Development and testing of a theory-based behavioural change intervention: a pilot investigation in a nursery school in a deprived area of Scotland.

OBJECTIVE: Investigate the effect of a theory-based intervention on oral-health knowledge, attitudes and behaviours of early years staff (EYS), parents and nursery children. METHODS: Qualitative research with staff and parents from eight nurseries through interviews/focus groups. An intervention was developed and piloted using pre-posttest design. CLINICAL SETTING: Nurseries in deprived communities in Dundee, Scotland. PARTICIPANTS: 111 children aged 3-5 years attending nursery, including 79 parents and 8 nursery staff. INTERVENTIONS: Staff session targeted outcome expectancies, subjective norms and self-efficacy for tooth-brushing in nursery using information provision, modelling and goal-setting, followed by a three-week intervention. Parent-child dyads received a leaflet with instructions for goal-setting, planning and monitoring home brushing. Tooth-brushing self-monitoring materials (e.g. two-minute timer, diaries) were used and certificates provided in the nursery as rewards. OUTCOME MEASURES: EYS knowledge, attitudes and behaviours were assessed before and after the intervention using self-report questionnaires. Parents completed interviews assessing beliefs about tooth-brushing and their children's tooth-brushing behaviour at baseline and post-intervention follow-up. RESULTS: Significant improvements in staff knowledge, but not attitudes, self-efficacy, or nursery tooth-brushing were reported. Parent-child dyads completing the intervention were not more likely to report their child carried out twice-daily tooth-brushing at home. The intervention did not improve parents' intentions to brush their child's teeth twice a day or beliefs about the ease of twice-daily tooth-brushing. CONCLUSION: Only past behaviour significantly predicted posttest brushing. Parents who found brushing easier at baseline were more likely to complete the intervention. Recommendations are made regarding implementing psychological theory and methods into oral-health interventions.

An investigation of the relationship between untreated decayed teeth and dental sepsis in 5-year-old children.

OBJECTIVES: To investigate the prevalence of dental sepsis in 5-year-old children in Scotland and the relationship between sepsis, treated and untreated decayed teeth, oral cleanliness (visible plaque on anterior teeth) and socio-economic deprivation. SUBJECTS AND METHODS: Six thousand, nine hundred and ninety-four children of mean age 5.3 years were examined as part of a survey conducted under the Scottish Health Board's Dental Epidemiological Programme. The presence of dental sepsis was recorded, in addition to caries status, and presence of plaque. Postal code information was used to obtain a measure of material deprivation. Relationships between sepsis and its possible contributory factors were explored using stepwise logistic regression. MAIN RESULTS: In the whole sample, 4.8% of children examined had dental sepsis, ranging from 2% in the most affluent areas to 11% in the most deprived. Children with sepsis had much higher caries experience (mean dmft 6.30) than those without sepsis (mean dmft 2.36). However, when these factors and the presence of plaque were entered into a logistic regression model to predict presence or absence of dental sepsis, the most important factor was not deprivation, but untreated decay. CONCLUSIONS: The proportion of children with sepsis increases markedly with caries experience. This disadvantage can be mitigated if more of the caries experience is treated. These findings would not support a policy of non-intervention for deciduous caries if oral sepsis is to be minimised.

Dissolved inorganic carbon utilization and the development of extracellular carbonic anhydrase by the marine diatom Phaeodactylum tricornutum.

