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Here, we describe the establishment of mutant‐specific polymerase chain reaction (PCR) for detection of a c‐KIT c.1430G>T mutation in feline mast cell tumours. Several mutations in feline c‐KIT have been identified, with the c.1430G>T mutation accounting for a significant portion of feline mast cell tumour mutations. The c.1430G>T mutation in c‐KIT exon 9 was detected in 15.7% (11 of 70) of samples by mutant‐specific PCR but in only 7.1% (5 of 70) by PCR–restriction fragment length polymorphism (RFLP) in the genomic DNA isolated from 70 formalin‐fixed paraffin‐embedded sections or cells collected by fine needle aspiration. Mutant‐specific PCR showed remarkably higher detection rate than did PCR–RFLP. DNA sequence analysis did not always yield identical results to those of mutant‐specific PCR, suggesting heterogeneity of tumour cells. Mutant‐specific PCR is a valid and efficient screening tool for detection of the c‐KIT c.1430G>T point mutation in feline mast cell tumours compared with PCR–RFLP and sequencing analysis. 相似文献
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Byung‐Joon Seung Seung‐Hee Cho Soo‐Hyeon Kim Min‐Kyung Bae Ha‐Young Lim Jung‐Hyang Sur 《Veterinary and comparative oncology》2021,19(1):132-139
Cutaneous mast cell tumours (MCTs) are the most frequent malignant skin tumours in dogs. Mutations in the c‐KIT proto‐oncogene are correlated with the pathogenesis and aggressiveness of MCTs. To date, studies have focused on c‐KIT mutations and KIT protein localization, with a general lack of mRNA‐level analyses. In this study, c‐KIT mRNA expression was investigated in canine MCTs by RNA in situ hybridization (RNA‐ISH). Furthermore, we evaluated associations between c‐KIT mRNA expression and the histological grade, KIT immunohistochemical staining pattern and other clinicopathological parameters. c‐KIT mRNA expression was observed in all MCT samples, appearing as clusters of dots in the cytoplasm of neoplastic cells. A significant correlation was detected between c‐KIT mRNA expression (quantified according to the H‐score and the percentage of positive cells) and the histological grade (determined using two‐and three‐tier grading systems; P < .05). We also found a significant positive correlation (all P < .05) between c‐KIT mRNA expression and the proliferation indices (mitotic index, Ki‐67, and Ag67). However, no significant associations with c‐KIT expression from RNA‐ISH were found with respect to different KIT staining patterns. Overall, these results demonstrate that c‐KIT mRNA expression might be an additional tool for measuring the c‐KIT status in canine cutaneous MCTs and could serve as a potential prognostic factor. Further studies should evaluate the prognostic significance of c‐KIT mRNA expression in a large and uniform cohort of canine MCTs. 相似文献
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Sena Kurita Ryo Miyamoto Hiroyuki Tani Masato Kobayashi Takashi Sasaki Kyoichi Tamura Makoto Bonkobara 《Journal of veterinary pharmacology and therapeutics》2019,42(6):673-681
One of the potential mechanisms underlying acquired resistance to toceranib in canine mast cell tumor (MCT) is the emergence of a secondary mutation in the KIT gene. Here, genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib were investigated in the toceranib‐susceptible canine MCT cell line VI‐MC, which carries a KIT‐activating mutation resulting in a predicted p.(Asn508Ile) amino acid change in the receptor tyrosine kinase protein KIT. Two sublines were cloned from VI‐MC and toceranib‐resistant sublines then were established by continuous exposure to toceranib. The mutation status of KIT in parental VI‐MC and its sublines was investigated using next‐generation sequencing (NGS). Additionally, effects of secondary mutations on toceranib sensitivity in p.(Asn508Ile)‐mutant KIT were examined. KIT secondary mutations, including those encoding p.(Asn679Lys)‐, p.(Asp819Val)‐, and p.(Asp819Gly)‐mutant KIT, that confer toceranib insensitivity to p.(Asn508Ile)‐mutant KIT emerged only in toceranib‐resistant VI‐MCs. These mutations were not detected by NGS in the parental VI‐MC line or in the toceranib‐naive cloned VI‐MCs, although the parental line and sublines exhibited genetic heterogeneity in KIT that may have been caused by genetic evolution during clonal expansion. VI‐MC clones with these secondary mutations in KIT appear to have arisen from subclones during treatment with toceranib rather than being pre‐existing. However, further study using a higher resolution technique will be needed to confirm the developmental mechanism of KIT secondary mutation in canine MCT cells with acquired resistance to toceranib. 相似文献
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Santiago Peralta Angela L. McCleary‐Wheeler Gerald E. Duhamel Kristiina Heikinheimo Jennifer K. Grenier 《Veterinary and comparative oncology》2019,17(3):439-445
