Expression of the paired box domain Pax7 protein in myogenic cells isolated from the porcine semitendinosus muscle after birth

The paired box domain gene Pax7 plays a pivotal role in satellite cell physiology and may represent one of the candidate genes influencing the dynamic stages of early post-natal growth observed in pig. Quiescent satellite cells express Pax7 and, when activated, they co-express the myogenic bHLH protein MyoD. The aims of this study were to investigate, by immunohistochemistry, the putative differential expression of Pax7 and to ascertain the amount of activated satellite cells (Pax7(+)/MyoD(+)) in myogenic cells isolated at different post-natal time points and in adults. Our results indicate that Pax7(+) cells represent between 10 and 15% of the whole myogenic cell population found at birth indicating that these cells provide a modest contribution to the development of new fibres. The number of activated satellite cells (Pax7(+)/MyoD(+)) was scarce after birth but it was higher respect to adults. An interesting result was that at 1 month after birth the number of Pax7(+) cells had increased within the pool of myogenic cells with respect to myogenic cells extracted at birth. We speculate that Pax7 might be one of the molecules involved in controlling the proliferation/differentiation ratio in the pool of satellite cells present in post-natal porcine skeletal muscles.     

Cytochrome P450 2J2*7 polymorphisms in Japanese, Mongolians and Ovambos

Human cytochrome P450 2J2 (CYP2J2) is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that have potent vasodilatory properties. Variability of the CYP2J2 gene is highly constrained except for its proximal promoter: there is a relatively common and functionally relevant single nucleotide polymorphism, indicated by -50G > T polymorphism (CYP2J2*7). Although genetic variation is known among ethnic groups, data for allele frequency are limited to a few Caucasian, Asian, and one African populations. In the present study, genotype distribution of CYP2J2*7 polymorphisms was investigated using polymerase chain reaction and restriction fragment length polymorphism assay in Japanese (n = 338), Mongolian (n = 118), and Ovambo (n = 186) populations and the findings compared with other populations. The mutant (CYP2J2*7) frequencies in the Japanese, Mongolians, and Ovambos were 0.0621, 0.0339, and 0.0672, respectively. Except for the Taiwanese, a general uniformity in the polymorphism in the Asian populations was observed. The mutation frequency of Ovambos was relatively lower than that of the African-American population. This study is the first to investigate the distribution of the CYP2J2*7 gene polymorphisms in Japanese, Mongolians, and Ovambos. These data will be informative and facilitate genetic association studies, in Asian and African populations for CYP2J2-related diseases such as cardiovascular disorders.     

Quantitative comparison of PTH1R in breast cancer MCF7 and osteosarcoma SaOS-2 cell lines

The aim of the present study was to compare the classical parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptors in MCF7 breast cancer cells with SaOS-2 osteosarcoma cell line. Quantitative binding showed that (125)I-PTHrP-1-34(Tyr) binds with a single binding site in both cells. However (125)I-PTHrP-1-34(Tyr) has higher affinity binding in MCF7 (K(D) = 1.88 +/- 0.08 nM) than in SaOS-2 cells (K(D) = 4.4 +/- 0.185 nM). The competitive binding using 3.3 nM (125)I-PTHrP-1-34(Tyr) with increasing amounts (0.33-33 nM) of unlabelled human PTHrP-1-34, PTHrP-7-34, PTHrP-1-86 His(5)-PTHrP-1-36, His(5)-Phe(23)-PTHrP-1-36 or PTH-1-34 revealed different displacements. In SaOS-2 the PTHrP-7-34 and PTHrP-1-86 caused similar displacement compared with 73% by PTH-1-34 and 70% by PTHrP-1-34. However, in MCF7, PTHrP-7-34, PTHrP-1-86 and PTH-1-34 displaced by 54%, 72% and 67%, respectively, compared to 87% by PTHrP-1-34. The His(5)-Phe(23)-PTHrP-1-36 caused an increase in the K(D) from 2.0 +/- 0.03 nM to 2.75 +/- 0.045 nM in MCF7 cells, but had no significant effect in SaOS-2 cells. The PTH/PTHrP receptor in both cell lines revealed a single 85 KDa band with different intensity. Our results suggest that the PTH/PTHrP receptor in MCF7 cells has higher binding affinity for PTHrP than PTH compared to the receptor in SaOS-2 cells.     

