Search for nucleon decay via n→ν[over ¯]π0 and p→ν[over ¯]π+ in Super-Kamiokande.

We present the results of searches for nucleon decay via n→ν[over ¯]π0 and p→ν[over ¯]π+ using data from a combined 172.8 kt·yr exposure of Super-Kamiokande-I,-II, and-III. We set lower limits on the partial lifetime for each of these modes: τn→ν[over ¯]π0>1.1×10(33) years and τp→ν[over ¯]π+>3.9×10(32) years at a 90% confidence level.

Search for dinucleon decay into kaons in Super-Kamiokande.

A search for the dinucleon decay pp → K+ K+ has been performed using 91.6 kton·yr data from Super-Kamiokande-I. This decay provides a sensitive probe of the R-parity-violating parameter λ112''. A boosted decision tree analysis found no signal candidates in the data. The expected background was 0.28±0.19 atmospheric neutrino induced events and the estimated signal detection efficiency was 12.6%±3.2%. A lower limit of 1.7×10(32) years has been placed on the partial lifetime of the decay O16 → C14K+ K+ at 90% C.L. A corresponding upper limit of 7.8×10(-9) has been placed on the parameter λ112''.

Evidence for the appearance of atmospheric tau neutrinos in super-Kamiokande.

Super-Kamiokande atmospheric neutrino data were fit with an unbinned maximum likelihood method to search for the appearance of tau leptons resulting from the interactions of oscillation-generated tau neutrinos in the detector. Relative to the expectation of unity, the tau normalization is found to be 1.42 ± 0.35(stat)(-0.12)(+0.14)(syst) excluding the no-tau-appearance hypothesis, for which the normalization would be zero, at the 3.8σ level. We estimate that 180.1 ± 44.3(stat)(-15.2)(+17.8) (syst) tau leptons were produced in the 22.5 kton fiducial volume of the detector by tau neutrinos during the 2806 day running period. In future analyses, this large sample of selected tau events will allow the study of charged current tau neutrino interaction physics with oscillation produced tau neutrinos.

Xylitol carryover effects on salivary mutans streptococci after 13 months of chewing xylitol gum.

To assess mutans streptococci (MS) during xylitol gum chewing (mean 3.8 g/day, 2.9 times/day) for 13 months and then for 15 months after the intervention, Japanese mothers with high salivary MS were randomized into two groups: xylitol gum (n = 56) and no gum (n = 51). The proportion of low MS levels was highest at 3 months of consumption (48.8%), but was significantly lower compared to baseline at the end of the intervention (p < 0.001). MS levels did not change during the postintervention period. The data suggest that in the xylitol group 23.3% showed persistent carryover effects by xylitol gum chewing in the postintervention period.

Refined human artificial chromosome vectors for gene therapy and animal transgenesis.

Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.

Search for differences in oscillation parameters for atmospheric neutrinos and antineutrinos at Super-Kamiokande.

We present a search for differences in the oscillations of antineutrinos and neutrinos in the Super-Kamiokande-I, -II, and -III atmospheric neutrino sample. Under a two-flavor disappearance model with separate mixing parameters between neutrinos and antineutrinos, we find no evidence for a difference in oscillation parameters. Best-fit antineutrino mixing is found to be at (Δm2,sin2 2θ)=(2.0×10(-3) eV2, 1.0) and is consistent with the overall Super-K measurement.

A method for rapid and complete substitution of the circulating erythrocytes in SCID mice with bovine erythrocytes and use of the substituted mice for bovine hemoprotozoa infections.

We have previously developed an in vivo experimental system for a bovine hemoprotozoan parasite, in which SCID mice were periodically transfused with bovine red blood cells (Bo-RBCs), followed by infection with the parasite. The SCID mice prepared by the original method, however, had both mouse and bovine RBCs in the circulation, and their proportion always fluctuated significantly. In the present study, we aimed to deplete the mouse RBCs circulating in SCID mice and, thereby, to create SCID mice having complete Bo-RBC substitution. An anti-erythropoietin rabbit serum, an anti-mouse RBC rabbit serum and 23 monoclonal anti-mouse RBC rat antibodies were prepared for this purpose. They were examined, after administration into SCID mice, for their ability to decrease hematocrit value and also for any other adverse effect. A monoclonal antibody, clone 2E11, was found to have potent ability to induce clearance of the mouse RBCs in SCID mice without causing toxic effects. SCID mice receiving this antibody together with periodic transfusion of Bo-RBCs had their circulating RBCs completely substituted with Bo-RBCs. Infection of Bo-RBC-SCID mice with bovine hemoprotozoan parasites demonstrated that elimination of the mouse RBCs from Bo-RBC-SCID mice resulted in augmentation of parasite growth.

