Catalytic and regulatory properties of phosphofructokinase (PFK) (EC 2.7.1.11), pyruvate kinase (PK) (EC 2.7.1.40), and creatine kinase (CK) (EC 2.7.3.2) from the heart of the red-eared turtle (Pseudemys scripta elegans) were studied. Particular attention was given to those properties of the enzymes which could help to explain the high glycolytic efficiency in this tissue and so provide insights into the selective forces involved in the evolutionary development of an extreme tolerance to anoxia. The control of glycolytic flux in turtle ventrical muscle has been vested primarily in phosphofructokinase and pyruvate kinase. Creatine phosphate and fructose diphosphate play pivotal roles in the channelling of carbon through the pathway and in the production of metabolic energy as ATP. The levels of these key metabolites are in turn tightly regulated. One of the enzymes studied (phosphofructokinase) controls the levels of fructose diphosphate produced and creatine kinase (itself modulated by the cell's redox balance) controls creatine phosphate levels. When oxygen levels are reduced, NADH accumulates because of a decrease in electron transport chain regeneration of NAD (129). This leads to an effective activation of creatine kinase by lowering the Kм for creatine phosphate. This activation causes a drop in creatine phosphate levels without the decline in ATP levels that are seen at the onset of hypoxia in other tissues ( 4 ). Since the high levels of creatine phosphate present in aerobic heart are responsible for the inhibition of phosphofructokinase by greatly increasing the for its substrate, fructose-6-phosphate, this drop in concentration deinhibits the enzyme and leads to a flush of its product, fructose diphosphate. The increase in fructose diphosphate (1) serves to activate phosphorructokinase by itself, and (2) further reduces the effect of creatine phosphate by a deinhibition of the enzyme. These two effects cause an autocatalytic increase in flux through this locus* Fructose diphosphate also feed-forward activates pyruvate kinase by decreasing the Km for its substrate, phosphoenolpyruvate, and serves to deinhibit this enzyme which is normally inactive due to alanine and ATP inhibition. The most important feature of such a regulatory system is that it is a kind of autocatalytic cascade. Once the activation of creatine kinase is initiated by the redox imbalance inherent in anaerobiosis, all the various regulatory interactions potentiate one another. Drops in inhibitor levels lead to increases in activator levels and these activators serve to further deinhibit. These interacting effects serve to potentiate anoxic production of energy to compensate for the temporary depletion of oxygen in diving stress and in long periods of hibernation in this turtle, Nature's premier vertebrate anaerobe. / Science, Faculty of / Zoology, Department of / Graduate

Anaerobiosis results in low ATP levels and cytoplasmic acidosis. Pyrophosphate (PPi) may play an important role in anaerobiosis as an energy source replacing ATP, as suggested by the hypoxic induction of PPi:fructose 6-phosphate 1-phosphotransferase and sucrose synthase in preference to phosphofructokinase and invertase. Here we show that vacuolar H$ sp+$-translocating pyrophosphatase (PPase) is also strongly induced by anoxia in rice seedlings. The PPase transcript abundance is increased within the first hours of anoxia, and decreases within 2 days after the return of seedlings to air, similarly to alcohol dehydrogenase-1 (Adh1). However, tissue studies show that the highest transcript induction for PPase is in the root whereas the highest induction of Adh1 is in the shoot. Assays of enzyme specific activity indicate a 75-fold increase in PPase activity over 6 days of anoxia, while the vacuolar ATPase changes only slightly. Return of seedlings to air results in rapid disappearance of enzyme activity. Chilling stress in rice seedlings also gives rise to an increase in immunoreactive PPase enzyme, and a progressive 20-fold increase in enzyme specific activity within 6 days. Upon return to room temperature both enzyme level and specific activity decrease. In corn, hypoxic stress results in a small induction in the PPase transcript, and no increase in PPase specific activity, which, however, is constitutively high in this material. It is suggested that in both species, H$ sp+$-PPase may play an important role in hypoxia and chilling stress, not only in conserving ATP, but also in limiting cytoplasmic acidosis.

Thesis (M.S.)--University of Wisconsin--La Crosse, 2002. / Includes bibliographical references.

