highly complex and heterogeneous tissue serving a multitude of functions in the organism. Non-coding RNAs (ncRNAs) are known to participate in skeletal muscle development as critical regulators. However, the regulatory mechanisms of ncRNAs on chicken muscle traits are not well understood. In the present study, we collected the leg muscle from male embryos of Tibetan chicken at embryonic (E) 10 and E18 for RNA sequencing. A total of 6,583 differentially expressed mRNAs (DEMs) including 3,055 down-regulated and 3,528 up-regulated were identified in E18. We identified 695 differentially expressed lncRNAs (DELs) (187 down-regulated and 508 up-regulated) and 1,906 differentially expressed circRNAs (DECs) (1,224 down-regulated and 682 up-regulated) in E18. Among the 130 differentially expressed miRNAs (DEMIs), 59 were up-regulated and 71 were down-regulated in E18. Numerous DEMs and target genes for miRNAs/lncRNAs were significantly enriched in the muscle system process and cell cycle. We constructed a miRNA-gene-pathway network by considering target relationships between genes related to skeletal muscle development and miRNAs. A competing endogenous RNA (ceRNA) network was also constructed by integrating competing relationships between DEMs, DELs, and DECs. Several DELs and DECs were predicted to regulate the ADRA1B, ATP2A2, ATP2B1, CACNA1S, CACNB4, MYLK2, and ROCK2 genes. We discovered the crosstalk between the ncRNAs and their competing mRNAs, which provides insights into ceRNA function and mechanisms in the skeletal muscle development of chicken.

MiRNA-seq analysis in skeletal muscle of chicken and function exploration of miR-24-3p

The regulation of skeletal muscle growth and development in chicken is complex. MicroRNAs (miRNAs) have been found to play an important role in the process, and more research is needed to further understand the regulatory mechanism of miRNAs. In this study, leg muscles of Jinghai yellow chickens at 300 d with low body weight (slow-growing group) and high body weight (fast-growing group) were collected for miRNA sequencing (miRNA-seq) and Bioinformatics analysis revealed 12 differentially expressed miRNAs (DEMs) between the two groups. We predicted 150 target genes for the DEMs, and GO and KEGG pathway analysis showed the target genes of miR-24-3p and novel_miR_133 were most enriched in the terms related to growth and development. Moreover, networks of DEMs and target genes showed that miR-24-3p and novel_miR_133 were the 2 core miRNAs. Hence, miR-24-3p was selected for further functional exploration in chicken primary myoblasts (CPMs) with molecular biology technologies including qPCR, cell counting kit-8 (CCK-8), 5-ethynyl-2’-deoxyuridine (EdU) and immunofluorescence. When proliferating CPMs were transfected with miR-24-3p mimic, the expression of cyclin dependent kinase inhibitor 1A (P21) was up-regulated and both CCK-8 and EdU assays showed that the proliferation of CPMs was inhibited. However, when the inhibitor was transfected into the proliferating CPMs, the opposite results were found. In differentiated CPMs, transfection with miR-24-3p mimic resulted in up regulation of MYOD, MYOG and MYHC after 48 h. Myotube areas also increased significantly compared to the mimic negative control (NC) group. When treated with inhibitor, differentiation CPMs produced the opposite effects. Overall, we revealed 2 miRNAs (novel_miR_133 and miR-24-3p) significantly related with growth and development and further proved that miR-24-3p could suppress the proliferation and promote differentiation of CPMs. The results would facilitate understanding the effects of miRNAs on the growth and development of chickens at the post-transcriptional level and could also have an important guiding role in yellow-feathered chicken breeding.

MiR-21-5p Regulates the Proliferation and Differentiation of Skeletal Muscle Satellite Cells by Targeting KLF3 in Chicken.

