Acid-base disturbances have serious clinical consequences and are particularly critical in patients whose cardiopulmonary function is compromised. Cellular transport of NH3 and NH4+ has important physiological significance in the regulation of acid-base balance. In the kidney, production and excretion of NH3/NH4+ is critical for net acid excretion. Recently, two non-erythroid glycoproteins (Rhbg and Rhcg) belonging to the Rh family were suggested to be involved in NH3/NH4+ transport. Thus far, the functional properties of these membrane proteins as transport mechanisms are not resolved. In this study, we expressed Rh proteins in Xenopus oocytes and demonstrated that they transport both NH4+ and NH3. As such, the Rh transporters are unique in being able to transport both the ionic and the neutral gaseous components of ammonia. Previous studies have shown that DIDS, a stilbene derivative known to inhibit anion exchangers, was shown to inhibit CO2 transport by AQP1. This led us to hypothesize that DIDS might also inhibit transport of other gases such as NH3 by Rh proteins. We therefore conducted the present study to test the effects of DIDS on NH4+ and NH3 transport by Rh glycoproteins. To do so we used ion-selective microelectrodes and two-electrode voltage clamp to measure changes in surface pH (pHs) and whole cell currents (I) induced by NH3/NH4+ and methyl ammonium (MA/MA+) with or without DIDS. All experiments were conducted in Xenopus oocytes expressing Rhbg. Rhbg was expressed by injecting the oocytes with cRNA of the cloned genes. Control oocytes were injected with H2O. Our results indicate that in oocytes expressing Rhbg, exposure to 5mM NH4Cl (NH3/NH4+) caused a decrease in surface pH (pHs) and an inward current. The decrease in pHs is caused by NH3 influx whereas the inward current is due to electrogenic NH4+ influx. In the presence of DIDS, exposure to 5mM NH4Cl caused a significantly smaller decrease in pHs and current. The %inhibition of pHs and ΔI were 33% and 49%, respectively (P<0.05). Similarly, exposing oocytes expressing Rhbg to 5mM MA/MA+ (a substitute to NH3/NH4+) caused a decrease in pHs and an inward current. In the presence of DIDS, the MA/MA+ induced changes in pHs and current were also inhibited (37% and 63%, respectively; P<0.05). DIDS had no effect on NH3/NH4+ transport in H2O-injected oocytes (not expressing Rhbg). In summary, our data support the following conclusions: 1) RhAG and Rhbg transport both the ionic NH4+ and the neutral NH3 species. 2) Transport of NH4+ is electrogenic. 3) RhAG and Rhbg expression both enhance MA transport, an electroneutral component. 4) Like Rhbg, RhAG also transports MA+, an electrogenic component. The charged MA+ seems to be a direct substrate for RhAG whose transport likely resembles that of NH4 +. 5) Rhcg is likely to be a predominantly NH3 transporter. 6) RhAG and Rhbg are unlikely to be NH4 +/H+ exchangers. Regarding the effect of DIDS, our data also indicate that 1) DIDS partially inhibits the transport of NH3 and MA by Rhbg without affecting endogenous NH3 and MA transport. 2) DIDS also inhibits the electrogenic transport of NH4 + and MA+ by Rhbg. 3) DIDS is the only inhibitor shown to block both gas (NH3) and ionic (NH4 +) transport by Rhbg. / acase@tulane.edu

Abstract Glycans, as a part of glycoproteins, glycolipids and other glycoconjugates, are involved in many vital intra- and inter-cellular tasks, such as protein folding and sorting, protein quality control, vesicular trafficking, cell signalling, immunological defence, cell motility and adhesion. Therefore, their correct construction is crucial for the normal functioning of eukaryotic cells and organisms they form. Most cellular glycans are constructed in the Golgi, and abnormalities in their structure may derive, for instance, from alkalinization of the Golgi lumen. In this work we show that Golgi pH is generally higher and more variable in abnormally glycosylating, i.e. strongly T-antigen (Gal-β1,3-GalNAc-ser/thr) expressing cancer cells, than in non-T-antigen expressing cells. We also confirmed that the Golgi pH alterations detected in cancer cells have the potential to induce glycosylation changes. A mere 0.2 pH unit increase in Golgi pH is able to induce T-antigen expression and inhibit terminal N-glycosylation in normally glycosylating cells. The mechanism of inhibition involves mislocalization of the corresponding glycosyltransferases. We also studied potential factors that can promote Golgi pH misregulation in health and disease, and found that cultured cancer cells, despite variation and elevation in Golgi pH, are fully capable of acidifying the Golgi lumen under the normal Golgi pH. Moreover, we introduce a Golgi localized Cl-/HCO3- exchanger, AE2a, that participates in Golgi pH regulation by altering luminal bicarbonate concentration and thus also buffering capacity. Participation of AE2a in Golgi pH regulation is especially intriguing, because it also provides a novel mechanism for expelling protons from the Golgi lumen.

