Nos récepteurs d'intérêt, les récepteurs à l'angiotensine AT1 et AT2, appartiennent à la classe A de la grande famille des récepteurs couplés aux protéines G (RCPG). Jusqu'à très récemment, la rhodopsine bovine était le seul RCPG dont la structure cristallographique était résolue. La structure de la rhodopsine est employée couramment comme modèle en modélisation par homologie des RCPG de classe A. La structure de la rhodopsine est constituée de sept hélices transmembranaires. La plupart de ces hélices ne sont pas droites, mais cassées ou incurvées. Pour comprendre quels sont les motifs structuraux possibles pour les cassures d'hélices, nous avons réalisé une étude exhaustive des motifs d'hélices cassées au niveau d'un seul résidu de jonction (motif HXH) grâce à une base de données de structures d'hélices cassées développée localement. Les résultats montrent que le résidu de jonction n'admet qu'un nombre limité de conformations conduisant à la classification de ces cassures en six motifs bien distincts. Un de ces motifs correspond à une cassure au niveau d'un renflement. Ce motif se retrouve dans l'hélice transmembranaire 2 (TMH2) de la rhodopsine où une cassure se fait au niveau d'un motif GG correspondant à un renflement p. Ce motif GG, situé aux positions 2.56-2.57, n'est pas conservé parmi les RCPG mais une proline est fréquemment observée aux positions 2.58, 2.59 ou 2.60. Nos récepteurs d'intérêt AT1 et AT2 possèdent une proline à la position 2.58. L'étude de l'évolution de l'hélice transmembranaire 2 au sein de la famille des RCPG suggère fortement que la position en 2.58 correspond à une délétion d'un résidu au niveau de la cassure. Ceci est confirmé par des analyses structurales de la Protein Data bank. Ces résultats indiquent que la structure de la rhodopsine peut être utilisée directement pour modéliser l'hélice 2 lorsque la proline est en position 2.59 ou 2.60 (renflement p)/ Lorsque la proline est en position 2.58, la rhodopsine peut aussi être utilisée comme modèle structural à condition de prendre en compte la délétion d'un résidu au niveau du renflement, pour obtenir une cassure proline en coude classique.

<p>This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an <em>N</em>-acyl-(4<em>R</em>)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated.</p> / Report code: LIU-TEK-LIC-2006:46.

Warm-season turf species are becoming increasingly popular for putting green use in the transition zone. Ultradwarf bermudagrass (Cynodon dactylon (L.)  C. transvaalensis Burtt-Davy) is the prevalent warm-season putting green species, but seashore paspalum (Paspalum vaginatum Swartz) and ‘Diamond’ zoysiagrass [Zoysia matrella (L.) Merr.] may also be grown in the transition zone. Warm-season species are susceptible to winter injury and may require different management regimes than cool-season species. Therefore, the objectives of this research were to assess the impacts of various management practices on warm-season putting green species and characterize the physiological basis for differences in freeze tolerance of various warm-season putting green species. Field studies determined sampling procedures form thatch-mat depth and soil organic matter content of warm-season putting greens and assessed the impact of various management practices on different warm-season putting green species/varieties. The relative freeze tolerance of ‘Champion’ and ‘TifEagle’ ultradwarf bermudagrass cultivars, ‘SeaDwarf’ seashore paspalum, and Diamond zoysiagrass were determined, along with the accumulation of proline and polyamines during cold acclimation, in growth chamber studies. All species/varieties required different sampling numbers for determination of thatch-mat depth and soil organic matter. More sand was incorporated into the turf canopy and surface hardness was increased with brushing and vibratory rolling TifEagle putting greens after sand topdressing application than either treatment alone. Putting green management programs with lower mowing heights and increased mowing frequencies increased ball roll distance on a MiniVerde putting green without negatively affecting turf quality. Weekly vertical mowing + daily grooming on TifEagle reduced thatch depth and turfgrass quality, while increasing topdressing incorporation over either treatment alone. Diamond was the most freeze tolerant species/variety, followed by TifEagle, Champion, then SeaDwarf. Cold acclimation increased proline concentration for all species/varieties except SeaDwarf, but had inconsistent effects on polyamines. Spermidine and putrescine concentrations differed with species/variety, but were not correlated to freeze tolerance.

No description available.

This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an N-acyl-(4R)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated. / Report code: LIU-TEK-LIC-2006:46.

