Multiple IgH Isotypes Including IgD, Subclasses of IgM, and IgY Are Expressed in the Common Ancestors of Modern Birds.

Although evolutionarily just as ancient as IgM, it has been thought for many years that IgD is not present in birds. Based on the recently sequenced genomes of 48 bird species as well as high-throughput transcriptome sequencing of immune-related tissues, we demonstrate in this work that the ostrich (Struthio camelus) possesses a functional δ gene that encodes a membrane-bound IgD H chain with seven CH domains. Furthermore, δ sequences were clearly identified in many other bird species, demonstrating that the δ gene is widely distributed among birds and is only absent in certain bird species. We also show that the ostrich possesses two µ genes (µ1, µ2) and two υ genes (υ1, υ2), in addition to the δ and α genes. Phylogenetic analyses suggest that subclass diversification of both the µ and υ genes occurred during the early stages of bird evolution, after their divergence from nonavian reptiles. Although the positions of the two υ genes are unknown, physical mapping showed that the remaining genes are organized in the order µ1-δ-α-µ2, with the α gene being inverted relative to the others. Together with previous studies, our data suggest that birds and nonavian reptile species most likely shared a common ancestral IgH gene locus containing a δ gene and an inverted α gene. The δ gene was then evolutionarily lost in selected birds, whereas the α gene lost in selected nonavian reptiles. The data obtained in this study provide significant insights into the understanding of IgH gene evolution in tetrapods.

Unstable expression of transgene is associated with the methylation of CAG promoter in the offspring from the same litter of homozygous transgenic mice.

Transgenic animals have been established for studying gene function, improving animals' production traits, and providing organ models for the exploration of human diseases. However, the stability of inheritance and transgene expression in transgenic animals has gained extensive attention. The unstable expression of transgene through DNA methyltransferase (DNMT) targeting to the methylation of transgenic DNA such as CAG promoter and Egfp coding region in homozygous transgenic animals is still unknown. In the present study, the offspring from the same litter of homozygous transgenic mice carrying ubiquitously expressed enhanced green fluorescence protein driven by CMV early enhancer/chicken ß-actin (CAG) promoter was observed to have unstable expression of transgene Egfp, quantitative PCR, western blot and bisulfite sequencing were conducted to quantify the expressional characteristics and methylation levels in various tissues. The correlation between transgene expression and methylation was analyzed. We have found that transgene expression is dependent on the methylation of CAG promoter, but not Egfp coding region. We have also characterized the correlation between the methylation of CAG promoter and DNMT, and found that only Dnmt3b expression is correlated with the methylation of CAG promoter. In conclusion, Dnmt3b-related methylation of CAG promoter can inhibit the transgene expression and may result in the unstable expression of transgene in the offspring from the same litter of homozygous transgenic mice.

Cloning and in vitro function analysis of codon-optimized FatI gene.

Currently, n-3 polyunsaturated fatty acids (n-3 PUFAs) have attracted great attention because of their biological significance to organisms. In addition, PUFAs show an obvious impact on prevention and treatment of various diseases. Because n-3 PUFAs cannot be endogenously synthesized by mammals, mammals have to rely on a dietary supplement for sufficient supply. The finding and application of the fatty acid dehydrogenase I (FatI) gene are expected to change the current situation because it can convert n-6 polyunsaturated fatty acids (n-6 PUFAs) to n-3 PUFAs. Meanwhile, the gradual maturation of transgenic technology makes it possible to produce transgenic animals that can synthesize n-3 PUFAs by themselves. In this study, the DNA coding sequence of FatI was synthesized by a chemical method after codon optimization according to the mammal's codon bias. The synthesized DNA sequence was introduced into Boer goat fetal fibroblasts by the constructed recombinant eukaryotic expression vector pcDNA3.1(+)-FatI. Boer goat fetal fibroblasts were transfected by electroporation, and the stable transfected cell lines were obtained by G418 selection. Genomic DNA PCR and Southern blot were applied to verify that the foreign gene FatI was integrated into the genome of the Boer goat fibroblasts. RT-PCR results showed the expression of FatI gene at the mRNA level. The fatty acid profile of cells carrying the FatI gene revealed an increase in total n-3 PUFAs (from 0.61 to 0.95), but a decrease in n-6 PUFAs (from 10.34 to 9.85), resulting in a remarkable increase in the n-3:n-6 ratio (from 0.059 to 0.096). The n-3:n-6 ratio had a 63.49 percent increase, which is a precursor of the response of n-3 desaturase activity of the FatI gene. The study may provide a practical tool for producing transgenic animals that can produce n-3 PUFAs by themselves, and we hope that the application will lay the foundation for animals producing n-3 PUFAs, which will benefit human nutrition and wellness.