The proliferation and differentiation of skeletal muscle satellite cells (SMSCs) play an important role in the development of skeletal muscle. Our previous sequencing data showed that miR-21-5p is one of the most abundant miRNAs in chicken skeletal muscle. Therefore, in this study, the spatiotemporal expression of miR-21-5p and its effects on skeletal muscle development of chickens were explored using in vitro cultured SMSCs as a model. The results in this study showed that miR-21-5p was highly expressed in the skeletal muscle of chickens. The overexpression of miR-21-5p promoted the proliferation of SMSCs as evidenced by increased cell viability, increased cell number in the proliferative phase, and increased mRNA and protein expression of proliferation markers including PCNA, CDK2, and CCND1. Moreover, it was revealed that miR-21-5p promotes the formation of myotubes by modulating the expression of myogenic markers including MyoG, MyoD, and MyHC, whereas knockdown of miR-21-5p showed the opposite result. Gene prediction and dual fluorescence analysis confirmed that KLF3 was one of the direct target genes of miR-21-5p. We confirmed that, contrary to the function of miR-21-5p, KLF3 plays a negative role in the proliferation and differentiation of SMSCs. Si-KLF3 promotes cell number and proliferation activity, as well as the cell differentiation processes. Our results demonstrated that miR-21-5p promotes the proliferation and differentiation of SMSCs by targeting KLF3. Collectively, the results obtained in this study laid a foundation for exploring the mechanism through which miR-21-5p regulates SMSCs.

In silico analysis of selenoprotein N (Gallus gallus): absence of EF-hand motif and the role of CUGS-helix domain in antioxidant protection.

Selenoprotein N (SEPN1) is critical to the normal muscular physiology. Mutation of SEPN1 can raise congenital muscular disorder in human. It is also central to maturation and structure of skeletal muscle in chicken. However, human SEPN1 contained an EF-hand motif, which was not found in chicken. And the biochemical and molecular characterization of chicken SEPN1 remains unclear. Hence, protein domains, transcription factors, and interactions of Ca2+ in SEPN1 were analyzed in silico to provide the divergence and homology between chicken and human in this work. The results showed that vertebrates' SEPN1 evolved from a common ancestor. Human and chicken's SEPN1 shared a conserved CUGS-helix domain with function in antioxidant protection. SEPN1 might be a downstream target of JNK pathway, and it could respond to multiple stresses. Human's SEPN1 might not combine with Ca2+ with a single EF-hand motif in calcium homeostasis, and chicken SEPN1 did not have the EF-hand motif in the prediction, indicating the EF-hand motif malfunctioned in chicken SEPN1.

H2S promotes a glycometabolism disorder by disturbing the Th1/Th2 balance during LPS-induced inflammation in the skeletal muscles of chickens

Hydrogen sulfide (H2S) is a common environmental pollutant. In humans, H2S enters the body and is transported to different tissues and organs, inducing various types of damage such as chronic inflammatory reactions. Glucose metabolism disorders have been shown to be closely associated with chronic inflammation. The goal of the present study was to investigate the effects and mechanisms of H2S on glycometabolism disorders and chronic inflammatory responses. A chronic inflammation model in the skeletal muscles of chickens was induced using lipopolysaccharide (LPS), after which the animals were exposed to exogenous H2S. Subsequently, the glucose metabolism and the pathways associated with chronic inflammation were analyzed. The pathological analysis showed that significant inflammatory injury to skeletal muscles occurred after animals exposed to H2S. The Th1/Th2 ratio imbalance was exacerbated after exposure to H2S with IFNγ downregulated and IL-1, IL-4, and IL-6 upregulated. In addition, the level of IκBα was suppressed and induced the expression of NF-κB, significantly activating the inflammatory pathway, while the expression of heat shock proteins was elevated. In addition, glucose metabolism factors were analyzed. IRS1 phosphorylation was inhibited in animals exposed to H2S, and the expression of insulin-like growth factor (IGF) signaling pathway-related factors was upregulated to promote insulin resistance, causing glucose metabolism disorders. The results of this study revealed that H2S can trigger changes in the ratio of Th1/Th2 to produce more proinflammatory cytokines that disturb the insulin signaling pathway, causing glycometabolism disorders during the inflammatory response in the skeletal muscles of chickens.

Copper (II) and/or arsenite-induced oxidative stress cascades apoptosis and autophagy in the skeletal muscles of chicken

