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The radiosensitizing effect of 5-aminolevulinic acid (5-ALA) has been demonstrated in glioma and melanoma in a number of studies. Enhancing the radiosensitivity of colorectal cancer may improve survival rates and lessen adverse effects. The present study assessed the radiosensitizing effect of 5-ALA in colorectal cancer using the human colon cancer cell line HT29 in vitro and in vivo. In vitro, cells were pretreated with 5-ALA and exposed to ionizing radiation. Cells pretreated with or without 5-ALA were compared using a colony formation assay. In vivo, HT29 cells were implanted into mice subcutaneously and subsequently exposed to ionizing radiation. 5-ALA was administrated by intraperitoneal injection. Subcutaneous tumors treated with or without 5-ALA were compared. Single-dose and multi-dose irradiations were applied both in vitro and in vivo. Cells exposed to multi-dose irradiation and pretreated with 5-ALA in vitro had a significantly lower surviving fraction compared with cells without 5-ALA pretreatment. Following multi-dose irradiation in vivo, the volume of the subcutaneous tumors treated with 5-ALA was significantly lower compared with that of tumors without treatment. These results suggest that radiotherapy with 5-ALA may enhance the therapeutic effect in colon cancer.

A 49-year-old man was referred to our hospital for close examination of a submucosal tumor with ulceration located in the upper gastric body. PET-CT showed FDG uptake both in the gastric tumor and an enlarged lymph node. Although routine biopsies and EUS-FNA of this tumor did not demonstrate any malignant findings, he underwent excisional biopsy of the enlarged lymph nodes to rule out malignant lymphoma. Histopathological examination revealed that the excised specimens were metastatic nodes of poorly differentiated carcinoma probably from the known gastric tumor. Subsequently, he underwent total gastrectomy with D2, and histopathological findings confirmed the final diagnosis of gastric carcinoma with lymphoid stroma(GCLS). We report a case of GCLS that was difficult to diagnose.

Epoxyeicosatrienoic acids (EETs) are known to have beneficial pharmacological effects on various cardiovascular events. However, EETs are biologically metabolized by soluble epoxide hydrolase (sEH) to less active metabolites. In our search for potent sEH inhibitors, we optimized a series of cyclopropyl urea derivatives and identified compound 38 as a potent sEH inhibitor with minimal CYP inhibition and good oral absorption in rats. Administration of 38 to DOCA-salt rats suppressed urinary albumin and MCP-1 excretion without affecting systolic blood pressure.

Central neurofibroma of the mandible is a rare tumour. This report is of a 21-year-old Japanese man with a symptomatic solitary neurofibroma of the left mandible. The tumour was excised with preservation of the mandibular nerve. The tumour consisted of proliferated dense collagen fibres and scattered spindle cells without mitotic figures or atypism. Immunohistochemistry showed that tumour cells were positive for vimentin, S100 protein, neurone specific enolase, and glial fibrillary acidic protein, but negative for desmin and α-smooth muscle actin. The diagnosis of neurofibroma was distinguished from a desmoplastic fibroma by the immunohistochemical findings.

The effects of SM-197378, 2-[[[amino(imino)methyl]amino]carbonyl]-1-methyl-4-trifluoromethyl-1H-indol-7-yl=hydrogen=sulfate monohydrate, a novel potent Na+/H+exchange inhibitor, on heart injury were studied using a rabbit model involving 30 min of myocardial ischemia and 5 h of reperfusion. Intravenous administration of SM-197378 before ischemia reduced the infarct size by approximately 30-50% in a dose-dependent manner. This anti-necrotic effect was achieved without significant hemodynamic changes. Moreover, administration of SM-197378 before reperfusion also resulted in a significant, approximately 30-40%, reduction in the infarct size. The anti-necrotic effect of pre-ischemic bolus treatment with SM-197378 was compared with that of nicorandil, a K+channel opener with nitrate-like activity, and ischemic preconditioning. With 30 and 60 min of ischemia, the anti-necrotic effects of SM-197378 and ischemic preconditioning were similar and superior to that of nicorandil. With 90 min of ischemia, the anti-necrotic effect of SM-197378 was superior to that of ischemic preconditioning.

Experimental studies for freezing phenomena of ethylene glycol solution on an inclined polyvinyl-chloride plate have been performed. It is found that the crystal ice formed on the inclined cooled plate is removed from the plate surface due to a buoyancy force acting on the crystal ice. It is shown that the crystal ice grows in a shape of sheet by joining with neighboring ice and that the ice slides along the inclined surface when it is removed. The onset of the ice removal condition is related to the cooling heat flux at the surface. The ice removal occurs more easily for the plate of large inclination than that of a small one.

The effect of a novel potent Na+/H+ exchange inhibitor, SM-20550 [N-(aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonic acid], on survival after myocardial infarction was studied. Anesthetized rats underwent occlusion of the coronary artery (30 min) followed by reperfusion (14 days). SM-20550 was administered intravenously before ischemia (1-day treatment group) or before ischemia and on the 2 days following (3-day treatment group). The infarct size was significantly reduced on the 14th day after surgery by approximately 17 and 20% in 1- and 3-day treatment groups, respectively. The survival rate on day 14 was significantly enhanced in both treatment groups (96%) compared with the vehicle-treated control group (70%). These results suggest that SM-20550 improved survival after myocardial infarction, at least due to its antinecrotic effect.

