Neuronal, cardiac, and skeletal muscle electrical signaling is achieved through the highly regulated activity of several types of voltage-gated ion channels to produce an action potential (AP). Voltage-gated potassium (Kv) channels are responsible for repolarization of the AP. Kv channels are uniquely and heavily glycosylated proteins. Previous reports indicate glycosylation modulates gating of some Kv channel isoforms; often, terminal sialic acid residues alter Kv channel gating. Here, we questioned whether alterations in glycosylation impact Kv channel gating, thus altering APs and cardiac excitability. ST3Gal-IV, a sialyltransferase expressed at uniform levels throughout the heart, adds sialic acids to N- and O-glycans through alpha 2-3 linkages. Electrocardiograms (ECGs) suggest that cardiac conduction/rhythm are altered in ST3Gal-IV(-/-) animals, which show an increased incidence of arrhythmic beats. AP waveform parameters and two components of IK, the transient outward, Ito, and the slowly inactivating, IK,slow, were compared in neonatal control versus ST3Gal-IV(-/-) and glycosidase treated atrial and ventricular myocytes. Action potential durations (APDs) measured from ST3Gal-IV(-/-) and glycosidase treated atrial myocytes were lengthened significantly (~25-150%) compared to control; however, ventricular APDs were unaffected by changes in glycosylation. Consistently, atrial Ito and IK,slow activation were shifted to more depolarized potentials (by ~9-17 mV) in ST3Gal-IV(-/-) and glycosidase treated myocytes, while ventricular K+ currents were unaltered. Those channels responsible for producing Ito and IK,slow were examined under conditions of full and reduced glycosylation. Sialylation and N-glycosylation uniquely and differently impact gating of two mammalian Shaker family Kv channel isoforms, Kv1.4 and Kv1.5; Kv1.4 gating was unaffected by changes in channel glycosylation, while N-linked sialic acids, acting through electrostatic mechanisms, fully account for glycan effects on Kv1.5 gating. In addition, sialic acids modulate the gating of three Kv channel isoforms that are not N-glycosylated, Kv2.1, Kv4.2, and Kv4.3, through apparent electrostatic mechanisms. Click chemistry was utilized to confirm that these three isoforms are O-glycosylated and sialylated; thus, O-linked sialylation modulates gating of Kv2.1, Kv4.2, and Kv4.3. This study suggests that regulated or aberrant glycosylation alters the gating of channels producing IK in a chamber-specific manner, thus altering the rate of cardiac repolarization and potentially leading to arrhythmias.

