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.

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.

Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.

DNA replication of histone gene repeats in Drosophila melanogaster tissue culture cells: multiple initiation sites and replication pause sites.

We showed previously that DNA replication initiates at multiple sites in the 5-kb histone gene repeating unit in early embryos of Drosophila melanogaster. The present report shows evidence that replication in the same chromosomal region initiates at multiple sites in tissue culture cells as well. First, we analyzed replication intermediates by the two-dimensional gel electrophoretic replicon mapping method and detected bubble-form replication intermediates for all fragments restricted at different sites in the repeating unit. Second, we analyzed bromodeoxyuridine-labeled nascent strands amplified by the polymerase chain reaction method and detected little differences in the size distribution of nascent strands specific to six short segments located at different sites in the repeating unit. These results strongly suggest that DNA replication initiates at multiple sites located within the repeating unit. We also found several replication pause sites located at 5' upstream regions of some histone genes.

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 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.

Flow cytometry analysis of alpha1-adrenoceptor subtypes.

To characterize the alpha1-adrenoceptor subtypes, we developed a flow cytometry method using the fluorescent ligand BODIPY-FL prazosin and the anti-peptide antibody against the alpha1b-adrenoceptor amino terminus (designated 1B-N1-C) as probes. Three alpha1-adrenoceptors (alpha1a, alpha1b and alpha1d) expressed in CHO cells were detected by BODIPY-FL prazosin; however, only alpha1b-adrenoceptor subtype was detected by the anti-peptide antibody 1B-N1-C. Furthermore, the flow cytometry analysis with 1B-N1-C specifically identified alpha1b-adrenoceptor in native cells of hamster DDT1-MF2 cells, rat hepatocytes and cardiomyocytes.

Serology of liver transplantation in the rat. I. Alloantibody responses and evidence for tolerance in a nonrejector combination.

The antibody response against class I and class II RT1 antigens has been studied in PVG rats grafted with DA liver. In this nonrejector combination, liver grafts survive permanently in all normal recipients and in about 50% of recipients presensitized by a DA skin graft, with concurrent induction of transplant tolerance for other DA organs and skin. Using a two-stage radioimmunoassay, the anti-class I (RT1Aa) levels in sera of normal PVG recipients of DA liver grafts were found to be low (maximal titer 1:50 serum dilution or less); after peaking at 2 weeks posttransplantation, they diminished to background levels by 6 weeks. The anti-RT1Aa response showed a close parallel to cell-mediated rejection events in the liver graft recipients. In contrast, anti-class II (RT1Ba/Da) responses reached much higher titers (over 1:1000), which were maintained for several weeks before declining after 4 months. Similar observations were made in presensitized recipients. The induction of tolerance in the alloantibody response was indicated by the inability of DA skin grafts to restimulate anti-RT1 antibody in liver recipients. The observations support the picture of "split tolerance" indicated by previous cellular studies in this combination.

Immunosuppressive activity of serum from liver-grafted rats. Passive enhancement of fully allogeneic heart grafts and induction of systemic tolerance.

Immunological enhancement of allogeneic heart graft survival by serum from rats tolerized by liver grafting has been studied. Serum taken from long-term-surviving PVG rats carrying orthotopic DA liver transplants (OLT serum) was able to increase the survival time of PVG.RT1a heterotopic heart grafts in PVG recipients. Administration of 1 ml of OLT serum at the time of heart grafting led to permanent survival of the grafts in all animals. The recipients became systemically tolerant of RT1a and several weeks later were able to accept permanently skin grafts from the same donor strain, while rejecting third-party grafts. Enhancement appeared to be mediated initially by IgG antibodies in the OLT serum against class II donor RT1a antigens; significant enhancement was produced by as little as 100 micrograms of antibody. Recipient alloantibody responses following enhancement were studied and showed selective suppression of the anti-class-I (RT1Aa) antibody levels, while the anti-class-II antibody response was apparently unaffected. The implications of these results for mechanisms of unresponsiveness following enhancement and liver transplantation are discussed.

A novel immunosuppressant, FTY720, with a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation.

