Determining growth inhibition of Candida albicans biofilm on denture materials after application of an organoselenium-containing dental sealant.

STATEMENT OF PROBLEM: Denture stomatitis is a chronic inflammatory condition caused by the formation of Candida albicans biofilm on denture bases. It is associated with aggravating intraoral pain, itching, and burning sensations. It can also potentiate cardiovascular diseases and aspiration pneumonia. The problem has thus far eluded efficient, toxic-free, and cost-effective solutions. PURPOSE: The purpose of this in vitro study was to investigate the effectiveness of organoselenium to inhibit the formation of C. albicans biofilm on the surface of acrylic resin denture base materials when it is either incorporated into the acrylic resin material or coated on the denture surface as a light-polymerized surface sealant. MATERIAL AND METHODS: Sixty heat-polymerized polymethyl methacrylate disks were fabricated and assigned to 4 groups (n=15): disks coated with a light-polymerized organoselenium-containing enamel surface sealant (DenteShield), disks impregnated with 0.5% organoselenium (0.5% selenium), disks impregnated with 1% organoselenium (1% selenium), and disks without organoselenium (control). C. albicans biofilm was grown on each disk which had been placed in a well of the microtiter plate containing 1-mL brain heart infusion broth inoculated with C. albicans. The plates were incubated aerobically at 37 °C for 48 hours. A confocal laser scanning microscope was used to determine the biofilm thickness, biomass, and live/dead cell ratio. Biofilm morphology was examined with scanning electron microscopy, whereas microbial viability was quantified by the spread plate method. The data were analyzed by using ANOVA and Tukey-Kramer multiple comparisons (α=.05). RESULTS: The microbial viability, biofilm thickness, biofilm biomass, and live/dead cell ratio were lower (P<.001) on disks in the test groups (DenteShield, 0.5% selenium, 1% selenium) when compared with the control group, with these variables being lowest in the 0.5% selenium and 1% selenium groups. The 0.5% selenium and 1% selenium groups did not differ significantly from each other in any of the variables (P>.05). Scanning electron microscope images showed inhibition of both biofilm growth and yeast to hyphae transition in the DenteShield, 0.5% selenium, and 1% selenium groups, with visible disruption of the biofilm morphology. CONCLUSIONS: The present study demonstrated that organoselenium, whether incorporated into or coated on the surface of an acrylic resin denture base material, has the potential to inhibit Candida albicans biofilm growth on denture surfaces and as such can be clinically useful for the prevention of denture stomatitis.

Inadequacy of self-Implemented preventive measures to control caries increment due to poor dietary habits in 6 and 12 years old children in Riga, Latvia.

AIM: This study investigated whether the self-implemented preventive measures practiced among the 6 and 12 years olds in Riga, Latvia can control the caries increment due to poor dietary habits among this age groups. MATERIAL AND METHODS: Caries examination was performed on Thirty-eight 6 and thirty-nine 12 years olds by visual and bitewing radiographic examination at baseline and after 3 years. All participants and/or their parents completed dietary habits questionnaires. The data was analyzed using t-test, chi-square test, ANOVA and frequency tables, Wilcoxon and Fisher's tests (α=0.05). RESULTS: The mean (SD) values of caries experience at baseline/3-year period in 6- vs.12-year olds were as follows. DMFS: 0.72 (1.02)/3.13 (3.13) (p=0.0000) vs. 6.79 (5.14)/14.79 (9.86) (p=0.0000); dmfs: 11.26(8.71)/7.74 (4.86) (p=0.078) vs. 3.57 (2.03)/1.5 (0.71) (p=0.317). The statistical significance was reported only for the consumption of soft drinks (p=0.032) and sugared tea (p=0.018) for the 6 years olds, and for sugared tea (p=0.017) and number of teaspoons of sugar added to tea (p=0.0095) for the 12-years olds. There was positive caries increment in all the 6 and 12 years olds that reported significant increase in consumption of soft drinks and sugared tea, and increase daily number of teaspoons of sugar used in tea. CONCLUSIONS: The present study demonstrated high cariogenic diet among the children in Riga, which is associated with increased caries experience that the currently practiced self-implemented oral hygiene measures was not capable of controlling.

Antimicrobial effect of herbal extract of Acacia arabica with triphala on the biofilm forming cariogenic microorganisms.

