PARVB overexpression increases cell migration capability and defines high risk for endophytic growth and metastasis in tongue squamous cell carcinoma.

BACKGROUND: Tongue squamous cell carcinoma (TSCC) is highly diverse, even in its early stages. This cancer is classified into three subtypes (superficial, exophytic, and endophytic) based on macroscopic appearance. Of these subtypes, the endophytic tumours have the worst prognosis because of their invasiveness and higher frequency of metastasis. METHODS: To understand the molecular mechanism underlying the endophytic subtype and to identify biomarkers, we performed a comprehensive gene expression microarray analysis of clinical biopsy samples and also confirmed the clinical relevance of differential gene expression. RESULTS: Expression of the parvin-beta (PARVB) gene and its encoded protein was significantly upregulated in endophytic-type TSCC. PARVB is known to play a critical role in actin reorganization and focal adhesions. Knockdown of PARVB expression in vitro caused apparent decreases in cell migration and wound healing, implying that PARVB has a crucial role in cell motility. Moreover, metastasis-free survival was significantly lower in patients with higher tumour expression of PARVB. CONCLUSIONS: These findings suggest that PARVB overexpression is a candidate biomarker for endophytic tumours and metastasis. This protein may be a clinically useful target for adjuvant TSCC therapy.

Development of a telediagnosis endoscopy system over secure internet.

OBJECTIVES: We developed a new telediagnosis system to securely transmit high-quality endoscopic moving images over the Internet in real time. This system would enable collaboration between physicians seeking advice from endoscopists separated by long distances, to facilitate diagnosis. METHODS: We adapted a new type of digital video streaming system (DVTS) to our teleendoscopic diagnosis system. To investigate its feasibility, we conducted a two-step experiment. A basic experiment was first conducted to transmit endoscopic video images between hospitals using a plain DVTS. After investigating the practical usability, we incorporated a secure and reliable communication function into the system, by equipping DVTS with "TCP2", a new security technology that establishes secure communication in the transport layer. The second experiment involved international transmission of teleendoscopic image between Hawaii and Japan using the improved system. RESULTS: In both the experiments, no serious transmission delay was observed to disturb physicians' communications and, after subjective evaluation by endoscopists, the diagnostic qualities of the images were found to be adequate. Moreover, the second experiment showed that "TCP2-equipped DVTS" successfully executed high-quality secure image transmission over a long distance network. CONCLUSIONS: We conclude that DVTS technology would be promising for teleendoscopic diagnosis. It was also shown that a high quality, secure teleendoscopic diagnosis system can be developed by equipping DVTS with TCP2.

GASDERMIN, suppressed frequently in gastric cancer, is a target of LMO1 in TGF-beta-dependent apoptotic signalling.

Defining apoptosis-regulatory cascades of the epithelium is important for understanding carcinogenesis, since cancer cells are considered to arise as a result of the collapse of the cascades. We previously reported that a novel gene GASDERMIN (GSDM) is expressed in the stomach but suppressed in gastric cancer cell lines. Furthermore, in this study, we demonstrated that GSDM is expressed in the mucus-secreting pit cells of the gastric epithelium and frequently silenced in primary gastric cancers. We found that GSDM has a highly apoptotic activity and its expression is regulated by a transcription factor LIM domain only 1 (LMO1) through a sequence to which Runt-related transcription factor 3 (RUNX3) binds, in a GSDM promoter region. We observed coexpression of GSDM with LMO1, RUNX3 and type II transforming growth factor-beta receptor (TGF-betaRII) in the pit cells, and found that TGF-beta upregulates the LMO1- and GSDM-expression in the gastric epithelial cell line and induces apoptosis, which was confirmed by the finding that the apoptosis induction is inhibited by suppression of each LMO1-, RUNX3- and GSDM expression, respectively. The present data suggest that TGF-beta, LMO1, possibly RUNX3, and GSDM form a regulatory pathway for directing the pit cells to apoptosis.

Stereoselective pharmacokinetics of oxybutynin and N-desethyloxybutynin in vitro and in vivo.