Anti-senescence effect of FatI gene in goat somatic cells.

The fatty acid dehydrogenase I (FatI) is able to express in mammalian cells and convert n-6 polyunsaturated fatty acids (PUFAs) to n-3 PUFAs. n-3 PUFA is an important component of the cell membrane and plays an important role in the prevention and control of a variety of human diseases. However, n-3 PUFAs cannot be endogenously synthesized by mammals because they lack the dehydrogenase that converts n-6 to n-3 PUFA. For the time being, gradually matured transgenic technology makes it possible to produce transgenic animals that are able to synthesize n-3 PUFAs by themselves. However, the transgenic technology itself may bring negative impacts. In this study, the eukaryotic expression vector pcDNA3.1-FatI was introduced into the genome of Boer goat fetal fibroblasts cultured in vitro, and the influence of biological characteristics of the fetal fibroblast was studied via overexpression of FatI. The results showed that the proliferation and apoptosis of cultured fetal fibroblast were not affected significantly by the overexpression of FatI using BrdU and TUNEL staining methods, respectively. Moreover, the overexpression of FatI significantly inhibited the senescence of somatic cells compared with enhanced green fluorescent protein (EGFP) transgenic cells (P < 0.01). Quantitative PCR revealed that the mRNA expression of P16 and P53 in the FatI transgenic cell group was significantly lower than that in the EGFP transgenic cell group (P < 0.01). In conclusion, the senescence of goat somatic cells was inhibited by the overexpression of the FatI gene.

Granulosa Cells Improved Mare Oocyte Cytoplasmic Maturation by Providing Collagens.

Assisted reproductive technology has important clinical applications and commercial values in the horse industry. However, this approach is limited largely by the low efficiency of oocyte in vitro maturation (IVM), especially cytoplasmic maturation. To improve the efficiency of mare oocyte IVM, we evaluated the effects of co-culture with cumulus-oocyte complexes (COCs) and granulosa cells (GCs) from follicles with small (<15 mm) and large diameters (>35 mm). Our results showed that oocyte nucleus maturation was not significantly improved by co-culturing with GCs. Interestingly, the cytoplasmic maturation of oocytes, defined by the distribution of cortical granules and mitochondria, as well as reactive oxygen species (ROS) levels, improved dramatically by co-culture with GCs, especially those derived from small follicles. Moreover, GCs promoted cumulus cell expansion by upregulating the expression of BMP15 in oocytes. To determine the mechanism underlying the effects of GCs, the transcriptomes of GCs from large and small follicles were compared. Expression levels of COL1A2, COL6A1, and COL6A2 were significantly higher in GCs from small follicles than in those from large follicles. These three genes were enriched in the extracellular matrix proteins-receptor interaction pathway and were involved in the regulation of collagens. Taken together, our results suggest that co-culture with GCs is beneficial to oocyte cytoplasmic maturation, and the increased expression of COL1A2, COL6A1, and COL6A2 improve the mare oocyte IVM system via the regulation of collagen.

Identification of Candidate Genes for Twinning Births in Dezhou Donkeys by Detecting Signatures of Selection in Genomic Data.

Twinning trait in donkeys is an important manifestation of high fecundity, but few reports are available elucidating its genetic mechanism. To explore the genetic mechanism underlying the twin colt trait in Dezhou donkeys, DNA from 21 female Dezhou donkeys that had birthed single or twin colts were collected for whole-genome resequencing. FST, θπ and Tajima's D were used to detect the selective sweeps between single and twin colt fecundity in the Dezhou donkey groups. Another set of 20 female Dezhou donkeys with single or multiple follicles during estrus were selected to compare concentrations of reproductive hormone including follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2) and progesterone (P4). Four candidate genes including ENO2, PTPN11, SOD2 and CD44 were identified in the present study. The CD44 gene had the highest FST value, and ENO2, PTPN11 and SOD2 were screened by two joint analyses (FST and θπ, θπ and Tajima's D). There was no significant difference in the LH, FSH and P4 levels between the two groups (p > 0.05); however, the serum E2 content in the multi-follicle group was significantly higher than that in the single-follicle group (p < 0.05). The identified candidate genes may provide new insights into the genetic mechanism of donkey prolificacy and may be useful targets for further research on high reproductive efficiency.

