Binding of αvß1 and αvß6 integrins to tenascin-C induces epithelial-mesenchymal transition-like change of breast cancer cells.

Tenascin-C (TNC), a large hexameric extracellular glycoprotein, is a pleiotropic molecule with multiple domains binding to a variety of receptors mediating a wide range of cellular functions. We earlier reported that TNC induces epithelial-mesenchymal transition (EMT)-like change in breast cancer cells. In the present study, we clarified TNC receptor involvement in this process. Among integrins previously reported as TNC receptors, substantial expression of αv, α2, ß1 and ß6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin ß6 mRNA was remarkably upregulated by transforming growth factor (TGF)-ß1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin αvß6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-ß1. The α2 and ß1 subunits were mainly localized at cell-cell contacts, αv being found near cell cluster surfaces. Immunoprecipitation showed increase in αvß1 heterodimers, but not α2ß1, after TNC treatment. Activated ß1 subunits detected by an antibody against the Ca(2+)-dependent epitope colocalized with αv in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against αv and ß1 blocked EMT-like change caused by TNC alone. In addition, anti-αv and combined treatment with anti-ß1 and anti-αvß6 inhibited TGF-ß1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits αv, ß1 and ß6, but not α2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against ß1 and αvß6 reduced αv subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of ß6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of αvß6 and αvß1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.

A case of lobular carcinoma in situ presenting as a solid mass.

A patient presented with a 2 cm lump in the lower outer quadrant of the left breast. Mammogram and ultrasonography showed a solid mass with a microlobulated contour, partially irregular border and microcalcifications. MRI showed an irregular mass with early enhancement and high signal intensity, and the late-phase image demonstrated a partial washout pattern. These findings suggest that the tumour was a malignant invasive carcinoma. Non-invasive ductal carcinoma was diagnosed after a fine needle aspiration and core needle biopsy followed by a partial breast excision and sentinel lymph node (SLN) biopsy. A pathological examination of the lesion displayed characteristic small monomorphic cells, solid proliferation and massive distension within the lobular unit. The tumour was immunohistochemically negative for E-cadherin and pure lobular carcinoma in situ (LCIS) was diagnosed. Pure LCIS is very rare and there have been no previous reports of pure LCIS forming a solid mass.

Mucoepidermoid carcinoma of the anal canal: an immunohistochemical study.

We present a case of mucoepidermoid carcinoma of the anal canal, with special reference to immunohistochemical analysis of the tumor to clarify its histogenesis. A 36-year-old man underwent surgery for mucoepidermoid carcinoma of the anal canal. Immunohistochemical analysis of the resected specimen was performed. Serial sections were stained immunohistochemically by the labeled streptavidin-biotin peroxidase method for various antigens, including epithelial membrane antigen (EMA); carcinoembryonic antigen (CEA); different types of cytokeratins, including CK10 and CAM 5.2; and p53 oncoprotein. The solid component of the tumor cells was immunohistochemically positive for EMA, CEA, and CAM 5.2, but negative for CK10. These staining patterns were different from those of anal squamous epithelium. These results confirm that mucoepidermoid carcinoma of the anus may arise from the anal transitional zone, and that it is biologically different from squamous cell carcinoma of the anus.

Gastric seromuscular and omental pedicle flap for bronchopleural fistula after pneumonectomy.

We report a case of postpneumonectomy bronchopleural fistula treated using a gastric seromuscular and omental pedicle flap and maintaining good postoperative respiratory function. A 76-year-old man underwent right pneumonectomy with regional lymph node dissection for squamous cell carcimoma of the lung. Five weeks later, a bronchopleural fistula occurred. Empyema with the bronchopleural fistula was diagnosed and chest tube drainage implemented immediately. Despite the drainage, signs of inflammation persisted and the patient's nutrition did not improve leading to surgery, on August 18, 1997. The bronchopleural fistula was closed by horizontal suture proximal to the stapling sutured line. A gastric seromuscular and omental pedicle flap was sutured as a cover over the bronchial stump. Postoperative analysis of respiratory function and arterial blood gas showed good results.

