The epidermal growth factor-seven transmembrane (EGF-TM7) receptor CD97 is required for neutrophil migration and host defense

The epidermal growth factor-seven transmembrane (EGF-TM7) family is a group of seven-span transmembrane receptors predominantly expressed by cells of the immune system. Family members CD97, EGF module-containing mucin-like receptor (EMR) 1, EMR2, EMR3, EMR4, and EGF-TM7-latrophilin-related protein are characterized by an extended extracellular region with a variable number of N-terminal EGF-like domains. EGF-TM7 receptors bind cellular ligands as demonstrated by the interaction of CD97 with decay accelerating factor (CD55) and dermatan sulfate. Investigating the effect of newly generated mAb on the migration of neutrophilic granulocytes, we here report for the first time in vivo data on the function of CD97. In dextran sulfate sodium-induced experimental colitis, we show that homing of adoptively transferred neutrophils to the colon was significantly delayed when cells were preincubated with CD97 mAb. The consequences of this defect in neutrophil migration for host defense are demonstrated in a murine model of Streptococcus pneumoniae-induced pneumonia. Mice treated with CD97 mAb to EGF domain 1 (1B2) and EGF domain 3 (1C5) displayed a reduced granulocytic inflammatory infiltrate at 20 h after inoculation. This was associated with a significantly enhanced outgrowth of bacteria in the lungs at 44 h and a strongly diminished survival. Together, these findings indicate an essential role for CD97 in the migration of neutrophils.     

Human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand on human macrophages and activated neutrophils

The epidermal growth factor (EGF)-TM7 subgroup of G-protein-coupled receptors is composed predominantly of leukocyte-restricted glycoproteins defined by their unique hybrid structure, in which extracellular EGF-like domains are coupled to a seven-span transmembrane moiety via a mucin-like stalk. The EGF-TM7 group comprises mouse F4/80, human EGF module-containing mucin-like hormone receptor (EMR) 1, human EMR2, and human and mouse CD97, the genes for which map to human chromosome 19p13 and the syntenic regions of the mouse genome. In this study we describe the cloning and characterization of EMR3, a novel human EGF-TM7 molecule, and show the existence of its cellular ligand. The EMR3 gene maps closely to the existing members of the EGF-TM7 family on human chromosome 19p13.1 and, in common with other EGF-TM7 genes, is capable of generating different protein isoforms through alternative splicing. Two alternative splice forms have been isolated: one encoding a 652-amino acid cell surface protein consisting of two EGF-like domains, a mucin stalk, and a putative G-protein-coupled receptor domain and the other encoding a truncated soluble form containing only two EGF-like domains. As with other members of the EGF-TM7 family, EMR3 mRNA displays a predominantly leukocyte-restricted expression pattern, with highest levels in neutrophils, monocytes, and macrophages. Through the use of soluble EMR3 multivalent probes we have shown the presence of a ligand at the surface of monocyte-derived macrophages and activated human neutrophils. These interactions suggest a potential role for EMR3 in myeloid-myeloid interactions during immune and inflammatory responses.     

The EGF-TM7 family: a postgenomic view

With the human and mouse genome projects now completed, the receptor repertoire of mammalian cells has finally been elucidated. The EGF-TM7 receptors are a family of class B seven-span transmembrane (TM7) receptors predominantly expressed by cells of the immune system. Within the large TM7 superfamily, the molecular structure and ligand-binding properties of EGF-TM7 receptors are unique. Derived from the processing of a single polypeptide, they are expressed at the cell surface as heterodimers consisting of a large extracellular region associated with a TM7 moiety. Through a variable number of N-terminal epidermal growth factor (EGF)-like domains, EGF-TM7 receptors interact with cellular ligands such as CD55 and chondroitin sulfate. Recent in vivo studies demonstrate a role of the EGF-TM7 receptor CD97 in leukocyte migration. The different number of EGF-TM7 genes in man compared with mice, the chimeric nature of EMR2 and the inactivation of human EMR4 point toward a still-evolving receptor family. Here we discuss the currently available information on this intriguing receptor family.     

