Genetic and epigenetic alterations of lysophosphatidic Acid receptor genes in rodent tumors by experimental models

Lysophosphatidic acid (LPA) is a bioactive mediator and induces several biological effects, including cell proliferation, migration, morphogenesis and differentiation. LPA interacts with at least six G protein-coupled receptors (GPCRs), including LPA receptor-1 (LPA(1)), LPA(2), LPA(3), LPA(4), LPA(5) and LPA(6). These receptors show different biological functions through the binding of LPA, depending on the type of cells. In human malignancies, a high level of LPA production was found in plasma and ascites in ovarian cancer cases. Moreover, aberrant expression levels of LPA receptor genes were detected in some cancer cells. Therefore, it is suggested that LPA receptors may be involved in the pathogenesis of tumor cells as well as LPA per se. Recently, we have reported that alterations of LPA receptor genes also occur in rodent tumors. In this review, we summarize the recent evidence in the investigations of LPA receptor alterations in rodent tumors by experimental models.     

Altered expression of thioredoxin reductase-1 in dysplastic bile ducts and cholangiocarcinoma in a hamster model

Thioredoxin reductase 1 (TrxR) is a homodimeric selenoenzyme catalyzing thioredoxin (Trx) in an NADPH-dependent manner. With regard to carcinogenesis, these redox proteins have been implicated in cell proliferation, transformation and anti-apoptosis. In the present study, using a hamster cholangiocarcinoma (ChC) model, we evaluated the immunohistochemical expression pattern of TrxR in precancerous lesions and ChCs as well as in normal bile ducts. The goal of this study was to determine the potential role and importance of TrxR in cholangiocarcinogenesis. For the ChC model, we obtained liver tissue specimens with dysplastic bile ducts prior to the development of ChC 8 weeks after initiation of the experiment and ChC samples at 27 weeks. The immunohistochemical analysis showed diffuse cytoplasmic overexpression of TrxR in the dysplastic bile duct epithelial cells as well as in cholangiocarcinoma; this was comparable to the negative or weakly positive in normal and type 1 hyperplastic bile ducts. However, TrxR appeared to be considerably down-regulated in the ChCs when compared to the higher expression observed in the dysplastic bile ducts. Therefore, these results suggest that TrxR overexpression followed by down-regulation might be an important event in cholangiocarcinogenesis, especially at early stages including the cellular transformation of candidate bile ducts. Further studies are however required to determine whether TrxR may be a potential target molecule for chemoprevention against cholangiocarcinogenesis. In addition, the molecular mechanism as well as the importance of the loss of TrxR in the development of cholangiocarcinoma, following dysplastic transformation of bile duct cells, also remains to be clarified.     

Novel insertion mutation of ABCB1 gene in an ivermectin-sensitive Border Collie

P-glycoprotein (P-gp) is encoded by the ABCB1 gene and acts as an efflux pump for xenobiotics. In the Border Collie, a nonsense mutation caused by a 4-base pair deletion in the ABCB1 gene is associated with a premature stop to P-gp synthesis. In this study, we examined the full-length coding sequence of the ABCB1 gene in an ivermectin-sensitive Border Collie that lacked the aforementioned deletion mutation. The sequence was compared to the corresponding sequences of a wild-type Beagle and seven ivermectin-tolerant family members of the Border Collie. When compared to the wild-type Beagle sequence, that of the ivermectin-sensitive Border Collie was found to have one insertion mutation and eight single nucleotide polymorphisms (SNPs) in the coding sequence of the ABCB1 gene. While the eight SNPs were also found in the family members'' sequences, the insertion mutation was found only in the ivermectin-sensitive dog. These results suggest the possibility that the SNPs are species-specific features of the ABCB1 gene in Border Collies, and that the insertion mutation may be related to ivermectin intolerance.     