Die Speicheldrüsen der Schmeißfliege Calliphora vicina produzieren bei Stimulierung mit dem Neurohormon Serotonin (5-Hydroxytryptamine, 5-HT) einen KCl-reichen Primärspeichel. Der transepitheliale K+-Transport wird durch eine apikal lokalisierte vakuoläre H+-ATPase (V-ATPase) energetisiert. Stimulierung der Speicheldrüsen mit 5-HT aktiviert die apikale V-ATPase, die Protonen aus der Zelle in das Drüsenlumen transportiert. Trotz des auswärts gerichteten Protonentransportes führt die 5-HT-Stimulierung kurioserweise zu einer intrazellulären Ansäuerung. Die Ursachen dieser 5-HT-induzierten Ansäuerung waren unzureichend untersucht. Deshalb war das Ziel dieser Arbeit die Identifikation aller Transporter, die an der intrazellulären pH-(pHi)-Regulation in unstimulierten Speicheldrüsen von Calliphora vicina beteiligt sind und an der Entstehung und Regulation der 5-HT-induzierten pHi-Änderungen mitwirken. Von besonderem Interesse war hierbei die funktionelle Mitwirkung der V-ATPase, deren Beteiligung an der pHi-Regulation in tierischen Zellen bisher wenig untersucht war. Wesentliche Ergebnisse dieser Arbeit waren: • Messungen des pHi-Wertes in der unstimulierten Drüse zeigten, dass vor allem die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter an der Aufrechterhaltung des Ruhe-pHi beteiligt sind. • Zur Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung (NH4Cl-Vorpuls) tragen ebenfalls im Wesentlichen die V-ATPase und mindestens ein Na+-abhängiger HCO3--Transporter bei. Der Na+/H+-Antiporter hat in der unstimulierten Drüse keinen messbaren Einfluss auf den Ruhe-pHi. • Die Wiederherstellung des Ruhe-pHi nach einer intrazellulären Alkalisierung (Na-acetat-Vorpuls) ist Cl--abhängig, aber auch unter extremen Bedingungen waren die Zellen noch in der Lage sich vollständig von einer intrazellullären Alkalisierung zu erholen. Einen entscheidenden Anteil daran hat offenbar die hohe intrazelluläre Puerkapazität. • Ein Na+-abhängiger Glutamat-Transporter ist per se kein pHi-regulierender Transporter, seine Aktivität hat jedoch Einfluss auf den Ruhe-pHi in der unstimulierten Speicheldrüse von Calliphora vicina. • 10 nM 5-HT induzieren in den Calliphora Speicheldrüsen eine intrazelluläre Ansäuerung. An dieser Ansäuerung ist der Na+/H+-Antiporter entscheidend beteiligt. Auch eine klare Cl--Abhängigkeit der 5-HT-induzierten Ansäuerung konnte beobachtet werden. Wahrscheinlich ist eine gekoppelte Aktivität von Na+/H+-Antiporter und Cl-/HCO3--Antiporter. • Messungen mit einem O2-empndlichen Fluoreszenzfarbstoff zeigten, dass Stimulierung der Speicheldrüsen mit 5-HT die Zellatmung aktivierte. Der cAMP- und der IP3/Ca2+-Weg tragen auf komplexe Weise zu der 5-HT-induzierten Aktivierung der Zellatmung und damit auch zu den 5-HT-induzierten pHi-Änderungen bei. • Mit molekularbiologischen Untersuchungen ist es gelungen den Na+-abhängigen Glutamat-Transporter, den Na+/H+-Antiporter, die Carboanhydrase und die Untereinheit C der V-ATPase in den Calliphora Speicheldrüsen direkt nachzuweisen. Zudem konnte erstmals der direkte Nachweis für die Expression eines nH+/K+-Antiporters in den Speicheldrüsen von Calliphora vicina erbracht werden. Diese Arbeit trug ganz wesentlich zum Verständnis der pHi-Regulation in der unstimulierten und stimulierten Speicheldrüse von Calliphora vicina bei. Mechanismen die zur Aufrechterhaltung und Wiederherstellung des Ruhe-pHi nach einer intrazellulären Ansäuerung bzw. Alkalisierung beitragen, konnten mit pHi-Messungen und auch molekularbiologisch nachgewiesen werden. Die Mechanismen, welche die 5-HT-induzierte intrazelluläre Ansäuerung verursachen, konnten ebenfalls aufgeklärt werden. Zudem wurde an den Calliphora Speicheldrüsen eine neue optische Methode zur Messung des O2-Verbrauchs in tierischen Geweben etabliert. / The tubular salivary glands of the blowfly Calliphora vicina consist of a single layer of epithelial cells. Stimulation with the neurohormone serotonin (5-hydroxytryptamine,5-HT) induces the secretion of a KCl-rich primary saliva. Transepithelial K+-transport is energized by a vacuolar-type H+-ATPase (V-ATPase) which is located in the apical membrane. 