Patched (Ptc) is a putative twelve transmembrane domain protein that is both a Hedgehog (Hh) receptor and transcriptional target of Hh. In this study, we isolated Xenopus Ptc cDNAs, Ptc-1 and Ptc-2, and carried out comparative analyses on their expression patterns. The putative Ptc-2 protein has a long C-terminal extension that has similarities in both length and sequence to those of Ptc-1 proteins in mouse, chick and human. In both early embryogenesis and hindlimb development, Ptc-2 expression is restricted to cells that receive a Hh signal, a pattern similar to that of Gli-1. Ptc-1, however, shows a broader distribution, mainly non-overlapping with that of Ptc-2. Despite the difference in their expression patterns, both are induced in animal cap explants synergistically by Shh and Noggin, showing a conserved regulation in their activation mechanisms.

The effects of N-(aminoiminomethyl)-1, 4-dimethyl-1H-indole-2-carboxamide methanesulfonic acid (SM-20550), a novel potent Na(+)/H(+) exchanger, and nicorandil, a K(+) channel opener with nitrate-like activity, were studied in a myocardial ischemia and reperfusion injury model. Anesthetized rabbits underwent occlusion of the coronary artery (30 min) followed by reperfusion (5 h). Intravenous administration of SM-20550 before ischemia reduced the infarct size by approximately 30-70% in a dose-dependent manner, with a significant reduction in serum creatine phosphokinase activity. Similarly, intravenous administration of nicorandil before ischemia reduced the infarct size by 33% with a significant reduction in serum creatine phosphokinase activity. Moreover, intravenous administration of SM-20550 after ischemia resulted in a significant, approximately 20-40% reduction in the infarct size, but the administration of nicorandil after ischemia did not reduce the infarct size. These results indicate that SM-20550 reduced myocardial necrosis when administered either before or after ischemia.

Three novel cDNAs encoding serine proteases, that may play a role in early vertebrate development, have been identified from Xenopus laevis . These Xenopus cDNAs encode trypsin-like serine proteases and are designated X enopus e mbryonic s erine p rotease (Xesp)-1, Xesp-2, and XMT-SP1, a homolog of human MT-SP1. Xesp-1 is likely to be a secreted protein that functions in the extracellular space. Xesp-2 and XMP-SP1 are likely to be type II membrane proteases with multidomain structures. Xesp-2 has eight low density lipoprotein receptor (LDLR) domains and one scavenger receptor cysteine-rich (SRCR) domain, and XMT-SP1 has four LDLR domains and two CUB domains. The temporal expressions of these serine protease genes show distinct and characteristic patterns during embryogenesis, and they are differently distributed in adult tissues. Overexpression of Xesp-1 caused no significant defect in embryonic development, but overexpression of Xesp-2 or XMT-SP1 caused defective gastrulation or apoptosis, respectively. These results suggest that these proteases may play important roles during early Xenopus development, such as regulation of cell movement in gastrulae.

The expression of calponin(CAL) and metallothionein(MT), markers of salivary gland myoepithelial cells and ductal basal cells, respectively, were evaluated in normal submandibular(n=4) and sublingual glands(n=3) and obstructive lesions of salivary glands(n=14) in humans. Immunoreactivity for CAL was observed in the normal myoepithelium in normal glands and reactive myoepithelium in the obstructive lesions. MT immunoreactivity was seen in the ductal basal cells but not in the myoepithelium of normal glands. During the degeneration of glandular cells in obstructive lesions, reactive myoepithelial cells expressed both the CAL and MT. The immunoreactivity for CAL and MT, however, was seen in the reactive myoepithelial cells of the duct like structures in the fibrotic glands during the late stages of obstructive lesions. The results of the present study reasonably allowed us to suggest that the reactive myoepithelial cells in obstructive salivary gland lesions may demonstrate cellular characteristics of both the myoepithelial/ductal basal cells of normal salivary glands with respect to phenotypic heterogeneity of expression of CAL and MT.

Calponin (CALP) is a 34kDa protein which interacts with actin, tropomyosin and calmodulin, and regulates contractive roles of smooth muscle cells. The present study deals with the expression of CALP and actin in myoepithelial cells (MECs) during postnatal development of rat salivary glands. In the postnatal development of MECs, the specimens obtained from submandibular (SMG) or sublingual glands (SLG) on 0 to 84days old rats were examined by immunocytochemistry.MECs detected by CALP and actin were observed from day 0 to mature salivary glands, and the expressions of actin immunoreaction in MECs of salivary glands on days 5 or 7 were higher than those of CALP, while in MECs of salivary glands of rats over 5 weeks old, the expressions of CALP were a little higher than in actin. MECs in SMGs and SLGs during the postnatal development were characterized by existence of CALP immunoreactivity, whereas actin immunostaining was strongly localized in the smooth muscle cells of the blood vessels. No MECs detected with CALP and actin were observed in ductal segments including striated ducts and granular convoluted tubules (GCT), suggesting that the secretion from GCT cells was not related to CALP and actin. The CALP immunoreactivity is a useful marker for MECs in salivary glands in the rat.

To study the expression and role of adhesion molecules in normal salivary glands and their tumors, we have studied simultaneously the expression of E-cadherin, and associated cytoplasmic proteins, α-catenin and β-catenin. Paraffin sections were evaluated using the streptavidin biotin peroxidase complex (SBPC) method for E-cadherin, and the SBPC method with tyramide signal amplification (TCA) for α- and β-catenin.Normal submandibular (n=10), parotid (n=4) and oral minor glands (n=4) showed cell membrane staining for E-cadherin and a positive reaction in intercalated and striated ducts for α - and β -catenin. Pleomorphic adenoma (n = 17) showed cell membrane staining for E-cadherin and α-catenin in luminal and non-luminal cells, but were negative for β-catenin. E-cadherin was also focally or irregularly positive in modified myoepithelial cells. Warthin's tumors (n = 4) expressed Ecadherin only and were negative for a- and β-catenins. Papillary adenocarcinoma (n = 4) showed predominant staining for E-cadherin, with no expression in the other tumor cells. Adenoid cystic carcinoma (n = 4) showed only cell membrane positivity for E-cadherin, with an absence of α- and β-catenins in the luminal cells. Non-luminal tumor cells of adenoid cystic carcinoma were negative for E-cadherin and positive for α- and β-catenins. The results were concluded as follows, 1) there was uniform cell membrane staining for E-cadherin in normal and benign tumor epithelium, but α- and β-catenins showed different expression and heterogeneous localization, 2) loss of E-cadherin reactivity was seen in malignant lesions, 3) there was no correlation between the distribution of Ecadherin, α-catenin and β-catenin in benign or malignant tumors of salivary gland origins. The results show variability and heterogeneous expression for E-cadherin, α- and β-catenins in salivary gland tumors.

