O gene TrCit do fungo filamentoso Trichoderma reesei codifica a proteína citrato sintase, uma enzima chave do ciclo de Krebs. Análise da região 5´ upstream de TrCit mostra que o gene está adjacente ao gene TrIcl (que codifica a proteína isocitrato liase, uma enzima do ciclo de glioxalato), em uma orientação cabeça-cabeça. A região promotora intergênica de 647 pb rica em G + C, apresenta uma ilha CpG, seqüência INR, caixas GC, caixas CAAT, sítios de ligação para diversos fatores de transcrição e é isenta de caixa TATA. O gene TrCit de 1573 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1422 pb produz uma proteína de 474 aminoácidos, com um peso molecular estimado de 52,3 kD. O gene TrIcl de 1880 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1788 pb produz uma proteína de 596 aminoácidos, com um peso molecular estimado de 65,4 kD. A atividade transcricional da região promotora foi analisada utilizando como repórter o gene de higromicina B fosfotransferase (hph). Uma região funcional necessária à transcrição de ambos os genes foi identificada na região central do promotor e contém uma caixa GC que liga o putativo fator de transcrição Sp1 de T. reesei (TrZnFSp1). O gene do putativo fator de transcrição “zinc-finger” TrZnFSp1 de 1500 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1344 pb produz uma proteína de 448 aminoácidos, com um peso molecular estimado de 48,4 kD. Os resultados mostram que ambos os genes são transcritos de forma divergente a partir de um promotor bidirecional que compartilha na região central uma caixa GC, necessária para a transcrição de ambos os genes. / The TrCit gene from the filamentous fungus Trichoderma reesei codes for the citrate synthase protein, a key enzyme in the Krebs cycle. Analysis of TrCit 5’ upstream region showed that it is adjacent to the TrIcl gene that codes for isocitrate lyase protein, an enzyme involved in the glyoxylate cycle. Both genes, on a head-to-head orientation, are separated by an intergenic GC-rich and TATA-less promoter region of 647 base pairs. This bidirectional promoter has diverse cis regulatory elements: a CpG island, two INR sequences, GC boxes, CAAT boxes and several putative interaction sites for different transcription factors. The TrCit gene, 1,573-base pair-long, has an open reading frame of 1,422 base pairs interrupted by two introns. The gene codes for a protein with an estimated molecular weight of 52.3 kD. The TrIcl gene, 1,880-base pair-long, contains 3 exons and 2 introns and a putative coding sequence of 1,788 base pairs. The estimated molecular weight of TrICL is 65.4 kD. he transcriptional activity of the intergenic promoter region was analyzed using hygromicin B phosphotransferase (hph) as a reporter gene. A functional region required for the transcription of both genes was identified in the centre of this promoter. It has a GC box that interacts with a putative transcription factor Sp1 from T. reesei (TrZnFSp1). The results presented in this work show that both genes are divergently transcribed from a bidirectional promoter that shares an essential central GC box.

No description available.

Hypoxia refers to any condition in which the water is less than fully saturated with oxygen. Although it is generally accepted that adults are more tolerant of hypoxic conditions than larval stages, there is little information to support this assumption. To determine whether reduced concentrations of dissolved oxygen (DO) affect fishes differently during various early life stages, I examined the responses of two species of fish (fathead minnows (Pimephales promelas) and rainbow trout (Oncorhynchus mykiss)) exposed to low dissolved oxygen concentrations at different ages during the first 100 days post-hatch. The changes in oxygen requirements and respiratory patterns that occur during ontogeny and exposure to hypoxia were observed. The results of this study suggest that the early larval stages appear to be at least as tolerant of short-term exposure to low dissolved oxygen concentrations as the older, more developed stages. Fathead minnows underwent a gradual transition from being metabolic conformers to regulators during development. Hemoglobin appeared to be playing a larger role in oxygen supply in the early post-hatch trout than in the minnows. Fathead minnow larvae produced relatively low concentrations of lactate upon exposure to hypoxia. Conversely, rainbow trout larvae exhibited significant increases in lactate concentration under similar conditions. This implies that there is a threshold oxygen concentration below which trout larvae utilize anaerobic metabolism to provide additional energy. Lactate dehydrogenase activity increased as the rainbow trout larvae aged, suggesting that they develop an anaerobic capacity which could be used to provide additional energy during hypoxia. The minnows did not exhibit this increase in activity. The ability of larval fishes to detect and avoid hypoxic conditions was also examined. The overall trends suggest that throughout this period of development, both fish species gradually leave an area as the dissolved oxygen concentration decline. Both species appeared to leave the hypoxic areas with deliberate motions, indicating that a directed sensor system allowed them to detect oxygen gradients. The results suggest that a combination of physiological, biochemical, and behavioral mechanisms may allow fishes to cope with hypoxia. / Ph. D.