A detailed linkage map of medaka, Oryzias latipes: comparative genomics and genome evolution.

We mapped 633 markers (488 AFLPs, 28 RAPDs, 34 IRSs, 75 ESTs, 4 STSs, and 4 phenotypic markers) for the Medaka Oryzias latipes, a teleost fish of the order Beloniformes. Linkage was determined using a reference typing DNA panel from 39 cell lines derived from backcross progeny. This panel provided unlimited DNA for the accumulation of mapping data. The total map length of Medaka was 1354.5 cM and 24 linkage groups were detected, corresponding to the haploid chromosome number of the organism. Thirteen to 49 markers for each linkage group were obtained. Conserved synteny between Medaka and zebrafish was observed for 2 independent linkage groups. Unlike zebrafish, however, the Medaka linkage map showed obvious restriction of recombination on the linkage group containing the male-determining region (Y) locus compared to the autosomal chromosomes.

Effect of pancreatic juice reflux into biliary tract on N-nitrosobis(2-oxopropyl)amine (BOP)-induced biliary carcinogenesis in Syrian hamsters.

To elucidate the possible role of pancreatic juice reflux into the biliary tract in promoting the development of biliary carcinoma, Syrian hamsters were subjected to cholecystoduodenostomy and ligation of the distal end of the common duct and then subcutaneously injected with N-nitrosobis(2-oxopropyl)amine (BOP) (experimental group). The incidences of gallbladder carcinoma and extrahepatic bile duct carcinoma in the experimental group was significantly higher than in the sham-operated group (P < 0.01, P < 0.05). The proliferating cell nuclear antigen (PCNA) labeling indices of both regions gradually increased with time, and were significantly higher in the experimental group at weeks 9 and 16 than in the sham-operated group at the same time. Trypsin and phospholipase A2 (PLA2) activities in bile and tissue levels of superoxide dismutase (SOD) in the gallbladder and extrahepatic bile ducts were higher in the experimental group than in the sham-operated group. These findings suggest that the carcinogenic effect of BOP was enhanced in biliary epithelium that had proliferated in response to and/or had been injured by activated pancreatic enzymes refluxing into the biliary tract and then increased free radical activity, leading to a high frequency of carcinoma development in the biliary tract.

Molecular cloning, expression and tissue distribution of canine lipopolysaccharide (LPS)-binding protein.

The plasma lipopolysaccharide (LPS)-binding protein (LBP) plays an important role in infection caused by Gram-negative bacteria. It is markedly increased during acute-phase responses. In this study, we cloned the full length of canine LBP cDNA and determined its amino-acid sequence and its expression in several canine tissues. The isolated LBP cDNA contained a 1443-bp coding region, which encodes a 25-amino-acid signal peptide, a 456-amino-acid mature LBP and a stop codon, and a 3'-noncoding region containing a TATTTAT motif, which is probably involved in the degradation and/or suppression of mRNA translation. The amino-acid sequence of the mature canine LBP showed 78%, 66% and 67% identity with that of human, rat and rabbit LBPs, respectively. In transient expression assays, canine and human LBPs accelerated the production of tumor necrosis factor-alpha induced by LPS in human monocytes. Northern blot analysis showed that LBP mRNA is mainly expressed in the liver and kidneys of normal dogs. In situ hybridization analysis revealed that the canine LBP mRNA is mainly located in hepatocytes and in epithelial cells of the proximal urinary tubules of the kidneys. These findings suggest that LBP is produced in organs readily exposed to LPS, where it probably plays an important role in bacterial infections, particularly in those occurring after major surgery.

Expression of fibronectin and tenascin-C mRNA by myofibroblasts, vascular cells and epithelial cells in human colon adenomas and carcinomas.

