Abstract The carbonic anhydrases (CAs) participate in the maintenance of pH homeostasis in various tissues of the human body by catalyzing the reversible reaction CO2 + H2O ⇔ HCO3- + H+. Carbonic anhydrase isoenzyme VI (CA VI) is secreted into the human saliva by the serous acinar cells of the parotid and submandibular glands. The present work was undertaken in order to gain an understanding of the physiological role of CA VI in the oral cavity. CA VI concentrations were compared with other salivary characteristics and with the clinical dental status of the subjects. Saliva samples were collected under strictly controlled conditions from 209 young, healthy men and their CA VI concentrations determined by means of a specific time-resolved immunofluorometric assay. Salivary secretion rate, pH, buffering capacity, α-amylase activity level and counts of lactobacilli and mutans streptococci were also determined. Salivary CA VI concentrations showed positive correlations with salivary secretion rate (r = 0.20, p = 0.003) and amylase activity level (r = 0.46, p < 0.001), but not with pH, buffering capacity, or counts of mutans streptococci or lactobacilli. Salivary CA VI concentration, pH and buffering capacity correlated negatively with the number of decayed, missing or filled teeth (DMFT index). The correlation between salivary CA VI concentration and DMFT index was closest in the subjects with poor oral hygiene. No correlation was found between salivary secretion rate or amylase activity and the DMFT index. The location of CA VI in the enamel pellicle, a thin layer of proteins on dental surfaces providing a protective interface between the tooth surface and the external environment, was demonstrated in samples of extracted teeth using immunostaining with anti-CA VI antibody. Immunostaining for salivary α-amylase, which was used as a positive control, produced virtually the same staining patterns. The presence of CA VI in the natural enamel pellicle was confirmed by Western blotting of pellicle proteins. Histochemical staining of enamel pellicle formed in vitro showed that the bound enzyme retains its CA activity. To determine whether CA VI is transferred into the circulation, blood and saliva samples were collected from four healthy male volunteers at 3-h intervals throughout a 24-h period and assayed for CA VI concentration. CA VI was present in all the serum samples, although its concentration was about 22 times lower than in the saliva. The presence of CA VI in serum was confirmed using a sensitive Western blotting method. Western blotting also showed that serum CA VI is associated with IgG, which may protect the enzyme from proteolytic degradation or target it to sites that do not contain CA VI. The present results suggest that salivary CA VI is not involved in regulation of the actual pH or buffering capacity of the saliva, but it does seem to have a specific role in the oral cavity. High salivary concentrations of CA VI appear to be associated with low caries experience. Since active CA VI is located in the enamel pellicle, it may function locally in the microenvironment of the dental surfaces and accelerate the neutralization of the acid metabolic products of bacterial plaque.

Cultivation in the laboratory is essential for understanding the phenotypic characteristics and environmental preferences of bacteria. However, basic phenotypic information is not readily accessible. Here, we compiled phenotypic and environmental tolerance information for > 5,000 bacterial strains described in the International Journal of Systematic and Evolutionary Microbiology (IJSEM) with all information made publicly available in an updatable database. Although the data span 23 different bacterial phyla, most entries described aerobic, mesophilic, neutrophilic strains from Proteobacteria (mainly Alpha-and Gammaproteobacteria), Actinobacteria, Firmicutes, and Bacteroidetes isolated from soils, marine habitats, and plants. Most of the routinely measured traits tended to show a significant phylogenetic signal, although this signal was weak for environmental preferences. We demonstrated how this database could be used to link genomic attributes to differences in pH and salinity optima. We found that adaptations to high salinity or high-pH conditions are related to cell surface transporter genes, along with previously uncharacterized genes that might play a role in regulating environmental tolerances. Together, this work highlights the utility of this database for associating bacterial taxonomy, phylogeny, or specific genes to measured phenotypic traits and emphasizes the need for more comprehensive and consistent measurements of traits across a broader diversity of bacteria. IMPORTANCE Cultivation in the laboratory is key for understanding the phenotypic characteristics, growth requirements, metabolism, and environmental preferences of bacteria. However, oftentimes, phenotypic information is not easily accessible. Here, we compiled phenotypic and environmental tolerance information for > 5,000 bacterial strains described in the International Journal of Systematic and Evolutionary Microbiology (IJSEM). We demonstrate how this database can be used to link bacterial taxonomy, phylogeny, or specific genes to measured phenotypic traits and environmental preferences. The phenotypic database can be freely accessed (https://doi.org/10.6084/m9.figshare.472392), and we have included instructions for researchers interested in adding new entries or curating existing ones.