BACKGROUND: Dental caries is a biofilm-related infectious disease with a multifactorial etiology, over five billion inhabitants have affected worldwide due to this disease. OBJECTIVE: Antimicrobial efficacy of a mixed herbal powder extract (MHPE) against cariogenic microorganisms was investigated. MATERIALS AND METHODS: MIC, MBC, kinetics of killing, biofilm disruption and anticaries effect of MHPE were determined. For biofilm disruption, biofilms of Streptococcus mutans, Lactobacillus casei, Actinomyces viscosus and Candida albicans were treated with MHPE for 30 min and attached cells were quantified after staining. For live/dead staining biofilm assay, S. mutans biofilm treated with MHPE for 1min, 5min and 1 h was examined with confocal laser scanning system after live/dead staining. Efficacy was experimented by structural quality using Scanning Electron Microscope (SEM). Anticaries effect was determined by formation of caries-like lesion in continuous flow biofilm model. RESULTS: MHPE exhibited inhibition zones ranging from 12.5 to 24.0 mm. The highest inhibition zone was recorded at concentration of 50 µg/ml. MIC for S. mutans was between 12.23 and 36.7 µg/ml, while the MBC values ranged from 36.7 to 110.65 µg/ml. Inhibitory concentration of MHPE was three fold higher than CHLX. Significant reduction of cell count (49-95%) was observed with increasing time and higher concentration. Percentage biofilm reduction compare with negative control was 96.9% (A. viscosus), 94% (C. albicans), 99.8% (L. casei) and 91.7% (S. mutans). For MHPE-treated biofilm, live/dead staining demonstrated significant (p < 0.05) higher in deceased red fluorescence areas in all kinetics points from 53.6% (1min) to 85% (1h). SEM confirmed the damage in the outer layers of S. mutans. MHPE has components with effective antibacterial activity against caries-inducing microorganisms. CONCLUSION: The anti-adherence and anti-biofilm effect as well as the faster killing activity suggests that MHPE formula has effective antibacterial activity and could be a useful source of anti-cariogenic agents in near future.

Factors influencing the caries experience of 6 and 12 year old children in Riga, Latvia.

AIM: Authors assessed the influence of drinking tea with sugar, level of cariogenic microflora and use of fluoride toothpaste and tablets on caries experience of 6 and 12 year old children in Riga, Latvia. METHODS: 141 six and 164 twelve year old children were examined clinically and with bitewing radiographs to determine their dmft/DMFT and dmfs/DMFS. Children or their parents responded to questionnaire on number teaspoons of sugar (TS) used per cup of tea, number of cups of tea consumed daily, using fluoride or non-fluoride toothpaste (TP), frequency of toothbrushing, using fluoride tablets or not (ft). Salivary mutans streptococci (MS) and lactobacilli (LB) levels were measured in children with dmft/DMFT>4.0 for age of 6 (73% (n=103)), and for age of 12 (54% (n=88)). Impact of variables in caries status was determined using frequency tables and ANOVA, while proportion differences were tested using chi-square test. RESULTS: In 6 year olds, statistically significant associations were observed between the salivary microflora (MS, LB) and dt/DT (p=0.032; dt=3.71, DT=0.42), use of F-toothpaste (p=0.020), and TS (p<0.001). In 12 year olds, statistically significant (p<0.01) associations were observed between salivary microflora and dt/DT, ds/DS and dmft/DMFT. In both age groups, significant (p<0.001) association was demonstrated between dmfs/DMFS and salivary microflora, F-toothpaste and TS. CONCLUSIONS: The present study indicated that the caries development in Latvian children was associated with consumption of sugary tea and use of non-fluoride toothpaste.

Prevalence and patterns of chronic disease multimorbidity and associated determinants in Canada.

INTRODUCTION: Multimorbidity is increasingly recognized as a key issue in the prevention and management of chronic diseases. We examined the prevalence and correlates of chronic disease multimorbidity in the general adult Canadian population in relation to age and other key determinants. METHODS: We extracted data from the Canadian Community Health Survey 2011/12 on 105 416 Canadian adults. We analysed the data according to the number of multimorbidities (defined as 2+ or 3+ diseases from a list of 9) and examined the determinants of multimorbidity using regression analyses. RESULTS: Our findings show that 12.9% of Canadians report 2+ chronic diseases and 3.9% report 3+ chronic diseases. Those reporting 3 or more chronic diseases were more likely to be female, older, living in the lowest income quintile and to have not completed high school. In the overall population, social deprivation is associated with a 3.7 odds of multimorbidity, but when examined across age groups, the odds of multimorbidity were notably higher in middle age, 7.5 for those aged 35 to 49 years and 5.4 for those aged 50 to 64 years. DISCUSSION: As the proportion of Canadians living with multiple chronic diseases increases, we need to assess chronic disease from a holistic perspective that captures multimorbidity and upstream factors, to facilitate broader and more context-appropriate associations with healthy living, quality of life, health care costs and mortality. Special consideration should be given to the role that social deprivation plays in the development of multimorbidity. Canadians living in the lowest socioeconomic group are not only more likely to develop multimorbidity, but the onset of multimorbidity is also likely to be significantly earlier.