To understand the mechanisms of tissue remodeling during cancer progression, it is important to know the type of cells that actively express extracellular matrix (ECM) proteins. Twenty-nine adenocarcinomas, 5 adenomas and non-neoplastic mucosa samples were therefore investigated to determine their fibronectin (FN) and tenascin-C (TN-C) expression using in situ hybridization (ISH) and immunohistochemical staining. In the non-neoplastic mucosa, no mRNA signals were found. Two of the adenomas demonstrated positive signals in peri-cryptal cells and the vessels. In the cancers, TN-C and FN mRNAs were found in 86% and 96% of the total cases, respectively. The signals were mainly detected in myofibroblasts, labeled with alpha-smooth muscle actin, in the cancer stroma. TN-C mRNA-positive cells were often observed in localized areas, such as in cancer stroma associated with invading edges and/or in host tissues surrounding the invading cancer front, but rarely in the center of the tumors. FN mRNA-positive cells were more widely spread throughout the cancer stroma, although they were also frequently observed at invading edges. Vascular cells in cancer tissues were also labeled. In 10 specimens, cancer cells themselves expressed FN and/or TN-C mRNA. Comparison with histo-pathological findings revealed positive relationships between the degree of mRNA expression of FN and TN-C and the depth of invasion as well as the frequency of metastasis to lymph nodes. The expression of FN and TN-C by myofibroblasts, vascular cells and cancer cells could be important for the remodeling process of neoplastic tissues during cancer development and progression.

Co-expression of tenascin and fibronectin in epithelial and stromal cells of benign lesions and ductal carcinomas in the human breast.

Tenascin (TN)-C and fibronectin (FN), which are glycoproteins of the extracellular matrix (ECM), are up-regulated in cancer tissues, including breast cancer. For assessment of their involvement in cancer invasion, it is important to know which cells are responsible for their production and secretion. The distribution of cells expressing TN and FN mRNAs in benign and malignant human breast tissues was therefore analysed by in situ hybridization, using digoxigenin-labelled cRNA probes, in addition to demonstrating the proteins immunohistochemically. Both mRNAs were expressed in epithelial cancer as well as in stromal cells in a large fraction of the tumours, with co-expression in individual cells. In cancers with intraductal components and/or those consisting of large nests, the mRNAs were more often expressed in the cancer than in the stromal cells. In scirrhous carcinomas, in contrast, the stromal cells were almost always positive for TN and FN mRNAs, while the cancer cells only rarely exhibited TN or FN expression. In benign lesions including adenosis, fibroadenoma and intraductal papilloma, the expression patterns also varied. These findings indicate that TN and FN co-expressed by cancer cells and stromal cells are probably involved in the intraductal extension and early invasion of cancer cells and in the remodelling of cancer stroma.

Expression of basic fibroblast growth factor in hyperplastic parathyroid glands from patients with multiple endocrine neoplasia type I.

Primary hyperparathyroidism was investigated using the presence of basic fibroblast growth factor (bFGF) from the immunohistochemical viewpoint with an anti-bFGF antibody in hyperplastic parathyroid glands of patients with multiple endocrine neoplasia type I (MEN-I) and of patients with non-MEN. The results corresponded well with the data from the DNA analysis. Twenty-five hyperplastic parathyroid glands from 11 patients with MEN-I and 38 glands from 20 patients with non-MEN primary hyperparathyroidism were stained immunohistochemically according to the avidin-biotin-peroxidase complex procedure. When 50% or more of the cells appeared uniformly stained, it was judged positively stained. In addition, 18 hyperplastic parathyroid glands from patients with MEN-I patients and 24 hyperplastic parathyroid glands from non-MEN patients were also analyzed for DNA using flow cytometry. The ratio of positively stained hyperplastic parathyroid glands was 72% in MEN-I patients and 18% in non-MEN patients. The difference between the two groups was significant (p < 0.01). The nodules consisted of oxyphilic cells in 7 of 25 hyperplastic parathyroid glands from MEN-I patients and in 10 of 38 hyperplastic parathyroid glands from non-MEN patients, and all the cells were positive for bFGF. There was no significant correlation between bFGF staining and the DNA ploidy pattern. bFGF possibly plays a role in the development of parathyroid gland hyperplasia, especially in MEN-I patients. The increase of oxyphilic cells may be correlated with the expression of bFGF.

Preoperative diagnosis of the follicular variant of papillary carcinoma of the thyroid: discrepancy between image and cytologic diagnoses.