Inhibition of fibroblast growth factor 2-induced apoptosis involves survivin expression, protein kinase C alpha activation and subcellular translocation of Smac in human small cell lung cancer cells

To investigate the mechanism by which fibroblast growth factor 2 (FGF-2) inhibits apoptosis in the human small cell lung cancer cell line H446 subjected to serum starvation, apoptosis was evaluated by flow cytometry, Hoechst 33258 staining, caspase-3 activity, and DNA fragmentation. Survivin expression induced by FGF-2 and protein kinase Cα (PKCα) translocation was detected by subcellular frac-tionation and Western blot analysis. In addition, FGF-2-in-duced release of Smac from mitochondria to the cytoplasm was analyzed by Western blotting and immunofluorescence. FGF-2 reduced apoptosis induced by serum starvation and up-regulated survivin expression in H446 cells in a dose-dependent andtime-dependentmanner, andinhibitedcaspase-3 activity. FGF-2 also inhibited the release of Smac from mitochondria to the cytoplasm induced by serum starvation and increased PKCα translocation from the cytoplasm to the cell membrane. In addition, PKC inhibitor inhibited the expression of survivin. FGF-2 up-regulates the expression of survivin protein in H446 cells and blocks the release of Smac from mitochondria to the cytoplasm. PKCα regulated FGF-2-induced survivin expression. Thus, survivin, Smac, and PKCα might play important roles in the inhibition of apoptosis by FGF-2 in human small cell lung cancer cells.     

Effect of mammogenic hormones on the expression of FGF7, FGF10 and their receptor in mouse mammary gland

To investigate the regulation of estrogen, progesterone and prolactin stimulating the development of mammary gland, the Kunming mice were used as experimental animals in this study. Through the experiment in vitro, the effect of mammogenic hormones were systematically investigated on expression of FGF7 and FGF10 and their receptor in different periods. The results are as follows: in mammary glands of mice, 17 beta-estradiol increased the expression of FGF7; progesterone did not affect the expression of FGF7; prolactin up-regulated the expression of FGF7 significantly in pregnancy and lactation. 17 beta-estradiol increased the expression of FGF10; progesterone and prolactin reduced the expression of FGF10 significantly in virgin; prolactin significantly increased the expression of FGF10 in pregnancy. When 17 beta-estradiol in the body was in relatively high proportion, it would lower the expression of KGFR; while 17 beta-estradiol in the body was in relatively low proportion, it would increase the expression of KGFR. Low concentration of progesterone increased the expression of KGFR and high progesterone did not affect the expression of KGFR. Prolactin increased the expression of KGFR significantly in pregnancy and lactation.     

Sexual dimorphism and androgen regulation of satellite cell population in differentiating rat levator ani muscle

The bulbocavernosus (BC) and levator ani (LA) muscles of rats show remarkable androgen-dependent sexual dimorphism. These muscles are additionally of interest because they are thought to indirectly mediate sexual differentiation of innervating spinal motoneurons. This sexual differentiation of the BC/LA is thought to be due to an increase in muscle units in the male rat during the first week after birth. We examined the cellular basis of this differentiation by studying satellite cells in the LA of postnatal day 2.5 rats, when sexual dimorphism is already prominent. Two experiments were performed in which LA satellite cells were measured: (1) wild-type (WT) males were compared with females and to Tfm androgen receptor mutant males, which are androgen insensitive despite producing masculine amounts of testosterone, and (2) females treated prenatally and/or postnatally with testosterone proprionate were compared with females receiving vehicle injections. Our results indicate that WT males have a larger LA and a greater number of satellite cells in the LA muscle than females or Tfm males. However, satellite cell density was similar for all three groups. Prenatal testosterone treatment masculinized LA size and resulted in a corresponding increase in satellite cell populations, while postnatal TP treatment resulted in a tendency for increased satellite cell density without a significant increase in LA size. Taken together, these studies indicate that satellite cells in the neonatal LA muscle are sexually dimorphic, and that this dimorphism likely results from perinatal actions of androgens on androgen receptors.     