The role of receptor oligomerization in modulating the expression and function of leukocyte adhesion-G protein-coupled receptors

The human leukocyte adhesion-G protein-coupled receptors (GPCRs), the epidermal growth factor (EGF)-TM7 proteins, are shown here to function as homo- and hetero-oligomers. Using cell surface cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer analysis of EMR2, an EGF-TM7 receptor predominantly expressed in myeloid cells, we demonstrate that it forms dimers in a reaction mediated exclusively by the TM7 moiety. We have also identified a naturally occurring but structurally unstable EMR2 splice variant that acts as a dominant negative modulator by dimerizing with the wild type receptor and down-regulating its expression. Additionally, heterodimerization between closely related EGF-TM7 members is shown to result in the modulation of expression and ligand binding properties of the receptors. These findings suggest that receptor homo- and hetero-oligomerization play a regulatory role in modulating the expression and function of leukocyte adhesion-GPCRs.     

Human EMR2, a novel EGF-TM7 molecule on chromosome 19p13.1, is closely related to CD97

The epidermal growth factor (EGF)-TM7 proteins [EMR1, (EGF-like molecule containing mucin-like hormone receptor 1) F4/80, and CD97] constitute a recently defined class B GPCR subfamily and are predominantly expressed on leukocytes. These molecules possess N-terminal EGF-like domains coupled to a seven-span transmembrane (7TM) moiety via a mucin-like spacer domain. Genomic mapping analysis has suggested a possible EGF-TM7 gene family on the human chromosome 19p13 region. In this study, a new member of the EGF-TM7 family, EMR2, which shares strikingly similar molecular characteristics with CD97, is described. In addition to mapping closely to CD97 on human chromosome 19p13.1, EMR2 contains a total of five tandem EGF-like domains and expresses similar protein isoforms consisting of various numbers of EGF-like domains as a result of alternative RNA splicing. Furthermore, EMR2 and CD97 exhibit highly homologous EGF-like domains and share identical gene organization, indicating that both genes are the products of a recent gene duplication event. The homologous EGF-like domains enable the identification of both EMR2 and CD97 by monoclonal antibodies (mAbs) raised against the first EGF-like domain of CD97, whereas mAbs directed against the extracellular spacer domain of CD97 are able to differentiate these two proteins. Both EMR2 and CD97 are highly expressed in immune tissues; however, unlike CD97, which is ubiquitously expressed in most cell types, EMR2 expression is restricted to monocytes/Mφ and granulocytes. EMR2 fails to interact with CD55, the cellular ligand for CD97, suggesting the possibility of a different cellular ligand(s). EMR2 may therefore have a unique function in cells of monocyte/Mφ and granulocyte lineages.     

EMR4, a novel epidermal growth factor (EGF)-TM7 molecule up-regulated in activated mouse macrophages,binds to a putative cellular ligand on B lymphoma cell line A20

A novel member of the EGF-TM7 family, mEMR4, was identified and characterized. The full-length mouse EMR4 cDNA encodes a predicted 689-amino acid protein containing two epidermal growth factor (EGF)-like modules, a mucin-like spacer domain, and a seven-transmembrane domain with a cytoplasmic tail. Genetic mapping established that mEMR4 is localized in the distal region of mouse chromosome 17 in close proximity to another EGF-TM7 gene, F4/80 (Emr1). Similar to F4/80, mEMR4 is predominantly expressed on resident macrophages. However, a much lower expression level was also detected in thioglycollate-elicited peritoneal neutrophils and bone marrow-derived dendritic cells. The expression of mEMR4 is up-regulated following macrophage activation in Biogel and thioglycollate-elicited peritoneal macrophages. Similarly, mEMR4 is over-expressed in TNF-alpha-treated resident peritoneal macrophages, whereas interleukin-4 and -10 dramatically reduce the expression. mEMR4 was found to undergo proteolytic processing within the extracellular stalk region resulting in two protein subunits associated noncovalently as a heterodimer. The proteolytic cleavage site was identified by N-terminal amino acid sequencing and located at the conserved GPCR (G protein-coupled receptor) proteolytic site in the extracellular region. Using multivalent biotinylated mEMR4-mFc fusion proteins as a probe, a putative cell surface ligand was identified on a B lymphoma cell line, A20, in a cell-binding assay. The mEMR4-ligand interaction is Ca2+-independent and is mediated predominantly by the second EGF-like module. mEMR4 is the first EGF-TM7 receptor known to mediate the cellular interaction between myeloid cells and B cells.     

