[Regulation of m

N^(6)-methyladenosine (m^(6)A) has been identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, and plays important biological roles in the regulation of cellular metabolic processes. However, its role in myogenic differentiation is unclear. Here, we altered the m^(6)A RNA methylation level by overexpression of METTL3, and explored the effect of m^(6)A RNA methylation on myogenic differentiation of murine myoblasts in vitro. The m6A RNA methylation level is regulated by exogenous methylation inhibitor cycloleucine (Cyc) and methyl donor betaine (Bet). Therefore, chemical reagents of Cyc and Bet were used to test the regulatory effect of m^(6)A RNA methylation on myogenic differentiation. Results showed that METTL3 and Bet positively regulated the m^(6)A RNA methylation levels, and Cyc negatively regulated m^(6)A RNA methylation levels. In addition, m^(6)A methylation positively regulated myogenic differentiation in murine myoblasts. These findings provide insight in the mechanisms underlying the effect of m^(6)A RNA methylation on myogenesis.

[MiR-222-3р Regulates the Proliferation and Differentiation of C2C12 Myoblasts by Targeting BTG2].

MiR-222-3р has been implicated in tumor cell proliferation and has an important role in the differentiation and maturation of myogenic cells. However, its role in skeletal myoblast proliferation is still unclear. In this study, we found that miR-222-3р expression increases initially and then decreases during C2C12 myoblast proliferation. Using synthetic miRNA mimics and inhibitors in gain- or loss-of-function experiments, we snowed that miR-222-3р overexpression in C2C12 cells promotes myoblast proliferation and represses myofiber formation, while miR-222-3р downregulation has the opposite effect. Using a prediction program, BTG2 was identified as a possible target gene of miR-222-3р. During myogenesis, miR-222-3р mimics repress BTG2 expression, while miR-222-3р inhibitors promote BTG2 expression. Using dual-luciferase reporter assay, we further demonstrated that miR-222-3р specifically targets BTG2. Additionally, we show that siRNA-mediated downregulation of BTG2 expression in C2C12 myoblasts promotes the proliferation and suppresses differentiation. In conclusion, we provide a novel insight into the mechanism by which miR-222-3р regulates the proliferation and differentiation of C2C12 myoblasts by targeting BTG2. This information contributes to our understanding of the role of miRNAs in skeletal muscle development.

Effects of muscle fiber type on glycolytic potential and meat quality traits in different Tibetan pig muscles and their association with glycolysis-related gene expression.

The myosin heavy chain (MyHC) composition, glycolytic potential, mitochondrial content, and gene expression related to energy metabolism were analyzed in eight muscles from Tibetan pigs, to study how meat quality develops in different muscle tissues. The muscles were classified into three clusters, based on MyHC composition: masseter, trapezius, and latissimus dorsi as 'slow-oxidative-type'; psoas major and semimembranosus as 'intermediate-type'; and longissimus dorsi, obliquus externus abdominis, and semitendinosus as 'fast-glycolytic-type'. The 'slow-oxidative-type' muscles had the highest MyHC I and MyHC IIA content (P < 0.01); 'intermediate-type' muscles, the highest MyHC IIx content (P < 0.01); and 'fast-glycolytic-type' muscles, the highest MyHC IIb content (P < 0.01). The pH values measured in 'slow-oxidative-type' muscles were higher than those in the other clusters were; however, the color of 'fast-glycolytic-type' muscles was palest (P < 0.01). Mitochondrial content increased in the order: fast-glycolytic-type < intermediate-type < slow-oxidative-type. In the 'slow-oxidative-type' muscles, the expression levels of genes related to ATP synthesis were higher, but were lower for those related to glycogen synthesis and glycolysis. Mitochondrial content was significantly positively correlated with MyHC I content, but negatively correlated with MyHC IIb content. MyHC I and mitochondrial content were both negatively correlated with glycolytic potential. Overall, muscles used frequently in exercise had a higher proportion of type I fibers. 'Slow-oxidative-type' muscles, rich in type I fibers with higher mitochondrial and lower glycogen and glucose contents, had a higher ATP synthesis efficiency and lower glycolytic capacity, which contributed to their superior meat quality.