A new compound with an immunosuppressive property was purified from culture filtrates of Isaria sinclairii and was chemically modified to FTY720. Rat spleen cells incubated with FTY720 demonstrated features characteristic of apoptosis--such as the absence of surface microvilli, chromatin condensation, and the formation of apoptotic bodies--by electron microscopy, and genemic DNA fragmentation by agarose gel electrophoresis. When FTY720 was administered in liver-allografted rats at a dose of 0.5 mg/kg from day 1 to day 14 after transplantation, the recipients survived significantly longer than the control group. Pretransplant treatment with 5 mg/kg of FTY720 one day before and on the day of grafting induced a remarkable prolongation of recipient survival, and three of 10 recipients survived for longer than 50 days. Furthermore, administration of FTY720 at 5 mg/kg on days 3 and day 4 after grafting also prolonged survival. In canine kidney allografting, a pretransplant 2-day course of FTY720 at 5 mg/kg prolonged graft survival. Daily administration of FTY720 in combination with CsA resulted in a significant prolongation of graft survival in a synergistic manner. In addition, FTY720 appeared to be nontoxic in canine recipients. These results demonstrated that FTY720, having a unique mechanism of action, induces long-term graft acceptance in rat and dog allotransplantation.

Lack of evidence that a transplanted liver causes acute graft-versus-host disease in rats. A comparison of liver and spleen grafts.

In this study, we used rat transplant models to investigate whether--and, if so, to what extent--transplanted liver had the potential to incite graft-versus-host disease, compared with the disease induced by a spleen graft. Livers from PVG(RT1c) rats were transplanted orthotopically into (DAxPVG)F1 (RT1a/c) rats and vascularized spleen grafts from PVG rats were transplanted heterotopically into (DAxPVG)F1 recipients. The intensity of the GVH disease was assessed by the recipients' morbidity and mortality, recipient-type serum class I (RT1Aa) antigen titer, and histological examination. The recipients of spleen grafts died within 14 and 23 days of transplantation; all animals had lost body weight and showed typical GVH signs, such as ear erythema, diarrhea, and alopecia. However, all the recipients bearing liver grafts survived indefinitely and did not demonstrate weight loss or the typical symptoms associated with GVH disease. The skins, tongues, and intestines of the liver-grafted rats were virtually normal at histological examination, whereas the livers, salivary glands, and skins of spleen-grafted rats were infiltrated by immunoblasts. The recipient-type serum RT1Aa antigen titer increased progressively until death in the spleen graft but not the liver graft recipients. These results provide evidence that suggest that transplanted liver is less likely than transplanted spleen to initiate the GVH disease in rats.

Identification of sperm antigenic determinants with phylogenetically diverse and limited distribution using monoclonal antibodies.

Mouse monoclonal antibodies capable of recognizing mouse sperm antigens were produced and the inter-species cross-reactivity of the antigenic determinants was examined. Of the seven clones independently established, it was found by indirect immunofluorescence staining that one was bound to the tail, one could recognize the head and five other antibodies reacted with the crescent-shaped anterior acrosome. Ultrastructural studies showed that the anti-head antibody recognized the sperm surface, and the anti-tail antibody reacted with the fibrous sheath. Five acrosomal antigenic determinants were found in the acrosome. These antibodies reacted with sperm from the male reproductive tract (testis, cauda epididymis, vas deferens), but not with somatic tissues. The anti-tail antibody reacted with sperm from four other mammalian species (musk shrew, rat, boar and human). The anti-head antibody and one of the anti-acrosome antibodies were bound only to mouse sperm. The other four acrosomal antigenic determinants showed various degrees of inter-species cross-reactivity. None of these antibodies reacted with chicken sperm.

An isochizomer of TaqI from Thermus thermophilus HB8.

A site-specific endonuclease has been isolated from Thermus thermophilus HB8 and named TthHB8I. It recognizes the same sequences as TaqI from Thermus aquaticus YT-1 does. The amount of TthHB8I in the cells was comparable to that of TaqI. T. thermophilus HB8 has an advantage over T. aquaticus YT-1 for preparation of a TaqI-like enzyme since it is easier to obtain T. thermophilus HB8 cells in quantity.

Characterization of antibody to 2'-5' linked oligoadenylic acid.

