Expression and genomic imprinting of the porcine Rasgrf1 gene

Imprinted genes play important roles in mammalian growth, development and behavior. The Rasgrf1 (Ras protein-specific guanine nucleotide exchange factor 1) gene has been identified as an imprinted gene in mouse and rat. In the present study, we detected its sequence, imprinting status and expression pattern in the domestic pigs. A 228 bp partial sequence located in exon 14 and a 193 bp partial sequence located in exon 1 of the Rasgrf1 gene in domestic pigs were obtained. A G/A transition, was identified in Rasgrf1 exon 14, and then, the reciprocal Berkshire × Wannan black F₁ hybrid model and the RT-PCR-RFLP method were used to detect the imprinting status of porcine Rasgrf1 gene at the developmental stage of 1-day-old. The expression profile results indicated that the porcine Rasgrf1 mRNA was highly expressed in brain, pituitary and pancreas, followed by kidney, stomach, lung, testis, small intestine, ovary, spleen and liver, and at low levels of expression in longissimus dorsi, heart, and backfat. The expression levels of Rasgrf1 gene in brain, pituitary and pancreas tissues were significantly different between the two reciprocal F₁ hybrids. Imprinting analysis showed that porcine Rasgrf1 gene was maternally expressed in the liver, small intestine, paternally expressed in the lung, but biallelically expressed in brain, heart, spleen, kidney, stomach, pancreas, backfat, testis, ovary, longissimus dorsi and pituitary tissues.     

Widespread differentiation stage-specific expression of the gene encoding phosphoprotein p19 (metablastin) in mammalian cells

p19 is a highly conserved 19 kD cytosolic protein that undergoes phosphorylation in response to diverse extracellular factors in mammalian cells. Its expression is abundant in brain and testis and is developmentally regulated. To gain insights regarding its function, we analyzed the expression of p19 mRNA in a variety of cell types during induction of differentiation. Murine erythroleukemia cells showed a moderate increase followed by a marked decrease in the abundance of p19 mRNA during induction of differentiation. In murine C2 myoblasts and primary fetal rat osteoblasts, p19 mRNA was abundant in replicating cells and decreased to undetectable levels during differentiation. In resting human peripheral blood lymphocytes, p19 mRNA was virtually undetectable but was strongly induced during blast transformation of both B and T cells. In rat liver, p19 mRNA was abundant on embryonic day 17 and decreased during early postnatal development. Upon fractionation of adult rat liver cells by centrifugal elutriation, p19 mRNA was not detected in hepatocytes while a low level was observed in a fraction enriched in non-parenchymal epithelial cells. CCl4-induced liver regeneration resulted in induction of p19 mRNA in hepatocytes. Primary cultures of embryonic and neonatal rat brain were analyzed by indirect immunofluorescence using co-staining with stage-specific markers. p19 expression was restricted to immature neurons and oligodendrocyte precursors. In contrast to the other cell types examined, the neuronal and glial precursors that express p19 were shown, using BrdU labeling, to be postmitotic both in primary culture and in vivo. The data demonstrate widespread, stage-specific expression of p19 and suggest that the protein exerts a general, lineage-independent function during induction of differentiation of mammalian cells. In view of the available evidence on the stimulation of serine phosphorylation of p19 by several growth factors, our working hypothesis is that phosphorylation of p19 may be involved in the mechanism by which growth factors control cell differentiation.     

Expression studies of neogenin and its ligand hemojuvelin in mouse tissues.