Arsenic (As) is a ubiquitous environmental toxin and robust inducer of oxidative stress (OxS). Copper (Cu) is an essential microelement, which participates in OxS as a cofactor for certain enzymes, with narrow optimal range between essential and toxic concentrations. However, their effects are rarely studied in chicken skeletal muscles, which have soaring per capita consumption andare susceptible to oxidative damage. In the present study, we demonstrated that the administration of copper sulfate (300 mg kg−1) or arsenite (30 mg kg−1) individually or their co-administration leads to varying degrees of OxS in the skeletal muscles of chickens. Corresponding to the protein expression pattern, the mRNA levels of caspase, B-cell lymphoma-2 (Bcl-2) families, and autophagy-related genes were also compromised in the experimental groups, indicating the involvement of both apoptotic and autophagic cell death. Additionally, rampant mitochondrial fission caused the vicious cycle between imbalanced mitochondrial dynamics and OxS, thus tethering intracellular homeostasis. The abovementioned muscle damage and index anomalies were time dependent, and more deteriorated effects were observed in Cu2+ and arsenite co-administered groups than those in groups administered Cu2+ and arsenite alone. Intriguingly, in the studied skeletal muscles, namely wing biceps brachii and leg gastrocnemius, there were conspicuous differences in oxidative toxicity susceptibility, which needs further study. The present study showed that Cu and/or As induce oxidative damage in chicken skeletal muscles and discussed its mechanism in terms of apoptosis, autophagy, and mitochondrial dynamics, thus voicing concerns about poultry breeding areas cross-contaminated with Cu2+ and arsenite.

MiRNA-223 upregulated by MYOD inhibits myoblast proliferation by repressing IGF2 and facilitates myoblast differentiation by inhibiting ZEB1

Skeletal muscle differentiation can be regulated by various transcription factors and non-coding RNAs. In our previous work, miR-223 is differentially expressed in the skeletal muscle of chicken with different growth rates, but its role, expression and action mechanism in muscle development still remains unknown. Here, we found that MYOD transcription factor can upregulate miR-223 expression by binding to an E-box region of the gga-miR-223 gene promoter during avian myoblast differentiation. IGF2 and ZEB1 are two target genes of miR-223. The target inhibition of miR-223 on IGF2 and ZEB1 are dynamic from proliferation to differentiation of myoblast. miR-223 inhibits IGF2 expression only in the proliferating myoblast, whereas it inhibits ZEB1 mainly in the differentiating myoblast. The inhibition of IGF2 by miR-223 resulted in the repression of myoblast proliferation. During myoblast differentiation, miR-223 would be upregulated owing to the promoting effect of MYOD, and the upregulation of miR-223 would inhibit ZEB1 to promote myoblast differentiation. These results not only demonstrated that the well-known muscle determination factor MYOD can promote myoblast differentiation by upregulate miR-223 transcription, but also identified that miR-223 can influence myoblast proliferation and differentiation by a dynamic manner regulates the expression of its target genes.

The IGF-1/Akt/S6 Signaling Pathway is Age-Dependently Downregulated in the Chicken Breast Muscle.

The skeletal muscle mass is known to be controlled by the balance between protein synthesis and degradation. The fractional rate of protein synthesis has been reported to decrease age-dependently from 1 to 4 weeks of age in the chicken breast muscle (pectoralis major muscle). On the other hand, age-dependent change of the fractional protein degradation rate was reported to be less in the skeletal muscle of chickens. These findings suggest that protein synthesis is age-dependently downregulated in chicken muscle. We herein investigated the age-dependent changes in protein synthesis or proteolysis-related factors in the breast muscle of 7, 14, 28, and 49-day old broiler chickens. IGF-1 mRNA level, phosphorylation rate of Akt, and phospho-S6 content were coordinately decreased in an age-dependent manner, suggesting that IGF-1-stimulated protein synthesis is downregulated with age in chicken breast muscle. In contrast, atrogin-1, one of the proteolysis-related factors, gradually increased with age at mRNA levels. However, plasma Nτ-methylhistidine concentration, an indicator of skeletal muscle proteolysis, did not coordinately change with atrogin-1 mRNA levels. Taken together, our results suggest that the IGF-1/Akt/S6 signaling pathway is age-dependently downregulated in the chicken breast muscle.

Identification of Suitable Reference Genes for Real Time Quantitative Polymerase Chain Reaction Assays on Pectoralis major Muscle in Chicken (Gallus gallus ).

Thirteen reference genes were investigated to determine their stability to be used as a housekeeping in gene expression studies in skeletal muscle of chickens. Five different algorithms were used for ranking of reference genes and results suggested that individual rankings of the genes differed among them. The stability of the expression of reference genes were validated using samples obtained from the Pectoralis major muscle in chicken. Samples were obtained from chickens in different development periods post hatch and under different nutritional diets. For gene expression calculation the ΔΔCt approach was applied to compare relative expression of pairs of genes within each of 52 samples when normalized to mitochondrially encoded cytochrome c oxidase II (MT-CO2) target gene. Our findings showed that hydroxymethylbilane synthase (HMBS) and hypoxanthine phosphoribosyl transferase 1 (HPRT1) are the most stable reference genes while transferrin receptor (TFRC) and beta-2-microglobulin (B2M) ranked as the least stable genes in the Pectoralis major muscle of chickens. Moreover, our results revealed that HMBS and HPRT1 gene expression did not change due to dietary variations and thus it is recommended for accurate normalization of RT-qPCR data in chicken Pectoralis major muscle.

