Novel Antigens of Oral Actinomyces Species Prepared From a Cell Wall Enzyme Lysate

Hamada, Shigeyuki; Okahashi, Nobuo; Kimura, Shigenobu; Imanishi, H; Koga, T.; Kawata, Seiichi; Michalek, Suzanne M.; McGhee, Jerry R.

doi:10.7883/yoken1952.35.171

Cited by 3 publications

(3 citation statements)

References 15 publications

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“…The Rantz-Randall antigen, a carbohydrate antigen, was prepared from S. anginosus NCTC 10713 as described by Hamada et al [13]. Brie¯y, S. anginosus whole cells 50 mg=ml in sterile saline were autoclaved at 1218C for 20 min and the centrifuged supernate (RRA) was collected.…”

Section: Methodsmentioning

confidence: 99%

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Antigenic characterisation of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal exudate cells

Sasaki

Ohara‐Nemoto

Tajika

et al. 2001

Journal of Medical Microbiology

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A novel antigen that induces nitric oxide (NO) synthesis by murine peritoneal exudate cells (PEC) was prepared from a culture supernate of Streptococcus anginosus NCTC 10713 in dialysed medium by column chromatography with DEAE-Sephacel followed by size-exclusion high performance liquid chromatography (HPLC). A chemical analysis of the S. anginosus antigen (SAA) revealed that it mainly consisted of carbohydrates (rhamnose, N-acetylglucosamine, glucose and galactose), smaller quantities of protein and a trace amount of phosphorus. The SAA stimulated PEC from C57BL/6N mice to produce NO and accumulate induced NO synthetase (iNOS) mRNA in a dose-dependent manner, reaching a plateau with 10±30 ìg=ml. Furthermore, a reverse transcription-PCR assay revealed that SAA 10 ìg=ml could induce mRNA accumulation of tumour necrosis factor-á, interleukin (IL)-1â and IL-6 as well as iNOS. In contrast, RantzRandall antigen (RRA), a carbohydrate antigen prepared from the organisms, could not induce NO synthesis or cause the accumulation of iNOS mRNA, although cytokine production was observed after stimulation. The SAA-induced NO synthesis, but not the cytokine production, was sensitive to heat. Furthermore, an immunoblot analysis of SAA indicated that the 43-kDa protein band reacted with anti-SAA but not anti-RRA antibodies. In immunodiffusion, SAA reacted with both anti-SAA and anti-RRA antibodies, and the precipitin bands formed crossing lines, suggesting that SAA could possess two different antigenic components ± one that reacts speci®cially with anti-SAA antibodies and another that has an identity similar to that of RRA. Taken together, SAA, a novel antigen of S. anginosus, was found to induce NO synthesis as well as produce in¯ammatory cytokines in murine PEC. It is suggested that the protein molecule of SAA may exclusively induce NO synthesis, and its carbohydrate component(s) could have a relationship to cytokine production.

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Section: Methodsmentioning

confidence: 99%

“…Rabbit antisera were prepared by subcutaneous immunisation with SAA and RRA emulsi®ed with Freund's incomplete adjuvant, as described previously [13]. Antigen-speci®c antibodies were puri®ed from the antisera by Protein A Sepharose (Amersham Pharmacia…”

Section: Antigenic Characterisation Of Saamentioning

confidence: 99%

Antigenic characterisation of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal exudate cells

Sasaki

Ohara‐Nemoto

Tajika

et al. 2001

Journal of Medical Microbiology

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“…Therefore, the use of Ml enzyme lysate for purification of S. mutans cell wall components could be a useful means for determining mechanisms of lymphocyte activation and differentiation. In this regard we have already prepared antigens from enzyme lysates of serotype c S. mutans (unpublished data), group A S. pyogenes (type 3) (28), and Actinomyces species (14).…”

Section: Properties Of the Peptidoglycan Portion Isolated From Cell Wmentioning

confidence: 99%

Isolation and Characterization of the Serotype g Carbohydrate Moiety from an Enzyme Lysate of Streptococcus mutans 6715 Cell Walls

