Molecular genetic and expression analysis of alpha-tocopherol transfer protein mRNA in German shepherd dogs with degenerative myelopathy

Degenerative Myelopathy (DM) is a progressive neurological disorder of the spinal cord preferentially occurring in German shepherd dogs. The pathogenesis of the disease is unknown. However, there are indications that vitamin E deficiency may be involved in the pathogenesis of DM. Therefore, we analyzed the expression and the nucleotide sequence of the canine alpha-tocopherol transfer protein (alpha Ttp) of German shepherd dogs with DM in order to determine whether a deficiency or a defect of the alpha Ttp could be a primary factor in the pathogenesis of DM, as found in human patients with Ataxia with vitamin E deficiency (AVED). The cDNA of the coding region of the canine alpha Ttp-mRNA was generated from total liver RNA using RT-PCR and 5' RACE technique. We determined the sequence of 707 out of 834 base pairs or 84.8% of the canine alpha Ttp coding region. Sequence comparison of canine alpha Ttp between affected and control dogs revealed no differences in either nucleotide or predicted amino acid sequence. Using Northern blot analysis alpha Ttp-mRNA expression was solely found in the liver of the dogs, rats and humans, while various other organs showed no alpha Ttp-mRNA expression. No significant differences in expression levels of canine alpha Ttp mRNA were found between DM and control dogs. Our data suggest that the canine alpha Ttp gene is unlikely to be involved in the pathogenesis of DM in German shepherd dogs.     

Isolation, characterization, and expression of stromelysin-1 in primary tumors of dogs

OBJECTIVE: To isolate and characterize the cDNA sequence of canine stromelysin-1 (matrix metalloproteinase [MMPI-3), screen various naturally developing primary tumors of dogs, and assess the effect of stromelysin-1 on survival of dogs with cancer. SAMPLE POPULATION: 3 canine cell lines and biopsy specimens of primary tumors collected from 54 dogs. PROCEDURE: 3 canine cell lines and biopsy specimens of primary tumors collected from 54 dogs at the University of Illinois Veterinary Teaching Hospital were used in the study. Primer sets based on human stromelysin-1 and consensus sequences were designed for expression, screening, and isolation. Two additional primer sets were designed for screening. Samples were assayed at least in duplicate. Data were analyzed for differences in expression of stromelysin-1 on the basis of sex, age, metastasis, malignant versus nonmalignant tissue origin, and duration of patient survival. RESULTS: A 1,479-bp cDNA nucleotide sequence was amplified from established canine cell lines. The open reading frame encoded a protein consisting of 478 amino acids. This sequence was 70% to 88% homologous with stromelysin-1 of other species at the amino acid level. Fifty-four samples were screened for stromelysin-1. Of these, 34 (63%) had positive results and 20 (37%) had negative results for expression. Stromelysin-1 and metastasis were associated with a poor prognosis for survival. CONCLUSIONS AND CLINICAL RELEVANCE: Stromelysin-1 is a potential activator of other members of the MMP family. Additional studies are needed to investigate the relationship between stromelysin-1 production and aggressive biological behavior of tumors in dogs.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

Molecular cloning of canine membrane-anchored inhibitor of matrix metalloproteinase, RECK

The reversion-inducing cysteine-rich protein with Kazal motifs (RECK) gene is one of the endogenous matrix metalloproteinase (MMP) inhibitors. It was reported that decreased RECK expression closely correlated with tumor malignancy. We determined the cDNA sequence of the canine RECK gene. The cDNA sequence and deduced amino acid of canine RECK were 2,913 bases and 971 residues, respectively. The predicted amino acid sequence of the protein showed 95.5% and 91.9% homology with human and mouse RECK, respectively. RECK mRNA expression was analyzed in various canine tissues and tumor cell lines by quantitative RT-PCR. The highest RECK expression was detected in lung and testis. In comparison with the tissues, a remarkably low expression level was detected in tumor cell lines. In addition, the RECK gene was transfected in the canine transitional cell carcinoma, and its influence on cell proliferation, migration, and invasion was analyzed. The transfected RECK gene suppressed only canine tumor invasion. These results showed that RECK might play an important role in tumor malignancy in dogs as well as in other mammalians.     

cDNA of Canine Heat Shock Protein 70 (HSP70)

Full-length canine HSP70 cDNA was sequenced and the expression of HSP70 mRNA was investigated. The full-length cDNA sequence of the HSP70 gene (2322 bp) contained a single long open reading frame (1920 bp) coding a protein of 640 amino acids. The amino acid sequence of the canine HSP70 gene shared about 90-95% sequence similarity with bovine, human and mouse HSP70 proteins. Southern blot analysis with HSP70 probe gave three distinct bands of 9.4 kb, 5 kb and 4.4 kb in BamHI digests and two distinct bands of 19 kb and 4 kb in EcoRI digests. Canine HSP70 mRNA was detectable in canine peripheral blood mononuclear cells and stomach but not in liver, kidney, spleen, small intestine, large intestine and skin of dogs.     