Juvenile hemochromatosis is a severe hereditary iron overload disease caused by mutations in the HJV (hemojuvelin) and HAMP (hepcidin) genes. Hepcidin is an important iron regulatory hormone, and hemojuvelin may regulate hepcidin synthesis via the multifunctional membrane receptor neogenin. We explored the expression of murine hemojuvelin and neogenin mRNAs and protein. Real-time RT-PCR analysis of 18 tissues from male and female mice was performed to examine the mRNA expression profiles. To further study protein expression and localization we used immunohistochemistry on several tissues from three mouse strains. Mouse Neo1 mRNA was detectable in the 18 tissues tested, the highest signals being evident in the ovary, uterus, and testis. Neogenin protein was observed in the brain, skeletal muscle, heart, liver, stomach, duodenum, ileum, colon, renal cortex, lung, testis, ovary, oviduct, and uterus. The spleen, thymus, and pancreas were negative for neogenin. The highest signals for Hjv mRNA were detectable in the skeletal muscle, heart, esophagus, and liver. The results indicate that Neo1 mRNA is widely expressed in both male and female mouse tissues with the highest signals detected in the reproductive system. Moreover, Hjv and Neo1 mRNAs are simultaneously expressed in skeletal muscle, heart, esophagus, and liver.     

Molecular cloning and characterization of rat and human calpain-5

Until today, 14 isoforms of mammalian calpains have been identified, including calpain-5. The C. elegans calpain-5 homologue tra-3 is reported to be essential for necrotic neuronal cell death. In this study, we cloned and characterized rat calpain-5, which is highly homologous to human and mouse sequences. The nucleotide sequence is 87% and 93% identical with human and mouse calpain-5, respectively. The protein sequence is well conserved, showing 96% identity in mouse and 92% in human. RT-PCR analysis revealed strong expression of calpain-5 in rat lungs, kidneys, and brain while week expression in heart, whereas in rat brain regions it is ubiquitously expressed. The mRNA expression in different human tissues showed equal expression. However, in human brain regions calpain-5 was strongly expressed in hypothalamus, thalamus, cerebellum, and frontal lobe. Western blot analysis on human neuroblastoma SH-SY5Y cells demonstrated calcium-dependent processing of calpain-5, despite the absence of calmodulin-like domain.     

Molecular cloning, expression profile and functional implications of clusterin in the pituitary gland of helmeted guinea fowl (Numida meleagris)

Clusterin is shown to contain putative amphipathic alpha-helices that mediate hydrophobic interactions with numerous types of molecules and may be involved in clearance of cellular debris caused by cell injury or death. To assess this function in vivo, we have cloned the full-length cDNA encoding guinea fowl (Numida meleagris) clusterin and studied its synthesis and expression pattern in specific cell types in pituitary. Quantity of clusterin mRNA expressed in pituitary and endocrine tissues was quantified by real-time PCR. Highest levels were detected in gonads. In situ hybridization showed clusterin mRNA in endocrine cells and folliculostellate cells. Clusterin protein detected by immunohistochemistry was observed in endocrine cells, folliculostellate cells and in colloid. The expression pattern suggests that clusterin is produced by endocrine cells for cytoprotection. Degenerating endocrine cells are phagocytosed by folliculostellate cells and digested by their lysosomal enzymes. In folliculostellate cells clusterin interacts and aggregates with by-products of digestion that subsequently become stored in colloid.     

Expression of the Low-Affinity p75 Nerve Growth Factor Receptor in the Developing Rat Pituitary Gland

We investigated, by means of in situ hybridization with a digoxigenin-labelled RNA probe, the expression of the low-affinity p75 nerve growth factor receptor (NGFR) in the developing pituitary primordium of the rat. In 13-day pc embryos, intense staining of p75 NGFR mRNA was present in the cytoplasm of all cells of Rathke's pouch. In day-17 pc embryos p75 NGFR expression was present primarily in the cells of the intermediate lobe. In the newborn rat pituitary only very weak staining was observed, predominantly in the intermediate lobe. In neural structures the staining at day 13 pc was comparable to that of day 17 pc. Since p75 expression is seen very early during pituitary development and declines during the time the expression of pituitary hormonal phenotypes are steadily increasing, we suggest that the p75 NGFR expression in Rathke's pouch may play a temporally defined role in the commitment rather than in the differentiation of the various pituitary cell types.     

