Kenichiro Shibata

Expression of angiopoietin-like 4 (ANGPTL4) in human colorectal cancer: ANGPTL4 promotes venous invasion and distant metastasis

There is strong evidence that the angiopoietin family is involved in the regulation of tumour progression. Recently, it has been reported that angiopoietin-like 4 (ANGPTL4) expression in cancer cells promotes the metastatic process by increasing vascular permeability. The present study was conducted to examine ANGPTL4 expression and its association with clinicopathological factors and prognosis in human colorectal cancers. We examined 144 cases of surgically-resected human colorectal adenocarcinomas by immunohistochemistry, RT-PCR and Western blot analysis. Also, overall survival was investigated. Among 144 cases of adenocarcinoma, 95 cases (66.0%) showed positive staining in the cytoplasm of the carcinoma cells for ANGPTL4. Histologically, well, moderately, poorly differentiated adenocarcinoma or mucinous carcinoma showed 55.2, 79.3, 61.5 or 44.4% expression of ANGPTL4, respectively. The expression of ANGPTL4 was correlated with the depth of tumour invasion (p<0.0005), Vienna classification (category 3-5)(p<0.00005), venous invasion (p<0.0005) and Dukes classification (p<0.005). However, ANGPTL4 expression was not correlated with overall survival. However, all patients (100%) with distant metastasis showed immunopositivity for ANGPTL4. The mRNA and the protein expression of ANGPTL4 were shown in four resected samples and cultured cell lines by RT-PCR or western blot analysis. These findings suggest that ANGPTL4 is one of the factors involved in the progression of human colorectal cancer, especially venous invasion and distant metastasis.

Clinicopathological significance of angiopoietin-like protein 4 expression in oesophageal squamous cell carcinoma

Background Angiopoietin-like protein 4 (ANGPTL4) is involved in regulating glucose homeostasis, insulin sensitivity, angiogenesis and lipid metabolism, and also acts as an apoptosis survival factor for vascular endothelial cells. The protein is also known to be induced in hypoxic environments characteristic of cancer tissue. Recently, ANGPTL4 was shown in cancer cells to facilitate the transendothelial passage of the cells, resulting in distant metastasis. Clinically, venous invasion resulting in distant metastasis is crucial for oesophageal cancer progression. Aims To determine ANGPTL4 expression and its association with clinicopathological factors and prognosis in human oesophageal squamous cell carcinoma (OSCC). Methods 104 cases of surgically-resected OSCC specimens were examined by immunohistochemistry. The association of ANGPTL4 expression with clinicopathological characteristics and postoperative survival rate was statistically analysed. Results Expression of ANGPTL4 was statistically correlated with the degree of differentiation, lymphatic invasion and venous invasion. Results of multivariate analysis, performed using multiple logistic regression, showed that lymph node metastasis, lymphatic invasion and ANGPTL4 expression were independent factors predicting venous invasion. Survival rates of patients with ANGPTL4-positive tumours tended to be statistically lower than those with ANGPTL4-negative tumours. Conclusions ANGPTL4 may play an important role in metastasis through lymphovascular invasion, and may potentially affect prognosis.

Aminopeptidase and caseinolytic activites ofMycoplasma salivarium

Aminopeptidase activity was demonstrated inMycoplasma salivarium (ATCC 23064) cells disrupted by sonic vibrations and lyophilized (crude enzymes), and weak endopeptidase or carboxypeptidase activity was also suggested.The crude enzymes were suspended in 0.1 M borate buffer, pH 8.0, containing 0.5% (w/v) sodium deoxycholate, and then the suspensions were centrifuged at 100,000g for 2 h. Thus separated, the supernatants were applied to a column of Sephacryl S-300. As a result, aminopeptidase activity was separated from caseinolytic activity, which had already been demonstrated in this organism. The aminopeptidase activity was inhibited byo-phenanthroline and stimulated by Mn2+, and the enzyme exhibited a strong affinity for leucine and arginine. On the other hand, the caseinolytic activity was inhibited considerably by o-phenanthroline and Ni2+ and slightly by diisopropyl fluorophosphate and Co2+. The caseinolytic activity was therefore believed to be due mainly to metalloproteinases and partly to serine proteinases.

Novel diagnostic procedure for determining metastasis to sentinel lymph nodes in breast cancer using a semi-dry dot-blot method

We developed an easy, quick and cost‐effective detection method for lymph node metastasis called the semi‐dry dot‐blot (SDB) method, which visualizes the presence of cancer cells with washing of sectioned lymph nodes by anti‐pancytokeratin antibody, modifying dot‐blot technology. We evaluated the validity and efficacy of the SDB method for the diagnosis of lymph node metastasis in a clinical setting (Trial 1). To evaluate the validity of the SDB method in clinical specimens, 180 dissected lymph nodes from 29 cases, including breast, gastric and colorectal cancer, were examined. Each lymph node was sliced at the maximum diameter and the sensitivity, specificity and accuracy of the SDB method were determined and compared with the final pathology report. Metastasis was detected in 32 lymph nodes (17.8%), and the sensitivity, specificity and accuracy of the SDB method were 100, 98.0 and 98.3%, respectively (Trial 2). To evaluate the efficacy of the SDB method in sentinel lymph node (SLN) biopsy, 174 SLNs from 100 cases of clinically node‐negative breast cancer were analyzed. Each SLN was longitudinally sliced at 2‐mm intervals and the sensitivity, specificity, accuracy and time required for the SDB method were determined and compared with the intraoperative pathology report. Metastasis was detected in 15 SLNs (8.6%), and the sensitivity, specificity, accuracy and mean required time of the SDB method were 93.3, 96.9, 96.6 and 43.3 min, respectively. The SDB method is a novel and reliable modality for the intraoperative diagnosis of SLN metastasis.

