Characterization of Bacteroides gingivalis by direct fluorescent antibody staining and cellular fatty acid profiles

Bacteroides gingivalis is a newly proposed species which includes strains isolated from the mouth. Thirteen strains of B. gingivalis isolated from three geographic locations in the United States and France were examined with direct fluorescent antibody staining and analysis of total cellular fatty acids and compared with 16 strains of B. asaccharolyticus of nonoral origin by the same methods. Bacteroides gingivalis strains reacted with the B. gingivalis conjugate (fluorescein isothiocyanate labeled antibody reagent) only, while the B, asaccharolyticus strains reacted with the B. asaccharolyticus conjugate only. The B. gingivalis strains showed negative fluorescence with fluorescein isothiocyanate conjugates for other black-pigmented Bacteroides species. The specificity of the B. gingivalis conjugate was demonstrated by its failure to stain 88 strains of aerobic and anaerobic bacteria other than B. gingivalis. The fatty acid profiles of B. gingivalis and B. asaccharolyticus were readily distinguishable. The B. gingivalis profile was also distinguishable from those of other pigmenting Bacteroides species on the basis of concentration ratios among the characteristic components. These results support the species separation of B. gingivalis and B. asaccharolyticus. Further, they indicate the usefulness of cellular fatty acid profiles as an adjunct to the use of specific fluorescent antibody conjugates for identification of Bacteroides species.     

Genetics of social behaviour in charrs (Salvelinus species)

Juvenile brook charr (Salvelinus fontinalis) are territorial and have high levels of agonistic behaviour. Juvenile lake charr (S. namaycush) rarely show any indication of territoriality and have very low levels of agonistic behaviour. The inheritance of behaviour in their reciprocal hybrids was investigated by multivariate stepwise discriminant analysis. Lateral display, charge, chase, and forage composed the first canonical axis (87.2% of total dispersion). Pooled hybrid means were closer to that of brook charr, suggesting either directional dominance or modification of the behaviour of hybrids in response to water flow. The mean values of reciprocal hybrids were skewed towards the maternal parent, suggesting a maternal effect in the phenotypic expression of behaviour in these fish.     

Field evaluation of a DNA hybridization assay for nuclear polyhedrosis virus in gypsy moth (Lepidoptera: Lymantriidae) larvae.

DNA hybridization assays were used to detect the presence of viral DNA in gypsy moth (Lymantria dispar L.) larvae collected weekly from high density populations or reared from field-collected egg masses. DNA was extracted from larvae, bound to nitrocellulose filters, and hybridized with digoxigenin-labeled L. dispar NPV (LdNPV) DNA probes. The virus incidence determined from DNA hybridization assays was compared with that determined with conventional microscopic examination of larvae for polyhedral inclusion bodies. Among neonates reared from field-collected egg masses, average mortality from LdNPV (15.4%) within 10 d after hatch was not significantly different from the percentage of extracts containing LdNPV DNA (14.8%) found among larvae frozen 5 d after hatch before any mortality occurred. Field-collected larvae were split into two groups: half were frozen immediately and probed for LdNPV DNA and the other half were reared on artificial diet. The proportion containing LdNPV DNA closely approximated the proportion that died within 6 d of collection, but the proportion that died within 13 d of collection was underestimated.     

Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid.

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.     

Preservation of RNA for in situ hybridization: Carnoy's versus formaldehyde fixation.

Tissues fixed with organic solvent fixatives such as Carnoy's solution are known to give poor and erratic results with in situ hybridization, whereas those fixed with paraformaldehyde produce more consistent results. To understand this difference and to improve the utility of Carnoy's-fixed tissue for in situ hybridization, we explored several parameters of RNA integrity and preservation. Carnoy's-fixed, paraffin-embedded livers and paraformaldehyde-fixed, paraffin-embedded livers of mice were compared for RNA extractability, degradation, and hybridizability. In addition, retention of RNA in tissue sections after sequential in situ hybridization treatments was compared. RNA was found to be easily extractable from Carnoy's-fixed liver and was well preserved, with only slight degradation of high molecular weight RNA. Conversely, only a small percentage of the RNA was extractable from paraformaldehyde-fixed liver unless the tissue was digested with protease. The extracted RNA was well preserved, without detectable degradation. Sections of tissue fixed in Carnoy's solution subjected to in situ hybridization retained only about 10% of their original RNA content and gave correspondingly weak in situ hybridization signals. Formaldehyde-fixed tissues retained much more of the RNA (about 45%) and produced strong in situ hybridization signals. Treatment of Carnoy's-fixed tissue sections with vaporous formaldehyde increased retention of RNA and provided in situ hybridization signals comparable with those of paraformaldehyde-fixed tissues.     

Bacteroides and Staphylococcus glycocalyx: chemical analysis, and the effects on chemiluminescence and chemotaxis of human polymorphonuclear leucocytes.