Isolation and characterization of Cox17p from porcine heart by determining its survival-promoting activity in NIH3T3 cells

We have found that the gel filtration fraction of porcine heart extract clearly promoted the survival of NIH3T3 fibroblast cells in the serum-free medium condition. A structural analysis showed that the active fraction contained a novel peptide, porcine Cox17p (p-Cox17p), which was recently reported by Chen et al. as dopuin (Z. W. Chen et al., Eur. J. Biochem. 249 (1997) 518-522). Porcine Cox17p/dopuin possesses high sequence homology to the product of human COX17 gene (h-Cox17p). Although Cox17p has been implied to be involved in copper recruitment to mitochondria and in the functional assembly of cytochrome oxidase in yeast, its role in mammalian cells is unknown. In this study, we chemically synthesized p-Cox17p to investigate its biological effects. Refolding experiments of synthesized linear p-Cox17p revealed the existence of mostly one pattern of three intrachain disulfide bridges similar to that of native p-Cox17p, because the main oxidized p-Cox17p was completely co-eluted with the natural product. The addition of heavy metal ions such as copper, zinc and cadmium significantly inhibited the formation of the oxidized form, suggesting that reduced p-Cox17p may interact directly with these metal ions. The reduced and oxidized forms of p-Cox17p were also confirmed to promote the survival of NIH3T3 cells in serum-free medium as observed with the natural product, indicating that Cox17p may be a bioactive peptide.     

Follistatin gene expression in the ovary and extragonadal tissues

Follistatin is a glycosylated single-chain protein originally isolated from porcine follicular fluid. It specifically inhibits the secretion of FSH from the pituitary. We have now isolated and characterized a cDNA for rat follistatin from the PMSG-stimulated ovarian library. The deduced amino acid sequence of the rat follistatin precursor is highly homologous (greater than 98%) to porcine and human follistatins including potential Asn-glycosylation sites. The genomic clone encoding rat follistatin was also isolated and revealed that the exon and intron organization of the follistatin gene structure is conserved among rat, porcine, and human. Northern analyses in rat tissues demonstrated that the follistatin gene is expressed not only in the ovary but also in the kidney and brain. In the immature rat ovary, the follistatin mRNA level is stimulated by PMSG injection (20 IU/rat), but is not affected by human CG (10 IU/rat) after PMSG administration. In situ hybridization studies revealed that the mRNA level in the ovary was low in primordial follicles, but dramatically increased in the granulosa cells of the growing secondary and tertiary follicles and then decreased in the mature preovulatory follicles. A strong follistatin mRNA signal was observed over the collecting tubules of the outer medulla of the kidney, and a weak to moderate signal was detected in brain. The broad tissue distribution of follistatin mRNA strongly suggests other physiological roles for follistatin besides the inhibition of pituitary FSH release.     

大鼠睾丸发育过程中促甲状腺激素释放激素受体mRNA的表达

为研究促甲状腺激素释放激素受体(TRHR)在大鼠睾丸组织中的表达规律和在生殖发育调节中的作用,依据大鼠垂体中的TRH-RcDNA设计引物,采用RT-PCR法从大鼠睾丸组织中获得了TRH-R的cDNA克隆,测序表明其核苷酸序列与大鼠垂体中的TRH-RcDNA序列完全一致.应用非放射性原位杂交(NR-ISH)技术观察TRH-RmRNA在大鼠睾丸中的定位,结果显示杂交信号集中在间质细胞中,生精细胞无杂交信号.利用实时动态定量RT-PCR法观察了TRH-R在不同发育阶段大鼠睾丸中的表达变化,发现在睾丸间质细胞发育的初期阶段(第8天),没有TRH-R的表达,但从第15天起能观察到TRH-R的表达,并且表达量在20天、35天、60天、90天逐渐增加.这些结果表明,大鼠睾丸组织间质细胞能特异性表达TRH-R,并且表达量与发育过程相关.     

Structure and expression of the chicken beta nerve growth factor gene.