Canine CD20 gene

The human CD20 antigen, a 35kDa cell surface nonglycosylated hydrophobic phoshpoprotein is expressed consistently on almost all human B-cells, and its monoclonal antibody is used for the therapy on human B-cell lymphoma. In the present study, canine CD20 gene was cloned and sequenced, and the expression of CD20 mRNA was investigated in canine peripheral blood mononuclear cells (PBMCs), and lymph nodes from healthy dogs, and canine lymphoma cells. Using canine cDNA as a template, full-length of canine CD20 gene was sequenced by 5'-RACE and 3'-RACE methods. The full-length of the cDNA sequence of canine CD20 was 1239bp encoding 297 amino acids. The amino acid sequences of canine CD20 showed 73 and 68% sequence similarities with those of human and mouse, respectively. Canine CD20 was predicted to contain domains of amino acid sequences consisting of two extracellular domains (EM), four transmembrane domains (TM), and three intracellular domains (IC) as in human CD20. Canine CD20 mRNA was detected in PBMCs and lymph node from healthy dogs, and B-cells of canine lymphoma, but not in T-cell lymphoma cells and non-T and non-B-cell lymphoma cells by RT-PCR analysis. From these results, canine CD20 might be targeted for monoclonal antibody therapy against B-cell lymphoma of dogs.     

Sequencing of the Von Hippel-Lindau Gene in Canine Renal Carcinoma

Background: Similarities in human and canine renal cell carcinoma (RCC) epidemiology and biologic behavior suggest that molecular mechanisms of tumorigenesis may be similar in both species. Approximately 75% of RCC in people are of the clear cell subtype, up to 85% of which are associated with mutation of the von Hippel-Lindau ( VHL ) gene. The canine VHL coding deoxyribonucleic acid (DNA) shares 90% identity with the human VHL gene.
Objective: To determine whether or not RCC in dogs are associated with VHL mutations, and if so determine the prevalence, type, and location of these mutations.
Animals: Thirteen dogs with RCC, 2 dogs with primary renal sarcomas, and 10 dogs without neoplastic kidney disease.
Methods: DNA was extracted from paraffin-embedded RCC tissue; DNA extracts from paraffin-embedded and snap-frozen nonneoplastic canine kidneys and canine whole blood were used as negative controls. Polymerase chain reaction and sequencing of the 3 VHL exons was performed, and results compared with the accessioned canine sequence.
Results: All VHL exons were amplified from 9 of 13 canine RCC samples, both renal sarcomas, 8 of 10 nonneoplastic kidney samples, and canine whole blood; only exon 2 could be amplified from 2 RCC samples. Mutations were not identified in any exons. A maximal prevalence of 33.6% for VHL mutations in canine RCC was determined.
Conclusion and Clinical Importance: Although similarities between canine and human RCC merit further investigation of the dog as a model for some subtypes of renal tumors, the lower prevalence of VHL mutations suggests that oncogenesis in these 2 species differs.     

PCR screening for candidate etiological agents of canine hepatitis

PROBLEM ASSESSED: Hepatitis, either acute or chronic, is a relatively common hepatic disease in dogs. Several forms of canine hepatitis can occur, some with a defined cause, most cases have an unknown etiology. The similarities between canine hepatitis and human viral hepatitis suggest that canine hepatitis may have a viral etiology too. OBJECTIVE: To test liver tissue of dogs with hepatitis for the presence of candidate agents based on their known association with hepatitis in other mammals. METHODS AND APPROACH: The following infectious agents were tested by PCR: Hepadnaviridae, Helicobacter spp., Leptospira spp., Borrelia spp., hepatitis A virus, hepatitis C virus and hepatitis E virus. Also canine adenovirus and parvovirus were included. Ninety-eight liver tissue samples of dogs with various histologically diagnoses forms of hepatitis were tested. Primers were designed on conserved regions in the genome of each of these agents, to increase the likelihood of detection by PCR. To further increase sensitivity, nested PCRs for all agents were designed. Finally, for each agent a nested short primer PCR (SPP) was performed. RESULTS: None of these agents were detected by nested PCR and nested SPP. However, in two acute hepatitis liver samples parvovirus was detected by nested PCR, and one of these was also detected by nested SPP. CONCLUSIONS: Hepatitis in dogs is not caused by agents with high homology to known infectious agents that cause hepatitis in other species.     

