Immunohistochemical Expression of Potential Therapeutic Targets in Canine Thyroid Carcinoma

Background

Thyroid carcinoma is a common endocrine tumor in the dog. Local invasive growth frequently precludes surgical excision and, in up to 38% of dogs, the tumor has already metastasized by the time of diagnosis. Therefore, it is important to investigate new treatment modalities that may be useful for the large number of dogs with inoperable tumors or metastatic disease.

Hypothesis/Objectives

To investigate the immunohistochemical expression of potential therapeutic targets in canine thyroid tumors.

Animals

74 dogs with thyroid neoplasia.

Methods

Immunohistochemistry was performed for thyroglobulin, calcitonin, vascular endothelial growth factor (VEGF), p53, cycloxygenase‐2 (cox‐2), and P‐glycoprotein (P‐gp).

Results

Fifty‐four (73%) tumors were classified as follicular cell thyroid carcinomas (FTCs) and 20 (27%) as medullary thyroid carcinomas (MTCs). Eighty percent of FTCs and all MTCs had a high percentage (76–100%) of neoplastic cells immunopositive for VEGF. Thirteen percent of FTCs and 50% of MTCs expressed cox‐2. Seven percent of FTCs and 70% of MTCs expressed P‐gp. No tumor was immunopositive for p53 expression. Expression of VEGF (P = .034), cox‐2 (P = .013), and P‐gp (P < .001) was significantly higher in MTCs compared to FTCs.

Conclusions and Clinical Importance

VEGF is a potential therapeutic target in both FTC and MTC in dogs. Cox‐2 and P‐gp may be useful molecular targets in canine MTC.     

cDNA cloning and characterization of the warm-temperature-acclimation-associated protein Wap65 from carp, Cyprinus carpio

We determined full-length cDNA of carp warm-temperature-acclimation-associated 65-kDa protein (Wap65). It encoded 439 amino acid residues with a signal peptide of 22 residues and showed an amino acid sequence identity of 88% to that of goldfish reported before (J. Biol. Chem. 1995. 270: 17087–17092). The number of potential N-linked glycosylation sites of carp Wap65 was two in contrast to three for goldfish. In addition, molecular mass determined by SDS-PAGE was apparently different from that of goldfish. These results suggest that the amount of oligosaccharide is different between the carp and goldfish protein. As in goldfish, carp Wap65 mRNA showed marked accumulation in hepatopancreas of the 30 °C- acclimated fish, which was 8-fold higher than that of the 10 °C-acclimated fish. Carp Wap65 showed 30% amino acid identity to mammalian hemopexins, which appeared to be considerably low in comparison with those among mammalian hemopexins (72 to 80%), or among carp Wap65 and rainbow trout hemopexin-like protein (70%). However, although mammalian hemopexins contain residues comprising the heme binding pocket, carp Wap65 lacked one of the two histidine residues to serve as heme axial ligands in hemopexins. Our data on carp protein substantiates the previous observation for goldfish and indicates that Wap65 might have some important functions in warm-temperature-acclimation of fish.     

猪繁殖与呼吸综合征病毒核衣壳蛋白与糖蛋白的表达纯化及鉴定

In order to gain nucleocapsid protein （N） and glycoprotein （E） of porcine reproductive and respiratory syndrome virus （PRRSV）, the total RNA was extracted,ORF7 and ORF5 genes were obtained by RT-PCR, inserted into pET-28a(+) vector, and then transformed into Escherichia coli BL21（DE3）,respectively. The expressed products were identified by SDS-PAGE and Western blotting, and purified by affinity chromatography. The results showed that N and E fusion protein were successfully expressed and the proteins had good reactionogenicities by Western blotting analysis. The purities of the purified proteins were 89% and 90%, respectively. This study could lay foundations for molecular biological function research and establishment of test methods for detection antibodies of PRRSV.     

动物水泡性口炎病毒的抗原性

动物水泡性口炎病毒(VSV)引起动物水泡性疾病.VSV有两个主要抗原群是核酸蛋白和G蛋白抗原.G蛋白因为能产生中和性抗体被认为是在发动抑制病毒感染方面起主要作用.已经证实VSV 的New Jersey 株中产生独立的中和性单克隆抗体的抗原决定蔟分布在G 蛋白的193～289氨基酸之间.     

