Porcine insulin receptor substrate 4 (<Emphasis Type="Italic">IRS4</Emphasis>) gene: cloning,polymorphism and association study

Using PCR and inverse PCR techniques we obtained a 4,498 bp nucleotide sequence FN424076 encompassing the complete coding sequence of the porcine insulin receptor substrate 4 (IRS4) gene and its proximal promoter. The 1,269 amino acid porcine protein deduced from the nucleotide sequence shares 92% identity with the human IRS4 and possesses the same domains and the same number of tyrosine phosphorylation motifs as the human protein. We detected substitution FN424076:g.96C<G in the promoter region that segregates in Meishan and a synonymous substitution FN424076:g.1829T<C in the coding sequence with allele C present only in Meishan. Linkage mapping placed the IRS4 gene at position 82 cM on the current USDA–USMARC linkage map of porcine chromosome X. Association analyses were performed on 555 animals of 12th–15th generation of the Meishan × Large White cross and showed that both SNPs were highly significantly associated with backfat depth (P = 0.0005) and that the SNP FN424076:g1829T<C was also associated with loin depth (P = 0.017). The Meishan alleles increased back fat depth and decreased loin depth. IRS4 can be considered a positional candidate gene for at least some of the QTL located at the centromeric region of porcine chromosome X.     

SERPINE2, an inhibitor of plasminogen activators,is highly expressed in the human endometrium during the secretory phase

Background  

SERPINE2, also known as protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent SERPINs that modulates the activity of plasminogen activators (PAs). PAs and their SERPIN inhibitors, such as SERPINB2 and SERPINE1, were expressed in the human endometrium and were implicated in implantation. However, expression data about SERPINE2 in the human endometrium is still unknown. Thus, we conducted an investigation to reveal the spatiotemporal and cellular expression of SERPINE2 in the human uterus during the menstrual cycle.     

Spatiotemporal expression of the serine protease inhibitor,SERPINE2, in the mouse placenta and uterus during the estrous cycle,pregnancy, and lactation

Background  

SERPINE2, also known as glia-derived nexin or protease nexin-1, belongs to the serine protease inhibitor (SERPIN) superfamily. It is one of the potent serpins that modulates the activity of the plasminogen activator (PA) and was implicated in tissue remodeling. In this study, we investigated the expression patterns of SERPINE2 in the mouse placenta and uterus during the estrous cycle, pregnancy, and lactation.     

Porcine NAMPT gene: search for polymorphism,mapping and association studies

NAMPT encodes an enzyme catalysing the rate‐limiting step in NAD biosynthesis. The extracellular form of the enzyme is known as adipokine visfatin. We detected SNP AM999341:g.669T>C (referred to as 669T>C) in intron 9 and SNP FN392209:g.358A>G (referred to as 358A>G) in the promoter of the gene. RH mapping linked the gene to microsatellite SW944. Linkage analysis placed the gene on the current USDA – USMARC linkage map at position 92 cM on SSC9. Association analyses were performed in a wild boar × Meishan F₂ family (W × M), with 45 traits recorded (growth and fattening, fat deposition, muscling, meat quality, stress resistance and other traits), and in a commercial Landrace × Chinese‐European (LCE) synthetic population with records for 15 traits (growth, fat deposition, muscling, intramuscular fat, meat colour and backfat fatty acid content). In the W × M, SNP 669T>C was associated with muscling, fat deposition, growth and fattening, meat quality and other traits and in the LCE with muscling, meat quality and backfat fatty acid composition. In the W × M, SNP 358A>G was associated with muscling, fat deposition, growth and other traits. After correction for multiple testing, the NAMPT haplotypes were associated in the W × M with, in descending order, muscling (q = 0.0056), growth (q = 0.0056), fat deposition (q = 0.0109), fat‐to‐meat ratio (q = 0.0135), cooling losses (q = 0.0568) and longissimus pH_U (q = 0.0695). The SNPs are hypothesized to be in linkage disequilibrium with a causative mutation affecting energy metabolism as a whole rather than fat metabolism alone.     