金黄地鼠Kcnq1基因的分子克隆与鉴定以及在多种器官组织中的表达差异分析

本研究对金黄地鼠Kcnq1基因进行分子克隆与鉴定以及在多种器官组织的表达差异进行分析,旨在研究Kcnq1基因在地鼠各组织器官中的功能.提取金黄地鼠心脏组织总RNA,根据大鼠Kcnq1的保守序列区域设计引物TK1,用RT-PCR化后的cDNA在T4连接酶的作用下与pMD18-T载体特异性连接,转化感受态大肠杆菌DH5a中,筛选重组子并酶切鉴定,将鉴定后的重组子进行DNA测序.提取心、肝、脾、肺、肾各组织总RNA,并反转,将各组织cDNA做荧光定量检测,检测各组织表达量的差异.结果,克隆出金黄地鼠Kcnq1基因477 bp的部分片段长度,推测出编码的159个氨基酸.与大鼠等物种Kcnq1基因比对,核苷酸和氨基酸序列均具有较高的同源性.荧光定量结果显示,Kcnq1在金黄地鼠心脏中表达量最高,在肺脏和肾脏中均有较高表达,在脾脏中低度表达,在肾脏中基本不表达.该研究结果为深入研究金黄地鼠KCNQ1基因功能奠定了基础.     

金黄地鼠Kcnq1基因的分子克隆与鉴定以及在多种器官组织中的表达差异分析

对金黄地鼠Kcnq1基因进行分子克隆与鉴定以及在多种器官组织的表达差异进行分析,旨在研究Kcnq1基因在地鼠各组织器官中的功能。提取金黄地鼠心脏组织总RNA,根据大鼠Kcnq1的保守序列区域设计引物TK1,用RT-PCR的方法从金黄地鼠心脏组织中扩增出Kcnq1cDNA片段,并且以心脏cDNA片段为模板,将纯化后的cDNA在T4连接酶的作用下与pMD18-T载体特异性连接,转化感受态大肠杆菌DH5α中,筛选重组子并酶切鉴定,将鉴定后的重组子进行DNA测序。提取心、肝、脾、肺、肾各组织总RNA,并反转录,将各组织cDNA做荧光定量检测,检测各组织表达量的差异。结果显示克隆出金黄地鼠Kcnq1基因477bp的部分片段长度,推测出编码的159个氨基酸。与大鼠等物种Kcnq1基因比对,核苷酸和氨基酸序列均具有较高的同源性。荧光定量结果显示,Kcnq1在金黄地鼠心脏中表达量最高,在肺脏和肾脏中均有较高表达,在脾脏中低度表达,在肾脏中基本不表达。该研究结果为深入研究金黄地鼠Kcnq1基因功能奠定了基础。     

Detection of APAF1 mutation in Holstein cows and mummified foetuses in Japanese dairy herds

Some of the highest genetic merit sires have been shown to harbour recessive mutations affecting fertility, which may spread rapidly in the population through AI. These disorders may result in abortion and decline in pregnancy per insemination in cows. This study was carried out on 240 Holstein‐Friesian cows and 15 mummified foetuses. Blood and tissue samples were collected from the cows and mummified foetuses, respectively, for DNA extraction. Allele‐specific PCR was designed for the detection of the cows and foetuses carrying the nonsense mutation (C/T) in apoptosis peptide activating factor 1 gene (APAF1). The mutant allele frequency of the APAF1 in carrier cows and mummified foetuses was calculated. Milk samples were taken from the carrier and non‐carrier cows for progesterone assay. The allele‐specific PCR reaction efficiently distinguished the C/T mutation in APAF1. Of 240 cows, seven cows (2.9%) were diagnosed to carry one copy of the mutant allele of APAF1. However, the carrier frequency was 33.3% in mummified foetuses (five of 15). The mutant allele frequency was 0.02 and 0.17 in the cows and mummified foetuses, respectively. Concentrations of progesterone did not differ between cows with APAF1 mutation and non‐carrier cows during 45 days post‐insemination. This study provided allele‐specific PCR for the detection of APAF1 mutation in cows. Moreover, it reports the carrier and mutant allele frequencies of APAF1 in dairy cows and mummified foetuses in Japan.     

A collision tumor consisting of granular cell tumor and adenocarcinoma in the uterus of an aged djungarian hamster

A neoplastic nodular lesion consisting of an admixture of granular cell tumor and adenocarcinoma was found in the uterus of a 26-month-old Djungarian hamster. Neoplastic cells of the uterine adenocarcinoma showed an epithelial nature in their growth patterns and by cytokeratin-immunopositive reaction, exhibiting nuclear pleomorphism. The granular cells had an abundant amount of fine granular eosinophilic cytoplasm and eccentric or central nuclei with no nuclear atypia; the granular structures were positive for periodic acid-Schiff with diastase resistance and were confirmed as lysosomes/autophagosomes by electron microscopy; immunohistochemically, the cells reacted to desmin, vimentin and α-smooth muscle actin and negatively for neurogenic, histiocyte/macrophage or epithelial markers, indicating smooth muscle origin. Because these tumors were generated from different cell origins, a diagnosis of collision tumor was made.     