Kinetics of the reactions of amoxycillin sodium and potassium clavulanate alone and in combination were investigated in the liquid and frozen states at selected pH values of 2.0, 4.6 and 7.0. A stability indicating HPLC assay was developed to perform simultaneous quantification of these compounds validated under stressed conditions.Amoxycillin and clavulanate degradation obeyed first-order kinetics under all conditions of this study. The effect of temperature, buffer, concentrations and complexation were investigated. Both compounds showed acceleration in rates due to general acid catalysis from buffer species. The buffer catalysis rate constants due to total phosphate and total acetate at 55 degrees celsius were 5.84x10(subscript)-1 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1 and 1.53 X10(subscript)-1 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1 for amoxycillin, 2.33 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1 and 4.4x10(subscript)-1 mol dm(subscript)-3)(subscript)-1 h(subscript)-1 for clavulanate respectively. The buffer independent rate constant values were obtained and interpreted according to the available literature data. Increase in the initial concentration of amoxycillin or clavulanate did not change the first-order rate constant values of these antibiotics significantly at liquid state temperatures. However in the buffer systems, the rate of hydrolysis of amoxycillin in the combination was significantly subject to clavulanate catalysis. This novel finding was influenced by phosphate buffer concentration. A kinetic model was proposed and the second-order catalytic rate constant values at pH 7.0 and 55 degrees celsius were estimated for clavulanate catalysis of amoxycillin (k(subscript)cvc) to be k(subscript)cvc = 1.75 X10(subscript)2 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1 and for phosphate catalyzed of clavulanate catalysis of ++ / amoxycillin (k(subscript)phccv) as k(subscript)phccv = 2.87 (mol dm(subscript)-3)(subscript)-1 h(subscript)-1.The temperature dependence of the rate of amoxycillin sodium degradation over the pH range evaluated did not change significantly. However the E(subscript)a values of potassium clavulanate decreased slightly with increase in pH. Both the compounds showed similar E(subscript)a values at pH 4.6 in acetate system. Hence 71.2 kJ mol(subscript)-1 for amoxycillin and 75.1 kJ mol(subscript)-1 for clavulanate.The investigation on complexation effects by HPbetaCD on the rate of hydrolysis of amoxycillin and clavulanate indicated no significant change in the rate of reaction of amoxycillin in the acetate buffer system. But the rate of clavulanate hydrolysis in combination was decreased by approximately 10%. The rate constant within the cyclodextrin complex and the stability constant of the complex obtained for clavulanate at pH 4.6 and 55 degrees celsius were k(subscript)c = 1.54x 10(subscript)-1 h(subscript)-1 and K(subscript)c = 74.2 (mol dm(subscript)-3)(subscript)-1.Extrapolation of the rate constant values to the frozen state from the liquid state data indicated marked acceleration of the rate of amoxycillin and clavulanate in all the pH values investigated. The highest acceleration in rate recorded was 15.0 fold for clavulanate in the hydrochloric acid system and the lowest value was 4.4 fold for amoxycillin at -7.3 degrees celsius. The rate constant values obtained were interpreted in terms of the concentration model (Pincock and Kiovsky 1966), phase-temperature relationship of the solutes, buffer catalysis, pH change and polymerization reactions.In the hydrochloric acid system a kinetic model was deduced providing adequate explanation of the experimental results. The stabilizing effect of sodium chloride used for maintaining constant ionic strength (mu=0.5) was ++ / enormous in this system. The shelf-life of amoxycillin was increased from 2.2 h to 58.3 h at -7.3 degrees celsius when sodium chloride was included in the system. It also stabilized the rates of the reactions significantly in the buffer systems.The buffer systems used in this study stabilized the rates of the reaction of both the drug compounds considerably. The shelf-life of amoxycillin in phosphate buffer was 621.3 h at -13.5 degrees celsius and in acetate buffer the shelf-life of clavulanate was 71.9 h at the same temperature. These are the highest shelf-life values recorded so far in the literature for amoxycillin and clavulanate at this frozen temperature.

Abstract not available

Thesis (Ph.D.)--University of Toledo, 2006. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Major advisor: Zi-Jian Xie. Includes abstract. Title from title page of PDF document. Bibliography: pages 64-70, 104-108, 121-158.

A field study was conducted to assess phosphorus (P) and potassium (K) concentrations of both lactating and dry cow diets on Oregon's dairies. Thirty-seven dairy farms, located in western Oregon, were grouped according to geographic region, valley (V) or coast (C), and herd size, small (S) or large (L). Farms were visited on three separate occasions. During each visit, lactating and dry cow diets were recorded and corresponding feed ingredients were collected and analyzed for P and K. For each diet recorded, fecal and urine samples were collected and analyzed for P and K. When available, milk production data was obtained. During the initial visit, a survey was issued to producers to assess P and K knowledge as well as gather herd data and management information. Survey responses received indicated that two-thirds of participants were knowledgeable about P and its affects within the environment. Knowledge of K appeared to be less than that of P. Average P concentration of the lactating cow diet was 0.40% (DM basis) and did not differ between region (P=0.12) or herd size (P=0.76). Fecal P excretion did not differ between region (P=0.08) or herd size (P=0.27), however, a trend for larger fecal excretion in V farms contributed to the lower calculated apparent P digestibility for V than C. Potassium in lactating cow diet was greater (P=0.01) for C than V, however, but no difference between herd size (P=0.10) was determined. Overfeeding of K also occurred in dry cow diet with no difference between region (P=0.40) and herd size (P=0.72). Combining the fecal and urinary fractions, an individual lactating cow consuming 0.40% P (89.8 g) and 1.71% K (384.3 g) per day will excrete 24.0 kg of manure P and 76.3 kg of manure K annually. The magnitude of plant available P and K produced; requires Oregon producers to acquire additional land, an additional two-tenths ha/cow, to be to apply P and K at agronomic rates. / Graduation date: 2004

