Evidence that Porphyromonas (Bacteroides) gingivalis fimbriae function in adhesion to Actinomyces viscosus.

Porphyromonas (Bacteroides) gingivalis adheres to gram-positive bacteria, such as Actinomyces viscosus, when colonizing the tooth surface. However, little is known of the adhesins responsible for this interaction. A series of experiments were performed to determine whether P. gingivalis fimbriae function in its coadhesion with A. viscosus. Fimbriae typical of P. gingivalis were isolated from strain 2561 (ATCC 33277) by the method of Yoshimura et al. (F. Yoshimura, K. Takahashi, Y. Nodasaka, and T. Suzuki, J. Bacteriol. 160:949-957, 1984) in fractions enriched with a 40-kDa subunit, the fimbrillin monomer, P. gingivalis-A. viscosus coaggregation was inhibited by purified rabbit antifimbrial immunoglobulin G (IgG) at dilutions eightfold higher than those of preimmune IgG, providing indirect evidence implicating P. gingivalis fimbriae in coadhesion. Three types of direct binding assays further supported this observation. (i) Mixtures of isolated P. gingivalis fimbriae and A. viscosus WVU627 cells were incubated for 1 h, washed vigorously with phosphate-buffered saline (pH 7.2), and subjected to electrophoresis. Transblots onto nitrocellulose were probed with antifimbrial antiserum. Fimbrillin labeled positively on these blots. No reaction occurred with the control protein, porcine serum albumin, when blots were exposed to anti-porcine serum albumin, (ii) A. viscosus cells incubated with P. gingivalis fimbriae were agglutinated only after the addition of antifimbrial antibodies. (iii) Binding curves generated from an enzyme immunoassay demonstrated concentration-dependent binding of P. gingivalis fimbriae to A. viscosus cells. From these lines of evidence, P. gingivalis fimbriae appear to be capable of binding to A. viscosus and mediating the coadhesion of these species.     

Adhesion of Actinomyces viscosus to Porphyromonas (Bacteroides) gingivalis-coated hexadecane droplets.

Interbacterial adhesion (coadhesion) is considered a major determinant of dental plaque ecology. In this report, we studied several aspects of the adhesion of Porphyromonas (Bacteroides) gingivalis to hexadecane in order to use the liquid hydrocarbon as a convenient substratum for coadhesion assays. Washed suspensions of hydrophobic P. gingivalis 2561 cells were vortexed with hexadecane to yield highly stable cell-coated droplets. Kinetics of coadhesion between Actinomyces viscosus cells and P. gingivalis-coated hexadecane droplets (PCHD) was subsequently studied. Aliquots of PCHD were added to A. viscosus suspensions, and the mixtures were gently rotated. Avid adhesion of A. viscosus cells to the immobilized P. gingivalis layer could be readily measured by the decrease in turbidity in the aqueous phase, following phase separation. Despite the ability of A. viscosus cells to adsorb to hexadecane following vigorous mixing, gentle mixing did not appreciably promote adhesion to bare hexadecane. Moreover, extensive microscopic examinations revealed that A. viscosus cells adhered exclusively to the bound P. gingivalis cells rather than to exposed areas of hexadecane. Coadhesion of A. viscosus to the PCHD appeared to follow first-order kinetics, attaining 80% levels within 30 min. Electron micrographs revealed A. viscosus cells adhering to the P. gingivalis cell layer adsorbed at the hexadecane-water interface. Interestingly, P. gingivalis cells did not appear to penetrate the hexadecane. A viscosus mutants lacking type 1 or type 2 fimbriae or both were still able to bind to the PCHD. No obvious correlation was observed between relative hydrophobicity of A. viscosus strains and their binding to PCHD. However, defatted bovine serum albumin, an inhibitor of hydrophobic interactions, was the most potent inhibitor among those tested. The data suggest that this approach provides a simple, quantitative technique for studying kinetics of bacterial coadhesion which is amenable to both light and electron microscopic observation.     

cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin

Summary The complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine. A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned cDNA insert, which was 1 kb in length, revealed a 432-bp open reading frame encoding an amino-terminal peptide of 15 amino acids and a mature enzyme of 129 amino acids identical in sequence to II. Forms I and II from kidney and liver were also analyzed using enzymatic amplification via PCR and direct sequencing; both organs contain mRNA encoding the two lysozymes. Evolutionary trees relating DNA sequences coding for lysozymesc and α-lactalbumins provide evidence that the gene duplication giving rise to conventional vertebrate lysozymesc and to lactalbumin preceded the divergence of fishes and tetrapods about 400 Myr ago. Evolutionary analysis also suggests that amino acid replacements may have accumulated more slowly on the lineage leading to fish lysozyme than on those leading to mammal and bird lysozymes.     

Numerical simulation of circulation in a Caribbean-type backreef lagoon

A numerical model based on the vertically intergrated equations of motion and continuity was used to simulate circulation in a shallow, well-mixed, Caribbean-type backreef lagoon. As the focus of previous research, Great Pond Bay, St. Croix, provided a suitable study site. Tides, wind, and the effect of waves impinging on the reef were incorporated within the model. Direction of simulated flow under various wind and wave regimes agrees well with patterns found during current meter and drogue tracking experiments. When momentum transfer over the reef due to wave breaking and set-up is added, the magnitude of flow within the lagoon, ranging 5–30 cm/s, also compares favorably to in situ measurements. Model results indicate a relatively rapid and realistic flushing period of less than one day. Results of this study indicate that numerical modeling techniques potentially offer an accurate and cost-effective means to predict the pattern of water movement within coral-reef lagoons.     

