Rebamipide, an anti-ulcerative drug, inhibits induction of salivary dysfunction by benzodiazepines.

OBJECTIVES: The purpose of this study was to determine whether rebamipide, an antistomach ulcer agent, ameliorated benzodiazepine-induced hyposalivation in rat parotid gland (PG) and submandibular gland (SMG). METHODS: Saliva was collected from PG and SMG through a capillary cannula inserted into the parotid duct and sublingual papillae, respectively, every 15 min for 1 h after stimulation with pilocarpine dissolved in physiological saline and intraperitoneally administered at 1 mg kg-1 . Diazepam (DZP) was administered intraperitoneally at a dose of 0.2 mg kg-1 twice daily for 7 days. Rebamipide was administered at 10, 20, 30, or 100 mg kg-1 concomitantly with DZP to determine its effect on hyposalivation. The effect of rebamipide on movement of intracellular calcium ([Ca2+ ]i) in isolated parotid acinar cells was analyzed using Fluo4, a fluorescent dye used to detect Ca2+ . RESULTS: Repetitive administration of DZP decreased salivary secretion in PG and SMG. This inhibitory effect was weakened by administration of rebamipide. Prior administration of DZP (10-6 M) significantly suppressed carbachol (10-7 M)-induced increase in [Ca2+ ]i. This inhibitory effect was ameliorated by combined use with rebamipide (5 × 10-4 M). CONCLUSION: This findings suggest that rebamipide weakens the downregulatory effect of DZP on salivary secretion by preventing DZP-induced suppression of increase in [Ca2+ ]i.

Biochemical and immunohistochemical characterization of the isoforms of myosin and actin in human placenta.

Human placenta has long been known to contain large quantities of smooth muscle-type myosin and actin, while precise isoform compositions of its contractile proteins are not known. To determine the isoform compositions, myosin and actin were extracted from human term placentas and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting by using isoform-specific monoclonal anti-myosin and anti-actin antibodies. The placental myosin was found to be composed of about 65% of a nonmuscle-type heavy chain isoform (MIIA), each about 15% of two smooth muscle-type heavy chain isoforms (SM1 and SM2) and about 5% of a brain/fetus-type heavy chain isoform (MIIB2). Whereas the MIIA isoform was present in both vascular and extravascular tissues, the SM1 isoform was localized almost only in the vascular tissue. Similarly, human term placenta was found to contain approximately 60, 30, and 10% of ß-nonmuscle, α-smooth muscle, γ-smooth muscle actin isoforms, respectively. The ß-nonmuscle actin was located primarily in the extravascular tissue, while the α-smooth muscle actin was located mostly in the vascular tissue. The extravascular tissue of the human term placenta thus appears to be composed of almost only nonmuscle-type isoforms of contractile proteins. The vascular tissue appears to be composed of both smooth muscle-type and nonmuscle-type isoforms of contractile proteins.

Convenient and reproducible in vivo gene transfer to mouse parotid glands.

OBJECTIVES: Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands. METHODS: The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen's ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (10(7) vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR. RESULTS: The chosen volume for mouse parotid vector delivery was 20µL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva. CONCLUSION: These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models.

Coordinate involvement of cell cycle arrest and apoptosis strengthen the effect of FTY720.

