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Whey protein is a by-product of cheese and casein manufacturing processes. It contains highly bioactive molecules, such as epidermal growth factor, colony-stimulating factor, transforming growth factor-&alpha, and -&beta, insulin-like growth factor, and fibroblast growth factor. Effects of whey protein on immune responses after antigen (hemagglutinin peptide) injection were evaluated in rats. Experimental diets were formulated based on NIH-31M and supplemented with 1% amino acids mixture (CON) or 1% whey protein concentrate (WPC) to generate isocaloric and isonitrogenous diets. Rats were fed the experimental diets for two weeks and then exposed to antigen two times (Days 0 and 14). Blood was collected on Days 0, 7, 14, and 21 for hematological analysis. The WPC group showed decreased IgA and cytotoxic T cells before the antigen injection (Day 0) but increased IgG, IL-2, and IL-4 after antigen injection due to increased B cells and T cells. Helper T cells were increased at Days 14 and 21, but cytotoxic T cells were not affected by WPC. WPC may activate adaptive immunity (IgG) against antigen by modulating helper T cells. Bioactive molecules might contribute to the immune-enhancing effects of whey protein concentrate.

Myogenesis occurs in both the prenatal and postnatal periods and the prenatal myogenesis is related to the postnatal myogenesis and the incidence of disease later in life. Glucocorticoids used as therapeutic agents for many diseases, but cause adverse effects on muscle homeostasis, including defects in fetal muscle development. The action of glucocorticoids on differentiated skeletal muscle was well studied, but their effects on myotube formation have not been well investigated. Dexamethasone (DEX) and cortisone (COR), two synthetic therapeutic glucocorticoids, suppress myotube formation in C2C12 cells. Both COR and DEX attenuated myotube formation through modulation of myogenic regulatory factors. In addition, they affected the IGF/PI3K/AKT/mTOR signaling pathway, resulting in increased proteolytic protein (atrogin-1 and MURF1) for muscle degradation and decreased ribosomal S6 phosphorylation. The current results conclude that COR and DEX inhibit myotube formation in C2C12 cells by modulating both the myogenic program via MRFs and protein metabolism via IGF/PI3K/AKT/mTOR signaling pathway. Glucocorticoids decrease myogenesis via AKT/mTOR signal pathway.

Quercetin (3,3',4',5,7-pentahydroxyflavone) is a major dietary flavonol found in diverse fruits and vegetables such as onions, cauliflower, apple skin, lettuce and chili peppers. In recent studies, quercetin is reported as a functional compound and shows a wide range of biological effects such as antioxidant, anti-inflammatory and antiangiogenic properties in obesity, diabetes, cardiovascular diseases and various cancers. However, to date, the therapeutic effect of quercetin on the progression of endometriosis, which is a common gynecological disease in reproductive-aged women and brings chronic pelvic pain and infertility, has not been examined in depth. Results of this study demonstrated that quercetin inhibited the proliferation and induced the cell cycle arrest in VK2/E6E7 and End1/E6E7 cells. Furthermore, it induced cell apoptosis with DNA fragmentation, loss of mitochondrial membrane potential and reactive oxygen species production. The effects accompanied down-regulation of ERK1/2, P38 MAPK and AKT signaling molecules. Additionally, the administration of quercetin indicated antiproliferative and anti-inflammatory effects on endometriosis autoimplanted mouse models. The mRNA expression of Ccnd1 significantly decreased in response to quercetin intraperitoneal injection when compared to that in vehicle-treated mice. The knockdown of CCND1 mRNA attenuated the proliferation with sub-G0/G1 cell cycle arrest and increased the apoptosis of VK2/E6E7 and End1/E6E7 cells. Furthermore, the treatment of quercetin induced miR-503-5p, miR-1283, miR-3714 and miR-6867-5p related to CCND1 in both cell lines and also stimulated miR-503-5p and miR-546 expression in the mouse model. Hence, quercetin may potentially act as a natural therapeutic to reduce and treat human endometriosis.

Oct4-Sox2-Nanog transcriptional networks are critical for the maintenance of embryonic stem (ES) cell self-renewal and induction of pluripotency. However, in transcription factor-induced reprogramming of somatic cells into induced pluripotent stem cells (iPSCs), Nanog is initially dispensable and Oct4 remains the sole factor that could not be substituted/omitted. Here, we show that mouse fibroblasts could be reprogrammed into iPSCs by Nanog and Bmi1, which replaces Sox2, Klf4, and c-Myc, in the absence of Oct4. Furthermore, we show that in the presence of shh agonists (oxysterol and purmophamine), which replaces the function of Bmi1, a single transcription factor, Nanog is sufficient to reprogram mouse fibroblasts into iPSCs. Nanog-induced iPSCs resemble mESCs in terms of morphology, global gene expression profiles, epigenetic status and pluripotency both in vitro and in vivo. These findings support that Nanog can replace the Oct4 for the somatic cell reprogramming and underlie the mechanisms of Nanog in reprogramming process.

