Antibacterial property of isoflavonoids isolated from Erythrina variegata against cariogenic oral bacteria.

The antibacterial property of 7 compounds, isolated from Erythrina variegata (Leguminosae) by repeated silica gel column chromatography, against cariogenic oral bacteria was investigated. Extensive spectroscopic study revealed that all were isoflavonoids. Among them, 3,9-dihydroxy-2,10-di(gamma,gamma-dimethylallyl)-6a,11a-dehydropterocarpan (erycristagallin) showed the highest antibacterial activity against mutans streptococci, other oral streptococci, Actinomyces and Lactobacillus species with a minimum inhibitory concentration (MIC) range of 1.56-6.25 microg/ml, followed by 3,6a-dihydroxy-9-methoxy-2,10-di(gamma,gamma-dimethylallyl)pterocarpan (erystagallinA) and 9-hydroxy-3-methoxy-2-gamma,gamma-dimethylallylpterocarpan (orientanol B) (MIC range: 3.13-12.5 microg/ml). The antibacterial effect of erycristagallin to mutans streptococci was based on a bactericidal action. Erycristagallin (6.25 microg/ml: MIC) completely inhibited incorporation of radio-labelled thymidine into Streptococcus mutans cells. Incorporation of radio-labelled glucose into bacterial cells was also strongly inhibited at MIC, and 1/2 MIC of the compound reduced the incorporation approximately by half. The findings indicate that erycristagallin has a potential as potent phytochemical agent for prevention of dental caries by inhibiting the growth of cariogenic bacteria and by interfering with incorporation of glucose responsible for production of organic acids.

Evaluation of co-aggregation among Streptococcus mitis, Fusobacterium nucleatum and Porphyromonas gingivalis.

AIMS: To develop a semi-quantitative method for evaluating co-aggregation reactions among three bacterial species, and to examine the influence of Fusobacterium nucleatum on the adherence of Porphyromonas gingivalis. METHODS AND RESULTS: The method involves coating hydroxyapatite (HAP) discs with streptococcal cells and treatment with radio-labelled bacterial cell suspensions. The sensitivity of the method was estimated by comparison with a turbidometric co-aggregation assay. Results from the two methods were in close agreement. Streptococcus mitis-coated HAP discs were immersed in a 3H-labelled Fus. nucleatum cell suspension and then a 14C-labelled P. gingivalis cell suspension. The discs were then pyrolysed to recover and quantify the released 3H and 14C radioactivity. The number of Fus. nucleatum cells on the discs increased with immersion time and this, in turn, resulted in elevated adherence of P. gingivalis. CONCLUSION: The data indicate that the method closely reflects co-aggregation characters, and that Fus. nucleatum has a positive effect on the adherence of P. gingivalis. SIGNIFICANCE AND IMPACT OF THE STUDY: The present method, which is designed to mimic the oral environment, should prove useful in the semi-quantitative evaluation of co-aggregation reactions.

Efficient generation of recombinant adenoviruses using adenovirus DNA-terminal protein complex and a cosmid bearing the full-length virus genome.

An efficient method of constructing recombinant adenoviruses (Ads) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique Swa I site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected into human embryonic kidney 293 cells together with the Ad DNA-terminal protein complex digested at several sites with Eco T22I or Ase I/EcoRI. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the above restriction digestion drastically reduced regeneration of the parent virus. Several hundred virus clones were readily obtained in each experiment, and about 70% of the clones were the desired recombinant viruses. Furthermore, because the cassette contained the full-length Ad genome, any position of the genome could be easily modified to develop a new vector design. We established construction systems for two types of Ad vectors, the E1-substitution type and the E4-insertion type. This method may greatly facilitate the application of recombinant Ads and should be useful for further improvement of Ad vectors.

Angiotensin II suppresses water absorption through the ventral skin of Japanese tree-frogs in vitro.

We previously described two different water absorption systems in the ventral skin of the Japanese tree-frog, Hyla arborea japonica: i.e., a rapid enhanced flow, which is observed in dehydrated tree-frogs or those stimulated by adrenaline beta-agonists or vasotocin, and a slow basal flow, which is observed in normally hydrated frogs during the non-breeding season. The rapid flow is completely blocked by ouabain, which has no effects on the slow basal flow. In the present experiment, we show that the vaso-constrictive hormone angiotensin II completely inhibits basal water absorption, but has no effect on rapid water absorption. These results confirm our previous finding that the two water absorption systems in the ventral skin of the Japanese tree-frog are independent of each other.

An immunocytochemical analysis of a class-specific antibody response against Trichinella spiralis in humans.

The response of different classes of antibodies against antigens of the muscle larvae of Trichinella spiralis was tested using an immunocytochemical approach. Ultrathin sections of resin-embedded larvae were treated with sera from patients with trichinosis, then exposed to a biotinated second antibody and stained with avidin-gold complex. Antibody of the M-class was a major component in the response against a slow-responding group of antigens that included stichocyte granules, the cuticle surface, and the esophagus-occupying substance; a minor component in the response against antigens of the rapid-responding group that included cuticle inner layers, hypodermis, hemolymph, and intestinal gland granules. The response of G-class antibody against the rapid-responding group of antigens was detected in all patients tested, while against the slow-responding group of antigens it was detected in only half of the patients, suggesting that an antibody shift from the M to the G class occurred in some patients. The results, obtained in humans, were similar to those we obtained previously in rats (J. Parasitology, 76,230-239, 1990), suggesting that the rat immune system can serve as an experimental model of human trichinosis.

Direct evidence that the cuticle surface of Trichinella spiralis muscle larvae shares antigenicity with stichocyte alpha-granules and the esophagus-occupying substance.

Antibodies against the cuticle surface of Trichinella spiralis muscle larvae were purified by means of immunoaffinity chromatography and incubated with ultrathin sections of muscle larvae. Major constituents of the parasite reactive with the purified antibodies included the cuticle surface, stichocyte alpha-granules, and the esophagus occupying substance of the muscle larvae. Thus the present data suggest that the cuticle surface is an antigenically different entity from the cuticle inner layers and its origin is likely stichocyte alpha-granules.