Antibody to 2'-5' linked triadenylate, A2'p5'A2'p5'A, was prepared by injecting bovine serum albumin conjugated with A2'p5'A2'p5'A into rabbits. The [8-14C]-A2'p5'A2'p5'A used in the study was synthesized by nonenzymatic template-directed condensation of [8-14C]AMP and 5'-phosphorimidazolide of A2'p5'A on poly U. The specificity of the antibody was determined by the inhibition of the binding of the radioisotope probe to the antibody by a competitor using the ammonium sulfate precipitation method. The cross reactivity of a number of modified oligonucleotides has been evaluated in order to gain information on the structural feature involved in the binding to the antibody. The adenine moiety and 2'-5' internucleotide linkage are crucial regions for the binding. The antibody reacted not only on A2'p5'A2'p5'A and A2'p5'A2'p5'A2'p5'A, but also on a cordycepin analogue of the trimer, (3'dA)2'p5'(3'dA)2'p5'(3'dA), significantly. Considerable cross reaction was observed with oligoadenylates bearing both 2'-5' and 3'-5' internucleotide linkages, while there was little cross reaction with 3'-5' linked oligoadenylates. The antibody showed very low or no affinity for adenosine, adenine mononucleotides, other nucleotides and other 2'-5' linked trinucleotides. The cross reactivity of the modified oligonucleotides is indicated to have relation to the biological activity of the corresponding 5'-phosphorylated oligonucleotides, so far reported. The antibody was further purified by affinity chromatography using RNA-Sepharose and AMP-Sepharose giving high specificity.

Acceleration of intradermal catabolism of guinea pig IgG1 and IgG2 antibodies by challenge with antigen.

The intradermal catabolism of antibodies injected in guinea pigs to provoke skin reactions was studied using 125I-labeled guinea pig IgG1 and IgG2 anti-ovalbumin antibodies. Disappearance of both the IgG1 and IgG2 antibodies from injected sites was accelerated by intravenous injection of the antigen. The antigen-antibody complexes produced in vitro were also catabolized more rapidly than free antibodies, when estimated using 125I-labeled antibodies. On the other hand, the catabolism of normal IgG2 was not influenced by local anaphylactic reaction elicited by IgG1 antiovalbumin antibody coexisting at the sites. Therefore, the enhanced catabolism of antibodies on challenge was not caused by increased vascular permeability due to anaphylactic reactions, but by more rapid elimination of immune complexes formed at the sites. The Fc parts of IgG1 and IgG2 antibodies played an essential role in the enhancement of catabolism since the catabolism of the F(ab')2 fragments was not accelerated by complex formation with ovalbumin, but rather reduced.

Comparative study on R-type pyocins of Pseudomonas aeruginosa.

Four R-type pyocins (R1, R2, R3, and R4) produced by different Pseudomonas aeruginosa strains were compared with respect to their structure and action methanism. It is known that these bacteriocins have structures similar to contractile bacteriophage tails and serologically cross-react with each other, but they are distinguished according to difference in range of sensitive strains. All these pyocins were shown to arrest the synthesis of protein and nucleic acid in sensitive cells, as previously reported for pyocin R1. Action of pyocin R3 was shown to require calcium ion. Although antiserum prepared against one pyocin neutralized other R-type pyocins as well as the homologous pyocin, some differences in antigen specificity were found between pyocin R1 and pyocin R2 and R3 antibody blocking assays. Electron microscopic observation of pyocin particles treated with the antiserum pre-absorbed with heterologous pyocin revealed that specific antigens were located on the distal portion of the fibers. Comparison of protein composition showed these pyocins to be composed of essentially similar subunit proteins but a subunit protein supposed to be a main constituent of the fiber was different in its molecular weight among these pyocins. These results suggest that the main structural difference of these pyocins is to be found in the fiber.

A DNA methylase from Thermus thermophilus HB8.

A DNA methylase was purified in a homogeneous state from a extremely thermophilic bacterium, Thermus thermophilus HB8, by chromatography on, successively, phosphocellulose, CM-cellulose, and heparin-Sepharose. The molecular weight of the enzyme was determined to be about 44,000 by gel filtration on a Sephadex G-100 column and 41,000 by SDS-poly-acrylamide gel electrophoresis, and these findings suggest a single polypeptide enzyme. The enzyme develops maximum activity around pH 7.4 and at 70 degrees C. Enzymatic activity is completely inhibited by 0.2 M NaCl or 2 mM HgCl2. The enzyme transfers methyl groups from S-adenosyl-L-methionine to a double stranded DNA. The sole product of the reaction was identified as N-6-methyl adenine after hydrolysis of the DNA with formic acid. The enzyme kinetics obey the Michaelis-Menten equation and Km values for S-adenosylmethionine and lambda phage DNA were determined to be 0.8 muM and 10 microgram/ml, respectively. The enzyme does not transfer methyl groups to TthHB8I endonuclease digested DNA as well as the host (T. thermophilus HB8) DNA. The number of methyl groups of the fully methylated phiX174 RF DNA was about twice as many as TthHB8I endonuclease sites on the DNA. The distribution of the methyl groups of phiX174 RF DNA among the HaeIII fragments was the same as that of TthHB8I endonuclease sites, suggesting that this DNA methylase is the other component of the modification-restriction system including TthHB8I endonuclease. The enzyme probably recognizes the sequence, 5'-TCGA-3', in a double stranded DNA and probably methylates adenine in the above sequence.

