The kynurenine pathway produces NAD+ from L-tryptophan. Metabolites known as the kynurenines are produced within the pathway. The effects of the kynurenines have been associated with a number of diseases including cancer, Alzheimer’s disease, Huntington’s disease, and acute pancreatitis. Kynurenine monooxygenase (KMO) is an enzyme that catalyses the conversion of L-kynurenine to 3-hydroxy-L-kynurenine, the downstream product of which is the neurotoxic quinolinic acid. L-kynurenine is positioned at a branching point within the pathway. Metabolism via KMO leads to quinolinic acid production whereas conversion via kynurenine aminotransferase (KAT) produces the neuroprotective kynurenic acid. Inhibition of KMO leads to an increase in kynurenic acid concentration. This has also been shown to ameliorate the symptoms of neurological diseases in a number of animal models as well as to protect against multiple organ dysfunction caused by acute pancreatitis in rodent models. These findings present KMO as a promising drug target. Due to the hydrophobic nature of human KMO (hKMO) it has been necessary to utilise other forms of KMO as models. Past studies have produced crystal structures of a truncated Saccharomyces cerevisiae KMO and of Pseudomonas fluorescens KMO (PfKMO). Previous work in this research group has resulted in the structure of variants of PfKMO bound to either inhibitor molecules or substrate. These structures identified residues involved in substrate binding and the presence of a highly mobile section of the C-terminus, giving rise to open and closed conformations. It was surmised the movement of the C-terminus was dependent upon the presence of substrate and an interactive network between the C-terminus and the rest of the protein. Using improved crystallising conditions high-resolution structures of PfKMO have been produced that allow for further study of residues involved in substrate binding and the interactive network within the C-terminus. The mutants R84K and Y404F showed severely decreased enzyme activity. Crystal structures of these proteins showed disrupted interactions between substrate and active site. These findings underline the importance of residues R84 and Y404 in substrate binding. An H320F mutation gives an analogous active site to hKMO. Crystallographic and kinetic study of this mutant proved very similar to PfKMO, supporting the use of PfKMO as a model for hKMO. Throughout the work each structure had a P21221 space group with two molecules in the asymmetric unit. The presence of an open and closed molecule within each structure, including substrate-free molecules refuted the connection between C-terminus and substrate. R386K and E372T mutations were separately introduced in order to interrupt the interactive network. The presence of both open and closed conformations in the structures of R386K and E372T refutes the necessity for the interactive network in C-terminus movement. The data analysed throughout the project suggest simple mobility and thermal motion as the cause of the movement of the C-terminus. This work, in conjunction with kinetic data from the thesis of Helen Bell, presents structural data to characterise the role of binding residues within the active site of KMO as well as the mechanistic role of the C-terminus. It also highlights the importance of certain binding residues and countered the previously held hypotheses surrounding the significance of the C-terminus. The mechanistic role of the C-terminus therefore remains unclear and requires further study.