Glycocalyx (or slime), which is an important virulence factor of many pathogenic bacteria, was isolated from Bacteroides fragilis, Bacteroides thetaiotaomicron and Staphylococcus epidermidis. Organisms were grown for 24 h in a chemically defined, dialysable liquid medium. Bacteria were centrifuged and the supernatant was concentrated and dialysed against distilled water. Total carbohydrate and protein were estimated using standard methods. Thin layer and gas-liquid chromatography of trifluoro acetic acid hydrolysed and non-hydrolysed samples provided evidence for the presence of polysaccharide, the absence of nucleic acids and lipopolysaccharide and for the identification of the individual sugar residues. Glucose, mannose and galactose (B. fragilis), glucose (B. thetaiotaomicron), and glucose and heptose (S. epidermidis) were the sugar residues detected. Uronic acid and hexosamine were detected in all species. Glycocalyx preparations (1 mg/ml) from Bacteroides and Staphylococcus significantly inhibited the chemiluminescence and chemotactic responses of viable human polymorphonuclear leucocytes (PMNL), but were not toxic for PMNL.     

“Australopithecus afarensis”: A composite species

In response to a critique byFerguson (1989),Leonard (1991) reiterates most of his original arguments for supporting “Australopithecus afarensis”Johanson, White, andCoppens, 1978 as a single species. He disregards the principle of morphological equivalence by comparing the dental metrics and morphology of a hominid with those of species of the Pongidae, which do not correspond with the degree of variation in hominids, instead of with those of species of the Hominidae. He fails to refute clear evidence that the range of variation of dental metrics and morphology in “A. afarensis” exceeds that seen in species of the Hominidae. On the basis of extreme variation, “A. afarensis” is, therefore, interpreted as representing a composite species.     

The extracellular matrix of lip wounds in fetal, neonatal and adult mice.

Wound healing in the fetus occurs rapidly, by a regenerative process and without an inflammatory response, resulting in complete restitution of normal tissue function. By contrast, in the adult, wounds heal with scar formation, which may impair function and inhibit further growth. The cellular mechanisms underlying these differing forms of wound healing are unknown but the extracellular matrix (ECM), through its effects on cell function, may play a key role. We have studied the ECM in upper lip wounds of adult, neonatal and fetal mice at days 14, 16 and 18 of gestation. The spatial and temporal distribution of collagen types I, III, IV, V and VI, fibronectin, tenascin, laminin, chondroitin and heparan sulphates were examined immunohistochemically. Results from the fetal groups were essentially similar whilst there were distinct differences between fetus, neonate and adult. Fibronectin was present at the surface of the wound in all groups at 1 h post-wounding. Tenascin was also present at the wound surface but the time at which it was first present differed between fetus (1 h), neonate (12 h) and adult (24 h). The time of first appearance paralleled the rate of wound healing which was most rapid in the fetus and slowest in the adult. Tenascin inhibits the cell adhesion effect of fibronectin and during development the appearance of tenascin correlates with the initiation of cell migration. During wound healing the appearance of tenascin preceded cell migration and the rapid closure of fetal wounds may be due to the early appearance of tenascin in the wound. Collagen types I, III, IV, V and VI were present in all three wound groups but the timing and pattern of collagen deposition differed, with restoration of the normal collagen pattern in the fetus and a scar pattern in the adult. This confirms that lack of scarring in fetal wounds is due to the organisation of collagen within the wound and not simply lack of collagen formation. The distribution of chondroitin sulphate differed between normal fetal and adult tissues and between fetal and adult wounds. Its presence in the fetal wound may alter collagen fibril formation. No inflammatory response was seen in the fetal wounds. The differences in the ECM of fetal and adult wounds suggests that it may be possible to alter the adult wound so that it heals by a fetal-like process without scar formation, loss of tissue function or restriction of growth.     

Functional reconstitution of the cardiac sarcoplasmic reticulum Ca2(+)-ATPase with phospholamban in phospholipid vesicles

The Ca2(+)-ATPase in cardiac sarcoplasmic reticulum (SR) is under regulation by phospholamban, an oligomeric proteolipid. To determine the molecular mechanism by which phospholamban regulates the Ca2(+)-ATPase, a reconstitution system was developed, using a freeze-thaw sonication procedure. The best rates of Ca2+ uptake (700 nmol/min/mg reconstituted vesicles compared with 800 nmol/min/mg SR vesicles) were observed when cholate and phosphatidylcholine were used at a ratio of cholate/phosphatidylcholine/Ca2(+)-ATPase of 2:80:1. The EC50 values for Ca2+ were 0.05 microM for both Ca2+ uptake and Ca2(+)-ATPase activity in the reconstituted vesicles compared with 0.63 microM Ca2+ in native SR vesicles. Inclusion of phospholamban in the reconstituted vesicles was associated with a significant inhibition of the initial rates of Ca2+ uptake at pCa 6.0. However, phosphorylation of phospholamban by the catalytic subunit of the cAMP-dependent protein kinase reversed the inhibitory effect on the Ca2+ pump. Similar findings were observed when a peptide, corresponding to amino acids 1-25 of phospholamban, was used. These findings indicate that phospholamban is an inhibitor of the Ca2(+)-ATPase in cardiac SR and phosphorylation of phospholamban relieves this inhibition. The mechanism by which phospholamban inhibits the Ca2+ pump is unknown, but our findings with the synthetic peptide suggest that a direct interaction between the Ca2(+)-ATPase and the hydrophilic portion of phospholamban may be one of the mechanisms for regulation.