Novel 1,4-chiral diamine ligand possessing a trans-9,10-dihydro-9,10-ethanoanthracene backbone was synthesized. The synthetic plan involves first LiAlH4 reduction of the Diels-Alder adduct obtained by reaction of dimenthyl fumarate and anthracene, which is followed by reacting the corresponding alcohol and subsequent attachment of mesylate and triflate units to get good leaving groups which are available substances for introducing nitrogen units via SN2 type reactions. Consequently, by using dimesyl ester and ditriflate esters five catalysts 27, 29, 30, 33 and 38 were synthesized. The first four catalysts 27, 29, 30 and 33 were used in transfer hydrogenation reactions with transition metal whereas catalyst 38 used as an organocatalyst in direct aldol reaction between acetone and p-nitrobenzaldehyde.

Vitamin B6, or more specifically the active form pyridoxal 5ʹ-phosphate (PLP), plays a crucial role as a cofactor for numerous enzymes linked to carbohydrate, fatty acid, and amino acid metabolism. There is a high prevalence of moderate vitamin B6 deficiency in the population that may be further exacerbated through the ingestion of vitamin B6 antagonists present in the food supply. For example, flaxseed contains the anti-pyridoxine factor 1-amino D-proline (1ADP) in the form of a dipeptide called linatine. In order to address these issues, the current study was designed to: 1) characterize and quantify the total amount of anti-pyridoxine factors present in flaxseed through the use of UPLC/ESI-MS analysis, 2) investigate the in vivo effects of synthetic and flaxseed-derived 1ADP on amino acid metabolism using a rat model of moderate B6 deficiency, and 3) identify novel biomarkers of vitamin B6 inadequacy using a LC-Qtof-MS based non-targeted metabolomics approach. The total anti-pyridoxine content, measured as 1ADP equivalents, in the flaxseed extract was found to be 177-437 μg/g of whole flaxseed, depending on the variety tested. Plasma biochemical analyses revealed that B6 vitamers, particularly PLP concentrations were reduced (P≤0.001), due to 1ADP ingestion (10 mg/kg diet) irrespective of the sources. Oral ingestion of flaxseed-derived 1ADP in moderately vitamin B6-deficient rats increased plasma cystathionine (P≤0.001), and decreased plasma α-aminobutyric acid (P≤0.001) and glutamic acid (P=0.017) concentrations compared to the controls. However, the ingestion of synthetic 1ADP elicited greater perturbations in amino acid profile compared to the flaxseed-derived 1ADP, which was predominantly in the form of the dipeptide linatine. Additionally, oral ingestion of the synthetic as well as the flaxseed-derived 1ADP significantly (P≤0.05) inhibited the activities of hepatic PLP-dependent enzymes involved in transsulphuration reactions of methionine metabolism. The use of a non-targeted metabolomics approach identified ten potential lipophilic markers of vitamin B6-insufficiency: glycocholic acid, glycoursodeoxycholic acid, murocholic acid, N-docosahexaenoyl GABA, N-arachidonoyl GABA, lumula, nandrolone, orthothymotinic acid, cystamine and 3-methyleneoxindole. These data serve to highlight potential deleterious effects of anti-pyridoxine factors linked to flaxseed in a population at risk for moderate vitamin B6 deficiency. / October 2015

Darbo tikslas – nustatyti endogeninių ir egzogeninių veiksnių poveikį žieminio rapso atsparumui šalčiui in vitro ir in vivo. Ginamieji disertacijos teiginiai: 1. Žieminio rapso atsparumas šalčiui didžiąją dalimi lemiamas endogeninio prolino, o ne tirpiųjų sacharidų kiekio augalų audiniuose. 2. Grūdinant ūglius in vitro egzogeniniai priedai (abscizo rūgštis, prolinas, tirtas aminorūgščių kompleksas) maitinamojoje terpėje didina atsparumą šalčiui. 3. In vitro sistemoje L-prolinas ir tirtas aminorūgščių kompleksas atstato atlydžio metu sumažėjusį užsigrūdinimo tvarumą. 4. Endogeninio prolino kiekis yra membranos stabilumo atsparumo šalčiui žymuo ir gali būti naudojamas kuriant atsparius šalčiui genotipus. / Aim of the work – to determine the effect of endogenous and exogenous factors on the cold tolerance of winter rapeseed in vitro and in vivo. Proposition to be defended: 1. Cold tolerance of winter rapeseed mainly determining by content of endogenous proline but not by soluble sugars. 2. Exogenous additives (abscisic acid, proline, amino acids) in nutrient medium increases the cold tolerance of rapeseed shoots in vitro. 3. In in vitro system the abscisic acid, proline and amino acids restore the reduced acclimation which occurs during de-acclimation period. 4. Endogenous proline content is the marker of membrane stability and cold tolerance and may be used to create cold-tolerant genotypes.