Made available in DSpace on 2016-12-08T16:24:18Z (GMT). No. of bitstreams: 1 PGCA14MA140.pdf: 91454 bytes, checksum: d7f6d38c8c463559f1c52096a57acd9b (MD5) Previous issue date: 2014-02-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / We describe the epidemiological, clinical signs and lesions of four outbreaks of acute pulmonary edema and emphysema in cattle (EEPAB) in the states of Santa Catarina and Paraná and its experimental reproduction of disease. Spontaneous disease occurred after transfer of mature cattle grazing and dry for another young and flourishing. All affected cattle were cows of the Dutch and Swiss brown races. The main clinical signs were dyspnea , labored abdominal breathing with the neck extended and mouth open and some had subcutaneous emphysema , besides the decrease in milk production and slow recovery or death . Necropsy findings were restricted to the lung which had dark red color, not collapsed, glossy and hipercriptante with marked interlobular emphysema. Histological lesions in the lung consisted mainly of alveolar and interlobular emphysema interspersed with areas of congestion and edema, hyaline degeneration of the wall of alveoli and infiltration of macrophages and eosinophils , moderate , diffuse . The experimental reproduction of the disease was performed in a beef with administration of 0.7 mg / kg / BW of L - Tryptophan orally in a single dose. The animal died on the seventh day of the experiment. The clinical signs and lesions were similar to those observed in naturally occurring disease / Descrevem-se os dados epidemiológicos, sinais clínicos e lesões de quatro surtos da doença do edema e enfisema pulmonar agudo de bovinos (EEPAB) nos estados de Santa Catarina e Paraná e sua reprodução experimental. A doença espontânea ocorreu após transferência de bovinos de pastagem madura e seca para outra jovem e viçosa. Todos os bovinos afetados eram vacas das raças holandês e pardo suíço. Os principais sinais clínicos foram dispnéia, respiração abdominal dificultosa com o pescoço estendido e boca aberta e alguns apresentavam enfisema subcutâneo, além de queda na produção de leite e morte ou recuperação lenta. Os achados de necropsia foram restritos ao pulmão o qual tinha coloração vermelho escuro, não colabado, de aspecto brilhante e hipercriptante com enfisema interlobular acentuado. As lesões histológicas no pulmão consistiam principalmente de enfisema alveolar e interlobular intercalado por áreas de congestão e edema, degeneração hialina da parede de alvéolos e infiltrado de macrófagos e eosinófilos, moderado, difuso. A reprodução experimental da doença foi realizada em um bovino, com administração de 0,7mg/kg/PV de L-Triptofano por via oral em dose única. O animal morreu no sétimo dia de experimento. Os sinais clínicos e lesões foram idênticos aos observados na doença espontânea

No description available.

<p>Two experimentswere  conducted  using  pigs  at different  life stages to  determine  the  effects  of dietary  tryptophan  and  water  delivered  oregano  essential  oil  on growth performance, rectal temperature, water use,intestinal integrity and gene expression of biomarkers in the face heat or transport stress. In the first experiment, 192 grow-finish pigs were used to investigate the effects of  water  supplementation  of  oregano  essential  oil  (OEO)  on  growth  performance,  water  intake, rectal temperature, intestinal integrity, and expression of genetic biomarkers during an acute heat challenge. Pigs were randomly allotted to 2 X 2 factorial arrangement of treatments with pigs being heat  stressed  or  not  and  being  supplemented  with  OEO  or  not with 8  replicate  pens  of  each treatment  with  6  pigs/pen  (4  barrows,  2  gilts  per  pen).  Water  treatments  were administered immediately, with  dosing at  47 μL/L of OEO. One-half  of  the  pigs  on  each  water  treatment remained under thermoneutral conditions (TN; 21.1C), while the other half was subjected to a 3 d diurnal, acute heat stress (HS) with 12 hours at 33.3 oC (7AM-7PM) and 12 hours at 26.7oC (7PM-7AM). Three days post-HS, temperatures were reduced back to TN for the rest of the study, and pigs remained on their water treatments. Rectal temperatures were collected in the morning and evening  of  the  heat  stress  period  on  one  barrow  and  one  gilt  in  each  pen. Jejunal  tissue was collected for subsequent histological examination and determination of gene expression. All data were  analyzed  using  the  GLM  procedure  of  SAS  (ver.  9.4).  Pigs  subjected  to  heat  stress  had reduced ADG (P < 0.003) and G:F (P < 0.008) during the 3d heat stress compared to pigs reared under thermoneutral conditions. However, post-heatstress, heat stressed pigs had compensatory gain resulting in increased ADG (P < 0.001) and G:F (P < 0.001) compared to thermoneutral reared pigs.   Overall,  there  was an  interaction  (P  <  0.006)  observed  between  water  and  heat  treatment with  OEO  increasing  ADG  in  thermoneutral  pigs  but  not  in  heat  stressed  pigs.  Similarly, interactions  between  water  and  heat  treatment  were  observed  for  ADFI  during  heat  stress  (P  < 0.004),  post  heat  stress  (P  <  0.01),  and  overall  (P  <  0.004)  from  increasing  OEO  intake  in thermoneutral pigs but not in heat stressed pigs. Rectal temperatures were higher (P < 0.001) for heat stressed pigs at the end of d 1 and 2 of the acute heat challenge compared to TN housed pigs. Pigs exposed to HS also used more water than pigs housed in a thermoneutral environment (P < 0.002). There were no differences between villi height, crypt depth or VH:CD between treatment groups  (P  >0.05).  There  was  also  no  difference  in  TP53  and  CDKNA1  gene  expression  among treatments (P > 0.10). In the second experiment, 36 barrows were used in an 18d experiment to investigate the effects of pre-weaning tryptophan supplementation on performance and intestinal integrity following  weaning  with  or  without  transport  stress  at  weaning.  Pigs  were  randomly allotted to 2 X 2 factorial arrangement of treatments of pre-weaning tryptophan supplementation or not and weaning transport or not. Pigs on the tryptophan treatment received 0.35, 0.45, and 0.55 g Trp/d in 5 day intervals, beginning 15 d prior to weaning.Tryptophan was dissolved in chocolate milk and administered by oral gavage with control pigs receiving milk only. At weaning, 4 pigs from each pre-weaning treatmentwere euthanized for collection of jejunal tissue.  Of the remaining pigs, half the pigs oneach treatment were transported for 12 h, and half were moved into individual pens  with  no  transport.  Following  transport,  all  pigs  were  individually  housed  and  provided  ad libitum  access  towater  andfeed  from  a  common  diet.  On  d  3  post-weaning,  all  pigswere euthanized for collection of jejunal tissue. Jejunal tissue was used for histological examination and for determination of gene expression. All data were analyzed using the GLM procedure of SAS (9.4).  No  effects  of  Trp  supplementation  were  observed  on  pre-weaning  (P  >  0.10)  growth. Pig BW and ADFI were unaffected (P > 0.10) by Trp supplementation and transport at weaning. Post-weaning, there was a tendency (P < 0.06) for an effect of transport on ADG as transported pigs lost weight in the 3 d post-weaning period while non-transported pigs gained slightly. Gain:Feed post-weaning  was  lower  (P  <  0.04)  for  transported  pigs  compared  to  non-transported  pigs.  No differences  were  observed  for villus base  and  mid  width,  villus  height,  crypt  depth  or  villus height:crypt   depth.   There   was   a   tendency   for   an   interaction   of   transportation   and   Trp supplementation  (P  <  0.06)  on  villi  base  width  driven  by  an  increased  villus  width  in  non-transported pigs given supplemental Trp but a decrease in villus width in transported pigs given supplemental Trp.These results conclude that these alleviating agents had minimal effects when pigs  were  stressed, however TN grow-finish pigs  benefitted  from  OEO  water  supplementation among growth performance.</p>