Immunoaffinity purification and properties of Drosophila melanogaster DNA polymerase alpha-primase complex.

Two hybrid cell lines (DM88-5E12 and DM88-4C9) secreting monoclonal antibodies against DNA polymerase alpha-primase complex from Drosophila melanogaster Kc cells were established by immunizing mice with the complex partially purified by a conventional method. The IgG subclasses of both antibodies were IgG1. Both antibodies immunoprecipitated the DNA polymerase alpha-primase complex from D. melanogaster Kc cells. The DNA-polymerizing activity was neutralized by 4C9 antibody, but not by 5E12 antibody. The DNA priming activity was not neutralized by either antibody. These antibodies did not cross-react to HeLa DNA polymerase alpha-primase complex. A rapid, two-step purification of DNA polymerase alpha-primase complex from D. melanogaster Kc cell was carried out by 5E12 antibody column chromatography followed by single-stranded DNA cellulose column chromatography. The immunoaffinity-purified enzyme had both DNA-polymerizing and DNA-priming activities with the specific activities of 50,000 and 2,000 units/mg, respectively. The effects of aphidicolin, NEM, ddTTP, BuPdGTP, and DMSO on the enzyme activity showed that the purified enzyme was DNA polymerase alpha, but not DNA polymerase beta, gamma, or delta. The purified enzyme consisted of polypeptides with apparent molecular weights of 180 (and 145, 140, 130 kDa), 72, 63, 51, and 49 kDa. The 5E12 antibody was shown to bind to all the high-molecular-weight polypeptides, 180, 145, 140, and 130 kDa, by immuno-Western blotting analysis.

Characterization of anti-ApApA antibodies.

Antibodies to a trinucleotide, ApApA, were prepared by injecting bovine serum albumin conjugated with ApApA into rabbits. The specificities of the antibodies were determined by estimating the inhibiton of the binding of [14C]ApApA to the antibodies by various nonradioactive mono-, oligo-, and polynucleotides, using the ammonium sulfate precipitation method. The antibodies were found to react with ApApN sequences and oligoadenylic acids, but also reacted slightly with polyadenylic acid, RNA and DNA. Significant crossreactions were observed with other oligonucleotides containing adenosine.

A new aspect of a restriction endonuclease Tth111 I. It has a degenerated specificity (Tth111 I).

We previously reported that Thermus thermophilus 111 contained two restriction enzymes, Tth111 I and Tth111 II. The former does not cleave phi X174RFDNA and the latter does. We have now found another endonuclease activity able to cleave phi X174RFDNA in the cell extract of T. thermophilus 111. The protein with this activity was purified in a homogeneous state by chromatography on cellulose phosphate, heparin-Sepharose 4B and hydroxylapatite, successively. However, this endonuclease activity was always accompanied with Tth111 I activity during the purification procedure and the purified protein also showed a strong Tth111 I activity, suggesting that the Tth111 I activity and the phi X174RFDNA-cleaving activity reside in a single molecule. The phi X174RFDNA-cleaving activity was enhanced more strongly with Mn2+ than with Mg2+ and seemed to be attributable to a relaxed specificity of Tth111 I activity as seen in the cases of EcoRI* and BamHI* Thus we designated the phi X174RFDNA-cleaving activity Tth111 I*. The molecular weight of the protein with both Tth111 I and Tth111 I* activities was determined to be about 76,000 by gel filtration on a Sephadex G-100 column and 39,000 by SDS-polyacrylamide gel electrophoresis, suggesting the enzyme to be a dimer consisting of identical polypeptide chains. The phi X174RFDNA sequences surrounding Tth111 I* cuts were determined by the chain terminator method of Sanger et al. The results confirmed that Tth111 I* recognized a degenerated form of the Tth111 I recognition sequence, i.e., a sequence such that one of the specified nucleotides in the Tth111 I recognition sequence, 5'GACNNNGTC3', was substituted with N (N stands for any of A, G, C, and T), such as 5'NACNNNGTC3', 5'GACNNNNTC3', 5'GACNNNGNC, and so on (arrows indicate cleavage sites).