A novel reagent, FTY720 (2-amino-2-[2-(4-octylphenyl)ethyl]-1,3-propanediol hydrochloride), has been shown to induce a significant decrease of lymphocytes and lymphoma cells and is expected to be a potent immunosuppressant and anti-tumor drug. The decrease in lymphocytes and lymphoma cells is mainly the result of FTY720-induced apoptosis. FTY720 directly affects mitochondria and induces cell death. Moreover, FTY720 activates protein phosphatase (PP) 2A and affects anti-apoptotic intracellular signal transduction proteins to attenuate the anti-apoptotic effect. In this study, we examined the relationship between FTY720-induced apoptosis and cell cycle regulation. FTY720 induced apoptosis significantly at the G0 / G1 phase and caused G0 / G1 cell cycle arrest of the human lymphoma cell lines HL-60RG and Jurkat. Simultaneously, retinoblastoma protein (pRB) was dephosphorylated, suggesting that dephosphorylation of pRB was related to FTY720-induced G0 / G1 cell cycle arrest. Because this dephosphorylation was completely blocked by a specific PP1 / 2A inhibitor, okadaic acid, it appears that FTY720-activated PP2A is essential for FTY720-induced cell cycle arrest. FTY720-induced apoptosis was inhibited by Bcl-2 overexpression in Jurkat cells, but this did not prevent FTY720-induced cell cycle arrest, suggesting that the mechanism of FTY720-induced cell cycle arrest is independent of the mechanism of FTY720-induced apoptosis. These two independent pathways strengthen the effect of FTY720.

A broad replication origin of Drosophila melanogaster, oriDalpha, consists of AT-rich multiple discrete initiation sites.

This work shows that the replication origin of Drosophila melanogaster, oriDalpha, consists of multiple discrete initiation sites. We attempted to map at high resolution the initiation sites in oriDalpha with a quantitative nascent DNA abundance assay using a competitive polymerase chain reaction (PCR) method. Nascent DNA was prepared from either cells blocked in very early S-phase and then labeled with 5-bromo-2'-deoxyuridine (BrdU), or asynchronously growing cells labeled briefly with BrdU. Denatured DNA was size-fractionated in alkaline sucrose gradients. BrdU-labeled nascent DNA was immuno-affinity purified using anti-BrdU antibodies. DNA was quantified with a competitive PCR method before and after immuno-purification. The results indicated that oriDalpha, whose size was presumed to be about 10 kb from two-dimensional gel electrophoretic analysis, contained four major initiation sites in its central 2.8 kb region, and six to approximately eight sites in 8.4 kb. All initiation sites corresponded with AT-rich sequences. Detailed analysis of one major initiation site indicated that its range was restricted to 700 bp.

Device for the removal of button batteries.

OBJECTIVE: There is an increasing number of accidents by erroneous ingestion of button batteries in recent years; the batteries arouse the interest of infants because of their attractive shape and luster. The batteries remaining in the gastrointestinal tract and discharging electric current over a long period of time may induce ulceration or perforation, thus must be carefully considered the selection of appropriate treatment. METHODS: We remove erroneously ingested button batteries with two tubes with ferrite magnets nearly the same size as the button batteries themselves. PATIENTS: Four cases of erroneous ingestion of button batteries. RESULTS: We easily removed button batteries from the stomach within 5 minutes in all cases with two magnet-attached tubes. CONCLUSION: We present this battery removal device together with a literature review, because it seems convenient and useful.

Automatic scoliosis detection based on local centroids evaluation on moiré topographic images of human backs.

This paper presents a technique for automating human scoliosis detection by computer based on moiré topographic images of human backs. Scoliosis is a serious disease often suffered by teenagers. For prevention, screening is performed at schools in Japan employing a moiré method in which doctors inspect moiré images of subjects' backs visually. The inspection of a large number of moiré images collected by the school screening causes exhaustion of doctors and leads to misjudgment. Computer-aided diagnosis of scoliosis has, therefore, been requested eagerly by orthopedists. To automate the inspection process, unlike existent three-dimensional techniques, displacement of local centroids is evaluated two-dimensionally between the left-hand side and the right-hand side of the moiré images in the present technique. The technique was applied to real moiré images to draw a distinction between normal and abnormal cases. According to the leave-out method, the entire 120 image data (60 normal and 60 abnormal) were separated into three data sets. The linear discriminant function based on Mahalanobis distance was defined on the two-dimensional feature space employing one of the data sets containing 40 moiré images and classified 80 images in the remaining two sets. The technique finally achieved the average classification rate of 88.3%.

The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage.