The presence of extracellular carbonic anhydrase (CA) in relation to medium composition was investigated using cultures of the marine diatom Phaeodactylum tricornutum Bohlin. Large-volume cultures, with low initial cell inocula were grown on ASP-2 (no dissolved inorganic carbon (DIC), 550 µM NOa - ), f/2 (2-0 mM DIC, 880µM NO3 - ) and modified f/2 (2.0 mM DIC, 20 µM NO3 - media. Cells growing on ASP-2 showed extracellular CA in the early stages of growth, whereas extracellular CA was not detected until partial depletion of total DIC in the stationary phase for cultures on f/2 or modified f/2. Both HCO3 - and CO2 were important in carbon limitation, extracellular CA being present when the free-CO2 concentration fell below 5 µM, but the HCO3 - concentration needed to be below 1 mM. When carbon-replete cells were transferred to carbon-limited conditions, extracellular CA was recorded within minutes, the process being light-dependent and completely inhibited by 3,3,4-dichlorophenyl-l, 1-dimethylurea (DCMU). The addition of DIC to carbon-limited cells resulted in a rapid decrease in extracellular CA activity. The membrane-impermeable inhibitor of carbonic anhydrase, dextran-bound sulphonamide (DBS) was used to inhibit extracellular CA activity in relation to photosynthetic rate in carbon-replete and carbon-limited cells. At the lowest DIC concentration (O'lOniM), for cells with maximum external CA activity, DBS gave over 80% inhibition of the photosynthetic rate, demonstrating the key role of external CA in maintaining high photosynthetic rate under conditions of carbon limitation. It is proposed that the key factor in the regulation of extracellular CA activity is the total Hux of inorganic carbon (C.) into the cell. This determines the Ci , flux into the chloroplast and when this is inadequate to support the photosynthetic rate attained by a carbon-replete chloroplast at optimum photon flux density, extracellular CA is activated. This mechanism would explain the observed interaction of CO2 and HCO3 - in the regulation of extracellular CA activity.

Utilization of dissolved inorganic carbon (DIC) and the response of the marine flagellate Isochrysis galbana to carbon or nitrogen stress.

The growth of the marine flagellate Isochrysis galbana was followed in batch cultures at four concentrations of dissolved inorganic carbon (DIC), from C- and N-replete lag phase into C- and/or N-deplete stationary phase. Organic buffers were omitted from the growth medium, and culture pH was maintained at 8.30±0.05 by the addition of acid or alkali. The responses of the flagellate to N stress included an increase in the C∶N ratio, and decreases in the ratios of glutamine (Gln)∶glutamate (Glu) and Chl a∶C, and the cell Chl a quota. Conversely, the responses to C stress included a decrease in the C∶N ratio, and increases in the ratios of Gln∶Glu and Chl a∶C, and the cell Chl a quota. The relationship between carbon-specific growth rate (C-µ), and the concentration of extracellular DIC, [DIC]ext , exhibited Michaelis-Menten type kinetics with a half saturation constant, KG(DIC) , of 81 µM. Comparative studies of the diatom Phaeodactylum tricornutum showed similar results, although the value of KG(DIC) was lower at 30 µM.

Carbon dioxide-concentrating mechanism and the development of extracellular carbonic anhydrase in the marine picoeukaryote Micromonas pusilla.

A range of marine photosynthetic picoeukaryote phytoplankton species grown in culture were screened for the presence of extracellular carbonic anhydrase (CAext ), a key enzyme in inorganic carbon acquisition under carbon- limiting conditions in some larger marine phytoplankton species. Of the species tested, extracellular carbonic anhydrase was detected only in Micromonas pusilla Butcher. The rapid, light-dependent development of CAext when cells were transferred from carbon-replete to carbon-limiting conditions was regulated by the available free- CO2 concentration and not by total dissolved inorganic carbon. Kinetic studies provided support for a CO2 - concentrating mechanism in that the K0.5 [CO2 ] (i.e. the CO2 concentration required for the half-maximal rate of photosynthesis) was substantially lower than the Km [CO2 ] of Rubisco from related taxa, whilst the intracellular carbon pool was at least seven fold greater than the extracellular DIC concentration, for extracellular DIC values ⩽1.0 mm. It is proposed that when the flux of CO2 into the cell is insufficient to support the photosynthetic rate at an optimum photon irradiance, the development of CAext increases the availability of CO2 at the plasma membrane. This ensures rapid acclimation to environmental change and provides an explanation for the central role of M. pusilla as a carbon sink in oligotrophic environments.