Cloning and antibody recognition analysis of the canine 5-lipoxygenase gene

5-Lipoxygenase cDNA was prepared from canine white blood cells revealing the full-length message using an oligonucleotide capping method. The sequenced 5-Lipoxygenase open reading frame revealed a 2031 base pair message encoding a 676 amino acid protein. The amino acid sequence showed mild variation with the presumed canine sequence, as well as differences in important residues of known phosphorylation observed in other species. The sequence had between 86 and 92% homology with other species, revealing a highly conserved sequence. Confirmation of gene product identity was achieved through transient transfection of the gene in a V5-Histidine tagged pcDNA 3.1 vector into a known canine cell line. Both V5 antibody and 5-lipoxygenase antibody confirmed the gene product using Western blotting and immunoflourescence.     

Genetic screening of the canine zinc finger protein multitype 2 (cZFPM2) gene in dogs with tetralogy of Fallot (TOF)

A recent genetic study in humans where the ZFPM2 gene was screened for mutations revealed a heterozygous mutation that was found to contribute to several sporadic cases of tetralogy of Fallot (TOF). We hypothesized that this would also hold true for a study group of dogs with TOF. We isolated the full-length cDNA of the canine ZFPM2 and aligned the deduced amino acid sequence against sequences from other species. Our analysis indicated that the canine ZFPM2 was phylogenetically much closer to the human ZFPM2 compared with the mouse or rat sequences. Further gene expression analysis also failed to identify differential gene expression between affected and unaffected dogs. We screened the cZFPM2 for mutations in a group of dogs with TOF but found no significant base changes in either the splice sites or the coding regions.     

Canine ABCB4: Tissue expression and cDNA structure

The ABCB gene subfamily of ABC (ATP-binding cassette) transporters is responsible for transporting a wide spectrum of molecules including peptides, iron, bile salts, drugs, and phospholipids. In humans, ABCB4 appears to be exclusively expressed on the apical membrane of hepatocytes where it translocates phosphatidylcholine from the inner to the outer leaflet of the canalicular membrane. Functional alterations in the ABCB4 transporter are associated with a number of cholestatic syndromes in humans. Because of its role in biliary lipid homeostasis in humans, investigation of the ABCB4 gene in dogs is warranted. Thus, the full cDNA sequence of canine ABCB4 was elucidated and its mRNA and protein expression levels in tissues were determined. Canine ABCB4 consists of 3804 nucleotides spanning 26 exons and is 89% identical to human ABCB4. Expression of ABCB4 in canine liver supports a potential role for the protein in normal biliary function similar to that in humans. The function of ABCB4 expressed in brain tissue has yet to be determined.     

Expression of thymic stromal lymphopoietin in canine atopic dermatitis

Background – In humans, thymic stromal lymphopoietin (TSLP) plays a central role in the development of allergic inflammation, such as atopic dermatitis (AD), but it is unknown whether it is involved in the pathogenesis of canine AD (CAD). Hypothesis/Objectives – Our aim was to characterize canine TSLP and to assess its expression in CAD. Methods – Canine TSLP was identified based on sequence homology with human TSLP and the complementary DNA (cDNA) cloned by RT‐PCR. Real‐time quantitative RT‐PCR was established to assess the expression of canine TSLP in cultured canine keratinocytes and in skin biopsy specimens from lesional and nonlesional skin of 12 dogs with CAD and eight healthy control dogs. Results – Partial canine TSLP cDNA was cloned and characterized. It contained four exons that shared 70 and 73% nucleotide identity with human and equine TSLP, respectively, encoding the signal peptide and full‐length secreted protein. We found significantly increased TSLP expression in lesional and nonlesional skin of dogs with CAD compared with healthy control dogs (P < 0.05), whereas no difference was measured between lesional and nonlesional samples. In cultured primary canine keratinocytes, we found increased TSLP expression after stimulation with house dust mite allergen extract or Toll‐like receptor ligands lipopolysaccharide and poly I:C. Conclusions and clinical importance – Increased TSLP expression in the skin of dogs with CAD supports an involvement of TSLP in the pathogenesis of CAD similar to that in humans. Further studies should elucidate the function and therapeutic potential of TSLP in CAD.     