Pseudomonas aeruginosa produces three types of bacteriocins: R-, F- and S-type pyocins. The S-type pyocin is a colicin-like protein, whereas the R-type pyocin resembles a contractile but non-flexible tail structure of bacteriophage, and the F-type a flexible but non-contractile one. As genetically related phages exist for each type, these pyocins have been thought to be variations of defective phage. In the present study, the nucleotide sequence of R2 pyocin genes, along with those for F2 pyocin, which are located downstream of the R2 gene cluster on the chromosome of P. aeruginosa PAO1, was analysed in order to elucidate the relationship between the pyocins and bacteriophages. The results clearly demonstrated that the R-type pyocin is derived from a common ancestral origin with P2 phage and the F-type from lambda phage. This notion was supported by identification of a lysis gene cassette similar to those for bacteriophages. The gene organization of the R2 and F2 pyocin gene cluster, however, suggested that both pyocins are not simple defective phages, but are phage tails that have been evolutionarily specialized as bacteriocins. A systematic polymerase chain reaction (PCR) analysis of P. aeruginosa strains that produce various subtypes of R and F pyocins revealed that the genes for every subtype are located between trpE and trpG in the same or very similar gene organization as for R2 and F2 pyocins, but with alterations in genes that determine the receptor specificity.

Immunosuppressant FTY720 induces apoptosis by direct induction of permeability transition and release of cytochrome c from mitochondria.

FTY720 has immunosuppressive activity in experimental organ transplantation and shows a prompt and protracted decrease of blood T lymphocytes upon oral administration. The blood lymphocyte decrease in vivo was mainly a result of FTY720-induced apoptosis. However, this apoptotic mechanism is not well understood. We examined the mechanism of FTY720-induced apoptosis in lymphoma. Western blotting and fluorescent caspase-specific substrate revealed that caspase-3 is involved in FTY720-induced apoptosis, whereas caspase-1 is not. Apoptotic cell death was inhibited by the pan-caspase inhibitor, Z-VAD-FMK, suggesting that caspase activation is essential for FTY720-induced apoptosis. FTY720 reduced mitochondrial transmembrane potential and released cytochrome c from the mitochondria of intact cells as well as in a cell-free system even in the presence of Z-VAD-FMK. As these mitochondrial reactions occurred before caspase activation, we concluded that FTY720 directly influences mitochondrial functions. The inhibition of mitochondrial permeability transition by Bcl-2 overexpression or by chemical inhibitors prevented all apoptotic events occurring in intact cells and in a cell-free system. Moreover, using a cell-free system, FTY720 did not directly affect isolated nuclei or cytosol. These results indicate that FTY720 directly affects mitochondria and triggers permeability transition to induce further apoptotic events.

[Value of upper gastrointestinal endoscopic examination of head and neck cancer patients].

Between January 1995 and March 1999, we performed the upper gastrointestinal endoscopic examinations on 287 patients with head and neck cancers and detected 23 cases (8%) of esophageal cancer and 8 cases (2.8%) of gastric cancer, showing how frequently esophageal cancer occurs in head and neck cancer. The esophageal cancer involved the oral cavity in 8 cases (9.5%), the oropharynx in 3 cases (8.6%), the hypopharynx in 10 cases (19.6%), and the larynx in 2 cases (2%). Esophageal cancer occurred most frequently in hypopharyngeal cancer, particularly the pyriform sinus type and the postcricoid type. We conclude that upper gastrointestinal endoscopic examination, including Lugol staining, is necessary in head and neck cancer patients.

[Study of matrix metalloproteinase-2 and -9 in thyroid papillary cancer].

