Thesis (Ph. D.)--Boston University, 1994. / The Escherichia coli mannitol permease (EIIMtl) is a 68 kilodalton (kDa) membrane-bound protein that carries out phosphoenolpyruvate-dependent transport and phosphorylation of D-mannitol. This protein also catalyzes a phosphoexchange reaction between mannitol and mannitol-1-phosphate and acts as a chemotactic receptor for mannitol in this bacterium. The gene that encodes this protein, mtlA, has been cloned and sequenced. A structural model for the EIIMtl has been previously proposed based upon hydropathy analysis of the deduced amino acid sequence of mtlA, proteolysis studies, and 'phoA fusion analysis. According to this model, the N-terminal domain (residues 1-334) is comprised of six or seven membrane-spanning alpha-helices and the C-terminal domain (residues 335-637), which contains two phosphorylation sites, is exposed to the cytoplasm. A series of mtlA deletion mutants was constructed for further analysis of structure/function relationships in the mannitol permease. In this study, several deletion mutants are selected for characterization of their mannitol binding activity, insertion/stability in the membrane, and oligomerization. The results showed that [TRUNCATED]

Oral antineoplastics (OAs) have become an emerging and rapidly growing field in cancer treatment. As with any chemotherapeutic agent, clinicians must be aware of potential drug interactions. Drug information databases are a common resource utilized to check for interactions between OAs and patient’s home medications. A major concern with OAs is that they are usually taken at home as well as picked up at a pharmacy by the patient themselves. The objective of this study was to determine the reliability of these databases for identifying potential interactions with OAs in a real-world setting. Hospital records were used to identify patients with common malignancies (leukemia, sarcoma, colon, lung, thyroid, prostate, kidney and liver cancers) treated with OAs from 2013 to 2014. To be included patients must have started an OA during the study period and have adequate records to evaluate OA use as well as home medications. The patient’s regimen is then entered into Drugs.com and Lexicomp™ interaction databases. In addition to documenting the number of interactions flagged by both databases, the severity of the interaction and disagreements between databases were analyzed. A major interaction was defined as either a “D” or “X” by Lexicomp™ and “major” by Drugs.com. As of this preliminary analysis, 407 of 876 subjects have been screened. Of the 407 screened, 9 patients (one patient with 3 different OAs) have been enrolled. Lexicomp™ flagged 34 interactions, of which 10 were major interactions. Drugs.com flagged 34 interactions, of which 6 were major interactions. Between the 2 databases there was only 60% agreement in flagging major interactions. These discrepancies are of concern in that clinicians hope resources they utilize are congruent with one another and allow them to practice in the safest manner in terms to avoid clinically significant drug interactions OA.

Several fungal endophytes (C. protuberata, F. culmorum, and C. magna) confer stress tolerance to plants in the presence of certain pressures. This relationship is known as habitat-adapted symbiosis; its mechanism is currently unknown. Here, sFTIR, FTIR coupled to an FPA detector, and Raman spectromicroscopy are used to examine whether any biochemical differences exist between different isolates of the same species: one that can confer stress tolerance to plants and the other that cannot. No major differences have been observed in spectra that can differentiate between those endophytes that confer stress tolerance and those that do not. However, some hy-phae from both isolates of C. protuberata have been found to contain mannitol; its pres-ence may be more common in geothermal rather than non-geothermal isolates. Mannitol is a compound involved in providing stress tolerance to fungi. Any role that it may have in the mechanism of habitat-adapted symbiosis will need further investigation.

A stainless steel, high temperature, phase change micro actuator has been demonstrated using the solid-liquid phase transition of mannitol at 168°C and In-Ag transient liquid phase diffusion bonding. Joints created with this bonding technique can sustain temperatures up to 695°C, while being bonded at only 180°C, and have thicknesses between 1.4 to 6.0 μm. Physical vapour deposition, inkjet printing and electroplating have been evaluated as deposition methods for bond layers. For actuation, cavities were filled with mannitol and when heated, the expansion was used to deflect a 10 μm thick stainless steel membrane. Bond strengths of the joints are found to be in the region of 0.51 to 2.53 MPa and pressurised cavities sustained pressures of up to 30 bar. Bond strength is limited by the bond contact area and the surface roughness of the bonding layers.