DIVING BEHAVIOR OF THE PACIFIC HARBOR SEAL (PHOCA VITULINA RICHARDII) IN MONTEREY BAY, CALIFORNIA

Physical environment and physiological characteristics of marine mammals potentially affect the duration and depth of diving. Härkönen (1987b) proposed a hypothesis that the harbor seal would gain maximum energy by foraging at intermediate depths. To investigate this hypothesis, we studied diving behavior of the Pacific harbor seal (Phoca vitulina ricbardii) during 1995 through 1997 in Monterey Bay, California. Dive depths (n = 13,063 dives) were recorded via time‐depth recorders. Approximately 80% of recorded dives were classified as square dives (type I), which typically were associated with foraging in pinnipeds. Approximately 11% of dives were V dives (type II; 1,402 dives), and the remainder (1,225 dives) were skewed dives (type III and IV). The deepest recorded dive was 481 m, while the greatest duration was 35.25 min. Body mass explained the variability of durations of long dives for females (95th percentile; D₉₅♂=‐5.47 + 0.18 × (mass♀), r²= 0.91, 95% CI for slope = [0.08, 0.28], n= 5) and for males (D₉₅♂=‐5.86 + 0.18 × (mass♀), r²= 0.83, 95% CI for slope = [0.12, 0.24], n= 11). The large proportion of variability in deep dives, however, was explained by body mass only for males (95th percentile; Z₉₅♂=‐363.9 + 6.05 × (mass♀), r²= 0.83, 95% CI for slope = [3.93, 8.17], n= 11) and not for females (Z₉₅,♂=?148.1 +3.11 × (mass♀), r²= 0.58, 95% CI for slope = [‐1.7, 7.9], n= 5, 95% CI for slope= [?1.7, 7.9]). Median depths of presumed foraging dives of harbor seals in the Monterey Bay area were between 5 and 100 m, which were within the range of the previously reported depths for other areas (< 100 m). Our findings generally supported Härkönen's hypothesis that harbor seals forage in the intermediate depth in their environment.     

Characterization of the porcine AMPK alpha 2 catalytic subunitgene (PRKAA2): genomic structure,polymorphism detection and association study

AMP‐activated protein kinase (AMPK), known as a key regulator of cellular energy homeostasis, plays an important role in regulation of glucose and lipid metabolism, and protein synthesis in mammals. The characterization of porcine PRKAA2 encoding the alpha 2 catalytic subunit of AMPK is reported in this study. PRKAA2 was assigned to porcine chromosome 6q by analysis of radiation hybrids (IMpRH panel), and its genomic structure was determined by BAC sequencing. PRKAA2 spans more than 62 kb and consists of nine exons and eight introns. A total of 25 polymorphisms were identified by re‐sequencing approximately 7 kb, including all the exons, exon–intron boundaries and 5′ and 3′ gene flanking regions using twelve founder animals of a Mangalitsa × Piétrain intercross. Neither of two single nucleotide polymorphisms (SNPs) found in the coding region caused an amino acid substitution. Two SNPs (NM_214266.1: c.236+142A>G and NM_214266.1: c.630C>T) in PRKAA2 were genotyped in the Mangalitsa × Piétrain F₂ cross (n = 589) and two commercial populations [Piétrain (n = 1173) and German Landrace (n = 536)] and evaluated for association with traits of interest (muscle development and fat deposition). Single SNP and haplotype analyses revealed weak associations between the PRKAA2 genotypes and loin muscle area in the investigated populations.     

Crossing the divide: gene flow produces intergeneric hybrid in feral transgenic creeping bentgrass population

Gene flow is the most frequently expressed public concern related to the deregulation of transgenic events ( Snow 2002 ; Ellstrand 2003 ). However, assessing the potential for transgene escape is complex because it depends on the opportunities for unintended gene flow, and establishment and persistence of the transgene in the environment ( Warwick et al. 2008 ). Creeping bentgrass (Agrostis stolonifera L.), a turfgrass species widely used on golf courses, has been genetically engineered to be resistant to glyphosate, a nonselective herbicide. Outcrossing species, such as creeping bentgrass (CB), which have several compatible species, have greater chances for gene escape and spontaneous hybridization (i.e. natural, unassisted sexual reproduction between taxa in the field), which challenges transgene containment. Several authors have emphasized the need for evidence of spontaneous hybridization to infer the potential for gene flow ( Armstrong et al. 2005 ). Here we report that a transgenic intergeneric hybrid has been produced as result of spontaneous hybridization of a feral‐regulated transgenic pollen receptor (CB) and a nontransgenic pollen donor (rabbitfoot grass, RF, Polypogon monspeliensis (L.) Desf.). We identified an off‐type transgenic seedling and confirmed it to be CB × RF intergeneric hybrid. This first report of a transgenic intergeneric hybrid produced in situ with a regulated transgenic event demonstrates the importance of considering all possible avenues for transgene spread at the landscape level before planting a regulated transgenic crop in the field. Spontaneous hybridization adds a level of complexity to transgene monitoring, containment, mitigation and remediation programmes.     