Preoperative diagnosis of the follicular variant of thyroid papillary carcinoma has not been examined sufficiently. The preoperative diagnosis of six patients with the follicular variant was investigated retrospectively as compared with 46 cases of the common type of papillary carcinoma (papillary structure dominant type), four of follicular carcinoma, 27 of follicular adenoma, and 42 of adenomatous goiter. Cervical soft radiography of the disease exhibited calcification in three of six cases (50%), all of which showed coarse calcification not accompanied by minute type. 201Tl scintigraphy of the disease revealed that four of six cases (66.7%) showed abnormal accumulation in the delayed phase. Four cases had favorable washout and one (16.7%) unfavorable washout. On ultrasonography, one of six cases (16.7%) was diagnosed as papillary carcinoma, four as follicular adenoma, and one as adenomatous goiter. In fine-needle aspiration cytology, two of five cases were diagnosed as papillary carcinoma of class V, two as papillary carcinoma of class IV, and one as follicular tumor suspected of class IIIa. On the other hand, true positive diagnostic rates of the histological types of tumors other than follicular carcinoma by ultrasonography or fine-needle aspiration cytology were approximately 80% and relatively satisfactory. Fine-needle aspiration cytology was the most useful method of diagnosing the follicular variant of papillary carcinoma. It seems necessary to consider the possibility of the follicular variant in diagnosis when papillary carcinoma is suspected from fine-needle aspiration cytology by preoperative diagnosis, while imaging suggests follicular tumor.

A family with hereditary high serum thyroxine-binding globulin.

A family with hereditary high serum thyroxine-binding globulin was studied. All the subjects studied were clinically euthyroid without goiter. The propositus (female), her mother and sister had high TBG, total T4 and total T3 with normal free T4, free T3 and TSH. Her father's thyroid function was within the normal range. Possible etiologic factors causing secondary hyper-TBG-nemia were ruled out in all the affected subjects. Isoelectric focusing demonstrated qualitatively normal microheterogeneity, ruling out possible TBG variations caused by liver diseases, estrogen therapy or pregnancy. Although the mechanism involved in the TBG increase awaits further investigation, it could be an abnormality in the gene controlling the synthesis of TBG.

Structural specificity of peptides in Z-DNA formation and energetics of the peptide-induced B-Z transition of poly(dG-m5dC).

The effects of oligopeptide binding in the conformation of double-stranded poly(dG-m5dC) have been examined by circular dichroism spectroscopy. Conversion from the right-handed B-form to the left-handed Z-form occurs very efficiently in the presence of peptides having amino acid sequences of the (Lys-X)n-Lys type, where X is a non-bulky amino acid residue such as Gly, Ala and Lys. The ability to induce the B-Z transition increases rapidly with increasing repeat number n, and the peptides with n = 2-3 readily convert the B-form DNA to the Z-form at a peptide/nucleotide molar ratio of 0.06 to 0.08 (one peptide per 13 to 17 nucleotides). The peptide-induced B-Z transition of poly(dG-m5dC) duplexes takes place even at a physiological salt concentration (150 mM NaCl). The activation energy and van't Hoff enthalpy of the B-Z conversion have been determined to be 124 (+/- 7) and 138 (+/- 12) kJ/mol, respectively, for the (Lys-Gly)2-Lys-induced transition. The van 't Hoff enthalpy of the peptide-induced B-Z transition is particularly small compared to those of the transitions caused by other inducers. The large enthalpic contribution to the Z-DNA formation indicates that the peptide binds preferentially to the Z-arranged DNA phosphate groups and stabilizes the Z-stretch significantly. It is pointed out that some DNA-binding proteins have amino acid sequences that favor Z-DNA.

[A clinical study of the significance of the gradient between feeding arterial tissue polypeptide antigen (TPA) and draining venous TPA in assessing the risk for recurrent breast cancers].

