Epidermal growth factor receptor expression in canine transitional cell carcinoma

Transitional cell carcinoma (TCC), a urinary bladder tumor with high mortality, is encountered commonly in dogs. Whereas overexpression of epidermal growth factor receptor (EGFR) is associated with development of human urinary bladder cancer, information on EGFR expression in canine TCC is lacking. In this study, EGFR protein and mRNA expression in canine normal bladder (n=5), polypoid cystitis (n=5) and TCC (n=25) were examined by immunohistochemistry and real-time polymerase chain reaction. EGFR protein expression was significantly higher in TCC than that in normal healthy bladder (P<0.001) and polypoid cystitis (P<0.005). High EGFR protein expression was significantly (P<0.01) associated with TCC with a sensitivity of 72% and specificity of 100%. Comparative analysis of protein and mRNA expression levels in TCC showed significant positive correlation (r=0.88, P<0.05) between mRNA and protein expression. These findings suggest that intense expression of EGFR protein could be used as a marker to help canine TCC diagnosis.     

转录因子CBF及其抗寒作用机制

从植物的抗寒方面综述了转录因子CBF（CRT／DRE-binding factor）的发现、分类、结构、在植物抗寒过程中发挥作用的分子机制,并介绍了调控CBF和受CBF调控的其他分子的信息,较为系统地归纳了CBF研究的最新进展。低温是影响植物生长、生产性能的重要因素之一。随着分子生物学的发展,植物抗逆分子机制成为当前植物学研究的一个热点。转录因子是转录起始过程中RNA聚合酶所需的辅助因子。真核生物RNA聚合酶自身无法启动基因转录,只有当转录因子结合在其识别的DNA序列上后,基因才开始转录。转录因子CBF广泛存在于各种植物中,可以识别COR基因中的CRT／DRE（C repeat/dehydration responsive element）元件,从而启动COR基因转录,是植物抗逆过程中一个重要的调节因子。     

Effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos <Emphasis Type="Italic">in vitro</Emphasis>

This study aimed to investigate the effect of nerve growth factor (NGF) on the development of preimplantation rabbit embryos in vitro. Zygotes were collected from superovulated New Zealand rabbits 19 h after injection of hCG and immediately mating and cultured in TCM-199 plus fatty-acid free BSA with different concentrations of NGF. Zygotes not treated with NGF served as control. At 24 h, 48 h, 72 h and 96 h of the culture, the numbers of the early cleavage stage, morulae, blastocysts and hatching blastocysts were determined. The intrazonal diameter of the blastocyst and the total cell numbers per blastocyst were measured after 96 h of culture. The results showed: (1) NGF at 100 ng/mL and 1000 ng/mL could improve the numbers of the hatching blastocysts which developed compared to the control treatment (p < 0.05); (2) All concentrations of NGF increased the total cell numbers in the blastocysts compared to the control treatment (p < 0.05); (3) NGF had no significant effect on the blastocyst intrazonal diameter of the blastocysts at 96 h of culture (p = 0.493); (4) The proportion in the early cleavage stage at 24 h of culture (p = 0.635), of morulae at 48 h of culture (p = 0.812) and of blastocysts at 72 h of culture (p = 0.812) in all treatments were not significantly different.     

Leucemia inhibitory factor; investigating the time‐dependent effect on viability of vitrified bovine embryos

Leucemia inhibitory factor (LIF) is involved in various reproductive processes, including sperm development, regulation of ovulation, as well as blastocyst formation, hatching and implantation in embryos. Moreover, LIF has also been shown significantly to enhance the blastocyst formation rates of bovine embryos, a finding that remains controversial. Our purpose was to investigate time‐dependent effect of LIF on bovine embryo culture, especially in terms of addition timing. Presumptive zygotes were cultured in five different groups. In this study, 100 ng/ml LIF was added to the culture medium were as follows; control: SOF alone, group A: at day 0 (fertilization day), group B: at day 4 post‐insemination (p.i.), group C: at day 4 to 7 (p.i. before vitrification) and group D: at day 8 (p.i. after thawing). Addition of LIF to the culture medium at day 4 significantly increased the percentage of blastocyst rate when compared day 0, day 4 at 6/7 and control group (41.8% versus 24.3%, 19.7%, 34.6%). In conclusion, the addition of LIF only on day 4 (p.i.) to the culture medium was found to be beneficial for bovine embryonic development based on several measures, including blastocysts rate, re‐expansion rate and cellular cryotolerance after vitrification.     