TITRE: Prévalence et profils de la multimorbidité au Canada et déterminants associés. INTRODUCTION: La prise en compte de la multimorbidité est de plus en plus reconnue comme un élément fondamental de la prévention et de la prise en charge des affections chroniques. Cette étude porte sur la prévalence et les corrélats de la multimorbidité chez les adultes canadiens en fonction de l'âge et de certains autres déterminants clés. MÉTHODOLOGIE: Nous avons extrait des données portant sur 105 416 adultes canadiens ayant répondu à l'Enquête sur la santé dans les collectivités canadiennes de 2011-2012. Nous les avons analysées en fonction du nombre d'affections concomitantes présentes (deux ou plus ou trois ou plus, sur une liste de neuf) et nous avons cherché à caractériser les déterminants de la multimorbidité à l'aide de régressions. RÉSULTATS: D'après notre analyse, 12,9 % des Canadiens souffraient de deux affections chroniques ou plus et 3,9 % de trois ou plus. Les répondants ayant déclaré souffrir de trois affections chroniques ou plus étaient plus susceptibles d'être des femmes, d'être plus âgés, de faire partie d'un ménage dont le revenu fait partie du quintile le plus faible et dont le niveau de scolarité le plus élevé de l'un des membres était inférieur aux études secondaires. La multimorbidité en lien avec le dénuement social était associée à une cote de 3,7 dans la population en général, mais de 7,5 chez les 35 à 49 ans et de 5,9 chez les 50 à 64 ans, soit la population d'âge moyen. ANALYSE: La proportion de Canadiens atteints de multiples affections chroniques étant en augmentation, nous devons adopter, pour étudier les affections chroniques et leurs facteurs en amont, une approche globale qui tienne compte de la multimorbidité, afin d'agir de manière globale et adaptée au contexte pour favoriser une vie saine et une meilleure qualité de vie et pour réduire les coûts des soins de santé et la mortalité. On devrait particulièrement tenir compte du rôle joué par le dénuement social dans l'apparition de la multimorbidité, car non seulement les Canadiens du groupe socioéconomique le plus démuni sont plus susceptibles de souffrir de multimorbidité, mais celle-ci surgira aussi probablement beaucoup plus précocement.

Antimicrobial activity of nanoemulsion on cariogenic planktonic and biofilm organisms.

INTRODUCTION: Nanoemulsions (NE) are a unique class of disinfectants produced by mixing a water immiscible liquid phase into an aqueous phase under high shear forces. NE have antimicrobial properties and are also effective anti-biofilm agents. MATERIALS AND METHODS: The effectiveness of nanoemulsion and its components was determined against Streptococcus mutans and Lactobacillus casei by live/dead staining. In vitro antimicrobial effectiveness of nanoemulsion against planktonic S. mutans, L. casei, Actinomyces viscosus, Candida albicans and mixed culture was determined by a serial dilution technique to obtain minimum inhibitory concentration and minimum bactericidal concentration (MIC/MBC). In addition, efficacy was investigated by kinetics of killing, adherence and biofilm assays. RESULTS: Compared to its components, nanoemulsion showed notable antimicrobial activity against biofilm organisms, up to 83.0% kill within 1min. NE dilutions ranging from 243 to 19683 were effective against planktonic S. mutans, L. casei, A. viscosus, C. albicans and mixed culture of these four strains as shown through MIC/MBC assays. NE showed antimicrobial activity against planktonic cells at high dilutions, confirmed by time kill studies. The level of adhesion on glass surface was reduced by 94.2-99.5% in nanoemulsion treated groups (p<0.001). 4-Day-old S. mutans, L. casei, A. viscosus, C. albicans and mixed cultures biofilms treated with NE showed reductions of bacterial counts with decreasing dilutions (p<0.001). CONCLUSION: These results suggest that nanoemulsion has effective anti-cariogenic activity against cariogenic microorganisms and may be a useful medication in the prevention of caries.

Antimicrobial activity of nanoemulsion on cariogenic Streptococcus mutans.