Analysis of carcass and meat quality traits and nutritional values of hybrid wild boars under different crossing systems.

The aim of this study was to analyze the results of two crossing systems between wild boars and different domesticated pig breeds. Hybrid wild boars were produced by crossing captured wild boars with Meishan pigs and LY sows according to the traditional production system. The resultant commercial hybrids were black and white in coat color, respectively. Significant differences were found in the carcass and meat quality traits and nutritional values between these two hybrid wild boars. Compared with the white hybrid wild boars, at the age of 300 days, the body weight of black hybrid wild boars was 9.41 kg lower, while percent lean was 2.51% less and percent fat 2.45% higher (P < 0.05). The black hybrid wild boars had higher pH2 (6.17 vs 6.09) and intramuscular fat (3.34 vs 2.52%), lower drip loss (2.21 vs 2.68%) and shear force (44.00 vs 52.23) (P < 0.05), and more unsaturated fatty acids and essential amino acids (P < 0.05). In conclusion, cross breeding was shown to be an effective method to improve the overall production performance of wild boars, but crossing with different dam line breeds caused different responses. Compared with the white hybrid wild boars, the black hybrid wild boars had worse growth rate and carcass traits, but better meat quality traits and nutritional values.

Distribution of H-FABP and ACSL4 gene polymorphisms and their associations with intramuscular fat content and backfat thickness in different pig populations.

Here, we analyzed the distribution of H-FABP/(HinfI, MspI, and HaeIII) and ACSL4/RsaI polymorphisms, and the associations of these 4 polymorphic loci with intramuscular fat (IMF) content and backfat thickness (BFT) in Yanan, Jinhua, Duroc, Landrace, Yorkshire, and Duroc x (Landrace x Yorkshire) (DLY) pigs. H-FABP/HinfI polymorphisms were present in all the 6 populations. At the ACSL4/RsaI locus, sows had 3 genotypes, whereas boars only had haplotype A or G, in Duroc, Landrace, Yorkshire, and DLY pigs. H-FABP/(MspI and HaeIII) and ACSL4/RsaI polymorphisms were absent in Yanan and Jinhua pigs. Linkage disequilibrium analysis indicated that the 3 loci (HinfI, MspI, and HaeIII) were separated. Association analysis showed that the H-FABP/HinfI locus significantly affected IMF content in DLY (P < 0.05) and Yanan (P < 0.001) pigs. The highest IMF content was recorded in the adH haplotype of the 3 H-FABP polymorphic loci (2.59%, P < 0.05) in DLY pigs. At the ACSL4/RsaI locus, higher IMF content was recorded for sows with a GG genotype or boars with a G haplotype compared to those with an AA genotype (2.53 vs 2.10%, P < 0.05) or A haplotype (2.48 vs 1.73%, P < 0.01) in DLY pigs. Significant differences were not obtained among these 4 polymorphic loci and BFT (P > 0.05). The results indicate that H-FABP and ACSL4 genes might serve as markers to improve IMF content (but not BFT) in the pig breeding system.

Effect of muscle-fiber type on glycogenin-1 gene expression and its relationship with the glycolytic potential and pH of pork.

