Angiotropic metastatic malignant melanoma in a canine mammary gland / 한국실험동물학회지

An eleven-year-old spayed female Yorkshire Terrier presented with a sublumbar mass and upon ultrasonographic examination, was revealed to have a mammary gland tumor. Black to reddish colored masses, located in the visceral peritoneum of the sublumbar region was observed on laparotomy with masectomy of the right side. In the laparotomy, we observed reddish masses multifocally located in the serosal membrane of the large intestine. Histopathologic examination of the intestinal and abdominal mass showed highly invasiveness into the muscle and metastasis of melanocytic tumor cells through the blood vessels. The mammary glands showed abnormal hyperplasia of melanocytes, destruction of the normal glands by tumor cells and infiltration of some lymphocytes in the pool of melanocytic cells. We have identified a malignant melanoma containing an angiotumoral complex in which tumor cells occupied a pericytic location along the microvessels with intravasation determined by immunohistochemistry for S100 protein and protein kinase C-alpha. Histologic findings in this dog lead to a diagnosis of an angiotropic metastatic malignant melanoma.

Eosinophilic myositis in a slaughtered Korean native cattle

Histopathological findings of eosinophilic myositis in the carcass of a slaughtered Korean native cow are presented. Lesions contained massive fibrous septae with vacuolar changes in some lesions, and the hypercontraction and rupturing of muscle bundles, with replacement by eosinophils. Necrosis and severe eosinophil infiltration were observed. Sarcoplasmic fragmentation and atrophy developed. Typical of granuloma, calcified myofibers were focally surrounded by macrophages and numerous inflammatory cells, and multinucleated giant cell formation was evident.

Modification,Expression and Purification of Human Endotoxin Binding Peptide Gene / 中国生物工程杂志

Objective: To modify EBP(endotoxin binding peptide), clone and express the mutate of EBP gene and gain purified mEBP.Method: mEBPgene was cloned by PCR site-directed mutagenesis. PinpointXa-3/mEBP expression vector was designed to express human mEBP as a fusion protein in BL21 (DE3) pLysS. Digested engineering bacteria by lysozyme and collected inclusion bodies.Fusion protein was purified by Pinpoint TM Xa purification system and cleaved by factorXa,mEBP was purified by RP-HPLC. Results: Mutations at residues 5 and 18(Gln→Lys) was obtained by PCR site-directed mutagenesis, expressed and purified mEBP successfully.Conclusions: Obtaining of purified mEBP lay a foundation for its biological activity research.

Study on the expression, purification and the anti-endotoxin activity of human endotoxin binding peptide and its mutant / 中华烧伤杂志

<p><b>OBJECTIVE</b>To express endotoxin binding peptide and its mutant in E coli DH5alpha and detect the antiendotoxin activity of the purified peptides.</p><p><b>METHODS</b>(1 ) E coli DH5at containing pinpointXa3-EBP and pinpointXa3-mEBP was activated by IPTG to express biotin fusion protein. The fusion proteins were purified, and then digested by factor Xa for isolation of EBP and mEBP. The target peptide was purified with affinity chromatography and reversed-phase HPLC. Sequences of 10 amino acids at N-terminal were used for identification of mEBP. (2) PBMCs were isolated from blood of normal people, and they were stimulated with 5 mg/L FITC-LPS plus 2.0,5. 0 and 12. 5 mg/L EBP or mEBP. Then the mean fluorescent intensity was detected. PBMC was also stimulated with 1 mg/L LPS plus 2.0, 5.0 and 12.5 mg/L EBP or mEBP for 5 hours for the detection of the TNF-alpha and IL-6 level in the supernatant. (3) Thirty-five Kunming mice were randomized into normal control ( n = 5, with intraperitoneal injection of 0. 2 ml isotonic saline) , model group(n = 5, with intraperitoneal injection of LPS and 20% TBSA full-thickness burns), and treatment group (n = 15, with intraperitoneal injection of 5 mg/kg PMB or 10 mg/kg EBP or mEBP after burns). The serum contents of TNF-a and IL-6, and TNF-alpha mRNA level in hepatic tissue in each group were determined 6 hours after treatment.</p><p><b>RESULTS</b>( 1 ) EBP and mEBP were obtained after Xa digestion of biotin fusion protein, with purity reaching above 98% . The sequence of 10 amino acid at N-terminal was in accord with what expected. (2) The fluorescent intensity was decreased followed by an increase in mEBP or EBP concentration. Compared with normal PMBC, IL-6 and TNF-alpha level in the supernatant were obviously lowered in 1 mg/L LPS + 12.5 mg/L EBP group and I mg/L LPS +2. O0 , 5. 0, 12. 5 mg/L mEBP groups ( P < 0.01). (3) The serum level of IL-6 and TNF-ca in the therapeutic groups were obviously lower than that in model group ( P < 0.01 ) , and the levels of these cytokines were significantly lower in 10 mg/kg mEBP group than that in 10 mg/kg EBP group ( P <0. 01) , but they were similar to that in 5 mg/kg PMB treatment group ( P >0.05). (4) Relative optical density of TNF-alpha. mRNA in control, model, 10 mg/kg mEBP, 10 mg/kg EBP and 5 mg/kg PMB groups was 0.25, 0.93, 0.51 , 0.77 and 0.43, respectively.</p><p><b>CONCLUSION</b>Endotoxin binding peptides can be obtained by procaryon expression. Both EBP and mEBP have anti-LPS activity, but mEBP is more effective.</p>

