No description available.

In Escherichia coli, the speC gene encodes biosynthetic ornithine decarboxylase (ODC), an enzyme that catalyzes the decarboxylation of ornithine to produce putrescine. The two polyamines, putrescine and spermidine, and the cyclic AMP (CAMP) - cAMP receptor protein (CRP) are known to inhibit the expression of ODC via undefined mechanisms. A single copy of the speC’-lacZ fusion plasmid pOL-1, containing an 843 base pair fragment including the spec promoter, was transferred to the E. coli CB806 chromosome to create E. coli λCBOL. In cell-free extracts prepared from E. coli λCBOL supplemented with cAMP, putrescine, or spermidine, the B-galactosidase activity encoded by the speC’-lacZ fusion was compared to the ODC activity encoded by spec. Only cyclic AMP and putrescine repressed the speC’-lacZ fusion. Cyclic AMP, putrescine, and spermidine all repressed the spec gene. A 444 bp AluI restriction fragment, containing a putative CRP binding site and a downstream open reading frame (ORF2) present on the strand complementary to speC, was fused to lacZ to create a transcriptional fusion, pCC2L. Analysis of E. coli CB806/pCC2L revealed that there was no detectable β8- galactosidase activity from the ORF2-lacZ fusion. However, promoter activity was detected in the opposite direction (3’ to 5’) of ORF2 as alkaline phosphatase activity, encoded on the same plasmid, increased in the presence of CAMP. A 678 bp DraI-AatII fragment, containing the CRP binding site and an adjacent open reading frame (ORF3) present on the speC coding strand, was subcloned into plasmid pBR322 to create pBCR. In the presence of 10 mM cAMP, E. coli CB806/pBCR exhibited an 18% inhibition in ODC activity encoded by spec. It is proposed that ORF3 encodes a protein that represses speC in the presence of CAMP. / Master of Science

In comparison to other Pseudomonas, P. aeruginosa grows poorly in L-lysine as a sole source of nutrient while fast growth mutants can be obtained. The proposed catabolic pathway involves lysine decarboxylation to cadaverine and its subsequent degradation through g-glutamylation pathway to d-aminovalerate and glutarate. The lysine decarboxylase A (ldcA) gene, previously identified as a member of the ArgR regulon of L-arginine metabolism, was found essential for L-lysine catabolism. The ldcA gene encodes a decarboxylase which takes L-lysine but not L-arginine as substrate. Contrarily, the ldcA expression was inducible by L-arginine but not by L-lysine. This peculiar arginine control on lysine utilization was also noted from uptake experiments. The lack of lysine-responsive control on lysine catabolism and its tight connection to arginine regulatory network provided an explanation of lysine as poor nutrient for P. aeruginosa. Catabolism of cadaverine, a product from lysine decarboxylation, was investigated and compared to that of putrescine, another diamine of similar biochemical properties that is derived from arginine and ornithine. While the g-glutamylation pathway was first reported in E. coli for putrescine utilization, an expanded version of this pathway was found in P. aeruginosa with redundant enzymes for polyamine degradation. The PauR protein was identified as a transcriptional repressor of genes for the catabolism of putrescine and cadaverine, as well as their corresponding downstream metabolites, g-aminobutyrate (GABA) and d-aminovalerate (AMV). PauR shows distinct dimer configuration after glutaraldehyde crosslinkage, and possible conformational changes could be triggered by the presence of putrescine and cadaverine, but not GABA. A newly identified ABC transport system, encoded by the agtABCD operon, was found important for the uptake of GABA and AMV; and expression of which is controlled by the AgtSR two-component system. The CbrAB two-component system was proposed to regulate the catabolite repression control protein Crc through a small RNA CrcZ. A consensus CbrB recognition sequence was proposed based on the conserved palindromic nucleotide sequence in the upstream activating sequence of the crcZ promoter. Genetic studies indicated utilization of arginine, lysine and diamines (but not histidine, GABA and AMV) might be under CbrAB regulation through the CbrAB/CrcZ/Crc system in P. aeruginosa.