Magister Scientiae Dentium - MSc(Dent) / Introduction: Saliva is a significant biological fluid involved in the maintenance of good oral health. Cigarette smoking exerts detrimental effects on oral health and has been shown to affect saliva, but with no consensus regarding its effect on the quantity (flow rate) and quality (pH) of the saliva. Aim: To assess the effect of cigarette smoking on the flow rate and pH of whole stimulated saliva. Method: A case control study was conducted using patients who presented at the UWC Oral Health Centre patient sifting/waiting area. The patients who agreed to participate were assessed for inclusion into the study until the sample size was (n=60), stratified by smoking (n=30) and non-smoking (n=30). Stimulated saliva samples were collected in specimen jars by asking patients to chew a sterilized rubber band for 5 minutes and spit the contents into the specimen jar provided at 1 minute intervals. The specimens were transported to the laboratory within 30 minutes to measure the salivary quantity and pH. Results: No statistically significant difference in the salivary flow rates was found between smokers and non smokers (p=0.5273). Smokers showed a statistically significant decrease in their pH compared to non smokers (p=0.028). Conclusion: Cigarette smoking reduces the salivary pH, thereby producing an acidic environment.

Mutant strains of the fern Ceratopteris richardii Brongn. were selected in vitro for tolerance to culture medium containing Al2(SO4)3-Na2EDTA at pH 4.4. Three strains, HαAT3, HαAT7, and HαAT29 showed enhanced growth relative to the wild type on selection medium. Because of the complex nature of the selection conditions, the mutants were tested for tolerance to low pH and other individual components of the selection medium. All three mutant strains were notably more tolerant to acidic conditions and slightly more tolerant to Na2EDTA, relative to the wild type. No consistent differences in response to Na2SO4 were evident. Genetic characterization indicated a single nuclear gene basis of inheritance for strain HαAT3. Strains HαAT7 and HαAT29 showed a nuclear basis of tolerance, but responses of gametophytes from F1 hybrids suggested segregation at two or more loci.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Polyhalite is a natural mineral that could be fed as an acidogenic product to induce a metabolic acidosis and prevent clinical hypocalcemia in dairy cows after calving. The overall objective of this study was to determine if the use of polyhalite mineral in the diets of pre-partum non-lactating dairy cows was effective as an acidogenic product. We measured the urine pH, dry matter intake, milk yield, and calcium and magnesium concentration of urine and serum in pre-partum and non-lactating dairy cows consuming diets containing a low dose of polyhalite (200 g/cow/day), a high dose of polyhalite (400 g/cow/day), calcium chloride (250 g/cow/day), or no acidogenic product. We hypothesized that including polyhalite mineral as an acidogenic product in the diets of pre-partum and non-lactating dairy cows will reduce urine pH and stimulate calcium metabolism mechanisms. We found that polyhalite effectively reduced urine pH and did not affect dry matter intake, and the stimulation of calcium metabolism was observed through an increase of calcium output in urine. In conclusion, feeding polyhalite mineral is an effective means for inducing metabolic acidosis without reducing dry matter intake. Based on these results, polyhalite should be fed at a dose of 400 g or more per cow per day to reduce urine pH. / Master of Science in Life Sciences / Low blood calcium concentration, also known as hypocalcemia, is one of the common metabolic disorders that affect dairy cows transitioning from the pre-partum to post-partum period. Reducing the dietary cation anion difference (DCAD) in cows during the close-up period is known to effectively reduce the probability of cows developing hypocalcemia after calving. Polyhalite is a natural mineral that could be fed as an acidogenic product to induce a metabolic acidosis and prevent hypocalcemia in dairy cows after calving. The overall objective of this study was to determine if the use of polyhalite mineral in the diets of pre-partum and non-lactating dairy cows was effective as an acidogenic product. We evaluated the urine pH, dry matter intake, milk yield, and calcium and magnesium concentration of urine and serum in pre-partum and non-lactating dairy cows consuming diets containing a low dose of polyhalite (200 g/cow/day) , a high dose of polyhalite (400 g/cow/day), calcium chloride (250 g/cow/day), or no acidogenic product. We hypothesized that including polyhalite mineral as an acidogenic product in the diets of pre-partum and non-lactating dairy cows will reduce urine pH and stimulate calcium metabolism mechanisms. We found that polyhalite effectively reduced urine pH and did not affect dry matter intake, and the stimulation of calcium metabolism was observed through an increase of calcium output in urine. In conclusion, feeding polyhalite mineral is an effective means for inducing metabolic acidosis without reducing dry matter intake. Based on these results, polyhalite should be fed at a dose of 400 g or more per cow per day to reduce urine pH.