This study analyzed the effect of muscle-fiber type composition on glycogenin-1 (GYG) gene expression and its impact on pH. The longissimus dorsi (LD) muscle contains more type IIB fibers (75.10%) than does the psoas major (PM) muscle (41.58%), while the PM has more type I (3.65 vs 0.94%), type IIA (34.15 vs 10.63%), and type IIX (20.62 vs 13.33%) fibers. Compared with PM, glycolytic potential (GP), pH45 min, and ΔpH from 45 min to 24 h post-mortem were all relatively higher in LD. Glycogen metabolites (lactate and GP) were negatively correlated with pH24 h and positively correlated with ΔpH. Expression of GYG was generally higher in LD. GYG expression was positively correlated with glycogen metabolite (lactate and GP) content and ΔpH, and was negatively correlated with pH24 h. These data confirm that the muscle-fiber type and GP have significant effects on ultimate pH and pH decline, and suggest that expression of GYG in muscles is related to the metabolism of glycogen and may impact GP, ΔpH, and ultimate pH. High expression of GYG was associated with a high glycogen content, large pH decline, and low ultimate pH in muscles post-mortem.

Inbreeding and genetic diversity in three imported Swine breeds in china using pedigree data.

The accumulation of inbreeding and the loss of genetic diversity is a potential problem in the modern swine breeds in China. Therefore, the purpose of this study was to analyze the pedigrees of Chinese Duroc (CD), Landrace (CL) and Yorkshire (CY) swine to estimate the past and current rates of inbreeding, and to identify the main causes of genetic diversity loss. Pedigree files from CD, CL and CY containing, 4529, 16,776 and 22,600 records, respectively, were analyzed. Pedigree completeness indexes of the three breeds, accounting for one generation back, were 83.72, 93.93 and 93.59%, respectively. The estimated average annual inbreeding rates for CD, CL and CY in recent three years were 0.21, 0.19 and 0.13%, respectively. The estimated average percentage of genetic diversity loss within each breed in recent three years was about 8.92, 2.19, and 3.36%, respectively. The average relative proportion of genetic diversity loss due to unequal contributions of founders in CD, CL and CY was 69.09, 57.95 and 60.57%, and due to random genetic drift was 30.91, 42.05 and 39.43%, respectively. The estimated current effective population size for CD, CL and CY was 76, 117 and 202, respectively. Therefore, CD has been found to have lost considerable genetic diversity, demanding priority for optimizing the selection and mating to control future coancestry and inbreeding. Unequal contribution of founders was a major cause of genetic diversity loss in Chinese swine breeds and random genetic drift also showed substantial impact on the loss of diversity.

Carcass and meat quality traits of four commercial pig crossbreeds in China.

We evaluated carcass and meat quality traits of two Chinese native crossbreeds Landrace x Meishan (LM) and Duroc x (Landrace x Meishan) (DLM) and two foreign crossbreeds Duroc x (Landrace x Yorkshire) (DLY) and PIC (an imported five-way crossbreed). One hundred and twenty weaned pigs (half castrated males and half females) were reared and slaughtered at a predestinated slaughter age. The general carcass and meat quality traits were measured and analyzed. The DLY and PIC crosses had significantly heavier live weights (93.39 and 96.33 kg, P < 0.01), significantly higher dressing percentages (80.65 and 79.39%, P < 0.05), significantly bigger loin areas (42.69 and 43.91 cm(2), P < 0.001), and significantly more lean carcasses (65.78 and 66.40%, P < 0.001) than LM and DLM. On the other hand, LM had a significantly lower live weight (70.29 kg, P < 0.01), significantly thicker back fat (3.54 cm, P < 0.001), significantly less lean carcasses (46.82%, P < 0.001), and significantly less ham and breech (26.53%, P < 0.05) than the other crossbreeds. Among meat quality parameters, LM had the highest intramuscular fat content (5.02%, P < 0.001) and the smallest fiber area (3126.45 µm(2), P < 0.01). However, PIC showed the lowest pH(1) (5.82, P < 0.01) and pH(2) (5.63, P < 0.01), the highest drip loss (2.89%, P < 0.01), and the lowest intramuscular fat (1.35%, P < 0.001). We concluded that LM and DLM had good meat quality traits but poorer carcass traits than DLY and PIC; DLY had good carcass and meat quality traits; PIC had good carcass traits, but it had less intramuscular fat, lower pH and higher drip loss.