Cloning,tissue expression and polymorphisms of chicken Krüppel‐like factor 7 gene

Krüppel‐like factor 7 (KLF7) has been extensively studied in mammalian species, but its role in birds is still unclear. In the current study, cloning and sequencing showed that the full‐length coding region of chicken KLF7 (Gallus gallus KLF7, gKLF7) was 891 bp long, encoding 296 amino acids. In addition, real‐time RT‐PCR analysis showed that gKLF7 was broadly expressed in all 15 chicken tissues selected, and its expression was significantly different in spleen, proventriculus, abdominal fat, brain, leg muscle, gizzard and heart between fat and lean broilers at 7 weeks of age. Additionally, one novel single nucleotide polymorphism (SNP), XM_426569.3: c. A141G, was identified in the second exon of gKLF7. Association analysis showed that this locus was significantly associated with fatness traits in Arbor Acres broiler random population and the eighth generation of Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) population (P < 0.05). These results suggest that gKLF7 might be a candidate gene for chicken fatness traits.     

Roles of the zona pellucida and functional exposure of the sperm‐egg fusion factor ‘IZUMO’ during in vitro fertilization in pigs

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm‐egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP‐intact and ZP‐free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44?0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti‐IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti‐IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP‐free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.     

Low expression of the antibacterial factor L‐amino acid oxidase in bovine mammary gland

In the mouse, L‐amino acid oxidase (LAO) produces hydrogen peroxide by utilizing free amino acids and is a proven antibacterial factor in mammary glands. Mastitis, a bacterial infection of the mammary gland, is the most frequent disease in dairy cattle. Here, we investigate whether LAO is expressed in the mammary gland of dairy cattle and is antibacterial. In dairy cattle, the expression level of LAO mRNA in the mammary gland was considerably lower than that in mice, and LAO activity was not observed in cattle milk that produced hydrogen peroxide. The expression of LAO mRNA was also low in Japanese Black cattle, the same as in Holstein cattle. A higher LAO mRNA expression was observed in the mastitis glands than in the lactating glands. Furthermore, spleen and lymph nodes expressed high levels of LAO mRNA in dairy cattle. We conclude that mammary glands in dairy cattle have lower ability to express the LAO gene compared to that in mice, which may result in a high incidence of mastitis.     

MicroRNAs as tumour suppressors in canine and human melanoma cells and as a prognostic factor in canine melanomas

Malignant melanoma (MM) is one of the most aggressive cancers in dogs and in humans. However, the molecular mechanisms of its development and progression remain unclear. Presently, we examined the expression profile of microRNAs (miRs) in canine oral MM tissues and paired normal oral mucosa tissues by using the microRNA‐microarray assay and quantitative RT‐PCR. Importantly, a decreased expression of miR‐203 was significantly associated with a shorter survival time. Also, miR‐203 and ‐205 were markedly down‐regulated in canine and human MM cell lines tested. Furthermore, the ectopic expression of miR‐205 had a significant inhibitory effect on the cell growth of canine and human melanoma cells tested by targeting erbb3. Our data suggest that miR‐203 is a new prognostic factor in canine oral MMs and that miR‐205 functions as a tumour suppressor by targeting erbb3 in both canine and human MM cells.     