The intertidal zone is characterized by persistent, tidally-driven fluctuations in both abiotic (e.g., temperature, [O2], salinity) and biotic (e.g., food availability, predation) conditions, which makes this a very physiologically challenging habitat for resident organisms. The magnitude and degree of variability of these environmental stressors differs between intertidal zones, with the most extreme physiological stress often being experienced by organisms in the high intertidal. Given that many of the fluctuating conditions in this environment are primary drivers of metabolic rate (e.g., temperature, [O2], food availability), we hypothesized that sessile conspecifics residing in different tidal zones would exhibit distinct ‘metabolic phenotypes,’ a term we use to collectively describe the organisms’ baseline metabolic performance and capacity. To investigate this hypothesis, we collected acorn barnacles (Balanus glandula) from low, mid, and high intertidal positions in San Luis Obispo Bay, CA and measured a suite of biochemical (whole-animal citrate synthase (CS) and lactate dehydrogenase (LDH) activity, aerial [lactate]), physiological (O2 consumption rates), morphological (body size), and behavioral (e.g., cirri beat frequency, % time operculum open) indices of metabolism. We found tidal zone-dependent differences in B. glandula metabolism that primarily related to anaerobic capacity, feeding behaviors and body size. Barnacles from the low intertidal tended to have a greater capacity for anaerobic metabolism (i.e., increased LDH activity), feed less when submerged, and be smaller in size compared to conspecifics in the high intertidal. We did not, however, see differences between barnacles from different tidal heights in whole-animal [lactate] following 24h of air exposure, which indicates that the enhanced capacity of low intertidal barnacles for anaerobic metabolism may have evolved to support metabolism during more prolonged episodes of emersion (>>24h) or during events other than emersion (e.g., coastal hypoxia, predation). There were also no significant differences in CS activity or baseline oxygen consumption rates (in air or seawater at 14˚C) across tidal heights, which implies that aerobic metabolic capacity may not be as sensitive to tidal position as anaerobic processes. Understanding how individuals occupying different shore heights differ in their metabolic capacity becomes increasingly interesting in the context of global climate change, given that the intertidal zone is predicted to experience even greater extremes in abiotic stress.

No description available.

In this study, a strategy called gene splicing by overlap extension, &ldquo / Gene SOEing&rdquo / , was used for the construction of the fusion proteins with the purpose of increasing the thermostability of mesophilic enzymes by incorporation of stability domain from a thermostable enzyme. Gene SOEing is a PCR-based approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. In fusion constructs, as the stability determinant Thermoplasma volcanium citrate synthase (CS) large domain has been used. This gene has recently been cloned in our laboratory. In two different fusions, as fusion partners, dehalogenase II (dehCII) gene of Pseudomonas sp. CBS3 and aminoglycoside-3&#039 / -phosphotransferase-II (APH(3&#039 / )-II) gene of E. coli were employed. Following the Gene SOEing, two fusion products, 1722 bp long CS Large Domain-dehCII and 1750 bp long CS Large Domain-APH(3&#039 / )-II were constructed. Also a 1586 bp long dehCII-APH(3&#039 / )-II fusion was prepared. Three fusion constructs were cloned in E. coli. Cloning was confirmed in each case, by restriction analysis of the isolated plasmids from recombinant colonies. APH(3&#039 / )-II gene associated with CS Large Domain-APH(3&#039 / )-II and dehCII-APH(3&#039 / )-II fusion constructs were successfully expressed in E. coli as revealed by enzyme assay and antibiotic agar plate assay. CS Large Domain-APH(3&#039 / )-II fusion protein retained 9.4% of the original APH(3&#039 / )-II activity after 10 minutes at 60&ordm / C. However, CS Large Domain-dehCII and dehCII-APH(3&#039 / )-II fusions did not display any dehalogenase activity.