Differential crosstalk between P2X7 and arachidonic acid in activation of mitogen-activated protein kinases

Accumulating evidence indicates that astroglial syncytium plays key role in normal and pathological brain functions. Astrocytes both in vitro and in situ respond to extracellular adenine-based nucleotides via the activation of P2 receptors. Massive release of ATP from neurons and glial cells occurs as a result of pathological conditions of the brain leading to neuroinflammation and involving P2X7 receptors. In this study, we investigated whether P2X7 stimulation on cultured cortical astrocytes promoted a differential activation of mitogen-activated protein kinases (MAPKs), and whether the second messenger arachidonic acid (AA), which is also a key modulator of neuroinflammation, affected the P2X7-mediated MAPK phosphorylation. The results show that the synthetic P2X7 receptor agonist 2′,3′-O-(4-benzoyl)benzoyl-ATP (BzATP), induced a concentration-dependent phosphorylation of MAPK ERK1/2, JNK and p38. Stimulation of ERK1/2, JNK and p38 phosphorylation was also obtained by pathophysiological levels of extracellularly applied AA. Interestingly, a robust potentiation of ERK1/2 phosphorylation was elicited by co-application of BzATP and AA, whereas no differences were observed in JNK or p38 phosphosignals. The kinases activation showed a differential dependence on the presence of extracellular Ca²⁺. The potentiation of BzATP-mediated ERK1/2 phosphorylation was also observed in human embryonic kidney cells (HEK293) stably transfected with rat P2X7, but not in HEK cells expressing truncated P2X7 receptor lacking the full cytoplasmic carboxy-terminal or in those carrying the structurally related rat P2X2. AA and BzATP synergism in ERK1/2 activation was abolished by cyclo-oxygenase and lipoxygenase pathway inhibitors.The result that ERK1/2-mediated transduction pathway is synergistically modulated by ATP and AA signalling depicts possible novel pharmacological targets for interfering with pathological activation of astroglial cells.     

Activated T cells complicate the identification of regulatory T cells in rheumatoid arthritis

Most cell surface markers for CD4⁺CD25⁺ regulatory T cells (Tregs) are also expressed by activated non-regulatory T cells. Recently, CD127 down-regulation was found to identify functional Tregs in healthy individuals, but there are no data from patients with inflammatory conditions. We examined peripheral blood mononuclear cells (PBMC) from rheumatoid arthritis patients with active inflammation and from healthy controls, and found that CD4⁺ T cells contained an equal proportion of CD25⁺CD127⁻/low cells in both groups. In patients, not all these cells expressed intracellular FOXP3. Upon activation by anti-CD3/anti-CD28, PBMC rapidly down-regulated CD127, while FOXP3 up-regulation was transitory and occurred in fewer cells. The activated cells were not anergic to restimulation and had no suppressive effects. The distinct kinetics indicate that the FOXP3⁻CD127⁻/low cells in rheumatoid arthritis patients most likely represent activated non-regulatory T cells. This complicates the use of CD127 for identification of Tregs in inflammatory diseases.     

Intracellular GSH levels rather than ROS levels are tightly related to AMA-induced HeLa cell death

Antimycin A (AMA) inhibits succinate oxidase and NADH oxidase, and also inhibits mitochondrial electron transport between cytochromes b and c. We investigated the involvement of ROS and GSH in AMA-induced HeLa cell death. AMA increased the intracellular H(2)O(2) and O(2)(*-) levels and reduced the intracellular GSH content. ROS scavengers (Tempol, Tiron, Trimetazidine and NAC) did not down-regulate the production of ROS and inhibit apoptosis in AMA-treated cells. Treatment with NAC and N-propylgallate showing the enhancement of GSH depletion in AMA-treated cells significantly intensified the levels of apoptosis. Calpain inhibitors I and II (calpain inhibitor III) and Ca(2+)-chelating agent (EGTA/AM) significantly reduced H(2)O(2) levels in AMA-treated HeLa cells. However, treatment with calpain inhibitor III intensified the levels of O(2)(*-) in AMA-treated cells. In addition, calpain inhibitor III strongly depleted GSH content with an enhancement of apoptosis in AMA-treated cells. Conclusively, the changes of ROS by AMA were not tightly correlated with apoptosis in HeLa cells. However, intracellular GSH levels are tightly related to AMA-induced cell death.