5-HT stimulates the apical V-ATPase which transports protons out of the cells into the lumen of the glands. Despite this outward directed proton transport, 5-HT stimulation leads to an intracellular acidication. The causes of this intracellular acidication were poorly understood. Therefore the aim of this thesis was the identication of all pHi regulating transporters which are involved in pHi regulation in the unstimulated salivary glands of Calliphora vicina and which contribute to the 5-HT-induced pHi changes. Of special interest was the functional role of the V-ATPase,whose contribution to pHi regulation in animal cells is, as yet, not well studied. Key results were: • pHi measurements in unstimulated glands showed that mainly the V-ATPase and at least one Na+-dependent HCO3--transporter are involved in maintenance of resting pHi. • V-ATPase and at least one Na+-dependent HCO3--transporter are also necessary for the recovery from an intracellular acidication (NH4Cl prepulse). • Recovery from an intracellular alkali load (Na-acetate prepulse) is partially Cl--dependent. • A Na+ dependent gluatamate-transporter is present in Calliphora salivary glands and its activity aects the resting pHi. • 10 nM 5-HT induce an intracellular acidication. This acidication is Na+-dependent, EIPA-sensitive and also Cl--dependent. No DIDS-sensitivity was observed. A coupled activity of a Na+/H+-antiporter and a Cl-/HCO3- -antiporter was suggested. • Using O2-sensitive fluorescent microbeads I could show that 5-HT stimulation of the Calliphora salivary glands activates cellular respiration. The cAMP and Ca2+-signalling pathways contribute in a complex manner to the 5-HT-induced activation of cellular respiration and consequently, also to the 5-HT-induced intracellular acidication. • The expression of a Na+ dependent glutamate-transporter, a Na+/H+-antiporter, a carbonic anhydrase, subunit C of the V-ATPase and a nH+/K+-antiporter were determined on mRNA level by RT-PCR. This thesis contributes signicantly to the understanding of pHi regulation in unstimulated and stimulated salivary glands of Calliphora vicina. Mechanisms which contribute to the maintenance and recovery of resting pHi were identied by using pHi measurements and molecular biological techniques. Mechanisms which are responsible for the 5-HT-induced intracellular acidication were also clarified. Furthermore a new optical method for measuring O2 consumption in animals cells was established by using the Calliphora salivary glands as a model.

Intracellular pH was monitored at the cytosolic surface of plasma membrane solute transporters (Na+/H+/nucleoside co-transporters, or Cl-/HCO3- exchangers), using pH-sensitive fluorescent proteins (FPs), dual emission green FP (deGFP4) and a monomeric red FP Nectarine (mNect), whose development and characterization are also reported here. Human concentrative nucleoside transporter, hCNT3, mediates Na+/H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in HEK293 cells. pH changes at the intracellular surface of hCNT3 were monitored by fusing mNect to the cytoplasmic N-terminus of hCNT3 (mNect.hCNT3) or an inactive hCNT3 mutant (mNect.hCNT3-F563C). Cells were incubated at the permissive pH for H+-coupled nucleoside transport, pH 5.5, under both Na+-free and Na+-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mM uridine, providing the first direct evidence for H+-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+. This suggests that in acidic Na+-containing conditions, 1 Na+ and 1 H+ are transported/uridine molecule, while in acidic Na+-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule and intestine, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Microdomains, discrete regions of altered cytosolic solute concentration, are enhanced by rapid solute transport and slow diffusion rates. pH-regulatory membrane transporters, like the Cl-/HCO3- exchanger AE1, could nucleate H+ microdomains, since AE1 has a rapid transport rate and cytosolic H+ diffusion is slow. As AE1 drives Cl-/HCO3- exchange, differences in pH, near and remote from AE1, were monitored simultaneously by deGFP4 fused to AE1 (deGFP4.AE1) and mNect.hCNT3-F563C. deGFP4.AE1-mNect.hCNT3-F563C distance was varied by co-expression of different amounts of the two proteins in HEK293 cells. As the deGFP4.AE1-mNect.hCNT3-F563C distance increased, mNect.hCNT3-F563C detected the cytosolic pH change with a time delay and reduced rate of pH change, compared to deGFP4.AE1. Carbonic anhydrase activity was essential for H+ microdomain formation. H+ diffusion along the plasma membrane was 60-fold slower than to the cytosolic ER-surface. During physiological HCO3- transport, a H+ microdomain 0.3 µm in diameter develops around AE1, which will affect nearby pH-sensitive processes.

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.