The polyamines are a group of natural compounds which have been found in almost all living tissues. N1- and N8- acetylspermidine have been known for a considerable time as normal constituents in human urine, but their physiological role is unknown. Recent studies have indicated the presence of enzymes in the tissues capable of converting spermidine into N1-acetylspermidine and N8-acetylspermidine and other enzyme activities which catalyze deacetylation and interconversion reactions. One approach for determining physiologic activity of an endogenous compound is to observe their pharmacologic and toxicologic effects. In the present study, the LD50 for N1-acetylspermidine, N8-acetylspermidine, spermidine, spermine and putrescine, were determined by intraperitoneal injection in mice. The LD50 for N1-acetylspermidine •2HCl and N8-acetylspermidine •2HCl were 1150 and 820 mg/kg, respectively, and the respective LD50 for spermidine •3HCl, spermine n#8226;4HCl and putrescine n#8226;2HCl were 870, 370, and 1400 mg/kg. The major difference between the toxicity of N1-acetylspermidine and N8acetylspermidine and that of spermidine, spermine and putrescine appeared to be the latency period for the time of death. While N1- and N8-acetylspermidine have very rapid effects in which animals died in the first ten minutes after the injection with very few delayed deaths, the effects of spermidine, spermine and putrescine occurred over a wider range of time intervals with some deaths occurring as late as ten days after treatment The other signs of toxicity with N1acetylspermidine, N8-acetylspermidine, spermidine, spermine, and putrescine were qualitatively quite similar as each compound produced hypothermia, sedation, muscle incoordination, decreased motor activity, decreased respiration, and clonic convulsions.

Alkaloids are a class of plant secondary metabolites produced in about 20% of plant families. Domesticated tobacco, Nicotiana tabacum produces nicotine as the predominant alkaloid. The biosynthesis of nicotine occurs exclusively in the roots of tobacco, yet accumulates in the leaves of tobacco where it is acts as a defense compound to deter insect herbivory. The research detailed in this dissertation addresses two aspects of nicotine physiology in tobacco: 1) an investigation of hypothesized protein-protein interactions among nicotine biosynthetic enzymes and 2) the characterization of a novel nicotine transporter. A hypothesized metabolic channel including the two nicotine biosynthetic enzymes putrescine N-methyltransferase (PMT), N-methylputrescine Oxidase (MPO) and the S-adenosylmethionine (SAM) recycling enzyme S-adenosylhomocysteine hydrolase (SAHH) has been proposed. To further explore this hypothesis, protein-protein interactions among nicotine biosynthetic enzymes PMT, MPO and SAHH were investigated using yeast two-hybrid assays and co-immunoprecipitation experiments. The yeast two-hybrid was conducted as both a directed screen to detect interactions between the hypothesized metabolic channel members and as a library screen to detect interactions between hypothesized metabolic channel members and proteins from a tobacco root cDNA library. Co-immunoprecipitation experiments were conducted using proteins produced in an in vitro transcription/ translation system and using native proteins from a tobacco root extract. The outcome of these experiments provided no further evidence of a nicotine metabolic channel and a discussion of the methods and outcomes of the experiments conducted is presented. The nicotine uptake permease, NUP1, was identified in tobacco roots and was shown to preferentially transport nicotine when expressed in Schizosaccharomyces pombe. This report presents the characterization of tobacco plants and hairy roots with diminished NUP1 transcripts created by using RNAi. The NUP1-RNAi hairy roots and plants showed a decreased level of nicotine and the hairy root cultures displayed an altered distribution of nicotine from the root to the culture medium. Additionally NUP1-GFP was used to determine that NUP1 localized to the plasma membrane of tobacco BY-2 protoplasts. Potential models for the role of NUP1 in nicotine physiology will be discussed. / Ph. D.

The aim of this thesis was to determine the content of biogenic amines (BA)and polyamines (PA)in selected types of cheese and the influence of storage time on the content of biogenic amines.