The 3' exon of the chicken beta nerve growth factor (NGF) gene was isolated by the use of a murine cDNA probe. DNA sequence analysis of the clone suggests a mature chicken NGF protein of 118 amino acids, showing approximately 85% homology to mouse and human NGF. In addition to this conservation of the mature NGF, parts of the propeptide and the untranslated 3' end of the NGF gene are also highly homologous in chicken, human and mouse. Therefore, these sequences probably subserve important functions. Expression of NGF mRNA in various chicken tissues was examined by RNA blot analysis with a chicken NGF probe. A single mRNA of 1.3 kb was detected at high levels in heart and brain of 10-week-old roosters, and, at lower levels in spleen, liver and skeletal muscle. These data suggest a correlation between NGF expression and the density of sympathetic innervation in peripheral organs, in analogy with findings for mammalian tissues. In the adult avian brain, NGF mRNA is found at higher concentration in the optic tectum and cerebellum than in the cortex and hippocampus. This pattern of NGF expression differs from that previously described for the rat brain. During late stages of development (day 18), NGF mRNA was expressed both in heart and brain of embryos but at lower levels than in the adult.     

Combined use of in situ hybridisation and immunocytochemistry for the investigation of prolactin gene expression in immature,pubertal, pregnant,lactating and ovariectomised rats

Summary We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a ³²P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland.     

cDNA sequence and tissue distribution of the mRNA for bovine and murine p11, the S100-related light chain of the protein-tyrosine kinase substrate p36 (calpactin I)

We have isolated and sequenced cDNA clones of bovine and murine p11 mRNAs. The nonpolyadenylated mRNAs are predicted to be 614 and 600 nucleotides, respectively. The p11 mRNAs both contain a 291 nucleotide open reading frame, preceded by a 5'-untranslated region of 73 nucleotides in bovine p11 mRNA and of 68 nucleotides in murine p11 mRNA. The deduced bovine p11 amino acid sequence is identical to the previously published partial bovine and complete porcine p11 protein sequence except for an additional COOH-terminal lysine residue. The bovine and murine p11 proteins are 92% homologous, whereas at the nucleotide level the conservation is 89% in the coding region and 75% in the 3'-untranslated region. Southern analysis of murine genomic DNA detected a single p11 gene, less than 10 kilobase pairs in size, containing as many as three introns. The p11 gene has been assigned to mouse chromosome 3 by analysis of interspecific hybrid cell panels and recombinant inbred mouse strains. The p11 gene is closely linked to the Xmmv-65 endogenous leukemia virus env gene and the guanylate binding protein-1 gene. Northern analyses of RNAs from mouse tissues and cell lines indicated that p11 mRNA levels vary widely. They are very low in liver, heart, and testes, moderate in brain, spleen, and thymus, and high in kidney, intestine, and lung. Analysis of the same RNA samples for p36 mRNA levels showed that expression of p11 and p36 mRNAs is not always coordinated. Brain and the mouse embryonal carcinoma cell line F9 contain moderate to high levels of p11 mRNA with very low levels of p36 mRNA. Sequence homology between p11 and the S100 proteins, and the serum-induced 2A9 gene product, as well as possible functions of p11 are discussed.     

The Vasculome of the Mouse Brain

The blood vessel is no longer viewed as passive plumbing for the brain. Increasingly, experimental and clinical findings suggest that cerebral endothelium may possess endocrine and paracrine properties – actively releasing signals into and receiving signals from the neuronal parenchyma. Hence, metabolically perturbed microvessels may contribute to central nervous system (CNS) injury and disease. Furthermore, cerebral endothelium can serve as sensors and integrators of CNS dysfunction, releasing measurable biomarkers into the circulating bloodstream. Here, we define and analyze the concept of a brain vasculome, i.e. a database of gene expression patterns in cerebral endothelium that can be linked to other databases and systems of CNS mediators and markers. Endothelial cells were purified from mouse brain, heart and kidney glomeruli. Total RNA were extracted and profiled on Affymetrix mouse 430 2.0 micro-arrays. Gene expression analysis confirmed that these brain, heart and glomerular preparations were not contaminated by brain cells (astrocytes, oligodendrocytes, or neurons), cardiomyocytes or kidney tubular cells respectively. Comparison of the vasculome between brain, heart and kidney glomeruli showed that endothelial gene expression patterns were highly organ-dependent. Analysis of the brain vasculome demonstrated that many functionally active networks were present, including cell adhesion, transporter activity, plasma membrane, leukocyte transmigration, Wnt signaling pathways and angiogenesis. Analysis of representative genome-wide-association-studies showed that genes linked with Alzheimer’s disease, Parkinson’s disease and stroke were detected in the brain vasculome. Finally, comparison of our mouse brain vasculome with representative plasma protein databases demonstrated significant overlap, suggesting that the vasculome may be an important source of circulating signals in blood. Perturbations in cerebral endothelial function may profoundly affect CNS homeostasis. Mapping and dissecting the vasculome of the brain in health and disease may provide a novel database for investigating disease mechanisms, assessing therapeutic targets and exploring new biomarkers for the CNS.     

