A novel selective medium for isolation of Streptococcus mutans

The aim of this study was to improve the selective medium of Streptococcus mutans. A new selective medium, designated MS-MUTV, was prepared by adding 10 mg/l valinomycin to the MS-MUT medium previously described. The average recovery of S. mutans was 72.1%, and the growth of S. sobrinus and S. anginousus group was inhibited on MS-MUTV, but allowed on MS-MUT. One hundred and thirty-nine human saliva samples were examined and counted for S. mutans and non-S. mutans colonies. The recovery of S. mutans on MS-MUTV was similar to that on MS-MUT. Eighty-two and 7.9 percent of the saliva samples obtained S. mutans pure cultures, with no bacterial growth on MS-MUTV, respectively. The remaining 10.1% were contaminated with non-S. mutans, with low-level CFU. MS-MUTV is useful for the isolation of S. mutans alone from clinical samples in routine examinations.     

Evaluation of activation treatments for blastocyst production and birth of viable calves following bovine intracytoplasmic sperm injection

The objective of this study was to compare the effectiveness of different methods of bovine oocyte activation following intracytoplasmic sperm injection (ICSI) in terms of oocyte cleavage and blastocyst rates, and calf production. Oocytes were harvested, post mortem, from the ovaries of Japanese Black heifers or cows. ICSI was carried out using a piezo-electric actuator. The injected or sham-injected oocytes that were assigned to three activation treatments, each replicated three times, were studied: (1) exposure to 5 microM ionomycin for 5 min (ionomycin); (2) exposure to 5 microM ionomycin for 5 min followed by culture in TCM199 for 3 h and a further 3h culture in 1.9 mM 6-dimethylaminopurine (DMAP-ionomycin+DMAP); (3) exposure to 7% ethanol in TCM199 for 5 min, 4 h after ICSI (ethanol). One or two blastocysts from the ionomycin+DMAP (8 recipients) and ethanol (17 recipients) oocyte activation treatments were non-surgically transferred into Holsteins for the study of calf production. The highest cleavage and blastocyst production rates were observed in the ionomycin+DMAP treatment (83.9% and 40.1%) by the ICSI. These rates were significantly (P<0.05) higher than those for the ionomycin oocyte activation treatment (57.6% and 18.2%) but did not differ from the ethanol treatment (75.6% and 29.4%). In the sham-injected, the highest blastocyst production rates were observed for the ionomycin+DMAP and ethanol treatments (10.7% and 11.3%). Pregnancy and birth rates for blastocysts derived from the ethanol oocyte activation treatment (58.8% and 47.4%) were significantly higher (P<0.05) than those of the ionomycin+DMAP treatment (12.5% and 9.2%). The results showed that post-ICSI oocyte activation with ethanol is more effective than activation with ionomycin alone or with ionomycin+DMAP for the production of viable blastocysts and calves.     

Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5

In order to find a unique proteinase, proteinase-producing bacteria were screened from fish sauce in Thailand. An isolated moderately halophilic bacterium was classified and named Filobacillus sp. RF2-5. The molecular weight of the purified enzyme was estimated to be 49 kDa. The enzyme showed the highest activity at 60 degrees C and pH 10-11 under 10% NaCl, and was highly stable in the presence of about 25% NaCl. The activity was strongly inhibited by phenylmethane sulfonyl fluoride (PMSF), chymostatin, and alpha-microbial alkaline proteinase inhibitor (MAPI). Proteinase activity was activated about 2-fold and 2.5-fold by the addition of 5% and 15-25% NaCl respectively using Suc-Ala-Ala-Phe-pNA as a substrate. The N-terminal 15 amino acid sequence of the purified enzyme showed about 67% identity to that of serine proteinase from Bacillus subtilis 168 and Bacillus subtilis (natto). The proteinase was found to prefer Phe, Met, and Thr at the P1 position, and Ile at the P2 position of peptide substrates, respectively. This is the first serine proteinase with a moderately thermophilic, NaCl-stable, and NaCl-activatable, and that has a unique substrate specificity at the P2 position of substrates from moderately halophilic bacteria, Filobacillus sp.     

Mechanism of apoptosis induced by doxorubicin through the generation of hydrogen peroxide