S100 proteins are calcium-binding proteins of the EF-hand superfamily and are involved in the regulation of a number of cellular processes. The present study deals with the immunohistochemical expression of S100A1 and S10100A6 in the rat submandibular and sublingual glands during postnatal development from day 0 to 12 weeks. Expression of S100A1 was particularly concentrated in pillar and transition cells in the granular convoluted tubule (GCT) and in striated duct cells of the submandibular gland age 4 weeks to maturity. None or only weak staining for S100A1 was observed in the duct segment at 0-5 days. On the contrary, immunostaining of S100A6 was present in proacinar cells in undifferentiated submandibular gland at age 3 days to 2 weeks. S100A6 immunoreactivity in rat submandibular gland coexisted with chromogranin reactivity. It is possible that S100A6 regulates secretion of chromogranin in proacinar cells. Secretion of growth factors and biologically active peptides in the GCT are regulated by calcium signals, and S100A1 may be involved in the secretory mechanism of granular cells. S100A1 and S100A6 are potentially of great importance in secretory functions of granular cells and proacinar cells, as well as in rat salivary glands.

As a part of a study on desirable structure of environmental planning in Asia and the Pacific region, questionnaire survey and stocktaking survey were conducted in order to identify the needs of thematic plan for regional cooperation on climate change.A questionnaire consists of the questions on current situation, constraints and needs of policy and measures for climate change was sent to the national focal point of 23 countries in this region.In stocktaking survey, various documents and reports from bilateral as well as multilateral cooperation agencies were collected.As a result of the questionnaire survey, a half of the counties have prepared GHGs inventory, national plan for sustainable development and policy&measures. Most countries are facing problems and constraints including lack of coordination mechanisms among government agencies, trained personal in public and private sector, cleaner technology and financial resources even multilateral as well as bilateral cooperation agencies have been stimulating technical and financial cooperation with counties in this region. In order to bridge the gap, following actions are identified as the needs of regional cooperation: creation of information network, capacity building, policy integration, activities in various regional forum, strengthening multilateral and bilateral cooperation, research and studies, awareness raising and role of mass media, and activities in private sector.Based on the analysis of the regional characteristics of GHGs emission structure, mitigation measures for land use change is identified as a area of study to be intensified.

The expression of human epithelial related antigen (hERA), a 40 kD transmembrane glycoprotein assigned to one of the small cell lung cancer antigen was evaluated in normal salivary glands and their tumors using immunohistochemical methods.In normal salivary gland acini, hERA was seen in lateral borders and basal plasma membrane of serous cells of the parotid and submandibular glands and in the basal layer of mucous cells of the sublingual and minor oral glands, the luminally projecting cell membrane being consistently unreactive. In ductal segments, hERA was seen in basal infoldings and lateral borders of striated duct cells, excretory ducts or interlobular ducts and the ductal basal cells in particular, were devoid of hERA immunostaining.Salivary pleomorphic adenoma showed luminal borders of tubulo-ductal structures immunoreactive for hERA whereas the nonluminal or the neoplastic myoepithelial cells were consistently unreactive. A similar pattern of immunoreactivity was observed in Warthin's tumor where lateral borders of luminally located tall columnar tumor cells showed reaction products whereas the basal cells were unreactive. Mucoepidermoid carcinoma showed hERA in epidermoid and intermediate tumor cells, but not in mucous secreting cells. In adenoid cystic carcinoma reaction product for hERA was seen in a limited number of lumen-lining cells and again the non-luminally located neoplastic myoepithelial cells were unreactive. The results of the present study suggest that hERA may be a useful marker to detect luminal cell components in salivary tumor and, apart from the neoplastic myoepithelial cells or their counterparts, these luminally located cells may have a potential implication in salivary gland tumorigenesis.

Japanese Borrelia strains FujiP2, AP83, NT24, NT29 and HT2 which had a 31-kilodalton protein non-reactive with monoclonal antibody (MAb) H5332 to outer surface protein A (OspA) were identified as B. garinii by the DNA hybridization method. MAb P3134 raised to strain NT24 reacted with OspA and the OspB-ranging protein of these isolates and cross-reacted with the OspB-ranging protein of some other isolates. Since the reactive protein was extracted by the Triton X-114 phase partitioning method, the MAb recognized the common epitope present in OspA and OspB. To our knowledge, this is the first report of an MAb reactive to both OspA and OspB.

E-cadherin is a calcium dependent cell adhesion molecule, important in cell to cell interactions in epithelial tissues. The present study examined E-cadherin expression in normal salivary glands, pleomorphic adenoma, Warthin's tumor, mucoepidermoid carcinoma, acinic cell carcinoma, adenoid cystic carcinoma and adenocarcinoma of salivary glands in paraffin sections using a monoclonal antibody (5H9). This study is firstly reported E-cadherin expression in the salivary gland and tumors.In normal submandibular and parotid gland, E-cadherin staining was confined to the cell membrane of acinar and ductal cells. In excretory ducts, high columnar cells showed the strongest reaction at the basal aspect and ductal basal cells were weakly positive or negative, the basement membrane zone lacked staining. No E-cadherin reaction was observed in modified myoepithelial cells and plasmacytoid cells. In pleomorphic adenoma and mucoepidermoid carcinoma, E-cadherin was positive at the lateral cell membrane or whole cell membrane of duct-like structure and epidermoid cells. Acinic cell carcinoma was negative for E-cadherin. In adenoid cystic carcinoma, the expression was only found in cribriform areas. In adenocarcinoma, the staining was seen in the cell membrane of duct like structures and occasional cells showed a strong cytoplasmic reaction. Positive E-cadherin staining in salivary gland tumors was higher in benign tumors than in malignant tumors.

