P53 Mice Research Articles

Modulation of Dhh signaling and altered Sertoli cell function in mice lacking the GPR37-prosaposin receptor.

The mammalian G-protein-coupled receptor 37 (GPR37) is expressed in brain, in adult testis, and during the early phase of gonad differentiation. Somatic Sertoli cells (SCs) are located within the seminiferous tubules where they support the germinal epithelium. An adequate number of SCs is required for the complete prepubertal differentiation of germ cells and adult fertility. This study shows that Gpr37 and its ligand prosaposin are both postnatally expressed by SCs, whose proliferation and maturation are affected in Gpr37-null mutant mice during postnatal testicular development. Mutant pups show a delayed timing in sperm cell development, with a partial arrest of spermatocytes at the meiotic pachytene (e.g., 1.5-fold increase in Gpr37(-/-) P21 pups) and their increased apoptosis (e.g., 1.8-fold and 3.5-fold increase in Gpr37(-/-) P21 and adult mice, respectively). Mutant adults have reduced testis weight (wild type, 299 ± 5 mg; knockout, 258 ± 16 mg; P < 0.05) and epididymal sperm count and motility (e.g., 1.5-fold and 1.45-fold decrease in Gpr37(-/-) mice, respectively). Lack of Gpr37 results in the reduction in androgen receptor levels during prepubertal testis development, alongside the altered expression of SC maturation markers. It also affects the prepubertal testis expression of desert hedgehog (Dhh) mitogenic cascade components (Dhh, 1.3-fold increase in Gpr37(-/-) P10 and P21 pups; Gli2, 1.4-fold and 1.6-fold increase in Gpr37(-/-) P10 and P21 pups, respectively) including patched homolog 1 (1.3-fold increase in Gpr37(-/-) P10 and P21 pups), which is found localized in prepubertal SCs and is associated with Gpr37 in cultured primary SC samples. These results indicate that Gpr37 is a specific modulator of murine testis Dhh mitogenic signaling and SC proliferation and maturation.

Exogenous IL-1Ra attenuates intestinal mucositis induced by oxaliplatin and 5-fluorouracil through suppression of p53-dependent apoptosis.

Chemotherapy-induced intestinal mucositis (CIM) is a major dose-limiting side effect of many chemoagents, resulting in weight loss, diarrhea, and even death. The current treatments for CIM are palliative and have limited benefit. Interleukin-1 receptor antagonist is a natural antagonist of interleukin-1. Our previous studies showed the protective effect of recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) on the intestine in mice after 5-fluorouracil chemotherapy. In this study, we further evaluated rhIL-1Ra in the treatment of CIM induced by different chemoagents and their combination. Normal as well as tumor-bearing mice were administered oxaliplatin (L-OHP), 5-fluorouracil, or their combination to induce intestinal mucositis and mortality. rhIL-1Ra administered after the chemotherapy, but not after the onset of diarrhea, significantly improved mouse survival, attenuated body weight loss, and reduced the incidence, severity, and duration of diarrhea. Histological examination showed that rhIL-1Ra-treated mice had a relatively intact mucosa structure, more proliferating crypt cells, and higher acid mucin content than the vehicle-treated mice. rhIL-1Ra suppressed crypt apoptosis by reducing the levels of proapoptotic proteins in wild-type, but not in IL-1RI or p53 mice. In addition, rhIL-1Ra was as effective as octreotide acetate in the treatment of chemotherapy-induced diarrhea, but with the advantage of reducing the epithelial apoptosis, the major cause of CIM. Importantly, the tumor sensitivity to chemotherapy was not affected by rhIL-1Ra. Thus, our data strongly suggest that rhIL-1Ra may be useful for the treatment of intestinal mucositis and improving the quality of life for cancer patients on chemotherapy.

Transplantation of Neuro2a Cells into the Developing Postnatal Mouse Eye.