Comparison of gut microflora of donkeys in high and low altitude areas.

Donkeys' gut microbe is critical for their health and adaptation to the environment. Little research has been conducted on the donkey gut microbiome compared with other domestic animals. The Tibetan Plateau is an extreme environment. In this study, 6 Qinghai donkeys (QH) from the Tibetan Plateau and 6 Dezhou donkeys (DZ) were investigated, and the contents of 4 parts-stomach, small intestine, cecum, and rectum-were collected. 16S rRNA sequencing and metagenomic sequencing were used to analyze the composition and diversity of gut microbial communities in donkeys. The results showed that the flora diversity and richness of the hindgut were significantly higher than those of the foregut (p < 0.01), with no sex differences, and the community structure and composition of the same or adjacent regions (stomach, small intestine, cecum, and rectum) were similar. Besides, the flora diversity and richness of QH on the Tibetan Plateau were significantly higher than those of DZ (p < 0.05). The major pathways associated with QH were signal transduction mechanisms and carbohydrate transport and metabolism, and Bacteroidales were the major contributors to these functions. Our study provides novel insights into the contribution of microbiomes to the adaptive evolution of donkeys.

A co-culture system with preantral follicular granulosa cells in vitro induces meiotic maturation of immature oocytes.

Development of technologies to mature oocytes in vitro is important for in vitro fertilization research. Here, we investigated the ability of preantral follicular granulosa cells (PAGCs) to restrain apoptosis and to promote the growth and meiotic resumption of immature murine oocytes in vitro. The oocytes of 55-65 µm derived from 12 to 14 days old juvenile mice were co-cultured with PAGCs in vitro. The results showed that the oocytes co-cultured with PAGCs for 7 days grew faster and 14.6% of immature oocytes were able to complete the first meiotic division and arrive at the MII stage. 71 oocytes co-cultured with PAGCs were fertilized and 16 embryos were able to form morula-blastocysts. Following the co-culture of immature oocytes with/without PAGCs for 7 days, the percentage of apoptotic oocytes were 33.5 and 51.4%, respectively (p < 0.01). Furthermore, the inhibition of apoptosis was communicated between oocytes and PAGCs through the GDF9-PI3 K-Akt signaling pathway. In conclusion, the co-culture with PAGCs has a beneficial effect on the growth and maturation of immature oocytes.

Co-culture of sperm with sertoli cells can improve IVF outcomes by increasing sperm motility in mice.

The micro-environment of spermatogenesis is important for the improvement of in vitro fertilization (IVF). Therefore, developing a co-culture system may be valuable to improve the rate of IVF. In this study, we aimed to investigate the secretions of testicular sertoli cells (SCs) to find whether it can improve the micro-environment of IVF, by which promote the efficiency of fertilization in mice. The results showed that the motility of sperms in CCSCF group (sperms co-culture with SCs) was significantly promoted and the rate of fertilization were significantly increased compared with the CTR group (control group: sperms not co-culture with SCs). Moreover, we found that the estrogen concentrations, the expression of estrogen receptor (ER) and the phosphorylation of AMPK in sperms were higher in the CCSCF group than in CTR group. In all, our results indicated that SCs co-cultured with sperms can improve the motility of sperms, E2 secreted by SCs can increase Ca2+ level in the intracellular and the level of phosphorylation of AMPK through Ca-MKKß in sperms.

[Effect of electroacupuncture at Baliao points on intrauterine residue and uterine volume restoration after uterine curettage of incomplete abortion].