Expression of the MDR1 gene and P-glycoprotein in canine mast cell tumor cell lines

Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity. P-glycoprotein (P-gp), which is encoded by the MDR1 gene, is considered to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. In the present report, we assessed the expression of MDR1 by RT-PCR in three canine mast cell tumor cell lines, TiMC, CoMS and LuMC, originating from a cutaneous tumor, an oral-mucosal tumor and a gastrointestinal tumor, respectively. P-gp expression was also examined by Western blot analysis, while the functional activity of P-gp was assessed by flowcytometric analysis of intracellular rhodamine-123 (Rhd-123) uptake. The results revealed that MDR1 gene and P-gp were both expressed in CoMS and LuMC cells, whereas neither was present in TiMC cells. In CoMS and LuMC cells, intracellular uptake of Rhd-123 increased in the presence of verapamil, a functional modulator of P-gp. In contrast, TiMC cells did not show any changes in the intracellular accumulation of Rhd-123 after the verapamil addition. These findings suggest that the expressions of MDR1 gene and P-gp probably contribute to cellular drug resistance in canine mast cell tumors.     

朝鲜鹌鹑黑素皮质素受体1基因多态性及其在不同羽色个体中的表达

通过混合样品DNA测序方法寻找黑素皮质素受体l（MC1R）基因的突变位点,采用Alu1-RFLP对突变位点在4种羽色（栗羽、黄羽、白羽、黑羽）鹌鹑群体中的基因分布进行了研究;利用qRT-PCR技术测定了MC1R基因在12日龄时4种羽色鹌鹑胚胎皮肤组织中的表达情况。结果表明,在鹌鹑MC1R基因上发现1个T/C突变位点,该位点没有导致编码蛋白氨基酸序列改变,A1u1-RFLP分析发现,该突变位点的不同基因型在4种羽色鹌鹑群体间的分布有显著差异（P〈0.05）。4种羽色鹌鹑皮肤组织中MC1R基因的表达量存在明显差异,栗羽鹌鹑皮肤组织中该基因的表达量明显高于黑羽鹌鹑皮肤中的表达量（栗羽〉黄羽〉白羽〉黑羽）。本试验没有发现导致日本鹌鹑黑羽突变的Glu92Lys突变位点,表明朝鲜鹌鹑的黑羽突变与报道的日本鹌鹑黑羽突变的机制不同,朝鲜鹌鹑的黑羽可能与其他基因的突变有关。     

NBT-PABA test to assess efficiency and kinetics of substituted proteolytic enzyme action in pancreatic duct ligated minipigs

The NBT-PABA test is an established method for diagnosis of pancreatic exocrine insufficiency. In the present study the NBT-PABA test was used to test and compare the efficacy of two multienzyme preparations (product A and B) differing in galenic preparation in minipigs in which pancreatic exocrine insufficiency (PEI) was induced by pancreatic duct ligation. Without enzyme substitution no distinct increase in PABA was found in blood after oral administration of NBT-PABA. Administration of both enzyme preparations led to a clear dose dependent rise in PABA-concentrations in blood. Interestingly, the two preparations showed different time curves of serum PABA concentration, indicating differences in the kinetic of proteolytic enzyme action. It is concluded that the NBT-PABA test can be a very useful test for indirectly evaluating proteolytic enzyme efficacy in vivo, and also gives information about the kinetics of enzyme action, not only the end-result of enzyme action (like digestibility trials which were used traditionally). A single test is performed in a few hours and there is no need for fistulated animals.     