In vitro studies and the multiple applications of an oxybutynin (OXY) transdermal delivery system to Japanese healthy volunteers were conducted to characterize the stereoselectivity in the pharmacokinetics of OXY and its metabolite, N-desethyloxybutynin (DEOB). In human liver microsomes, (R)-OXY and (R)-DEOB were eliminated slightly slower than the corresponding (S)-enantiomers. The production of DEOB from OXY for the (R)-enantiomer was also slower than that for the (S)-enantiomer. In human P450-expressing liver microsomes, OXY was metabolized mainly by CYP3A4 among five cytochrome P450s (CYPs) tested (CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5) and the kinetics were slightly different for the enantiomer. The unbound fraction of (R)-OXY in plasma was almost two times higher than that of (S)-OXY, whereas (R)-DEOB was bound to plasma protein more than (S)-DEOB. No differences were observed in the blood-plasma concentration ratios for the enantiomers. After multiple applications of the transdermal delivery system, the plasma concentrations of (R)-OXY were lower than those of (S)-OXY. These data indicate that for the stereoselectivity of OXY, the unbound fraction of each OXY enantiomer was a major factor and the metabolism in liver had a minimal effect.

Development of the real-time control (RTC) system for Tokyo sewage system.

Tokyo Metropolitan government has decided to make the maximum possible use of the existing facilities while ensuring safety against inundation and to promote measures also from a software approach by introducing a system capable of minimizing combined sewer overflow, the real-time control system (RTC). A pilot RTC system was installed in August 2002 for the Shinozaki Pumping Station. The RTC system monitors the precipitation volume and the water level in the pipe. Simulations were carried out on the basis of these data. From the results, it was found that with the use of the RTC it is possible to reduce CSO by roughly 50% for small rainfalls with a total precipitation level of 20 mm or less by strong rainwater in the pipe routes at the beginning of the rain. It has also been shown that CSO can be reduced by about 80% through the use of rainfall forecasting.

Telecytology in Hokkaido Island, Japan: results of primary telecytodiagnosis of routine cases.

In 1997, an Internet-based static image telepathology system was built at Sapporo National Hospital, Japan. We can exchange high-resolution microscopical images through a file transfer protocol server and discuss cytological findings and diagnosis on an electronic mailing list. We applied the system to primary telecytodiagnosis. From May 1997 to April 1999 we have made diagnoses of 614 daily cases only by looking at the video monitor images transmitted from the cytotechnologist of Wakkanai Municipal Hospital 300 km distant from Sapporo. The concordance between telecytodiagnosis and glass slide diagnosis was 88.6%. Kappa statistics for cervical smears was 0.919 and that for specimens other than uterine cervix was 0.810. The accuracy of telecytodiagnosis was 91.4%, and was not substantially different from that of the conventional mail-based cytology in a previous year. We had five cases with a severely inappropriate diagnosis in telecytology, all of which however were quickly corrected by follow-up histological or cytological specimens. With the use of an electronic mailing list the participants had quick and sufficient discussions. We conclude that telecytology is very useful for primary cytodiagnosis in regional medicine and that it may raise the accuracy of cytodiagnosis in future, if we make consistent efforts to reflect the benefits of telecytology in daily practices. This is the first report of clinical results of telecytology from Japan.

Hippocampal heme oxigenase-1 in a murine cardiac arrest model.

Heme oxygenase-1 (HO-1) is a member of the heat shock protein family (HSP-32). It responds to thermal stress in cultured glial cells. To our knowledge. nothing is known about the expression and response of the HO-1 in cerebral ischemia. Therefore, we show here the induction of HO-1 in the brain of mice after global cerebral ischemia. HO-1-like immunoreactivity was detected at 12, 24, and 48 hours after ischemia recirculation. The HO-1-like immunoreactive cells were observed in astrocytes in the hippocampal dentate gyrus and CA1. The peak level of HO-1-like immunoreactivity was found 48 hours after the recirculation. HO-1-like immunoreactivity was observed in GFAP-positive astrocytes by use of a double immunostaining method. These results provide direct evidence for the induction and localization of HO-1 immunoreactivity in vivo in a mouse cerebral ischemia. We suggest that HO-1, produced in astrocytes after ischemia-recirculation, may directly affect neurons to protect from cell death.

Virtual reality system using force feedback device for molecular modeling.

We designed two new concept of computer aided molecular modeling system using virtual reality technologies. Although it is still a prototype, the most characteristic function of the system is enabling its user to "touch" and feel the electrostatic potential field of a protein or a drug molecule. User can scan surface of a protein using a globular probe, which is given an electrostatic charge, controlled by a force feedback devise. The electrostatic force between the protein and the probe is calculated in real time, and immediately fed back into the force feedback device. The user can easily search interactively for positions where the probe is strongly attracted to the force field. Such positions can be regarded as candidate sites where small chemical groups corresponding to the probe, functional parts of lead compounds, can bind to the target protein. We also developed a binding simulator which we can bind a small molecule (drug) to a large molecule (protein). In this system, we placed a camera, so that we can not only "feel" but also "see" how drug fits into the binding site. Our prototype system has the potential to serve as a new application method as well as being applicable to conventional VR technologies, especially to force feedback technologies.