Ameloblastoma is a locally aggressive odontogenic tumour that occurs in humans and dogs. Most ameloblastomas (AM) in humans harbour mutually‐exclusive driving mutations in BRAF, HRAS, KRAS, NRAS or FGFR2 that activate MAPK signalling, and in SMO that activates Hedgehog signalling. The remarkable clinical and histological similarities between canine acanthomatous ameloblastoma (CAA) and AM suggest they may harbour similar driving mutations. In this study, aimed at characterizing the mutational status of SMO, BRAF, HRAS, KRAS, NRAS and FGFR2 in CAA, we used RNA sequencing, Sanger sequencing and restriction fragment length polymorphism assays to demonstrate that 94% of CAA (n = 16) harbour a somatic HRAS p.Q61R mutation. The similarities in MAPK‐activating mutational profiles between CAA and AM implicate conserved molecular mechanisms of tumorigenesis, thus, qualifying the dog as a potentially useful model of disease. Given the relevance of RAS mutations in the pathogenesis of odontogenic tumours and other types of cancer, the results of this study are of comparative, translational, and veterinary value. 相似文献
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JI Yun HJ Park MH Park MS Kim JH Choi E Lee SP Gong JM Lim ST Lee 《Reproduction in domestic animals》2014,49(5):705-710
Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats. 相似文献
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Characterization of key genes of the renin–angiotensin system in mature feline adipocytes and during in vitro adipogenesis 
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下载免费PDF全文 J. Riedel B. Badewien‐Rentzsch B. Kohn L. Hoeke R. Einspanier 《Journal of animal physiology and animal nutrition》2016,100(6):1139-1148
Obesity is a growing health problem in humans as well as companion animals. In the development and progression of obesity‐associated diseases, the members of the renin–angiotensin system (RAS) are proposed to be involved. Particularly, the prevalence of type 2 diabetes mellitus in cats has increased enormously which is often been linked to obesity as well as to RAS. So far, reports about the expression of a local RAS in cat adipocytes are missing. Therefore, we investigated the mRNA expression of various RAS genes as well as the adipocyte marker genes adiponectin, leptin and PPAR‐γ in feline adipocytes using quantitative PCR. To characterize the gene expression during adipogenesis, feline pre‐adipocytes were differentiated into adipocytes in a primary cell culture and the expression of RAS key genes measured. All major RAS components were expressed in feline cells, but obvious differences in the expression between pre‐adipocytes and the various differentiation stages were found. Interestingly, the two enzymes ACE and ACE2 showed an opposite expression course. In addition to the in vitro experiments, mature adipocytes were isolated from subcutaneous and visceral adipose tissue. Significant differences between both fat depots were found for ACE as well as AT1 receptor with greater expression in subcutaneous than in visceral adipocytes. Visceral adipocytes had significantly higher adiponectin and PPAR‐γ mRNA level compared to the subcutaneous fat cells. Concerning the nutritional status, a significant lower expression of ACE2 was measured in subcutaneous adipocytes of overweight cats. In summary, the results show the existence of a potentially functional local RAS in feline adipose tissue which is differentially regulated during adipogenesis and dependent on the fat tissue depot and nutritional status. These findings are relevant for understanding the development of obesity‐associated diseases in cats such as diabetes mellitus. 相似文献
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Objective To investigate the prevalence of feline calicivirus (FCV) infection in relation to ocular surface lesions in cats with upper respiratory tract diseases (URTD). Animals studied Ninety‐nine cats with ocular surface infection and symptoms or recent history of URTD were examined at various rescue shelters and hospitals. Procedure A complete general and ophthalmic examination was performed including Schirmer tear test, slit‐lamp biomicroscopy, fluorescein and lissamine green staining. Clinical and ocular symptoms were scored and recorded. Conjunctival samples were collected using a cytobrush, and nucleic acid extraction using RT‐PCR was carried out to analyze for the presence of various infectious agents. Results RT‐PCR detected either FCV, feline herpes virus type 1 (FHV‐1), Chlamydophila felis or Mycoplasma spp. in 63/99 samples. 30/63 samples were positive for FCV, 23/63 for C. felis, 21/63 for Mycoplasma spp., and 16/63 for FHV‐1. Out of the 30 FCV‐positive samples, 11 were positive only for FCV and in 19 samples FCV was seen in combination with other agents. FCV infection was highest in animals examined at the rescue centers and in the age group of 0–2 months. Erosive conjunctivitis was an important ocular finding. Oral ulcers were detected in all FCV‐infected cats. Conclusion Results indicate that FCV is highly prevalent in cats with URTD either as a sole infectious agent or in combination with other pathogens and therefore is a potential cause for ocular surface lesions during the URTD. 相似文献