No

No / Vitiligo is characterized by a mostly progressive loss of the inherited skin color. The cause of the disease is still unknown, despite accumulating in vivo and in vitro evidence of massive oxidative stress via hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) in the skin of affected individuals. The most favored hypothesis is based on autoimmune mechanisms. Since depletion of the essential amino acid l-tryptophan (Trp) severely affects various immune responses, we here looked at Trp metabolism and signaling in these patients. Our in vivo and in vitro data revealed total absence of epidermal Trp hydroxylase activities and the presence of H2O2/ONOO− deactivated indoleamine 2,3-dioxygenase. Aryl hydrocarbon receptor signaling is severely impaired despite the ligand (Trp dimer) being formed, as shown by mass spectrometry. Loss of this signal is supported by the absence of downstream signals (COX-2 and CYP1A1) as well as regulatory T-lymphocytes and by computer modeling. In vivo Fourier transform Raman spectroscopy confirmed the presence of Trp metabolites together with H2O2 supporting deprivation of the epidermal Trp pool by Fenton chemistry. Taken together, our data support a long-expressed role for in loco redox balance and a distinct immune response. These insights could open novel treatment strategies for this disease.—Schallreuter, K. U., Salem, M. A. E. L., Gibbons, N. C. J., Maitland, D. J., Marsch, E., Elwary, S., Healey, A. R. Blunted epidermal l-tryptophan metabolism in vitiligo affects immune response and ROS scavenging by Fenton chemistry, part 2: epidermal H2O2/ONOO−-mediated stress in vitiligo hampers indoleamine 2,3-dioxygenase and aryl hydrocarbon receptor-mediated immune response signaling.