Several fungal endophytes (C. protuberata, F. culmorum, and C. magna) confer stress tolerance to plants in the presence of certain pressures. This relationship is known as habitat-adapted symbiosis; its mechanism is currently unknown. Here, sFTIR, FTIR coupled to an FPA detector, and Raman spectromicroscopy are used to examine whether any biochemical differences exist between different isolates of the same species: one that can confer stress tolerance to plants and the other that cannot. No major differences have been observed in spectra that can differentiate between those endophytes that confer stress tolerance and those that do not. However, some hy-phae from both isolates of C. protuberata have been found to contain mannitol; its pres-ence may be more common in geothermal rather than non-geothermal isolates. Mannitol is a compound involved in providing stress tolerance to fungi. Any role that it may have in the mechanism of habitat-adapted symbiosis will need further investigation.

Methacholine (MCh) and mannitol challenges are tests used to assess airway responsiveness. It has been shown that airway responsiveness to direct bronchoconstrictors like MCh tends to increase following exposure to allergen but the response to mannitol an indirect stimuli, is not known. Furthermore, the provocative concentration causing a 20% decrease in Forced Expiratory Volume in one second (FEV1) for adenosine 5’ monophosphate (AMP) correlates better to sputum eosinophilia than MCh PC 20. Hence, we hypothesized that airway responsiveness will be greater when measured with mannitol than MCh. We studied airway responsiveness to MCh and mannitol first at 3 hours and then later at 24 hours after allergen challenge. The 3-hour study yielded results contrary to our hypothesis therefore a twenty-four hour study was undertaken. Ten mild atopic asthmatics who had a positive MCh challenge and an allergic response to allergen extracts such as cat, horse, and house dust mite completed the 3-hour study. Eleven mild atopic asthmatics with the criteria above completed the 24-hour study. Both studies were non-blinded, randomized clinical trials. Airway responsiveness to MCh was quantitated by changes in PC20. Airway responsiveness to mannitol was quantitated as PD15 in the 3-hour study and dose response ratio (DRR) in the 24-hour study. In both studies, the allergen challenges were separated by 14 days. Fractional exhaled nitric oxide measurements (FENO) were collected in both studies at varying time points to track airway inflammation. In the 3-hour study, the geometric mean MCh PC20 decreased significantly after allergen exposure from 0.88 mg/ml to 0.50 mg/ml (p = 0.02) indicating airway responsiveness to MCh increased. Conversely, the geometric mean mannitol PD15 increased significantly from 174 mg to 284 mg (p =0.02) indicating a decrease in airway responsiveness to mannitol. In the 24-hour study, the geometric mean MCh PC20 again decreased significantly from 5.9 mg/ml to 2.2 mg/ml (p= 0.01) after allergen exposure. The mannitol DRR increased significantly from 63 mg/∆%FEV1 to 158 mg/∆%FEV1 (p = 0.03). FENO levels increased significantly in MCh arm but not mannitol arm. That is pre allergen challenge versus 24 hours after allergen challenge (for MCh arm: 26 ppb pre to 55 ppb post; for mannitol arm: 31 ppb pre to 39 ppb post). In conclusion, at three and twenty-four hours after allergen challenge, a time when the airways are more responsive to MCh, there is a significant decrease in airway responsiveness to mannitol.

