Molecular cloning of feline hepatocyte growth factor (HGF) cDNA

Hepatocyte growth factor (HGF) is a pleiotropic cytokine responsible for regeneration, development and maintenance of various organs, and growth, invasion and metastasis of tumor cells. A full-length feline HGF cDNA was cloned and sequenced by RT-PCR from cat liver. Feline HGF consists of 728 amino acid and contains alpha- and beta-chains encoded in a single open reading frame. The predicted amino acid sequence of feline HGF showed 93.2, 93.3 and 93.3% homology with those of human, mouse and rat HGF, respectively. The putative proteolytic processing site, all cysteine residues, and four potential glycosylation sites are conserved in all species. Therefore, feline HGF is expected to have a similar three-dimensional structure to human, mouse and rat HGF.     

Molecular cloning of the canine c-Met/HGF receptor and its expression in normal and regenerated liver

The c-Met proto-oncogene is the receptor for hepatocyte growth factor (HGF), which is a member of the tyrosine kinase family. Activation of the HGF/c-Met signal pathway leads to cell proliferation, motility, regeneration, and morphogenesis. In this study, the complete nucleotide sequence of complementary DNA (cDNA) of canine c-Met was cloned, and its distribution was determined in tissues. The canine c-Met cDNA clone had an open reading frame of 4419 bp that encoded a putative polypeptide of 1383 amino acids. The c-Met mRNA was expressed in a variety of canine tissues including peripheral blood mononuclear cells (PBMC), bone marrow, liver, kidney, lung, stomach, uterus, testis, thymus, lymph node, small intestine, colon, adrenal gland, thyroid gland, heart, muscle, skin, pancreas, ovary, prostate, spleen, fat, cerebrum, and cerebellum. In addition, the c-Met mRNA expression in normal and regenerated liver was examined. The levels of the mRNA increased 2-fold in regenerated liver compared to that found in normal liver, indicating that c-Met is involved in various functions including remodeling of canine hepatocytes.     

Isolation and expression of five-amino-acid-deleted variant of feline hepatocyte growth factor (HGF) cDNA

Hepatocyte growth factor (HGF) is a pleiotropic cytokine that stimulates a wide array of cellular targets, including hepatocytes and other epithelial cells, melanocytes, endothelial and hematopoietic cells. We have cloned a different form of cDNA, with a deletion of 15 base pairs predicted to result in the loss of 5 amino acids from the first kringle domain. To investigate the biological activity, original and deleted variant of feline HGF cDNAs were transiently expressed in COS-7 cells. Both recombinant feline HGFs showed almost the same dose-response curves in the stimulation of the growth of BNL CL.2 cells (a mouse hepatocyte cell line) and scatter activity of Madin-Darby canine kidney (MDCK) cells. The findings reported here suggest that the deleted variant of feline HGF has almost the same biological activity as the original in terms of the proliferation and scatter activity.     

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in canine simple mammary gland adenocarcinomas

The expression of 5 markers associated with angiogenesis, proliferation, and apoptosis was studied in 26 canine simple mammary gland adenocarcinomas (SMGAs). The adenocarcinomas were graded histologically, and tissue sections were immunohistochemically stained for the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2), intra-tumor microvessel density, and tumor proliferation (PI) using antibodies against VEGF, VEGFR-2, von Willebrand factor, and Ki-67 antigen, respectively. Apoptotic indices (AI) were determined by an apoptosis assay. Markers VEGF and VEGFR-2 were detected in 96% and 100% of SMGAs, respectively. A high correlation between histologic grade and PI (r = 0.73), a moderate correlation between VEGF and histologic grade (r = 0.33), and between VEGF and PI (r = 0.42) were found. There was a significant difference in median PI among the 3 histologic grade groups (r < 0.05). Vascular endothelial growth factor may stimulate tumor cell proliferation through an autocrine loop, since VEGF and VEGFR-2 were expressed in most tumors.     

Bovine intelectins: cDNA sequencing and expression in the bovine intestine

Intelectins (Itlns) are lectins with potential roles in innate immunity, capable of binding bacteria via galactofuranose residues. Itlns also function as intestinal receptors for the antimicrobial glycoprotein lactoferrin (Lf). Since Lf binds strongly to enterohemorrhagic Escherichia coli O157:H7 (EHEC), we aimed to determine the expression of Lf receptor in terminal rectum, the site of predilection of EHEC in cattle. We sequenced two bovine intelectins (Itln1 and Itln2) and showed that both were expressed in abomasum and rectum, but expression appeared minimal in the jejunum. There was significantly higher expression of Itln2 in terminal rather than proximal rectum. Lactoferrin was expressed in all samples examined. Thus, we have demonstrated two novel bovine Itlns and shown that they are expressed along with Lf in the gastrointestinal tract, where they may interact with microbial pathogens.     