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Enterococcus faecalis is a major cause of nosocomial infections in humans and has been linked to severe extra‐intestinal infections in poultry. A zoonotic potential has been suggested and the aim of the present study was to investigate similarities in virulence gene profiles of E. faecalis originating from infections in humans and poultry respectively. A total of 106 isolates of E. faecalis [26 human clinical isolates, 60 poultry clinical isolates (including two small‐colony variants (SCVs) and 20 poultry cloacal isolates] were investigated for presence of seven virulence‐associated genes: ace, asa1, cylA, efaA, EF0591, esp and gelE. For each gene, the PCR‐amplification product was sequenced from one isolate in each group to explore intragenic variations between genes of human and poultry origin. Haemolytic and protease activities were assessed and isolates were assigned a sequence type (ST). Three of the seven genes investigated (ace, efaA and gelE) were present in all isolates. The asa1 was detected in 63/80 and 13/26 isolates of poultry and human origin respectively. For cylA, the numbers were 46/80 and 14/26 respectively. Among poultry isolates, esp and EF0591 were the least frequently observed genes (1/80 and 20/80 respectively); the prevalences among human isolates were 1/26 and 18/26 respectively. A high degree of similarity between genes in human and poultry isolates were confirmed by sequencing of amplification products. None of the cylA‐positive isolates demonstrated haemolytic activity, while the phenotypic expression of gelatinase varied. The ST16 was the only ST shared by human and poultry isolates. The SCV isolates did not show a unique virulence profile or phylogeny. In conclusion, regardless of the distinct phylogenetic background of most E. faecalis isolates of human and poultry origin, we found major similarities in virulence gene profile and gene sequences in isolates from the two sources, supporting the zoonotic risk associated with this organism. 相似文献
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L. Fésüs A. Zsolnai & I. Komlósi 《Zeitschrift für Tierzüchtung und Züchtungsbiologie》2005,122(2):127-130
Two F2 generations of an intercross between Hampshire boars and Hungarian Large White sows were produced to estimate the effects of the porcine KIT genotypes (II, Ii and ii) on quantitative and qualitative haematological indices, on piglet birth weight and growth performance until weaning. Piglets carrying the I allele had significantly fewer lymphocytes (p = 0.041) than the ii homozygotes, heterozygotes had measures between the two homozygotes. KIT genotypes did not influence white blood cells, red blood cells, haemoglobin and haematocrite. II genotype piglets were significantly lighter at birth than the ones carrying the recessive i allele, the effect of KIT genotypes on gain until weaning was not significant, but II piglets tended to gain less. The results of this study support the hypothesis of M. Johansson, H. Ellegren, L. Marklund, U. Gustavsson, E. Ringmar‐Cederberg, K. Andersson, I. Edfors‐Lilja and L. Andersson [(1992) Genomics, 14 , 965] that the pleiotrophic effect of the porcine KIT mutations on haematopoietic cells must be mild. 相似文献
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S. Sabattini G. Barzon M. Giantin R. M. Lopparelli M. Dacasto D. Prata G. Bettini 《Veterinary and comparative oncology》2017,15(3):1051-1061
This study investigated Kit receptor dysregulations (cytoplasmic immunohistochemical expression and/or c‐KIT mutations) in cats affected with splenic mast cell tumours. Twenty‐two cats were included. Median survival time was 780 days (range: 1–1219). An exclusive splenic involvement was significantly (P = 0.042) associated with longer survival (807 versus 120 days). Eighteen tumours (85.7%) showed Kit cytoplasmic expression (Kit pattern 2, 3). Mutation analysis was successful in 20 cases. Fourteen missense mutations were detected in 13 out of 20 tumours (65%). Eleven (78.6%) were located in exon 8, and three (21.6%) in exon 9. No mutations were detected in exons 11 and 17. Seven mutations corresponded to the same internal tandem duplication in exon 8 (c.1245_1256dup). Although the association between Kit cytoplasmic expression and mutations was significant, immunohistochemistry cannot be considered a surrogate marker for mutation analysis. No correlation was observed between c‐Kit mutations and tumour differentiation, mitotic activity or survival. 相似文献
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Xiaodong Zhang Liang Zhang Jinnan Shang Qiangqiang Tao Mi Tian Yingchun Ma Yiliang Xu Yueyun Ding Rong Zhou Kui Li Zongjun Yin 《Reproduction in domestic animals》2019,54(7):1018-1025