Many plants accumulate organic osmolytes in response to the imposition of environmental stresses that cause cellular dehydration. Of these, proline is the most extensively studied. Conclusive demonstration that this imino acid acts as a compatible solute which mediates osmotic adjustment has yet to be achieved, although a causal relationship between increased proline synthesis and plant tolerance of hyperosmotic stresses has previously been demonstrated. It is proposed that in many plants, the metabolic implications of the regulated increase in proline synthesis and/or a decline in proline degradation during stress may play a more important role in acclimation to adverse conditions than the simple accumulation of the end-product of these adjustments. In particular, the stress-induced increase in the transfer of reducing equivalents into proline by Δ¹-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) may be a protective mechanism whereby many species ameliorate shifts in cellular redox potential which accompany all biotic and abiotic stresses which cause proline accumulation, including those that do not cause cellular dehydration. The presence of several putative stress-regulated promoter elements in the AtP5CS1, AtP5CS2 and AtP5CR genes of Arabidopsis thaliana strongly implicates an adaptive role for stress-induced increases in proline synthesis in this species. Sequence homologies of several regions within the 5' untranslated regions of these genes to promoter elements which have been shown to participate in redox control of gene expression, the actions of phytochrome and hormones, and tissue-specific regulation of gene expression are also identified. These provide useful indicators both of the mechanisms by which proline synthesis is regulated and how these may relate to its importance in maintaining metabolic homeostasis. In an attempt to resolve the functionality of proline accumulation under stress, chimeric antisense genes comprising 1050 bp and 999 bp fragments of Arabidopsis cDNAs encoding AtP5CS1 and AtP5CR respectively were inserted in the reverse orientation between the CaMV 35S promoter and the GUS gene (encodes β-glucuronidase) in the plant transformation vector pBI121. These constructs were introduced separately into Arabidopsis by cocultivation with Agrobacterium tumefaciens strains carrying the pBI-P5CS1 (AS) and pBI-P5CR(AS) plasmids. Transgenic plants, which were selected on the basis of kanamycin resistance, regenerated at a low frequency in the presence of 1 mM proline. Transformation of 13 pBI-P5CS1(AS) and 7 pBI-P5CR(AS) lines was confirmed by PCR-mediated amplification of gene fragments within the introduced T-DNA. Segregation ratios for kanamycin resistance indicated that most of the lines have multiple T-DNA insertions. Transformants were characterised with respect to their growth rates and free proline content. In at least two pBI-P5CS1(AS) transformants and two pBI-P5CR(AS) transformants, a reduction in root growth rates in the presence of inhibitory concentrations of NaCI correlated with reduced β-glucuronidase activity relative to transgenic lines that were no more sensitive to NaCI than were controls. A reduction in root growth rate both in the absence and presence of hyperosmotic stress was noted in two pBI-P5CS1(AS) transformants, designated A5 and B12. In 14 day-old plants of the T₂ generation of both A5 and B12, free proline levels were significantly lower than in wild-type plants both in the absence of stress and following 24 h incubation in either 250 mM NaCI or 550 mM sorbitol or at 5 °C. In both lines, reduced growth rates in the absence of osmotic stress could be restored by exogenous proline, but not by exogenous glutamate. When used at isosmotic concentrations, sorbitol caused a larger reduction in free proline levels in both A5 and B12 than did NaCI. This observation may relate