(a) The Availability of Lysine Autoclaved in the Presence of a Carbohydrate. Heat treatment has long been known to improve the value of soybean oilmeal for chick growth. However, more recently it has been demonstrated that too much heat or too long a heating period lowers the availability of certain of the amino acids present. This lowered availability was accompanied by a brown coloration. Investigators had shown that such a brown coloration occurs in other materials as a result of a reaction between reducing sugars and amino acids. As a result this study was undertaken to determine whether a similar reaction was responsible for the darkening in colour of the soybean during heating and whether or not such a reaction affected the nutritive value of the meal. The results of the study showed that a decrease in nutritive value did accompany the browning of soybean oilmeal. It was also shown that this decrease was mainly due to a lowering of the available methionine. As the lysine gave the greatest growth response, the study was continued using crystalline lysine and cerelose. It was shown the reaction was affected by the time of heating, the ratio of lysine to carbohydrate present, and the presence of water. Assays were carried out both biologically and micro-biologically. (b) The Animal Protein Factor in Relation to the Nutrition of the Chick. A series of three experiments were carried out to determine, if possible, the value of Animal Protein Factor Concentrates in proactical chick rations when fed to chicks which were not depleted of the factor(s). Experiment I was set up using a basal ration which would correspond to a practical starter ration using wheat and a mixture of coarse grains as the cereals. The ration was fed with and without a source of animal protein and was supplemented at various levels with the A.P.F. produced by-Merck and Company. Experiment II was set up using a high energy type of diet containing corn and soybean such as is used for broiler production. Males and females were fed separately to determine if there was a separate response of cockerels and pullets to the A.P.F. Supplementation. Experiment III was set up again using a corn soybean ration. The purpose of this experiment was to compare the Animal Protein Factor Concentrates produced by Merck and Lederle respectively. From the results it appears that supplementation with A.P.F. concentrates is practical but that the results vary with the level and the type of diet used. Apparently there is an optimum level above which further supplementation exerts a depressing effect upon chick growth. This is also true where fishmeal is included in the diet. Lederle gave a greater response than did the Merck product both in growth and feed efficiency. / Land and Food Systems, Faculty of / Graduate

Experiments were conducted to study the effect of dietary lysine level on thyroid activity and body temperature in White Leghorn cockerel chicks. Thyroid activity was measured in birds 4-to-5 weeks of age and body temperature was measured in birds 4-to-8 weeks of age. Three diets based on corn and sesame meal and containing 20% protein and 0.64% (basal diet), 1% (control diet) or 2% of L-lysine respectively were used. These diets contained deficient, optimal and excessive amounts of L-lysine respectively. Thyroidal experiments were conducted at different times of the year. Thyroid activity was assessed by measuring the level of I¹³¹ in the plasma and thyroid or thyroid only at various times after an intravenous injection of 0.55 μc of I¹³¹ 100 gm of body weight. In August-September, thyroidal response to diet was investigated in birds fed the test diets for either 1 or 17 days prior to administration of I¹³¹. When the test diets were fed for 1 day prior to the injection of I¹³¹, data for per cent I¹³¹ per unit of thyroid weight and for the ratio of thyroidal to plasma I¹³¹ (T/P ratio) indicated that either a deficiency or an excess of dietary lysine enhanced thyroid activity. Feeding the test diets for 17 days appeared to attenuate the responses observed in the 1-day trial. In January, thyroidal response to diet was assessed in birds fed test diets either 1 day before or 3 days after the injection of I¹³¹ . Only the diets containing 0.64% and 1% of L-lysine were fed. When appropriate, 0.2% thiouracil was added to block thyroidal uptake of recycled I¹³¹. When all four diets were fed, I¹³¹ level per unit of thyroid weight and T/P ratio calculated from data obtained 5 minutes, 1 and 3 hours after I¹³¹ administration indicated that dietary lysine deficiency under the circumstances of the experiments reduced thyroidal uptake of the isotope. When I¹³¹ was injected 3 days prior to imposition of the test diets, the values for I¹³¹ per unit of thyroid weight after the feeding trials started indicated that the lysine deficiency reduced the rate of release of thyroidal I¹³¹. In a further experiment, levels of thyroidal I¹³¹ measured 5, 10, 24, 48, and 72 hours after I¹³¹ injection verified that the lysine deficiency reduced thyroid activity. In March, the effect of ambient temperature on thyroidal response to dietary lysine level was investigated. Thyroidal I¹³¹ was measured 6 hours after I¹³¹ administration to birds which had been fed the deficient diet for 1 day. Dietary lysine level and environmental temperature had an interacting effect on total thyroidal I¹³¹ and per cent I¹³¹ per 100 gm of body weight. Under the conditions of the experiment, lysine deficiency enhanced I¹³¹ uptake in a warm environment but has no effect or depressed activity in a cold environment. Temperatures were measured with thermistor probes inserted into the cloaca, the brain and the pectoral is muscle. When fed for either 18-to-30 hours or for 2 weeks, the lysine deficient diet increased muscle temperature. The diet had no apparent effect on brain or cloacal temperature but detection of a response may have been obviated by the technique used to measure temperatures in these two locations. Feeding sub-optimal or excessive levels of dietary lysine for only 1 day caused a change in thyroid activity. In a warm environment, both sub-optimal and excessive levels of lysine enhanced thyroid activity. Environmental temperature appeared to modify the thyroidal response to dietary lysine level. In a warm environment, a deficiency of lysine increased muscle temperature. / Land and Food Systems, Faculty of / Graduate