Memoria para optar al título profesional de Ingeniera en Recursos Naturales Renovables / La temperatura es un parámetro importante para el desarrollo de la digestión anaeróbica, que es la base de la producción de biogás; puede limitar sectores para aplicar la tecnología, regular la eficiencia, ayudar en la eliminación de patógenos, afectar el tiempo de retención de la materia e incrementar la producción de biogás. Este trabajo se desarrolló para evaluar la dinámica de producción de biogás en función del régimen térmico, estableciendo la relación entre las temperaturas que afectan a un biodigestor tipo chino y proponer zonas en Chile que posean mayor aptitud para aplicar esta tecnología. El biodigestor utilizado con capacidad de 10 m3, se alimentó tres veces por semana con una mezcla de relación 3:1 agua/estiércol (v/v). Se realizaron monitoreos en dos periodos, febrero a julio (2011) y enero a agosto (2012); en este último se instalaron sensores de registro continuo (temperatura y humedad) y se realizaron análisis de la composición del biogás generado cada semana. En el primer periodo se aplicaron correlaciones lineales a las variables, obteniendo que la temperatura del aire afectó en mayor proporción a la temperatura de descarga, estanque y la del biodigestor. Para el segundo periodo se analizaron las tendencias de las variables, se aplicaron análisis de anomalías para series de tiempo, promedios móviles y ajustes funcionales de las variables más relevantes en el estudio. De los resultados se obtuvo, que la temperatura interna del biodigestor mantuvo una temperatura promedio de 22°C y la temperatura media del suelo se ajustó mejor a la temperatura media máxima del aire. Los análisis de composición fluctuaron de 55 a 85% de CH4 (metano) y de 16 a 35% de CO2 (dióxido de carbono). La zonificación se realizó aplicando el “índice térmico” propuesto en un estudio previo, pero esta vez con información térmica actualizada para todo el país; obteniendo que a partir de la región de Los Lagos y a lo largo de la cordillera de Los Andes, no es apta la aplicación de esta tecnología. Así es posible afirmar que estos biodigestores favorecen la estabilidad térmica interna y que Chile posee un importante potencial para su implementación. / Temperature is an important parameter for the development of anaerobic digestion, base of biogas production. Limit sectors to apply technology, regulate efficiency, assist in the elimination of pathogens, affect the retention time and increase the biogas production. This work was developed to assess, the dynamics of biogas production based on the thermal regime, linking between temperatures affecting a chinese digester and propose areas in Chile that have greater ability to apply this technology. A digester with 10 m3 of capacity was fed three times a week with a 3:1 mixture of water/manure. Monitorings were applied in two periods, february to july (2011) and january to august (2012); installing continuously recording sensors and analyzes compositional of biogas generated weekly. In the first period were applied linear correlations to the variables, it was found that the air temperature affects mainly the discharge, the pond and the digester temperature. In the second period was performed trend analysis, anomalies, moving averages and functional adjustements, it was found that temperature inside of digester was 22°C average and the soil average temperature is more similar with the air average temperature. In the analysis of composition, methane ranged from 55 to 85% and 16 to 35% of carbon dioxide. The zoning was performed by “thermal index”, it was found that since the region of Los Lagos and Andes mountain, range is not suitable the application of this technology. So it can be said that these digesters favor internal thermal stability and that Chile has significant potential for implementation.

No description available.

No description available.

