Loss of Neutrophil Homing to the Periodontal Tissues Modulates the Composition and Disease Potential of the Oral Microbiota.

Periodontal disease is considered to arise from an imbalance in the interplay between the host and its commensal microbiota, characterized by inflammation, destructive periodontal bone loss, and a dysbiotic oral microbial community. The neutrophil is a key component of defense of the periodontium: defects in their number or efficacy of function predisposes individuals to development of periodontal disease. Paradoxically, neutrophil activity, as part of a deregulated inflammatory response, is considered an important element in the destructive disease process. In this investigation, we examined the role the neutrophil plays in the regulation of the oral microbiota by analysis of the microbiome composition in mice lacking the CXCR2 neutrophil receptor required for recruitment to the periodontal tissues. A breeding protocol was employed that ensured that only the oral microbiota of wild-type (CXCR2+/+) mice was transferred to subsequent generations of wild-type, heterozygote, and homozygote littermates. In the absence of neutrophils, the microbiome undergoes a significant shift in total load and composition compared to when normal levels of neutrophil recruitment into the gingival tissues occur, and this is accompanied by a significant increase in periodontal bone pathology. However, transfer of the oral microbiome of CXCR2-/- mice into germfree CXCR2+/+ mice led to restoration of the microbiome to the wild-type CXCR2+/+ composition and the absence of pathology. These data demonstrate that the composition of the oral microbiome is inherently flexible and is governed to a significant extent by the genetics and resultant phenotype of the host organism.

Horizontal and Vertical Transfer of Oral Microbial Dysbiosis and Periodontal Disease.

One of the hallmark features of destructive periodontal disease, well documented over the last 50 y, is a change to the quantitative and qualitative composition of the associated microbiology. These alterations are now generally viewed as transformational shifts of the microbial populations associated with health leading to the emergence of bacterial species, which are only present in low abundance in health and a proportionate decrease in the abundance of others. The role of this dysbiosis of the health associated microbiota in the development of disease remains controversial: is this altered microbiology the driving agent of disease or merely a consequence of the altered environmental conditions that invariably accompany destructive disease? In this work, we aimed to address this controversy through controlled transmission experiments in the mouse in which a dysbiotic oral microbiome was transferred either horizontally or vertically into healthy recipient mice. The results of these murine studies demonstrate conclusively that natural transfer of the dysbiotic oral microbiome from a periodontally diseased individual into a healthy individual will lead to establishment of the dysbiotic community in the recipient and concomitant transmission of the disease phenotype. The inherent resilience of the dysbiotic microbial community structure in diseased animals was further demonstrated by analysis of the effects of antibiotic therapy on periodontally diseased mice. Although antibiotic treatment led to a reversal of dysbiosis of the oral microbiome, in terms of both microbial load and community structure, dysbiosis of the microbiome was reestablished following cessation of therapy. Collectively, these data suggest that an oral dysbiotic microbial community structure is stable to transfer and can act in a similar manner to a conventional transmissible infectious disease agent with concomitant effects on pathology. These findings have implications to our understanding of the role of microbial dysbiosis in the development and progression of human periodontal disease.

Serum pharmacokinetics and tissue and milk residues of oxytetracycline in goats following a single intramuscular injection of a long-acting preparation and milk residues following a single subcutaneous injection.

Separate groups of goats were used to determine drug depletion patterns in serum (n=10), tissue (n=20) and milk (n=8) following a single intramuscular (i.m.) dose of 20 mg/kg of a long-acting oxytetracycline (OTC) formulation (Liquamycin LA-200). Milk residues were also determined following a subcutaneous (s.c.) administration of the same product at the same dose. Serum samples were taken for 24 h post-treatment and tissues (fat, liver, kidney, muscle and injection site) collected at 4, 7, 14, 21 and 28 days following injection. Milk from lactating goats was collected every 12 h for 8 days following both the i.m. and s.c. treatments utilizing an intervening 5-week washout period. Residues in serum and tissue were measured using a microbial inhibition assay, while milk residues were measured using both a microbial inhibition assay and a validated HPLC method. The serum pharmacokinetic parameters of OTC in goats were determined, with a mean AUC=67.4 microg h/mL, mean terminal half-life=14.4 h, and apparent clearance=0.33 L/kg h. Tissue half-lives could not be determined with confidence because the collection times provided only two points at which residues could be measured for most tissues. Oxytetracycline residues in all goat tissue samples measured less then cattle tissue tolerance by 96 h postdosing. One-compartment model describing milk depletion data for i.m. and s.c. dosing had terminal slope half-lives of 20.1 and 36.1 h, respectively. By 96 h post-treatment none of the milk samples contained OTC residues in excess of the cattle milk tolerance (0.3 p.p.m.). For both milk and tissue, the upper-bound 99% confidence intervals for the samples taken from goats 96 h postdosing were lower than approved cow milk and tissue tolerances.