Expression of colonic antigens by goblet and columnar epithelial cells in ileal pouch mucosa: their association with inflammatory change and faecal stasis.

AIMS: To investigate colonic metaplasia of goblet and columnar epithelial cells in ileal pouch mucosa; to correlate this with the degree of morphological and inflammatory change; and to assess whether such changes are related to the presence of faecal stasis. METHODS: Biopsy specimens of ileal pouch mucosa were taken from 31 patients (30 with ulcerative colitis, one with familial adenomatous polyposis) either before (eight patients) or after (23 patients) ileostomy closure. A simple morphological technique was used to assess changes in villous height. Inflammatory change was estimated using an established scoring system for pouchitis, and acquisition of colonic antigens was determined by immunohistochemistry using three monoclonal antibodies which recognise components of the two major epithelial cell types in the colorectum. The degree of staining with the monoclonal antibodies was graded and the grades correlated with an index of villous atrophy and with the inflammatory scores. RESULTS: Five of eight (63%) pre-closure and 15 of 23 (65%) post-closure biopsy specimens showed increased staining with an antibody against components of columnar epithelial cells. One of eight (12%) pre-closure and 15 of 23 (65%) post-closure biopsy specimens stained with an antibody for colonic mucin. Although both types of staining showed a positive correlation with the pouchitis score, they also occurred in the absence of inflammation. CONCLUSIONS: Both goblet and columnar cells acquire colonic characteristics which are incomplete, but may represent a true adaptive response as they can develop in the absence of inflammation. As the change in goblet cells occurs after ileostomy closure, faecal stasis is likely to be a major contributory factor. Changes in columnar cells may occur before ileostomy closure in the absence of faecal stasis.

Ileal pouch dialysate is cytotoxic to epithelial cell lines, but not to CaCo-2 monolayers.

OBJECTIVE: All patients with ileal pouch-anal anastomosis (IPAA) have some degree of villous atrophy, mucin changes and chronic inflammation. The mechanism underlying these changes is unknown. This study investigates the hypothesis that luminal factor(s) may affect epithelial cells in in-vitro studies. DESIGN: IPAA dialysate from eight patients with prior ulcerative colitis was assessed. METHODS: The effect of the dialysate on epithelial cell (i-407, HT-29 and CaCo-2) proliferation (3H-thymidine incorporation) and cytotoxicity (51-chromium release) was determined. Bile acid(s) at concentrations measured in IPAA dialysate were assessed in isolation and in combination for cytotoxicity against CaCo-2 cells. The effect of dialysate and bile acids on immature and mature CaCo-2 monolayer cytotoxicity, transepithelial electrical resistance (TEER) and histology was investigated. RESULTS: IPAA dialysate is antiproliferative and cytotoxic to the cell lines. At concentrations present in dialysate chenodeoxycholic acid and deoxycholic acid were cytotoxic to CaCo-2 cells. IPAA dialysate was not cytotoxic to mature CaCo-2 cell monolayers. TEER was maintained across monolayers with dialysate but not with control Rheomacrodex (P = 0.02). Bile acids at concentrations present in dialysate were cytotoxic to monolayers. CONCLUSION: Ileal pouch dialysate is cytotoxic to epithelial cells due (in part) to bile acids. Bile acids in isolation are cytotoxic to both CaCo-2 cells and monolayers. Despite the presence of bile acid the dialysate is not cytotoxic to CaCo-2 monolayers and preserves epithelial resistance and histological structure.

Ion accumulation in lucerne plants regenerated from salt-adapted suspension cultures compared with recultured cells from these plants.

Lucerne (Medicago media cv. Rambler) plants, regenerated from cell suspension cultures adapted to 200 mM NaCl, and cell cultures derived from the leaflets of regenerated plants showed enhanced Na+ exclusion compared with the unselected plants and cell cultures. There was a decrease in the K+ concentration in response to NaCl treatment but the selected plants and cell cultures maintained a significantly higher K+ concentration and showed much lower Na+/K+ ratios than unselected plants and cell cultures. The proline concentration increased in the shoots and cell cultures in response to NaCl stress but the selected plants and cell cultures showed a significantly higher proline concentration than unselected plants and cell cultures.