OBJECTIVE: To observe the clinical therapeutic effect of the combination of electroacupuncture (EA) at Baliao points (bilateral Shangliao [BL 31], Ciliao [BL 32], Zhongliao [BL 33] and Xialiao [BL 34]) and oral administration of mifepristone tablets and its influence on uterine volume restoration after uterine curettage of incomplete abortion as compared with simple oral administration of mifeprstone tablets. METHODS: A total of 58 patients after uterine curettage of incomplete abortion were randomized into an EA group and a western medication group, 29 cases in each one. In the western medication group, mifepristone tablets were administered orally, 2 tablets each time, once daily. In the EA group, on the base of the treatment as the western medication group, EA was applied to Baliao points, with disperse-dense wave, once daily, 50 min each time. The treatment for 3 days was as one course and 2 courses of treatment were required, at the interval of 1 day in the two courses. Before and after treatment, the area of intrauterine residue and blood flow signal positive rate of color Doppler flow imaging (CDFI) were recorded in patients of the two groups respectively. The days of vaginal bleeding and the rate of second operation were recorded after treatment in patients of the two groups. Using the three-dimensional ultrasound B reconstruction, the uterine endometrial volume after menstruation resumption was measured in patients of the two groups, and the clinical therapeutic effect was evaluated. RESULTS: After treatment, the intrauterine residue area and CDFI blood flow signal positive rate were all reduced as compared with the values before treatment in patients of the two groups (P<0.05). After treatment, the intrauterine residue area and CDFI blood flow signal positive rate in the EA group were less than those in the western medication group (P<0.05). After treatment, the days of vaginal bleeding in patients of the EA group were less than that in the western medication group and the rate of second operation was lower than the western medication group (P<0.05). The uterine endomentrial volume after menstruation resumption in the EA group was larger than that in the western medication group after treatment (P<0.05). The total effective rate was 55.2% (16/29) in the EA group, higher than 37.9% (11/29) in the western medication group (P<0.05). CONCLUSION: The combined treatment of electroacupuncture at Baliao points and oral administration of mifepristone tablets effectively promotes uterine contraction, softens and discharges intrauterine residue and contributes to uterine volume restoration in the patients after uterine curettage of incomplete abortion. The therapeutic effect of this combined therapy is better than simple oral administration of mifepristone tablets.

Ancient Patrilineal Lines and Relatively High ECAY Diversity Preserved in Indigenous Horses Revealed With Novel Y-Chromosome Markers.

Extremely low nucleotide diversity of modern horse Y-chromosome has been reported, and only poor phylogenetic resolution could be resulted from limited Y-chromosome markers. In this study, three types of horse Y-chromosome markers, including Single-nucleotide polymorphisms (SNPs), copy number variants (CNVs), and allele-specific CNVs, were developed by screening more than 300 male horses from 23 indigenous Chinese horse populations and 4 imported horse breeds. Fourteen segregating sites including a novel SNP in the AMELY gene were found in approximately 53 kb of male-specific Y-chromosome sequences. CNVs were detected at 11 of 14 sites, while allele-specific CNVs at 6 polymorphic sites in repeated fragments were also determined. The phylogenetic analyses with the SNPs identified in this study and previously published 51 SNPs obtained mainly from European horses showed that indigenous Chinese horses exhibit much deeper divergence than European and Middle Eastern horses, while individuals of Chinese horses with the C allele of the AMELY gene constituted the most ancient group. Via SNPs, CNVs, and allele-specific CNVs, much higher diversity of paternal lines can be detected than those identified with merely SNPs. Our results indicated that there are ancient paternal horse lines preserved in southwestern China, which sheds new light on the domestication and immigration of horses, and suggest that the priorities of the conservation should be given to the ancient and rare paternal lines. These three marker types provided finer phylogenetic resolution of horse patrilineal lines, and the strategies used in the present study also provide valuable reference for the genetic studies of other mammalian patrilineages.

Maternal imprinting during mouse oocyte growth in vivo and in vitro.

Epigenetic regulation of gene expression is critical for oogenesis in mammals. In this study, a simple and efficient method was used to obtain the oocytes from cultured fetal mouse ovaries of 12.5dpc. The methylation pattern of these oocytes was examined. The results showed that the establishment of imprinting of Igf2r and Peg3 in oocytes derived from cultured fetal mouse germ cells in vitro follows a slower time course than that of oocytes in vivo. However, oocytes in vitro and in vivo share similar methylation patterns. Igf2r was gradually de novo methylated, and the methylation covers 80% CpG sites in oocytes cultured for 28days. However, only 45% of the CpG sites is methylated in Peg3 at the same stage. Furthermore, it demonstrated that the degree of DNA methylation is positively correlated with the size of oocytes in vitro and in vivo, indicating a progressive methylation process during oocyte growth.