The main anticancer action of doxorubicin (DOX) is believed to be due to topoisomerase II inhibition and free radical generation. Our previous study has demonstrated that TAS-103, a topoisomerase inhibitor, induces apoptosis through DNA cleavage and subsequent H(2)O(2) generation mediated by NAD(P)H oxidase activation [H. Mizutani et al. J. Biol. Chem. 277 (2002) 30684-30689]. Therefore, to clarify whether DOX functions as an anticancer drug through the same mechanism or not, we investigated the mechanism of apoptosis induced by DOX in the human leukemia cell line HL-60 and the H(2)O(2)-resistant sub-clone, HP100. DOX-induced DNA ladder formation could be detected in HL-60 cells after a 7 h incubation, whereas it could not be detected under the same condition in HP100 cells, suggesting the involvement of H(2)O(2)-mediated pathways in apoptosis. Flow cytometry revealed that H(2)O(2) formation preceded the increase in Delta Psi m and caspase-3 activation. Poly(ADP-ribose) polymerase (PARP) and NAD(P)H oxidase inhibitors prevented DOX-induced DNA ladder formation in HL-60 cells. Moreover, DOX significantly induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, in HL-60 cells at 1 h, but not in HP100 cells. DOX-induced apoptosis was mainly initiated by oxidative DNA damage in comparison with the ability of other topoisomerase inhibitors (TAS-103, amrubicin and amrubicinol) to cause DNA cleavage and apoptosis. These results suggest that the critical apoptotic trigger of DOX is considered to be oxidative DNA damage by the DOX-induced direct H(2)O(2) generation, although DOX-induced apoptosis may involve topoisomerase II inhibition. This oxidative DNA damage causes indirect H(2)O(2) generation through PARP and NAD(P)H oxidase activation, leading to the Delta Psi m increase and subsequent caspase-3 activation in DOX-induced apoptosis.     

Opisthorchis viverrini antigen induces the expression of Toll-like receptor 2 in macrophage RAW cell line

Opisthorchis viverrini infection induces inflammation in and around the bile duct, leading to cholangiocarcinoma in humans. To examine the mechanism of O. viverrini-induced inflammatory response, we assessed the expression of Toll-like receptors (TLRs) in RAW 264.7 macrophage cell line treated with an extract of O. viverrini antigen. Flow cytometry and immunocytochemistry showed that O. viverrini antigen induced the expression of TLR2 but not TLR4. Western blotting and immunocytochemistry revealed that nuclear factor-kappaB (NF-kappaB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were expressed in RAW 264.7 cells treated with O. viverrini antigen in a dose-dependent manner. These results suggest that O. viverrini induces inflammatory response through TLR2-mediated pathway leading to NF-kappaB-mediated expression of iNOS and COX-2.     

Mechanism of site-specific DNA damage induced by ozone

Ozone has been shown to induce lung tumors in mice. The reactivity of ozone with DNA in an aqueous solution was investigated by a DNA sequencing technique using 32P-labeled DNA fragments. Ozone induced cleavages in the deoxyribose-phosphate backbone of double-stranded DNA, which were reduced by hydroxyl radical scavengers, suggesting the participation of hydroxyl radicals in the cleavages. The ozone-induced DNA cleavages were enhanced with piperidine treatment, which induces cleavages at sites of base modification, but the inhibitory effect of hydroxyl radical scavengers on the piperidine-induced cleavages was limited. Main piperidine-labile sites were guanine and thymine residues. Cleavages at some guanine and thymine residues after piperidine treatment became more predominant with denatured single-stranded DNA. Exposure of calf thymus DNA to ozone resulted in a dose-dependent increase of the 8-oxo-7,8-dihydro-2'-deoxyguanosine formation, which was partially inhibited by hydroxyl radical scavengers. ESR studies using 5,5-dimethylpyrroline-N-oxide (DMPO) showed that aqueous ozone produced the hydroxyl radical adduct of DMPO. In addition, the fluorescein-dependent chemiluminescence was detected during the decomposition of ozone in a buffer solution and the enhancing effect of D2O was observed, suggesting the formation of singlet oxygen. However, no or little enhancing effect of D2O on the ozone-induced DNA damage was observed. These results suggest that DNA backbone cleavages were caused by ozone via the production of hydroxyl radicals, while DNA base modifications were mainly caused by ozone itself and the participation of hydroxyl radicals and/or singlet oxygen in base modifications is small, if any. A possible link of ozone-induced DNA damage to inflammation-associated carcinogenesis as well as air pollution-related carcinogenesis is discussed.     

Autoimmunity against a tissue kallikrein in IQI/Jic Mice: a model for Sjogren's syndrome

We have recently characterized IQI/Jic mice as a model for Sjogren's syndrome (SS), a chronic autoimmune disease in humans. In SS, local lymphocytic infiltrations into salivary and lacrimal glands frequently develop to the involvement of systemic exocrine and nonexocrine organs, and the mechanism for progression of this disease remains obscure. Herein, we report identification of an autoantigen shared by various target organs in IQI/Jic mice. Polypeptides identified based on immunorecognition by autoantibodies in sera from IQI/Jic mice affected with autoimmune disease (>12 weeks of age) were tissue kallikrein (Klk)-1 and -13 and were cross-reactive to the autoantibodies. Interestingly, Klk-13, but not Klk-1, caused a proliferative response of splenic T cells from IQI/Jic mice from the age of 4 weeks onward. In addition, remarkably enhanced expression of Klk-13 was observed in the salivary glands of the mice in accordance with the development of inflammatory lesions. These results indicate that Klk-13 acts as an autoantigen and may increase T cells responsive to organs commonly expressing Klk-13, playing a pivotal role in the etiology of progression of disease in IQI/Jic mice. Our findings provide insights into the contributions of autoantigens shared by multiple organs in the progress of SS from an organ-specific to a systemic disorder.