Entomopathogenic fungi (EPF) are the one of the potential cause of the morbidity and mortality of insects. In agro-forestry uses, they are applied mainly in the form of conidial preparations in dry, aqueous or oil formulations. This approach, while practical, works in a hit and miss fashion leading to a frustrating dilemma of why successes and failure perpetuate. The fundamental solution is to bridge gaps in our knowledge about conidia of EPF in varied environments where they confront a diversity of insect hosts to start their pathogenesis.<p>This thesis was undertaken to examine the effects of hydration and the regulatory role of ambient pH on proteases which are the primary pathogenicity determinants in Beauveria bassiana and Metarhizium anisopliae. The approaches used were those of biochemical, proteomics and functional proteomics. <p>Novel aspects of pH regulation/homeostasis during the soaking of conidia in water, (type II water, which had a maximum electrical conductivity of 1ìS/ cm at 298K/ 25° C) were identified. Hydrated conidia showed swelling in type II water as assessed by (Multisizer IIITM (Coulter CounterTM). Release of proteases, metabolic activity through liberation of ammonia and citrate and synthesis of protein, RNA and DNA was established. It was deduced that conidial enzymes are either attached by loose hydrogen bonding or were associated to the spore membranes. Water soaked or hydrated conidia can secrete citrate and ammonia to modify the ambient pH and maximize the activity of secreted proteases. <p>Pr1- and- Pr2-like proteases were liberated by washing conidia in tween (Tw), water (Ww) and buffer. The washing of conidia in buffers (pH 4-10) affected the release/activity of Pr1 and Pr2. The thesis shows a newly designed native IPG strip zymography to identify the release of 4 and 8 isoforms of proteases, respectively from conidia. The 2-DE zymography (copolymerized gelatin) of protease from Tw of <i>B. bassiana</i> and <i>M. anisopliae </i> indicated one band (Mr 70 kDa; pI 6.3) and six isozymes (Mr 115-129 kDa; pI 3.7-9.0), respectively, which were identified using mass spectrometry (MALDI-TOF) as a serine-like protease. <p>Six metalloprotease isozymes from <i>M. anisopliae</i> but only one from <i>B. bassiana</i> was documented by 1-DE native zymography combined with 2-D spot densitometry scans. Cationic PAGE native zymography separated two basic protease isozymes from Tw extract of M. anisopliae depending upon the pH of the incubation buffer. However, one activity band was identified from <i>B. bassiana</i>. Furthermore, only one activity band was apparent during 1st and 2nd Ww up to day 2 for both EPF. SDS PAGE (non-dissociating) zymogram of secreted protease isozymes from Tw of <i>B. bassiana</i> revealed three bands of Mr100, 60, and 36.3 kDa. The isozymes observed at day 2 and 3 had a Mrs of 35.4 and 25 kDa, and 24.7 and 20.3 kDa at day 4. The SDS PAGE zymograms for <i>M. anisopliae</i> indicated two isozymes of Mr 103 and 12 kDa, respectively. During the 1st Ww and incubation of spores at day 2 and 3, a 12 kDa band was observed. These results confirm the presence of diversity of proteases and their isozymes with unique molecular sizes.<p>This thesis research discovered and characterized a diversity of proteins/enzymes not previously reported from any other fungi. A newly designed enzyme overlay membrane (EOM) technique revealed three isoforms of Pr1-like subtilisin from Tw of <i>M. anisopliae </i>(pI 8.1-9.7) and <i>B. bassiana</i> (pI 8.4-9.7). Conversely, only one isoform of Pr2-like trypsin was identified from <i>M. anisopliae</i> and no Pr2-like activity was observed from <i>B. bassiana</i>. Use of metalloprotease (MEP) inhibitors in conjunction with EOM analysis revealed their release during treatment in Tw. In <i>M. anisopliae</i> four activities (pI 4.4-7.5) of thermolysin-like MEP were observed. However, Tw of <i>B. bassiana</i> showed one activity band (pI 5.5). In addition, an isozyme of neutral MEP containing Zinc from <i>M. anisopliae </i>(pI 6.1) and one from <i>B. bassiana</i> (pI 6.5-7.6), respectively, was identified. MALDI-TOF and Q-TOF analysis revealed the presence of proteins similar to ROD 1, Ü- and â-glucanases, elastase, lipase 5 and galectin 7, which are important during the initial phase of germination and pathogenesis. <p>In addition subtilisin (Pr1-like), trypsin (Pr2-like) and NAGase synthesis from the germinating conidia and mycelia under the supply of different carbon and nitrogen (C/ N) sources was studied. The regulation of the synthesis of cuticle-degrading enzymes (CDE) from germinating conidia and mycelia was hypothesized to be controlled through regulatory derepression and nutritional starvation. Pr1 and Pr2 are regulated in a different manner in conidia and mycelia. Both enzymes are regulated through a multiple control mode. It was concluded that C/ N repression occurs only when it is necessary for infective structures to establish a nutritional relationship with the host cuticular structures. In addition, C/ N sources have a significant effect upon pH modulation, ammonia production and protease secretion. Furthermore, the synthesis of Pr1 and Pr2 from germinating conidia was affected by the (inducer pH) pHi of the growth media. Growing mycelia of <i>B. bassiana</i> under acidic (4.0), neutral (7.0) and basic (11.0) pH conditions produce ammonia which modifies the pH thereby creating environments suitable for protease. Growth, morphology, radial extension rate and conidiation at different pHi revealed that both EPF modify the pH of growth medium effectively as opposed to the saprophytic fungus, <i>Aspergillus nidulans</i>. <p>The presence of MEPs and Pr2-like trypsin suggests that these enzymes can act as a back up system for Pr1 to breach the cuticle and facilitate penetration before appressoria formation. The diversity of isozymes released from conidia suggests that the EPF are pre-adapted to pathogenic mode of life style, further contributing complexity to their interaction with host insects. Such isozymes can circumvent protease inhibitors present in the insect cuticle and the hemolymph. In addition, these isozymes may offer selective advantages in exploring new habitats (substrates) either as pathogen or saprophyte.