Polymorphonuclear leukocytes (PMN) are directly involved in development of ischemic myocardial injury. Adhesion of PMN to endothelial cells is an initial step that triggers a sequential process leading to acute inflammatory responses. Interaction between P-selectin and its oligosaccharide ligand, sialyl Lewis x (sLe x), plays an important role in the early stage of the adhesion. To examine the role of P-selectin in various animal disease models especially in rats, we have cloned rat E- and P-selectin cDNAs and established monoclonal antibodies against these rat selectins. In this report, we describe the generation and characterization of anti-rat P-selectin antibodies (ARPs). These antibodies detect cell surface P-selectin on thrombin-stim ulated rat platelets. More importantly, intravenous administration of ARP2-4 reduced infarction developed after 30 min of ischemia followed by 24 h of reperfusion in a rat myocardial injury model. In addition, similar protective effect was also observed by administratio n of a sLe 1oligosaccharide. These results indicate that cell adhesion mediated via P-selectin is involved in the development of ischemia and reperfusion injury in rat heart

For liquid-shaking culture of bulbs of Liliurn japonicum Thunb., we analyzed the concentration changes in macro-inorganic ions in the culture media to clarify their relationships with bulb growth.1. Inorganic ions, SO42-, NH4+, and especially H2PO4- were consumed in large quantities by bulbs during culture, whereas the consumption of K+, Mg2+, and Ca2+ was very slow.2. The optimum concentration of NH4+ for bulb enlargement was 10-20 mM. The application of NO3- alone or NH4+ higher than 30 mM as nitrogen source retarded the bulb growth. Root elongation was promoted by application of NO3- as the sole nitrogen source.3. Root growth was poor in the medium containing a high ratio of NH4+ /NO3-. Application of glutamine instead of NH4+ promoted root growth.4. The in vitro rooted bulbs sprouted and bolted abundantly after planting in the soil. Based on the above results, bulbs should be cultured first in a medium containing NH4+ for bulb enlargement and then transferred to a NH4+ free medium for root development. These treatments improved shoot growth after transplanting.5. Although the absorption rate of H2PO4- from the medium with the high concen-tration of NaH2PO4 was higher than that from the medium with the low concentration, bulb enlargement was not promoted.6. High concentration of SO42- in the medium inhibited bule growth.

培養中のユリ鱗片上に形成した不定芽を0℃で約21日間, 低温馴化 (Cold-hardening) した後, 茎頂を約1mm角の大きさで摘出した. 次に, この茎頂を0.3Mショ糖を含むMS固形培地に置床し, 16～24時間, 前培養した. 更に, 2Mグリセリンと0.4Mショ糖を含むアルギン酸ビーズで包埋してから, ガラス化液 (PVS2液) で0℃, 100分間の脱水によりビーズガラス化した. その後, 液体窒素中で急速冷却し, 40℃の温水で急速に加温した結果, 約95%のシュート形成率を示した. 茎頂をビーズで包埋した後, 乾燥するビーズ乾燥法と比べ, ビーズガラス化法では, 脱水時間が著しく短縮されるほか, シュート形成率が20～30%高く, その上, 小球の生体重が重いという利点がある. しかもビーズガラス化法は, ビーズ包埋しない従来のガラス化法と比べて, 多量の茎頂を同時に処理することが可能である. 以上のことから, ユリにおいてもビーズガラス化法は, 効率的で実用的な茎頂の超低温保存法になり得ると考えられる.

Objectives: In adenomotous hyperplasia (AH) and oncocytic adenomatous hyperplasia (OAH) of the intranodal heterotopic salivary gland tissue and Warthin's tumours of the parotid glands, the expression of various intermediate filament proteins and other tissue markers in the epithelial components were compared, since they share common histopathological characteristics with both eosinophilic luminal cells and basal cells.Materials and Methods: Tissue specimens from 79 intraparotid and extraparotid cervical lymph nodes containing heterotopic salivary gland tissue and 10 Warthin's tumours of parotid glands were evaluated for the expression of cytokeratins, vimentin, actin, epithelial membrane antigen (EMA), S-100 protein, lysozyme (Ly), lactoferrin (La), α1 -antichymotrypsin (α1-ACT), α1 -antitrypsin (α1 -AT), and human gastric and lung cancer antigens by using the three stage streptavidin-biotin immunoperoxidase method.Results and Conclusions: A similar profile of positive immunoreactivity for cytokeratins, EMA, Ly, La, α1 -ACT, α1 -AT and human lung and gastric cancer antigens was observed in the epithelial components of AH, OAH and Warthin's tumours, while vimentin, actin and S-100 protein were negative in all of them. The histopathological and immunohistochemical profiles of AH and OAH in the intranodal heterotopic salivary gland tissue were closely mimicked by the epithelial tumour cells in Warthin's tumours. These findings suggest that Warthin's tumours may sometimes, but not always, arise from the ductal cells of heterotopic salivary gland tissue included in the lymph nodes.