Apical membrane localization of glycogen synthase kinase 3β protein in normal colon epithelium and aberrant distribution in colorectal cancer

Glycogen synthase kinase 3beta (GSK-3beta) was subsequently shown to function in a wide range of cellular processes. GSK-3beta is a multifunctional serine/threonine kinase which performs a role in several signaling pathways including Wnt signal transduction. Recently, the activity of membrane-localized GSK-3beta has been shown to be crucial for initiation of Wnt cascade. In our study, the membrane localization of GSK-3beta was found on the apical membrane of normal epithelium, where co-localized and directly bound with MUC1. In colorectal cancer, depolarized cells showed the aberrant distribution of GSK-3beta on the cellular membrane with beta-catenin nuclear accumulation. The aberrant distribution of the membrane-localized GSK-3beta may contribute to the development of colorectal cancer.

Hemagglutinating Activity of Mycoplasma salivarium and Its Attachment to Sheep Red Blood Cells

Mycoplasma salivarium (ATCC 23064) and 10 other strains isolated from human saliva agglutinated red blood cells of rabbits and human types A and O weakly, and those of sheep (SRBC) and human type B strongly. Glycoproteins on the surface of the organism cells and N‐acetylneuraminic acid residues and some sugars on SRBC were suggested to be involved in agglutination of SRBC. Protein A‐like activity was detected in the organism cells. The organism cells were also shown to attach to SRBC in PPLO broth (Difco) supplemented with 10% horse serum, and bivalent metal ions were suggested to be involved in the attachment. The organism cells attaching to SRBC activated complement through the alternative pathway and lyzed the SRBC.

Chemical analyses, local Shwartzman reactivity, and body weight-decreasing activity of aqueous-phenol extracts of Mycoplasma salivarium cells

Aqueous-phenol extracts of Mycoplasma salivarium ATCC 23064 cells (APM) showed demonstrable differences from lipopolysaccharides (LPSs) of Veillonella rodentium ATCC 17743. These were as follows: smaller amounts of amino sugars and an absence of 2-keto-3-deoxyoctonate; local Shwartzman reactivity and body weight-decreasing activity, even though the activities were rather weak compared with those of LPSs. Therefore, phenol-water extractive components of Mycoplasma salivarium might be of pathogenic importance in mediating damaging effects on the periodontium.

Validation of a Novel Diagnostic Kit Using the Semidry Dot-Blot Method to Detect Metastatic Lymph Nodes in Breast Cancer: Distinguishing Macrometastases From Nonmacrometastases

Micro‐Abstract We evaluated 159 lymph nodes using the kits targeted cytokeratin 19 protein compared with pathology results. The kits could distinguish macrometastases from nonmacrometastases in breast cancer, with 94.4% sensitivity, 96.2% specificity, and 95.6% accuracy. Diagnosis was achieved in approximately 20 minutes using the kits, at a cost within

D6 – PROTEOLYTIC ACTIVITIES

30 USD. The kits are accurate, fast, cost‐effective, and a dual detection method. Background: The semidry dot‐blot method is a diagnostic procedure for detecting lymph node (LN) metastases using the presence of cytokeratin (CK) in lavage fluid from sectioned LNs. We evaluated 2 novel kits that use newly developed anti‐CK‐19 antibodies to diagnose LN metastases in breast cancer. Patients and Methods: We examined 159 LNs dissected that we sliced at 2‐mm intervals and washed with phosphate‐buffered saline. The suspended cells in the lavage were centrifuged and lysed to extract protein. This extracted protein was used with a low‐power and a high‐power kit to diagnose LN metastasis. Diagnoses on the basis of the kits were compared with pathological diagnoses. Results: Of the 159 LNs, 68 were assessed as positive and 91 as negative in permanent section examination. Sensitivity, specificity, and accuracy of the low‐power kit for detecting LN metastases was 83.8%, 100%, and 93.1%, respectively. Those of the high‐power kit were 92.6%, 92.3%, and 92.5%, respectively. Combining the low‐ and high‐power kit results, those for distinguishing macrometastases were 94.5%, 95.2%, and 95.0%, respectively. Diagnosis was achieved in approximately 20 minutes, at a cost of less than

Pulmonary thrombotic microangiopathy caused by gastric carcinoma

30 USD. Conclusion: The kits were accurate, fast, and cost‐effective in diagnosing LN metastases without the loss of LN tissue.