QTL alleles on chromosome 7 from fatty Meishan pigs reduce fat deposition

For detecting QTL in the whole swine genome, 1068 pigs from three F₂ populations constructed by crossing European Wild boar and Pietrain (W×P), Meishan and Pietrain (M×P), and Wild Boar and Meishan (W × M) were genotyped for genetic markers evenly spaced at approximately 20 cM intervals. AQTL analysis was performed using a least-squares method. Here the results of the QTL analysis on the porcine chromosome 7 are presented. QTL for carcass composition (e.g. head weight, carcass length, backfat depth, abdominal fat and bacon meat) were mapped in the chromosomal region CYPA/CYPD-TNFB-S0102 in M×P and W×M, but not in W×P. The QTL explained 5.3%–27.2% of the F₂ phenotypic variance in the two F₂ populations. Most traits affected by the mapped QTL were related to carcass fatness. The mode of gene action of QTL was additive. Surprisingly, in contrast to the parental phenotype, the QTL alleles from fatty Meishan were associated with thinner backfat than Pietrain and Wild Boar alleles, suggesting that the genome of the fatty Meishan pig contains genes which can reduce fat content of carcass substantially.     

Effects of Acetylcholine, Cytochalasin B and Amiprophosmethyl on Phloem Transport in Radish （Raphanus sativas）

We investigated the role of the ＂sieve tube-companion cell complex＂ lining the tube periphery, particularly the microfilament and microtubule, in assisting the pushing of phloem sap flow. We made a simple phloem transport system with a living radish plant, in which the conducting channel was exposed for local treatment with chemicals that are effective in modulating protoplasmic movement （acetylcholine, （ACh） a neurotransmitter in animals and insects; cytochalasin B, （CB） a specific inhibitor of many cellular responses that are mediated by microfilament systems and amiprophos-methyl, （APM） a specific inhibitor of many cellular responses that are mediated by microtubule systems）. Their effects on phloem transport were estimated by two experimental devices： （i） a comparison of changes in the amount of assimilates in terms of carbohydrates and ^14C-labeled photosynthetic production that is left in the leaf blade of treated plants; and （ii） distribution patterns of ^14C-labeled leaf assimilates in the phloem transport system. The results indicate that CB and APM markedly inhibited the transfer of photosynthetic product from leaf to root via the leaf vein, while ACh enhanced the transfer of photosynthetic product in low concentrations （5.0×10^-4 mol/L） but inhibited it in higher concentrations （2.0×10^-3 mol/L） from leaf to root via the leaf vein. Autoradiograph imaging clearly reveals that ACh treatment is more effective than the control, and both CB and APM treatments effectively inhibit the passage of radioactive assimilates. All of the results support the postulation that the peripheral protoplasm in the sieve tube serves not only as a passive semi-permeable membrane, but is also directly involved in phloem transport.     

Streptokinase variants from Streptococcus pyogenes isolates display altered plasminogen activation characteristics – implications for pathogenesis

Streptococcus pyogenes (group A streptococcus, GAS) secretes streptokinase, a potent plasminogen activating protein. Among GAS isolates, streptokinase gene sequences (ska) are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type‐1 and cluster type‐2) with cluster type‐2 being further divided into sub‐clusters type‐2a and type‐2b. In this study, far‐UV circular dichroism spectroscopy indicated that purified streptokinase variants of each type displayed similar secondary structure. Type‐2b streptokinase variants could not generate an active site in Glu‐plasminogen through non‐proteolytic mechanisms while all other variants had this capability. Furthermore, when compared with other streptokinase variants, type‐2b variants displayed a 29‐ to 35‐fold reduction in affinity for Glu‐plasminogen. All SK variants could activate Glu‐plasminogen when an activator complex was preformed with plasmin; however, type‐2b and type‐1 complexes were inhibited by α₂‐antiplasmin. Exchanging ska_type‐2a in the M1T1 GAS strain 5448 with ska_type‐2b caused a reduction in virulence while exchanging ska_type‐2a with ska_type‐1 into 5448 produced an increase in virulence when using a mouse model of invasive disease. These findings suggest that streptokinase variants produced by GAS isolates utilize distinct plasminogen activation pathways, which directly affects the pathogenesis of this organism.     