Antimicrobial susceptibility of the Lyme disease Borrelia, strain HP1 vi, isolated from the tick ixodes persulcatus in Hokkaido, Japan, was determined in vitro and in vivo. A broth dilution technique was used to determine the minimum inhibitory concentrations (MICs) and minimum borreliacidal concentrations (MBCs) of five antimicrobial agents. Strain HP1 vi was most susceptible to minocycline (MBC, 0.2 micrograms/ml). The other antimicrobial agents tested, aspoxicillin, cefmetazole sodium, imipenem cilastatin sodium, and panipenem betamipron, had higher MBCs of 12.5 micrograms/ml, 25 micrograms/ml, > 25 micrograms/ml, and > 25 micrograms/ml, respectively. In vivo antibiotic susceptibility study using a ddY mouse model demonstrated that minocycline and amoxicillin were effective; minocycline had a lower 50% effective dose (ED50) value (6.25 mg/kg) than amoxicillin (30 mg/kg).

Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Lilium japonicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25°C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.

Primer reactivities of 25 Borrelia burgdorferi sensu lato isolates from the ticks, Ixodes persulcatus and I. ovatus, in Japan and 10 isolates in Europe and North America were investigated. The methods used in this study were the polymerase chain reaction (PCR) on the flagellin structural gene (fla), the outer surface protein A gene (osp A) and the outer surface protein B gene (osp B), and the restriction fragment length polymorphism (RFLP) analysis of PCR products from osp A and osp B, The flagellin PCR primer set reacted with all the Borrelia strains tested. Four genospecies, B. burgdorferi sensu stricto, B. garinii, B. afzelii and B. japonica, were differentiated by PCR using osp A and osp B primers combined with RFLP analysis. Some Japanese isolates from I. persulcatus were identified as B. garinii or B. afzelii. The other isolates from I. persulcatus did not fit in any of the 4 genospecies. These results suggested that Japanese isolates from I. persulcatus are highly heterogeneous in their osp A and osp B structures. Furthermore, PCR primers targeting fla are applicable to the gene diagnosis for Lyme disease in Japan and osp A and osp B primers can be used to classify B. burgdorferi sensu lato isolates into genospecies by PCR and RFLP analyses.

Antibiotic susceptibilities of twelve borrelial isolates from skin of patients with erythema migrans (EM) and ticks (Ixodes persulcatus and I. ovatus) in Japan were examined by in vitro microdilution MIC method and macrodilution MBC method. Nine EM isolates and 3 tick isolates were susceptible to amoxicillin, erythromycin, and minocycline. MICs for Japanese isolates were 0.038-0.30 microgram/ml, < 0.012 microgram/ml, and < 0.012-0.05 microgram/ml, respectively. MBCs were as follows: 0.038-0.88 microgram/ml, < 0.012-0.10 microgram/ml, and < 0.025-0.78 microgram/ml, respectively. These antibiotics could be recommended for treatment of patients in early stage of Lyme disease in Japan.

Histatin is a group of histidine-rich polypeptides specific to parotid and submandibular gland secretions with several different biological functions such as stabilization of mineral-solute interaction in oral fluid, and antibacterial and antifungal actions. The authors generated polyclonal antibody to histatin by using purified histatin 5 as an immunogen and assayed the immunoreactivity by enzyme linked immunosorbent assay (ELISA) and immunoblotting. The antibody was further used to localize histatin in normal and tumors of salivary glans, pleomorphic adenoma, Warthin's tumor, adenoid cystic, acinic cell, mucoepidermoid, papillary cystadenocarcinoma and undifferentiated carcinoma by immunohistochemical methods. The normal major and minor human salivary glands showed an intense immunoreactivity in ductal cells, trace immunoreactivity in serous acini and no immunoreactivity in mucous acinar cells, suggesting histatin in mainly produced by ductal cells and to a lesser extent by serous cells. A consistent immunoreactivity of histatin in ductal segments of normal glands and a variable expression in the tumor cells of all the neoplastic lesions examined may implicate a role of this polypeptide in normal salivary gland function and salivary tumors. In addition, the findings may implicate common precursor cells, however, differing in the state of differentiation in normal and neoplastic conditions.

The immunoreactivity of monoclonal anti-neuron-specific enolase (MoAb NSE) on 10% formalin-fixed sections of normal human salivary glands and tumors were examined by the avidin-biotin-peroxidase complex (ABC) method. MoAb NSE staining of ductal and acinar cells of the normal salivary glands were, negative, and peripheral nerve fiber in the gland tissue showed strongly positive staining. In salivary gland tumors, pleomorphic adenoma (34 in total 68), myoepithelioma (15 in 35), monomorphic adenoma (7 in 10), adenolymphoma (15 in 23), mucoepidermoid carcinoma (3 in 16), acinic cell carcinoma (1 in 7), adenoid cystic carcinoma (12 in 20) and sialocarcinoma (4 in 15), stained focally for MoAb NSE staining. Frequency of occurrence for positive NSE activity varies among benign and malignant salivary gland tumors. In addition to MoAb NSE, S-100 protein and GFAP also demonstrated positive reactions in pleomorphic adenoma (Simpson et al Cancer 54: 1364-1369, 1984). The outer layer cells and/or peripheral cells of tubulo-ductal structure as well as modified myoepithelial cells and the cells of solid structure coexpressed with NSE, S-100 protein and GFAP. It is postulated that the salivary gland tumors particularly pleomorphic adenoma may be neuroectodermal in origin, arising from stem cells in intercalated duct segments.