Metastasis of cancer starts with the penetration of cancer cells through the membrane surrounding the cancer focus into the stroma (extracellular matrix). The focal membrane consists of mainly type-IV collagen. An immunochemical study of 28 patients with benign thyroid nodular diseases and 27 patients with papillary carcinoma revealed the fragmentation of type-IV collagen in 4 patients with papillary carcinoma. Matrix metalloprotease (MMP)-2 and MMP-9 are the major enzymes which decompose type-IV collagen, and they have been suggested to be related to cancer metastasis. Therefore, we conducted biochemical and immunohistochemical studies to determine the relationship between these MMPs and the degree of malignancy in thyroid diseases. The concentration of MMP-2 in the serum of patients with papillary carcinoma and patients with benign nodules was 526.0 +/- 96.6 and 522.7 +/- 114.6 ng/ml, respectively, and that of MMP-9 was 53.8 +/- 40.3 and 39.9 +/- 36.0 respectively. There was no significant difference between the two groups in the concentration of either enzyme. The concentration of TIMP-2 in the serum was below the detectable level. On the other hand, the concentration of MMP-2 in the tissue of papillary carcinoma, benign nodules and normal tissue was 12.1 +/- 8.1, 5.7 +/- 4.3, and 0.6 +/- 0.5 ng/mg tissue protein, respectively, and that of MMP-9 was 4.2 +/- 4.1, 2.1 +/- 1.7, and 0.4 +/- 0.3 ng/mg tissue protein, respectively. Concentrations of both enzymes were significantly higher in the papillary carcinoma tissue. Immunohistochemical studies revealed a diffuse granular distribution of MMP-2 in the cytoplasm of the tumor cells. These findings imply that MMP-2 and MMP-9 are related to the degree of malignancy of cancer, especially metastasis.

Evidence that FTY720 induces T cell apoptosis in vivo.

The immunosuppressant FTY720 induces a drastic decrease in blood lymphocytes, especially T cells; a decrease which is assumed to be the immunosuppressive mechanism of this drug. FTY720 causes cell death in vitro in lymphocytes and leukemia cells. However, the deletion mechanism of blood lymphocytes in vivo remains unclear. We investigated whether administration of FTY720 induced lymphocyte apoptosis in blood circulation. A marked decrease in the number of blood lymphocytes was observed within an hour after a single oral administration of FTY720 at doses of 5-10 mg/kg in rats and mice. Experiments using fluorescein isothiocyanate (FITC)-Annexin V and APO-BRDU methods revealed that FTY720 induced blood lymphocyte apoptosis in a dose-dependent manner. On the other hand, lymphocyte homing to Peyer's patches was proposed as the mechanism underlying the blood lymphocyte decrease at these doses. However, similar results were obtained when using aly/aly mice, which lack Peyer's patches and lymph nodes. Thus, we concluded that apoptosis of blood lymphocytes was induced immediately after administration of FTY720, and the cells could be immediately scavenged by phagocytes or the reticuloendothelial system in addition to Peyer's patches homing. We also concluded that T cells were highly sensitive to FTY720, which induced apoptosis in vivo.

Chromosomal imbalances in adult T-cell leukemia revealed by comparative genomic hybridization: gains at 14q32 and 2p16-22 in cell lines.

Comparative genomic hybridization was used to identify chromosomal imbalances in eight cell lines and 12 blood samples from patients with adult T-cell leukemia/lymphoma (ATL). The chromosomes most often over-represented in the cell lines were 2p (6 cases), 7q (4 cases), and 14q (4 cases), with minimal common regions at 2p16-22, 7q21-36, and 14q32, respectively. Distinct imbalances were detected in only 7 of the clinical samples. Chromosomes 14q32 and 2p16-22 harbor TCL1 and a transcription factor, HTLF (human T-cell leukemia virus enhancer factor), respectively. FISH analysis revealed that TCL1 did not juxtapose to TCRA, and we detected no expression of TCL1 in any of the ATL cell lines despite the 14q32 amplifications. Moreover, expression of HTLF was not elevated in the ATL cell lines bearing multiplication of 2p. These results suggest that chromosomal regions 2p16-22 and 14q32 harbor genes other than HTLF and TCL1 that are involved in cellular immortalization or in the pathogenesis of ATL.

Gains, losses, and amplifications of genomic materials in primary gastric cancers analyzed by comparative genomic hybridization.