Plasminogen, absorbed by Escherichia coli expressing curli or by Salmonella enteritidis expressing thin aggregative fimbriae, can be activated by simultaneously captured tissue-type plasminogen activator (t-PA)

Curli are fimbrial structures expressed by Escherichia coli that specifically interact with matrix proteins such as fibronectin and laminin. Similar structures are also expressed by Salmonella enteritidis and have been denoted thin aggregative fimbriae. Bacteria expressing curli and thin aggregative fimbriae were found to bind radiolabelled plasminogen as well as the tissue-type plasminogen activator (t-PA). By contrast, E. coli carrying a gene locus with an insertionally inactivated chromosomal curlin subunit were unable to bind the two human proteins. The purified subunit polypeptides of curli and thin aggregative fimbriae bound plasminogen and t-PA with high affinity (1 × 10⁸ to 2 × 10⁸ M^-1). The binding of plasminogen and t-PA to curli-expressing E. coli was only partially inhibited by fibronectin and laminin. Plasminogen absorbed from human plasma by curli-expressing E. coli was readily converted to plasmin by t-PA; both plasmin and t-PA were functionally active when bound to the bacteria. A simultaneous binding of fibrinolytic proteins and matrix proteins to fimbriae of E. coli and S. enteritidis could provide these pathogens with both adhesive and invasive properties.     

Studies on Thermoelectric Properties of n‐type Polycrystalline SnSe1‐xSx by Iodine Doping

Iodine‐doped n‐type SnSe polycrystalline by melting and hot pressing is prepared. The prepared material is anisotropic with a peak ZT of ≈0.8 at about 773 K measured along the hot pressing direction. This is the first report on thermoelectric properties of n‐type Sn chalcogenide alloys. With increasing content of iodine, the carrier concentration changed from 2.3 × 10¹⁷ cm^?3 (p‐type) to 5.0 × 10¹⁵ cm^?3 (n‐type) then to 2.0 × 10¹⁷ cm^?3 (n‐type). The decent ZT is mainly attributed to the intrinsically low thermal conductivity due to the high anharmonicity of the chemical bonds like those in p‐type SnSe. By alloying with 10 at% SnS, even lower thermal conductivity and an enhanced Seebeck coefficient were achieved, leading to an increased ZT of ≈1.0 at about 773 K measured also along the hot pressing direction.     

Genetic analysis of artificial pigmentation mutants in Porphyra yezoensis Ueda (Bangiales, Rhodophyta)

Porphyra yezoensis Ueda artificial pigmentation mutants, yel (green), fre (red‐orange) and bop (pink), obtained by treatment with /V‐methyl‐/V′‐nitro‐N‐nitrosoguanidine, were genetically analysed. The mutations associated with color phenotypes are recessive because all of the heterozygous conchocelis resembled the wild type color when they were crossed with the wild type (wt). In the reciprocal crosses of yel × wt, both parental colors and eight types of blades appeared in the F₁ gametophytic blades from the heterozygous conchocelis. Both colors segregated in the sectored F₁ blades in a 1:1 ratio, indicating that the color pheno‐type of yel resulted from a single mutation in the nuclear gene. In the reciprocal crosses of fre × wt, however, four colors and more than 40 types of blades appeared in the F₁ blades from the heterozygous conchocelis, indicating that the color phenotype of fre resulted from two mutations in different genes. In the reciprocal crosses of bop×wt, three colors and 12 types of blades were observed in the F₁ blades from the heterozygous conchocelis. Both parental colors appeared far more frequently than the third new color. These results indicated that the color phenotype of bop resulted from two closely linked mutations in different genes, and the epistasis occurred in the F₁ blades. The mutants, yel, fre and bop, differ from the spontaneous green (C‐O), the red (H‐25) and the violet (V‐O) mutants of P. yezoensis, respectively.     