A pigmented nevus is defined as a hamartomatous proliferation of nevus cells, and it is a most common skin lesion. Pigmented nevi of the oral mucosa are considered to a rare lesion with only a few cases being reported in Japan. Here, an intradermal nevus on the palate is described together with the results of immunohistochemical studies. A 44-yearold man had noted pigmentation and swelling of the palate for few years, but it was asymptomatic and not treated previously. The lesion was diagnosed to be an intradermal nevus histopathologically, and numerous melanocytic masses were seen in the connective tissue under the palatal epithelia. The melanin pigment stained with Masson-Fontana stain. Immunohistochemically, the following antibodies were tested: PoAb S-100 protein, MoAb S-100α, MoAb S-100β, MoAb or PoAb neuronspecific enorase (NSE), PoAb substance P, MoAb chromogranin A, keratin (MoAb K8.12, KL1), MoAb vimentin, and MoAb glial fibrillary acidic protein (GFAP). The melanocytes were positive to PoAb S-100 protein, MoAb S-100/β and MoAb vimentin.

MAM-3 and MAM-6 antigens of human milk fat globule membrane designated as human polymorphic epithelial mucin (PEM) were detected immunohistochemically in a total of 86 specimens from 39 cases of oral squamous cell carcinoma (SCC) and 6 cases of normal oral mucosa. The two antigens were identified by MoAbs 67D11 and 115G3 (MAM-3 antigen) and by MoAbs 115D8 and 115F5 (MAM-6 antigen) in paraffin sections. MoAb 115G3 staining was found in well keratinized foci; whereas, MoAb 115D8 and 115F5 staining was very low in keratinized tumor cells. The cytochemical distribution of the antigens was classified into 3 types; (i) cell membrane positive type, (ii) whole cell positive type and (iii) antigen negative type. Reduction and disappearance of the two antigens were correlated to the mode of tumor invasion and suggested that glycocalyx complex in cell membrane properties was changed for malignant transformation.

The distribution pattern of epidermal growth factor receptor (EGFR) was investigated in paraffin sections of squamous epithelial lesions, including benign and malignant lesions in the oral mucosa and skin. Normal oral epithelium showed a distribution of cell membrane positive type of EGFR staining; whereas, keratinized cells were negative. Benign lesions, papilloma, condyloma, and keratoacanthoma, also showed similar distribution of cell membrane positive type. Leukoplakia in the oral mucosa, senile keratosis and Bowen's disease displayed a mixed pattern with cell membrane positive type (CMPT) and focal cell membrane positive type (FCMPT). Paget's cells in Paget's disease showed whole plasma positive type distribution, as non cell membrane positive type. Squamous-cell carcinoma in the oral cavity indicated a reduction of EGFR staining. Distribution profiles were divided into 3 patterns: CMPT, FCMPT, and non cell membrane positive type (NCMPT), and occasionally, mixing of 2 or 3 patterns was found in the same squamouscell foci. Histologic, keratinized or malignancy grades in squamous cell carcinomas were not directly related to EGFR expression and distribution.

Immunohistochemical features of mucoepidermoid carcinoma (19 cases) in human salivary glands were studied by means of the detection of polymorphic epithelial mucin (PEM) assessed with the MAM-3 antigen using MoAbs 67D11 and 115G3, and the MAM-6 antigen using MoAbs 115D8 and 115F5. Immunohistochemically, the distribution of PEM was divided into three types; 1) cell membrane positive type, 2) luminal surface positive type, and 3) tumor cell cytoplasm positive type. Epidermoid tumor cells exhibited the distribution of the cell membrane positive type for MoAb 115G3 staining, however, lacked such a distribution for MoAb 67D11. Intermediate tumor cells showed a staining of luminal surface positive type with MoAb 115F5 and 115D8, negative staining for MoAb 67D11, and an irregular positive staining for MoAb 115G3. Mucin producing cells lacked PEM staining, but reacted strongly with the PAS reagent. Tumor cells of high grade malignancy displayed a heterogeneity for PEM expressions, in particular for MoAb 115G3 staining. The highest intensity of MoAb 115G3 staining appeared in tumor cells intermingled with negative tumor foci. The identification of the MAM-6 antigen showed a distribution of the irregular tumor cell cytoplasm positive type. The histogenesis of mucoepidermoid carcinoma in human salivary glands was implicated in terms of the immunohistochemical assessment of PEM detection.

The expression of epidermal growth factor receptor (EGFR) was studied in benign and malignant tumors of the breast using a monoclonal antibody of this receptor. The benign lesions were obtained from mastopathy (14 cases), fibroadenoma (12 cases), and malignant lesions, and from papillo-tubular (17 cases), solid-tubular (15 cases), and scirrhous carcinomas (10 cases). The highest incidence of positive EGFR staining was found in the scirrhous carcinomas (70%). The incidence was moderate in tubular carcinomas (60-65%), and low in solid or papillary type (13-35%). Immunolocalization of EGFR staining was divided into 1) luminal-border positive type, 2) cell-membrane positive type, and 3) cytoplasm positive type. In benign lesions, EGFR expression was usually of the luminal border or cell membrane positive type. A heterogeneity of EGFR staining was found in malignant tumors, but evidence suggests in scirrhous carcinoma that EGFR expression is positively correlated with tumor cell proliferation.

Summary 26 human fetuses were examined to elucidate the immunohistochemical distributions of lysozyme, lactoferrin, α 1 -antichymotrypsin, and α 1 -antitrypsin in prenatal salivary glands. Development of fetal salivary glands was divided into 4 stages: The early developmental stage (EDS) , the early intermediate developmental stage (EIDS) , the late intermediate developmental stage (LIDS) , and the late developmental stage (LDS) and were used to compare antigen localization during salivary gland development. Lysozyme (LY) staining was prominent in serous or demilune cells of the mucous acinar compartment. Lactoferrin (LF) was rarely seen in the fetal glands; only trace amounts were seen in serous cells. α 1 -antichymotrypsin (α 1 - ACT ) was diffusely positive particularly in glandular ducts. α 1 -antitrypsin (α 1 - AT ) was also diffusely distributed in all salivary gland elements and was more abundant in ductal cells than acinar cells. During the EDS, immunohistochemical staining of LY, LF, α 1 -ACT, and α 1 -AT could be observed with glandular intensity increases corresponding to the advance of cytodifferentiation of granular epithelium occuring in the subsequent EIDS and LIDS. Staining intensities were continuous during the LDS even though the amount of those materials in the fetal salivary glands was not of the extent seen in the adult salivary gland. These results suggest that production of LY, LF, α 1 -ACT, and α 1 -AT was positive during prenatal development of human salivary glands. The present study discusses the protective roles and defense mechanisms of LY, LF, α 1 -ACT, and α 1 -AT in developing human salivary glands.

