Development of a novel matrix-assisted laser desorption/ionization time-of-flight mass spectrum (MALDI-TOF-MS)-based typing method to identify meticillin-resistant Staphylococcus aureus clones.

BACKGROUND: Mass spectrum analysis enables species- and subspecies-level identification, and can be used as an epidemiological tool in outbreak management. However, its reliability at clonal level has yet to be established. AIM: To establish a matrix-assisted laser desorption/ionization time-of-flight mass-spectrum-based method that enables bacterial clone identification with accuracy equivalent to pulsed-field gel electrophoresis/phage open-reading frame typing (PFGE/POT). METHODS: Meticillin-resistant Staphylococcus aureus (MRSA) was used in this study. Mass spectra were obtained from a standard strain of S. aureus (ATCC29213) and 57 clinically isolated strains, categorized according to POT. Peaks associated with MRSA clone identification (N = 67) were extracted. Based on this peak information, the feasibility of MRSA clone identification was examined by cluster analysis. FINDINGS: In addition to the 58 strains used for peak extraction, mass spectrum analysis of 24 clinically isolated outbreak strains revealed that peak data could be used for successful identification of clones. These typing results were fully consistent with the PFGE and POT results. CONCLUSION: This novel method enables simple and rapid typing with accuracy equivalent to PFGE/POT. This method would be suited to rapid outbreak analysis, offering accurate information to combat infectious diseases.

Dental caries area of rat molar expanded by cigarette smoke exposure.

OBJECTIVES: Passive smoking is the involuntary inhalation of cigarette smoke (CS) and has an adverse impact on oral health. We examined the effect of CS exposure on caries risk and experimental dental caries. METHODS: Experimental dental caries was induced in rat maxillary molars which were inoculated orally with Streptococcus mutans MT8148 and maintained on a cariogenic diet (diet 2000) and high sucrose water during the experimental period. CS-exposed rats were intermittently housed in an animal chamber with whole-body exposure to CS until killed. Whole saliva was collected before CS exposure (day 0) and for 30 days after the start of CS exposure. Saliva secretion was stimulated by administration of isoproterenol and pilocarpine after anesthesia. Maxillary molars were harvested on day 31. RESULTS: The increase in body weight of the CS-exposed rats was less than that of the control rats. Salivary flow rate, concentration of S. mutans in the stimulated saliva and caries activity score did not significantly differ between 0 and 30 days after the start of CS exposure. Histological examination of the caries-affected area on maxillary molars 30 days after CS exposure showed expansion compared to control rats. In the electron probe microanalysis, no differences were observed between the mineral components of the CS-exposed teeth and the control teeth. CONCLUSION: These results suggest that CS exposure expands the caries-affected area in the maxillary molars of the rat.

Cellular locations of proteinases and association with vesicles in Porphyromonas gingivalis.

We found that locations of arginine-specific gingipain (RGP) in the cellular fractions in the crude extract, envelope, vesicles, and culture supernatants were 48%, 16%, 17%, and 31%, respectively, and the corresponding values of lysine-specific gingipain (KGP) were 47%, 10%, 7%, and 36%, respectively. Although the molecular mass of RGP in the culture supernatant had been determined as 43 kDa, and that of KGP had been as 48 kDa, molecular masses of both proteinases solubilized from the vesicles were estimated to be over 1,500 kDa, since they eluted in the void volume of the column in the gel filtration on Sephacryl S-300. There was no reduction of molecular size by the following treatment with SDS, high-concentration NaCl, or urea. Interestingly, the occurrence of the macromolecular forms could not observed in other enzymes tested such as monopeptidyl, dipeptidyl, and tripeptidyl peptidases, as well as alkaline phosphatase. Therefore, occurrence of the macromolecular forms may be restricted to the proteinases. When the vesicle and culture supernatants containing free RGP and KGP were mixed and incubated, neither RGP nor KGP seemed to bind to vesicles. RGP bound to the vesicle was found to be more stable to heat treatment than the free form, suggesting that association of RGP with the vesicle caused heat stability of this enzyme.

Hemoglobin binding activity and hemoglobin-binding protein of Prevotella nigrescens.

Prevotella nigrescens, lacking siderophores was found to bind to the hemoproteins. The binding was observed also in the envelope which was prepared by sonication of the cell. The binding occurred in the pH-dependent manner; the binding was observed below neutral pHs of the incubation mixtures but only slightly observed in the neutral and alkaline pHs. Furthermore, hemoglobin bound to the envelope was dissociated at high pHs buffers. Maximum amounts of hemoglobin bound to 1 mg envelope was 51.2 mug. Kd for the reaction at pH 5.0 was 2.1 x 10¹° M (210 pM). From the dot blot assay, hemoglobin could bind to a protein solubilized from the envelope by a detergent, referred to as hemoglobin-binding protein (HbBP), then it was purified by the sequential procedures of ion exchange chromatography, affinity chromatography and isoelectric focusing. Molecular weight and isoelectric point of the HbBP were 46 kDa and 6.1, respectively.