Molecular cloning of canine Mcl-1 gene and its expression in tumor cell lines

The canine Mcl-1 gene was cloned and sequenced. Canine Mcl-1 clone was 2694 base pairs in length and encoded 350 amino acids. The predicted amino acid sequence was 87.7%, 77.1% and 75.7% homologous to predicted human, mouse and rat Mcl-1, respectively. RT-PCR analysis revealed that canine Mcl-1 mRNA was expressed in PBMCs (peripheral blood mononuclear cells), bone marrow cells, MDCK (Madin-Darby canine kidney) and GL-1 (canine B cell leukemia) whereas undetectable in CL-1 (canine T cell lymphoma) cell line.     

猪感染口蹄疫亚洲Ⅰ型病毒分离及其VP1基因的变异比较

对口蹄疫病毒的分子流行病学的研究通常是以VP1基因序列分析为依据的。本试验分离到1株猪源口蹄疫亚洲Ⅰ型毒株,对其主要抗原基因VP1进行了扩增和测序,并与国内外报道序列进行比对,发现与国内报道珠同源性在83.3%~86.4%之间,与国外报道株的同源性在81.4%~98.4%之间。比较发现本次分离株与国内外报道株的差异的较大,只有2株报道株与其同源性在90%以上。     

Novel genotyping assay for the nt230 (del4) ABCB1 gene mutation and its allele frequency in Border Collie dogs in Mexico

A 4-bp deletion in the ATP-binding cassette subfamily B member 1 (ABCB1) gene, also referred to as the multidrug resistance gene (MDR1), produces stop codons that cause premature termination of P-glycoprotein 1 (P-gp) synthesis. Dogs with the homozygous mutation do not express functional P-gp, which increases their sensitivity markedly to many common veterinary drugs. We detected the nt230 (del4) ABCB1 mutation in Border Collie dogs in western Mexico with a simple and affordable primer-introduced restriction analysis PCR (PIRA-PCR). PIRA-PCR clearly identified all genotypes in our sample of 104 dogs. Genotype frequencies were 0.952 (wild/wild), 0.029 (wild/mut) and 0.019 (mut/mut). Allele frequencies were 0.033 (mutant alleles) and 0.966 (wild-type alleles). In this small subset of the Mexican dog population, we found a higher prevalence of the nt230 (del4) MDR1/ABCB1 gene mutation than reported in other countries.     

Association between novel variants in BMPR1B gene and litter size in Mongolia and Ujimqin sheep breeds

Prolificacy is an important trait of animals, specifically for sheep. The Bone morphogenetic protein receptor 1B (BMPR1B) is a major gene affecting the litter size of many sheep breeds. The well-known FecB mutation (Q249R) was associated fully with the hyper prolific phenotype of Booroola Merino. However, the identification of variation in all exonic regions of BMPR1B was rare. In this study, we sequenced all exonic regions of BMPR1B gene of Mongolia sheep breed, and ten novel variants were detected by direct sequencing. Among them, the litter size of the Mongolia ewes with the CC genotype was significantly higher (0.34 additional lambs, p < .05) than those with the TT genotype of the g.29346567C>T single nucleotide polymorphism (SNP). The litter size of the Mongolia ewes with the TT genotype was significantly higher (0.19 additional lambs, p < .05 and .31 additional lambs, p < .01, respectively) than those with the GT and GG genotypes of the c.1470G>T SNP. The silent c.1470G>T mutation is predicted to increase the stability of the mRNA secondary structure through reducing minimum free energy and is predicted to change the mRNA secondary structure of BMPR1B. Our findings may give potentially useful genetic markers for increasing litter size in sheep.     

Prognostic and predictive significance of KIT protein expression and c‐kit gene mutation in canine cutaneous mast cell tumours: A consensus of the Oncology‐Pathology Working Group

One of the primary objectives of the Oncology‐Pathology Working Group (OPWG), a joint initiative of the Veterinary Cancer Society and the American College of Veterinary Pathologists, is for oncologists and pathologists to collaboratively generate consensus documents to standardize aspects of and provide guidelines for oncologic pathology. Consensus is established through critical review of peer‐reviewed literature relevant to a subgroup's particular focus. Subsequent acceptance and approval of the document by the OPWG membership at large establishes consensus. The intent of this publication is to help educate practitioners and pathologists on the value of diagnostics related to the KIT receptor tyrosine kinase for canine cutaneous mast cell tumours and to provide a guide for the use of these tests in veterinary medicine. This document represents the opinions of the OPWG and the authors and does not constitute a formal endorsement by the American College of Veterinary Pathologists or the Veterinary Cancer Society.     