NAMPT,GRN, and SERPINE1 signature as predictor of disease progression and survival in gliomas

Adipose tissue is an important source of adipokines involved in anti- and pro-inflammatory effects. Their involvement in certain cancers such as breast and colon cancer is known but in gliomas it remains unexplored till date. The aim of this study was to assess the status of adipokines as prognostic markers of gliomas (low grade gliomas [LGG] and glioblastoma mutiforme [GBM]). Expression status (messenger RNA [mRNA]), overall survival (OS) and disease-free survival (DFS) was identified using gene expression profiling interactive analysis server. Clinicopathological analysis and correlation between different adipokines was performed using Xena server. Protein expression status was analyzed using tissue sections from the Human Protein Atlas. Out of 11 adipokines studied visfatin (NAMPT), apelin (APLN), granulin (GRN), serpin peptidase inhibitor/plasminogen activator inhibitor type 1 (PAI-1) member 1 (SERPINE1), and chemokine (C-C motif) ligand 2 (CCL2) mRNA levels were significantly upregulated in both LGG and GBM. Interleukin 6 (IL6) mRNA was found be significantly upregulated only in GBM. NAMPT, GRN, SERPINE1, and IL6 showed reduced OS as well as worst DFS for patients having higher mRNA expression in LGG. Increased expression of CCL2 showed worst OS in LGG patients while resistin (RETN) and GRN showed the worst OS in GBM patients. Higher expression of RETN, GRN, IL6, SERPINE1, and CCL2 were found to be positively correlated with shorter DFS in GBM. In the clinicopathological analysis, NAMPT, GRN, IL6, SERPINE1, and CCL2 expressions were significantly associated between the neoplasm histological G2 and G3 grades. Furthermore, expression of NAMPT, GRN, tumor necrosis factor, IL6, SERPINE1, and CCL2 were significantly associated with histological type in LGG patients. NAMPT, GRN, SERPINE1, CCL2, and RETN expression were found to be correlated with each other in gliomas. Finally, NAMPT, GRN, and SERPINE1 were also found to be upregulated using immunohistochemistry in a lower grade and high grade gliomas as compared to normal cells. In conclusion, we have identified key adipokines, namely NAMPT, GRN, and SERPINE1 as potential diagnostic and prognostic markers that might be instrumental in the development and progression of gliomas.     

Zinc Effects on Growth, Pigmentation and Antibacterial Activity of Monascus purpureus

Growth, pigmentation and antibacterial activity of Monascus purpureus Went (starch fungus) were affected by zinc. Zinc at concentrations of 2 × 10^-3M and 3 × 10^-3M nearly stopped the growth, pigmentation and antibiotic production of both wild type and strain NI IS in liquid medium. Relatively vigorous growth, high pigment production, and strong antibacterial activity of NIIS still occurred on solid medium with the same zinc concentrations. Wild type was hardly affected by zinc concentrations lower than 2 × 10^-3M. The growth of strain NI IS was significantly inhibited even by 5 × 10^-5M zinc, but pigment production and antibacterial activity were highly promoted by 5 × 10^-5M zinc. Increase of glucose concentration also promoted pigment and antibiotic productions of strain NI IS. Glucose effects were intensified as 1 × 10^-3M zinc was added. Zinc may act as a growth inhibitor and concomitantly as a stimulant for glucose uptake and for the synthesis of metabolites such as pigments and antibiotics.     

Environmental risk assessment for the small tortoiseshell Aglais urticae and a stacked Bt-maize with combined resistances against Lepidoptera and Chrysomelidae in central European agrarian landscapes

The cultivation of Lepidoptera‐resistant Bt‐maize may affect nontarget butterflies. We assessed the risk posed by event MON89034 × MON88017 (expressing Cry1A.105 and Cry2Ab2 against corn borers) to nontarget Lepidoptera. Using the small tortoiseshell Aglais urticae, a butterfly species common in central Europe, as a test organism we (i) assessed the toxicity of Bt‐maize pollen on butterfly larvae; (ii) measured pollen deposition on leaves of the host plant Urtica dioica; (iii) mapped the occurrence and distribution of host plants and larvae in two arable landscapes in Germany during maize anthesis; and (iv) described the temporal occurrence of a 1‐year population of A. urticae. (i) Larvae‐fed 200 Bt‐maize pollen grains/cm² had a reduced feeding activity. Significant differences in developmental time existed at pollen densities of 300 Bt‐maize pollen grains/cm² and in survival at 400 grains/cm². (ii) The highest pollen amount found was 212 grains/cm² at the field margin. Mean densities were much lower. (iii) In one region, over 50% of A. urticae nests were located within 5 m of a maize field, while in the other, all nests were found in more than 25 m distance to a maize field. (iv) The percentage of larvae developing during maize anthesis was 19% in the study area. The amount of pollen from maize MON89034 × MON88017 found on host plants is unlikely to adversely affect a significant proportion of larvae of A. urticae. This paper concludes that the risk of event MON89034 × MON88017 to populations of this species is negligible.     