Primary structure of 3-phosphoglycerate kinase from horse muscle. II. Amino acid sequence of cyanogen bromide peptides CB1-CB4 and CB6-CB14, sequence of methionine-containing regions, and complete sequence of the enzyme.

The amino acid sequences of 13 of the 14 cyanogen bromide peptides of horse muscle 3-phosphoglycerate kinase have been determined. These peptides together constitute 75% of the structure of the enzyme. Except for the smallest peptides, automated sequence analysis of the parent peptide was employed together with the automated or manual (5-dimethylaminonaphthalene-1-sulfonyl (dansyl)-Edman method) sequencing of relevant peptides obtained by proteolytic digestion. In the case of the five smallest peptides, all between 6 and 11 residues in length, sequences were derived from analyses of the intact peptides. CB14a, a peptide produced in low yield during the preparation of the cyanogen bromide fragments, was helpful in the sequence analysis of CB14. This peptide appeared to have arisen as the result of a cyanogen bromide cleavage on the carboxyl side of a tryptophanyl residue. The sequences of these 13 cyanogen bromide peptides, together with that of CB1, reported previously (Hardy, G. W., Darbre, A., and Merrett, M. (1981) J. Biol. Chem. 256, 10284-10292), have been combined with data obtained from the sequence analysis of tryptic peptides derived from the methionine-containing regions of the enzyme to determine the complete sequence. Isolation of these overlap peptides was facilitated by the labeling of methionyl residues with iodo[2-14C]acetic acid prior to tryptic digestion. Tryptophan-containing tryptic peptides were also isolated, and their sequence analysis enabled the positions of the four tryptophanyl residues to be established. In this way, the problem caused by tryptophan degradation during the preparation of the cyanogen bromide fragments was overcome. The NH2 terminus of CB1, the NH2-terminal cyanogen bromide peptide, was identified as N-acetyl serine by mass spectrometry. The alignment of the cyanogen bromide fragments of horse muscle 3-phosphoglycerate kinase described here is in complete agreement with that based on X-ray analysis of the enzyme reported previously (Banks, R. D., Blake, C. C. F., Evans, P. R., Haser, R., Rice, D. W., Hardy, G. W., Merrett, M., and Phillips, A. W. (1979) Nature 279, 773-777). The complete structure is comprised of 416 residues and has a molecular weight of 44,519.

Enzyme levels in relation to obligate phototrophy in chlamydobotrys.

During the transition from photoheterotrophic growth on acetate to phototrophic growth on carbon dioxide, there is a decrease in isocitrate lyase and increase in ribulose-1,5-diphosphate carboxylase activity in Chlamydobotrys stellata cultures. The increase in ribulose-1,5-diphosphate carboxylase activity is the result of protein synthesis, there being a close correlation between increase in enzyme activity and protein precipitated by antibody to ribulose-1,5-diphosphate carboxylase. The purified ribulose-1,5-diphosphate carboxylase was similar to the constitutive enzyme from other green algae having a molecular weight of 530,000 and composed of two types of subunit of molecular weight 53,000 and 14,000.Enzyme assays demonstrated an incomplete tricarboxylic acid cycle in cells growing photoheterotrophically on acetate or phototrophically on carbon dioxide. Although these cells lack alpha-ketoglutarate dehydrogenase and succinate thiokinase, a cyclic flow of acetate carbon is possible in the presence of the glyoxylate cycle enzymes but the yield of adenosine triphosphate from acetate oxidation may be insufficient to support heterotrophic growth, so rendering Chlamydobotrys an obligate phototroph.

Ileal pouches: adaptation and inflammation.