Antisense repression of sucrose synthase in carrot (Daucus carota L.) affects growth rather than sucrose partitioning.

To unravel the roles of sucrose synthase in carrot, we reduced its activity in transgenic carrot plants by an antisense approach. For this purpose, the cDNA for the main form of carrot sucrose synthase was expressed in antisense orientation behind the 35S promoter of cauliflower mosaic virus. In independent antisense plant lines grown in soil, sucrose synthase activity was reduced in tap roots but not in leaves. In the sink organs, sucrose utilization was markedly decreased and higher levels of sucrose but lower levels of UDP-glucose, glucose, fructose, starch and cellulose were found. The phenotype of the antisense plants clearly differed from that of control plants. Both leaves and roots were markedly smaller, and the antisense line with the lowest sucrose synthase activity also developed the smallest plants. In most of the plant lines, the leaf-to-root dry weight ratios were not changed, suggesting that sucrose synthase in carrot is a major determinant of plant growth rather than of sucrose partitioning. In contrast to the acid invertases, which are critical for partitioning of assimilated carbon between source leaves and tap roots (Tang et al., Plant Cell 11: 177-189 (1999)), sucrose synthase appears to be the main sucrose-cleaving activity, feeding sucrose into metabolism.

Antisense repression of vacuolar and cell wall invertase in transgenic carrot alters early plant development and sucrose partitioning.

To unravel the functions of cell wall and vacuolar invertases in carrot, we used an antisense technique to generate transgenic carrot plants with reduced enzyme activity. Phenotypic alterations appeared at very early stages of development; indeed, the morphology of cotyledon-stage embryos was markedly changed. At the stage at which control plantlets had two to three leaves and one primary root, shoots of transgenic plantlets did not separate into individual leaves but consisted of stunted, interconnected green structures. When transgenic plantlets were grown on media containing a mixture of sucrose, glucose, and fructose rather than sucrose alone, the malformation was alleviated, and plantlets looked normal. Plantlets from hexose-containing media produced mature plants when transferred to soil. Plants expressing antisense mRNA for cell wall invertase had a bushy appearance due to the development of extra leaves, which accumulated elevated levels of sucrose and starch. Simultaneously, tap root development was markedly reduced, and the resulting smaller organs contained lower levels of carbohydrates. Compared with control plants, the dry weight leaf-to-root ratio of cell wall invertase antisense plants was shifted from 1:3 to 17:1. Plants expressing antisense mRNA for vacuolar invertase also had more leaves than did control plants, but tap roots developed normally, although they were smaller, and the leaf-to-root ratio was 1.5:1. Again, the carbohydrate content of leaves was elevated, and that of roots was reduced. Our data suggest that acid invertases play an important role in early plant development, most likely via control of sugar composition and metabolic fluxes. Later in plant development, both isoenzymes seem to have important functions in sucrose partitioning.

In vitro increases in plasmid DNA supercoiling by hydrostatic pressure.

By conducting topoisomerase I-mediating supercoiling assays, effects of elevated pressure on DNA supercoiling were investigated for the first time. It was found that pressure elevations induced a progressive increase in plasmid DNA linking numbers, winding the DNA duplex by a magnitude of 1.1-1.6x10(-3) angular degree/base/MPa. Implications for the findings were discussed in terms of disturbance of the tertiary structure of DNA by elevated pressure.

Evidence for genetic linkage between the ure and trh genes in Vibrio parahaemolyticus.