Structural and functional characterization of a novel T cell receptor co-regulatory protein complex, CD97-CD55

CD97, the archetypal member of the EGF-TM7 protein family, is constitutively expressed on granulocytes and monocytes and rapidly up-regulated on T and B cells following activation. The key isoform of CD97 expressed on leukocytes binds the complement regulatory protein CD55 (also termed decay-accelerating factor). CD97 has been shown recently to mediate co-stimulation of T cells via CD55. Here, we demonstrate that blocking the interaction between CD55 on monocytes and CD97 on T cells leads to inhibition of proliferation and interferon-gamma secretion. This implies that bidirectional interactions between CD97 and CD55 are involved in T cell regulation. Structural studies presented here reveal the molecular basis for this activity. We have solved the structure of EMR2, a very close homolog of CD97, using x-ray crystallography. NMR-based chemical shift mapping of the EMR2-CD55 interaction has allowed us to generate a model for the CD97-CD55 complex. The structure of the complex reveals that the T cell and complement regulatory activities of CD55 occur on opposite faces of the molecule. This suggests that CD55 might simultaneously regulate both the innate and adaptive immune responses, and we have shown that CD55 can still regulate complement when bound to CD97.     

Molecular analysis of the epidermal growth factor-like short consensus repeat domain-mediated protein-protein interactions: dissection of the CD97-CD55 complex

Epidermal growth factor-like (EGF) and short consensus repeat (SCR) domains are commonly found in cell surface and soluble proteins that mediate specific protein-protein recognition events. Unlike the immunoglobulin (Ig) superfamily, very little is known about the general properties of intermolecular interactions encoded by these common modules, and in particular, how specificity of binding is achieved. We have dissected the binding of CD97 (a member of the EGF-TM7 family) to the complement regulator CD55, two cell surface modular proteins that contain EGF and SCR domains, respectively. We demonstrate that the interaction is mediated solely by these domains and is characterized by a low affinity (86 microm) and rapid off-rate (at least 0.6 s(-1)). The interaction is Ca(2+) -dependent but is unaffected by glycosylation of the EGF domains. Using biotinylated multimerized peptides in cell binding assays and surface plasmon resonance, we show that a CD97-related EGF-TM7 molecule (termed EMR2), differing by only three amino acids within the EGF domains, binds CD55 with a K(D) at least an order of magnitude weaker than that of CD97. These results suggest that low affinity cell-cell interactions may be a general feature of highly expressed cell surface proteins and that specificity of SCR-EGF binding can be finely tuned by a small number of amino acid changes on the EGF module surface.     

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith–Lemli–Opitz syndrome

Smith–Lemli–Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3β-hydroxysterol-Δ⁷-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4β-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d₇-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25 μmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.     

7-Dehydrocholesterol-derived oxysterols and retinal degeneration in a rat model of Smith-Lemli-Opitz syndrome

Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease characterized by markedly elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in tissues and fluids of affected individuals, due to defective 3β-hydroxysterol-Δ(7)-reductase (Dhcr7). Treatment of Sprague Dawley rats with AY9944 (an inhibitor of Dhcr7) leads to similar biochemical features as observed in SLOS. Eighteen oxysterols previously have been identified as oxidation products of 7-DHC (most of them distinct from cholesterol (Chol)-derived oxysterols) in solution, in cells, and in brains obtained from Dhcr7-KO mice and AY9944-treated rats, formed either via free radical oxidation (peroxidation) or P450-catalyzed enzymatic oxidation. We report here the identification of five 7-DHC-derived oxysterols, including 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), 4α- and 4β-hydroxy-7-DHC, 24-hydroxy-7-DHC and 7-ketocholesterol (7-kChol, an oxysterol that is normally derived from Chol), in the retinas of AY9944-treated rats by comparing the retention times and mass spectrometric characteristics with corresponding synthetic standards in HPLC-MS analysis. Levels of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol were quantified using d(7)-DHCEO as an internal standard. Among the five oxysterols identified, only 7-kChol was observed in retinas of control rats, but the levels of 7-kChol in retinas of AY9944-rats were 30-fold higher. Intravitreal injection of 7-kChol (0.25μmol) into a normal rat eye induced panretinal degeneration within one week; by comparison, contralateral (control) eyes injected with vehicle alone exhibited normal histology. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the retinal degeneration associated with the SLOS rat model and in SLOS patients.     

