草鱼MYH mRNA表达量分析中采用的内参基因稳定性比较

本研究分别以β-actin、18S rRNA和GAPDH为内参基因,采用实时荧光定量PCR对草鱼早期发育时期肌球蛋白重链(myosin heavy light,MYH)基因的mRNA表达量进行分析,并比较不同内参基因对MYH基因mRNA表达水平检测结果的准确性.研究结果表明,以β-actin和GAPDH作为内参,MYH基因mRNA表达水平完全一致,其表达量从原肠到仔鱼阶段逐次递增,仔鱼与原肠期阶段相比表达量差异显著;当采用18S rRNA作为内参时,MYH基因mRNA在发育阶段的表达量呈不稳定状态.因此,β-actin和GAPDH均可作为内参基因,用于草鱼早期发育中MYH基因mRNA的相对定量研究:而18S rRNA作为内参时,可能会对检测结果造成偏差.本研究不仅准确的揭示了草鱼MYH基因mRNA的表达特征,并且为荧光定量PCR技术在鱼类基因表达研究方面提供了有价值的参考.     

Validation of internal controls for gene expression analysis in the intestine of rats infected with Hymenolepis diminuta

The non-invasive parasitic cestode Hymenolepis diminuta induces hypertrophy, hyperplasia and other changes in cell activity in the intestine of rats which are indicated in the expression of mRNA. We have investigated various house-keeping genes (GAPDH, β-actin, 18S and HPRT) and other internal controls (total RNA/unit biomass, total RNA/unit length of intestine) to validate gene expression in the rat intestine after cestode infection and drug-induced neuromodulation. Variation in GAPDH, β-actin, 18S and HPRT expression was observed in rat jejunal tissue according to treatment. Total RNA/unit length of intestine was found to be the most suitable internal control for normalizing target gene mRNA expression in both infected and/or drug-induced rat intestine. This normalization method may be applied to studies of gene expression levels in intestinal tissue where hypertrophy, hyperplasia, rapid growth and cell differentiation generally occur.     

Regulation of the Epithelial Adhesion Molecule CEACAM1 Is Important for Palate Formation

Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at various developmental stages before, during, and after palate fusion using GeneChip® microarrays. Ceacam1 was one of the highly up-regulated genes during palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was present in prefusion palatal epithelium and was degraded during fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1 ^−/−) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1 ^−/− mice. TGFβ3 expression, apoptosis, and cell proliferation in palatal epithelium were not affected in the palate of Ceacam1^−/−mice. However, CEACAM1 expression was retained in the remaining MEE of TGFβ-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion.     

鳜鱼基因表达转录分析中的内参选择比较

目前基因表达的转录分析多采用单一或多个看家基因作为内参来校正目的基因的表达量。该实验以鳜鱼6个不同组织和5个不同胚胎发育阶段为研究对象,应用实时荧光定量PCR技术,观察了GAPDH、β-actin和18S rRNA三个看家基因mRNA水平的表达情况。geNorm统计分析表明,胚胎发育阶段β-actin表达最为稳定;不同的组织样品间,GAPDH表达最为稳定;而18S rRNA 的表达在不同的发育阶段不稳定。当利用多基因作为内参时,使用两个最稳定表达的看家基因即可对目的基因的表达进行准确校正。该结果证实了基因表达转录分析中内参基因选择的必要性,同时为鳜鱼等鱼类基因表达分析时内参基因的选择提供有价值的参考     

Differential expression of 11beta-hydroxysteroid dehydrogenase types 1 and 2 mRNA and glucocorticoid receptor protein during mouse embryonic development