Current feeding strategies for dairy cows focus on meeting the energy requirements for high levels of milk production. However a major concern is the effect that these feeding regimes might have on rumen pH, which can have harmful effects on the cow and rumen microbial population. Several interventions have been used to counteract the effects of low rumen pH such as the use of probiotics e.g. yeast (Saccharomyces cerevisiae). However benefits have been inconclusive due to large individual animal variation in responses to treatment observed. The use of novel monitoring technologies can help assess the effect that different dietary interventions have on performance, rumen pH and rumen health. Data from three on-farm dairy cow trials (Trial 1 standard diet plus yeast; Trial 2 standard diet plus acidotic challenge plus yeast; Trial 3 cows grazing grass plus yeast) was used to evaluate the use of rumination collars (RC), rumen pH boluses, a whole cow dynamic mechanistic simulation model (SM) and the effect that different feeding strategies have on performance rumen pH dynamics and rumination time. No statistically significant differences between Control (no yeast) and Treatment (addition of yeast) diets were observed on any of the parameters measured. The lack of animal response to yeast supplementation observed in the three feeding Trials could be attributed to the stage of lactation, as the cows were passed peak lactation. Comparison of rumination time obtained with the RC and visual observations (obtained directly and from video recordings) suggest that the RC can be used to determine rumination time in housed cows. However its poor performance in grazing environments makes its use not advisable in cows outside at grass. The rumen pH boluses provided detailed and accurate data on circadian rumen pH. Highly varied individual responses to the feeding strategies were observed. This resulted in a diverse degree of risk of individual cows which experienced sub-acute rumen acidosis. The SM was able to accurately predict circadian pH, compared against the data obtained from Trials 1 and 2. The model provided pH values that were in agreement with those obtained with the rumen boluses. The use of new technologies to monitor cows individually could aid in whole-herd management, for example by setting thresholds for rumen pH and rumination time related to individual cow status, and then trigger appropriate interventions.

The regulation of pH is a critical homeostatic function of plant cells. In addition to acting as the primary cationic species responsible for energizing the plasma membrane, protons likely act as an important regulator and messenger. Despite this importance, few studies have thoroughly described cytosolic pH patterns as the plant cell progresses through the cell cycle. To investigate pH in plant cells, I chose Nicotiana tabacum (tobacco) Bright Yellow-2 (BY-2) cells as a model system. My research has two aims. First, I will measure and report the interphase cytosolic pH of BY-2 cells. Next, I will assay the cytosolic pH as BY-2 cells progress through mitosis and cytokinesis. I hypothesize that pH patterns are be temporally or spatially associated with structures such as the mitotic spindle or the phragmoplast. To investigate cytosolic pH in BY-2 cells, I will develop a cytosolic pH reporter based on a pH sensitive ratiometric fluorescent dye. This dye will be able to resolve both temporal and spatial changes in pH throughout the cytosol while imposing a minimal amount of stress on BY-2 cells. I found that pH-GFP, a variant of eGFP, had qualities of a robust pH reporter. To introduce the dye, explored biolistic bombardment, Agrobacterium mediated transient transformation, and polyethylene glycol mediated transformation as methods for introducing the pH-GFP gene into BY-2 cells. I observed very few transformation events using these methods and my observations did not support these approaches as suitable for introducing pH-GFP into BY-2 cells.