Canine renal failure syndrome in three dogs

Three dead dogs were brought to the College of Veterinary Medicine, Kyungpook National University for study. Clinically, all the dogs showed emaciation, anorexia, depression, hemorrhagic vomiting and diarrhea for 7~10 days before death. All the clinical signs were first noted for about one month after feeding the dogs with commercial diets. At necropsy, all 3 dogs had severe renal damage with the same green-yellowish colored nephroliths in the renal pelvis. They also showed systemic hemorrhage and calcification of several organs, which might have been induced by uremia. Microscopically, necrosis, calcification and calculi were detected in the renal tubules, and especially in the proximal convoluted tubules and collecting ducts of the kidney. These findings were supportive of a mycotoxic effect, and especially on their kidneys. However, the precise cause of the toxic effect in these cases of canine renal failure could not be determined.

Multiple intestinal lymphomatous polyposis in a Jindo dog

A male, 5-year-old Jindo dog underwent enterectomy and enteroanastomosis due to ileus of the intestine at a local veterinary hospital. Grossly, the excised intestine showed markedly thickened multinodular masses in the serosal layer of the upper part, and soft-to-firm, creamcolored neoplastic masses that displayed extensive nodular mucosal protuberances into the lumen. The neoplastic masses were filled with large round cells that were ovoid in shape and they had pale and/or hyperchromatic nuclei. The neoplastic cells had mainly infiltrated into the mucosal and submucosal layers, and they had diffusely invaded the muscular and serosal layers. Therefore, the diagnosis of canine multiple intestinal malignant lymphomatous polyposis was made based on the gross and histopathological findings. The origin of these tumor cells was determined to be B-cells since they were positive for anti-CD20.

Selection of a peptide mimic the neutralization epitope of hepatitis E virus with phage peptide display technology / 生物工程学报

Hepatitis E is an acute hepatitis casused by hepatitis E virus (HEV) in developing countries, where it occurs as cases sporadic and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. HEV is icosahedron non-enveloped virus, and its genome is a single-stranded, positive-sense, 3'-polyadenylated RNA about 7.5 kb in length. It contains three open reading frames (ORFs). Of which ORF1 codes for a polyprotein of 1693 amino acids and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicasre, and an RNA-dependent RNA polymerase, besides the most hypervariable region of the HEV genome. And ORF3 codes for a 123-amino-acide-long polypeptide with unknown function. While the major viral capsid protein (pORF2, ORF2 codes) of 660 amino acid was showed to contain the protective epitope. The bacterially expressed polypeptide disignated as NE2 has been proved to be a protective antige. And the anti-NE2 monoclonal antibodies (mAb) was screend, two of these mAbs 8C11 and 8H3 were showed to be against separate conformational neutralization epitope of hepatitis E virus (HEV). And these two mAb were used to screen for binding peptides from a 7-peptides phage display library. After four rounds of panning, tweenty-one positive monoclonal phages (11 for 8C11, and 10 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptide 8C11 (N'-His-Pro-Thr-Leu-Leu-Arg-Ile-C', named 8C11A) and 8H3 (N'-Ser-Ile-Leu-Pro-Tyr-Pro-Tyr-C', named 8H3A) were then synthesized and cloned to insert between amino acid 78 to 83 of hepatitis B core antigen (HBcAg), then expressed in E. coli. The recombinant proteins aggregate into homodimer or polymer on SDS-PAGE, and could bind with mAb 8C11 and 8H3 in Western blotting. Respectively, the recombinant protein C8C11A showed to be dimer mainly, which can bind with mAb 8C11. The monomer and dimer of C8H3A are in the same amount on SDS-PAGE, but only the dimer could bind with mAb 8H3 on Western blotting. The renatured recombinant proteins were all showed to aggregate into virus like particles which were similar as HBcAg on transmission electron micrograph. The dominant peptide 8H3A (N'-Ser-Ile-Leu-Pro-Tyr-ProTyr-C') that selected out by mAb 8H3 was further chemo-synthesized, and its binding activity was confirmed by BIAcore biosensor. The result showed that this 7-peptide can bind with mAb 8H3 in a big Ka and Kd form, which means the binding is not stable. These results implicated that conformational dependent neutralization epitope could be partially modeled by short peptide, which provided a feasible route for subunit vaccine development.