No / Vitiligo is characterized by a progressive loss of inherited skin color. The cause of the disease is still unknown. To date, there is accumulating in vivo and in vitro evidence for massive oxidative stress via hydrogen peroxide (H2O2) and peroxynitrite (ONOO−) in the skin of affected individuals. Autoimmune etiology is the favored theory. Since depletion of the essential amino acid l-tryptophan (Trp) affects immune response mechanisms, we here looked at epidermal Trp metabolism via tryptophan hydroxylase (TPH) with its downstream cascade, including serotonin and melatonin. Our in situ immunofluorescence and Western blot data reveal significantly lower TPH1 expression in patients with vitiligo. Expression is also low in melanocytes and keratinocytes under in vitro conditions. Although in vivo Fourier transform-Raman spectroscopy proves the presence of 5-hydroxytryptophan, epidermal TPH activity is completely absent. Regulation of TPH via microphthalmia-associated transcription factor and l-type calcium channels is severely affected. Moreover, dopa decarboxylase (DDC) expression is significantly lower, in association with decreased serotonin and melatonin levels. Computer simulation supports H2O2/ONOO−-mediated oxidation/nitration of TPH1 and DDC, affecting, in turn, enzyme functionality. Taken together, our data point to depletion of epidermal Trp by Fenton chemistry and exclude melatonin as a relevant contributor to epidermal redox balance and immune response in vitiligo.

<p>  </p> <p>In commercial settings, piglet weaning and transportation occur concurrently due to the expansive application of multi-site production systems across the United States and the combination of these events can be defined as an early life stressor. Early life stress is known to reduce pig performance, efficiency, and immune resilience contributing to reduced welfare and increased production losses. To combat the deleterious effects of stress on pigs, the essential amino acid tryptophan (Trp) supplemented above current National Research Council (NRC 2012) recommended levels, improves neuroendocrine responses to stress, lowers plasma cortisol and norepinephrine concentrations, and improves hypothalamic-pituitary-adrenal axis recovery time following stress. However, there are discrepancies concerning the Trp requirement for nursery pigs, suggesting the 2012 recommendations for Trp may be inaccurate for optimizing growth performance and health in modern pigs. Three experiments were conducted to determine the effects of supplemented standard ileal digestible Trp above NRC (2012) recommended levels on performance, feed efficiency, immune vigor, and stress tolerance. The objective of experiment 1 was to eliminate or reduce short- and long-term, transport-induced reductions in piglet feed efficiency and growth by supplementing Trp above NRC (2012) recommendations pre-weaning and/or during the nursery phase. An oral gavage of Trp or a control milk carrier was provided to pre-allotted piglets beginning at day 5 of lactation and continuing to weaning. At weaning all pigs were blocked by sex, weaning weight and pre-wean treatment and randomly assigned to transport and post-wean treatments. Pigs were fed in four nursery phases with diets containing 1X or 2X NRC recommended concentrations of SID Trp and a common grower diet fed in 6 phases during the grow-finish period. At market, loin characteristics were measured via ultrasound and carcass data was collected from the packing plant. Pre-weaning Trp supplementation had no effect on pre-weaning growth performance; however, post-wean Trp increased overall body weight and average daily gain in nursery pigs when Trp was supplemented pre-wean. The objective for experiment 2 was to mitigate the adverse effects of early life transport stress on subsequent immune challenges by providing supplemental Trp during the nursery period. At weaning, pigs were transported for 8 hours and assigned to treatments of vaccine-induced immune challenge and dietary treatment. Pigs were fed standard nursery diets, in four phases, over 35 days with pigs receiving 1X or 2X the NRC (2012) recommended Trp concentration. Half the pigs on each dietary treatment were subjected to a 3-wk vaccine challenge consisting of circovirus, mycoplasma, and influenza vaccines administered in wk 2, 3, and 4 post-wean, respectively. At market, loin characteristics were measured via ultrasound and carcass data was collected.  At the culmination of the nursery period, unchallenged pigs supplemented with Trp were heavier compared to control pigs. This resulted from an overall improvement in average daily gain for 2X Trp fed pigs. However, increasing Trp did not affect market weight, loin eye area, or lean percent in market pigs, although 2X Trp increased back fat. The objective of experiment 3 was to determine what ratio of SID tryptophan, relative to lysine, maximizes growth performance and feed efficiency in weaned pigs during the nursery period. Pigs were blocked by sex and weaning weight and randomly allotted to one of five dietary treatments with Trp increasing stepwise by 0.33%-units, respectively. Pigs were fed standard nursery diets, in four phases, over 35 days with pigs receiving 1X, 1.33X, 1.66X, 2X, or 2.33X the NRC (2012) recommended Trp concentration.  Overall, there were no differences in performance or efficiency across dietary treatments during a 35-day nursery period in unstressed healthy pigs.  In conclusion, increasing Trp in swine diets prior to and/or following stress events can mitigate stress-related perturbations in performance and efficiency.      </p>