The Bo-RBC-SCID mouse model for evaluating the efficacy of anti-theilerial drugs.

We have previously developed a mouse model which allowed the proliferation of Theileria sergenti in severe combined immunodeficiency (SCID) mice with circulating bovine erythrocytes (Bo-RBC). In the present study, this model was utilized to test the efficacy of anti-theilerial drugs. Bo-RBC-SCID mice were created by giving periodic transfusions of T. sergenti-free Bo-RBC, and subsequently infecting with T. sergenti. Three anti-protozoal compounds, Pamaquine (Yamanouchi Pharmaceutical Co. Ltd), Ganaseg (Japan CIBA-GEIGY Ltd) and Buparvaquone (Coopers Animal Health Ltd), were subcutaneously administered into the mice at doses recommended for cattle therapy. Blood examinations demonstrated that all three drugs significantly reduced the level of parasitemia although Ganaseg was effective only at a dose five times higher than that recommended for cattle therapy. Administration of the drugs neither caused any sign of acute toxicity nor changed the rate of Bo-RBC in the SCID mice's circulating blood cells. The results indicate that the Bo-RBC-SCID mouse model may offer a useful in vivo system for evaluating the efficacy of anti-protozoal drugs against T. sergenti.

Inhibitory effect of heparin on red blood cell invasion by Theileria sergenti merozoites.

Theileria sergenti infection has been one of the most serious infectious diseases of cattle in Japan. A major component in the pathogenesis of T. sergenti is anaemia. The erythrocytic life-cycle, which is responsible for all of the clinical manifestations of T. sergenti infection, is initiated by invasion of bovine red blood cells (RBCs) by merozoites. Here we have focused on the effect of heparin, which has an inhibitory effect on RBC invasion by Plasmodium falciparum, and demonstrated for the first time that bovine RBC invasion by T. sergenti was inhibited by heparin. Further, analysis of this mechanism showed that bovine RBC agglutination, by purified T. sergenti merozoites, was inhibited by heparin and low molecular weight (LMW) heparin. Moreover, hemagglutination was inhibited by treatment of the merozoites with heparinase. These results suggest that merozoites have heparin-like molecules on their surface which may be one of the important factors for attachment to RBCs.

Sequence analysis of the major piroplasm surface protein gene of benign bovine Theileria parasites in east Asia.

Relatively benign Theileria parasites are widespread among cattle in East Asia. Although the parasites are presumed to be of the Theileria sergenti/Theileria buffeli/Theileria orientalis group, their taxonomic status and epidemiology have not been well defined. In the present study, theilerial DNA samples were collected from various East Asian countries, including Japan, Korea, Taiwan, and China. DNA sequences encoding a major piroplasm surface protein were amplified by polymerase chain reaction, followed by cloning into a plasmid vector. More than 20 DNA clones derived from parasite DNA of a single infected animal were examined for their restriction-fragment-length polymorphism, showing that they were classified into four major types. Sequence analysis revealed six types of DNA sequences encoding major piroplasm surface protein with homologies of between 75 and 91%. Of the six sequences, four were identical to those previously reported, while the other two appeared to be new sequences. Among the DNA clones derived from a single infected animal, two to three distinct sequences were often found. Phylogenetic analysis of the six major piroplasm surface protein sequences indicates that five of the six are closely related to each other, and that all are distantly related to the homologous genes of Theileria annulata and Theileria parva. The results suggest that, in addition to those described as T. sergenti/T. buffeli/T. orientalis, there may be some undefined Theileria species distributed in East Asia, and that many cattle are infected with mixed populations of geographically variable Theileria parasites.

Babesia canis infection in canine-red blood cell-substituted SCID mice.