The present study aimed to investigate the influence of the host retinal microenvironment on cell migration and differentiation using Neuro2a (N2a) cells transduced with green fluorescent protein. N2a cells were transplanted into the vitreous cavities of developing mouse eyes (C57BL/6) on postnatal days 1, 5, and 10 (P1, 5, and 10). To analyze the effects of the host microenvironment on neural differentiation of N2a cells in vitro, cells were treated with a conditioned medium (CM) collected from retinal cells cultured at each developmental stage. We observed that numerous cells transplanted into P5 mice eyes migrated into all layers of the host retina, and the presence of processes indicated morphological differentiation. Some transplanted N2a cells expressed several neural markers. However, cells transplanted into the P1 and 10 mice eyes only proliferated within the vitreous cavity. Neurite length increased in N2a cells treated with CM collected from the cultured retinal cells from P5 and 10 mice, while western blotting revealed that the levels of proteins related to neural differentiation were not significantly altered in N2a cells treated with CM. We show that the migration and differentiation capacities of transplanted cells were differentially influenced by the microenvironment of the retinal postnatal ontogeny.

Tuning of neocortical astrogenesis rates by Emx2 in neural stem cells.

Tuning of neocortical astrogenesis rates by Emx2 in neural stem cells.

In Newborn Mice, Tpo Signaling Is Required for Normal Megakaryocyte Proliferation, Maturation, Platelet Formation, and Platelet Survival

Congenital amegakaryocytic thrombocytopenia (CAMT) is a disorder caused by c-mpl mutations that block or severely reduce thrombopoietin (Tpo) signaling, leading to amegakaryocytic thrombocytopenia and ultimately aplastic anemia. In neonates and infants, CAMT can have different clinical manifestations than in adults, and cases have been described of newborn infants who presented with severe thrombocytopenia but normal numbers of immature-appearing megakaryocytes (MKs) in the bone marrow. To elucidate the potential mechanisms underlying the developmental differences in the manifestations of Tpo deficiency, we evaluated MKs and platelets in newborn c-mpl-/- mice on postnatal day 1 (P1). Since the liver is the main site of megakaryopoiesis at that developmental stage (Liu et al., Blood 2014), liver MKs in P1 mice were immunohistochemically stained for VWF, quantified, measured, and ultrastructurally evaluated using transmission electron microscopy (TEM), as previously described. Histological analysis showed that MKs in newborn c-mpl-/- mice were present at a concentration of 30% and were significantly smaller compared to WT pups, a finding previously reported in adult mice. Unlike in adult c-mpl-/- mice, however, which have ultrastructurally normal MKs, TEM analysis of neonatal c-mpl-/- liver MKs revealed a significant shift toward immature cells and maturational abnormalities in 68% of MKs, including a disorganized demarcation system, reduced granules, and an abnormally wide peripheral zone. Ultrastructural analysis of platelets from c-mpl-/- P5 mice also revealed a larger open canalicular system and more glycosomes compared to age-matched WT platelets. These findings were not associated with increased levels of baseline P-selectin expression, suggesting that they were not the result of activation during processing. Next, we serially evaluated platelet counts in WT and c-mpl-/- mice during the transition from fetal/neonatal to adult megakaryopoiesis. At P2, c-mpl-/- platelet counts were 170±54x103/µl, representing 26% of those in P2 WT mice (648±137x103/µl). Over the first two weeks of life, WT platelet counts doubled and reached nearly adult levels, as previously described. In contrast, c-mpl-/- platelet counts failed to increase, and remained stably low at P14. Since we previously showed that the rise in platelet counts during this period was associated with a one-day prolongation of the neonatal platelet lifespan (Liu et al., Blood 2014), we evaluated platelet survival in c-mpl-/- compared to WT mice, using in vivo biotinylation. These studies suggested that the platelet lifespan was significantly shorter in c-mpl-/- compared to WT newborn (P2) mice. In contrast, we found no differences in platelet lifespan between c-mpl-/- and WT adult mice. Taken together, our findings suggest that, in fetal and neonatal life, Tpo signaling is required for normal MK proliferation, maturation, platelet formation, and platelet survival. This is in contrast to adult life, when Tpo deficiency leads only to reduced MK and platelet counts. We hypothesize that these differences reflect the lack of Tpo-independent pathways of MK maturation in the environment of the fetal/neonatal liver, and have implications in the developmental stage-specific clinical manifestations of CAMT. DisclosuresNo relevant conflicts of interest to declare.

The selective loss of the type 2 iodothyronine deiodinase in mouse thyrotrophs increases basal TSH but blunts the thyrotropin response to hypothyroidism.