鲢鱼肉肌原纤维乳糖蛋白的制备及其溶解性

研究了乳糖与鲢鱼肉肌原纤维蛋白(Mf)在不同反应条件下通过美拉德反应制备的肌原纤维乳糖蛋白的功能特性。测定了其在0．1mol／L Nacl溶液中的溶解性，并对其进行了SDS聚丙烯酰胺凝胶电泳分析。乳糖与Mf以1：1、2：1、4：1和6：1的质量比混合，冷冻干燥后，在50和55℃，相对湿度为65％的条件下反应0～48h制备的肌原纤维乳糖蛋白。其溶解性较Mf有不同程度的提高。乳糖与Mf质量比为1：1，50℃反应48h后，肌原纤维乳糖蛋白在0．1m01／L NaCl溶液中的溶解性从反应前的18％提高到63％；55℃反应24h，其溶解性达到89％。结果表明．乳糖与鲢鱼肉Mf发生美拉德反应后生成的肌原纤维乳糖蛋白在低离子强度溶液中的溶解性得到了有效提高，乳糖能有选择性地与肌球蛋白重链相结合。     

牛精子膜麦芽凝集素结合糖蛋白的分离与定位

牛精子膜用非离子去污剂 NP-40增溶,经固相凝集素亲和层析,初步分离了精子膜麦芽凝集素结合糖蛋白,经 SDS-聚丙烯酰胺凝胶电泳、银染色显示出了四条蛋白质带,分子量分别为78,60.42和33 kD。用分离到的糖蛋白制备抗血清,对牛精子进行免疫细胞化学标记,结果顶体区显示为强阳性,与麦芽凝集素亲和细胞化学的染色一致。用抗血清处理精子再进行亲和细胞化学染色时,顶体区的麦芽凝集素结合糖残基多数被封闭,揭示麦芽凝集素结合糖残基是构成抗原决定簇的成分。     

甘薯糖蛋白对小鼠血脂水平的影响

为了研究甘薯糖蛋白对小鼠血脂水平的影响。先用含8.0%猪油的高脂鼠料诱导小鼠成为高脂血,再将其平均分为四组,一组用普通鼠料饲养作为对照,其他三组分别用含0.5%、1.0%及1.5%甘薯糖蛋白的鼠料饲养,在第15天和第30天对每只小鼠采血,用全自动生化分析仪测定其中的甘油三酯及血清胆固醇的浓度。结果发现用含1.0%甘薯糖蛋白的鼠料饲养15d就能明显降低高脂血模型小鼠血液中的血清总胆固醇水平,继续饲养30d能显著降低其中的甘油三酯水平;如果用含1.5%甘薯糖蛋白的鼠料饲喂,则只需15d就能同时降低两者的水平。因此甘薯糖蛋白具有显著降低高脂血模型小鼠血脂的作用。     

一个重要的精子受体—猪透明带糖蛋白β-D-半乳糖残基

 【目的】证明末端半乳糖残基在猪透明带糖蛋白中的分布以及它在精子结合和侵入中的作用。【方法】体外成熟的猪卵母细胞去除周围卵丘细胞，用异硫氰酸荧光素(fluorescein isothiocyanate，FITC) 络合西非单叶豆凝集素(Bandeiraea simplicifolia lectin-I，BS-I) 或蓖麻凝集素(Ricinus communis agglutinin，RCA-I)处理30min，分别观察末端-D-半乳糖残基或-D-半乳糖残基在透明带中的定位分布。荧光显微镜观察显示，BS-I和RCA-I都标记于整个透明带上且比较集中于透明带的外层，其中BS-I显示微弱荧光反应，而RCA-I呈强荧光反应且在透明带的外层反应尤为强烈。植物凝集素处理的卵母细胞用于体外受精，分别在受精2h或12h观察精-卵结合和精子入卵情况。【结果】RCA-I处理组卵母细胞平均精子结合数显著低于对照组（18 vs 95），而精子入卵则完全被阻断；可是BS-I处理组的卵母细胞的精子结合数少量被抑制（59 vs 95），而且精子入卵率与对照组无显著差异。【结论】猪透明带末端-D-半乳糖残基作为精子受体的重要组成成分，参与精-卵结合和受精。     

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.     

减毒沙门氏菌为载体传递DNA疫苗诱导对新城疫病毒的免疫保护

探讨了以减毒鼠伤寒沙门氏茵为栽体传递新城疫病毒DNA疫苗的安全性、免疫原性和可行性。将含新城疫病毒(NDV)F48E9株融合蛋白(F)基因的真核表达质粒pcDNA3-F的重组减毒鼠伤寒沙门氏菌ZJ111株(ZJ111／pcD-NA3一F菌株)，以10^8CFU进行首免，2周后二免，三免后4周攻击强毒株F48E9，观察其安全性和免疫原性，同时设只含空载体pcDNA3的ZJ111／pcDNA3菌株对照及口服PBS对照。结果表明：重组ZJ111／pcDNA3-F菌株具有良好的安全性。对强毒株攻击的保护率达64．7％。重组ZJ111／pcDNA3-F菌株不仅能诱导雏鸡产生NDVELISA抗体，而且诱导产生的法氏囊B淋巴细胞和胸腺T淋巴细胞增殖反应显著高于ZJ111／pcDNA3时照组。这些结果提示，减毒沙门氏菌为载体不仅可直接将NDVF基因呈递给鸡体细胞进行表达，产生抗NDV的体液免疫，而且还可诱导细胞免疫应答。