Background: Cultured gingival substitute has been found to be a useful graft material for treatment of gingival recession. However, such substitutes include xenograft derivative materials that involve concomitant risk of viral contamination. To eliminate this risk, we designed new gingival substitutes made of recombinant human collagen types I and III sponges and cultured these substitutes in animal-free media (HFDM-1). Methods: Gingival fibroblasts were seeded onto sponges of type I or III recombinant collagen. These sponges were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS), HFDM-1 with 2% human serum (HS), or HFDM-1. Fibroblast proliferation in these samples was compared using the cell-counting kit assay. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) released into the cultured media were examined by enzyme-linked immunosorbent assay. Results: The fibroblasts proliferated significantly in all six combinations of collagen and medium types. The fibroblast growth rate after 9 days of culture was equal between HFDM-1 with 2% HS and DMEM with 10% FBS. The type III collagen sponge showed a higher fibroblast growth rate than the type I sponge. VEGF concentrations in HFDM-1 with 2% HS were higher than those in other media. The highest HGF levels were detected in DMEM with 10% FBS. Conclusions: The new cultured gingival substitute containing no animal-derived materials produced good cell proliferation and VEGF release. The results suggested that the substitute may provide a new tool for the treatment of gingival recession.

The effects of 3-[2-({[amino(imino)methyl]amino}carbonyl)-4-chloro-1H-indol-1-yl]-1-propanesulphonic acid monohydrate (SM-198110), a novel potent Na+/H+ exchange inhibitor, and cariporide (Hoe642), another Na+/H+ exchange inhibitor, were studied in a myocardial ischaemia and reperfusion injury model. Anaesthetized rabbits were subjected to occlusion of the coronary artery for 30 min followed by reperfusion for 5 h. SM-198110 or cariporide was administered before ischaemia and before reperfusion. We also assessed the anti-necrotic effect of SM-198110 when given before reperfusion, both alone and together with glibenclamide, a K(ATP) channel blocker, 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) channel-selective blocker and 8-(p-sulphophenyl)-theophylline (8-SPT), an adenosine receptor blocker. The infarct size was reduced dose-dependently by i.v. administration of SM-198110 before ischaemia, with a significant reduction in serum creatine phosphokinase activity. Infarct sizes, normalized to the size of the area-at-risk (means+/-SE) were: vehicle 56.6+/-3.7%; low-dose SM-198110 39.2+/-6.3%; mid-dose 32.8+/-7.4% (P0.05); high-dose 22.1+/-6.7% (P0.01). This anti-necrotic effect of SM-198110 was achieved without significant haemodynamic changes. Cariporide given before ischaemia also reduced infarct size significantly and dose-dependently. SM-198110 administered before reperfusion also resulted in a dose-dependent reduction in the infarct size. Infarct sizes were: vehicle 56.6+/-3.7%; low-dose SM-198110 44.5+/-5.7%; mid-dose 36.3+/-6.6% (P0.01); high-dose 34.7+/-3.8% (P0.01). In contrast, cariporide given before reperfusion did not reduce infarct sizes significantly. The anti-necrotic effect of SM-198110 was observed even when given 10 min after the beginning of reperfusion. Glibenclamide and 5-HD abolished the anti-necrotic effect of treatment before reperfusion with SM-198110. However, the co-administration of 8-SPT with SM-198110 did not affect infarct size. These results suggest that, in addition to Na+/H+ exchange inhibition, mitochondrial and/or sarcolemmal K(ATP) channels contribute to the anti-necrotic effect of SM-198110 when the latter is given before reperfusion.

The protective effects of the Na+-H+ exchange (NHE) inhibitors SM-198110 (2-[[(aminoiminomethyl) amino] carbonyl]-4-chloro-1H-indole-1-propanesulfonic acid monohydrate) and SM-197378 (N-(aminoiminomethyl)-1-methyl-7-(sulfooxy)-4-(trifluoromethyl)-1H-indole-2-carboxamide monohydrate) were investigated in perfused Langendorff guinea-pig hearts subjected to ischemia (40 min) and reperfusion (40 min). The recovery of left ventricular developed pressure (LVDP) from ischemia by reperfusion was 39.0% in the control, while in the hearts pretreated with SM-198110 or SM-197378 (10(-7) M), it was about 100%. The ATP level, monitored simultaneously by (31)P-nuclear magnetic resonance spectrometry, was already higher than the control value at the end of the ischemic period, and the elevation in Na+ or Ca2+ fluorometric signals induced during ischemia was suppressed. In post-treated hearts, the LVDP recovery rate was significantly higher with SM-198110 than with SM-197378. By in vitro electron paramagnetic resonance spectrometry, SM-197378 was found to directly quench the active oxygen radical, whereas SM-198110 had no effect. Numbers of apoptotic cardiomyocytes after ischemia (1 h) followed by reperfusion (5 h) were significantly lower in SM-197378-treated than in SM-198110-treated hearts, consistent with the level of activity of caspase-3. These results suggest that the antioxidant effects of NHE inhibitors have an important role in apoptosis during ischemia-reperfusion, but apoptosis is not a major manifestation of cardiac function during postischemic recovery, and that NHE-sensitive mechanisms of reperfusion injury promote both necrotic and apoptotic processes death.