The type 2 iodothyronine deiodinase (D2) is essential for feedback regulation of TSH by T4. We genetically inactivated in vivo D2 in thyrotrophs using a mouse model of Cga-driven cre recombinase. Pituitary D2 activity was reduced 90% in the Cga-cre D2 knockout (KO) mice compared with control Dio2(fl/fl) mice. There was no growth or reproductive phenotype. Basal TSH levels were increased 1.5- to 1.8-fold, but serum T4 and T3 were not different from the controls in adult mice. In hypothyroid adult mice, suppression of TSH by T4, but not T3, was impaired. Despite mild basal TSH elevation, the TSH increase in response to hypothyroidism was 4-fold reduced in the Cga-cre D2KO compared with control mice despite an identical level of pituitary TSH α- and β-subunit mRNAs. In neonatal Cga-cre D2KO mice, TSH was also 2-fold higher than in the controls, but serum T4 was elevated. Despite a constant TSH, serum T4 increased 2-3-fold between postnatal day (P) 5 and P15 in both genotypes. The pituitary, but not cerebrocortical, D2 activity was markedly elevated in P5 mice decreasing towards adult levels by P17. In conclusion, a congenital severe reduction of thyrotroph D2 causes a major impairment of the TSH response to hypothyroidism. This would be deleterious to the compensatory adaptation of the thyroid gland to iodine deficiency.

P21(WAF1) (/Cip1) limits senescence and acinar-to-ductal metaplasia formation during pancreatitis.

Trans-differentiation of pancreatic acinar cells into ductal-like lesions, a process defined as acinar-to-ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre-malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down-regulation of p21(WAF1) (/Cip1) , a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down-regulation is implicated in controlling the early events of acinar cell trans-differentiation and ADM formation. p21-mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein-induced pancreatitis, using wild-type (WT) and p21-deficient (p21(-/-) ) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up-regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down-regulation is associated with ADM formation. In support of this hypothesis, p21(-/-) mice showed a significant increase in number and size of metaplasia. In addition, p21 over-expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell-autonomous manner. p21(-/-) mice displayed increased expression and relocalization of β-catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate-keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration.

Mcph1/Brit1 deficiency promotes genomic instability and tumor formation in a mouse model.

MCPH1, also known as BRIT1, has recently been identified as a novel key regulatory gene of the DNA damage response pathway. MCPH1 is located on human chromosome 8p23.1, where human cancers frequently show loss of heterozygosity. As such, MCPH1 is aberrantly expressed in many malignancies, including breast and ovarian cancers, and the function of MCPH1 has been implicated in tumor suppression. However, it remains poorly understood whether MCPH1 deficiency leads to tumorigenesis. Here, we generated and studied both Mcph1−/− and Mcph1−/−p53−/− mice; we showed that Mcph1−/− mice developed tumors with long latency, and that primary lymphoma developed significantly earlier in Mcph1−/−p53−/− mice than in Mcph11+/+p53−/− and Mcph1+/−p53−/− mice. The Mcph1−/−p53−/− lymphomas and derived murine embryonic fibroblasts (MEFs) were both more sensitive to irradiation. Mcph1 deficiency resulted in remarkably increased chromosome and chromatid breaks in Mcph1−/− p53−/− lymphomas and MEFs, as determined by metaphase spread assay and spectral karyotyping analysis. Additionally, Mcph1 deficiency significantly enhanced aneuploidy as well as abnormal centrosome multiplication in Mcph1−/−p53−/− cells. Meanwhile, Mcph1 deficiency impaired double strand break (DSB) repair in Mcph1−/−p53−/− MEFs as demonstrated by neutral Comet assay. Compared with Mcph1+/+p53−/− MEFs, homologous recombination and non-homologous end joining activities were significantly decreased in Mcph1−/−p53−/− MEFs. Notably, reconstituted MCPH1 rescued the defects of DSB repair and alleviated chromosomal aberrations in Mcph1−/−p53−/− MEFs. Taken together, our data demonstrate MCPH1 deficiency promotes genomic instability and increases cancer susceptibility. Our study using knockout mouse models provides convincing genetic evidence that MCPH1 is a bona fide tumor suppressor gene. Its deficiency leading to defective DNA repair in tumors can be utilized to develop novel targeted cancer therapies in the future.