The incidence of postoperative recurrence of primary breast cancers in 73 patients was correlated with the blood levels of tissue polypeptide antigen (TPA) in peripheral veins (V-TPA), feeding arteries (A-TPA), draining veins (V-TPA) and the gradient between the TAP levels in draining veins and feeding arteries (V-A TPA). With progression in the stage of the cancers, all parameters were increased. In stages II and III, the mean level of V-TPA was significantly higher than the mean level of A-TPA (p less than 0.05). In each stage, each parameter was increased in the patients with recurrences when compared with the levels in the patients with no recurrences. In patients with V-A TPA gradients greater than 30 U/L, the rate of recurrence was significantly higher when compared with patients with gradients less than 30 U/L (p less than 0.05). The clinical significance of this finding is that patients with V-A TPA gradient greater than 30 U/L should be closely followed because of the increased risk of recurrences.

B-Z transition of poly(dG-m5dC) induced by binding of Lys-containing peptides.

Effects of oligopeptides containing Lys residues on the conformation of poly(dG-m5dC) have been investigated by circular dichroism spectroscopy. Lys-Ala-Lys (KAK) and its longer analogs with Lys-Ala repeats are found to convert the B-form polynucleotide to the Z form very efficiently. The ability to induce the B-Z transition is characteristic of alternating Lys-Ala sequences and increases exponentially with increasing number of the repeats. The heptapeptide KAKAKAK has an ability comparable with that of spermine, one of the most effective inducers hitherto known. The present results provide the first example of the B-Z transition of poly(dG-m5dC) induced by peptide binding.

[Study on composite resin cores. Part 2. Effect of pressing composite resin].

In this study, we investigated effects of pressing in building composite resin cores on compressive strength, retention of prefabricated post and sealing ability. 1. Compressive strength increased by pressing in composite resin cores. 2. Prefabricated post to the retentive forces was not influenced by pressing, but increased when bonding agent was used. 3. Pressing was very effective to improve sealing ability of composite resin to tooth structure. 4. Sealing ability of light-cured composite resin was not influenced by pressing but by curing types of bonding agent.

[Effect of soft laser on pain].

Two kinds of semiconductor laser, STOMA-LASER and Four-Luck, were used for patients having pain in the mouth and their effective rates were compared in 93 patients (133 cases). As a result, the following results were obtained. 1. The effective rate was 70.7% at the stage just after radiation. 2. Age, sex and oral hygiene were not significant for the effective rate. 3. The kinds of diseases were remarkably significant. 4. The kind of apparatus did not play any role in the effective rate as a whole, but was significant for the kinds of disease.

[Study on fatigue toughness of dental materials. 1. Compressive strength on various luting cements and composite resin cores].

In this study, we investigated compressive strength of various luting cements and composite resin cores in both dry and wet condition, and then influences of repeating load on compressive strength in wet condition of distilled water at 37 degrees C. As frequency of repeating load increased, compressive strength of all materials decreased. It means that the repeated load cycling test used in this study is adequate for evaluating durability of various dental materials. The results were as follows: 1. In the condition of no loading in both dry and wet condition, resin cement indicated the highest compressive strength of all cements examined and was followed by glass ionomer cement, zinc phosphate cement and polycarboxylate cement. Glass ionomer cement was notably influenced in wet condition. 2. After 10,000 cycles of loading in wet condition, resin cement indicated the highest compressive strength again and was followed by glass ionomer cement, polycarboxylate cement and zinc phosphate cement. In particular, compressive strength of zinc phosphate cement decreased remarkably. 3. In the condition of no loading, visible light-cured composite resin core was superior to chemical one. Visible light one was notably influenced in wet condition. 4. After 10,000 cycles of loading, visible light-cured composite resin core was superior to chemical one.

[Composite resin cores. Part 1. Comparison of various composite resin cores].

This study evaluated compressive strength, sealing ability and tensile bond strength of various materials of composite resin cores. 1. Compressive strength of the various materials, after they were placed in air for 24 hours, underwent thermal cycling testing, or were soaked in water for 2 weeks, ranged from 2000 kgf/cm2 to 2900 kgf/cm2. 2. The sealing ability after thermal cycling testing and being soaked in water for 2 weeks varied among the materials. 3. The bond strength of all composite resin core materials to dentin was less than 50 kgf/cm2. 4. In this study, no relationship was found between sealing ability and tensile bond strength.