Arginase distribution in tissues of domestic avian species

Arginase catalyzes the hydrolysis of L-arginine to yield L-ornithine and urea. Its wide tissue distribution in mammals, important roles in different biochemical pathways, and incompetence of information about the distribution of this enzyme in tissues of different avian species directed us to design this study. Arginase activity was assayed by diacetyl monoxime method for determination of enzymatically formed urea in tissue homogenates of different tissues of few species of birds. The protein content of tissues was determined by Lowry method. Arginase is present in almost all organs of avian species studied, albeit in different levels. The heart and skeletal muscles of chicken were the richest sources of arginase followed by different parts of small intestine, while in pigeon and quail, only the heart contained significant activity. The results of this study will be discussed in terms of the possible involvement of arginase in several biochemical and physiological functions in these species and candidate this enzyme as a biomarker for cardioskeletal disorders but not an indicator for monitoring hepatic injuries.

Functional conservation between rodents and chicken of regulatory sequences driving skeletal muscle gene expression in transgenic chickens

BackgroundRegulatory elements that control expression of specific genes during development have been shown in many cases to contain functionally-conserved modules that can be transferred between species and direct gene expression in a comparable developmental pattern. An example of such a module has been identified at the rat myosin light chain (MLC) 1/3 locus, which has been well characterised in transgenic mouse studies. This locus contains two promoters encoding two alternatively spliced isoforms of alkali myosin light chain. These promoters are differentially regulated during development through the activity of two enhancer elements. The MLC3 promoter alone has been shown to confer expression of a reporter gene in skeletal and cardiac muscle in transgenic mice and the addition of the downstream MLC enhancer increased expression levels in skeletal muscle. We asked whether this regulatory module, sufficient for striated muscle gene expression in the mouse, would drive expression in similar domains in the chicken.ResultsWe have observed that a conserved downstream MLC enhancer is present in the chicken MLC locus. We found that the rat MLC1/3 regulatory elements were transcriptionally active in chick skeletal muscle primary cultures. We observed that a single copy lentiviral insert containing this regulatory cassette was able to drive expression of a lacZ reporter gene in the fast-fibres of skeletal muscle in chicken in three independent transgenic chicken lines in a pattern similar to the endogenous MLC locus. Reporter gene expression in cardiac muscle tissues was not observed for any of these lines.ConclusionsFrom these results we conclude that skeletal expression from this regulatory module is conserved in a genomic context between rodents and chickens. This transgenic module will be useful in future investigations of muscle development in avian species.

Mitochondrial Oxidative Damage in Chicken Skeletal Muscle Induced by Acute Heat Stress

Mitochondria have long been known to play a critical role in maintaining bioenergetic status under physiological conditions. Our previous studies have shown that acute heat stress increases production of mitochondrial reactive oxygen species (ROS) in the skeletal muscle of chickens. This increased ROS production may lead to nonspecific modification of lipids and proteins, which may then result in bioenergetic dysfunctions. If increased mitochondrial ROS production in heat-stressed chickens causes oxidative damage, changes in mitochondrial peroxidized lipids and oxidatively modified proteins can be detected. To study this, 3-week-old male broiler chickens (n=4-8) were exposed to acute heat stress (34°C for 18h) while control chickens were kept at thermoneutral condition (25°C). Skeletal muscle subsarcolemmal mitochondria were isolated and used to study mitochondrial malondialdehyde (MDA) and protein carbonyl groups. Mitochondrial thiobarbituric acid reactive substances (TBARS) formation was measured colorimetrically with 0.01% butylated hydroxytoluene (BHT) and expressed as MDA equivalent. To detect oxidation-sensitive mitochondrial proteins, polypeptides resolved by two-dimensional (2D) electrophoresis were immunostained with DNP-specific antibodies for carbonylated proteins using Western blotting. In heat-stressed chickens, mitochondrial MDA was 2.7-fold higher, and 82 mitochondrial proteins were oxidized when compared to that of control chickens. These results suggest that in heat-stressed chickens, increased mitochondrial ROS production leads to oxidative damage to mitochondrial lipids and proteins.