Ileal pouch-anal anastomosis (IPAA) has become the operation of choice following proctocolectomy for ulcerative colitis (UC) and familial adenomatous polyposis. Functioning ileal pouch mucosa undergoes histological changes resembling the colon (colonic metaplasia). The possible role of stasis and luminal factors--bile acids, short-chain fatty acids and bacteria--are discussed. It seems likely that colonic metaplasia is an adaptive response to the new luminal environment in IPAA. Inflammation in the ileal reservoir ('pouchitis') is the most significant late complication in IPAA. It occurs in 20-30% of patients and is virtually confined to those with prior UC. The clinical picture in pouchitis is highly variable; however, it can be easily categorized into three groups. Nevertheless, in most cases it is likely to represent recurrent UC in the ileal pouch. Current treatments and possible preventative strategies for pouchitis have been outlined.

Glycollate-pathway enzymes in mitochondria from phototrophic, organotrophic and mixotrophic cells of Euglena.

Glycollate dehydrogenase and NADFH-glyoxylate reductase are constitutive enzymes in Percoll-purified mitochondria from phototrophic, mixotrophic and organotrophic cells of Euglena gracilis Klebs strain z Pringsheim. Glycollate oxidation by isolated mitochondria is stimulated four-fold by the addition of glutamate but rates of glycine oxidation are low in mitochondria from all cell types, the ratio of malate to glycine oxidation always being greater than 4:1. Measurement of the rate of NADPH oxidation in intact mitochondria and mitoplasts showed that the outer mitochondrial membrane is impermeable to NADPH and in the absence of NADPH-dehydrogenase activity the oxidation of NADPH by mitoplasts is dependent on the presence of glyoxylate for NADPH-glyoxylate-reductase activity. It is concluded that glycollate oxidation in the mitochondrion provides glyoxylate which, in the presence of a suitable amino-donor, can be converted to glycine by glutamate-glyoxylate amino-transferase so providing essential intermediates for biosynthesis. Glycollate oxidation outside the mitochondrion is concerned with photorespiratory metabolism and the inability of mitochondria to oxidise exogenous glycine at appreciable rates means that the separation of photorespiratory metabolism from the biosynthesis of essential intermediates is effected.

The regulation of synthesis of mitochondrial enzymes in regreening and division-synchronized Euglena cultures.

The effect of light and carbon nutrition on the synthesis of citrate synthase (EC 4.1.3.7) and malate dehydrogenase (EC 1.1.1.37) in dark-grown resting (carbon deficient) and in phototrophic division-synchronized cultures of Euglena gracilis Klebs strain z were investigated. Exposure of dark-grown Euglena to white or red light produced a transient increase in the specific activities of citrate synthase and malate dehydrogenase but blue light (of equal energy) was ineffective. Citrate-synthase activity increased at the end of the light phase and in early dark phase in phototrophic cultures division-synchronized by a regime of 14 h light-10 h dark. The addition of ethanol or malate produced a twofold increase in citrate-synthase activity compared with phototrophic cultures. White and blue light, but not red light, produced a transient repression of the metabolite-induced increase in citrate-synthase activity in division-synchronized cultures. Since only red light could effect a transient increase in the specific activity of mitochondrial enzymes, and the blue-red plastid receptor should respond to both blue and red light, the synthesis of mitochondrial enzymes in regreening cultures may be under the control of a new photoreceptor responding only to red light. In division-synchronized phototrophic cells the primary effector of synthesis of mitochondrial enzymes is not light but carbon nutrition.

Regulation of synthesis of citrate synthase in regreening Euglena gracilis.

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.

Citrate-synthase mRNA in relation to enzyme synthesis in division-synchronized Euglena cultures.

The regulation of citrate-synthase (EC 4.1.3.7) synthesis in division-synchronized cultures of Euglena gracilis Klebs strain z was investigated. Citrate-synthase activity increased approximately two fold at the end of the light phase and in early dark phase in phototrophic cells synchronized by a 14 h light-10 h dark regime. Anti-(citrate synthase) was used to demonstrate that this increase in activity resulted from an increase in citrate-synthase protein. The amount of polyadenylated RNA per aliquot of culture increased midway through the light phase (before DNA replication) and had doubled by the end of this phase. Anti-(citrate synthase) was used to detect precursor citrate synthase in translations of total polyadenylated RNA in rabbit reticulocyte lysates. Citratesynthase mRNA was found to be present in cells at each stage of a division cycle, so that a stagespecific production of this mRNA to coincide with an increase in enzyme protein is unlikely. It is suggested that a post-transcriptional control operates in the regulation of citrate-synthase synthesis.