Datura metel is a herbaceous plant found in almost all tropical parts of the world. It belongs to the family Solanaceae whose members, viz. Duboisia, Atropa, Hyoscyamus and Datura plants are known to produce tropane alkaloids- hyoscyamine and scopolamine which are most noted for their therapeutic use as anti-cholinergic agents. Since these alkaloids are produced in very low amounts in plants, alternative sources and methods of production for these alkaloids have been crucial in meeting the demands for these drugs. Endophytic fungi inhabiting a plant may have the potential to produce the same compounds as the host plants. The aim of the present study was to search for tropane alkaloid producing endophytic fungal isolates from Datura metel. Eighteen endophytic fungi were isolated from various tissues of Datura metel and screened for the presence of three tropane alkaloid biosynthetic genes- putrescine N-methyltransferase (PMT), tropinone reductase I (TRI) and hyoscyamine 6β-hydroxylase (H6H) using PCR-based screening approach. Six endophytic fungal isolates were found to possess the PMT, TR1 and H6H genes. The fungi were identified using molecular taxonomy as Col letotrichum boninense, Phomopsis sp., Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col letotrichum gloeosporioides and the identity was confirmed using colony and spore morphology. The production of tropane alkaloids hyoscyamine and scopolamine by the fungi has been ascertained using various techniques like TLC, HPLC and ESI-MS/MS by comparison with the authentic reference standards. The amount of tropane alkaloids produced by all six fungi in liquid cultures was quantified using HPLC analysis. Among the six tropane alkaloid-producing fungi Col letotrichum incarnatum gave the highest yields of hyoscyamine and scopolamine which were 3.906 mg/L and 4.13 mg/L, respectively. With an aim to characterize the tropane alkaloid biosynthetic genes in these fungi, the PMT gene was isolated from five of the endophytic fungi- Col letotrichum boni-nense, Fusarium solani, Col letotrichum incarnatum, Col letotrichum siamense and Col-letotrichum gloeosporioides for the first time and the sequence analysis showed high ho-mology (98%) to the Datura metel PMT cDNA. The gene was found to be devoid of introns in the fungi. Further phylogenetic analysis of the full length PMT sequence from the fungi strongly supports the hypothesis of horizontal gene transfer between the host plant and endophytic fungi. For further in detail characterization of fungal PMT, the Col letotrichum boninense PMT gene was taken as a representative. CbPMT gene was cloned in pRSET A expres-sion vector and heterologously expressed in E. coli and biochemically characterized. For optimal yield of soluble protein upon heterologous expression different conditions such as IPTG concentration, temperature and time post induction were optimized. Optimal yield was obtained by inducing the culture by 0.25 mM IPTG once it had reached and O.D. of 0.6 and incubating at 37◦ C for 3 h. The recombinant CbPMT enzyme expressed as histidine tagged fusion protein was purified using Ni-NTA affinity chromatography. Gel elution studies were carried out to determine molecular weight of the protein and it was found that the protein exists as a homodimer in solution with some amount also present as a monomer. Catalytic activity of the purified recombinant enzyme was studied for its dependence on both substrates putrescine as well as S-adenosylmethionine (SAM). The Km and Vmax values for putrescine were found to be 464 µM and 18.55 nkat/mg, respectively, while those for S-adenosylmethionine were found to be 628 µM and 18.63 nkat/mg, respectively. Optimum temperature for activity was found to be 37◦ C and optimum pH range was found to be 8-9. Fluorescence spectroscopy was used to study the binding affinity of both the sub-strates to the enzyme. Fluorescence quenching data for each substrate was analysed by using a nonlinear regression curve fit and Kd values were found to be 0.309 mM for pu-trescine and 0.118 mM for SAM, respectively. Circular dichroism spectrum of the enzyme indicated a pattern typical for alpha helix in the secondary structure. Binding of either substrate led to increase in ellipticity of the protein. Fluorescence quenching studies with collisional quenchers- acrylamide, potassium iodide, and cesium chloride indicated that the native protein is folded in a conformation that allows tryptophan residues to be acces-sible for quenching. The fraction of tryptophan residues (fa ) accessible for quenching by acrylamide (1.06) was found to be higher than that for potassium iodide (0.54) while that cesium ions was the least (0.38). The neutral quencher acrylamide could access all the tryptophans meaning that none of tryptophans are completely buried inside hydrophobic cores. the differential accessibility to the charged quenchers, however, indicates that more of the tryptophans are surrounded by positively charged amino acids. The unfolding of the protein was studied with the aid of chaotropic agents guanidine-HCl and urea and thermodynamic parameters were determined. The denaturant m-values were found to be 2.313 kcal/mol/M for Gdn-HCl and 2.345 kcal/mol/M for urea respectively. The free energy of unfolding was estimated to be 2.635 kcal/mol for Gdn-HCl and 4.630 kcal/mol for urea. Since no reports are available about the thermodynamics of folding and unfolding of PMT from any plant source, this study contributes towards the understanding of protein stability. Although a lot of reports are available on the biochemical characterization of PMT from different plant sources, the crystal structure of PMT is not yet available. In the current work, homology based modelling studies on CbPMT were carried out to get some idea about the protein tertiary structure. Homology based modelling studies showed that a significant amount of protein is present as α-helices which are present on the surface while the β-sheets are present in the interior of the protein. Each monomer of the protein is capable of binding both the substrates and hence the dimerization property of the enzyme could be a purely structural one leading to more stability and solubility of the protein. In conclusion, this study has shown for the first time that endophytic fungi have significant potential to be used for tropane alkaloid production and six such fungal strains have been identified. Although the production of tropane alkaloids by endophytic fungi is not very high, it can be scaled up by over-expressing the biosynthetic gene putrescine N-methyltransferase in the highest producer- Col letotrichum incarnatum to further increase the yield. These endophytic fungi have significant potential to be applied in fermentation technology to meet the demands for these drugs economically.