Self-renewal, differentiation, and tumorigenicity characterize cancer stem cells (CSCs), which are rare and maintained by specific cell fate regulators. CSCs are isolated from glioblastoma multiforme (GBM) and may be responsible for the lethality of incurable brain tumors. Brain CSCs may arise from the transformation of undifferentiated, nestin-positive neural stem or progenitor cells and GFAP-expressing astrocytes. Here, we report a role of Nanog in the genesis of cancer stem-like cells. Using primary murine p53-knockout astrocytes (p53−/− astrocytes), we provide evidence that enforced Nanog expression can increase the cellular growth rate and transform phenotypes in vitro and in vivo. In addition, Nanog drives p53−/− astrocytes toward a dedifferentiated, CSC-like phenotype with characteristic neural stem cell/progenitor marker expression, neurosphere formation, self-renewal activity, and tumor development. These findings suggest that Nanog promotes dedifferentiation of p53-deficient mouse astrocytes into cancer stem-like cells by changing the cell fate and transforming cell properties.

Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by the transcription factors Oct4, Sox2, and Klf4 in combination with c-Myc. Recently, Sox2 plus Oct4 was shown to reprogram fibroblasts and Oct4 alone was able to reprogram mouse and human neural stem cells (NSCs) into iPS cells. Here, we report that Bmi1 leads to the transdifferentiation of mouse fibroblasts into NSC-like cells, and, in combination with Oct4, can replace Sox2, Klf4 and c-Myc during the reprogramming of fibroblasts into iPS cells. Furthermore, activation of sonic hedgehog signaling (by Shh, purmorphamine, or oxysterol) compensates for the effects of Bmi1, and, in combination with Oct4, reprograms mouse embryonic and adult fibroblasts into iPS cells. One- and two-factor iPS cells are similar to mouse embryonic stem cells in their global gene expression profile, epigenetic status, and in vitro and in vivo differentiation into all three germ layers, as well as teratoma formation and germline transmission in vivo. These data support that converting fibroblasts with Bmi1 or activation of the sonic hedgehog pathway to an intermediate cell type that expresses Sox2, Klf4, and N-Myc allows iPS generation via the addition of Oct4.

MicroRNAs (miRNAs) are an abundant class of small regulatory RNAs that regulate the stability and translation of cognate mRNAs. Although an increasing number of porcine miRNAs has recently been identified, the full repertoire of miRNAs in pig remains to be elucidated. To identify porcine miRNAs potentially involved in myogenesis and adipogenesis, we constructed small RNA cDNA libraries from skeletal muscle and adipose tissue and identified 89 distinct miRNAs that are conserved in pig, of which 15 were new. Expression analysis of all newly identified and selected known porcine miRNAs revealed that some miRNAs were enriched in a tissue-specific manner, whereas others were expressed ubiquitously in the porcine tissues examined. Our results expand the number of known porcine miRNAs and provide useful information for further investigating the biological functions of miRNAs associated with growth and development of skeletal muscle or adipose tissue in pig.

CD127 (the alpha chain of the IL-7 receptor) is an 80 kDa type I membrane glycoprotein that mediates the signal for the differentiation, maturation, and survival of lymphocyte development. In this study, the full-length cDNA of pig Cd127 was cloned and sequenced. Pig CD127 cDNA contains an open reading frame (1,380 bp) encoding 459 amino acids. Phylogenetic analysis revealed that pig CD127 is most homologous to that of cow (Bos taurus) with 87% amino acid identity and less homologous to that of chicken (Gallus gallus) with 36% amino acid identity among those of species. Structurally, pig CD127 has well-conserved cytoplasmic tail that transmits a signal and little diversity of extracellular domain, compared with those of other mammalian species. Expression analysis indicates that the mRNA transcript of pig CD127 is mainly detected in lymphoid tissues, but still detected in nonlymphoid tissues. Future studies will be focused on determining a more defined expression pattern of pig CD127 and the role of CD127 during the lymphocyte development in pigs.