OBJECTIVE: To assess antimicrobial activities of nanoemulsion (NE) to control the adhesion and biofilm formation by Streptococcus mutans by in vitro. DESIGN: In vitro antimicrobial susceptibility of nanoemulsion was determined as per National Committee for Clinical Laboratory Standards guidelines and agar diffusion, serial dilution technique for the determination of minimum inhibitory concentration and minimum bactericidal concentration (MIC/MBC). Efficacy was tested by kinetics of killing, biofilm assay and scanning electron microscopy. RESULTS: : NE concentrations ranging from 1:100 to 1:10,000 dilutions were effective against S. mutans as shown through MIC/MBC assays. NE showed antimicrobial activity against planktonic cells at high dilutions, confirmed by time kill studies. 4-day-old S. mutans biofilms were treated with NE; subsequent reductions of bacterial cell counts were noticed with decreasing dilutions. Staining of NE-treated biofilms with LIVE/DEAD BacLight resulted in dead cell areas of up to 48% in 1 min, 84% at 1h and significant (<0.05) increases in dead cell counts at all time points. Damage to cell membranes and cell walls of S. mutans by NE was demonstrated using scanning electron microscopy (SEM). CONCLUSION: These results suggest that nanoemulsion has effective antibacterial activity against S. mutans and may be a useful medication in the prevention of dental caries.

Anti-cariogenic effect of a cetylpyridinium chloride-containing nanoemulsion.

OBJECTIVES: The aim of this pilot study was to investigate the anticaries activity of a nanoemulsion composed of soybean oil, water, Triton X-100 and cetylpyridinium chloride. METHODS: Tooth blocks (3 mm length x 3 mm width x 2 mm thickness) were cut from smooth surfaces of selected molar teeth using a water-cooled diamond wire saw. The blocks were randomly assigned to three experimental groups: (A) nanoemulsion, (B) 0.12% chlorhexidine gluconate, and (C) no treatment. The formation of dental caries in human tooth enamel was tested using a continuous flow dual-organism (Streptococcus mutans and Lactobacillus casei), biofilm model, which acts as an artificial mouth and simulates the biological and physiological activities observed within the oral environment. Experimental groups A and B were treated with their respective solutions once daily for 30 s on each occasion, while group C received no treatment. 10% sucrose was supplied every 6 h for 6 min to simulate meals and pH cycling. The experiment lasted for 5 days, and the tooth blocks were harvested and processed for demineralization assessment using transverse microradiography (TMR). RESULTS: For both lesion depth and mineral loss, statistical analysis indicated that Emulsion was significantly lower than Control and Chlorhexidine, and Chlorhexidine was significantly lower than Control. CONCLUSIONS: We conclude that cetylpyridinium-containing nanoemulsions appear to present a feasible means of preventing the occurrence of early caries.

Integrated hospital emergency care improves efficiency.

BACKGROUND: There is uncertainty about the most efficient model of emergency care. An attempt has been made to improve the process of emergency care in one hospital by developing an integrated model. METHODS: The medical admissions unit was relocated into the existing emergency department and came under the 4-hour target. Medical case records were redesigned to provide a common assessment document for all patients presenting as an emergency. Medical, surgical and paediatric short-stay wards were opened next to the emergency department. A clinical decision unit replaced the more traditional observation unit. The process of patient assessment was streamlined so that a patient requiring admission was fully clerked by the first attending doctor to a level suitable for registrar or consultant review. Patients were allocated directly to specialty on arrival. The effectiveness of this approach was measured with routine data over the same 3-month periods in 2005 and 2006. RESULTS: There was a 16.3% decrease in emergency medical admissions and a 3.9% decrease in emergency surgical admissions. The median length of stay for emergency medical patients was reduced from 7 to 5 days. The efficiency of the elective surgical services was also improved. Performance against the 4-hour target declined but was still acceptable. The number of bed days for admitted surgical and medical cases rose slightly. There was an increase in the number of medical outliers on surgical wards, a reduction in the number of incident forms and formal complaints and a reduction in income for the hospital. CONCLUSIONS: Integrated emergency care has the ability to use spare capacity within emergency care. It offers significant advantages beyond the emergency department. However, improved efficiency in processing emergency patients placed the hospital at a financial disadvantage.

Development of a quantitative method to monitor the effect of a tooth whitening agent.