PACAP activates PKA, PKC and Ca(2+) signaling cascades in rat neuroepithelial cells.

Several studies have reported that the PAC(1) receptor (PAC1-R), the specific receptor for PACAP, is expressed at early developmental stages. Here, we describe that the cytosolic Ca(2+) concentration ([Ca(2+)](i)) was increased by PACAP, but not VIP, in a concentration range from 10(-12) to 10(-8) M via the PAC(1)-R in isolated single cells from the rat neural fold. This activation of the cells by PACAP was mimicked by agonists and inhibited by antagonists of the cAMP/PKA and PLC/PKC cascades. These data indicate that PACAP/PAC(1)-R is linked to [Ca(2+)](i) signaling via two G-protein-coupled protein kinase pathways and may thereby play an important role in early neurodevelopment.

A BAC-based STS-content map spanning a 35-Mb region of human chromosome 1p35-p36.

We have devised a mapping method for rapid assembly and ordering of bacterial artificial chromosome (BAC) clones on a radiation hybrid (RH) panel, using sequence-tagged sites (STSs) and PCR. The protocol consists of two rounds of two-dimensional screening from a limited number of BACs to correspond each to an STS. In the first round, STSs are assembled in the RH bins and ordered according to PCR signals derived from 384-well microtiter plates (MTPs) in which BAC clones have been arrayed. In the second round, individual BAC clones are isolated from the MTPs to build a contig. We applied this method to a 35-Mb region spanning human chromosome 1p35-p36 and assembled 1366 BACs in 11 contigs, the longest being about 20 Mb. The working draft sequences of the human genome have been integrated into the contigs to validate the accuracy.

Japanese experience of telemedicine in oncology.

We started telemedicine projects from 1990 with a telepathology system within Tsukiji Campus of National Cancer Center. In 1994, we connected Tsukiji Campus and Kashiwa Campus by 6 Mbps optical fiber leased line using IP protocol for data transmission, for teleconference, telepathology, and teleradiology projects. We also started connection of regional cancer centers and are now forming a cancer center network of 14 cancer centers. We are at present organizing 130 teleconferences per year with an attendance of more than 16000 people as summary. We have also used a high-resolution image transferring system, such as SHD (2000 pixelsx2000 pixels resolution) system on one side, and an economical telemedicine system using JAVA and a WWW browser (NCC_image) on the other side. We think that providing information is another field of telemedicine. We began the experimental gopher and WWW service in 1993. We are now providing official up-to-date cancer information for patients and healthcare professionals. We are getting more than 400000 hits per month. We are also providing a teleconference video session which is held every week on the Internet using a Real Video system with synchronized slide presentation on the WWW browser. We are also organizing a Cancer Image Reference Database System including DICOM images with viewer software. This paper is a summary of the telemedicine projects performed at the National Cancer Center.

The effect of cardiac arrest on the blood-testis barrier to albumin, tumor necrosis factor-alpha, pituitary adenylate cyclase activating polypeptide, sucrose, and verapamil in the mouse.

Impotence commonly occurs after events such as acute myocardial infarction, coronary bypass, head trauma, and cerebral bleeding, including subarachnoid hemorrhage. We hypothesize that the hypoxia accompanying these events could damage the blood-testis barrier (BTB) and so cause testicular dysfunction, a possible cause of impotence. We examined the effect of cardiac arrest in mice on testis weight and various aspects of BTB function. Testis weight was decreased by about 24% 12 hours after cardiac arrest but had recovered fully by day 7. The testis/serum ratio for albumin was increased 12 hours after arrest, showing a disruption in the vascular BTB with recovery by 24 hours. The testis/serum ratio for sucrose was not consistently elevated, showing that the Sertoli cell BTB remained intact. The testis/serum ratio for verapamil was increased on day 3 of cardiac arrest, suggesting impaired function of the BTB's p-glycoprotein efflux transporter. Transporters for pituitary adenylate cyclase activating polypeptide and tumor necrosis factor-alpha were not affected by cardiac arrest. These results show that cardiac arrest affects testis weight and some aspects of BTB function. Such changes might have long-term effects on testicular function.