No description available.

This thesis is missing pages 87, 172 and 255. No other copies of the thesis have these pages. -Digitization Centre / This thesis examined the effects of exhaustive exercise on acid-base and metabolite status in the intracellular and extracellular compartments of two very different fish species: the active, pelagic rainbow trout, and the sluggish, benthic starry flounder. In both species, exhaustive exercise resulted in an acidosis in the extracellular compartment of mixed respiratory and metabolic origin. Despite the reduction in pHe, red blood cell pHi was well regulated, though more precisely in trout than in flounder. Catecholamines were mobilized into the blood after exercise in trout but not in flounder. Circulating catecholamines may play an important role in regulating red blood cell (RBC) pHi in trout after exercise. In trout, lactate appeared in the blood in excess of H+ ; the reverse pattern was observed in flounder. H+ appearance was similar in both species. Differential release of lactate from the muscle mass was apparently responsible for this discrepancy. After exhaustive exercise, both trout and flounder experienced a severe intracellular acidosis in the white muscle, as measured by 14c-DMO (5,5-dimethyl -2,4-oxazolidinenione) distribution. H+ and lactate were not produced in equimolar quantities, with H+ produced in excess of lactate. Muscle lactate and H+ production was about 3-fold lower in flounder than in trout. The muscle intracellular acid-base disturbance was corrected more rapidly in flounder (4-8h) than in trout (8-12h). In flounder, this occurred prior to , but in trout after , correction of the extracellular acidosis. In flounder, a more rapid correction of muscle metabolite status was associated with the more rapid correction of the intracellular acidosis. After exercise there was a reduction in the whole body extracellular fluid volume and expansion of the intracellular fluid volume, largely reflecting changes within the muscle. This fluid shift resulted in a general hemoconcentration. Exercise led to a transient increase in net H+ excretion in both trout and flounder. Negligible amounts of lactate were transferred to the water. In flounder, about 20% of the total H+ load produced passed through the extracellular space and was transiently stored in the water, which appeared to hasten correction of the intracellular acid-base disturbance. In contrast, in trout, a much smaller portion of the acid load (about 6%), though about the same absolute amount as in flounder, was transferred to the water. This appeared to expedite correction of the extracellular acidosis.The results of this thesis argue against a prominent role for the Cori cycle in the final disposition of the lactate burden produced during exercise. Instead, it is suggested that the bulk of the lactate was metabolized in situ, either by oxidation or glyconeogenesis. In flounder, this was almost the sole fate of lactate, as very little appeared in the blood space. In trout, a significant portion of the lactate was exported to the blood, which was taken up and metabolized by aerobic tissues. / Thesis / Doctor of Philosophy (PhD)

No description available.