The aim of this study was to explore the expression difference of miRNAs and mRNAs between the follicular phase (FP) and luteal phase (LP) in porcine ovaries and provide a theoretical basis for the research on mammalian reproductive regulation. RNA‐Seq and miRNA‐Seq were used to identify differentially expressed genes (DEGs) and miRNAs (DEMs) between the FP and LP in ovaries of six sows (3‐year‐old Yorkshire pigs with similar weights and same parities). Bioinformatic analysis was used to screen potential genes and miRNAs related to porcine ovarian function. Real‐time qualitative PCR was used to validate the sequencing results. RNA‐Seq results showed that 3,078 genes were up‐regulated, and 1,444 genes were down‐regulated in the LP compared with the FP, and DEGs were significantly enriched in 242 Gene Ontology (GO) terms and 33 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. miRNA‐Seq identified 112 DEMs, of which 25 were up‐regulated and 87 were down‐regulated in the LP compared with the FP. We obtained 186 intersection genes (IGs) between the 4,522 DEGs and 2,444 target genes predicted from the 112 DEMs. After constructing a miRNA‐gene‐pathway network, we identified key miRNAs and genes including miR‐17‐3p, miR‐214, miR‐221‐5p, miR‐125b, FGF1, YWHAG, YWHAZ, FDFT1 and DHCR24, which are enriched in Hippo and PI3K‐Akt signalling pathways, and various metabolic pathways. These results indicate that these key genes and miRNAs may play important roles in the developmental transition from FP to LP in porcine ovaries and represent candidate targets for further study. 相似文献
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Characterization of HOX gene expression in canine mammary tumour cell lines from spontaneous tumours 下载免费PDF全文
下载免费PDF全文 P. DeInnocentes E. C. Graff F. M. Lutful Kabir R. Curtis Bird 《Veterinary and comparative oncology》2015,13(3):322-336
Spatial/temporal controls of development are regulated by the homeotic (HOX) gene complex and require integration with oncogenes and tumour suppressors regulating cell cycle exit. Spontaneously derived neoplastic canine mammary carcinoma cell models were investigated to determine if HOX expression profiles were associated with neoplasia as HOX genes promote neoplastic potential in human cancers. Comparative assessment of human and canine breast cancer expression profiles revealed remarkable similarity for all four paralogous HOX gene clusters and several unlinked HOX genes. Five canine HOX genes were overexpressed with expression profiles consistent with oncogene‐like character (HOXA1, HOXA13, HOXD4, HOXD9 and SIX1) and three HOX genes with underexpressed profiles (HOXA11, HOXC8 and HOXC9) were also identified as was an apparent nonsense mutation in HOXC6. This data, as well as a comparative analysis of similar data from human breast cancers suggested expression of selected HOX genes in canine mammary carcinoma could be contributing to the neoplastic phenotype. 相似文献
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There are three pigments that affect the color of an eggshell: protoporphyrin, biliverdin and biliverdin‐zinc chelate. Protoporphyrin is the main pigment in brown and light‐brown eggshells, whereas very little protoporphyrin is found in white eggshells. Eggshell protoporphyrin is derived from the heme formation in birds. Coproporphyrinogen III oxidase (CPOX) and ferrochelatase (FECH) represent rate‐limiting enzymes for the heme‐biosynthetic pathway. Breast cancer resistance protein (BCRP), feline leukemia virus receptor (FLVCR), and heme‐responsive gene‐1 (HRG1) serve as primary transporters for both protoporphyrinogen and heme. Finally, four organic anion transporting polypeptide family members (including solute carrier organic anion transporter family, SLCO1C1, SLCO1A2, SLCO1B3 and LOC418189) may affect pigment transport within eggshells. Here we measured gene expression levels in key tissues of egg‐producing hens. We analyzed three different types of hens that generated distinct eggshell colors: white, pink or brown. Our data revealed three ways in which eggshell color was genetically influenced. First, high‐level expression of CPOX generated more protoporphyrinogen and a brown eggshell color. In contrast, high expression of FECH likely converted more protoporphyrinogen into heme, reduced protoporphyrinogen levels within the eggshell and generated a light color. Second, heme transporters also affected eggshell color. High‐level expression of BCRP, HRG1 and FLVCR were associated with brown, white and generally lighter eggshell colors, respectively. Finally, protoporphyrin precipitation also affected eggshell color, as high expression of both SLCO1A2 and SLCO1C1 were associated with brown eggshell color. As such, we have identified seven genes in which expression levels in different tissues were associated with eggshell color. 相似文献