to an ABM-mediated post-transcriptional effect on AtP5CS1 gene expression which affects NaCI-, but not sorbitol-mediated proline accumulation in Arabidopsis. Post-transcriptional regulation of the expression of the genes involved in proline biosynthesis may account, at least partly, for the absence of dramatic phenotypic effects in any of the pBI-P5CS1(AS) or pBI-P5CR(AS) lines. Under the premise that regulation of shifts in proline metabolism regulate cellular redox potential under conditions of stress may be mirrored by the involvement of proline metabolism in modulating metabolism during normal growth and development, the effects of exogenous proline on Arabidopsis seed germination, seedling growth and in vitro shoot organogenesis were investigated. A dose-dependent inhibition of radicle emergence by millimolar concentrations of proline could be overcome by the artificial oxidants methylene blue and phenazine ethosulphate. Assays of the rate-limiting dehydrogenases of the oxidative pentose phosphate pathway (OPPP), as well as changes in the contributions of ¹⁴C₁ - and ¹⁴C₆ -labelled glucose to respired CO₂ during germination, are consistent with activation of the OPPP during Arabidopsis seed germination. An approximately four-fold increase in free proline, which peaked at the time of radical emergence, was not parallelled by changes in other amino acids and could not be ascribed to degradation of seed storage proteins. Delayed radical emergence in T₂ generation seeds of the pBI-P5CS1(AS) lines A5 and B12 correlated with an approximately 35% reduction in the maximal concentration of proline accumulated during germination. Millimolar concentrations of exogenous proline had a dose-dependent inhibitory effect on Arabidopsis seedling growth both in the light and in darkness. This reduction in growth arises at least in part from a decline in cell elongation. Accordingly, exogenous proline increased total extractable peroxidase activity in Arabidopsis seedlings through the selective induction of peroxidase isoforms. Histochemical analysis of the hypocotyls of plants grown in the presence of exogenous proline suggested that proline increased the levels of lignin and/or the phenolic precursors thereof. A dose-dependent decrease in extractable chlorophyll and damage to chloroplastic and mitochondrial ultrastructure was observed in 21 day-old Arabidopsis seedlings grown in the presence of millimolar concentrations of exogenous proline. In vitro shoot organogenesis from Arabidopsis hypocotyl explants was stimulated by 1 mM proline, and to a lesser extent by 5 mM proline, but inhibited by inclusion of 10 mM proline in the hormonallysupplemented regeneration media. The ability of low concentrations of proline analogues (azetidine-2-carboxylate and thioproline) to overcome the stimulatory effect of 1 mM proline, and a slight increase in the stimulative effects of 1 mM proline by D-proline, are consistent with an important role for the interconversions of proline and its precursors in regulating cell division and differentiation. Together, these data strongly support an important role for the interconversions of proline and its precursors in the regulation of intermediary metabolism under both normal and stressful conditions. These findings draw into question the widely accepted, although poorly investigated, hypothesis that proline is an inert compatible solute that can be accumulated to high levels with minimal effects on cellular metabolism. The novel proposal that stress-induced changes in proline metabolism exert a regulatory effect though an influence on the level of reduction of the cellular NADP pool is discussed in relation to recent evidence that a signal related to proline synthesis and/or degradation selectively increases the expression of stress-induced plant genes. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