This study demonstrated a quantitative method for assessing the effect of a tooth whitening agent. Forty human teeth were stained with a tea solution, and randomly assigned to two groups (A, B) of twenty teeth. The teeth were subsequently treated with either sodium hypochlorite (NaOCL) or deionized distilled water (DDW) by intermittent immersion (60 seconds on each occasion) in a 1:10 dilution of NaOCL (group A) or DDW (group B). Prior to whitening and following each immersion, the color of the teeth at the stained spot was measured using ShadeEye-Ex Dental Chroma Meter and quantitative light-induced fluorescence (QLF). ShadeEye-Ex instantly gave a numerical value for the stain intensity, chroma (C), which is the average of three measurements taken automatically by the machine. QLF gave a quantitative value for the stain, delta Q (% mm2), following analysis of the fluorescence image of the tooth. Immersion was stopped after four readings when one specimen, in group A, was observed to have regained its natural color. There was a good correlation between C and delta Q with either NaOCL (Pearson correlation coefficient (r) = 0.974; p < 0.05) or DDW (r = 0.978; p < 0.05). With NaOCL, an inverse relationship observed between stain measurements, C (Linear fit correlation (R) = -0.982; p < 0.05) or delta Q (R = -0.988; p < 0.05) and exposure time correlated to a linear fit, but not with DDW. ANOVA showed a significant difference between the means (n = 20) of the reading at the measurement intervals (0, 60, 120 and 180 seconds) for both C (p < 0.001) and delta Q (p < 0.001) with NaOCL but not with DDW. In conclusion, the study highlighted the potential of ShadeEye-Ex Dental Chroma Meter as a tool for the quantitative assessment of the gradual change in shade of discolored teeth by tooth whitening products.

Nitric oxide contributes to control of effusion in experimental otitis media.

OBJECTIVES/HYPOTHESIS: Nitric oxide (NO) is a small, short-lived free radical involved in cellular signaling and known to play a role in inflammation. It is generated on demand by the enzyme nitric oxide synthase (NOS) on arginine. We have previously found that mRNA encoding NOS is produced in the middle ear during otitis media. The role of NO was therefore explored in an experimental model of immune-mediated otitis media. STUDY DESIGN AND METHODS: Guinea pigs were systemically immunized and later challenged in the middle ear with the same antigen. One ear of each animal was challenged with antigen alone. In the opposite ear, antigen was combined with a potent inhibitor of NOS, N(G)-amino-L-arginine (L-NAA). After survival for 24, 48, or 72 hours, the middle ears were evaluated for otitis media. RESULTS: Inhibition of NOS resulted in significantly increased middle ear effusion at all three time periods. This increase was blocked by the addition of excess 1-arginine, which bypasses the inhibitory effects of L-NAA. The infiltration of cells into the middle ear lumen and the hyperplasia of the middle ear mucosa were unaffected by L-NAA administration. CONCLUSIONS: The results suggest that NO is involved in regulating the permeability of the middle ear vascular, the transudation of serum into the middle ear mucosa, and/or the movement of extracellular fluid across the middle ear mucosal epithelium.

Stress as a diagnostic challenge for postconcussive symptoms: sequelae of mild traumatic brain injury or physiological stress response.

There is considerable controversy regarding whether persistent postconcussive symptoms (PCS) are injury-specific, in a subgroup of individuals after mild traumatic brain injury (TBI). The following findings have contributed to this controversy: (1) The base rate of PCS in mild TBI is comparable to uninjured controls; and (2) The severity of PCS covary with daily stress levels (Gouvier, Cubic, Jones, Brantley, & Cutlip, 1992). We examined this relationship further by evaluating the effects of experimental conditions of stress or relaxation on individuals with TBI and uninjured control subjects, with low and high PCS endorsement. We evaluated psychophysiological parameters, neuropsychological performance, and changes in PCS and stress perception. In our study, subjects with TBI increased PCS after engaging in cognitively challenging tasks, and demonstrated significant autonomic changes in the stress condition. Symptomatic TBI subjects exposed to high stress had increases in PCS complaints, decreased speed of information processing, and subtle memory deficits. Our results suggest that PCS are injury-specific and that individuals with a history of TBI are susceptible to the effects of stress. Relaxation training including breathing retraining may be an effective means of decreasing PCS and cognitive complaints in subjects with mild TBI.

Protein kinase C mediates P2U purinergic receptor inhibition of K+ channel in apical membrane of strial marginal cells.