No description available.

The present thesis deals with different aspects of the chemistry and photo-biology of various ferrocene-conjugated metal complexes, their interaction with double helical DNA, DNA photocleavage and photo-enhanced cytotoxicity in visible light. Phenyl analogues of the active complexes have been synthesized and used for comparison in biological assays. Chapter I provides an introduction to the potential of metal complexes as photochemotherapeutic agents with special reference to organometallic compounds. A brief overview of Photodynamic Therapy (PDT) as a new modality of cancer treatment has been given. Various modes of non-covalent interactions of small molecules with duplex DNA are mentioned. Recent reports on the metal-based photocytotoxic and DNA cleaving agents including photoactivatable organometallic compounds are discussed. The objective of the present investigation is also presented in this chapter. Chapter II presents the synthesis, characterization, structure, DNA binding, DNA photocleavage, photocytotoxicity, mechanism of cell death and cellular localization of ferrocene-conjugated L-methionine reduced Schiff base Cu(II) complexes of phenanthroline bases. To explore the role of the ferrocenyl moiety the phenyl analogues of the ferrocenyl complexes are synthesized and used as controls for comparison purpose. Chapter III deals with the photo-induced DNA cleavage and photo-enhanced cytotoxicity of ferrocene-appended L-tryptophan Cu(II) complexes of heterocyclic bases. The synthesis, characterization, structural comparisons, DNA binding, DNA photocleavage, photocytotoxic activity and cell death mechanism in visible light are discussed in detail. Chapter IV describes the synthesis, characterization and structure of ferrocenylmethyl-L-tyrosine Cu(II) complexes of phenanthroline bases. The complexes are evaluated for DNA binding, DNA photocleavage and photocytotoxic activity in visible light. The cellular localization of the complexes and the mechanism of cell death induced by the complexes are also discussed. Chapter V presents the photocytotoxic effect of ferrocene-conjugated L-amino acid reduced Schiff base Cu(II) complexes of anthracenyl/pyrenyl imidazophenanthroline. The ability of the complexes to bind to double helical DNA and cleave it under photo-illumination conditions is described. Evaluation of the complexes as photochemotherapeutic agents and comparison with currently clinically available drug Photofrin are presented. The mechanism of cancer cell death and cellular localization of the complexes are studied by fluorescence microscopy. Chapter VI describes the synthesis, characterization and photochemotherapeutic efficacy of Cu(II) complexes having ferrocene-appended L-amino acid reduced Schiff base ligands and the naturally occurring polyphenol curcumin. Stabilization of curcumin by complexation to metal for improved photodynamic effect in cancer cells is described with comparison to the parent dye and clinically used drug Photofrin. The mechanism of cell death induced by the copper complexes and their localization in cancer cells are also presented. Finally, the summary of the dissertation and conclusions drawn from the present investigations are presented. The references in the text have been indicated as superscript numbers and compiled at the end of each chapter. The complexes presented in this thesis are represented by bold-faced numbers. Crystallographic data of the structurally characterized complexes are given in CIF format in the enclosed CD (Appendix-I). Due acknowledgements have been made wherever the work described is based on the findings of other investigators. Any unintentional omission that might have happened due to oversight or mistake is regretted.