Vesicular glutamate transporter 3 is strongly upregulated in cochlear inner hair cells and spiral ganglion cells of developing circling mice

Vesicular glutamate transporter 3 (VGLUT3) plays a major role in hearing, and mice lacking the VGLUT3 are congenitally deaf due to absence of glutamate release at the inner hair cell afferent synapses. However, whether VGLUT3 is expressed normally in the cochleae of developing circling mice (homozygous (cir/cir) mice), the animal model for human deafness type DFNB6, has not been established. In this study, we investigated the developmental expression of VGLUT3 in cochlear inner hair cells (IHCs) and spiral ganglion cells (SGCs) of homozygous (cir/cir) mice from postnatal day (P)1 to P14 using immunofluorescence (IF) staining and Western blot. VGLUT3 immunoreactivity (IR) and protein expression increased progressively with age in homozygous (cir/cir) and control mice (heterozygous (+/cir) mice and ICR mice). The rank order of VGLUT3 IR in IHCs and SGCs in P14 mice was homozygous (cir/cir) mice=heterozygous (+/cir) mice>ICR mice. The rank order of total protein expression was homozygous (cir/cir) mice>heterozygous (+/cir) mice=ICR mice at P14. IF staining and Western blot analysis indicated that developmental VGLUT3 expression in cochleae was most prominent in homozygous (cir/cir) mice. The possible contribution of VGLUT3 upregulation in the cochlear degeneration is discussed.

LRP4 is critical for neuromuscular junction maintenance.

The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.

Abstract 89: Development of preclinical lung adenocarcinoma model

Abstract Lung cancer is a leading cause of cancer death in worldwide. 85% of lung cancers are classified as non-small cell lung cancers (NSCLC), and adenocarcinoma (ADC) type is the most prevalent cancer in NSCLC. Approximately 50% of non-small cell lung cancer (NSCLC) patients have mutations in some oncogenes. The K-ras activation due to the mutant is notorious in many kinds of cancer types, including pancreatic cancer and lung adenocarcinoma, as is generally accepted as driving mutation for lung malignancy of lung adenocarcinoma. Another predominantly alternated gene in lung cancer is the EGFR that would hit the similar signaling with K-ras activation, to which small molecules and antibodies target to halt the tumor progression. The tumor suppressor p53 is also often altered in almost all kinds of cancers and lung cancer. In this reason, K-ras activated mouse is utilized as lung cancer mouse model. K-ras activated mouse eventually develops lung adenocarcinoma, which can be accelerated with mutated p53. Due to the gain-of-function, mutant p53 protein contributes to tumor metastasis and shortening the latency of tumor progression. Thereby activated K-ras/mutant p53 mouse is preferentially used as lung adenocarcinoma preclinical model. Here, we have used mouse tumor cells derived from activated K-ras/mutant p53 mouse to establish syngeneic lung cancer mouse model, allowing in vivo imaging. We asked whether the gain-of-function metastatic phenotype observed in the mouse is responsible for the formation of lung tumors when inoculated intravenously. Tumor cells from the mice could efficiently develop tumors in four weeks at the same genetic background. Tumors from the mice were histologically identical with original lung adenocarcinoma, suggesting that these cells are the efficient tools to generate preclinical lung tumor mouse model. We have examined this model for evaluating the efficiency of chemotherapeutics and signaling pathways involved in tumor growth regulation. Citation Format: Young-Ah Suh, Minsuh Kim, Sun-Hye Lee, Chae Lim Jung, Hye-Min Mun, Ju-Hee Oh, Eun Kyung Choi, Se Jin Jang. Development of preclinical lung adenocarcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 89. doi:10.1158/1538-7445.AM2014-89

Expression and localization of somatostatin receptor types 3, 4 and 5 in the wild-type, SSTR1 and SSTR1/SSTR2 knockout mouse cochlea.

Somatostatin (SST) is a peptide hormone that exerts inhibitory effects mediated through binding to specific cell surface G protein-coupled receptors, of which five distinct subtypes (SSTR1-SSTR5) have been characterized. Our study performed on mouse cochlear hair cells shows the expression and localization of the three receptors (SSTR3-SSTR5) in wild-type (WT), single-knockout (SSTR1 KO) and double-knockout SSTR1/SSTR2 (DKO) mice. Similar SSTRs expression were observed in the inner hair cells (IHC), outer hair cells (OHC) and supporting cells of cultivated P7 mouse organ of Corti (OC) explants as well as in cultivated cochlear neuroepithelial supporting cells (NEsc). We found differences in the expression of SSTR3-5 in WT, SSTR1 KO and DKO mouse cochlea, which might be explained as a compensatory effect in the cochlea after the loss of SSTR1 and/or SSTR2.