Porphyromonas gingivalis mutant defective in a putative extracytoplasmic function sigma factor shows a mutator phenotype.

INTRODUCTION: Porphyromonas gingivalis is implicated as a major pathogen in the development and progression of chronic periodontitis. P. gingivalis must possess the ability to tolerate stress signals outside the cytoplasmic membrane by transcriptional activation of genes encoding proteins involved in defense or repair processes. Some bacteria utilize a distinct subfamily of sigma factors to regulate extracytoplasmic function (hence termed the ECF subfamily). METHODS: To elucidate their role in P. gingivalis, a chromosomal mutant carrying a disruption of an ECF sigma factor PG1318-encoding gene was constructed. Hemagglutination and proteolytic activities were measured in the PG1318-defective mutant. Reverse transcription-polymerase chain reaction (RT-PCR) analysis and southern blot analysis were used to assess transcription of kgp in the PG1318-defective mutant. Frequency of spontaneous mutation that conferred resistance to l-trifluoromethionine was measured in the PG1318-defective mutant. RESULTS: The PG1318-defective mutant formed non-pigmented colonies on blood agar plates at a relatively high frequency. Arginine-specific and lysine-specific proteinase activities of the non-pigmented variants were remarkably decreased compared with those of the parent strain and the pigmented variants. RT-PCR analysis showed that kgp was not transcribed in some non-pigmented variants and southern blot analysis revealed that there was a deletion in their kgp region. Frequency of mutation conferring resistance to l-trifluoromethionine was significantly higher in the PG1318-defective mutant than in the wild-type. CONCLUSION: These results suggest that PG1318 plays a role in the regulation of mutation frequency in the bacterium.

Inorganic polyphosphate: a possible stimulant of bone formation.

Inorganic polyphosphates [Poly(P)] are often distributed in osteoblasts. We undertook the present study to verify the hypothesis that Poly(P) stimulates osteoblasts and facilitates bone formation. The osteoblast-like cell line MC 3T3-E1 was cultured with Poly(P), and gene expression and potential mineralization were evaluated by reverse-transcription polymerase chain-reaction. Alkaline phosphatase activity, von Kossa staining, and resorption pit formation analyses were also determined. The potential role of Poly(P) in bone formation was assessed in a rat alveolar bone regeneration model. Poly(P) induced osteopontin, osteocalcin, collagen 1alpha, and osteoprotegerin expression and increased alkaline phosphatase activity in MC 3T3-E1 cells. Dentin slice pit formation decreased with mouse osteoblast and bone marrow macrophage co-cultivation in the presence of Poly(P). Promotion of alveolar bone regeneration was observed locally in Poly(P)-treated rats. These findings suggest that Poly(P) plays a role in osteoblastic differentiation, activation, and bone mineralization. Thus, local poly(P) delivery may have a therapeutic benefit in periodontal disease.

Dipeptidyl peptidase IV of Streptococcus anginosus: purification and characterization.

We found that N-unblocked nine p-nitroanilde derivatives of amino acids or peptides were hydrolyzed by the crude cell extracts of Streptococcus anginosus NCTC 10713. Then dipeptidyl peptidase IV was purified 323-fold by the procedures including ammonium sulfate concentration, anion exchange chromatography (twice), gel filtration (twice), hydrophobic interaction chromatography, and isoelectric focusing. The molecular weight was calculated as 84 kDa, and the isoelectric point was 4.9. The enzyme hydrolyzed mainly dipeptides containing proline residues at P1 position. It was strongly inhibited by serine enzyme inhibitors. General protease inhibitors, metal chelators, thiol alkylating agent, reducing agent, and several metal ions had no effect on the enzyme activity. Optimum pH for the activity was found at 7.0. The enzyme was mostly inactivated by heating at 50 degrees C for 15 min.

[A successful management of acute myocardial infarction with severe left main trunk lesion using beating heart coronary artery bypass grafting: report of a case].