By means of comparative genomic hybridization (CGH), we screened 58 primary gastric cancers for changes in copy number of DNA sequences. We detected frequent losses on Ip32-33 (21%), 3p21-23 (22%), 5q14-22 (36%), 6q16 (26%), 9p21-24 (22%), 16q (21%), 17p13 (48%), 18q11-21(33%), and 19(40%). Gains were most often noted at I p36 (22%), 8p22-23 (24%), 8q23-24 (29%), 11q12-13 (24%), 16p(21%), 20p (38%), 20q (45%), Xp21-22(38%), and Xq21-23 (43%), with high-level amplifications at 6p21(2%),7q31(10%), 8p22-23(5%), 8q23-24 (7%), 11q13(4%), 12p12-13(4%), 17q21(2%), 19q12-13(2%), and 20q13(2%). High-level amplification at 8p22-23 has never been reported in any other cancer type and its frequency was as high as that reported for the MYC, MET, and KRAS genes. We narrowed down the smallest common amplicon to 8p23.1 by reverse-painting FISH to prophase chromosomes. Southern blot analysis using one EST marker (D38736) clearly demonstrated that amplification of this exon-like sequence had occurred in all three tumors in which amplifications at 8p22-23 had been detected by CGH. Our data provide evidence for several, previously undescribed, genomic aberrations that are characteristic of gastric cancers.

Comparative genomic hybridization of squamous cell carcinoma of the esophagus: the possible involvement of the DPI gene in the 13q34 amplicon.

We investigated copy number aberrations in 29 primary tumors and 12 cell lines of esophageal squamous cell carcinoma (ESC) using comparative genomic hybridization. In the primary tumors, the most common sites of copy number gains were 3q26.3-27 (45%), 8q24 (41%), 5p15 (38%), Xq27-28 (38%), 14q32 (31%), 11q13 (28%), and 20q13.3 (28%). High-level gains (HLGs) indicative of gene amplifications were identified at 11q13 in two cases, and in one case each at 2q33-34, 3q25-29, 5p15.1-15.2, 7q21-22, 11p11.2, 12p11.2-12, and 13q34. Recurrent losses were observed only at 9p13(17.2%). In the 12 ESC cell lines, the most common sites of HLGs were 5p15.1-15.3 (four cases), 11q13 (four cases), 8q24.1-24.2 (three cases), 20q13.2-13.3 (three cases), 3q26.3 (two cases), and 7p15-22 (two cases). Less frequent HLGs (one case each) were observed at 2p16-22, 3q25, 7p12-14, 7q21-22, 9q34, 10q21, 11p11.2, 14q13-14, 14q31-32, 15q22-26, and 17p11.2. Chromosomes and chromosome arms that showed frequent losses in the cultured lines were 18q (58%), 4 (50%), 9p (50%), and 3p (42%). These findings provide evidence for a number of previously unknown genomic aberrations in ESC, suggesting target regions for positional cloning of genes relevant to carcinogenesis in the esophagus. In particular, we identified a significant amplification of the DPI gene (TFDPI), a transcription factor that forms heterodimers with E2FI, in the single primary tumor that exhibited HLG at 13q34.

Identification of a novel gene, MASL1, within an amplicon at 8p23.1 detected in malignant fibrous histiocytomas by comparative genomic hybridization.

We used comparative genomic hybridization to study malignant fibrous histiocytomas (MFHs) from 19 patients to detect changes in the copy number of DNA sequences, along entire chromosomes. Together with losses and gains in various chromosomal regions, distinct high-level amplifications were found at six loci (4q12-21, 8p21-pter, 8q24.1-qter, 9q12-13, 12p11.2-pter, and 15q11.2-15), suggesting that those regions may contain unknown (proto) oncogenes. We focused on the 8p amplicon, where detailed characterization allowed us to determine that the minimal common amplified region lay between markers D8S1819 and D8S550 at 8p23.1. A novel gene designated MASL1 (MFH-amplified sequences with leucine-rich tandem repeats 1) was isolated from within this narrowly defined region. Expression of the MASL1 gene was enhanced significantly in MFH tumors bearing the 8p amplicon. The primary structure of its deduced product revealed an ATP/GTP-binding site, three leucine zipper domains, and a leucine-rich tandem repeat, all of which are important structural or functional elements for interactions among proteins related to the cell cycle. These features suggest that overexpression of MASL1 might well be oncogenic with respect to MFH.