Strial marginal cells (SMC) electrogenically secrete K+ via slowly activating K+ (I[sK]) channels, consisting of I(sK) regulatory and KvLQT1 channel subunits, and the associated short circuit current (I[sc]) is inhibited by agonists of the apical P2U receptor [Liu et al., Audit. Neurosci. 2 (1995) 331-340]. Measurements of relative K+ flux (JK) with a self-referencing K+-selective probe demonstrated a decrease in JK after apical perfusion of 100 microM ATP. On-cell macro patch recordings from the apical membrane of gerbil SMC showed a decrease of the I(sK) channel current (I[IsK]) by 88 +/- 8% during pipette perfusion of 100 microM ATP. The magnitude of the decrease of L(sc) by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (20 nM) decreased I(IsK) (gerbil: by 62 +/- 10%; rat: by 72 +/- 6%) in perforated-patch whole-cell recordings while the inactive analog, 4alphaPMA, had no effect. By contrast, elevation of cytosolic [Ca2+] by A23187 increased the whole-cell I(IsK). The expression of the isk gene transcript was confirmed and the serine responsible for the species-specific response to PKC was found to be present in the gerbil I(sK) sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the I(sK) channel of SMC in response to activation of the apical P2U receptor and predict that the secretion of endolymph in the human cochlea may be controlled by PKC in the same way as in our animal model.

Regulation of the taurine transporter gene in the S3 segment of the proximal tubule.

Traditionally, bulk amino acid reabsorption in the kidney has been thought to be localized to the early portions of the proximal nephron. Adult Sprague-Dawley rats were fed diets with low, normal, and high taurine content for two weeks. Kidneys were hybridized with an 35S-radiolabeled complementary RNA probe to the rB16a subclone encoding the extracellular and transmembrane domains of the rat brain taurine transporter. Identical fragments were generated by RT-PCR from rat brain and kidneys as confirmed by DNA sequencing. Hybridization was localized to the outer zone of the medulla of all the kidneys. In the normal diet animals, taurine transporter mRNA was localized to the S3 segment of the proximal tubule, to the loop of Henle in the medulla, and to the glomerular epithelial cell layer. With taurine restriction, taurine transporter mRNA expression was up-regulated predominantly in the S3 segment and was virtually absent in this segment in animals supplemented with taurine. These experiments have precisely localized the rat kidney taurine transporter gene, demonstrating regulation that is limited to the S3 segment of the proximal tubule.

Attenuation by creatine of myocardial metabolic stress in Brattleboro rats caused by chronic inhibition of nitric oxide synthase.

1. The present experiment was undertaken to investigate: (a) the effect of nitric oxide synthase (NOS) inhibition, mediated by oral supplementation of the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), on measures of myocardial energy metabolism and function: (b) the effect of oral creatine supplementation on these variables, in the absence and presence of L-NAME. 2. In one series of experiments, 4 weeks oral administration of L-NAME (0.05 mg ml-1 day-1 in the drinking water) to Brattleboro rats caused significant reductions in myocardial ATP, creatine, and total creatine concentrations and an accumulation of tissue lactate when compared with control animals. Administration of creatine (0.63 mg ml-1 day-1 in the drinking water) for 4 weeks elevated myocardial creatine and total creatine concentrations and reduced lactate accumulation, but did not significantly affect ATP or phosphocreatine (PCr). Concurrent treatment with creatine and L-NAME prevented the reduction in creatine and total creatine concentrations, and significantly attenuated the accumulation of lactate and the reduction in ATP seen with L-NAME alone. 3. In a second series of experiments, 4 weeks treatment with L-NAME and creatine plus L-NAME increased mean arterial blood pressure in conscious Brattleboro rats. Hearts isolated from these animals showed decreased coronary flow and left ventricular developed pressure (LVDP), and total mechanical performance. Treatment with creatine alone had no measurable effect on either mean arterial blood pressure or coronary flow in isolated hearts. However, there was an increase in LVDP, but not in total mechanical performance, because there was a bradycardia. 4. These results indicate that creatine supplementation can attenuate the metabolic stress associated with L-NAME administration and that this effect occurs as a consequence of the action of creatine on myocardial energy metabolism.

A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.

Nuclear-encoded proteins targeted to the chloroplast are typically synthesized with N-terminal transit peptides which are proteolytically removed upon import. Structurally related proteins of 145 and 143 kDa copurify with a soluble chloroplast processing enzyme (CPE) that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein and have been implicated in the maturation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and acyl carrier protein. The 145- and 143-kDa proteins have not been found as a heterodimer and thus may represent functionally independent isoforms encoded by separate genes. Here we describe the primary structure of a 140-kDa polypeptide encoded by cDNAs isolated by using antibodies raised against the 145/143-kDa doublet. The 140-kDa polypeptide contains a transit peptide, and strikingly, a His-Xaa-Xaa-Glu-His zinc-binding motif that is conserved in a recently recognized family of metalloendopeptidases, which includes Escherichia coli protease III, insulin-degrading enzyme, and subunit beta of the mitochondrial processing peptidase. Identity of 25-30%, concentrated near the N terminus of the 140-kDa polypeptide, is found with these proteases. Expression of CPE in leaves is not light dependent. Indeed, transcripts are present in dark-grown plants, and the 145/143-kDa doublet and proteolytic activity are both found in etioplasts, as well as in root plastids. Thus, CPE appears to be a necessary component of the import machinery in photosynthetic and nonphotosynthetic tissues, and it may function as a general stromal processing peptidase in plastids.