Accumulating evidence suggests that the actions of glucocorticoids in target tissues are critically determined by the expression of not only the glucocorticoid receptor (GR) but also the glucocorticoid-metabolizing enzymes, known as 11β-hydroxysteroid dehydrogenase types 1 and 2 (11β-HSD1 and 11β-HSD2). To gain insight into the role of glucocorticoids in fetal development, the expression patterns of the two distinct 11β-HSD isozymes and GR were studied in the mouse embryo from embryonic day 12.5 (E12.5, TERM = E19) to postnatal day 0.5 (P0.5) by in situ hybridization and immunohistochemistry, respectively. 11β-HSD1 mRNA was detected in the heart as early as E12.5 and maintained thereafter. In the lung and liver, 11β-HSD1 mRNA was first detected between E14.5 and E16.5, increased to high levels towards term and maintained after birth. Relatively low levels of 11β-HSD1 mRNA were also detected in the kidney, adrenal glands and gastrointestinal tract at E18.5. However, the mRNA for 11β-HSD1 was undetectable in all other embryonic tissues including the brain. In contrast, kidney was the only organ that expressed appreciable levels of 11β-HSD2 mRNA during embryonic life. The level of 11β-HSD2 mRNA in the kidney increased dramatically in the newborn, which coincided with expression of 11β-HSD2 mRNA in the whisker follicle, tooth and salivary gland. Distinct from the profiles of 11β-HSD1 and 11β-HSD2 mRNA, GR protein was detectable in all tissues at all ages studied except for the thymus, salivary gland, and bone. Taken together, the present study demonstrates that tissue- and developmentally-stage specific expression of 11β-HSD1 and 11β-HSD2 as well as GR occurs in the developing mouse embryo, thus highlighting the importance of these two enzymes and GR in regulating glucocorticoid-mediated maturational events in specific tissues during murine embryonic development.     

Gene-specific oxidative lesions in aged rat brain detected by polymerase chain reaction inhibition assay

An exposure of isolated rat brain genomic DNA to oxidative stress in the form of iron salts (Fe²⁺) and ascorbate results in gene-specific DNA lesions detectable by a quantitative polymerase chain reaction (PCR) based assay in which PCR amplification efficiency of the affected genes (e.g. β-actin and p53) is grossly impaired. Such oxidative DNA lesions are prevented by hydroxyl radical scavengers like mannitol (20 mM) and sodium benzoate (20 mM) or by the antioxidant enzyme catalase (50 μg/ml) present in the incubation mixture during exposure to Fe²⁺ and ascorbate. When brain DNA isolated from young (4-6 months of age) and aged (20-24 months of age) rats are analyzed similarly by the PCR based method, the amplification levels of β-actin and p53 genes are noticeably decreased in the case of aged rat indicating an accumulation of gene-specific DNA lesions during brain aging.     

Change of actin isomers during differentiation of smooth muscle

Changes of actin isomers during development and differentiation of chicken gizzard were investigated by polyacrylamide gel electrophoresis. The two-dimensional gel electrophoresis with SDS-polyacrylamide gels in the presence of urea as the second dimension clearly separated three actin isomers which appear during development of the smooth muscle. The three actin isomers change the relative concentrations during development as follows: (1) gizzard-type γ-actin begins to be synthesized late on the 7th day of embryogenesis and increases in amount until hatching, (2) nonmuscle-type γ-actin exists only at earlier stages (before 15 days of embryogenesis), and (3) the amount of β-actin increases in proportion to the decrease of nonmuscle type γ-actin, the amount of nonmuscle actin in gizzards then becoming constant. Actin composition of gizzard before 7 days of embryonic age was nonmuscle type and consisted of β-actin and nonmuscle-type γ-actin. These observations indicate that developmental process of gizzard smooth muscle cells are classified as three stages: nonmuscle, intermediate and smooth muscle stages.     

Role of GSK-3β in the osteogenic differentiation of palatal mesenchyme

Introduction

The function of Glycogen Synthase Kinases 3β (GSK-3β) has previously been shown to be necessary for normal secondary palate development. Using GSK-3ß null mouse embryos, we examine the potential coordinate roles of Wnt and Hedgehog signaling on palatal ossification.