Einleitung: Ruminal werden Futterpflanzen zu kurzkettigen Fettsäuren (SCFAs) abgebaut. Diese bilden die Hauptenergiequelle für den Wiederkäuerorganismus. Da diese Fettsäuren jedoch auch maßgeblich die pH-Homöostase der Vormagenschleimhaut beeinflussen, muss das Pansenepithel in der Lage sein, Änderungen im Substrat- und Protonenangebot festzustellen und anschließend regulative Prozesse anzupassen, um Stoffwechselentgleisungen und so auch einer Pansenazidose vorzubeugen. In anderen Spezies erwiesen sich sogenannte „Freie Fettsäurerezeptoren“ (FFARs) als potenzielle Sensoren veränderter SCFA-Mengen im Darmlumen, die u. a. durch Modulation der intrazellulären Spiegel an zyklischem Adenosinmonophosphat (cAMP) ihre Wirkung vermitteln. Ziele der Untersuchungen: Es sollte in der vorliegenden Arbeit untersucht werden, ob FFARs im Pansenepithel des Schafes vorkommen und durch SCFAs aktiviert sowie intrazelluläre Signalwege über cAMP moduliert werden können. Im Anschluss sollte erarbeitet werden, inwiefern der nachgewiesene Einfluss von Butyrat auf die epithelialen cAMP-Spiegel Auswirkungen auf die epitheliale pH-Modulation infolge einer veränderten Aktivität von Monocarboxylattransportern (MCTs) und Na+/H+-Austauschern (NHEs) hat. Tiere, Material und Methoden: Sämtliche Untersuchungen wurden an Geweben des Vormagens von Schafen (Ovis gmelini aries) durchgeführt. Mittels Reverse-Transkriptase-Polymerase-Kettenreaktion (RT-PCR) und immunhistochemischer Färbungen wurde das Vorliegen verschiedener FFARs in nativem Pansengewebe untersucht. Zur funktionellen Charakterisierung wurden Epithelstücke aus dem ventralen Pansensack in Ussing-Kammern inkubiert und anschließend die cAMP-Spiegel im Epithel mittels einer quantitativen, kompetitiven Analyse bestimmt. Dabei wurde der Einfluss von Forskolin (ein Stimulator der cAMP-synthetisierenden Adenylylzyklasen), von Butyrat sowie von Niacin (ein FFAR-Agonist) betrachtet. Mithilfe von radioaktiv markiertem Azetat wurde der Effekt variierender cAMP-Spiegel auf die Transportaktivität von MCTs unter Zuhilfenahme von zwei verschiedenen MCT-Hemmstoffen (Cyanohydroxyzimtsäure und p-Hydroxymercuribenzoesäure) in Ussing-Kammern evaluiert. Die Aktivität der NHEs wurde an kultivierten Pansenepithelzellen durch Messung des intrazellulären pH-Wertes mittels Spektrofluorometrie unter Einfluss des NHE-Inhibitors 5-N-Ethyl-N-Isopropyl Amilorid ermittelt. Auch hierbei wurden in den Zellen unterschiedliche cAMP-Spiegel durch Forskolin-Applikation induziert. Die Daten der verschiedenen Untersuchungen wurden an 5-8 Tieren je Versuchsansatz erhoben. Die Normalverteilung wurde mittels Kolmogorov–Smirnov-Test ermittelt. Ein Friedman-Test mit anschließendem Dunn-Test wurde für die Analyse der cAMP-Experimente genutzt. MCT und NHE Experimente wurden mithilfe einer einfachen, geblockten Varianzanalyse und anschließendem Tukey-Test ausgewertet. Ergebnisse: Die FFARs GPR109A und FFAR2 konnten an allen untersuchten Lokalisationen (Netzmagen, Pansenvorhof, dorsaler und ventraler Pansensack, Psalter) über RT-PCR bzw. im ventralen Pansensack auch über die immunhistochemischen Färbungen detektiert werden, wohingegen FFAR3 lediglich als mRNA im Vorhof nachweisbar war. Dies lässt die beiden Rezeptoren GPR109A und FFAR2 als mögliche Strukturen zur Detektion von SCFAs im Pansenepithel erscheinen. Die Analyse der intrazellulären cAMP-Spiegel in Epithelien aus dem ventralen Pansensack konnte einen hemmenden Einfluss von Butyrat auf diesen Botenstoff darlegen, was auf eine Beteiligung der genannten FFARs hindeutet. Die Applikation des GPR109A-Agonisten Niacin hatte jedoch keinen Effekt auf die cAMP-Spiegel, sodass eine Wirkungsvermittlung von Butyrat über diesen Rezeptor unwahrscheinlich scheint. Mit Blick auf die funktionellen Auswirkungen dieser cAMP-Modulation hatten variierende cAMP-Level im Kontrast zu Erkenntnissen aus Nicht-Wiederkäuerspezies keinen Einfluss auf die Transportaktivität des ruminalen MCT1 unter den gewählten in vitro-Versuchsbedingungen. Andererseits konnte die Regulation des intrazellulären pH-Wertes von kultivierten Pansenepithelzellen tendenziell durch erhöhte cAMP-Spiegel gehemmt werden, was auf einer Hemmung von NHEs durch den second messenger beruhen könnte. Schlussfolgerungen: Die Expression