Novel oxysterols observed in tissues and fluids of AY9944-treated rats: a model for Smith-Lemli-Opitz syndrome

Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ(7)-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d(7)-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Genome Sequence of Aureobasidium pullulans AY4, an Emerging Opportunistic Fungal Pathogen with Diverse Biotechnological Potential

Aureobasidium pullulans AY4 is an opportunistic pathogen that was isolated from the skin of an immunocompromised patient. We present here the draft genome of strain AY4, which reveals an abundance of genes relevant to bioindustrial applications, including biocontrol and biodegradation. Putative genes responsible for the pathogenicity of strain AY4 were also identified.     

ETL, a novel seven-transmembrane receptor that is developmentally regulated in the heart. ETL is a member of the secretin family and belongs to the epidermal growth factor-seven-transmembrane subfamily

Using differential display of rat fetal and postnatal cardiomyocytes, we have identified a novel seven-transmembrane receptor, ETL. The cDNA-predicted amino acid sequence of ETL indicated that it encodes a 738-aa protein composed of a large extracellular domain with epidermal growth factor (EGF)-like repeats, a seven-transmembrane domain, and a short cytoplasmic tail. ETL belongs to the secretin family of G-protein-coupled peptide hormone receptors and the EGF-TM7 subfamily of receptors. The latter are characterized by a variable number of extracellular EGF and cell surface domains and conserved seven transmembrane-spanning regions. ETL mRNA expression is up-regulated in the adult rat and human heart. In situ hybridization analyses revealed expression in rat cardiomyocytes and abundant expression in vascular and bronchiolar smooth muscle cells. In COS-7 cells transfected with Myc-tagged rat ETL, rat ETL exists as a stable dimer and undergoes endoproteolytic cleavage of the extracellular domain. The proteolytic activity can be abolished by a specific mutation, T455A, in this domain. In transfected mammalian cells, ETL is associated with cell membranes and is also observed in cytoplasmic vesicles. ETL is the first seven-transmembrane receptor containing EGF-like repeats that is developmentally regulated in the heart.     

The effect of the hypocholesteremic drug, AY 9944 on the synthesis of bile salts in rat liver

1. The compound trans-1,4 bis-(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride (AY9944) blocks cholesterol synthesis at a late stage. This leads to a decrease in cholesterol and accumulation of cholesta-5,7-diene-3-beta-ol (7-dehydrocholesterol) in tissues and plasma. 2. The effect of AY9944 on bile salt synthesis in rat liver was studied. The synthesis of conjugated cholic and chenodeoxycholic acids was measured in hepatocytes isolated from rats 2 h, 24 h and 48 h after administration of a single oral dose of AY9944. Production of the two bile salts was inhibited by 70-80% in hepatocytes from AY9944-treated as compared to untreated animals. 3. When AY9944 was added to the incubation medium in vitro of hepatocytes prepared from untreated rats the synthesis of conjugated cholic and chenodeoxycholic acids was not inhibited during the first hour of incubation, probably because of the presence of endogenous cholesterol. However when hepatocytes from untreated rats were incubated with AY9944 for periods of 2 h or longer, bile salt production was decreased markedly. 4. Bile salt synthesis is stimulated when rats are subjected to total biliary drainage for 24 h. The effect of AY9944 on this stimulation was studied. The content of conjugated cholic and chenodeoxycholic acid in the bile was measured as an indicator of bile salt synthesis. 5. In control animals the rate of secretion of biliary bile salts began to increase after about 24 h of total biliary drainage and reached a maximum after approximately 36 h. A single oral dose of AY9944 given 2 h after the start of total biliary drainage delayed and reduced this response. 6. The results show that inhibition of cholesterol synthesis by AY9944 resulting in the replacement of cholesterol by 7-dehydrocholesterol decreases but does not completely prevent bile salt synthesis.     

Regulation of bile acid synthesis. V. Inhibition of conversion of 7-dehydrocholesterol to cholesterol is associated with down-regulation of cholesterol 7 alpha-hydroxylase activity and inhibition of bile acid synthesis.