CD82, which was originally referred to as KAI1 (kangai 1), is a member of the tetraspanin protein family, which contains four transmembrane domains. CD82 is implicated in a variety of biological processes, including apoptosis, cell adhesion, and cell migration. In this study, the full-length cDNA of pig CD82 was cloned and sequenced. Pig Cd82 cDNA contains an open reading frame (801 bp) encoding 266 amino acids. Sequence alignment results indicated that pig CD82 cDNA evidenced 85.45%, 85.63%, 77.03%, and 77.78% identity with human, cattle, rat, and mouse, respectively. In the expression study, the constitutive expression of swine Cd82 mRNA was detected in a variety of tissues, including lymphoid tissues as well as nonlymphoid tissues. Future studies will be focused on the functional role of CD82 during the course of pig infectious diseases or tumor development.

This study characterizes the factors responsible for the cholesterol reduction by Lactobacillus acidophilus ATCC 43121. In addition, two-dimensional gel electrophoresis (2-DE) and protein profiling was also used to study the response of ATCC 43121 at the proteome level in the presence of cholesterol. The results show that the cell-free supernatant (CFS) produced by ATCC 43121 in the presence of bile salts could also reduce the cholesterol in the broth, whereas all previous reports have suggested a mechanism by live cells. The active fraction was partially purified by 60% ammonium sulfate precipitation, and subsequent Sephacryl S-100 column chromatography. The molecular weight of the component with cholesterol-reducing activity was estimated to be approximately 12 kDa by SDS-PAGE. These results suggest that the novel protein isolated from CFS may be an important factor in the mechanism for cholesterol reduction by ATCC 43121. In addition, the proteins expressed by ATCC 43121 in the presence of cholesterol micelles were detected by 2-DE, five protein spots with at least a 2.5-fold increase in amounts being identified. The responsible proteins may be involved in the stress-response, translation, and metabolic processes. These results may suggest a new possibility for the mechanism underlying cholesterol reduction by lactic acid bacteria (LAB), differing from the conclusions of previous reports.

Recently, Bmi1 was shown to control the proliferation and self-renewal of neural stem cells (NSCs). In this study, we demonstrated the induction of NSC-like cells (NSCLCs) from mouse astrocytes by Bmi1 under NSC culture conditions. These NSCLCs exhibited the morphology and growth properties of NSCs, and expressed NSC marker genes, including nestin, CD133, and Sox2. In vitro differentiation of NSCLCs resulted in differentiated cell populations containing astrocytes, neurons, and oligodendrocytes. Following treatment with histone deacetylase inhibitors (trichostatin A and valproic acid), the potential of NSCLCs for proliferation, dedifferentiation, and self-renewal was significantly inhibited. Our data indicate that multipotent NSCLCs can be generated directly from astrocytes by the addition of Bmi1.

Weaning is a stressful condition for mammals and negatively affects growth performance by dystrophy of intestinal villi and crypts. Dietary plasma protein (PP) has shown the increased growth perfor...

An experiment was conducted to examine the effects of different types of vitamin D3 on skeletal muscle development using growing rats. Three-week-old rats were fed a vitamin D3-deficient diet for 4 weeks and vitamin D3-replete diets for the next 4 weeks as each of vitamin D3 (vit D3, 1,000 IU/kg diet), 25-hydroxyvitamin D3 (25-OH D3, 400 IU/kg diet), and 1α-hydroxyvitamin D3 (1α-OH D3, 4 IU/kg diet) was supplemented. Negative control group was fed a vitamin D3-deficient diet, and positive control group was fed a vitamin D3-supplemented diet (1,000 IU/kg diet) for the whole experimental period, 8 weeks. In results, body weight, feed intake, and body composition (lean and fat) were not affected by vitamin D3 dose and type. Quantitative gene expression of skeletal muscle differentiation markers (MyoD, Myogenin, Mrf4, Myf5, and Myostatin) also showed no difference. However, skeletal muscle fiber type distribution (myofibrillar ATPase staining) was changed in bicep femoris (BF) of the rats fed different types ...

Laboratory of Cell Function Regulation, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea Cell Therapy Center, College of Medicine, Yonsei University, Seoul, Republic of Korea Department of Pathology, College of Medicine, Korea University Guro Hospital, Seoul, Republic of Korea Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea

The hypocholesterolemic effects of Lactobacillus acidophilus 43121 (43121) and a mixture of Lactobacillus casei and Bifidobacterium longum (MIX) were studied in hypercholesterolemia-induced pigs. Serum total cholesterol was decreased by supplementation of either 43121 or MIX, although, high-density lipoprotein cholesterol was not changed. The hypocholesterolemic effect of 43121 and MIX was mainly due to bile acid dehydroxylation, this effect being supplementation-time dependent.