Specification of regions of DNA replication initiation during embryogenesis in the 65-kilobase DNApolalpha-dE2F locus of Drosophila melanogaster.

In the early stage of Drosophila embryogenesis, DNA replication initiates at unspecified sites in the chromosome. In contrast, DNA replication initiates in specified regions in cultured cells. We investigated when and where the initiation regions are specified during embryogenesis and compared them with those observed in cultured cells by two-dimensional gel methods. In the DNA polymerase alpha gene (DNApolalpha) locus, where an initiation region, oriDalpha, had been identified in cultured Kc cells, repression of origin activity in the coding region was detected after formation of cellular blastoderms, and the range of the initiation region had become confined by 5 h after fertilization. During this work we identified other initiation regions between oriDalpha and the Drosophila E2F gene (dE2F) downstream of DNApolalpha. At least four initiation regions showing replication bubbles were identified in the 65-kb DNApolalpha-dE2F locus in 5-h embryos, but only two were observed in Kc cells. These results suggest that the specification levels of origin usage in 5-h embryos are in the intermediate state compared to those in more differentiated cells. Further, we found a spatial correlation between the active promoter regions for dE2F and the active initiation zones of replication. In 5-h embryos, two known transcripts differing in their first exons were expressed, and two regions close to the respective promoter regions for both transcripts functioned as replication origins. In Kc cells, only one transcript was expressed and functional replication origins were observed only in the region including the promoter region for this transcript.

Identification of amplified DNA sequences on double minute chromosomes in a leukemic cell line KY821 by means of spectral karyotyping and comparative genomic hybridization.

Double minute chromosomes (dmin) are cytogenetic hallmarks of amplified genes. Using spectral karyotyping (SKY) and comparative genomic hybridization (CGH), we identified the origin of amplified DNA in a leukemic cell line, KY821, that harbors numerous dmin. The SKY revealed that the DNA sequences of dmin are derived from materials of chromosome 8, and CGH showed a high degree of overrepresentation only at 8q22-24, indicating that in KY821 only chromosomal material of 8q22-24, containing MYC, is amplified in dmin. An approach combining SKY with CGH should facilitate efforts to identify novel chromosomal regions of gene amplification and contribute information about genetic lesions that underly neoplastic tumors.

The DNA replication-related element (DRE)/DRE-binding factor system is a transcriptional regulator of the Drosophila E2F gene.

Two mRNA species were observed for the Drosophila E2F (dE2F) gene, differing with regard to the first exons (exon 1-a and exon 1-b), which were expressed differently during development. A single transcription initiation site for mRNA containing exon 1-b was mapped by primer extension analysis and numbered +1. We found three tandemly aligned sequences, similar to the DNA replication-related element (DRE; 5'-TATCGATA), which is commonly required for transcription of genes related to DNA replication and cell proliferation, in the region upstream of this site. Band mobility shift analyses using oligonucleotides containing the DRE-related sequences with or without various base substitutions revealed that two out of three DRE-related sequences are especially important for binding to the DRE-binding factor (DREF). On footprinting analysis with Kc cell nuclear extracts and a glutathione S-transferase fusion protein with the N-terminal fragment (1-125 amino acid residues) of DREF, all three DRE-related sequences were found to be protected. Transient luciferase expression assays in Kc cells demonstrated that the region containing the three DRE-related sequences is required for high promoter activity. We have established transgenic lines of Drosophila in which ectopic expression of DREF was targeted to the eye imaginal disc cells. Overexpression of DREF in eye imaginal disc cells enhanced the promoter activity of dE2F. The obtained results indicate that the DRE/DREF system activates transcription of the dE2F gene.