von GPR109A und FFAR2 lassen diese zwei FFARs als potenzielle Sensoren der intraruminalen bzw. intraepithelialen Nährstoffkonditionen erscheinen. Dabei deuten die vorliegenden Untersuchungen auf eine Aktivierung des FFAR2 durch Butyrat und dessen Metaboliten in den basalen Schichten des Pansenepithels hin. Infolge der Rezeptoraktivierung kommt es vermutlich zu einer Verminderung der intraepithelialen cAMP-Spiegel, welche wiederum einen (schwachen) Einfluss auf die Regulation des intrazellulären pH-Wertes mithilfe von NHEs zu haben scheinen. Entgegen unserer Ausgangshypothese scheinen aber die FFARs des ovinen Pansenepithels die pH-Homöostase des Epithels nur geringfügig zu beeinflussen. Ihre genaue physiologische Bedeutung – insbesondere des GPR109A – bleibt somit noch spekulativ.:1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für den Wiederkäuer 3 2.2 Transport kurzkettiger Fettsäuren über das Pansenepithel 3 2.2.1 Apikale Aufnahme in das Pansenepithel 4 2.2.2 Basolaterale Ausschleusung in den Blutstrom 6 2.3 Metabolisierung kurzkettiger Fettsäuren im Pansenepithel 8 2.4 pH-Homöostase 9 2.4.1 pH-Regulation des Pansenlumens 9 2.4.2 pH-Regulation des Pansenepithels 10 2.5 Anpassungsmechanismen des Pansenepithels 12 2.6 Rolle des Butyrats 15 2.7 Fettsäurerezeptoren 16 2.7.1 G-Protein-gekoppelte Rezeptoren 17 2.7.2 GPRs für SCFAs 17 2.7.2.1 FFAR2 17 2.7.2.2 FFAR3 18 2.7.2.3 GPR109A 19 2.7.3 FFARs im Wiederkäuerorganismus 20 2.8 Monocarboxylattransporter 22 2.8.1 Die Familie der MCTs 22 2.8.2 Regulation der MCTs 23 2.8.3 MCTs im Pansenepithel 24 2.9 Natrium-Protonen-Austauscher 26 2.9.1 Die Familie der NHEs 26 2.9.2 Regulation der NHEs 27 2.9.3 NHEs im Pansenepithel 28 2.10 Fragestellungen der vorliegenden Arbeit 30 3 Publikationen 32 3.1 Publikation 1 32 3.2 Publikation 2 41 3.2.1 Supporting Information 56 4 Diskussion 57 4.1 Nachweis von FFARs im Pansenepithel 57 4.1.1 Regulation intrazellulärer Signalwege durch FFARs 59 4.1.2 GPR109A als potenzieller Butyrat-Rezeptor im Pansenepithel 62 4.1.3 FFAR2 als potenzieller Rezeptor für Butyrat 63 4.2 Seitenabhängigkeit der Butyrat-Effekte 64 4.3 pH-Abhängigkeit der cAMP-Spiegel 66 4.4 Einfluss von cAMP auf die Aktivität der MCTs 68 4.5 Einfluss von cAMP auf die NHE-Aktivität 70 4.6 Schlussfolgerungen 73 5 Zusammenfassung 75 6 Summary 77 7 Literaturverzeichnis 79 8 Anhang 101 8.1 Im Rahmen dieser Dissertation gehaltene Präsentationen 101 Danksagung 103 / Introduction: Forage plants are ruminally degraded to short chain fatty acids (SCFAs). These serve as the main energy source for ruminants. As SCFAs also influence the pH-homeostasis of the ruminal mucosa, the epithelium must be able to detect changes of both substrate and proton accumulation and adapt transport processes accordingly, in order to prevent metabolic dysfunction and thus the risk of ruminal acidosis. Studies in non-ruminant species detected so-called ‘free fatty acid receptors’ (FFARs) as potential SCFA-sensors in the gut lumen. It has been shown that these receptors transduce their information by modulation of intracellular levels of cyclic adenosine monophosphate (cAMP). Aim: This study intended to investigate if FFARs are located in the ovine ruminal epithelium. It should further be evaluated if FFARs can be stimulated by SCFAs leading to a modulation of intracellular pathways via cAMP. Finally, the study aimed to elucidate the influence of low epithelial cAMP-levels after butyrate application on the regulation of pH-homeostasis in the ruminal epithelium by modulating the activity of transport proteins such as monocarboxylate transporters (MCTs) and Na+/H+ exchangers (NHEs). Animals, material, and methods: All experiments were conducted with ovine (Ovis aries) ruminal tissues. The expression of different FFARs was investigated in native tissues using a reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining. For functional analysis, epithelial cAMP levels were determined by a quantitative and competitive assay after incubation of epithelia of the ruminal ventral sac in Ussing chambers. The influence of forskolin (a stimulator of the adenylyl cyclases), butyrate, as well as niacin (an FFAR agonist) was evaluated. Further, the effect of varying cAMP levels on transport activity of MCTs