The aims of this thesis were to: 1) develop a new method for the determination of interstitial pH at rest and during exercise in vivo, 2) systematically explore the effects of different ingestion regimes of 300 mg.kg-1 sodium citrate on blood and urine pH at rest, and 3) to combine the new interstitial pH technique with the findings of the second investigation in an attempt to provide a greater understanding of H+ movement between the extracellular compartments. The purpose of the first study was to develop a method for the continuous measurement of interstitial pH in vastus lateralis was successfully developed using microdialysis and 2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). To avoid the presence of an artificial alkalosis during exercise, it was necessary to add 25 mM HCO3- to the perfusate. The outlet of the probe was cut less than 10 mm from the skin and connected to a stainless steel tube completing the circuit to a microflow-through cuvette (8 fÝl) within a fluorescence spectrophotometer. This prevented the loss of carbon dioxide from the dialysate and any subsequent pH artefact. Interstitial pH was collected from six subjects before, during and after five minutes of knee-extensor exercise at three intensities 30, 50, and 70 W. Mean,,bSEM interstitial pH at rest was 7.38,,b0.02. Exercise reduced interstitial pH in an almost linear fashion. The nadir value for interstitial pH at 30, 50 and 70 W exercise was 7.27, 7.16 and 7.04, respectively. The lowest pH was obtained 1 min after exercise, irrespective of workload, after which the interstitial pH recovered in a nearly exponential manner. The mean half time of interstitial recovery was 5.2 min. The changes in interstitial pH exceeded the changes in venous blood pH. This study demonstrated that interstitial pH can be measured using microdialysis and that it is continuously decreased during muscle activity. The purpose of the second study was to establish an optimal ingestion regime for the ingestion of 300 mg.kg-1 of sodium citrate and maximise the alkalotic effect while minimising any side effects. Increasing the effectiveness of alkali ingestion may lead to further increases in muscle performance. Ingesting 300 mg.kg-1 sodium citrate at a rate of 300 mg.min-1 was identified as the optimal ingestion regime to maximise alkalosis at rest, which occurred 3.5 h post-ingestion. This was determined by monitoring eight human subjects ingesting 300 mg.kg-1 sodium citrate at five different rates, control (no ingestant), bolus, 300, 600 and 900 mg.kg.min-1 on five days separated by at least 48 hours. Sodium citrate was ingested in capsule form with water ad libitum, with the exception of bolus, which was combined with 400 ml less than 25 percent orange juice and consumed in less than 1 min. Arterialised blood (mean 71.3,,b3.5 mmHg) acid-base and electrolyte status was assessed via the withdrawal of ~5 ml of blood every 30 min across an eight hour duration, placed on ice and analysed within five minutes. No alkalotic difference was found between ingestion rates (mean 7.445,,b0.004, 7.438,,b0.004 and 7.442,,b0.004 for 300, 600 and 900 mg.min-1, respectively). All experimental ingestion regimes were associated with elevations in [HCO3-] (29.6, 29.7, 29.8, 29.9 and 26.3 mmol.l-1 for bolus, 300, 600, 900 and control, respectively). The 300 ingestion regime had the greatest impact on [H+], a 0.66 meq.l-1,,e10-8 change. Bolus ingestion (3.93,,b0.08 mmol.l-1) of sodium citrate had no effect on control (4.06,,b0.08 mmol.l-1) blood [K+], however, 300 mg.min-1 decreased blood [K+] (p less than 0.05). There was no effect of sodium citrate on blood [Cl-], but after 2.5 h blood [Cl-] was lower than pre-ingestion values (p less than0.05). All ingestion rates of sodium citrate increased (p less than 0.05) urine pH above control. This is the first study to investigate the effect of varying ingestion rates on acid-base status at rest in humans. The results suggest that ingesting sodium citrate in small