No / Two force fields, the GROMOS53A5/53A6 (united atom) and the AMBER95 (all atom) parameter sets, coupled with partial atomic charges derived from quantum mechanical calculations were evaluated for their ability to reproduce the known crystalline forms of the polyols mannitol and sorbitol. The force fields were evaluated using molecular dynamics simulations at 10 K (which is akin to potential energy minimization) with the simulation cell lengths and angles free to evolve. Both force fields performed relatively poorly, not being able to simultaneously reproduce all of the crystal structures within a 5% deviation level. The parameter sets were then systematically optimized using sensitivity analysis, and a revised AMBER95 set was found to reproduce the crystal structures with less than 5% deviation from experiment. The stability of the various crystalline forms for each of the parameter sets (original and revised) was then assessed in extended MD simulations at 298 K and 1 bar covering 1 ns simulation time. The AMBER95 parameter sets (original and revised) were found to be effective in reproducing the crystal structures in these more stringent tests. Remarkably, the performance of the original AMBER95 parameter set was found to be slightly better than that of the revised set in these simulations at 298 K. The results of this study suggest that, whenever feasible, one should include molecular simulations at elevated temperatures when optimizing parameters.

Chez Bacillus subtilis l’expression de l’opéron mtl pour l’utilisation du mannitol est contrôlé par MtlR. MtlR est un activateur transcriptionnel qui appartient à la famille des régulateurs DeoR composé d’un domaine HTH suivi de deux PRDs, un domaine EIIBGat et un domaine EIIAMtl-like.Le mécanisme général de la régulation de l’activité de MtlR est basé sur sa phosphorylation par des composants du PTS. La phosphorylation sur la Cystéine 419 du domaine EIIBGat par P~EIIAMtl a un effet négatif majeur sur l’activité de MtlR. Par conséquent, dans un mutant mtlF où EIIAMtl est délétée MtlR est constitutivement actif.Dans cette étude nous avons mis en évidence un nouveau phénomène de régulation de MtlR impliquant la protéine du PTS, EIIBMtl.Nous avons observé que lorsque on déléte l’opéron mtl ou EIIBMtl et EIIAMtl, l’activité constitutive de MtlR dans un mutant mtlF déjà observée est abolie d’où notre hypothèse que EIIBMtl aura un effet sur l’activité de MtlR. Par des expériences de double hybride nous avons montré une interaction directe, spécifique et bidirectionnelle entre les deux protéines EIIBMtl et EIIBGatEIIAMtl-like de MtlR. D’une manière comparable à la cellule où EIIBMtl est fusionnée à la protéine EIICMtl nous avons démontré que seulement la forme EIIBMtl fusionné à la perméase EIICMtl est capable d’activer MtlR mais nous avons également démontré que ce n’est pas EIICMtl qui est essentielle à l’interaction entre EIIBMtl et MtlR mais c’est le voisinage de la membrane qui est essentielle pour l’établissement de cette interaction et l’activation de MtlRUn modèle de régulation de l’activité du régulateur MtlR est proposé. Dans ce modèle l’induction de l’opéron mtl via l’activation de MtlR requiert la phosphorylation de PRDII de MtlR par P~His-HPr, la déphosphorylation de EIIBGat de MtlR par EIIAMtl et la présence de la forme non-phosphorylée de l’EIIBMtl qui est dominante en présence du substrat inducteur, le mannitol. Ainsi, l’EIIBMtl non-phosphorylée séquestre MtlR déphosphorylé sur sa cystéine 419 à la membrane, l’active et induit l’expression de l’opéron mtl. / The Bacillus subtilis mtl operon encodes the enzymes necessary for mannitol utilization. Its expression is controlled by MtlR, a transcriptional activator belonging to the DeoR family. MtlR contains a HTH domain followed by two PTS regulation domains (PRDs), an EIIBGat domain and an EIIAMtl-like domain.The general mechanism of the regulation of MtlR activity is based on its phosphorylation by components of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS). The phosphorylation of EIIBGat on cysteine 419 by P~EIIAMtl has a major negative effect on the activity of MtlR. The absence of EIIAMtl in a mtlF mutant therefore leads to constitutively active MtlR.In this study a new mechanism of MtlR regulation based on the interaction of the PTS component EIIBMtl with MtlR is presented.We observed that the deletion of the entire mtlAFD operon or of mtlF and only the 3’-part of mtlA (encoding the EIIBMtl domain) abolishes the constitutive MtlR activity of the mtlF mutant, suggesting that MtlR activity depends on functional EIIBMtl. By carrying out yeast two-hybrid experiments we could establish a direct, specific and bidirectional interaction between EIIBMtl and the EIIBGatEIIAMtl-like part of MtlR.Complementation of the above mutants was possible with entire MtlA, but not with the EIIBMtl domain. EIIBMtl is normally fused to the membrane protein EIICMtl; we therefore fused EIIBMtl to another membrane, which indeed restored MtlR function in the absence of EIICMtl. The EIICMtl domain is therefore not essential for the interaction between EIIBMtl and MtlR; it is rather the vicinity of the membrane which is required for the activation of MtlR.A regulation model of MtlR activity is proposed. In this model, the MtlR-mediated induction of the mtlAFD operon requires the phosphorylation of PRDII by P~His-HPr and the dephosphorylation of EIIBGat by EIIAMtl. The presence of unphosphorylated EIIBMtl, which prevails when the inducer mannitol is present, is also required. Under these conditions unphosphorylated EIIBMtl sequesters MtlR dephosphoryled on cysteine 419, but phosphorylated at His-342, to the membrane thereby activating the transcription activator, which leads to increased expression of the mtlAFD operon.