Effects of chronic infusions of alpha-trinositol on regional and cardiac haemodynamics in conscious rats.

1. Male, Long Evans rats (350-450 g) were chronically instrumented for the measurement of renal, mesenteric and hindquarters haemodynamics, and were given three consecutive, 24 h infusions of vehicle (sterile saline at 0.3 ml h-1, n = 8) or alpha-trinositol (D-myo-inositol-1,2,6-triphosphate) at 5, 20 and 80 mg kg-1 h-1 (0.3 ml h-1; n = 9). During infusion of alpha-trinositol at 5 or 20 mg kg-1 h-1, cardiovascular changes were little different from those seen during saline infusion. However, during infusion of alpha-trinositol at 80 mg kg-1 h-1 there were increases in hindquarters vascular conductance, renal flow and vascular conductance, that were all significantly different from the changes seen in the saline group. Infusion of alpha-trinositol at the high dose in naive rats (n = 8) had even more marked vasodilator effects. 2. Two groups of rats (n = 8 in each), chronically instrumented for the measurement of cardiac haemodynamics, were given 48 h infusions of saline (0.3 ml h-1) or alpha-trinositol (2 mg kg-1 bolus, 80 mg kg-1 h-1 infusion at 0.3 ml h-1). During the infusion of saline, there were slight reductions in heart rate, cardiac index, peak aortic flow, dF/dtmax and central venous pressure. In the animals receiving alpha-trinositol, with the exception of central venous pressure, all the above variables, together with total peripheral conductance, increased. 3. These results, collectively, indicate that incremental infusions of alpha-trinositol do not reveal its full vasodilator potential, possibly due to concurrent activation of counter-regulatory vasoconstrictor mechanisms. However, infusion of alpha-trinositol at a high dose causes substantial increases in renal,mesenteric and hindquarters flows and vascular conductances, supported by significant increases in indices of cardiac inotropism. Such effects, in the absence of significant hypotension, tachycardia or signs of desensitization, give alpha-trinositol a unique cardiovascular profile.

Magnesium sulphate reverses the carotid vasoconstriction caused by endothelin-I, angiotensin II and neuropeptide-Y, but not that caused by NG-nitro-L-arginine methyl ester, in conscious rats.

1. Magnesium sulphate (MgSO4) has been used for many years in the prevention of eclamptic seizures, but its mechanism of action has never been elucidated. Recent studies suggest that cerebral vasospasm is an important feature of eclampsia and we have developed and tested the hypothesis that MgSO4 can reverse cerebral vasoconstriction. 2. Studies were performed in conscious, male Long Evans rats with pulsed Doppler probes sutured around both common carotid arteries after the external carotid artery had been ligated on the left, thus allowing simultaneous measurement of changes in common and internal carotid blood flow. Intravascular catheters were placed in the abdominal aorta for measurement of systemic blood pressure and in the right jugular vein for administration of drugs. Mean arterial blood pressure and mean Doppler shift signals were used to calculate percentage changes in common and internal carotid vascular conductance. 3. After a period of recovery the animals were infused with endothelin-1, angiotensin II, neuropeptide-Y or NG-nitro-L-arginine methyl ester alone or in combination, and MgSO4 in low or high dose was infused when the effects of the vasoconstrictors had become established. 4. MgSO4 itself, at the low dose, had no effect on carotid vascular conductance. Endothelin-1, angiotensin II and neuropeptide-Y all reduced common and internal carotid vascular conductance and this effect was significantly attenuated by low dose MgSO4. The carotid vasoconstrictor action of endothelin-1 was completely abolished by high dose MgSO4.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the haemodynamic effects of angiotensin II in conscious rats by AT2-receptor antagonists given after the AT1-receptor antagonist, EXP 3174.