In the chronic bile fistula rat, the administration of a bolus dose of mevinolinic acid, an inhibitor of HMG-CoA reductase, was followed by rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and a decrease in bile acid synthesis. These observations suggested that either newly synthesized cholesterol or some other metabolite of mevalonate may be involved in the regulation of bile acid synthesis. In order to distinguish between these two alternatives, we carried out experiments in which cholesterol synthesis was blocked by AY9944, a compound that inhibits the conversion of 7-dehydrocholesterol to cholesterol, a last step in the cholesterol biosynthesis pathway. Rats underwent biliary diversion for 72 h at which time they were given intravenously either a bolus dose of AY9944 (1 mg/kg) or control vehicle. At 0 (pre-treatment control), 0.5, 1.5, and 3 h post bolus, livers were harvested and specific activities of cholesterol 7 alpha-hydroxylase were determined. At 1.5, 3, and 6 h post bolus, AY9944 inhibited bile acid synthesis by 19 +/- 6%, 40 +/- 4%, and 41 +/- 6%, respectively, as compared to pretreatment baseline. Cholesterol 7 alpha-hydroxylase activity determined at 0.5, 1.5, and 3 h was decreased by 44 +/- 6%, 44 +/- 2%, and 36 +/- 2%, respectively, as compared to the control value. In in vitro experiments using microsomes from livers of control bile fistula rats, the addition of AY9944 (up to 100 microM) failed to inhibit cholesterol 7 alpha-hydroxylase activity. The results of this study demonstrate that, in the chronic bile fistula rat, acute inhibition of cholesterol synthesis at either early or late steps leads to a rapid down-regulation of cholesterol 7 alpha-hydroxylase activity and decrease in bile acid synthesis.     

AY9944 A-7 promotes meiotic resumption and preimplantation development of prepubertal sheep oocytes maturing in vitro

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. FF-MAS is converted to testis meiosis-activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this study was to determine the effects of AY9944 A-7 supplementation to oocyte maturation media on prepubertal sheep oocyte meiotic resumption and subsequent preimplantation development of embryos. Prepubertal sheep oocytes isolated at the germinal vesicle stage from their follicles were cultured with 0, 10, 20, 30, and 40 μM AY9944 A-7 for 24 hours in media with or without a meiotic inhibitor hypoxanthine (Hx, 4 mM). The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. After maturation for 24 hours, oocytes with PBI were inseminated in vitro, and the percentages developing to the two-cell stage and blastocyst stage were measured as indicators of early embryonic developmental competence. AY9944 A-7 induced maturation of sheep cumulus-oocyte complexes with optimal concentrations of 10 and 20 μM both in Hx-inhibited meiotic maturation and spontaneous maturation, whereas AY9944 A-7 with any concentrations had no significant effect on that of denuded oocytes and split cumulus-oocyte complexes. Furthermore, maturing oocytes treated with either 10 or 20 μM AY9944 A-7 dramatically increased the percentages of ovine embryos developing to the two-cell stage and blastocyst stage. Higher concentrations of AY9944 A-7, 30 and 40 μM, were detrimental to oocytes and led to their degeneration. The present findings indicated for the first time that AY9944 A-7 was not only able to promote meiotic maturation, both Hx-inhibited and spontaneous, but also enhanced preimplantation developmental competence of prepubertal sheep oocytes maturing in vitro.     

绵羊钙调蛋白调节基因Calponin h1 的分子克隆

在一项研究中我们发现雌激素体在胚胎发育后期对绵羊子宫平滑肌Calponin (CaP) 基因的活动有明显上调作用,而CaP一直被作为观察其他基因表达水平变化的基准参照基因（Reference Gene）。迄今为止, 绵羊CaP尚未完整克隆,为进一步了解其结构和功能,根据人、小鼠和家猪的同源保守区序列设计锚定寡核苷酸引物,通过5′-RACE及3′-RACE方法克隆了绵羊子宫平滑肌组织全长CaP h1 cDNA (GenBank登录号： AY327118), 在cDNA序列的基础上, 又通过PCR-SSP方法获得了CaP h1基因除内含子1、2之外的其余4个内含子全部序列 （GenBank登录号分别为：AY771807,AY771808, AY771809, AY771810） 。DNA序列测定和分析表明,绵羊子宫平滑肌CaP h1 cDNA全长1499bp, 编码297个氨基酸,5′-UTR及3′-UTR分别为79bp和529bp。CaP h1基因组DNA的克隆和序列分析表明,绵羊CaP全长约8kb,由 7个外显子和6个内含子组成。 同源序列比较发现,该基因外显子在不同物种间相对保守;与人类、野猪、小鼠、大鼠和鸡Calponin mRNA同源性分别为88％、92％、81％、79％和81％,但不同物种间内含子存在较大差异(>50%)。本研究填补了绵羊CaP基因分子克隆的空白,为进一步研究该基因的功能及子宫平滑肌收缩的调节机理奠定了基础。