Superoxide radical production in chicken skeletal muscle induced by acute heat stress

Heat stress is of major concern for poultry, especially in the hot regions of the world because of the resulting poor growth performance, immunosuppression, and high mortality. To assess superoxide (O2*-) production in mitochondria isolated from skeletal muscle of chickens (n = 4 to 8) exposed to acute heat stress, electron spin resonance (ESR) spectroscopy using 5,5-dimethyl-1-pyrroline N-oxide (DMPO) as a spin trap agent and lucigenin-derived chemiluminescence (LDCL) method were applied. ESR spectra of suspensions containing mitochondria from control and acute heat-treated meat-type chickens showed similar hyperfine coupling constants (aN = 1.44 mT, aHbeta = 0.12 mT, and aHbeta = 0.11 mT) to those of DMPO-O2*- adducts observed in a hypoxanthine-xanthine oxidase system. Heat exposure resulted in enhancement of the DMPO-O2*- signal. The results using LDCL showed significantly enhanced superoxide production in heat stress-treated skeletal muscle mitochondria of meat-type chickens, whereas no such increase was observed in laying chickens. The enhancement of superoxide production in the former case was associated with heat-induced increments in rectal and muscle temperatures, leading to significant body weight loss. In contrast, the latter case showed no increase in temperatures, although there was a slight decrease in body weight gain. Percentage increases of superoxide production in the presence of carboxyatractylate, a specific inhibitor of adenine nucleotide translocator (ANT), were the same for skeletal muscle mitochondria from meat- and laying-type chickens from the control or heat stress-treated group. This finding suggests the irrelevance of ANT in the regulation of reactive oxygen species flux under heat stress conditions. The study provides the first evidence of superoxide anion production in the skeletal muscle mitochondria of meat-type chickens in response to acute heat stress.

The fastest contracting muscles of nonmammalian vertebrates express only one isoform of the ryanodine receptor

The skeletal muscles of chickens, frogs, and fish have been reported to express two isoforms (alpha and beta) of the sarcoplasmic reticulum calcium release channel (ryanodine receptor or RYR), while mammals express only one. We have studied patterns of RYR isoform expression in skeletal muscles from a variety of fish, reptiles, and birds with immunological techniques. Immunoblot analysis with a monoclonal antibody that recognizes both nonmammalian RYR isoforms and a polyclonal antibody specific to the alpha isoform show two key results: (a) two reptilian orders share with mammals the pattern of expressing only the alpha (skeletal) RYR isoform in skeletal muscle; and (b) certain functionally specialized muscles of fish and birds express only the alpha RYR isoforms. While both isoforms are expressed in the body musculature of fish and birds, the alpha isoform is expressed alone in extraocular muscles and swimbladder muscles. The appearance of the alpha RYR isoform alone in the extraocular muscles and a fast-contracting sonic muscle in fish (toadfish swimbladder muscle) provides evidence that this isoform is selectively expressed when rapid contraction is required. The functional and phylogenetic implications of expression of the alpha isoform alone are discussed in the context of the mechanism and evolution of excitation-contraction coupling.

Intracellular targeting of isoproteins in muscle cytoarchitecture.

Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment.

Microtubule organisation in fibroblasts from dystrophic chickens and persons with Duchenne muscular dystrophy

In spite of many attempts to correlate the expression of the Duchenne muscular dystrophy (DMD) genotype with defined phenotypic changes in both muscle and non-musde cells from affected individuals1–4, the molecular basis of this X-linked genetic defect has not yet been identified. However, Shay and Fuseler reported recently5 that interphase cells derived from the muscle of newborn dystrophic chickens had greatly diminished cytoplasmic microtubule complexes, and they speculated that this might be due to a specific defect in the microtubule-organising centres. Insofar as microtubules are a major component of the intracellular cytoskeleton6, a network thought to interact with the cell-surface membrane7, the possibility that a microtubule defect might explain the membrane alterations associated with muscular dystrophies1–4 makes this finding extremely interesting. To assess the generality of this finding with respect to chicken dystrophy and to extend the approach to DMD, we used indirect immunofluorescence with an antiserum to purified brain tubulin to examine the organisation of microtubules in cells from the muscle of dystrophic chickens and in skin fibroblasts from persons with DMD. Unlike Shay and Fuseler5, we find no lack of a microtubular network in cells derived from cardiac or skeletal muscle of chickens carrying the dystrophic gene (am). Furthermore, our examination of fibroblasts from persons with DMD revealed a similar extensive network of microtubules in each case.