The Potency of Sernai (Wedelia biflora) Leaf Water Extract as Antipyretic Compared to Amino Fenol Derivate and Salicylic Acid on Mice (Mus Musculus)

The study was conducted to screen the antipyretic activity of aqueous of extract of the leaves of sernai (Wedelia biflora) with split-plot design. Eighteen healthy mice weighing between (20-25 g), aged 2-3 months were divided into six groups of three animals each: negative control group were given distilled water (P0), P1, and P2 as a positive control respectively given salicylic acid 42.25 mg/kg body weight, the amino phenol 25 mg/kg body weight, group P3, P4, and P5 given of sernai leaf aqueous extract at a concentration of 50, 75, and 100% respectively. The initial rectal temperature of each animal was recorded by digital thermometer. The pyrexia was induced by injecting a solution of 12.5% peptone. The difference in temperature was observed every 30 minutes for 3 hours and respective time interval was found out by statistical method. The results showed that the water extract of leaves sernai with various concentrations significantly different (P0.01) with an observation time. Average temperatures in the P5 mice was significantly different (P0.05) with P0. P3 and P4 is not significantly different from P0, but P3 and P4 significantly different (P0.05) with P2 and not significantly different from P1. P5 was not significantly different from the P2 and P1 significantly different. It was concluded that the aqueous extract of leaves sernai potential as an antipyretic in mice and at concentrations of 50 and 75% effective in reducing fever in mice tend equivalent to the amino phenol, while the concentration of 100% with salicylic acid.Key words: amino fenol, antipyretic, salicylic acid, sernai

Cisplatin-induced renal injury is independently mediated by OCT2 and p53.

Tubular secretion of cisplatin is abolished in mice deficient for the organic cation transporters Oct1 and Oct2 (Oct1/2(-/-)mice), and these animals are protected from severe cisplatin-induced kidney damage. Since tubular necrosis is not completely absent in Oct1/2(-/-)mice, we hypothesized that alternate pathways are involved in the observed injury. Studies were done in wild-type, Oct1/2(-/-), or p53-deficient animals, all on an FVB background, receiving cisplatin intraperitoneally at 15 mg/kg. Cisplatin metabolites were analyzed using mass spectrometry, and gene expression was assessed using Affymetrix microarrays and RT-PCR arrays. KEGG pathway analyses on kidneys from mice exposed to cisplatin revealed that the most significantly altered genes were associated with the p53 signaling network, including Cdnk1a and Mdm2, in both wild-type (P = 2.40 × 10(-11)) and Oct1/2(-/-)mice (P = 1.92 × 10(-8)). This was confirmed by demonstrating that homozygosity for a p53-null allele partially reduced renal tubular damage, whereas loss of p53 in Oct1/2(-/-)mice (p53(-/-)/Oct1/2(-/-)) completely abolished nephrotoxicity. We found that pifithrin-α, an inhibitor of p53-dependent transcriptional activation, inhibits Oct2 and can mimic the lack of nephrotoxicity observed in p53(-/-)/Oct1/2(-/-)mice. These findings indicate that (i) the p53 pathway plays a crucial role in the kidney in response to cisplatin treatment and (ii) clinical exploration of OCT2 inhibitors may not lead to complete nephroprotection unless the p53 pathway is simultaneously antagonized.

Effects of cell-type-specific expression of a pan-caspase inhibitor on renal fibrogenesis

The caspase family of enzymes is grouped into two major sub-families, namely apoptotic and inflammatory caspases, which play central roles in the induction of apoptosis, regulation of inflammation and immunity, and cellular differentiation. The role of caspase activation in tubular epithelium and interstitial cells of 3 lines of transgenic mice with obstructed nephropathy was examined: p35 mice bearing the pan-caspase inhibitor protein expressed by the p35 gene separated from the universal CAG promoter by a floxed STOP sequence were crossed with γGT.Cre and FSP1.Cre mice that express Cre recombinase in the cortical tubular epithelium and FSP1(+) interstitial cells, respectively. The γGT.Cre;p35, FSP1.Cre;p35 and p35 control mice were then challenged with unilateral ureter obstruction (UUO). Proinflammatory parameters such as protein levels of active IL-1β subunit and mRNA levels of TNF-α and NOD-like receptor pyrin domain containing-3, and profibrogenic parameters such as interstitial matrix deposition and mRNA levels of fibronectin EIIIA isoform and α1 chain of procollagen type I in the kidneys were significantly increased at 7days in the FSP1.Cre;p35- and p35-UUO mice, but not in the γGT.Cre;p35-UUO mice. These changes paralleled the numbers of apoptotic nuclei in tubules, but not in interstitial cells, and the protein levels of active caspase-3 subunit in the kidneys of FSP1.Cre;p35-, p35- and γGT.Cre;p35-UUO mice. This study provides evidence of the critical role of caspase activation in the tubular epithelium, but not in FSP1(+) interstitial cells, in apoptosis and inflammasome induction, leading to proinflammatory and profibrogenic processes in fibrous kidneys with UUO.