The purpose of this investigation was to compare the ventilatory threshold (T(vent)) with the lactate threshold (T(lact)) during 60 minutes of steady-state exercise at the calculated thresholds. Eight trained, male cyclists (mean age=23.3 yrs, ht=176.4 cm, wt=70.7 kg, VO₂max=61.02 ml/kg‧minˉ¹) performed a 23 W/min progressive intensity cycling test for determination of T(lact) and T(vent). T(vent) was determined by the non-linear increase in excess CO₂ (ExCO₂) while T(lact) was calculated by the 'individual anaerobic threshold' (IAT) method. Subsequently, subjects performed up to 60 minutes steady-state exercise at the threshold workloads. Results at T(vent) and T(lact) indicate significant differences (p<0.01; T(lact)>T(vent)) between VO₂, ExCO₂, HR, [BLa] and workload as calculated by Hotelling's T²-test. During the steady state exercise at each specified workload, VO₂, [BLa], heart rate and ExCO₂ were measured at 15 minute intervals. All subjects completed the steady-state exercise at T(vent) (VSS) while only 2 subjects completed the steady-state exercise at T(lact) (LSS) (avg time=48.4 min). Comparison of metabolic variables using MANOVA and multiple comparisons revealed significant differences between VSS and LSS for HR and VO₂ at all time intervals, for [BLa] at 30 and 45 minute intervals and for ExCO₂ at the 30 minute interval. Furthermore, examination of [BLa] over time using trend analysis revealed a stabilization during VSS ([formula omitted]=3.05 mmol‧Lˉ¹) whereas [BLa] continuously increased over time during LSS. Findings indicate that T(lact) (IAT method) overestimates the ability to perform prolonged work over 45 min. while T(vent) (ExCO) allows for steady-state exercise greater than 60 minutes. / Education, Faculty of / Curriculum and Pedagogy (EDCP), Department of / Graduate

Despite the clinical importance of anaerobic bacteria in otitis media and the uncertainty regarding the proper treatment of the anaerobic focal infection, few experimental studies focused upon the role of these microorganisms in otitis media have been publis­hed. In the present investigation a guinea-pig model for the induc­tion of anaerobic monoinfections in the middle ear was described. Bacteroides fragilis and Propionibacterium acnes (4.0-10x10 colony forming units) injected via the tympanic membrane were capable of inducing clinical and histological otitis media with persistent seguele in the middle ear cavity. Bacteroides asacc-harolyticus, Peptostreptococcus micros and Peptost repto- coccus anaerobius failed to induce otitis media. B. fragilis otitis was accompanied by increased serum IgG and IgM antibody titres against the challenge organism, whereas P. acnes and P. anaerobius did not induce a humoral immune response. The results suggested true virulence of B. fragilis in guinea-pig middle ear monoinfections. Metronidazole was found to accelerate the elimination of B.fragilis from the middle ear. However, even high doses of metro­nidazole were nojt fully effective perhaps reflecting an incomplete anaerobiosis at the site of infection in some instances. At pre­sent, nitroimidazoles in chronic otitis media must be regarded as a possible alternative reguiring further study, particularly with regard to the dosage. In order to gain further knowledge of the interaction between poly­morphonuclear leukocytes and bacteria under anaerobic conditions an in vitro model was established. It was shown that P. acnes was readily phagocytosed with the aid of C3 activated either via the classical or alternative pathway and that killing of P. acnes was inefficient during anaerobiosis. The results suggest that P.acnes is maintained in the pus in chronic otitis media because it sur­vives phagocytosis. Finally, the interaction between the most common pathogen in acute purulent otitis media, Streptococcus pneumoniae, and human poly­morphonuclear leukocytes under anaerobic conditions was studied. Since purulent maxillary sinus effusion (and probably also purulent middle ear effusion), invariably has a pO^ approaching zero, such studies are highly relevant with regard to the host defence in sinuitis and perhaps also in otitis media. S. pneumoniae was killed by the phagocytes under anaerobic condi­tions although at a slower rate than in air. Degradation of pneumo­coccal teichoic acid, DNA and RNA took place after phagocytosis under aerobic as well as anaerobic conditions, whereas degradation of unsaturated cell membrane lipids took place only under aerobic conditions. Furthermore, the pneumococcal autolytic system did not participate in the killing or the degradation of the bacteria. / <p>Diss. Umeå, Umeå universitet, 1984, härtill 6 uppsatser</p> / digitalisering@umu

No description available.