Hamada

Torii

Okahashi

et al. 1983

Microbiology and Immunology

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The serotype-specific carbohydrate moiety of Streptococcus mutans was isolated by mild degradation of purified cell walls with a cell-wall lytic enzyme. Cell walls of serotype g S. mutans strain 6715 were digested with M1 enzyme, an endo-N-acetylmuramidase purified from culture supernatants of Streptomyces globisporus strain 1829. The enzyme lysate of the cell walls was applied to a CM Sephadex C-25 column to remove the M1 enzyme from the cell wall lysate and then subjected to Sephadex G-100 column chromatography. Carbohydrate antigens with serotype g specificity, designated M1g, and a peptidoglycan--polysaccharide complex lacking serotype specificity (M1PG) were separated. Purified serotype g antigen was also obtained by autoclaving the S. mutans 6715 whole cells in saline at 120 C for 30 min. The extract was applied to a DEAE Sephadex A-25 column to remove nucleic acids and teichoic acids. The unbound peak fraction was concentrated and re-chromatographed on a Bio-Gel P-100 column. The void volume fraction contained serotype g carbohydrate and was designated RRg antigen. M1g and RRg antigens formed a band of identity with anti-serotype g serum by immunodiffusion. These antigens were composed mainly of galactose, glucose, and rhamnose at an approximate weight ratio of 8 : 4: 1, while constituent sugars of M1PG consisted of rhamnose and glucose, with no detectable galactose. M1g also contained peptidoglycan residues other than threonine, an interpeptide bridge component of the native cell wall peptidoglycan. Marked inhibition of the quantitative precipitin reaction between M1g and anti-serotype g serum was obtained with melibiose and galactose, which suggests that the immunodeterminant of the serotype g carbohydrate is an alpha-linked galactose-glucose terminal linkage.

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Lymphoid Cell Responses to Bacterial Cell Wall Components: Murine B‐Cell Responses to a Purified Cell Wall Moiety of Actinomyces

et al. 1983

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A mitogenic component, designated fraction C (Fr C), has been purified from a mutanolysin enzyme digest of Actinomyces cell walls by CM Sephadex C-25 ion-exchange and G-100 gel filtration chromatography. Good mitogenic responses were obtained with Fr C over a broad dose range with peak mitogenesis seen with 500 micrograms/culture. Fraction C (mol. wt. = 35,000-40,000) consists of 75% carbohydrate and 23% protein, is non-dialysable, resistant to heat, lysozyme or protease treatment, and partially sensitive to base, and all mitogenic activity is destroyed by either periodate or acid treatment. Fraction C is a B-cell mitogen since it induced responses in nude (nu/nu) and nu/+ BALB/c spleen cell cultures and purified splenic B-cell cultures, but did not stimulate purified splenic T-cell cultures. Similar mitogenic fractions for B cells have been obtained from cell walls of A. naeslundii and from a human isolate of A. viscosus. Good polyclonal IgM synthesis and plaque-forming cell responses to hapten or erythrocytes were obtained in vitro with the purified cell wall fractions derived from all three Actinomyces strains studied. These results indicate that the Actinomyces cell wall possesses a carbohydrate-rich component which activates B cells and may represent a common determinant of this genus.

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Novel Antigens of Oral Actinomyces Species Prepared From a Cell Wall Enzyme Lysate

Cited by 3 publications

References 15 publications

Antigenic characterisation of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal exudate cells

Antigenic characterisation of a novel Streptococcus anginosus antigen that induces nitric oxide synthesis by murine peritoneal exudate cells

Isolation and Characterization of the Serotype g Carbohydrate Moiety from an Enzyme Lysate of Streptococcus mutans 6715 Cell Walls

Lymphoid Cell Responses to Bacterial Cell Wall Components: Murine B‐Cell Responses to a Purified Cell Wall Moiety of Actinomyces

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