Changes in reproductive function and white blood cell proliferation induced in mice by injection of a prolactin-expressing plasmid into muscle

Prolactin (PRL) is a pituitary hormone involved in various physiological processes, including lactation, mammary development, and immune function. To further investigate the in vivo and comparative endocrine roles of PRL, mouse PRL cDNA fused to the cytomegalovirus promoter, was introduced into muscle by direct injection. Previously we studied the function of rat PRL using the same protocol. PRL mRNA was detected in the muscle following injection by RT-PCR and subsequent Southern blot analysis. PRL was also detected and Western blot analysis revealed a relatively high level of serum PRL. In the pCMV-mPRL-injected female mice, the estrous cycle was extended, especially in diestrus stage and the uterus thickening that was shown in normal estrous stage was not observed. In the pCMV-mPRL-injected male mice, new blood vessels were first found at 5 weeks of age and fully developed blood vessels were found after 8 weeks in the testis. The number of Leydig cells increased within the testis and the testosterone level in serum was observed high. Finally, the number of white blood cells (WBCs) increased in the pCMV-mPRL-injected mice. The augmentation of WBCs persisted for at least 20 days after injection. When injection was combined with adrenalectomy, there was an even greater increase in number of WBCs, especially lymphocytes. This increase was returned normal by treatment with dexamethansone. Taken together, our data reveal that intramuscularly expressed mouse PRL influences reproductive functions in female, induces formation of new blood vessels in the testis, and augments WBC numbers. Of notice is that the Leydig cell proliferation with increased testosterone was conspicuously observed in the pCMV-mPRL-injected mice. These results also suggest subtle difference in function of PRL between mouse and rat species.     

小鼠不同组织及缺氧损伤后的大鼠心肌细胞中RIP3的表达

目的:检测小鼠组织中受体相互作用丝氨酸/苏氨酸蛋白激酶家族(RIPs)表达谱,并检测RIP3在大鼠心肌细胞缺氧损伤后的表达。方法:①采用荧光实时定量PCR分别检测RIPs家族基因在小鼠组织(心、肝、肺、肾、脑、小肠、骨骼肌、脾和主动脉)中的mRNA表达谱,并采用Western blot进一步检测RIP3在小鼠组织的蛋白表达谱。②将培养的大鼠心肌细胞分为缺氧组和对照组,缺氧组置于缺氧环境中培养48 h,采用western blot检测其中RIP3的表达变化。结果:①mRNA水平:RIP1 mRNA在脑组织中表达最高,心脏、肺、肾、骨骼肌较低;RIP2在心脏和肺表达量较其他组织高;RIP3在肠中表达较其他组织高出4倍以上,脑组织中未检测到RIP3表达;RIP4的表达以肺最高,而骨骼肌、脑和血管中表达量低。②蛋白水平:在小鼠组织中,RIP3表达以脑、骨骼肌中最高,心脏、肝、肺中表达较低。③培养的大鼠心肌细胞中,缺氧组心肌细胞的RIP3表达量显著高于对照组(P0.05)。结论:RIPs在小鼠组织中呈现差异表达,而在培养的大鼠心肌细胞缺氧损伤后RIP3表达升高。