The localization of glycollate-pathway enzymes in Euglena.

Isolation of organelles from broken-cell suspensions of phototrophically grown Euglena gracilis Klebs was achieved by isopycnic centrifugation on sucrose gradients. 2. Equilibrium densities of 1.23g/cm3 for peroxisome-like particles, 1.22g/cm3 for mitochondria and 1.17g/cm3 for chloroplasts were recorded. 3. The enzymes glycollate dehydrogenase, glutamate-glyoxylate aminotransferase, serineglyoxylate aminotransferase, aspartate-alpha-oxoglutarate aminotransferase, hydroxy pyruvate reductase and malate dehydrogenase were present in peroxisome-like particles. 4. Unlike higher plants glycollate dehydrogenase and glutamate-glyoxylate aminotransferase were present in the mitochondria of Euglena. 5. Rates of glycollate and D-lactate oxidation were additive in the mitochondria, and, although glycollate dehydrogenase was inhibited by cyanide, D-lactate dehydrogenase activity was unaffected. 6. Glycollate oxidation was linked to O2 uptake in mitochondria but not in peroxisome-like particles. This glycollate-dependent O2 uptake was inhibited by antimycin A or cyanide. 7. The physiological significance of glycollate metabolism in Euglena mitochondria is discussed, with special reference to its role in photorespiration in algae.

Induction potential for glyoxylate cycle enzymes during the cell cycle of Euglena gracilis.

In light/dark synchronized cultures of Euglena gracilis Klebs Z the enzymes malate synthase, isocitrate lyase and acetate thiokinase were induced upon addition of acetate at all stages of the cell cycle. Cycloheximide and p-fluorophenylalanine inhibited the development of enzyme activity, showing that induction was dependent on protein synthesis. The maximum rate of induction for all three enzymes was constant for much of the cell cycle but doubles in a single step during the period of DNA replication. Although these data indicate that enzyme potential was regulated by gene dosage and that the structural gene for each enzyme was continuously available for transcription during the cell-cycle it was not possible by using inhibitors of RNA synthesis, to demonstrate concurrent transcription during enzyme induction.

The glycolate pathway and photosynthetic competence in euglena.

The development of glycolate pathway enzymes has been determined in relation to photosynthetic competence during the regreening of Euglena cultures. Phosphoglycolate phosphatase and glycolate dehydrogenase rapidly reached maximal levels of activity but the complete development of ribulose 1,5-diphosphate carboxylase and concomitant photosynthetic carbon dioxide fixation were not attained until 72 hours of illumination. Specific inhibitors of protein synthesis showed that the formation of ribulose 1,5-diphosphate carboxylase in both division-synchronized and regreening cultures was prevented by both cycloheximide and d-threo-chloramphenicol, whereas phosphoglycolate phosphatase formation was only inhibited by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide. Since cycloheximide prevented ribulose diphosphate carboxylase synthesis and photosynthetic carbon dioxide fixation without affecting phosphoglycolate phosphatase synthesis during regreening, it was concluded that photosynthetic competence was not necessary for the development of the glycolate pathway enzymes. The inhibition of phosphoglycolate phosphatase synthesis by d-threo-chloramphenicol but not by l-threo-chloramphenicol or cycloheximide shows that the enzyme was synthesized exclusively on chloroplast ribosomes, whereas protein synthesis on both chloroplast and cytoplasmic ribosomes was required for the formation of ribulose 1,5-diphosphate carboxylase. Although light is required for the development of both Calvin cycle and glycolate pathway enzymes during regreening it is concluded that the two pathways are not coordinately regulated.