Methods

Palates were harvested from GSK-3β, embryonic days 15.0–18.5 (e15.0–e18.5), and e15.5 Indian Hedgehog (Ihh) null embryos, and their wild-type littermates. The phenotype of GSK-3β null embryos was analyzed with skeletal whole mount and pentachrome stains. Spatiotemporal regulation of osteogenic gene expression, in addition to Wnt and Hedgehog signaling activity, were examined in vivo on GSK-3β and Ihh +/+ and −/− e15.5 embryos using in situ hybridization and immunohistochemistry. To corroborate these results, expression of the same molecular targets were assessed by qRT-PCR of e15.5 palates, or e13.5 palate cultures treated with both Wnt and Hedgehog agonists and anatagonists.

Results

GSK-3β null embryos displayed a 48 percent decrease (*p<0.05) in palatine bone formation compared to wild-type littermates. GSK-3β null embryos also exhibited decreased osteogenic gene expression that was associated with increased Wnt and decreased Hedgehog signaling. e13.5 palate culture studies demonstrated that Wnt signaling negatively regulates both osteogenic gene expression and Hedgehog signaling activity, while inhibition of Wnt signaling augments both osteogenic gene expression and Hedgehog signaling activity. In addition, no differences in Wnt signaling activity were noted in Ihh null embryos, suggesting that canonical Wnt may be upstream of Hedgehog in secondary palate development. Lastly, we found that GSK-3β −/− palate cultures were “rescued” with the Wnt inhibitor, Dkk-1.

Conclusions

Here, we identify a critical role for GSK-3β in palatogenesis through its direct regulation of canonical Wnt signaling. These findings shed light on critical developmental pathways involved in palatogenesis and may lead to novel molecular targets to prevent cleft palate formation.     

H-2 histocompatibility region influences the inhibition of arachidonic acid cascade by dexamethasone and phenytoin in mouse embryonic palates

We have reported that susceptibility to glucocorticoid- and phenytoin-induced cleft palate and glucocorticoid receptor levels in mice are influenced by the H-2 histocompatibility complex on chromosome 17. Phenytoin competes with glucocorticoids for the glucocorticoid receptor and inhibits production of prostaglandins and thromboxanes. In this paper, we have investigated whether glucocorticoids and phenytoin inhibit arachidonic acid release and prostaglandin biosynthesis directly in the embryonic palates and whether the H-2 gene complex influences the degree of inhibition. Using congenic strains varying only in the H-2 region, we demonstrate here that both glucocorticoids and phenytoin inhibit the release of 3H-arachidonic acid and prostaglandin biosynthesis from embryonic palatal tissue, prelabeled with 3H-arachidonic acid. The degree of inhibition of arachidonic acid release and of prostaglandin biosynthesis is greater in the strain with H-2a (A/Wy) than in its corresponding congenic partner H-2b (A.BY). Thus, these results provide further evidence for a similar genetic and biochemical pathway for the teratogenic action of both phenytoin and glucocorticoids.     

��-Actin Association with Endothelial Nitric-oxide Synthase Modulates Nitric Oxide and Superoxide Generation from the Enzyme

Protein-protein interactions represent an important post-translational mechanism for endothelial nitric-oxide synthase (eNOS) regulation. We have previously reported that β-actin is associated with eNOS oxygenase domain and that association of eNOS with β-actin increases eNOS activity and nitric oxide (NO) production. In the present study, we found that β-actin-induced increase in NO production was accompanied by decrease in superoxide formation. A synthetic actin-binding sequence (ABS) peptide 326 with amino acid sequence corresponding to residues 326–333 of human eNOS, one of the putative ABSs, specifically bound to β-actin and prevented eNOS association with β-actin in vitro. Peptide 326 also prevented β-actin-induced decrease in superoxide formation and increase in NO and l-citrulline production. A modified peptide 326 replacing hydrophobic amino acids leucine and tryptophan with neutral alanine was unable to interfere with eNOS-β-actin binding and to prevent β-actin-induced changes in NO and superoxide formation. Site-directed mutagenesis of the actin-binding domain of eNOS replacing leucine and tryptophan with alanine yielded an eNOS mutant that exhibited reduced eNOS-β-actin association, decreased NO production, and increased superoxide formation in COS-7 cells. Disruption of eNOS-β-actin interaction in endothelial cells using ABS peptide 326 resulted in decreased NO production, increased superoxide formation, and decreased endothelial monolayer wound repair, which was prevented by PEG-SOD and NO donor NOC-18. Taken together, this novel finding indicates that β-actin binding to eNOS through residues 326–333 in the eNOS protein results in shifting the enzymatic activity from superoxide formation toward NO production. Modulation of NO and superoxide formation from eNOS by β-actin plays an important role in endothelial function.     