A capillary electrophoretic procedure employing contactless conductivity detection (C4 D) has been developed for direct determination of the glycerol and mannitol polyalcohols in biological and pharmacological samples. Both glycerol and mannitol are fully separated from the sample matrix within very short times of 3.0 and 3.9 min., respectively, when using the optimized background electrolyte, 60 mM H3BO3 + 30 mM LiOH (pH 9.1). The limits of detection amount to 0.5 µM for glycerol and 0.3 µM for mannitol. The repeatability of the glycerol determination in real biological materials is characterized by the coefficient of variation values, 0.5 % and 3.2 %, for the migration time and the peak area, respectively. The procedure has been used to monitor the free glycerol concentration in adipose tissue microdialyzates. A physiological study has demonstrated that the lipolysis occurring during a sporting action can be stimulated by local application of adrenaline. The procedure has further been utilized to determine mannitol in a pharmacological preparation.

<p>Farm animals face a variety of stressors during commercial production practices, and economic necessities and ethical considerations require mitigation strategies to help animals cope with stressors that might otherwise reduce animal performance or lead to morbidity. In swine production, arguably the most stressful period of a pig’s life is the first several weeks following weaning (nursery period), where pigs must rapidly adapt to a host of environmental and physiological stressors. In broiler chickens, exposure to environmental temperatures above their comfort zone is a considerable stressor. Accordingly, several studies were conducted with the objective of developing alternative ways to measure physiological stress in nursery pigs and broiler chickens. These alternative methods may improve the ability of animal scientists to measure physiological stress and thereby aid in development of mitigation strategies. Nursery pigs frequently struggle with diarrhea and other intestinal diseases characterized by increases in intestinal permeability. Therefore, several studies were conducted to evaluate the use of non-metabolizable carbohydrates (lactulose and mannitol) as a non-invasive measure of intestinal health in weanling pigs. In Exp 2.1 and Exp 2.2, an aspirin-induced model of intestinal permeability was explored and the excretion pattern of lactulose and mannitol in urine over a 48 h urine collection period was determined. Aspirin at 15 mg/kg BW increased the excretion of lactulose over that of pigs given 0 or 30 mg/kg of aspirin, and therefore has potential to be used to induce intestinal permeability in nursery pigs. The excretion of lactulose and mannitol peaked at 4 h post-oral dose with a solution of lactulose and mannitol and was primarily complete by 8 h post-oral dose. In the few published reports of the lactulose and mannitol test of intestinal health in weanling pigs, the dose of lactulose and mannitol has varied considerably, raising questions as to the comparability of the results. Accordingly, in Exp 3.1, the impact of variation in the dose of lactulose and mannitol was explored, and pigs given the lowest dose at 0.2 g/kg BW lactulose and 0.02 g/kg BW mannitol had large numerical decreases in lactulose excretion warranting further investigation. Pigs in Exp 3.1 also demonstrated low urine recovery rates (50% successful urine collection averaged over the 3 urine collection time points) which were postulated to be due to the stresses associated with urine collection in metabolism cages combining with the stresses of weaning. Therefore, in Exp 3.2, an alternative urine collection procedure was developed that utilized a urine collection pad held in place by an elastic wrap. Results from Exp 3.2 with urine collected either by metabolism cages or via the urine collection pads indicated that the urine collection pad held promise as an alternative urine collection method that would not require the use of metabolism cages. Accordingly, the use of the collection pad was evaluated as part of a lactulose and mannitol test of intestinal health in group-housed nursery pigs in Exp 5.1. In brief, in Exp 5.1, pigs were weaned, transported for 12 h in a livestock trailer, and fed five treatment