Specificity of assays used by regulatory agencies to detect antibiotic residues in tissues of culled dairy cows.

OBJECTIVE: To determine percentage of false-positive test results for assays used by regulatory agencies to detect antibiotic residues in tissues. DESIGN: Prospective study. ANIMALS: 426 dairy cows. PROCEDURE: Dairy cows scheduled for culling that were identified as being unlikely to have antibiotic residues in tissues on the basis of strict inclusion criteria were used. A sample of kidney obtained from each cow at slaughter was tested on-site, using the swab test on premises (STOP; 97 samples) or the fast antibiotic screening test (FAST; 329 samples). Frozen samples (n = 1,278) of liver, muscle, and kidney were thawed and retested at a federal laboratory, using the same screening assays. Kidney and liver samples (n = 852) were also tested using the 7-plate bioassay confirmation test used for confirmation and identification of antibiotic residues. RESULTS: Results of screening assays performed onsite were negative. When frozen samples were retested, 20 (12 liver, 7 kidney, and 1 muscle) had positive FAST results, but none had positive STOP results. Of the samples tested with the 7-plate bioassay confirmation test, 4 liver samples had results indicating a tetracycline (n = 3) or an unidentified microbial inhibitor (1) as a residue. CLINICAL IMPLICATIONS: Results suggest it is unlikely that regulatory action will be taken against producers sending untreated cattle to market. However, because results of the FAST and 7-plate bioassay confirmation test were positive when applied to frozen tissue, use of assays based on microbial inhibition may not be valid for confirmation of residues.

The Food Animal Residue Avoidance Databank (FARAD). Past, present and future.

During the last one-and-one-half decades, FARAD has established an unparalleled compilation of residue and pharmacokinetic information for veterinary species. In order to fulfill its mission, FARAD has become as much a research project as an educational one. Pressing problems, such as disease-altered kinetics, minor-species drug use, and industrial contaminants in livestock, require the new methods of analysis FARAD is developing. The data upon which this work is based can be greatly augmented by participation by other nations. In the United States, it was the cooperation of both academic and regulatory organizations that made the success of FARAD possible. Similar international cooperation can facilitate use of the FARAD model in other countries for the economic benefit of all participants, enhancement of food safety, and promotion of animal welfare.

New Zealand adolescents' perceptions of self and others.

As part of a larger study of adolescents' stress, 1,799 12- to 18-yr.-olds in New Zealand compared themselves with other young people of their age on five general issues. Girls were significantly more likely than boys to say they think they worry more and have more health problems than most people of their age; younger adolescents were more likely than older ones to say they think they are under more peer pressure than most people of their age. On all items, however, most participants rated themselves physically and psychologically healthier than most of their peers.

Biphasic binding kinetics between FepA and its ligands.

The Escherichia coli FepA protein is an energy- and TonB-dependent, ligand-binding porin that functions as a receptor for the siderophore ferric enterobactin and colicins B and D. We characterized the kinetic and thermodynamic parameters associated with the initial, energy-independent steps in ligand binding to FepA. In vivo experiments produced Kd values of 24, 185, and 560 nM for ferric enterobactin, colicin B, and colicin D, respectively. The siderophore and colicin B bound to FepA with a 1:1 stoichiometry, but colicin D bound to a maximum level that was 3-fold lower. Preincubation with ferric enterobactin prevented colicin B binding, and preincubation with colicin B prevented ferric enterobactin binding. Colicin B release from FepA was unexpectedly slow in vivo, about 10-fold slower than ferric enterobactin release. This slow dissociation of the colicin B.FepA complex facilitated the affinity purification of FepA and FepA mutants with colicin B-Sepharose. Analysis of a fluorescent FepA derivative showed that ferric enterobactin and colicin B adsorbed with biphasic kinetics, suggesting that both ligands bind in at least two distinct steps, an initial rapid stage and a subsequent slower step, that presumably establishes a transport-competent complex.

Ligand-specific opening of a gated-porin channel in the outer membrane of living bacteria.

Ligand-gated membrane channels selectively facilitate the entry of iron into prokaryotic cells. The essential role of iron in metabolism makes its acquisition a determinant of bacterial pathogenesis and a target for therapeutic strategies. In Gram-negative bacteria, TonB-dependent outer membrane proteins form energized, gated pores that bind iron chelates (siderophores) and internalize them. The time-resolved operation of the Escherichia coli ferric enterobactin receptor FepA was observed in vivo with electron spin resonance spectroscopy by monitoring the mobility of covalently bound nitroxide spin labels. A ligand-binding surface loop of FepA, which normally closes its transmembrane channel, exhibited energy-dependent structural changes during iron and toxin (colicin) transport. These changes were not merely associated with ligand binding, but occurred during ligand uptake through the outer membrane bilayer. The results demonstrate by a physical method that gated-porin channels open and close during membrane transport in vivo.