Involvement of Glucocorticoids in the Development of the Secondary Palate

Genetic differences between various inbred strains of mice in the levels of glucocorticoid receptors embryonic in maxillary mesenchyme cells appear to be reflected in the magnitude of the responses to steroids in these cells. High levels of glucocorticoids cause significant growth inhibition in maxillary mesenchyme cells with subsequent alterations in the production of extracellular matrix components. The presence of higher levels of cytoplasmic glucocorticoid receptor proteins may be one factor which could predispose those strains such as A/J to a greater inhibition of craniofacial growth in vivo by glucocorticoids and therefore increase the frequency of cleft palate production. Furthermore, women with infertility treated with glucocorticoids to support pregnancy give birth to infants with a marked decrease in birth weight [98]. Pharmacologic doses of glucocorticoids can also cause a dramatic reduction in the growth of a number of fetal tissues in mice and humans. In fact, there is evidence that glucocorticoids may be a causative factor in the production of cleft palate in primates [52]. The nature of the molecular elements which determine the biochemical and physiologic responses to glucocorticoids in the palate still remains largely unknown. Although in the mouse there is some evidence to suggest that the major histocompatibility locus (H-2) might be involved, the level(s) at which this control is exerted is unknown. It is possible that this locus may regulate in some manner the level of glucocorticoid receptors and the response to glucocorticoids in the secondary palate. Moreover, there is evidence to suggest that other genes distinct from, but closely linked to the H-2 locus may be important in determining both the strain-dependent differences in susceptibility to glucocorticoid-induced cleft palate and the intracellular levels of cyclic AMP in the secondary palate. It is also apparent that glucocorticoids in conjunction with other hormones or growth factors such as epidermal growth factor and agents which regulate cyclic nucleotide metabolism are essential for the normal development of the secondary palate. Excesses or deficiencies in either the level of these growth regulators and/or in their receptors in specific fetal tissues at defined periods in development are likely to lead to certain fetal malformations. Definition and integration of the genetic, biochemical, and endocrine factors which are involved in the control of cellular growth as influenced by alterations in the composition of cell surface and extracellular matrix components should provide some insights into the events associated with normal palatogenesis.     

Lack of renal 11 beta-hydroxysteroid dehydrogenase type 2 at birth, a targeted temporal window for neonatal glucocorticoid action in human and mice

Background

Glucocorticoid hormones play a major role in fetal organ maturation. Yet, excessive glucocorticoid exposure in utero can result in a variety of detrimental effects, such as growth retardation and increased susceptibility to the development of hypertension. To protect the fetus, maternal glucocorticoids are metabolized into inactive compounds by placental 11beta-hydroxysteroid dehydrogenase type2 (11βHSD2). This enzyme is also expressed in the kidney, where it prevents illicit occupation of the mineralocorticoid receptor by glucocorticoids. We investigated the role of renal 11βHSD2 in the control of neonatal glucocorticoid metabolism in the human and mouse.

Methods

Cortisol (F) and cortisone (E) concentrations were measured in maternal plasma, umbilical cord blood and human newborn urine using HPLC. 11βHSD2 activity was indirectly assessed by comparing the F/E ratio between maternal and neonatal plasma (placental activity) and between plasma and urine in newborns (renal activity). Direct measurement of renal 11βHSD2 activity was subsequently evaluated in mice at various developmental stages. Renal 11βHSD2 mRNA and protein expression were analyzed by quantitative RT-PCR and immunohistochemistry during the perinatal period in both species.

Results

We demonstrate that, at variance with placental 11βHSD2 activity, renal 11βHSD2 activity is weak in newborn human and mouse and correlates with low renal mRNA levels and absence of detectable 11βHSD2 protein.