We have developed a mouse model for Babesia canis infection using severe combined immunodeficiency (SCID) mice whose circulating red blood cells had been substituted with canine red blood cells. Substitution of red blood cells in SCID mice was achieved by repetitive transfusions of canine red blood cells, together with administration of an antimouse red blood cell monoclonal antibody. Following inoculation of canine-red blood cell-SCID mice with B. canis, parasites proliferated in the canine red blood cells that had been transfused into the SCID mice, resulting in much higher parasitaemia than that observed in dogs. In an attempt to demonstrate the utility of this mouse model, three antiprotozoal drugs, diminazene diaceturate, clindamycin and oxytetracycline, were examined for their efficacy to inhibit the growth of B. canis in canine-red blood cell-SCID mice. The mouse model clearly showed that diminazene diaceturate and oxytetracycline were capable of eliminating B. canis from the canine-red blood cell-SCID mice, whereas clindamycin exhibited only a static effect as parasitaemia relapsed upon cessation of drug administration.

Detection of Theileria sergenti schizonts in bovine lymph node.

The growth of Theileria sergenti schizonts in bovine lymph nodes was examined by experimental infection. Six naive Holstein calves were inoculated with a sporozoite stabilate into the superficial cervical and subiliac lymph nodes. Biopsy samples from the inoculated calves were collected every 24 h for 10 days. Histological examination demonstrated the schizont of T. sergenti on day 4 post-inoculation. The growth of schizonts in the lymph node was observed on days 4-8 post-inoculation. Electron microscopic observation disclosed that the core of the schizont has a high density central core surrounded by numerous electron-dense spherules.

Structure determination of galacto-oligosaccharides by pyridylamination and NMR spectroscopy.

Galacto-oligosaccharides formed from lactose by the action of some beta-galactosidases were subjected to gel chromatography on Bio-Gel P-2, and the resulting oligosaccharide fractions were converted into pyridylamino (PA) derivatives. Each PA-oligosaccharide fraction, which consisted of several isomers in a given size-class, was then subjected to HPLC on an ODS column. Twenty-one individual galacto-oligosaccharide components were isolated in this way. The structures of most of these compounds, namely six disaccharides, five trisaccharides, two tetrasaccharides, and a pentasaccharide, were determined by 13C-NMR spectroscopy. The results obtained will be useful for the study of the activity of various galacto-oligosaccharides on the growth of Bifidobacterium species.

Clearance of Theileria sergenti-infected bovine red blood cells in severe combined immune deficiency mice.

Clearance of Theileria sergenti-infected bovine red blood cells (Bo-RBCs) from the blood circulation of severe combined immune deficiency (SCID) mice was studied to help understand the mechanisms of anemia developing in cattle infected with T. sergenti. For the clearance test, Bo-RBC samples having 2%, 58%, and 76% parasitemia and, as a control, parasite-free Bo-RBCs were prepared in the Bo-RBC-SCID mouse model. The T. sergenti-infected Bo-RBCs and the uninfected control Bo-RBCs were separately labeled with two, green and red, fluorescent dyes, mixed together, and injected intravenously into SCID mice. The blood samples collected at various time points were observed under a fluorescent microscope, and the numbers of green and red fluorescing RBCs were counted differentially to determine the clearance rates of T. sergenti-infected and uninfected Bo-RBCs. This test clearly demonstrated that the Bo-RBC samples having higher parasitemias were cleared faster from the blood circulation of SCID mice. The results suggest that the intravascular clearance system in SCID mice may have a mechanism by which T. sergenti-parasitized and non-parasitized Bo-RBCs are recognized and cleared differentially.

Stimulation of host-defense mechanism with synthetic adjuvants and recombinant cytokines against viral infection in mice.

The efficacy of synthetic immunoadjuvants and recombinant cytokines for the potentiation of host-resistance against virus infection was investigated using mouse models infected with Sendai virus and herpes simplex type 1 virus (HSV). The synthetic MDP derivative, MDP-Lys(L18), and recombinant cytokines, IL-1 beta, IFN-gamma, G-CSF and GM-CSF were shown to be effective for the stimulation of nonspecific protection against Sendai virus infection in mice. Both MDP-Lys(L18) and GM-CSF were effective for the protection against HSV infection in cyclophosphamide (CY)-treated mice. B30-MDP was suggested to be useful as an immunoadjuvant for the potentiation of antigenicity of recombinant or component vaccines.