Dystrophic chicken muscle: altered synaptic acetylcholinesterase.

Individual motor endplates in the skeletal muscles of chickens genetically homozygous for muscular dystrophy have been compared with those in normal chickens. Measurements were made there, by specific autoradiographic techniques, of the numbers of total cholinesterase-like molecules and of acetylcholinesterase molecules. The acetylcholinesterase is distinctly decreased at the endplates in dystrophic muscles. The various data available on these muscles are compatible with the concept that a neural factor which determines the synaptic acetylcholinesterase, along with a number of other characters in the muscle cell, is defective in this disorder.

Aflatoxicosis: The Presence of Aflatoxins or Their Metabolites in Livers and Skeletal Muscles of Chickens

INTRODUCTIONFOLLOWING the discovery of aflatoxins, which are produced by certain strains of Aspergillus flavus (Sargeant et al., 1961), interest has developed in mycotoxicological investigations.Aflatoxin is composed of at least 4 difurano coumatin derivatives which, when separated by thin layer chromatography (TLC), fluoresce under long-wave ultraviolet light in descending order as blue (B1 and B2) and green (G1 and G2) spots (Asao et al., 1963Asao et al., 1965; de Iongl et al., 1962; Hartley et al., 1963). Of the 4 derivatives, B1 is the most toxic, with an LD50 of 18.2 μg. in day old ducklings, (Carnaghan et al., 1963). Metabolism of aflatoxin occurs primarily in the liver, but metabolic pathways are not fully understood.Although numerous reports have been published on the biological, chemical and toxic properties of aflatoxin, its presence in food of animal origin and destined for human consumption has not been adequately established (Hesseltine et al., 1966).…

Investigation of Myosin Heterogeneity Observed during Chromatography on Diethylaminoethyl Cellulose

A procedure is described for obtaining a purified, stable, polyribonucleotide-free myosin fraction from skeletal muscle of chicken. The method involves extraction by pyrophosphate, differential ultracentrifugation of the extract, treatment with pancreatic ribonuclease, gel filtration of the treated extract, and chromatography on diethylaminoethyl cellulose. A myosin obtained by the pyrophosphate extraction and gel filtration appears to be homogeneous by electrophoresis or analytical ultracentrifugation. This material yields, on DEAE-cellulose chromatography, five fractions (I to V), eluting at ionic strengths corresponding to the following compositions of the elution buffers: I, 0.02 m potassium pyrophosphate; II, 0.125 m KCl-0.003 m potassium pyrophosphate; III, 0.18 m KCl-0.005 m potassium pyrophosphate; IV, 0.36 m KCl-0.01 m potassium pyrophosphate; and V, 0.90 m KCl-0.02 m potassium pyrophosphate. Fraction I consists mainly of myoglobin, Fractions II to IV show the characteristics of myosin, and Fraction V is a ribonucleoprotein. The two myosin fractions eluted at lower ionic strength contain polyribonucleotide and have a high Ca2+ adenosine triphosphatase activity, while Fraction IV, which is eluted at high ionic strength, is free of polyribonucleotide and has a low Ca2+ ATPase activity. The sedimentation values of the chromatographic myosin fractions are similar, but their electrophoretic behavior differs. All fractions give negative results for ATP sensitivity and Mg2+ ATPase activity. Results obtained following treatment with RNase or chelating agents of the extract or myosin eluted at lower ionic strength on DEAE-cellulose suggest that the chromatographic heterogeneity observed is due to the complex formation between polyribonucleotide and myosin and mediated by divalent ions. Pretreatment with these agents results in a single myosin fraction eluted from DEAE-cellulose at the same ionic strength as the myosin free from polyribonucleotide. Treatment of chromatographic myosin fractions containing polyribonucleotide by this same procedure gives the same polyribonucleotide-free fraction upon chromatography. Consideration of the results obtained by electrophoresis and ultracentrifugation of the Sephadex-excluded fractions in relation to the chromatographic behavior suggests that pyrophosphate extraction results in myosin which is in complex with polyribonucleotide by means of divalent ions. ATPase activity was found to be lowest in the myosin fraction which was free of polyribonucleotide when compared to the fractions which did contain polyribonucleotide. No explanation of this observation is given at the present time.