Double mutagenesis of a positive charge cluster in the ligand-binding site of the ferric enterobactin receptor, FepA.

Siderophores and colicins enter bacterial cells through TonB-dependent outer membrane proteins. Using site-directed substitution mutagenesis, we studied ligand recognition by a prototypic Escherichia coli siderophore receptor, FepA, that binds the iron chelate ferric enterobactin and colicins B and D. These genetic experiments identified a common binding site for two of the three ligands, containing multiple positive charges, within cell surface residues of FepA. Elimination of single residues in this region did not impair the adsorption or transport of ferric enterobactin, but double mutagenesis in the charge cluster identified amino acids (Arg-286 and Arg-316) that participate in siderophore binding and function in FepA-mediated killing by colicins B and D. Ferric enterobactin binding, furthermore, prevented covalent modification of FepA within this domain by either a fluorescent probe or an arginine-specific reagent, corroborating the involvement of this site in ligand recognition. These results identify, for the first time, residues in a TonB-dependent outer membrane protein that participate in ligand binding. They also explain the competition between ferric enterobactin and the colicins on the bacterial cell surface: all three ligands interact with the same arginine residues within FepA during their penetration through the outer membrane.

Some effects of sex, age, and household structure on family drawings of Barbadian children.

An analysis of the family drawings of a nonclinical sample of 502 Barbadian children aged 7-11 years is reported. The inclusion or omission of figures and the size and positioning of the figures of parents and self were examined with reference to sex, age, and household structure. The view that cultural values about the structure of the family unit and parental roles are reflected in children's representations of their parents in family drawings was supported.

Effect of soluble tissue factor on the kinetic mechanism of factor VIIa: enhancement of p-guanidinobenzoate substrate hydrolysis.

The mechanism by which the protein cofactor, tissue factor, enhances the activity of its cognate serine protease, coagulation factor VIIa (FVIIa), has been studied using the fluorogenic ester substrate 4-methylumbelliferyl p'-guanidinobenzoate (MUGB). Kinetic data were collected at pH 8.4 and pH 7.6 in the presence and absence of soluble tissue factor (sTF; recombinant human tissue factor containing only the extracellular domain). Pre-steady-state techniques allowed the determination of the individual rate constants for acylation (k2) and deacylation (k3) of the sTF.FVIIa complex as well as the dissociation constant for the noncovalent Michaelis complex with MUGB. Alternative methods were required for determination of these parameters for free FVIIa due to extremely slow hydrolysis of MUGB in the absence of sTF. Under all experimental conditions, deacylation was found to be rate-limiting. The major effect of sTF was to raise the affinity of FVIIa for MUGB (31-fold at pH 8.4 and 36-fold at pH 7.6); only minor changes in k2 and k3 were observed. Thus, we conclude that for the ester substrate MUGB, sTF exerts greater allosteric effects on substrate binding than on the later steps involved in the catalytic pathway.

Acid-base catalytic mechanism and pH dependence of fructose 2,6-bisphosphate activation of the Ascaris suum phosphofructokinase.

A form of phosphofructokinase (PFK) from Ascaris suum desensitized to hysteresis in the reaction time course and ATP allosteric inhibition has been used to study the activation by fructose 2,6-bisphosphate (F26P2) at varied pH in both reaction directions. In the direction of phosphorylation of F6P, V and V/KMgATP are constant over the pH range 6-9, while V/KF6P decreases at low pH, giving a pK value of 7.0, and at high pH, giving a pK of 8.9. V and V/KMgATP are insensitive to the presence of F26P2, but V/KF6P is increased by a constant amount in the presence of saturating F26P2 over the entire pH range studied. The concentration of F26P2 that gives half the change in V/KF6P, Kact, increases as the pH decreases, giving a pK of 7.4, reflecting an enzyme group that must be unprotonated for optimum binding of F26P2. In the direction of phosphorylation of MgADP, V and V/KMgADP are pH-independent, and both are insensitive to the presence of F26P2. V/KFBP decreases at high pH, giving a pK of about 7.3, and is increased by a constant amount in the presence of F26P2 over the entire pH range studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Performance of various tests used to screen antibiotic residues in milk samples from individual animals.