Polyamines, specifically putrescine, spermidine, and spermine, are small cationic molecules found in all organisms. Cells can biosynthetically make these molecules, or alternatively, they can be transported from the extracellular environment. Malignant cells have been shown to require relatively high amounts of polyamines. There is a chemotherapeutic agent, DFMO, used to block the biosynthesis of polyamines. Many malignant cells can circumvent DFMO therapy by activating their transport system. A potential solution is to simultaneously block biosynthesis and transport of polyamines. However, little is known about the polyamine transport system in higher eukaryotes. This thesis aims to add to the basic biological understanding of the polyamine transport system, as well as contribute to our understanding of the way in which malignant cells are able to sustain rapid growth. This was done by screening six candidate genes believed to be involved in the polyamine transport system. These six genes were identified using various bioinformatics databases. They were screened using RNAi to knock down each gene of interest and by using an assay developed in our lab. One of the genes, RabX6, may play a possible role in the transport of putrescine.

The thesis deals with the use and effectiveness of some less common methods of conservation of fish meat. The formation of biogenic amines in meat is observed in connection with the non-traditional preservative methods. Amines can serve as indicators of protein degradation. The quality of fish was considered in connection with the increasing content of selected biogenic amines (putrescine, cadaverine, spermidine, spermine, 2-fenylathylamine, histamine, tyramine and tryptamine). Ultra performance liquid chromatography (UPLC) was used as the method for determination of biogenic amines. Amines were derivatized by dansylchloride before their UPLC separation. The fish flesh was vacuum-packed. Samples were stored for several weeks in a thermostat at the selected storage temperature after the application of selected preservative technique. Beta-irradiation and high hydrostatic pressure were used for the preservation of fish flesh. Control samples were not exposed to the â-irradiation and high pressure. The organoleptic properties were studied for all samples (smell/odor, insight and shape). Beta-irradiation was applied to fish meat of common carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss). Both these species of freshwater fish are economically significant. Carp and trout are the species being mostly consumed in the Czech Republic. Fish meat is considered to be provided the flesh is fresh. A testing series of samples was created with common carp to determine the appropriate dose of â-irradiation. The maximum permissible dose of irradiation for fish meat is 3 kGy. Fish samples were exposed this dose in the first experiment. The dose of irradiation was reduced in following experiments based on the experience from the initial experiment. The doses of 0.25, 0.5, 0.75, 1.0 and 2.0 kGy were applied to rainbow trout. The value of 0.75 kGy of â-irradiation or higher (1.0, 2.0 and 3.0 kGy) prolonged the shelf life of fish meat, which was stored for three months (98 days). Prolonging of the shelf life of fish meat to approximately 98 days at 3.5 °C is redundant from technical point of view. For that reason lower doses 0.25, 0.5 and 0.75 kGy were tested in more detail in the repeated experiment with carp meat. Lower doses of â-irradiation are considered to be more acceptable and-at the same time-sufficiently effective for delaying the beginning of degradation processes. 6 High hydrostatic pressure was applied to meat of common carp (Cyprinus carpio), rainbow trout (Oncorhynchus mykiss) and pike (Exos lucius). Pike is another very popular kind of freshwater fish. Pike flesh is very tasty, but in spite of this, pike is not so much popular among consumers compared to carp and trout. The cause is its high price. Samples of pike were stored at standard temperature 3.5 °C and also at higher temperature 12 °C (unlike experiments with â-irradiation). Lower temperature of storage (3.5 °C) followed the conditions of storing of fish meat in industrial refrigeration facilities and households. The high pressure might not be sufficient for preservation at higher storage temperatures. This assumption was based on available information. Samples were treated by high pressure and stored at both 3.5 °C and 12 °C to verify this assumption. Higher temperature simulated either failure of refrigeration equipment or unsuitable store temperature of meat. In all species selected freshwater fish two levels of high pressure were applied ? 300 and 500 MPa. Both levels had significantly reduced the formation of biogenic amines, especially in samples stored at 3.5 °C. At this temperature, the effect of 300 and 500 MPa delayed start of degradation processes in fish meat by 3?4 weeks. At 12 °C and 500 MPa, high pressure extended the sustainability of meat by no more than one week. 500 MPa is effective treatment at the lower temperature of 3.5 °C. High pressure is not reliable preservative techniques at higher temperature.

No description available.