The expression of carbonic anhydrase in canine mammary gland and mammary gland tumor

Carbonic anhydrase (CA), a metallo-enzyme containing zinc, broadly distributes in mammalian tissues and participates in physiological regulation such as respiration, acid-base balance, ion transport, bone resorption, as well as the development of tumor by the reversible hydration of carbon dioxide. However the expression of CA in the tissue of mammary gland tumor was not documented. In this study we examine the histolocalization and gene expression of CA in both normal canine mammary gland tissue and mammary gland tumor by histochemical examination, and RT-PCR. Four mRNA expression of CA isoenzymes, such as CA II, IV, VI and IX were found under RT-PCR analysis and different band patterns were found between normal canine mammary tissue and canine mammary gland tumor tissue. CA II, IV, VI and IX gene mRNA expression were found in the normal mammary gland tissue, indicating CA II, IV, VI and IX are likely to be the essential enzymes to maintain the normal physiological condition of canine mammary gland tissue cells. However the expression of CA IV was not found in the tissue of malignant mammary gland tumor that may become the marker for the prognostic recognition of canine mammary gland tumor.     

Localization of fibroblast growth factor I (acid fibroblast growth factor) and its mRNA in the bovine mammary gland during mammogenesis, lactation and involution

Growth factors are involved in development and function of the mammary gland. The aim of this study was the localization of fibroblast growth factor 1 (FGF-1) and its mRNA in the bovine mammary gland during different developmental and functional stages. Mammary tissue was obtained from German Brown Swiss cows (n = 23) during defined stages of mammogenesis (before and during pregnancy), lactogenesis, peak lactation and involution. The distribution of FGF-1 mRNA was studied using non-radioactive in situ hybridization, the corresponding FGF-protein was analysed using immunohistochemistry [avidin-biotin peroxidase complex (ABC)-method]. A moderate to distinct staining for FGF-mRNA was found in the epithelium of ducts and developing alveoli during mammogenesis. Post-partum at the same cellular locations, a considerable amount of FGF-1 mRNA, was seen that decreased during lactation. Also during early involution clear staining for FGF-mRNA could still be observed. Immunoreactive FGF-1 was found in considerable concentration in the epithelium of the mammary gland in heifers. The staining intensity generally decreased somewhat during mammogenesis and lactation, but could be always clearly demonstrated in the secretory epithelial cells of alveoli and glandular ducts. Also during the first day after the end of milking, the epithelium displayed a moderate to distinct epithelial immunostaining. Notably, After 4 weeks of involution, in many alveoli a shedding of the FGF-1 positive luminal cell layer was found. In our localization studies, no strict correlation between FGF-1 mRNA and its corresponding protein was found. The various reasons for this finding are discussed.     

萨能奶山羊乳腺组织5-HTR1基因的克隆与序列分析

根据GenBank其他物种5-HT受体-1(5-HTR1)基因序列的同源序列对奶山羊5-HTR1基因设计1对克隆引物,通过奶山羊乳腺组织提取的总RNA进行RT-PCR,将PCR产物克隆、测序。结果显示,奶山羊5-HTR1基因与牛、鼠的同源性分别为97.3%和87.2%,推测的氨基酸序列信号肽为1～45aa,SapB结构域均为78～145aa,结构特征与牛、鼠的5-HT1结构特征相一致。克隆了奶山羊乳腺组织中5-HTR1基因,为进一步研究牛乳腺组织中5-HT受体基因的分布和提高其泌乳量方面提供理论基础。     

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.     

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue remodeling processes. The ability to separate these properties, using mutant IGFBPs, is currently under investigation.     