In Arabidopsis thaliana (L.) Heyhn, the size of the pool of free proline increases up to 27-fold in response to osmotic stress. The magnitude of this accumulation is dependent upon the rate of imposition of the stress. Numerous reports have suggested a role for proline accumulation as a general adaptation to environmental stress. However, controversy surrounds the beneficial effect of proline accumulation in plants under adverse environmental conditions. Stress-induced proline accumulation in plants occurs mainly by de novo synthesis from glutamate. The final and only committed step of proline biosynthesis in plants is catalysed by Δ¹-pyrroline-5-carboxylate reductase (P5CR). The sequence of an incomplete 999 bp cDNA encoding P5CR from A. thaliana was determined. This enabled a preliminary molecular study of the structure and function of both the gene and the corresponding enzyme. The 999 bp cDNA insert in the clone Y AP057 was sequenced on the sense and antisense strands following subcloning of four sub-fragments in appropriate orientations. Comparison with known plant P5CR sequences revealed that Y AP057 does not encode the first 23 N-terminal amino acids of P5CR from Arabidopsis. However, it does encode the remaining 253 amino acid residues of Arabidopsis P5CR The cDNA Y AP057 is complete on the 3' end as indicated by the presence of a poly(A) tail. The nucleotide sequence determined shows complete homology to the corresponding exons of the genomic copy of a bona fide gene encoding P5CR in A. thaliana (Verbruggen et al, 1993). The only difference observed between the sequence of Y AP057 and that of a cDNA sequenced by these workers is that polyadenylation was initiated seven nucleotides earlier in Y AP057 than in the sequence of the published cDNA. Genomic Southern analysis suggests the presence of only a single copy of the gene encoding P5CR in Arabidopsis. Restriction mapping and sequencing the ends of another incomplete Arabidopsis P5CR cDNA clone FAFJ25 (664 bp) indicated that the regions sequenced were completely homologous to the corresponding portions of Y AP057. Analysis of codon usage in the Arabidopsis gene encoding P5CR revealed it to closely resemble the consensus pattern of codon usage in A. thaliana. This suggests that the gene is moderately. expressed. Expression of the gene encoding P5CR in Arabidopsis is not likely to be subject to translational control. Although P5CR from A. thaliana has a fairly high composition of hydrophobic amino acid residues, it does not possess any stretches of hydrophobic amino acids of sufficient length to act as membrane-spanning domains or to anchor the enzyme in a membrane. Neither does it contain an N- terminal leader sequence capable of directing it to either the plastid or mitochondrion. The enzyme therefore appears to be cytosolic. The nucleic acid and deduced amino acid sequences of Arabidopsis P5CR were compared with those from·eleven other organisms for which P5CR sequences are currently available. Except among the three different plants examined, P5CR sequences displayed less identity at the amino acid level than at the nucleotide level. The deduced amino acid sequence of Arabidopsis P5CR exhibits high similarity to the corresponding genes and amino acid sequences of P5CR from soybean and pea. Lower but significant similarity was observed to the amino acid sequences of P5CRs from human, Saccharomyces cerevisiae and the bacteria Escherichia coli, Pseudomonas aeruginosa, Thermus thermophilus, Mycobacterium leprae; Treponema pallidum and Methanobrevibacter smithii. Similarity was also observed to the translational product of a gene from Bacillus subtilis with high homology to the E. coli proC gene. However, construction of a phenogram indicating the relatedness of the various P5CR enzymes suggests that sequence analysis of this enzyme is not a good indicator of evolutionary relatedness of organisms from different biological kingdoms. Multiple alignment of the twelve known P5CR sequences indicated homology between the sequences across their entire lengths. Homology was particularly high in the C-terminal portions of the P5CRs studied. It is speculated that this region may be of importance in binding of the substrate Δ¹-pyrroline-S-carboxylate (P5C). Another region displaying high sequence conservation was found in the central portion of all P5CRs. All P5CRs studied, with the exception of PSCR from T. pallidum contained an N-terminal domain capable of binding a nicotinamide dinucleotide cofactor. Comparison of this region with consensus sequences for NADH and NADPH binding sites in proteins suggests that NADPH is the preferred reductant used by P5CRs from plants and human. In contrast, the N-terrninal domains of P5CRs from S. cerevisiae, M smithii, T. thermophilus and M leprae display greater similarity to a consensus NADH-binding site. The definite preference of plant P5CRs for NADPH in comparison with NADH suggests that P5CR may be involved in regulating the redox potential within plant cells and that this step in proline biosynthesis from glutamate may be of importance in overall metabolic regulation. Three amino acid residues are universally conserved in all P5CRs studied. All are found within blocks of high sequence similarity. These residues are likely to be of importance in the structure or catalytic mechanism of P5CR. A number of other residues are common to several of the enzymes examined. These may also be of importance in subsequent manipulation of Arabidopsis P5CR at the molecular level. Prediction of the putative secondary structures of A. thaliana, soybean, pea, human and E. coli indicated a high degree of similarity between the enzymes. This was particularly evident in the region of the putative P5C-binding domain. Considerable similarity exists in hydrophobicity profiles of P5CRs from these five organisms. Proline levels in reproductive organs of unstressed Arahidopsis plants were considerably higher than those in vegetative tissues. This suggests differential expression of enzymes involved in proline metabolism in these organs. In situ hybridisation studies indicated an increase in levels of mRNA transcripts encoding P5CR in stem tissues in response to water deprivation stress. Regulation of levels of mRNA transcript encoding P5CR in Arabidopsis therefore appears to be an osmotically sensitive process. Furthermore, this accumulation of transcript occurred in a tissue-specific manner. In particular, an increase in levels of transcript encoding P5CR was observed in the cortical parenchyma, phloem, vascular cambium and pith parenchyma in the vicinity of the protoxylem. The significance of these findings in contributing to a better understanding of the role of proline in adaptation to environmental stress is discussed. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.