Conclusions

We provide evidence for a weak or absent expression of neonatal renal 11βHSD2 that is conserved among species. This temporal and tissue-specific 11βHSD2 expression could represent a physiological window for glucocorticoid action yet may constitute an important predictive factor for adverse outcomes of glucocorticoid excess through fetal programming.     

冬虫夏草菌实时荧光定量PCR内参基因的筛选

以冬虫夏草单子囊孢子分离得到的菌株TZ8-1的3种菌丝形态为实验材料,提取RNA,经反转录获取cDNA,选择了 11个持家基因为候选内参基因(18SrRNA、APRTase、β-TUB、RPL2、EF1-α、PGI、PGM、H+-ATPase、ACT1、UBQ和GAPDH),根据该菌基因组注释结果来设计引物,采用实时荧...     

Lineage diversion of T cell receptor transgenic thymocytes revealed by lineage fate mapping

Background

The binding of the T cell receptor (TCR) to major histocompatibility complex (MHC) molecules in the thymus determines fates of TCRαβ lymphocytes that subsequently home to secondary lymphoid tissue. TCR transgenic models have been used to study thymic selection and lineage commitment. Most TCR transgenic mice express the rearranged TCRαβ prematurely at the double negative stage and abnormal TCRαβ populations of T cells that are not easily detected in non-transgenic mice have been found in secondary lymphoid tissue of TCR transgenic mice.

Methodology and Principal Findings

To determine developmental pathways of TCR-transgenic thymocytes, we used Cre-LoxP-mediated fate mapping and show here that premature expression of a transgenic TCRαβ diverts some developing thymocytes to a developmental pathway which resembles that of gamma delta cells. We found that most peripheral T cells with the HY-TCR in male mice have bypassed the RORγt-positive CD4⁺8⁺ (double positive, DP) stage to accumulate either as CD4⁻8⁻ (double negative, DN) or as CD8α⁺ T cells in lymph nodes or gut epithelium. Likewise, DN TCRαβ cells in lymphoid tissue of female mice were not derived from DP thymocytes.

Conclusion

The results further support the hypothesis that the premature expression of the TCRαβ can divert DN thymocytes into gamma delta lineage cells.     

Glucocorticoid-mediated inhibition of angiogenic changes in human endothelial cells is not caused by reductions in cell proliferation or migration

Background

Glucocorticoid-mediated inhibition of angiogenesis is important in physiology, pathophysiology and therapy. However, the mechanisms through which glucocorticoids inhibit growth of new blood vessels have not been established. This study addresses the hypothesis that physiological levels of glucocorticoids inhibit angiogenesis by directly preventing tube formation by endothelial cells.

Methodology/Principal Findings

Cultured human umbilical vein (HUVEC) and aortic (HAoEC) endothelial cells were used to determine the influence of glucocorticoids on tube-like structure (TLS) formation, and on cellular proliferation (5-bromo-2′-deoxyuridine (BrdU) incorporation), viability (ATP production) and migration (Boyden chambers). Dexamethasone or cortisol (at physiological concentrations) inhibited both basal and prostaglandin F_2α (PGF_2α)-induced and vascular endothelial growth factor (VEGF) stimulated TLS formation in endothelial cells (ECs) cultured on Matrigel, effects which were blocked with the glucocorticoid receptor antagonist RU38486. Glucocorticoids had no effect on EC viability, migration or proliferation. Time-lapse imaging showed that cortisol blocked VEGF-stimulated cytoskeletal reorganisation and initialisation of tube formation. Real time PCR suggested that increased expression of thrombospodin-1 contributed to glucocorticoid-mediated inhibition of TLS formation.

Conclusions/Significance

We conclude that glucocorticoids interact directly with glucocorticoid receptors on vascular ECs to inhibit TLS formation. This action, which was conserved in ECs from two distinct vascular territories, was due to alterations in cell morphology rather than inhibition of EC viability, migration or proliferation and may be mediated in part by induction of thrombospodin-1. These findings provide important insights into the anti-angiogenic action of endogenous glucocorticoids in health and disease.