Sole expression of laminin gamma 2 chain in invading tumor cells and its association with stromal fibrosis in lung adenocarcinomas.

Laminin-5 (LN-5), an important basement membrane (BM) protein consisting of laminin alpha3, beta3 and gamma 2 chains, has been suggested to be involved in tumor cell invasion and tissue repair. In this study, the distribution of the LN-5 subunits in atypical adenomatous hyperplasia (AAH) and different types of adenocarcinomas of the lung was examined by immunohistochemical analysis. In AAH and non-sclerosing, well-differentiated adenocarcinomas, the LN gamma 2 chain was frequently detected along with the continuous BMs. These BMs were also positive for both LN alpha3 and beta3 chains, suggesting that LN-5 had been deposited. In contrast, the cytoplasmic staining for the LN gamma 2 chain was frequently observed in tumor cells of sclerosing, well-differentiated adenocarcinomas, as well as of moderately and poorly differentiated adenocarcinomas, without any evidence of co-expression of the LN alpha3 and beta3 chains. This staining pattern of the LN gamma 2 chain was prominent in carcinoma cells invading into interstitial stroma and was associated with the formation of a central scar in the tumor tissues. These results suggest that the LN gamma 2 chain monomer could be an important indicator of progression of lung adenocarcinoma.

Immunohistochemical distribution of laminin-5 gamma2 chain and its developmental change in human embryonic and foetal tissues.

Immunohistochemical distribution of laminin gamma2 chain, a subunit of the basement membrane protein laminin-5, was examined in 19 cases of human embryos and foetuses ranging from 4 to 25 weeks of gestation. Laminin gamma2 was first detected in the basement membranes underlying ectodermal epithelial tissues, such as the skin and tooth, as early as 5-6 weeks of gestation. Between 6-7 and 12-13 weeks, laminin gamma2 was detected in the basement membranes of various endodermal epithelial tissues, such as the bronchus, oesophagus, stomach, intestines, urinary bladder, gallbladder and hepatopancreatic duct. The deposition of laminin gamma2 in basement membrane was associated with the process of morphogenesis. In the small intestine, laminin gamma2 first appeared in the basement membrane of the primitive short villi, and its level gradually increased in the villus region but decreased in the cryptic region during the maturation of the organ. In addition, non-basement membrane immunoreactivity for laminin gamma2 was detected in some mesoderm-derived tissues, such as the cartilage and skeletal and smooth muscle fibres. These results suggest a common role of laminin-5 and some specific roles of its gamma2 chain in the morphogenesis of human tissues.

Localization of the pituitary adenylate cyclase-activating polypeptide receptor and its mRNA in the rat adrenal medulla.

We examined the localization of the pituitary adenylate cyclase-activating peptide (PACAP) receptor (PAC1-R) and its mRNA with immunocytochemistry and in situ hybridization, respectively. PAC1-R immunoreactivity and its transcript were detected in both chromaffin cells and ganglion cells but not detected in the adrenal cortex. In addition, strong PAC1-R immunoreactivity was found beneath the plasma membrane of the immunoreactive medullary cells. Electron microscopic immunocytochemistry revealed that PAC1-R was predominantly expressed in adrenaline-containing cells. This report supports the notion that PACAP is an activator and modulator of catecholamine secretion as well as synthesis in the adrenal medulla.

Isolation and activity of proteolytic fragment of laminin-5 alpha3 chain.

Laminin-5 (alpha3beta3gamma2) is an important component of epithelial basement membranes. The 190-kDa alpha3 chain undergoes extracellular cleavage within the carboxyl (C) terminus consisting of five globular domains (G1 to G5), producing the mature laminin-5 with the 160-kDa alpha3 chain. To understand the physiological meaning of this processing, we isolated the C-terminal fragments of the alpha3 chain from the conditioned media of two kinds of human cell lines. The amino-terminal sequence of the fragments suggested that the cleavage occurs at Gln(1337)-Asp(1338) in the spacer region between the G3 and G4 domains. The G4-G5 fragment itself did not show significant activity, but it stimulated cell migration in the presence of a low concentration of the mature laminin-5, suggesting its regulatory role in cell migration.

Splice variants of PAC(1) receptor during early neural development of rats.