Abstract 287: Chamber Specific Function of p38 MAP Kinases in Right Ventricle Development, Hypertrophy, and Dysfunction

Background: Postnatal heart maturation is a well orchestrated process involving chamber specific changes in myocyte proliferation and growth. While the left ventricle undergoes significant growth, the right ventricle regresses relative to the left in terms of both myocyte number and size. While much attention has been focused on the left ventricular myocyte proliferation and hypertrophy, right ventricle specific changes are poorly studied. p38 MAP kinase is a well established signaling pathway for stress response and also plays an important role in cell differentiation, viability and proliferation. Its role in chamber specific postnatal heart maturation, however, has not been demonstrated Results and Methods: We observed RV specific induction of myocyte apoptosis and suppression of myocyte proliferation in neonatal hearts, coinciding with a transient induction of p38 activity significantly higher in the RV relative to the LV at P3 and P7 wild-type mouse hearts. In cardiomyocyte specific p38 MAP kinase alpha&amp;beta double KO (p38ab cdKO) mice, echocardiographic and histological analyses revealed RV specific chamber enlargement and dysfunction associated with significant postnatal lethality. Apoptotic activity in the RV was suppressed at P1 and myocyte proliferation and hypertrophy were significantly elevated at P3 and P7 in the p38ab cdKO hearts compared to the wild-type. In contrast, no differences were observed in the LV between the p38ab cdKO and wild-type. Consequently, the surviving p38ab cdKO mice showed significant and RV specific enlargement and dysfunction, leading to eventual RV failure and pulmonary hypertension. Conclusion: p38 a&amp;b MAP kinases are essential to RV specific maturation in postnatal hearts. p38ab cdKO mice represent a unique RV-specific hypertrophy and heart failure model for further studies.

Pla2g16 phospholipase mediates gain-of-function activities of mutant p53.

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value <0.05. Of particular interest, Pla2g16, which encodes a phospholipase that catalyzes phosphatidic acid into lysophosphatidic acid and free fatty acid (both implicated in metastasis), was increased in p53(R172H/+) osteosarcomas. Functional analyses showed that Pla2g16 knockdown decreased migration and invasion in mutant p53-expressing cells, and vice versa: overexpression of Pla2g16 increased the invasion of p53-null cells. Furthermore, Pla2g16 levels were increased upon expression of mutant p53 in both mouse and human osteosarcoma cell lines, indicating that Pla2g16 is a downstream target of the mutant p53 protein. ChIP analysis revealed that several mutant p53 proteins bind the Pla2g16 promoter at E26 transformation-specific (ETS) binding motifs and knockdown of ETS2 suppressed mutant p53 induction of Pla2g16. Thus, our study identifies a phospholipase as a transcriptional target of mutant p53 that is required for metastasis.

Apoptotic potential role of Agave palmeri and Tulbaghia violacea extracts in cervical cancer cells.