猪Sar1b基因的分子克隆、序列分析及原核表达

根据GenBank已发表的猪Sar1b基因序列（GenBank登录号：AY819557）设计1对引物,以猪肝脏组织总RNA的反转录产物为扩增模板,用RT-PCR方法扩增出猪Sar1b基因cDNA全长编码区,经过EcoRI-SalI双酶切后定向克隆于pET28a原核表达载体,获得pET28a-Sar1b重组原核表达载体。将携带有重组原核表达载体的大肠杆菌BL21（DE3）通过1 mmol/LITPG进行诱导表达,经过SDS-PAGE电泳检测,显示诱导表达蛋白大小大约为26 ku,与预期表达蛋白大小一致。Western blot检测显示该蛋白为His融合蛋白,表明重组原核表达载体在大肠杆菌中成功表达出了目的融合蛋白。猪Sar1b基因的克隆和表达研究,为进一步研究该基因的生物学功能奠定了基础。     

Nucleotide sequence of the canine alphaIIb gene from platelet-derived cDNA

OBJECTIVE: To determine the nucleotide sequence of the alphaIIb gene from canine platelet-derived cDNA. ANIMALS: 3 adult dogs. PROCEDURE: First-strand cDNA was prepared from total RNA isolated from canine platelets. The cDNA was amplified, using specific primers in polymerase chain reaction (PCR), and the nucleotide sequence was obtained from purified PCR products. RESULTS: Except for the nucleotide at position 694, results of all sequencing reactions of alphaIIb were identical for canine platelet-derived cDNA. Canine alphaIIb had 3 fewer codons than alphaIIb of humans. The nucleotide and deduced amino acid sequences of full-length canine alphaIIb shared > or = 83% similarity with the sequences established for humans. Segments of canine alphaIIb nucleotide and deduced amino acid sequences were > or = 78% similar to alphaIIb associated with 7 functional domains (extracellular, transmembrane, cytoplasmic, and 4 calcium-binding domains) in humans, with the highest degree of similarity correlating with the sequences of the 4 calcium-binding domains. Amino acid residues associated with development of alloantibodies in humans (Met837, Val837, Ile843, Ser843) are not encoded by canine alphaIIb. CONCLUSIONS AND CLINICAL RELEVANCE: The nucleotide variation at position 694 of canine alphaIIb may represent a polymorphism. The species differences in the alphaIIb sequence may contribute to variations in receptor-li gand interactions. The high degree of alphaIIb sequence conservation of the 4 calcium-binding domains implies functional importance. Some disorders associated with alphaIIbbeta3 in dogs are clinically analogous to diseases in humans, and results indicate that dogs are an appropriate model for the evaluation of gene therapy and other treatments of platelet-associated disorders.     

Expression of leukemia inhibitory factor and leukemia inhibitory factor receptor in the canine pituitary gland and corticotrope adenomas

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 family that activates the hypothalamic-pituitary-adrenal axis and promotes corticotrope cell differentiation during development. The aim of this study was to investigate the expression of LIF and its receptor (LIFR) in the canine pituitary gland and in corticotrope adenomas, and to perform a mutation analysis of LIFR. Using immunohistochemistry, immunofluorescence, and quantitative expression analysis, LIF and LIFR expression were studied in pituitary glands of control dogs and in specimens of corticotrope adenoma tissue collected through hypophysectomy in dogs with pituitary-dependent hypercortisolism (PDH, Cushing's disease). Using sequence analysis, cDNA was screened for mutations in the LIFR. In the control pituitary tissues and corticotrope adenomas, there was a low magnitude of LIF expression. The LIFR, however, was highly expressed and co-localized with ACTH_1-24 expression. Cytoplasmatic immunoreactivity of LIFR was preserved in corticotrope adenomas and adjacent nontumorous cells of pars intermedia. No mutation was found on mutation analysis of the complete LIFR cDNA. Surprisingly, nuclear to perinuclear immunoreactivity for LIFR was present in nontumorous pituitary cells of the pars distalis in 10 of 12 tissue specimens from PDH dogs. These data show that LIFR is highly co-expressed with adrenocorticotropic hormone (ACTH) and α-melanocyte-stimulating hormone (α-MSH) in the canine pituitary gland and in corticotrope adenomas. Nuclear immunoreactivity for LIFR in nontumorous cells of the pars distalis may indicate the presence of a corticotrope adenoma.