Until recently, occurrence of milk fever (MF) has been attributed to prepartum calcium intake. However, researchers in Iowa have concluded that high prepartum dietary potassium (K) is the major cause of MF. Potassium concentrations have been increasing on manure fertilized soils over the last 20 years. Grasses grown on these fields mirror the increase in available soil K. When high K forages are fed to dry cows, it has been associated with increased MF in some, but not all cases. Our objective was to identify factors that differed between low and high occurrence of disorders when high K forages were fed. For 1 yr, monthly interviews were conducted on 10 dairies in Western Oregon. Close-up rations, dystocia rating, crowding, and cow comfort data were collected. Cows were diagnosed healthy or having one or more of the following metabolic diseases: MF, retained fetal membranes (RFM), and left displacement of the abomasum (LDA). Feedstuffs were collected each month and analyzed for dry matter, crude protein (CP), acid detergent fiber (ADF), and macrominerals. Of the multiparous cows (n=3,587) included, incidences of MF, RFM and LDA were 3.6, 11.3, and 1.5%, respectively. Increasing MF occurrence was associated with uncomfortable conditions, dystocia, increasing prepartum dietary Na and ADF, and increasing Ca to P ratios; there was also a dietary K by Mg concentration interaction. Increased dietary concentrations of Mg can prevent MF if dietary K is <2.6%. In addition, dietary conditions of K >2.6% and Mg >0.4% increase the occurrence of MF. Inversely, high concentrations of K can prevent MF in a Mg deficient diet. Dietary factors associated with an increase in the occurrence of RFM are increased dietary concentrations of Ca, S, CP, use of anionic salts, and the K forage source. Factors associated with an increase in hypocalcemia in the postpartum cow are crowding, uncomfortable housing, and a dietary K by Mg concentration interaction. In conclusion, improved cow comfort, and the addition of Mg to high K forage diets could decrease the risk of the cow exhibiting MF, RFM, and hypocalcemia. / Graduation date: 1999

R.H. Kingston. / "May 10, 1951." "This report is essentially the same as a doctoral thesis in the Department of Physics, M.I.T." / Bibliography: p. 26-30. / Army Signal Corps Contract No. DA36-039 sc-100 Project No. 8-102B-0. Dept. of the Army Project No. 3-99-10-022.

Sponges are filter feeders that lack nerves and muscle but are nonetheless able to respond to changes in the ambient environment to control their feeding current. Cellular sponges undergo coordinated contractions that effectively expel debris. Syncytial sponges propagate action potentials through their tissue, causing immediate flagellar arrest. Understanding the basis of this coordination in sponges is of interest for the insight it provides on mechanisms of coordination in early branching animals. However, when I began this thesis no ion channels had been described from the Porifera. I adopted a multifaceted approach to studying the conduction system of sponges. This included cloning and characterizing potassium channels as a means to understanding the underlying ionic currents, and monitoring regulation of the sponge feeding current in response to environmental stimuli. The latter experiments provided a functional context. The glass sponges Rhabdocalyptus dawsoni and Aphrocallistes vastus arrest feeding in response to mechanical disturbance and to sediment in the incurrent water suggesting a protective role. Monitoring patterns of feeding current arrests also revealed several features of the glass sponge conduction system: pacemaker activity, mechanosensitivity, distinct excitability thresholds, and tolerance to repeated stimuli. With access to the genome of the demosponge Amphimedon queenslandica I have also cloned and characterized the first sponge ion channels. Inward rectifier potassium (Kir) channels were prioritized for their role in regulating excitability. Kir channels cloned from A. queenslandica shared critical residues and a strong rectifying phenotype with Kir channels typically expressed in excitable cells. A variety of potassium channels from A. queenslandica indicate great diversity and a foundation for coordination at the dawn of the Metazoa / Physiology, Cell and Developmental Biology