A 56-year-old male was admitted for sudden onset of severe chest pain and diagnosed as acute myocardial infarction (AMI). Emergency coronary angiography revealed complete obstruction of the left anterior descending artery (LAD) and 99% stenosis of left circumflex coronary artery (LCX), which was almost same with obstruction of the left main trunk coronary artery. Emergency coronary artery bypass grafting (CABG) to LAD and LCX was performed under a beating-heart. Left ventricular wall motion was improved after bypass grafting. CABG under a beating-heart is a new strategy for critical cases such as acute coronary syndrome. The better patency rate and the more complete revascularization that may be achieved with the new generation of stabilizers and retractors may improve long-term results.

Comparison of ability of apoptosis induction by lipopolysaccharide of Porphyromonas gingivalis with Escherichia coli.

We compared effects of Porphyromonas gingivalis LPS with Escherichia coli LPS to the murine peritoneal macrophage. E. coli LPS possessed a threshold dose between 100 micro g and 10 micro g, the higher dose induced apoptosis at the murine peritoneal macrophage while the lower dose did not. The ability of apoptosis induction at the murine peritoneal macrophage of P. gingivalis LPS was weaker than E. coli LPS. P. gingivalis LPS did not induce significant apoptosis in all tested dose. However, the morphology of the peritoneal macrophage treated by P. gingivalis LPS was obviously different from that of the unstimulated cells.

Metabolism of 2-nitrofluorene, an environmental pollutant, by liver preparations of sea bream, Pagrus major.

1. The in vitro metabolism of 2-nitrofluorene (NF), an environmental pollutant, was examined in fish, focusing on nitro-reduction followed by N-acylation and hydroxylation. 2. When NF was incubated with liver microsomes or cytosol of sea bream, Pagrus major, in the presence of NADPH or 2-hydroxypyrimidine, 2-aminofluorene (AF) was formed. 3. When AF was incubated with liver cytosol in the presence of acetyl-CoA or N-formyl-L-kynurenine, 2-acetylaminofluorene (AAF) or 2-formylaminofluorene (FAF) was formed, respectively. AAF and FAF thus formed were deacylated to the parent AF by the liver preparations. 4. AF, AAF and FAF were oxidized to 7-hydroxy or 5-hydroxy derivatives by the liver microsomes. 5. Nitro-reduction, N-acylation and ring-hydroxylation of NF and the metabolites were also observed in rat liver preparations. These activities in sea bream livers were lower than those of rat liver. However, the order of magnitude of these activities in fish was the same as in rat. 6. It is suggested that NF is effectively reduced to AF by the cytochrome P450 system or aldehyde oxidase, and the acylated metabolites, AAF and FAF, generated by arylamine acetyltransferase and formamidase were hydroxylated by the cytochrome P450 system in fish in the same way as in rat. Further, the acetylamino and formylamino derivatives were interconverted via amino derivatives in the fish.

Renal damage and salt-dependent hypertension in aged transgenic mice overexpressing endothelin-1.

The recent development of endothelin-1 (ET-1) antagonists and their potential use in the treatment of human disease raises questions as to the role of ET-1 in the pathophysiology of such cardiovascular ailments as hypertension, heart failure, renal failure and atherosclerosis. It is still unclear, for example, whether activation of an endogenous ET-1 system is itself the primary cause of any of these ailments. In that context, the phenotypic manifestations of chronic ET-1 overproduction may provide clues about the tissues and systems affected by ET-1. We therefore established two lines of transgenic mice overexpressing the ET-1 gene under the direction of its own promoter. These mice exhibited low body weight, diminished fur density and two- to fourfold increases in the ET-1 levels measured in plasma, heart, kidney and aorta. There were no apparent histological abnormalities in the visceral organs of young (8 weeks old) transgenic mice, nor was their blood pressure elevated. In aged (12 months old) transgenic mice, however, renal manifestations, including prominent interstitial fibrosis, renal cysts, glomerulosclerosis and narrowing of arterioles, were detected. These pathological changes were accompanied by decreased creatinine clearance, elevated urinary protein excretion and salt-dependent hypertension. It thus appears that mild, chronic overproduction of ET-1 does not primarily cause hypertension but triggers damaging changes in the kidney which lead to the susceptibility to salt-induced hypertension.

A MATE family multidrug efflux transporter pumps out fluoroquinolones in Bacteroides thetaiotaomicron.

We cloned a gene, bexA, that codes for a multidrug efflux transporter from the chromosomal DNA of Bacteroides thetaiotaomicron ATCC 29741 by using an Escherichia coli DeltaacrAB DeltaacrEF mutant as a host. Although the initial recombinant construct contained other open reading frames, the presence of bexA alone was sufficient to confer to the E. coli host elevated levels of resistance to norfloxacin, ciprofloxacin, and ethidium bromide. Disruption of bexA in B. thetaiotaomicron made the strain more susceptible to norfloxacin, ciprofloxacin, and ethidium bromide, showing that this gene is expressed in this organism and functions as a multidrug efflux pump. The deduced BexA protein sequence was homologous to the protein sequence of Vibrio parahaemolyticus NorM, a multidrug efflux transporter, and thus, BexA belongs to the multidrug and toxic compound extrusion (MATE) family.