Retinol and α-tocopherol concentrations in whole fish commonly fed in zoos and aquariums

Retinol (n = 17 spp.) and α-tocopherol (n = 9 spp.) concentrations in whole fish utilized for captive animal feeding programs were determined by high-performance liquid chromatography (HPLC) following routine storage and preparation after commercial purchase by two zoological institutions. Vitamin A activity was calculated from retinol values and ranged from 55 IU/100 g (immature herring) to >2,000 IU/100 g (salmon) on an as-fed basis. α-Tocopherol values, a measure of vitamin E activity, ranged from 0.9 IU/100 g (butterfish) to 12.3 IU/100 g (tilapia) on a wet basis. Vitamin levels in whole fish were intermediate to values previously quantified for muscle or liver tissues alone. Vitamin concentrations in fish livers were quantified separately in seven of these species; liver contributed 35–63% of total retinol measured and 8–34% of total α-tocopherol. Based on these analyses, whole fish commonly fed in zoos, aquariums, and marine zoological parks would appear to meet vitamin A requirements established for most species without additional supplementation, whereas levels of vitamin E quantified indicate a need for supplementation of diets for piscivores.     

Inheritance of the group I rDNA intron in Tetrahymena pigmentosa

We have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes of Tetrahymena was acquired by different Tetrahymena species at different times during evolution. We have now approached the question of intron mobility experimentally by crossing intron⁺ and intron^? strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron in T. pigmentosa is inherited as a neutral character and that intron⁺ and intron^? alleles segregate in a Mendelian fashion with no sign of intron homing. In an analysis of vegetatively growing cells containing intron⁺ and intron^? rDNA, initially in the same macronucleus, we similarly find no evidence of intron homing. During the course of this work, we observed to our surprise that progeny clones from some crosses contained three types of rDNA. One possible explanation is that T. pigmentosa has two rdn loci in contrast to the single locus found in T. thermophila. Some of the progeny clones from the genetic analysis were expanded for several hundred generations, and allelic assortment of the rDNA was demonstrated by subcloning analysis. © 1992 Wiley-Liss, Inc.     

Biological Control of Damping-Off of Alfalfa Seedlings with Bacillus cereus UW85

We explored the potential of biological control of alfalfa (Medicago sativa L.) seedling damping-off caused by Phytophthora megasperma f. sp. medicaginis by screening root-associated bacteria for disease suppression activity in a laboratory bioassay. A total of 700 bacterial strains were isolated from the roots of field-grown alfalfa plants by using Trypticase soy agar. A simple, rapid assay was developed to screen the bacteria for the ability to reduce the mortality of Iroquois alfalfa seedlings that were inoculated with P. megasperma f. sp. medicaginis zoospores. Two-day-old seedlings were planted in culture tubes containing moist vermiculite, and each tube was inoculated with a different bacterial culture. Sufficient P. megasperma f. sp. medicaginis zoospores were added to each tube to result in 100% mortality of control seedlings. Of the 700 bacterial isolates tested, only 1, which was identified as Bacillus cereus and designated UW85, reduced seedling mortality to 0% in the initial screen and in two secondary screens. Both fully sporulated cultures containing predominantly released spores and sterile filtrates of these cultures of UW85 were effective in protecting seedlings from damping-off; filtrates of cultures containing predominantly vegetative cells or endospores inside the parent cell had low biocontrol activity. Cultures grown in two semidefined media had significantly greater biocontrol activities than cultures grown in the complex tryptic soy medium. In a small-scale trial in a field infested with P. megasperma f. sp. medicaginis, coating seeds with UW85 significantly increased the emergence of alfalfa. The results suggest that UW85 may have potential as a biocontrol agent for alfalfa damping-off, thus providing an alternative to current disease control strategies.     

Histology and histochemistry of the testis and ovary of the platyfish,Xiphophorus maculatus,from birth to sexual maturity

Summary The gonads of 3-day- to 7-month-old male and female platyfish (Xiphophorus maculatus) were examined for the presence of 5-3-hydroxysteroid dehydrogenase (3-HSD) and glucose-6-phosphate dehydrogenase (G6PD) by histochemical means. In 3-day-old males a positive response for both enzymes is localized in the Leydig cells. With subsequent testicular development, these cells increase in number and display greater activity at the periphery of the testis and around the efferent ducts. In 3-day-old females 3-HSD and G6PD are localized in the stromal cells of the ovary. These cells increase in number and activity as the animals become sexually mature. Sertoli cells, efferent duct epithelium, and ovarian granulosa cells are negative at all stages of development examined. Our findings suggest that the gonads of neonatal fish possess the potential for steroidogenesis. The role played by sexsteroid hormones in the maturation of the brain-pituitary-gonad axis is discussed.     

Thermal acclimation and hardening in tadpoles of the bullfrog, Rana catesbeiana

1. 1.|Critical thermal maxima (CTMax) and minima (CTMin) were measured to evalute thermal hardening in Rana catesbeiana.

2. 2.|Tadpoles show heat hardening and CTMax acclimation, and both responses are influenced by developmental stage.

3. 3.|The first evidence of cold hardening in vertebrates is reported here.

4. 4.|Heat hardening significantly reduces cold tolerance, but there is otherwise no evidence of a cross-hardening effect.

The effect of blue light on free and esterified phenolic acids in etiolated gherkin tissues

The major phenolic acid found in gherkin tissues is p-coumaric acid, although cinnamic and caffeic acids are also present; these occur both free an