doses in quick succession induce a greater blood alkalosis than the commonly practised bolus protocol. Using the interstitial pH technique described above and the optimal ingestion regime (300 mg.min-1) identified above, the final experiment was designed to assess the influence of sodium citrate ingestion on interstitial pH at both rest and during exercise. Five subjects ingested 300 mg.kg-1 sodium citrate at 300 mg.min-1 again in capsule form with water ad libitum. Prior to ingestion, each subject had a cannula placed into their cephalic vein and one microdialysis probe (CMA-60) inserted into their left thigh, orientated along the fibres of vastus lateralus. This probe was used for the measurement of pH as described above. At the end of this period, an exercise protocol required five subjects to perform light exercise (10 W) for 10 min, before starting an intense exercise period (~90-95% leg VO2peak) to exhaustion followed by a 15 min recovery period. Dialysate and blood samples were collected across all periods. Mean,,bSEM interstitial pH for placebo and alkalosis were 7.38,,b0.12 and 7.24,,b0.16, respectively. Sodium citrate ingestion was not associated with an interstitial alkalosis. An exercise induced acidosis was observed in the interstitium during placebo but not during alkalosis (p less than 0.05). Mean,,bSEM venous pH were 7.362,,b0.003 and 7.398,,b0.003 for placebo and alkalosis, respectively. Sodium citrate ingestion was not associated with a venous alkalosis. Sodium citrate ingestion was associated with an increase in mean,,bSEM venous [HCO3-] (placebo 25.5,,b0.2, alkalosis 28.1,,b0.2). This increase in the blood bicarbonate buffer system was not associated with an increase in time to exhaustion (placebo 352,,b71, alkalosis 415,,b171). This was the first study to investigate the effects of sodium citrate ingestion on interstitial pH. The results of this study demonstrated that an interstitial alkalosis does not ensue after alkali ingestion, however, it was associated with the lack of an exercise induced acidosis suggesting an improved pH regulation during exercise.

BACKGROUND:The glycolytic nature of malignant tumors contributes to high levels of extracellular acidity in the tumor microenvironment. Tumor acidity is a driving force in invasion and metastases. Recently, it has been shown that buffering of extracellular acidity through systemic administration of oral bicarbonate can inhibit the spread of metastases in a mouse model for metastatic breast cancer. While these findings are compelling, recent assessments into the use of oral bicarbonate as a cancer intervention reveal limitations.METHODS:We posited that safety and efficacy of bicarbonate could be enhanced by dichloroacetate (DCA), a drug that selectively targets tumor cells and reduces extracellular acidity through inhibition of glycolysis. Using our mouse model for metastatic breast cancer (MDA-MB-231), we designed an interventional survival study where tumor bearing mice received bicarbonate, DCA, or DCA-bicarbonate (DB) therapies chronically.RESULTS:Dichloroacetate alone or in combination with bicarbonate did not increase systemic alkalosis in mice. Survival was longest in mice administered bicarbonate-based therapies. Primary tumor re-occurrence after surgeries is associated with survival rates. Although DB therapy did not significantly enhance oral bicarbonate, we did observe reduced pulmonary lesion diameters in this cohort. The DCA monotherapy was not effective in reducing tumor size or metastases or improving survival time. We provide in vitro evidence to suggest this outcome may be a function of hypoxia in the tumor microenvironment.CONCLUSIONS:DB combination therapy did not appear to enhance the effect of chronic oral bicarbonate. The anti-tumor effect of DCA may be dependent on the cancer model. Our studies suggest DCA efficacy is unpredictable as a cancer therapy and further studies are necessary to determine the role of this agent in the tumor microenvironment.