by Tam Fung-ping. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 254-264). / Chapter I. --- Title page --- p.I / Chapter II. --- Abstract --- p.II / Chapter III. --- Acknowlegements --- p.III / Chapter IV. --- Table of contents --- p.IV / Chapter V. --- List of tables --- p.V / Chapter VI. --- List of figures --- p.VI / Chapter VII. --- Abbreviations --- p.VII / Chapter Chapter 1. --- Literature Reviews / Chapter 1.1 --- Modes of energy generation in facultative bacteria --- p.1 / Chapter 1.1.1 --- Difference in energy generation mechanism between respiratory and fermentative pathways --- p.2 / Chapter 1.1.2 --- Difference in carbon metabolism during anaerobiosis --- p.6 / Chapter 1.2 --- Repression and derepression of genes during anaerobiosis --- p.8 / Chapter 1.3 --- Global regulatory network for respiratory control --- p.8 / Chapter 1.3.1 --- Fnr-regulated gene expression --- p.10 / Chapter 1.3.2 --- NarL-regulated gene expression --- p.11 / Chapter 1.3.3 --- Crp-regulated gene expression --- p.12 / Chapter 1.3.4 --- ArcA-regulated gene expression --- p.13 / Chapter 1.3.5 --- Overlapping control of gene expression --- p.14 / Chapter 1.3.6 --- Regulatory mechanism of respiratory control --- p.16 / Chapter 1.4 --- Other regulatory systems in respiratory control --- p.19 / Chapter 1.5 --- The puzzle of regulatory network in anaerobiosis --- p.22 / Chapter 1.6 --- ArcA-ArcB system in Escherichia coli --- p.24 / Chapter 1.6.1 --- Arc A and ArcB for aerobic respiratory control --- p.24 / Chapter 1.6.2 --- arcA/dye/msp/fex/sfrA/cpxC gene are on identical genetic locus --- p.26 / Chapter 1.6.3 --- Arc function and Sfr function of Arc A protein are separately regulated --- p.28 / Chapter 1.6.4 --- ArcB-ArcA as sensor regulator in two component system for respiratory control --- p.29 / Chapter 1.7 --- Objectives and strategies of present study --- p.37 / Chapter Chapter 2. --- Materials / Chapter 2.1 --- Bacterial strains --- p.41 / Chapter 2.2 --- Culture mediums --- p.44 / Chapter 2.3 --- "Buffers, chemicals and antibiotics" --- p.46 / Chapter 2.4 --- DNA primers --- p.53 / Chapter Chapter 3. --- Primer extension analysis for locating the transcriptional start point of anaerobic inducible arcA in pFS --- p.34 / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Methods --- p.57 / Chapter 3.2.1 --- Preparation of total RNA --- p.59 / Chapter 3.2.2 --- Formaldeyde agarose gel electrophoresis of RNA --- p.60 / Chapter 3.2.3 --- Spectrometric estimation of RNA --- p.61 / Chapter 3.2.4 --- End-labelling of arcAusp primer with 32P --- p.62 / Chapter 3.2.5 --- Precipitation of arcAusp primer with samples RNA --- p.63 / Chapter 3.2.6 --- Primer extension reaction --- p.63 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Preparation of RNA --- p.67 / Chapter 3.3.2 --- Determination of transcription start site by primer extension --- p.67 / Chapter 3.4 --- Discussions --- p.76 / Chapter 3.4.1 --- Selective activations of aerobic and anaerobic transcripts in response to oxygen level --- p.76 / Chapter 3.4.2 --- The arcA promoter is a sigma-70 dependent promoter --- p.77 / Chapter 3.4.3 --- Experimental design --- p.77 / Chapter Chapter 4. --- In vitro chemical mutagensis for finding some important regulatory elements of arcA in pFS --- p.34 / Chapter 4.1 --- Introduction / Chapter 4.2 --- Methods --- p.84 / Chapter 4.2.1 --- Large scale preparation of pFS34 plasmid --- p.84 / Chapter 4.2.2 --- PCR-mediated chemical mutagenesis of pFS34 --- p.86 / Chapter 4.2.3 --- Restriction enzyme digestion of PCR-amplified arcA insert after phenol