The basic helix-loop-helix myogenic regulatory factors play critical roles in skeletal myogenesis. Among the myogenic regulatory factors (MRFs), MRF4 shows a biphasic expression pattern during the formation of myotomes, although its function remains unclear. In this study, we used BEF (spontaneously immortalized bovine embryonic fibroblast that shows myogenic differentiation by overexpression of MyoD) and C2C12 cells to investigate the function of MRF4. Ectopic expressions of MRF4 did not stimulate myogenic differentiation in the BEF and C2C12 cells, but did show a marked increase of cell proliferation, upregulation of cyclin E, and downregulation of p21WAF1. Furthermore, MRF4 was found to induce degradation of the MyoD protein, which acts as a transcriptional activator for p21WAF1, and thus indicates that MRF4 accelerates cell proliferation by suppressing MyoD-dependent p21WAF1 expression. However, forced expression of MyoD in the MRF4-overexpressing cells inhibited cell proliferation and partially induced myogenic differentiation, which suggests that MyoD is a potential negative intercessor of MRF4 in the regulation of the cell cycle. Taken together, these results indicate that MRF4 and MyoD play competitive roles in myogenesis by stimulating cell proliferation and differentiation, respectively.

Background The pig, Sus scrofa domestica includes both the miniature and commercial domestic breed. These animals have influenced the human life and economies and have been studied throughout history. Although the miniature breeds are more recent and have increasingly been used in a variety of biomedical studies, their cell lines have rarely been established. Therefore, we sought to establish primary and immortal cell lines derived from both the miniature and domestic pig to better enable insight into possible in vivo growth differences. Results The in vitro lifespan of primary domestic pig fibroblast (PF) and miniature pig fibroblast (MPF) cells using a standard 3T3 protocol was determined. Both of the primary PF and MPF cells were shown to have a two-step replicative senescence barrier. Primary MPF cells exhibited a relatively shorter lifespan and slower proliferation rate compared to those of primary PF cells. Beyond senescence barriers, lifespan-extended PF and MPF cells were eventually established and indicated spontaneous cellular immortalization. In contrast to the immortalized PF cells, immortal MPF cells showed a transformed phenotype and possessed more frequent chromosomal abnormalities and loss of p53 regulatory function. The lifespan of primary MPF and PF cells was extended by inactivation of the p53 function using transduction by SV40LT without any detectable senescent phenotype. Conclusion These results suggest that p53 signaling might be a major determinant for the replicative senescence in the MPF cells that have the shorter lifespan and slower growth rate compared to PF cells in vitro.

A rapid molecular approach was developed for the initial identification of Lactobacillus acidophilus strains which are difficult to identify using a single biochemical test. The 16S-23S rRNA intergenic spacer regions and flanking 23S rRNA genes of 19 strains of lactobacilli were amplified and the nucleotide sequences and restriction site polymorphisms were analyzed. AluI was the most useful of the restriction enzymes analyzed and produced reproducible digestion profiles in the L. helveticus, L. plantarum, and L. casei groups, as well as in L. acidophilus. This restriction fragment length polymorphism method may be useful for the identification of L. acidophilus strains in dairy products.

Recent evidence has suggested that human skin fibroblasts may represent a novel source of therapeutic stem cells. In this study, we report a 3-stage method to induce the differentiation of skin fibroblasts into insulin- producing cells (IPCs). In stage 1, we establish the isolation, expansion and characterization of mesenchymal stem cells from human labia minora dermis- derived fibroblasts (hLMDFs) (stage 1: MSC expansion). hLMDFs express the typical mesenchymal stem cell marker proteins and can differentiate into adipocytes, osteoblasts, chondrocytes or muscle cells. In stage 2, DMEM/F12 serum-free medium with ITS mix (insulin, transferrin, and selenite) is used to induce differentiation of hLMDFs into endoderm-like cells, as determined by the expression of the endoderm markers Sox17, Foxa2, and PDX1 (stage 2: mesenchymal-endoderm transition). In stage 3, cells in the mesenchymal- endoderm transition stage are treated with nicotinamide in order to further differentiate into self-assembled, 3-dimensional islet cell-like clusters that express multiple genes related to pancreatic β-cell development and function (stage 3: IPC). We also found that the transplantation of IPCs can normalize blood glucose levels and rescue glucose homeostasis in streptozotocin- induced diabetic mice. These results indicate that hLMDFs have the capacity to differentiate into functionally competent IPCs and represent a potential cell-based treatment for diabetes mellitus.