The 10-point Milk and Dairy Beef Quality Assurance Program was developed collaboratively by the National Milk Producers Federation and the American Veterinary Medical Association and is designed to promote and document the responsible use of antibiotics in the dairy industry. One area of emphasis in this program is testing of individual animals for antibiotic residues after a specified post-treatment withdrawal time. We examined the performance of various assay systems on milk samples from individual cows. These assays are used at present on bulk tank milk samples by regulatory agencies, processing plants, producers, and veterinarians to detect the presence of beta-lactam antibiotics. A high proportion of false-positive results was obtained for both the pretreatment milk samples from cows with clinical mastitis and the milk samples obtained 21 days after initial therapy (nonantibiotic and antibiotic) for the treatment of mastitis. A high proportion of false-positive outcomes was obtained from the milk of clinically normal cows that had not received any medication for at least 30 days prior to evaluation. The results indicate a serious problem in the use of some assays that were designed to evaluate residues bulk tank milk samples to analyze samples from individual cows. This error in assay specificity results in the unjustifiable discarding of milk that meets regulatory standards and may be misused to accuse the producer or veterinarian of not adhering to regulatory guidelines. Maintaining a safe, high-quality milk supply is a constant goal of the dairy industry, which must be provided the appropriate tools and techniques to meet this challenge.

Corporal punishment in elementary education: views of Barbadian schoolchildren.

Most previous research has suggested that children often express little unconditional disapproval of the use of corporal punishment in schools. However, this might be expected to change when pupils become aware that such treatment is no longer permitted in many countries, or hear it labelled as "abuse." This paper reports on research conducted in elementary schools in the Caribbean island of Barbados, where head teachers (or their authorized deputies) are still permitted by law to use corporal punishment. Findings indicated that approximately three-quarters of pupils surveyed still approved use of corporal punishment with their own age group, although their comments also suggested that a considerable amount of routine (and illegal) "flogging" or "lashing" by regular classroom teachers occurred, which many wished to see stopped. The growing risk of clashes between parents and schools was also identified. While this and other recent studies in Barbados provide little evidence of support for the total abolition of corporal punishment within the educational system, it is hoped that research may have some role to play in exerting pressure on schools to eliminate some of their more ritualized and pedagogically counterproductive practices.

Fructose 2,6-bisphosphate and AMP increase the affinity of the Ascaris suum phosphofructokinase for fructose 6-phosphate in a process separate from the relief of ATP inhibition.

Kinetic data have been collected suggesting that heterotropic activation by fructose 2,6-bisphosphate and AMP is a result not only of the relief of allosteric inhibition by ATP but is also the result of an increase in the affinity of phosphofructokinase for fructose 6-phosphate. Modification of the Ascaris suum phosphofructokinase at the ATP inhibitory site produces a form of the enzyme that no longer has hysteretic time courses or homotropic positive (fructose 6-phosphate) cooperativity or substrate inhibition (ATP) (Rao, G.S. J., Wariso, B.A., Cook, P.F., Hofer, H.W., and Harris, B.G. (1987a) J. Biol. Chem. 262, 14068-14073). This form of phosphofructokinase is Michaelis-Menten in its kinetic behavior but is still activated by fructose 2,6-bisphosphate and AMP and by phosphorylation using the catalytic subunit of cyclic AMP-dependent protein kinase (cAPK). Fructose 2,6-bisphosphate activates by decreasing KF-6-P by about 15-fold and has an activation constant of 92 nM, while AMP decreases KF-6-P about 6-fold and has an activation constant of 93 microM. Double activation experiments suggest that fructose 2,6-bisphosphate and AMP are synergistic in their activation. The desensitized form of the enzyme is phosphorylated by cAPK and has an increased affinity for fructose 6-phosphate in the absence of MgATP. The increased affinity results in a change in the order of addition of reactants from that with MgATP adding first for the nonphosphorylated enzyme to addition of fructose 6-phosphate first for the phosphorylated enzyme. The phosphorylated form of the enzyme is also still activated by fructose 2,6-bisphosphate and AMP.

Barbadian adolescents' views of the "ideal" family.

An instrument examining the perceptions of "ideal" family functioning was administered to 536 adolescents aged 12 to 18 yr. in Barbados. Analysis indicated fairly extensive differences between the views of male and female students, somewhat fewer differences in age and type of school attended, and very few differences associated with household composition and religious affiliation.

A rapid purification procedure and computer-assisted sulfide ion selective electrode assay for O-acetylserine sulfhydrylase from Salmonella typhimurium.

An improved method for purifying O-acetylserine sulfhydrylase from Salmonella typhimurium is described as well as a new computer-controlled assay making use of the sulfide ion selective electrode. The purification method uses gradient elution from Q-Sepharose Fast Flow and phenyl-Sepharose columns to give 75 mg (50% yield) of the enzyme starting from 300 g of starting material in 3 days. The sulfide electrode assay makes use of sulfide and calomel electrodes attached to a signal buffer which serves as an impedance match. The output of the signal buffer is linked in parallel to a strip chart recorder and a Keithley Model 575 data acquisition and control system. The system 575 is interfaced to a Packard-Bell AT computer. In addition, two BASIC computer programs have been written to convert potential measured by the electrode to sulfide concentration and to convert the time course data to rates.