Theileria sergenti proliferates in SCID mice with bovine erythrocyte transfusion.

The unavailability of in vitro or in vivo experimental systems has been the major factor hampering the progress of research studies on Theileria sergenti, causative agent of theileriosis, a major disease of cattle in Japan. We report the first successful propagation of T. sergenti in SCID mice into which uninfected bovine erythrocytes (Bo-RBC) were supplied periodically. The infectivity of T. sergenti proliferated in an SCID mouse was ascertained by successful transfer of infection into another SCID mouse into which uninfected Bo-RBC were supplied periodically.

Candida guilliermondii infection in SCID mice in association with the acceleration of the elimination of transfused human red blood cells.

An acceleration of the elimination of transfused human (Hu) red blood cells (RBC) was found in C.B-17 scid (SCID) mice that were kept in our facility. Yeast like organisms were isolated from their tap water just as a pure culture and the two isolates SW5 and SW6 were assigned to be Candida guilliermondii by analysing their generic small subunit ribosomal RNA sequences. To test whether the isolates are infectious in mice, we inoculated SCID and BALB/c mice orally with SW5 and observed them for 63 and 48 days, respectively. The yeasts were frequently recovered from oral swabs, feces and their tap water throughout the experiment. Although none of the mice developed clinical signs or histopathological changes, a positive sero-conversion was confirmed in 4 of 5 SW5-inoculated BALB/c mice. Moreover, a significant acceleration of Hu-RBC elimination in all of the SW5-infected SCID mice was demonstrated. We believe this to be the first report of an inapparent but significant outbreak of C. guilliermondii infection in mice.

Experimental and clinical studies on the effects of synthetic muramyldipeptide derivatives on viral and opportunistic infections.

The efficacy of strearoyl-MDP derivatives, L18-MDP and MDP-Lys-18 against bacterial and viral infections in mice was examined. Both L18-MDP and MDP-Lys-L18 stimulated nonspecifically host-resistance against opportunistic infection with C. kutscheri in hydrocrotisone-treated mice against Sendai virus infection in mice.

Transfusion with xenogeneic erythrocytes into SCID mice and their clearance from the circulation.

We have previously demonstrated that the red blood cells (RBCs) in the blood circulation of SCID mice could be almost completely substituted with bovine RBCs by means of repeated transfusions. In the present study, transfusion experiments were carried out with RBCs from various animal species to investigate the ability of SCID mice to accept xenogeneic RBCs. Bovine (Bo), equine (Eq), human (Hu) and murine (Mu) RBCs were labeled with a fluorescent dye to trace their trafficking in the blood stream. Following the intravenous injection these RBCs were cleared from the circulation at various rates; the 1/100 reduction time in the labeled cell counts was 2 to 7 days, 4 to 7 hr and 1 to 2 hr with Bo-, Hu- and Eq-RBCs, respectively. In contrast, labeled Mu-RBCs from not only syngeneic but also allogeneic mouse strain were able to stay over 50 days. The difference in clearance rates was attributable to the difference in uptake of the RBCs by the splenic macrophages. The clearance rates of labeled RBCs were significantly decreased by co-transfusion with unlabeled RBCs of the same species, indicating that xenogeneic RBCs were recognized by the reticulo-endothelial system of SCID mice in a species-specific fashion. Furthermore, at least in the case of Hu-RBC, the complement component 3 may play a role in the Hu-RBC clearance in SCID mice since C3 deposition was observed on Hu-RBCs but not on Bo- and Eq-RBCs.

Assignment of the bacterial agent of urinary calculus in young rats by the comparative sequence analysis of the 16S rRNA genes of corynebacteria.

Comparative 16S rRNA gene sequencing was used to assign four isolates of spontaneous urinary calculus in young laboratory rats. The phylogenetic relationships among the rat isolates and selected species of corynebacteria were also inferred. Based on the homology and evolutionary distance analysis, the 16S rRNA genes of the rat isolates were almost identical with that of Corynebacterium renale ATCC 19412. Also the results of the phylogenetic analysis showed a close relationship among the isolates and C. renale, but they were clearly different from C. pilosum, C. cystitidis, C. kutscheri and Rhodococcus equi. The results of the present study and previously published biochemical data demonstrate that the organism involving urinary infections in young rats is identified to be C. renale.