Psychopharmaca are a large group of drugs widely used not only in psychiatry. Their systemic administration affects both the main diagnosis and the organism as a whole. The subject of our experiments is the effect of psychopharmaca on the changes in mitochondrial functions, which is beneficial for understanding of molecular mechanisms of therapeutic and adverse effects of drugs. The aim of this thesis was to study the in vitro effects of selected drugs on the cell energy metabolism. Selected antipsychotics (chlorpromazine, levomepromazine, haloperidol, risperidone, ziprasidone, zotepine, aripiprazole, clozapine, olanzapine, and quetiapine), antidepressants (bupropion, fluoxetine, amitriptyline, imipramine) and mood stabilizers (lithium, valproate, valpromide, lamotrigine, carbamazepine) were tested. In vitro effects of selected psychopharmaca were measured on isolated pig brain mitochondria. The activities of citrate synthase (CS) and electron transport chain (ETC) complexes (I, II+III, IV) were measured spectrophotometrically. Drug-induced changes of mitochondrial respiration rates linked to complex I (supported by malate and pyruvate) and complex II (supported by succinate) were evaluated by high resolution respirometry. Complex I was significantly inhibited by lithium, carbamazepine, fluoxetine,...

No description available.

The supply of precursors from primary metabolism is often overlooked when engineering secondary metabolism for increased product yields. This is because precursor supply may be assumed to be non-limiting, and it is considered difficult to engineer primary metabolism, because control of carbon flow (flux) is generally distributed among most enzymes of the pathway. The aim of this thesis was to increase the production of sterols, part of the isoprenoid class of secondary metabolites, in tobacco (Nicotiana tabacum) Bright Yellow 2 (BY-2) cell cultures. This was achieved by genetically engineering increased activity of mitochondrial citrate synthase, an enzyme of the tricarboxylic acid (TCA) cycle that is involved in the provision of cytosolic acetyl coenzyme A, the primary metabolite precursor to sterols. Metabolic flux analysis revealed that citrate synthase exerts significant control over cyclic TCA cycle flux in BY-2 cells and suggested that increasing the activity of downstream enzymes within secondary metabolism could lead to a further redirection of TCA-cycle-derived precursors into sterol biosynthesis. Attempts were made to achieve this by genetically engineering increased activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), a key enzyme of secondary metabolism involved in sterol biosynthesis. Consistent with previous research, transgenic lines had increased sterol levels. However, the high sterol phenotype was unstable, and attempts to co-express HMGR and citrate synthase genes were unsuccessful. The thesis demonstrates that increasing the provision of precursors to secondary metabolites can result in increased yields of those secondary metabolites but suggests that in most cases the activity of enzymes within secondary metabolism has a greater effect on those yields. It also reveals that single enzymes can exert significant control of flux within primary metabolism, although the control exerted by specific enzymes probably changes with the demands placed on metabolism.

Adaptation to chronic hypoxia induces endogenous cardioprotection and increases the heart resistance to ischemia/reperfusion injury. The heart mitochondria, which produce reactive oxygen species (ROS) in addition to ATP, play an important role in these processes. During ischemia/reperfusion, ROS are produced in excessive amounts and damage the cells. However, in lower concentrations, ROS are involved in the signalling pathway of cardioprotection induced by adaptation to chronic hypoxia. In the heart, two mitochondrial subpopulations have been observed, subsarcolemmal mitochondria (SSM) and intermyofibrillar mitochondria (IMFM), which differ in cell localization as well as in morphological and biochemical properties. The aim of this work was to introduce the method of SSM and IMFM isolation in our laboratory and to analyse their antioxidative capacity after adaptation to chronic hypoxia. Adult male Wistar rats were kept either under normoxic conditions or exposed to intermittent high-altitude hypoxia (IHA; 7000 m, 5 days a week/8 hours a day, totally 25 exposures). Mitochondrial subpopulations were isolated from heart left ventricle and their functionality was verified by measuring oxygen consumption and enzyme activities. The IMFM had higher oxygen consumption in comparison with SSM and activities...