Although V. parahaemolyticus does not generally produce urease, several studies have reported urease-positive V. parahaemolyticus isolates from clinical sources. Recently, studies have shown a complete coincidence between the urease-producing phenotype of V. parahaemolyticus strains and the possession of the thermostable direct haemolysin (TDH)-related haemolysin (TRH) gene (trh). TRH, like TDH, is considered to be an important virulence factor in the pathogenesis of V. parahaemolyticus gastroenteritis. The present study attempted to identify the gene ure encoding urease in V. parahaemolyticus to clarify the relationship between urease production and possession of trh. The polymerase chain reaction with mixed oligonucleotide primers targeted for conserved sequences of reported ure genes from other species was used to prepare a DNA probe to detect the V. parahaemolyticus ure gene. Colony hybridisation with this ure probe demonstrated that all the ure-positive strains produced urease. Considering the coincidence between production of urease and possession of trh in V. parahaemolyticus, it was concluded that the presence or absence of the ure gene is completely coincident with that of the trh gene in V. parahaemolyticus strains. Furthermore, the relative location of ure and trh on V. parahaemolyticus chromosomal DNA was analysed by pulsed-field gel electrophoresis. The results showed that, in all the strains examined, ure and trh were detected on the same NotI fragment, showing that the two genes localise within a relatively small portion of the chromosome DNA. These results suggest that the ure and trh genes are genetically linked in V. parahaemolyticus strains.

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studies using [gamma-32P] ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppressed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.

Ca(2+)-independent cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin (TDH) on Intestine 407, a cell line derived from human embryonic intestine.

The effects of Vibrio parahaemolyticus thermostable direct hemolysis on Intestine 407, a cell line derived from the intestine of human embryos, were investigated. The hemolysin was shown to be cytotoxic to Intestine 407. This cytotoxicity is accompanied by the damage of plasma membrane and lysosomes, as well as cellular degeneration in the form of large transparent blebs. Although an increase in cytosolic free Ca2+ due t the influx of extracellular Ca2+ was observed in cells treated with thermostable direct hemolysin, it was found to be irrelevant to any of the above effects. These results suggest that the effects of thermostable direct hemolysin observed in this study on Intestine 407 are not mediated by Ca(2+)-dependent pathways.

Isolation of mutant toxins of Vibrio parahaemolyticus hemolysin by in vitro mutagenesis.

Thermostable direct hemolysin produced by Vibrio parahaemolyticus is a major virulence factor of the organism. The hemolysin has a variety of biological activities such as lethality to mice, cytotoxicity to cultured cells, cardiotoxicity, and fluid accumulating activity in rabbit ileal loop test. In this study, we attempted to isolate less hemolytic mutant toxins of the thermostable direct hemolysin to use them for analysis of mode of action of the hemolysin. Six mutant toxins were obtained by in vitro mutagenesis of the cloned gene for the hemolysin. Characterization of the mutant toxins demonstrated that single amino acid substitutions at Gly62, Trp65, Thr67, Gly86, Glu116 and Glu138 resulted in a loss or lowering of the hemolytic activity. Two of the mutant toxins inhibited hemolysis by wild-type toxin on rabbit blood agar plates, while their hemolytic activity was below the detectable level. These mutant toxins would be useful for identifying the as yet unknown receptor for the hemolysin on the target cell membrane.

A mutant toxin of Vibrio parahaemolyticus thermostable direct hemolysin which has lost hemolytic activity but retains ability to bind to erythrocytes.

A mutant toxin, R7, of thermostable direct hemolysin (TDH) with a single amino acid substitution at glycine 62 was analyzed. The hemolytic activity of R7 decreased to less than 1/1,000 of that of wild-type TDH, and its mouse lethality was undetectable. This mutant toxin, however, showed a marked inhibitory effect on hemolysis by wild-type TDH. Enzyme immunoassay and flow cytometric analysis demonstrated that R7 retained approximately 50% of the ability to bind to erythrocytes compared with that of wild-type TDH, suggesting that its inhibition of hemolysis by wild-type TDH might be due to blocking the binding sites on the erythrocyte membrane. Wild-type TDH affected the erythrocyte membrane by causing an influx of calcium and propidium iodide, while R7 showed no detectable effects of these kinds. These results suggest that hemolysis by TDH consists of at least two steps, binding and postbinding, and that R7 is likely to be a postbinding activity-deficient mutant toxin of TDH.