was characterised on Ussing chamber-mounted epithelia with radioactively labelled acetate and two MCT inhibitors (cyano-hydroxycinnamic acid and p-hydroxymercuribenzoic acid). Finally, the activity of NHEs was assessed in cultured ruminal epithelial cells. The intracellular pH was evaluated by spectrofluorometry while the cells were incubated with forskolin (to modify intracellular cAMP levels) or the NHE inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. The data for the different set-ups were acquired from 5-8 animals each. Kolmogorov–Smirnov test was used for testing normality. For cAMP level analyses, the Friedman test followed by Dunn's test was performed. MCT and NHE measurements were analysed using one-way randomized block analysis of variance followed by Tukey's test. Results: GPR109A and FFAR2 were detected in all ovine ruminal epithelia examined (reticulum, atrium ruminis, ruminal ventral and dorsal sac, omasum) by RT-PCR and in ruminal ventral sac also by immunohistochemical staining. FFAR3, however, was detected solely on mRNA level in tissues of the ovine atrium ruminis. Thus, the two immunohistochemically detected receptors may serve as potential sensors for SCFAs in the ruminal epithelium. The analysis of intraepithelial cAMP levels revealed an inhibiting influence of butyrate application on cAMP pointing to an activation of FFARs by this SCFA. Nonetheless, the incubation with the GPR109A agonist niacin did not show any effect on cAMP levels. This finding contradicts the theory of an activation of GPR109A by butyrate. Looking at functional consequences of varying cAMP levels, in contrast to studies on non-ruminant species ruminal MCT1 activity was not influenced by different cAMP levels, at least under the conditions chosen in this in vitro study. However, regulation of intracellular pH in cultured ruminal epithelial cells tended to decrease with augmented cAMP levels. This might be mediated by an inhibition of NHEs. Conclusions: The expression of GPR109A and FFAR2 points at a participation of these receptors in sensing intraruminal and intraepithelial energy status. The present data hint at an activation of FFAR2 by butyrate or its metabolites in the basal layers of the epithelium. Activation of the receptor leads to decreased cAMP levels. This in turn seems to slightly modify the regulation of intracellular pH via NHEs. Contradicting our initial hypothesis, ovine ruminal FFARs seem to play only a minor role in modulation of epithelial pH homeostasis. The main physiological role of ruminal FFARs – especially of GPR109A – remains to be clarified.:1 Einleitung 1 2 Literaturübersicht 3 2.1 Bedeutung kurzkettiger Fettsäuren für den Wiederkäuer 3 2.2 Transport kurzkettiger Fettsäuren über das Pansenepithel 3 2.2.1 Apikale Aufnahme in das Pansenepithel 4 2.2.2 Basolaterale Ausschleusung in den Blutstrom 6 2.3 Metabolisierung kurzkettiger Fettsäuren im Pansenepithel 8 2.4 pH-Homöostase 9 2.4.1 pH-Regulation des Pansenlumens 9 2.4.2 pH-Regulation des Pansenepithels 10 2.5 Anpassungsmechanismen des Pansenepithels 12 2.6 Rolle des Butyrats 15 2.7 Fettsäurerezeptoren 16 2.7.1 G-Protein-gekoppelte Rezeptoren 17 2.7.2 GPRs für SCFAs 17 2.7.2.1 FFAR2 17 2.7.2.2 FFAR3 18 2.7.2.3 GPR109A 19 2.7.3 FFARs im Wiederkäuerorganismus 20 2.8 Monocarboxylattransporter 22 2.8.1 Die Familie der MCTs 22 2.8.2 Regulation der MCTs 23 2.8.3 MCTs im Pansenepithel 24 2.9 Natrium-Protonen-Austauscher 26 2.9.1 Die Familie der NHEs 26 2.9.2 Regulation der NHEs 27 2.9.3 NHEs im Pansenepithel 28 2.10 Fragestellungen der vorliegenden Arbeit 30 3 Publikationen 32 3.1 Publikation 1 32 3.2 Publikation 2 41 3.2.1 Supporting Information 56 4 Diskussion 57 4.1 Nachweis von FFARs im Pansenepithel 57 4.1.1 Regulation intrazellulärer Signalwege durch FFARs 59 4.1.2 GPR109A als potenzieller Butyrat-Rezeptor im Pansenepithel 62 4.1.3 FFAR2 als potenzieller Rezeptor für Butyrat 63 4.2 Seitenabhängigkeit der Butyrat-Effekte 64 4.3 pH-Abhängigkeit der cAMP-Spiegel 66 4.4 Einfluss von cAMP auf die Aktivität der MCTs 68 4.5 Einfluss von cAMP auf die NHE-Aktivität 70 4.6 Schlussfolgerungen 73 5 Zusammenfassung 75 6 Summary 77 7 Literaturverzeichnis 79 8 Anhang 101 8.1 Im Rahmen dieser Dissertation gehaltene Präsentationen 101 Danksagung 103