Mechanism of the inhibitory effect of angiostatin on plasminogen activation by its physiologic activators

The influence of angiostatin K1-4.5, a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis, on kinetic parameters (k _Pg and K _Pg) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect on the k _Pg value, but increases the value of K _Pg plasminogen activation by urokinase. A decrease in the k _Pg value and an increase in the K _Pg value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is a competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA with a mixed type. Such an influence of angiostatin on the kinetic constants of the plasminogen activation by urokinase suggests that angiostatin dose-dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In the case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with the formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex, which leads to a decrease in k _Pg and an increase in K _Pg of the plasminogen activation. Inhibition constants by angiostatin (K _i) of plasminogen-activator activities of uPA and tPA determined by the Dixon method were found to be 0.59 ± 0.04 and 0.12 ± 0.05 μM, respectively.     

Effects and mechanisms of ghrelin on cardiac microvascular endothelial cells in rats

Ghrelin is thought to directly exert a protective effect on the cardiovascular system, specifically by promoting vascular endothelial cell function. Our study demonstrates the ability of ghrelin to promote rat CMEC (cardiac microvascular endothelial cell) proliferation, migration and NO (nitric oxide) secretion. CMECs were isolated from left ventricle of adult male Sprague—Dawley rat by enzyme digestion and maintained in endothelial cell medium. Dil‐ac‐LDL (1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine‐labelled acetylated low‐density lipoprotein) intake assays were used to identify CMECs. Cells were split into five groups and treated with varying concentrations of ghrelin as follows: one control non‐treated group; three ghrelin dosage groups (1×10^?9, 1×10^?8, 1×10^?7 mol/l) and one ghrelin+PI3K inhibitor group (1×10^?7 mol/l ghrelin+20 μmol/l LY294002). After 24 h treatment, cell proliferation capability was measured by MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide] assay and Western blot for PCNA (proliferating cell nuclear antigen) protein expression. Migration of CMECs was detected by transwell assays, and NO secretion of CMECs was measured via nitrate reduction. Protein expression of AKT and phosphorylated AKT in CMECs was measured by Western blot after exposure to various concentrations of ghrelin and the PI3K inhibitor LY294002. Our results indicate that ghrelin significantly enhanced cell growth at concentrations of 10^?8 mol/l (0.271±0.041 compared with 0.199±0.021, P=0.03) and 10^?7 mol/l (0.296±0.039 compared with 0.199±0.021, P<0.01). However, addition of the PI3K/AKT inhibitor LY294002 inhibited the ghrelin‐mediated enhancement in cell proliferation (0.227±0.042 compared with 0.199±0.021, P=0.15). At a concentration between 10^?8 and 10^?7 mol/l, ghrelin caused a significant increase in the number of migrated cells compared with the control group (126±9 compared with 98±7, P=0.02; 142±6 compared with 98±7, P<0.01), whereas no such change could be observed in the presence of 20 μmol/l of the PI3K/Akt inhibitor LY294002 (103±7 compared with 98±7, P=0.32). Ghrelin treatment significantly enhanced NO production in a dose‐dependent fashion compared with the untreated control group [(39.93±2.12) μmol/l compared with (30.27±2.71) μmol/l, P=0.02; (56.80±1.98) μmol/l compared with (30.27±2.71) μmol/l, P<0.01]. However, pretreatment with 20 μmol/l LY294002 inhibited the ghrelin‐stimulated increase in NO secretion [(28.97±1.64) μmol/l compared with (30.27±2.71) μmol/l, P=0.37]. In summary, we have found that ghrelin treatment promotes the proliferation, migration and NO secretion of CMECs through activation of PI3K/AKT signalling pathway.