Prevalence of the AMHR2 mutation in Miniature Schnauzers and genetic investigation of a Belgian Malinois with persistent Müllerian duct syndrome

Persistent Müllerian duct syndrome (PMDS) is a sex‐limited disorder in which males develop portions of the female reproductive tract. Important consequences of PMDS are cryptorchidism and its sequelae of infertility and increased risk of testicular cancer. Anti‐Müllerian hormone (AMH) and its receptor (AMHR2) induce the regression of the Müllerian ducts in male embryos. In Miniature Schnauzer dogs, the genetic basis has been identified as an autosomal recessive nonsense mutation in AMHR2, but the allele frequency of the mutation is unknown. Thus, the primary objective of this study was to estimate the prevalence of the AMHR2 mutation in North American Miniature Schnauzers, in order to ascertain the value of genetic testing in this breed. An additional objective was to determine whether mutations in AMH or AMHR2 were responsible for PMDS in a Belgian Malinois; this would aid development of a genetic test for the Belgian Malinois breed. Genomic DNA from 216 Miniature Schnauzers (including one known PMDS case) was genotyped for the AMHR2 mutation, and DNA from a single PMDS‐affected Belgian Malinois was sequenced for all coding exons of AMH and AMHR2. The Miniature Schnauzer cohort had an AMHR2 mutation allele frequency of 0.16 and a carrier genotypic frequency of 0.27. The genetic basis for PMDS in the Belgian Malinois was not determined, as no coding or splicing mutations were identified in either AMH or AMHR2. These findings support a benefit to AMHR2 mutation testing Miniature Schnauzers used for breeding or with cryptorchidism.     

Establishment and characterization of dengue virus type 2 nonstructural protein 1 specific T cell lines

Immunity against dengue viruses (DENV) infection may include cellular immune responses which involve in the immunopathology of DENV infection hosts. This study was to establish short-term dengue virus type 2 (DENV2) nonstructural protein 1 (NS1) specific T cells from splenocytes from BALB/c mice immunized with DENV2 NS1 in vitro, which may be used to identify immunopathologic mechanism of dengue. Nine DENV2 NS1 specific T cell lines were successfully established by using limiting dilution methods and maintained for 20 weeks by re-stimulated with DENV2 NS1, recombinant mouse IL-2 and antigen presenting cell weekly. Phenotypically, these cells were mainly composed of CD3⁺CD4⁺ T cells. The culture supernatants of these cells contained large amounts of TNF-α and IFN-γ. Vascular tissue pathological change could be found in the mice adoptive transferred with DENV2 NS1 specific T cells. The results indicate that DENV2 NS1 specific T cells could be established and maintained with syngeneic T cell growth factors in vitro. Meanwhile, DENV2 NS1 specific T cells might contribute to the immunopathology of vascular leakage of dengue.     

丝羽乌骨鸡腺苷单磷酸脱氨酶1(AMPD1)基因多态性及其与肌苷酸含量相关研究

以丝羽乌骨鸡为研究素材,萧山鸡、白耳鸡、北京油鸡、茶花鸡、隐性白羽肉鸡为对照组,研究肌苷酸合成代谢过程中主要的催化酶鸡腺苷单磷酸脱氨酶1(AMPD1)基因在6个鸡品种中AMFD1基因序列多样性,结果表明：在525bp的片段中共存在10个多态位点,其中120位的A→G、355位的A→G的碱基变化仅在肌苷酸含量较高的鸡品种如丝羽乌骨鸡、北京油鸡、茶花鸡出现,推测这2个位点与肌苷酸含量密切相关。     

半番鸭POU1F1基因序列的克隆与生物信息学分析

根据GenBank中公布的鸭POU1F1基因序列,设计了6对引物,以半番鸭血液基因组DNA为模板,采用PCR方法扩增出POU1F1基因完整的cDNA序列,全长2209bp。对该序列进行生物信息学分析,结果表明该基因编码335个氨基酸,具有POU-specific(POUs)和Homeobox结构域,与鸡、猪、牛、人和小鼠的POU1F1基因氨基酸序列分别具有96.3%、89.5%、89.2%、90.6%和87.5%的同源性。