Effect of mercury chloride on oxidative stress and nuclear factor erythroid 2‐related factor 2 signalling molecule in liver and kidney of laying hens

This study investigated the effects of mercury chloride (HgCl₂) on the deposition of mercury (Hg), histopathology and oxidative stress in liver and kidney of laying hens. The gene expressions of antioxidant enzymes and nuclear factor erythroid 2‐related factor 2 (Nrf2)‐Kelch‐like ECH‐associated protein 1 (Keap1) were further studied to uncover the molecular mechanism. A total of 960 40‐week‐old Hyline brown laying hens were randomly allocated to five treatments with eight pens per treatment and 24 hens per pen. The hens were fed with five experimental diets containing graded levels of Hg at 0.270, 1.250, 3.315, 9.405 and 27.230 mg/kg respectively. Results revealed that both deposition of Hg and score of injury in liver and kidney were significantly increased as dietary Hg dosage up to 27.230 mg/kg diet. Deposition of Hg was positively related to score of injury in liver and kidney of laying hens. Besides, the activities of superoxidative dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione peroxidase (GSH‐Px), and glutathione (GSH) content all significantly decreased (p < 0.05), while malondialdehyde (MDA) content significantly increased (p < 0.05) after Hg exposure in liver and kidney of laying hens. In addition, positive relationships occurred between antioxidant enzyme activities and antioxidant enzyme gene expressions except between SOD activity and manganese superoxide dismutase (MnSOD) gene expression in liver. Meanwhile, Nrf2 gene expression was positively related to antioxidant gene expressions and negatively connected with Keap1 gene expression. Negative relationships occurred between Nrf2 and Keap1 protein levels in liver and kidney. In conclusion, Hg could dose‐dependently damage liver and kidney and induced hepatic and renal oxidative stress by means of suppressing Nrf2‐Keap1 signalling molecule in laying hens.     

Hypothalamo‐pituitary‐adrenocortical axis in stress‐susceptible and stress‐resistant pigs: endocrine responses to corticotrophin‐releasing factor and vasopressin*

Swiss Landrace pigs selected into genetically well‐characterized low and high tissue fat lines (f and F respectively) react differently to exogenous and endogenous stressors. Response of the hypothalamo‐pituitary‐adrenocortical (HPA) axis to i.v. administered ovine corticotrophin‐releasing factor (oCRF) and lysine vasopressin (LVP) in young females, intact and pretreated with dexamethasone or metyrapone, leads to the conclusion that different stress susceptibility of the two lines correlates with the sensitivity of pituitary corticotrophs to oCRF stimulation. Total amount of ACTH released after stimulation with submaximal oCRF doses was roughly equal in both lines, and cortisol level is even lower in the f‐line, most likely due to the considerably enhanced metabolic clearance rate of cortisol (lower half‐life of plasma cortisol compared with F‐line). LVP‐stimulated ACTH release is comparable with that of oCRF is stronger in the f‐line. Combined effect of oCRF and LVP is rather additive than synergistic but the half‐life ratio cortisol/ACTH after this stimulation is about four times higher than for stimulation by LVP and oCRF separately. In cases of externally stimulated HPA axis, cortisol plasma concentration tightly cross‐correlates with that of ACTH.     

Use of routine histopathology and factor VIII‐related antigen/von Willebrand factor immunohistochemistry to differentiate primary hemangiosarcoma of bone from telangiectatic osteosarcoma in 54 dogs

Hemangiosarcoma (HSA) of bone and telangiectatic osteosarcoma (tOSA) can appear similar histologically, but differ in histogenesis (malignant endothelial cells versus osteoblasts), and may warrant different treatments. Immunohistochemistry (IHC) for endothelial cell marker factor VIII‐related antigen/von Willebrand factor (FVIII‐RAg/vWF) is a well‐documented ancillary test to confirm HSA diagnoses in soft tissues, but its use in osseous HSA is rarely described. Archived samples of 54 primary appendicular bone tumours previously diagnosed as HSA or tOSA were evaluated using combination routine histopathology (RHP) and IHC. Approximately 20% of tumours were reclassified on the basis of FVIII‐RAg/vWF immunoreactivity, typically from an original diagnosis of tOSA to a reclassified diagnosis of HSA. No sample with tumour osteoid clearly identified on RHP was immunopositive for FVIII‐RAg/vWF. RHP alone was specific but not sensitive for diagnosis of HSA, compared with combination RHP and IHC. Routine histopathological evaluation in combination with FVIII‐RAg/vWF IHC can help differentiate canine primary appendicular HSA from tOSA.     