猪IL-6 cDNA的克隆及在大肠杆菌中的高效表达

运用RT—PCR方法,从经刀豆球蛋白A(ConA)诱导的猪外周血单核细胞(PMBcs)总RNA中扩增得到了猪IL-6(porcine IL-6,pIL-6)的cDNA,并将其克隆到pGEM—T载体上。DNA序列测定表明,克隆得到的pIL-6cDNA与国外报道的完全一致,包括终止密码子在内其编码区的长度为639bp,编码212个氨基酸残基的蛋白质。将pIL-6成熟肽段编码区亚克隆至pET-28(a)中,构建成原核表达质粒pETPIL6。该质粒的BL21(DE3)LysS转化菌在IPTG的诱导下可高效表达plL-6,表达量占菌体总蛋白的30．60%-38．35％。     

犬乳腺肿瘤组织病理学观察及腱糖蛋白C表达的检测

为了探讨腱糖蛋白C(TNC)表达与犬乳腺肿瘤生物学关系,对23例犬乳腺肿瘤临床病例进行病理学观察及分类,然后采用免疫组化SABC方法检测TNC蛋白在23例犬乳腺肿瘤和2例犬正常乳腺组织中的表达情况。结果显示:23例病例中,良性肿瘤有7例,占30.43%;恶性肿瘤有13例,占56.52%;增生病变有3例,占13.05%。在良性肿瘤中良性混合瘤最多,为3例,其余2例为导管内乳头状腺瘤,1例复杂腺瘤及1例纤维腺瘤。恶性肿瘤中,5例未分化癌,3例癌肉瘤,2例筛状癌,2例固体癌,1例纤维肉瘤,1例混合瘤引起的癌。TNC蛋白主要表达于细胞外基质。犬恶性乳腺肿瘤中,TNC蛋白阳性表达率为46.15%(6/13);良性乳腺肿瘤中,其阳性表达率为42.85%(3/7);乳腺增生病变和正常乳腺组织中,未见其表达。研究结果表明,TNC蛋白可以为犬乳腺肿瘤的鉴别诊断提供参考。     

Changes in oestrogen,progesterone and epidermal growth factor receptor concentrations and affinities during the oestrous cycle in the normal mammary gland and uterus of dogs

Changes in the concentrations and affinities of receptors for oestrogen (ER), progesterone (PR) and epidermal growth factor (EGF-R) were studied in mammary glands of healthy bitches with regard to age, the location in the mammary chain and the stage of the oestrous cycle. Uterus was used as the reference tissue for the evaluation of steroid receptors. Mammary and uterine samples from 7 healthy bitches were taken at five stages of the oestrous cycle in such a way that all the locations in the mammary chain were represented at each stage of the cycle (10 samples/dog). ER, PR and EGF-R were detected by biochemical assays using increasing concentrations of tritiated (steroids) or iodinated (EGF) ligands. A significant direct correlation was found between the ER and PR concentrations for mammary and uterine samples. No significant correlation was found between the steroid receptors and EGF-R concentrations. Mammary ER concentrations were significantly higher in bitches of 5 years of age or older than in younger ones; in posterior glands (4th and 5th pairs) than in anterior glands; and in the mid-luteal phase. Mammary PR did not vary significantly with age or location but was significantly lower in the early luteal phase than in other phases. A similar decrease in PR concentrations was observed in the uterus during the early luteal phase and uterine ER and PR concentrations were very low in the mid-luteal phase. Mammary EGF-R were not significantly higher in the early or mid-luteal phase than in pro-oestrus or anoestrus.The differences observed between the uterine and mammary steroid receptor concentrations during the oestrous cycle could be due to different mechanisms for regulating steroid receptor expression in the two tissues. Mammary EGF-R concentrations may be linked, as in other species, to cellular proliferation and/or to the serum progesterone concentrations.Abbreviations BSA bovine serum albumin - EGF epidermal growth factor - EGF-R receptor for epidermal growth factor - ER oestrogen receptor - K _d dissociation constant - LH luteinizing hormone - p probability of error - PR progesterone receptor     

Clinical significance of circulating vascular endothelial growth factor in dogs with mammary gland tumors

Increase in circulating vascular endothelial growth factor (VEGF) is suggested as a prognostic indicator in human patients with malignant tumors. The purpose of this study was to evaluate the clinical significance of circulating VEGF in dogs with mammary gland tumors (MGT). Both plasma and serum VEGF were significantly higher in dogs with MGT when compared with those in the healthy dogs. In dogs with MGT, the plasma and serum VEGF of the malignant group increased significantly compared with those of the benign group. Additionally, there was a significant difference between the plasma and serum VEGF in the groups with postoperative metastasis and no metastasis. Circulating VEGF is expected to be clinically available for the determination of prognosis in canine MGT.