Cervical cancer, a gynaecological malignant disorder, is a common cause of death in females in Sub-Saharan Africa, striking nearly half a million of lives each year worldwide. Currently, more than 50 % of all modern drugs in clinical use are of natural products, many of which have an ability to control cancer cells (Madhuri and Pandey, Curr Sci 96:779-783, 2009; Richter, Traditional medicines and traditional healers in South Africa, 2003). In South Africa, plants used to treat cancer are rare even though majority of our population continue to put their trust in traditional medicine. In this study we aimed to screen Agave palmeri (AG) and Tulbaghia violacea (TV) for potential role in inducing cell death in cervical cancer cell lines HeLa and ME-180, and in normal human fibroblast cell line KMST-6 cell lines. To achieve this, AG and TV crude extracts were utilized to screen for apoptosis induction, inhibition of cell proliferation followed by elucidation of the role of Bax, Bcl-2, p53, Rb, RBBP and Mdm2 genes in cervical cancer. In brief, plant leaves and roots were collected, crushed and methanolic extracts obtained. Different concentrations of the stock extracts were used to treat cancer cells and measure cell death using the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and flow cytometry. Western blot was applied to measure gene expression at protein level using RBBP6, p53, Mdm2, Rb, Bax, Bcl-2 and β-actin mouse monoclonal primary antibodies (IgG) and goat anti mouse coupled with horseradish peroxidase secondary antibody from Santa Cruz Biotechnology and real time-PCR was used for mRNA expression level. Plant extracts of AG and TV were time (24 h) and dose (50, 100, 150 μg/ml) dependent in their induction of cell death with an IC50 ~ 150 μg/ml. A further mixed respond by several genes was observed following treatment with the two plant extracts where RBBP6 was seen to be spliced in cancer cells while Bax was induced and Bcl-2 was inhibited with the levels of p53 remaining the same. The two plant extracts do induce cell death, in a p53 independent manner.

Habitual exercise plus dietary supplementation with milk fat globule membrane improves muscle function deficits via neuromuscular development in senescence-accelerated mice

We examined the effects of habitual exercise plus nutritional intervention through consumption of milk fat globule membrane (MFGM), a milk component, on aging-related deficits in muscle mass and function in senescence-accelerated P1 mice. Combining wheel-running and MFGM (MFGMEx) intake significantly attenuated age-related declines in quadriceps muscle mass (control: 318 ± 6 mg; MFGMEx: 356 ± 9 mg; P < 0.05) and in contractile force (1.4-fold and 1.5-fold higher in the soleus and extensor digitorum longus muscles, respectively). Microarray analysis of genes in the quadriceps muscle revealed that MFGMEx stimulated neuromuscular development; this was supported by significantly increased docking protein-7 (Dok-7) and myogenin mRNA expression. Treatment of differentiating myoblasts with MFGM-derived phospholipid or sphingolipid fractions plus mechanical stretching also significantly increased Dok-7 mRNA expression. These findings suggest that habitual exercise plus dietary MFGM improves muscle function deficits through neuromuscular development, and that phospholipid and sphingolipid in MFGM contribute to its physiological actions.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-339) contains supplementary material, which is available to authorized users.

The atypical structure and function of newborn arterial endothelium is mediated by Rho/Rho kinase signaling.

Endothelium of fetal or newborn arteries is atypical, displaying actin stress fibers and reduced nitric oxide (NO)-mediated dilatation. This study tested the hypothesis that Rho/Rho kinase signaling, which promotes endothelial stress fibers and inhibits endothelial dilatation, contributed to this phenotype. Carotid arteries were isolated from newborn [postnatal day 1 (P1)], P7, and P21 mice. Endothelial dilatation to acetylcholine (pressure myograph) was minimal at P1, increased at P7, and further increased at P21. Inhibition of Rho (C3 transferase) or Rho kinase (Y27632, fasudil) significantly increased dilatation to acetylcholine in P1 arteries but had no effect in P7 or P21 arteries. After inhibition of NO synthase (N(G)-nitro-l-arginine methyl ester), Rho kinase inhibition no longer increased acetylcholine responses in P1 arteries. Rho kinase inhibition did not affect dilatation to the NO donor DEA-NONOate. The endothelial actin cytoskeleton was labeled with phalloidin and visualized by laser-scanning microscopy. In P1 arteries, the endothelium had prominent transcytoplasmic stress fibers, whereas in P7 and P21 arteries, the actin fibers had a significantly reduced intensity and were restricted to cell borders. Phosphorylation of myosin light chains, a Rho kinase substrate, was highest in P1 endothelium and significantly reduced in P7 and P21 endothelium (laser-scanning microscopy). In P1 arteries, inhibition of Rho (C3 transferase) or Rho kinase (Y27632) significantly reduced the intensity of actin fibers, which were restricted to cell borders. Similarly, in P1 arteries, Rho inhibition significantly reduced endothelial levels of phosphorylated myosin light chains. These results indicate that the atypical function and morphology of newborn endothelium is mediated by Rho/Rho kinase signaling.