1. Conscious, Long Evans rats (n = 10), chronically instrumented for the measurement of regional haemodynamics, were studied on 3 consecutive experimental days to assess responses to angiotensin II (AII) (125 pmol kg-1, i.v.) and noradrenaline (1 nmol kg-1, i.v.) in the absence and presence of the AT2-receptor antagonist, PD 123319 (10 mg kg-1, i.v.) (day 1), the AT1-receptor antagonist, EXP 3174 (1 mg kg-1, i.v.) (day 2), and PD 123319 (10 mg kg-1, i.v.) given 24 h after EXP 3174 (day 3). 2. In naive rats (day 1), PD 123319 did not antagonize the haemodynamic effects of AII or noradrenaline. EXP 3174 (day 2) caused a marked, prolonged blockade of the haemodynamic effects of AII but not those of noradrenaline. Twenty four h after administration of EXP 3174 (day 3) there was still significant attenuation of the haemodynamic effects of AII. However, administration of PD 123319 at this time caused a further inhibition (lasting 1 h) of the effects of AII but not those of noradrenaline. 3. An identical 3 day protocol was used in a separate group of rats (n = 6) in which the AT2-receptor antagonist, PD 123177, was given instead of PD 123319, and the results were essentially the same, i.e., PD 123177 significantly attenuated the haemodynamic effects of AII but only when given 24 h after EXP 3174.4. In a separate group of rats (n = 4), a low dose of EXP 3174 (60 pg kg-' i.v.) was given to naive rats in order to simulate the degree of inhibition of the effects of All seen after administration of AT2-receptor antagonists in animals pretreated with EXP 3174. This low dose of EXP 3174 did not produce a sustained inhibition of the effects of All and the time course of recovery of All responses was similar to that seen with PD 123319 or PD 123177 given after the high dose of EXP 3174.5. The apparent inhibition of the effects of AII by the AT2-receptor antagonists, PD 123319 and PD 123177, when these were administered 24 h after the AT,-receptor antagonist, EXP 3174, may have been due to the functional activation of AT2-receptors and/or loss of AT2-receptor antagonist selectivity,and/or the displacement of nonspecifically bound EXP 3174 by AT2-receptor antagonists. While the latter explanation seems the most likely, these results raise the possibility that nonpeptide, All-receptor antagonists that act at both AT,- and AT2-receptors may have therapeutic advantages over selective AT,-receptor antagonists.

Effects of NG-nitro-L-arginine methyl ester on vasodilator responses to adrenaline or BRL 38227 in conscious rats.

1. Conscious, Long Evans rats, chronically instrumented for the measurement of regional haemodynamics, were used to assess responses to 3 min infusions of the potassium channel opener, BRL 38227 (1 and 10 micrograms kg-1 min-1) or adrenaline (0.05 and 0.5 microgram kg-1 min-1) in the absence and in the presence of NG-nitro-L-arginine methyl ester (L-NAME; 3 mg kg-1 h-1), an inhibitor of nitric oxide biosynthesis. 2. In the absence of L-NAME, the low dose of BRL 38227 caused slight hypotension and tachycardia, accompanied by small increases in mesenteric and hindquarters blood flow only. However, there were increases in renal, mesenteric and hindquarters vascular conductances. L-NAME had no effect on any of these responses. 3. The high dose of BRL 38227 caused substantial hypotension and tachycardia. Renal and hindquarters flows did not change significantly, but there was a marked increase in mesenteric flow. There were only modest increases in renal and hindquarters vascular conductances but a substantial mesenteric vasodilatation. In the presence of L-NAME, there was a slight reduction of the latter but no other changes in the responses to BRL 38227. 4. In the absence of L-NAME, the low dose of adrenaline caused slight hypotension but a marked tachycardia. There were no changes in renal or mesenteric blood flow but a clear-cut increase in hindquarters flow. Renal and mesenteric vascular conductances showed only small rises, in contrast to the substantial hindquarters vasodilatation. In the presence of L-NAME, there was significant attenuation of the tachycardia and of the increases in hindquarters flow and vascular conductance in response to adrenaline.5. The high dose of adrenaline caused marked hypotension and tachycardia. Renal flow did not change, but there was a fall in mesenteric and a marked rise in hindquarters flow. Renal vascular conductance showed a slight increase but mesenteric vascular conductance did not change significantly, whereas there was a substantial hindquarters vasodilatation. In the presence of L-NAME, adrenaline caused an increase in blood pressure but no significant change in heart rate; the renal vasodilatation was abolished, there was a mesenteric vasoconstriction, and the hindquarters vasodilatation was markedly reduced. L-NAME also attenuated the tachycardia induced by adrenaline in animals with no cardiac baroreflexes.6. The present results indicate that L-NAME-sensitive mechanisms are involved in the vasodilator and tachycardic effects of adrenaline. The relative lack of effect of L-NAME on responses to BRL 38227 indicates that the changes in the responses to adrenaline were not non-specific or due to changes in haemodynamic status caused by L-NAME. The results raise the possibility that the 'hypertensinogenic' properties of endogenous adrenaline could be amplified when nitric oxide biosynthesis is impaired.