Gestational diabetes promotes germ cell cCyst breakdown and primordial follicle formation in newborn mice via the AKT signaling pathway.

Type 1 diabetes (T1D) is a common disease in which pancreatic ß cells are impaired due to auto-immunity, pregnancy in women with it is associated with increased risk of neonatal morbidity, mortality. However, the effects of gestational diabetes on the reproduction of newborn offspring are still poorly understood. Here, we determined the cyst breakdown and primordial follicle formation in neonatal offspring born by streptozotocin (STZ)-induced diabetic or non-diabetic female mice, and found that the germ cell cyst breakdown was promoted in neonatal offspring of STZ -induced diabetic mice at postnatal Day 1, which sequentially accelerated the primordial follicle formation. Further investigation revealed that, the expression level of PI3K and p-AKT were significantly increased in ovaries of offspring born by T1D mice. These results indicated that STZ -induced gestational diabetes promotes germ cell cyst breakdown and primordial follicle formation by regulating the PI3K/AKT signaling pathway in the newborn offspring. In addition, this effect can be rescued by an insulin supplement. Taken together, our results uncover the intergenerational effects of gestational diabetes on neonatal offspring folliculogenesis, and provide an experimental model for treating gestational diabetes and its complications in neonatal offspring.

[Genetic co-adaptability among structural genes under the condition of genetic disequilibrium].

Genetic co-adaptability of colony gene bank is formed in the process of system evolvement, and it is a part of the attribute of gene bank, just as the gene frequency of every locus. With the genetic co-adaptability of loci combination under the condition of genetic disequilibrium, the genesis evolution and the system status of colony could be analyzed; through the study of genetic co-adaptability, the veracity of the economy character mark could be improved. We summarized about the concept, the basic and the actuality of genetic co-adaptability.

Three IgH isotypes, IgM, IgA and IgY are expressed in Gentoo penguin and zebra finch.

Previous studies on a limited number of birds suggested that the IgD-encoding gene was absent in birds. However, one of our recent studies showed that the gene was definitely expressed in the ostrich and emu. Interestingly, we also identified subclass diversification of IgM and IgY in these two birds. To better understand immunoglobulin genes in birds, in this study, we analyzed the immunoglobulin heavy chain genes in the zebra finch (Taeniopygia guttata) and Gentoo penguin (Pygoscelis papua), belonging respectively to the order Passeriformes, the most successful bird order in terms of species diversity and numbers, and Sphenisciformes, a relatively primitive avian order. Similar to the results obtained in chickens and ducks, only three genes encoding immunoglobulin heavy chain isotypes, IgM, IgA and IgY, were identified in both species. Besides, we detected a transcript encoding a short membrane-bound IgA lacking the last two CH exons in the Gentoo penguin. We did not find any evidence supporting the presence of IgD gene or subclass diversification of IgM/IgY in penguin or zebra finch. The obtained data in our study provide more insights into the immunoglobulin heavy chain genes in birds and may help to better understand the evolution of immunoglobulin genes in tetrapods.

Human lactoferrin transgenic rabbits produced efficiently using dimethylsulfoxide-sperm-mediated gene transfer.

Transgenic animal mammary gland bioreactors are used to produce recombinant proteins. However, it is difficult to validate whether these transgenic domestic animals are able to express the recombinant protein efficiently in their mammary glands before the birth of transgenic offspring. In the present study, a simple and efficient method was established to evaluate the functionality of animal mammary gland tissue-expressed cassettes. The gene transfer vector pGBC2LF was constructed, and the expression of human lactoferrin (LF) gene was controlled by the goat beta-casein gene 5' flanking sequence. To obtain the most efficient transfection, the influence of DNA concentration, dimethylsulfoxide (DMSO) concentration, and the ratio of linear-to-circular DNA required for associating DNA with spermatozoa were evaluated. Transfection of exogenous DNA into rabbit spermatozoa was found to be efficient using 30 microg mL(-1) DNA, DMSO at a final concentration of 3%, and a 3 : 1 ratio of linear-to-circular DNA, with 29 of 85 (34.1%) in vitro-fertilised embryos being transgenic. Using DMSO-sperm-mediated gene transfer (DMSO-SMGT), 89 rabbit offspring were produced, with 46 of these (57.1%) being transgenic. As mammary gland bioreactor models, 17 of 21 (81%) transgenic female rabbits could express human LF protein in their glands. During lactation of the transgenic rabbits, the highest level of human LF protein expressed was 153 +/- 31 microg mL(-1), and the mean expression level in all of the transgenic rabbits was 103 +/- 20 microg mL(-1) in the third week, declining gradually after this time. Our results demonstrate that transgenic rabbits produced by DMSO-SMGT were able to express human LF protein in the correct tissue.