extraction --- p.90 / Chapter 4.2.4 --- Large scale preparation of vector pFZYl and restriction enzyme digestion --- p.91 / Chapter 4.2.5 --- Ligation of EcoRI-SalI digested pFS34 fragment and vector pFZYl --- p.91 / Chapter 4.2.6 --- Preparation of electrotcompetent cell Salmonella typhymurium JR502 and electro-transformation --- p.92 / Chapter 4.2.7 --- Screening of transformed clones by LB-amp50-xgal plates --- p.93 / Chapter 4.2.8 --- Screening of recombinants colonies by Polymerase chain reaction (PCR) --- p.94 / Chapter 4.2.9 --- Screening of single-point mutated clones by PCR-single stranded conformational polymorphism (PCR-SSCP) technique --- p.96 / Chapter 4.2.10 --- Screening of mutated pFS34 clones with altered promoter activities byβ-gal assay --- p.98 / Chapter 4.2.11 --- Sequencing of mutated clones --- p.101 / Chapter 4.2.11.1 --- Recombinant M13 single-stranded sequencing of the mutated clones --- p.101 / Chapter 4.2.11.2 --- pUC18 double-stranded DNA sequencing of mutated clones --- p.105 / Chapter 4.3 --- Results --- p.108 / Chapter 4.3. --- l PCR-mediated chemical mutagenesis of pFS34 --- p.108 / Chapter 4.3.2 --- Screening of transformed clones by LB-amp50-xgal plate --- p.112 / Chapter 4.3.3 --- Screening of recombinants colonies by polymerase chain reaction (PCR) --- p.112 / Chapter 4.3.4 --- Screening of single-point mutated clones by PCR-single stranded conformational polymorphism (PCR-SSCP) technique --- p.114 / Chapter 4.3.5 --- Screening of mutated pFS34 clones with altered promoter activities byβ-gal assay --- p.117 / Chapter 4.3.6 --- Sequencing of mutated clones --- p.123 / Chapter 4.4 --- Discussions --- p.135 / Chapter 4.4.1 --- The possible mechanisms in anaerobic transcription --- p.135 / Chapter 4.4.2 --- The possible mechanisms in aerobic transcription --- p.143 / Chapter 4.4.3 --- Experimental design --- p.146 / Chapter Chapter 5 --- Investigation of the effect of integration host factor (IHF) and autoregulation on the expression of pFS34 / Chapter 5.1 --- Introduction --- p.152 / Chapter 5.2 --- Methods --- p.154 / Chapter 5.2.1 --- Construction of Escherichia coli mutant --- p.155 / Chapter 5.2.2 --- PCR check of mutant for the presence of pFS34 and pFZYl plasmid --- p.157 / Chapter 5.2.3 --- β-galactosidase assay of aerobic and anaerobic activities change of pFS34 --- p.157 / Chapter 5.3 --- Results / Chapter 5.3.1 --- Effect of integration factor (IHF) on pFS34 --- p.158 / Chapter 5.3.1.1 --- PCR analysis of E. coli. himA and himD mutant for the presence of pFS34 and pFZYl plasmid --- p.158 / Chapter 5.3.1.2 --- β-galatosidase assay of aerobic and anaerobic activities of pFS34 in E. coli. himA and himD mutant --- p.158 / Chapter 5.3.2 --- Autoregultion on expression of pFS34 --- p.162 / Chapter 5.3.2.1 --- PCR analysis of E. coli. arcA mutant for the presence of pFS34 plasmid --- p.162 / Chapter 5.3.2.2 --- β-galctosidase assay of aerobic and anaerobic activities of pFS34 (arcA-lacZ) in E. coli. arcA mutant --- p.162 / Chapter 5.4 --- Discussions --- p.167 / Chapter 5.4.1 --- Effect of IHF on aerobic and anaerobic expression of arcA --- p.167 / Chapter 5.4.1.1 --- Possible regulatory mechanism of IHF on aerobic transcription --- p.167 / Chapter 5.4.1.2 --- Possible regulatory mechanism of IHF on anaerobic transcription --- p.170 / Chapter 5.4.1.3 --- Affinity binding of IHF depends on topological state of arcA --- p.172 / Chapter 5.4.1.4 --- Possible role of IHF in global regulation of anaerobiosis --- p.173 / Chapter 5.4.1.5 --- Experimental design --- p.174 / Chapter 5.4.2 --- Autoregulatory