Epidermal tissue-derived T-cell growth factor, colony stimulating factor and nerve growth factor in chickens.

The purpose of this study was to determine the mechanism of the local cytokine-mediated immune response in the skin of chickens. The incorporation of 3H-thymidine into spleen T lymphocytes from 9-to 10-week-old chickens was augmented by the addition of epidermal tissue culture supernatant (ESN) from 11-day-old embryos. The colony formation of neonatal chicken bone marrow cells in the methylcellulose medium was also significantly increased by addition of ESN. When axonal outgrowth in matrigel was investigated, the embryonal sympathetic ganglion was found to grow axons outwards towards the epidermal tissue specimens. The above results suggest that chicken epidermal cells (probably keratinocytes) produce T-cell growth factor (corresponding to IL-1), colony-stimulating factor for macrophages (M-CSF) and granulocytes (G-CSF), and nerve growth factor (NGF).     

Analysis of genomic mutation and immunohistochemistry of platelet‐derived growth factor receptors in canine vascular tumours

We examined whether mutation of the platelet‐derived growth factor receptor protein tyrosine kinase (PDGFR)‐α and PDGFR‐β genes contributes to their overexpression in canine vascular tumours. Genomic sequences of trans‐ or juxtamembrane regions of PDGFR‐α and PDGFR‐β were analysed with immunohistochemical staining and polymerase chain reaction‐direct sequencing using DNA from paraffin‐embedded neoplastic tissues of 27 hemangiosarcomas (HSAs) and 20 hemangiomas (HAs). Immunohistochemically, 75% of the HA cases were positive for PDGFR‐α and almost most of the HA cases were negative for PDGFR‐β. Of the HSA cases, 55.6% were negative for PDGFR‐α and 63% were strongly positive for PDGFR‐β. Among the HA cases, 1 missense mutation was detected in PDGFR‐α exon 18 and 1 in PDGFR‐β exon 17. Two HSA cases had missense mutations in exon 14 and 1 in exon 17 of PDGFR‐β. Thus, genomic mutation of trans‐ or juxtamembrane regions of PDGFRs was not the main mechanism driving the activation of receptors in HSA and HA.     

Muscle type‐specific effect of myostatin deficiency on myogenic regulatory factor expression in adult double‐muscled Japanese Shorthorn cattle

To clarify muscle type‐specific effect of myostatin on myogenic regulatory factors (MRFs), we examined mRNA expression of MRFs in five skeletal muscles of normal (NM) and myostatin‐deficient double‐muscled (DM) adult Japanese Shorthorn cattle by quantitative reverse‐transcribed PCR. Among the four MRFs, namely, Myf5, MyoD, myogenin, and MRF4, MyoD expression was different among the muscles of the DM cattle (P < 0.01) but not of the NM cattle. Meanwhile, MyoD expression was significantly elevated only in masseter (MS) muscle in the DM cattle due to the myostatin deficiency (P < 0.05). Myf5 and MRF4 expression in semitendinosus (ST) was higher in the DM than in the NM cattle (P < 0.05). According to analysis of myosin heavy chain (MyHC) isoform expression, more MyHC‐2x and ‐2a and less ‐slow isoforms were expressed in the longissimus and ST muscles compared to the MS muscle in both cattle (P < 0.05), but no significant difference in MyHC expression was observed between the NM and DM cattle. Taken together, myostatin has influences on Myf5 and MRF4 expression in faster‐type muscles and on MyoD expression in slower‐type muscles, suggesting a possible muscle type‐specific effect of myostatin in skeletal muscle growth and maintenance.