The specific pituitary adenylate cyclase-activating polypeptide (PACAP) receptor, PAC(1)-R, consists of at least seven isoforms, and they are differentially coupled to signal transduction pathways by alternative splicing. We have found that the major splice variants of the PAC(1) receptor seen during development are the short splice isoform, PAC(1)-R-s (which does not contain either the "hip" or "hop" cassette), and another form, PAC(1)-R-hop (which contains the "hop" cassette). We also have applied an innovative molecular histochemical technique, in situ reverse transcription-polymerase chain reaction (RT-PCR), and determined that these two splice isoforms are colocalized in the neuroepithelia from the primitive streak stage.

Morphological evidence for neural interactions between leptin and orexin in the hypothalamus.

Both leptin and orexin have been recently discovered as peptides involved in feeding regulation. The morphological evidence of neural interaction between leptin and orexin, one considered to inhibit food intake and the other to stimulate it in the central nervous system (CNS), was studied by use of double immunostaining method. The leptin receptor-like immunoreactive (LR-LI) neurons in the hypothalamic arcuate nucleus and ventromedial nucleus were innervated by orexin-like immunoreactive (OX-LI) neurons. The distribution of LR-LI neurons in the hypothalamus was very similar to that of OX-LI neurons. These results may suggest that leptin and orexin are intimately correlated with each other and that they reciprocally regulate feeding at the hypothalamic level.

The functions of exogenous and endogenous laminin-5 on corneal epithelial cells.

Recent evidence suggests that the basement membrane not only separates basal cells from Bowman's layer, but also has a crucial role in the proliferation, differentiation and migration of corneal epithelial cells. The basement membrane is composed of a mixture of matrix components including collagens, laminins and heparan sulfate proteoglycans. In these extracellular matrixes, laminin is a major component of the basement membrane. Of 11 laminin isoformes, laminin-5 is a variant, composed of three nonidentical subunits alpha3, beta3, gamma2 and is a major component of the corneal basement membrane. However, little is known about the interactions of laminin-5 with corneal epithelial cells. In this study, we investigated the functions of laminin-5 on SV-40 transfected human corneal epithelial cells (HCE cells). We also revealed different functions between exogenous and endogenous laminin-5 on HCE cells. Laminin-5 is synthesized initially as a 490 kDa molecule that undergoes specific processing to cleavaged isoforms after being secreted. The alpha3 subunit is processed from 200-190 kDa to 160 kDa/145 kDa. The gamma2 subunit is processed from 150 kDa to 105 kDa/80 kDa. The beta3 subunit (140 kDa) is not processed. Exogenously added laminin-5 (soluble form) in this study was purified from a serum-free, conditioned medium of a human gastric carcinoma cell line STKM-I. This soluble laminin is a processed isoform containing alpha3 (160 kDa), beta3 (140 kDa) and gamma2 (105 kDa) chains. On the other hand, immunocytochemical analysis showed that HCE cells themselves secreted laminin-5 endogenously. Western blotting analysis revealed that HCE cells initially produced unprocessed isoform containing 190 kDa alpha3, 140 kDa beta3 and 150 kDa gamma2 chains and that after being secreted, the alpha3 chain was processed to 160 kDa/145 kDa and the gamma2 chain was processed to 105 kDa. Initially we investigated the functions of exogenous (processed) laminin-5 on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion via alpha3beta1 integrin, cell spreading, assembly of hemidesmosomes and mildly inhibited cell migration. Next we estimated the effect of endogenous (unprocessed) laminin-5 on HCE cells. Using an anti laminin-5 monoclonal antibody (mAb) or anti integrin alpha3beta1 mAbs, the blocking of the interaction between endogenously secreted laminin-5 and HCE cells caused strong inhibition of cell migration. Integrin alpha3beta1 and alpha6beta4 were expressed in HCE cells. These integrins are receptors of laminin-5. But, anti integrin alpha6beta4 mAbs did not have any blocking ability against cell migration. These results indicated that endogenous (unprocessed) laminin-5 has a crucial role in cell migration on HCE cells via alpha3beta1 integrin. In conclusion, structural differences between exogenous (processed) and endogenous (unprocessed) laminin-5 regulated their functions on HCE cells. Exogenously added laminin-5 strongly promoted cell adhesion, cell spreading and assembly of hemidesmosomes. Endogenously secreted laminin-5 had a crucial role in cell migration. In the future, processed soluble laminin-5 could be a useful drug for the prevention of recurrent corneal erosion, and unprocessed soluble laminin-5 could be applied for the treatment of prolonged corneal epithelial defects.