expression of arcA in pFS34 --- p.176 / Chapter Chapter 6. --- PCR walking of arcA from Salmonella typhimurium LT2 / Chapter 6.1 --- Introduction --- p.177 / Chapter 6.2 --- Methods --- p.186 / Chapter 6.2.1 --- Preparation of chromosomal DNA from Salmonella typhimurium LT2 --- p.186 / Chapter 6.2.2 --- Amplification of genomic arcA by linear PCR with arcAcds primer --- p.187 / Chapter 6.2.3 --- Low stringency PCR amplification of single-stranded arcA gene fragment and genomic DNA with anchor- random primer (delC-32R & delC-34R) --- p.188 / Chapter 6.2.4 --- High stringency PCR amplification with arcAcds primer and delC-23 primer --- p.189 / Chapter 6.2.5 --- High stringency PCR amplification with arcAusp2 and delC-23 primer --- p.190 / Chapter 6.2.6 --- "High stringency PCR amplification with delC-23 primer only, arcAusp2 primer only and mixture of delC-23 and arcAusp2 primer" --- p.191 / Chapter 6.2.7 --- High stringency PCR amplification with arcAusp2 only and Sau3A restriction enzyme digestion of PCR products --- p.192 / Chapter 6.2.8 --- Cloning of PCR walking products into pUC18 and heat shock transforming into E.coli. JM83 --- p.193 / Chapter 6.2.9 --- Confirmation of inserts in the clones and estimation of inserts size by PCR --- p.194 / Chapter 6.2.10 --- Dideoxy sequencing of PCR walking arcA fragments in pUC18 --- p.194 / Chapter 6.2.11 --- Subcloning of arcA fragment into pFZYl and PCR analysis for insertion of one insert with proper orientation --- p.195 / Chapter 6.2.12 --- arcA-galactosiadase assay of PCR walking arcA fragment-lacZ fusion --- p.196 / Chapter 6.3 --- Results --- p.198 / Chapter 6.3.1 --- Preparation of chromosomal DNA from Salmonella typhimurium LT2 --- p.198 / Chapter 6.3.2 --- Amplification of genomic arcA by linear PCR with arcAcds primer --- p.198 / Chapter 6.3.3 --- Low stringency PCR amplification of single-stranded arcA gene fragment and genomic DNA with anchor- random primer (delC-32R and delC-34R) --- p.200 / Chapter 6.3.4 --- High stringency PCR amplification with arcAcds primer and delC-23 primer --- p.200 / Chapter 6.3.5 --- High stringency PCR amplification with arcAusp2 、 primer and delC-23 prime --- p.203 / Chapter 6.3.6 --- "High stringency PCR amplification with delC-23 primer only, arcAusp2 primer only and mixture of delC-23 and arcAusp2 primer to check for flanking ends of bands" --- p.205 / Chapter 6.3.7 --- High stringency PCR amplification with arcAusp2 primer and Sau3A restriction enzyme digestion of PCR products --- p.207 / Chapter 6.3.8 --- Cloning of PCR walking products into pUC18 and heat-shock transforming into E. coli. JM83 --- p.210 / Chapter 6.3.9 --- Confirmation of inserts in the clones and estimation of inserts size by PCR --- p.210 / Chapter 6.3.10 --- Dideoxy sequencing of arc A PCR walking fragment: :pUC18 --- p.210 / Chapter 6.3.11 --- Subcloning of arcA fragment into pFZYl and PCR check for right insertion of single insert with proper orientation --- p.226 / Chapter 6.3.12 --- β-galactosidase assay --- p.232 / Chapter 6.4 --- Discussions --- p.227 / Chapter 6.4.1 --- PCR based gene walking strategy --- p.227 / Chapter 6.4.2 --- Confirmation of cloned arcA gene in pFS34 was a geniune arcA gene of S. typhimurium --- p.240 / Chapter 6.4.3 --- Promoter activity of further upstream arcA clones - AU87::pFZYl --- p.241 / Chapter Chapter 7. --- Overall Discussion --- p.244 / Chapter 7.1 --- Summary --- p.244 / Chapter 7.2 --- Proposed Model of regulation of arcA in Salmonella typhimurium --- p.249 / Chapter 7.3 --- Further Studies --- p.251 / References --- p.254