diets for 14 d post-weaning. The treatment diets were designed to evaluate the additive effects of L-glutamine and a combination of prebiotics and probiotics as potential antibiotic alternatives to aid in mitigating stress associated with weaning and transport. After two weeks of treatment diets, common diets were fed through market-weight. Urine collection pads were used on 40 pigs (8 pigs per treatment) on d 5 and d 12 post-weaning, with pigs allowed to remain in their familiar group pen during the urine collection period, and urine collection success rates averaged 84% across collection days. This improvement over that of Exp 3.1 may indicate that the use of a urine collection pad may improve the ability to obtain urine from pigs shortly after weaning. Considerable variation in the excretion of lactulose and mannitol was still observed similarly to that seen in Exp 3.1 and precluded statistical differences among dietary treatments. An increase in urine collection length was postulated to be one potential way to reduce that variation. Additional responses to the nursery diets fed in Exp 5.1 are discussed in Chapters 4 and 5. In broiler chickens, measurement of physiological heat stress is limited by the existing techniques which have considerable disadvantages. Therefore, in Chapter 6 a simple surgical procedure is detailed that allows small data loggers to be placed into the abdominal cavity of anesthetized broiler chickens. After a period to allow the chickens to recover from the surgery, these data loggers can record the internal temperature of the chicken at user-defined intervals and have the ability to gather large amounts of internal body temperature data in a wide variety of research settings. A more traditional measure of physiological heat stress in chickens is measurement of the temperature of the cloaca with a thermometer. Therefore, to compare the two methods, values from implanted data loggers were compared to values obtained by measuring the temperature of the cloaca in Exp 6.1 and Exp 6.2, and in general, the two methods yielded similar results. Since surgery to implant data loggers is not always possible or practical, in Chapter 7 the development and evaluation of an equation to predict the internal temperature of broiler chickens was investigated. In Exp 7.1, broiler chickens were exposed to four ambient temperatures each day for four days. The surface temperature of the chicken’s face was measured with a thermal imaging camera, while internal temperature was measured with data loggers as in Chapter 6. The resulting prediction equation contained the significant explanatory variables of the surface temperature of the face, the sex of the chicken, and the number of days of heat stress exposure. Accordingly, the accuracy of the prediction equation was evaluated in Exp 7.2, where chickens were exposed to the same 4 ambient temperatures and temperatures of the face and the internal body measured as in Exp 7.1. The prediction equation developed in Exp 7.1 was then used with the inputs of the facial surface temperature, sex of the birds, and number of days of heat stress exposure from chickens in Exp 7.2 to calculate a predicted internal temperature. This predicted internal temperature was then compared to the internal temperature as measured with data loggers in Exp 7.2. While the accuracy varied by experimental day and ambient temperature, the predicted internal temperature averaged 0.32°C greater than the measured internal temperature. Therefore, while the prediction equation shows considerable promise as a non-invasive metric of physiological heat stress in broiler chickens, refinement of the equation may be required in future studies before internal temperature may be predicted with the accuracy desired in a research setting. In conclusion, the lactulose and mannitol urinary test of intestinal health requires more research before wide-spread use but has considerable promise as a non-invasive test. Surgical implantation of data loggers to measure internal temperature of broiler chickens enables precise measurement of physiological heat stress in broiler chickens, and further research may enable accurate prediction of internal body temperature of broiler chickens without requiring invasive measurement techniques. <br></p><p> </p>