Metabolism of 2-nitrofluorene, 2-aminofluorene and 2-acylaminofluorenes in rat and dog and the role of intestinal bacteria.

1. The in vivo metabolism of 2-nitrofluorene (NF), an environmental pollutant, and 2-aminofluorene (AF) and its acylated derivatives, 2-formylaminofluorene (FAF) and 2-acetylaminofluorene (AAF), was examined in rat and dog. 2. 7-Hydroxy-2-nitrofluorene, 5-hydroxy-2-nitrofluorene, AF, AAF, FAF, 7-hydroxy-2-aminofluorene, 5-hydroxy-2-aminofluorene, 7-hydroxy-2-acetylaminofluorene, 5-hydroxy-2-acetylaminofluorene, 7-hydroxy-2-formylaminofluorene and 5-hydroxy-2-formylaminofluorene were identified as urinary and faecal metabolites of NF in rat and dog. 3. AAF and its hydroxylated derivatives were detected as major metabolites of NF in rat, but FAF and its hydroxylated metabolites were mainly excreted in dog. 4. AF, AAF, FAF and their hydroxylated metabolites were also identified as urinary and faecal metabolites of AF, AAF or FAF in rat, suggesting that AAF and FAF are interconverted via AF. 5. Treatment of rat and dog with antibiotics significantly decreased the urinary and faecal excretion of AF and its derivatives after oral administration of NF, and partly decreased the excretion of acylated metabolites after an oral dose of AF. 6. The caecal contents of untreated rats and some species of intestinal bacteria exhibited nitro-reductase activity toward NF, and acylating activity toward AF, affording AAF and FAF.

Application of the piezo-micromanipulator for injection of embryonic stem cells into mouse blastocysts.

Microinjection of embryonic stem (ES) cells into mouse blastocysts is one of the most important techniques for production of knockout or transgenic mice. However, skillful manipulation techniques and tremendous effort are required for this method. To overcome this difficulty, we applied a piezo-micromanipulator (PMM), which has been used for intracytoplasmic sperm injection in mice and production of cloned mice, for the injection of ES cells into blastocysts. When ES cells were injected by using a conventional method, 91% of the blastocysts were manipulated successfully. Using the PMM significantly (P < 0.01) increased the success rate of ES injection to 97%. The number of embryos manipulated in an hour increased from 9.7 embryos with the conventional method to 27.0 embryos with the PMM method. The injected ES cells did not show any detrimental effects due to a pulse from the PMM. After embryo transfer of the manipulated blastocysts, 39% of the newborns were chimeric mice with the conventional method, whereas 42% of the neonates were chimeric after the PMM method. These results indicate that microinjection of the ES cells into blastocysts is more efficient by the PMM method than the conventional method.

Alpha-tocopherol transfer protein is important for the normal development of placental labyrinthine trophoblasts in mice.

Alpha-tocopherol transfer protein (alpha-TTP), a cytosolic protein that specifically binds alpha-tocopherol, is known as a product of the causative gene in patients with ataxia that is associated with vitamin E deficiency. Targeted disruption of the alpha-TTP gene revealed that alpha-tocopherol concentration in the circulation was regulated by alpha-TTP expression levels. Male alpha-TTP(-/-) mice were fertile; however, placentas of pregnant alpha-TTP(-/-) females were severely impaired with marked reduction of labyrinthine trophoblasts, and the embryos died at mid-gestation even when fertilized eggs of alpha-TTP(+/+) mice were transferred into alpha-TTP(-/-) recipients. The use of excess alpha-tocopherol or a synthetic antioxidant (BO-653) dietary supplement by alpha-TTP(-/-) females prevented placental failure and allowed full-term pregnancies. In alpha-TTP(+/+) animals, alpha-TTP gene expression was observed in the uterus, and its level transiently increased after implantation (4.5 days postcoitum). Our results suggest that oxidative stress in the labyrinth region of the placenta is protected by vitamin E during development and that in addition to the hepatic alpha-TTP, which governs plasma alpha-tocopherol level, the uterine alpha-TTP may also play an important role in supplying this vitamin.

Mutagenicity of 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in the new gpt delta transgenic mouse.