Recent studies suggest that extra-embryonic tissues may be essential sources of early organizing signals for the mouse embryo. In vitro studies of human amniotic epithelial cells (HAEC) have shown that the amnion can produce various biologically active substances. In this study, the synthesis and release of activin A and noggin, and the activin signaling pathway, was investigated in HAEC. Conditioned medium from cultured HAEC contained activin A which was functionally active in Xenopus laevis animal cap assays. Immunohistochemistry, western blotting and reverse transcription-polymerase chain reaction confirmed that HAEC also synthesize and release noggin. Noggin transcripts were induced by the addition of recombinant activin A, and activin A was inhibited by activin antibody except in the presence of cycloheximide (CHX). These data demonstrate that noggin mRNA expression is induced directly by activin A without new protein synthesis, indicating that noggin is a primary response gene. The results suggest that there is an activin signaling pathway in HAEC, and that the human amnion might therefore be involved in neural formation during early development.

The effect of an Na+/H+ exchange inhibitor, SM-20550, on the adhesion, emigration and accumulation of leukocytes was studied in ischemia and reperfusion injury models using rat mesenteric venules or rabbit heart. Anesthetized rats underwent occlusion of the superior mesenteric artery (20 min) followed by reperfusion (60 min). After ischemia and reperfusion, the numbers of adherent and emigrated leukocytes increased significantly. Bolus intravenous administration of SM-20550 reduced the numbers of adherent and emigrated leukocytes in a dose-dependent manner, with statistical significance at dosages >0.1 mg/kg. Anesthetized rabbits underwent occlusion of the coronary artery (30 min) followed by reperfusion (5 h). Intravenous administration of SM-20550 before ischemia significantly reduced the neutrophil accumulation in the area-at-risk by 46% and reduced the infarct size by 38%. These results suggest that the inhibitory effect of SM-20550 on the neutrophil accumulation could be one of the mechanisms by which this drug limits the severity of infarctions.

The control mechanism of the anteroposterior axis specification in Xenopus epidermis was studied by comparing the expression of a novel anterior marker, Xepsin, with that of a panepidermal marker, type I keratin. Xepsin mRNA, which encodes a novel Xenopus serine protease, is transcribed zygotically with the expression peak in neurula stages. In normal development, its expression is limited to the anterior and anterior–dorsal portions within epidermis during neurula and tailbud stages, respectively. In UV-irradiated ventralized embryos (dorsoanterior index, DAI 0 and 1), an expression boundary for Xepsin is apparently formed within the epidermis. In contrast, Xepsin expression was observed throughout the epidermis in LiCl-treated dorsalized embryos (DAI 10), as seen from an expression pattern indistinguishable from that of type I keratin. These data suggest that posteriorizing signals which suppress the transcription of Xepsin are present in nonaxial regions and absent in the anterior dorsal mesoderm. That posteriorizing signals were present in nonaxial regions was also supported by a conjugation experiment in which Xepsin expression was suppressed in ectodermal explants conjugated with lateral or ventral marginal zone. Moreover, the partly suppressed expression of Xepsin in the epidermal region of exogastrulae indicates that the signals may travel horizontally within the plane of the epidermis. We also present data showing that both treatment with retinoic acid and the overexpression of a constitutively active form of a retinoic acid receptor caused the suppression of Xepsin mRNA transcription, suggesting that anterior–posterior patterning in the central nervous system and in the epidermis may share common endogenous factors, i.e., retinoids, in the Xenopus embryo.

The expression of P-selectin on postischemic endothelium after reperfusion has been shown to trigger neutrophil attachment and the subsequent inflammatory responses. Extensive studies have demonstrated that P-selectin is involved in the progression of neutrophil-mediated myocardial infarction and no-reflow phenomenon. In the present study, we examined the effects of selectin inhibitors, sialyl Lewis X-oligosaccharide and anti-P-selectin monoclonal antibody, PB1.3 on neutrophil-dependent left ventricular dysfunction in isolated rat heart. The hearts were subjected to global ischemia for 20 min and then reperfused for 45 min with rat plasma in the presence of human neutrophils during the first 5 min of the reperfusion. Left ventricular developed pressure and other parameters of the left ventricular function deteriorated throughout the reperfusion period in a neutrophil-dependent manner. In contrast, the coronary flow was reduced early on (< 15 min) but recovered to the level in the hearts reperfused with no neutrophils 45 min after the reperfusion. We examined the effects of selectin inhibitors under experimental conditions in which the hearts were perfused with 30 million neutrophils. The treatment with sialyl Lewis X-oligosaccharide at a dose of 0.3 mg/min resulted in amelioration of left ventricular developed pressure to 57.2 +/- 14%, compared to 26.1 +/- 4.3% in the saline-treated group (P < 0.05). Similarly, the treatment with mouse anti-human P-selectin monoclonal antibody (IgG1) PB1.3 at a dose of 0.6 mg/min resulted in the prominent recovery of left ventricular developed pressure after 45 min of reperfusion (59.9 +/- 9.3% vs. 26.1 +/- 4.3% in the saline-treated group, P < 0.05). PB1.3 also attenuated the elevation of left ventricular end-diastolic pressure compared to that of the saline-treated group during the reperfusion period. Moreover, the treatment with PB1.3 ameliorated the recovery of coronary flow until 10 min after the reperfusion and the recovery of coronary flow 10 min after the reperfusion was 55.2 +/- 9.2%, as compared to 28.2 +/- 7.7% in saline-treated hearts (P < 0.05). To our knowledge, this is the first direct demonstration that the specific inhibition of P-selectin results in the inhibition of neutrophil-mediated left ventricular dysfunction or myocardial stunning.