[Nuclear transfer of goat somatic cells transgenic for human lactoferrin].

Transgenic animal mammary gland bioreactors are being used to produce recombinant proteins with appropriate post-translational modifications, and nuclear transfer of transgenic somatic cells is a more powerful method to produce mammary gland bioreactor. Here we describe efficient gene transfer and nuclear transfer in goat somatic cells. Gene targeting vector pGBC2LF was constructed by cloning human lactoferrin (LF) gene cDNA into exon 2 of the milk goat beta-casein gene, and the endogenous start condon was replaced by that of human LF gene. Goat fetal fibroblasts were transfected with linearized pGBC2LF and 14 cell lines were positive according to PCR and Southern blot. The transgenic cells were used as donor cells of nuclear transfer, and some of reconstructed embryos could develop to blastocyst in vitro.

[Accelerated somatic cell senescence and changes in p16INK4a expression after exogenous DNA transfection].

During the transfection of mouse embryonic fibroblasts (MEF), we found these cells became senescent, appearing enlarged with hollow cytoplasm and multinucleated. The telomere lengths of these senescent MEFs were significantly shorter than primary untransfected MEFs. In senescent MEF cells, DNA methylation of p16INK4a 5'-regulatory region showed gradual reduction as cells aged. RT-PCR and Northern blot demonstrated that the expression of p16INK4a in transfected senescent cells was 12-16 times more than primary cells. The senescent transfected MEF cells as donors of nuclei could support the early development of cloned embryos after nuclear transfer.

Construction and Analysis of an Adipose Tissue-Specific and Methylation-Sensitive Promoter of Leptin Gene.

DNA methylation plays a very important role in the regulation of gene expression. Under general situations, methylation in a gene promoter region is frequently accompanied by transcriptional suppression, and those genes that are highly methylated display the phenomenon of low expression. In contrast, those genes whose methylation level is low display the phenomenon of active expression. In this study, we conducted DNA methylation analysis on the CpG sites within the promoter regions of five adipose tissue-specific transcriptional factors-Adiponectin, Chemerin, Leptin, Smaf-1, and Vaspin-and examined their messenger RNA (mRNA) expression levels in different mouse tissues. We also performed analyses on the correlation between the DNA methylation levels of these genes and their mRNA expression levels in these tissues. The correlation coefficient for Leptin was the highest, and it displayed a high expression in an adipose tissue-specific manner. Thus, we cloned the regulatory region of Leptin gene and incorporated its promoter into the eukaryotic expression vector pEGFP-N1 and constructed a recombinant plasmid named pEGFP-N1-(p-Lep). This recombinant plasmid was first verified by DNA sequencing and then transfected into mouse pre-adipocytes via electroporation. Measurement of the activity of luciferase (reporter) indicated that p-Lep was capable of driving the expression of the reporter gene. This study has paved a solid basis for subsequent studies on generating transgenic animals.

A C-terminally truncated mouse Best3 splice variant targets and alters the ion balance in lysosome-endosome hybrids and the endoplasmic reticulum.

The Bestrophin family has been characterized as Cl(-) channels in mammals and Na(+) channels in bacteria, but their exact physiological roles remian unknown. In this study, a natural C-terminally truncated variant of mouse Bestrophin 3 (Best3V2) expression in myoblasts and muscles is demonstrated. Unlike full-length Best3, Best3V2 targets the two important intracellular Ca stores: the lysosome and the ER. Heterologous overexpression leads to lysosome swelling and renders it less acidic. Best3V2 overexpression also results in compromised Ca(2+) release from the ER. Knocking down endogenous Best3 expression in myoblasts makes these cells more excitable in response to Ca(2+) mobilizing reagents, such as caffeine. We propose that Best3V2 in myoblasts may work as a tuner to control Ca(2+) release from intracellular Ca(2+) stores.