Gender differences and organ specificity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced mutagenesis were examined with the new gptdelta transgenic mouse (T. Nohmi, M. Katoh, H. Suzuki, M. Matsui, M. Yamada, M. Watanabe, M. Suzuki, N. Horiya, O. Ueda, T. Shibuya, H. Ikeda, T. Sofuni, A new transgenic mouse mutagenesis test system using Spi-and 6-thioguanine selections (Environ. Mol. Mutagen. 28 (1996) 465-470). In this mouse model, two distinct selections are employed to efficiently detect different types of mutations, i.e 6-thioguanine (6-TG) selection for point mutations and Spi-selection for deletions, respectively. In both selections, the highest mutant frequencies were observed in colon, followed by in spleen and liver. No increases in mutations were observed in testis, brain and bone marrow in PhIP-treated male mice. No significant differences in 6-TG and Spi- mutant frequencies were observed in colon and liver between male and female treated mice. The correlation between PhIP-induced mutagenesis and carcinogenesis in colon is discussed.

Spectra of gpt mutations in ethylnitrosourea-treated and untreated transgenic mice.

We have established a new transgenic mouse mutagenicity assay for the efficient detection of point mutations and deletions in vivo (Nohmi et al. [1996] Env. Mol. Mutagen. 28:465-470). In this assay, the gpt gene of Escherichia coli is used as a reporter for the detection of point mutations. Treatment of mice with ethylnitrosourea (ENU, 150 mg/kg) enhances by several-fold the mutant frequency of gpt in bone marrow. Here, we report the mutation spectra of the gpt gene recovered from bone marrow of ENU-treated and untreated transgenic mice. In the gpt mutants rescued from ENU-treated mice, more than 90% of the mutations were base change mutations; the predominant types were A:T to T:A transversions and G:C to A:T transitions. On the contrary, in the mutants rescued from untreated mice, 54% were base substitutions and the remainders were short deletions and insertions. Among untreated mice, the most frequently observed base substitution was G:C to A:T transitions (7/14 mutants). Three of these occurred at 5'-CpG-3' sites. Interestingly, the mutation spectra of the gpt gene were different from those of the gpt gene in ENU-treated and untreated E.coli, whereas they were similar to those of the lacZ and lacI genes in ENU-treated and untreated other transgenic mice or cultured mammalian cells. We also report the establishment of homozygous transgenic mice that have transgene lambdaEG10 DNA in both chromosome 17 of C57BL/6J mouse.

Spi(-) selection: An efficient method to detect gamma-ray-induced deletions in transgenic mice.

Despite the importance of genome rearrangement in the etiology of cancer and human genetic disease, deletion mutations are poorly detectable by transgenic rodent mutagenicity tests. To facilitate the detection and molecular analysis of deletion mutations in vivo, we established a transgenic mouse model harboring a lambdaEG10 shuttle vector that includes the red and gam genes for Spi(-) (sensitive to P2 interference) selection [Nohmi et al. (1996] Environ. Mol. Mutagen. 28:465-470]. This selection has a great advantage over other genetic systems, because phage deletion mutants can be preferentially selected as Spi(-) plaques, which can then be subjected to molecular analysis. Here, we show nucleotide sequences of 41 junctions of deletion mutations induced by gamma-irradiation. Unlike spontaneous deletion mutants, more than half of the large deletions occurred between short homologous sequences from one to eight bp. The remaining junctions had no such homologous sequences. Intriguingly, two Spi(-) mutants had P (palindrome)-like nucleotide additions at the breakpoints, which are frequently observed in the coding junctions of V(D)J recombination, suggesting that broken DNA molecules with hairpin structures can be intermediates in the repair of radiation-induced double-strand breaks. We conclude that Spi(-) selection is useful for the efficient detection of deletion mutations in vivo and that most rearrangements induced by gamma-rays in mice are mediated by illegitimate recombination through DNA end-joining.

Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation.

Familial Alzheimer disease mutations of presenilin 1 (PS-1) enhance the generation of A beta1-42, indicating that PS-1 is involved in amyloidogenesis. However, PS-1 transgenic mice have failed to show amyloid plaques in their brains. Because PS-1 mutations facilitate apoptotic neuronal death in vitro, we did careful quantitative studies in PS-1 transgenic mice and found that neurodegeneration was significantly accelerated in mice older than 13 months (aged mice) with familial Alzheimer disease mutant PS-1, without amyloid plaque formation. However, there were significantly more neurons containing intracellularly deposited A beta42 in aged mutant transgenic mice. Our data indicate that the pathogenic role of the PS-1 mutation is upstream of the amyloid cascade.