The rapid accumulation of municipal solid waste is a significant environmental concern in our rapidly growing world. Due to its low cost, high energy recovery and limited environmental impact anaerobic digestion (AD) is a promising solution for stabilizing the organic fraction of municipal solid waste (OFMSW). Hydrolysis is often the rate-limiting step during AD of wastes with high solid content; this step can be accelerated by pretreatment of waste prior to AD. This thesis presents the results of alkaline pretreatment of OFMSW using NaOH and KOH. Four different pH levels 10, 11, 12 and 13 at two temperatures 23±1°C and 80±1°C were examined to study the effects of the pretreatment on (i) enhancing the solubility of the organic fraction of the waste, and (ii) enhancing the AD process and the biogas production. The effects on solubility were investigated by measuring changes in the soluble COD (SCOD) concentrations of pretreated wastes and the enhanced AD was investigated by measuring volatile solids (VS) destruction, total COD (TCOD) and SCOD removal in addition to biogas and methane production using biochemical methane potential (BMP) assay and semi-continuous laboratory reactor experiments. Pretreatment at pH 13 at 80±1°C demonstrated the maximum solubility for both NaOH and KOH pretreated samples; however the BMP analysis demonstrated that pretreatment at pH 12 at 23±1°C showed the greatest biogas yield relative to the removed VS for both chemicals. Thus pretreatment at pH 12 at 23±1°C using NaOH and KOH were examined using semi-continuous reactors at three different HRTs: 10, 15 and 20 days. Pretreatment demonstrated a significant improvement in the AD performance at SRTs of 10 and 15 days.

Potassium ion channels play a key role in the generation and propagation of action potentials. The S4-S5 linker peptide (L45) is believed to be responsible for the anesthetic/alcohol response of voltage-gated K+ channels. We investigated this region to define the structural basis of 1-alkanol binding site in dShaw2 K+ channel. L45 peptides derived from dShaw2 and hKv3.4 K+ channel, which, if part of the complete channel, demonstrate different sensitivity to 1-alcohols. Specifically, dShaw2 is alcohol sensitive and hKv3.4 is alcohol resistant. Structural analysis of L45 with NMR and CD suggested a direct correlation between alpha-helicity and the inhibition of dShaw2 channel by 1-butanol. We used CD and NMR to determine the structure of L45 peptides in micelles and vesicles. We measured spin-lattice relaxation time (T1) and determined the location and surface accessibility of L45 in micelles. These experiments confirm that L45 of dShaw2 adopts an α-helical conformation, partially buried in the membrane and parallel to the surface. The binding and accumulation of rev proteins to an internal loop of RRE (rev responsive element) of unspliced mRNA precursors is a key step of propagation of human immunodeficiency (HIV) virus. Molecules that interfere with this process can be expected to show anti-HIV activity. Our work is based on an assumption that zinc fingers could compete with rev proteins, therefore impeding the life cycle of HIV and stopping its infection. We studied the influence of different cations, anions, and the concentration of salts and osmolytes on the binding affinity with Polyacrylamide Gel Electrophoresis (PAGE) and Isothermal Titration Calorimetry (ITC). We conclude that the types of anions and/or cations and their concentrations affect the enthalpy and entropy of the binding interacitons. Using a gel assay, we confirm that there are three products in RNA-Protein reaction, and both EDTA and salts (and their concentrations) in the gel or samples interfere with RNA-protein complex mobility.