The purpose of this study was to evaluate the Lys and Thr responses of TP-16 male broilers. Experiment 1 (14-28 d of age) showed no significant live performance effects. In Experiment 2, Lys and Thr (P=0.021) interacted to affect 28 to 42 d body weight gain and (P=0.004) feed intake. Lys main effects improved body weight gain (P=0.002), and feed conversion (P=0.009). Thr fed at the 0.68% level improved body weight gain and feed conversion. Mortality did not differ among treatments and averaged 0.09% across all treatments. The study indicated the sensitivity of the Thr:Lys ratio when broilers are fed diets containing marginal Lys. Although Thr X Lys interactions occurred in this study, the Lys levels in the test diets may have been too high for determining a ratio to an amino acid in a factorial study. Interaction results indicate that the Thr:Lys ratio in broilers from 28 to 42 days of age is between 0.57 to 0.68.

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of L-lysine and meso-diaminopimelate biosynthesis, which is the condensation of (S)-aspartate-β-semialdehyde (ASA) and pyruvate into dihydrodipicolinate via an unstable heterocyclic intermediate, (4S)-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. DHDPS has been an attractive antibiotic target because L-lysine and meso-diaminopimelate are cross-linking components between peptidoglycan heteropolysaccharide chains in bacterial cell walls. Studies revealed that mutant auxotrophs for diaminopimelate undergo lysis in the absence of diaminopimelate in the medium; therefore the assumption is that strong inhibition of DHDPS would result in disruption of meso-diaminopimelate and L-lysine biosynthesis in bacteria and would stop or decrease bacterial growth (eventually leading to bacterial death). In this work, the DHDPS inhibitor design is focused on the allosteric site of the enzyme. It was proposed that a compound mimicking binding of two L-lysine molecules at the allosteric site at the enzyme’s dimer-dimer interface would be a more potent inhibitor than the natural allosteric inhibitor of this enzyme, L-lysine. This inhibitor (R,R-bislysine) was synthesized as a racemic mixture, which was then separated with the aid of chiral HPLC. The mechanism of feedback inhibition of DHDPS from Campylobacter jejuni with its natural allosteric modulator, L-lysine, and its synthetic mimic, R,R-bislysine, is studied in detail. It is found that L-lysine is a partial uncompetitive inhibitor with respect to pyruvate and a partial mixed inhibitor with respect to ASA. R,R-bislysine is a mixed partial inhibitor with respect to pyruvate and a noncompetitive partial inhibitor with respect to ASA, with an inhibition constant of 200 nM. Kinetic evaluation of each DHDPS mutants (Y110F, H56A, H56N, H59A and H59N) has revealed amino acids responsible for the inhibitory effect of L-lysine, R,R-bislysine, and we have found that R,R-bislysine is a strong submicromolar inhibitor of Y110F, H56A, H56N and H59N.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains viii, 89 p. : ill. Includes abstract. Includes bibliographical references (p. 67-73).

Thesis (M.S.)--West Virginia University, 2000. / Title from document title page. Document formatted into pages; contains viii, 92 p. : ill. Vita. Includes abstract. Includes bibliographical references.

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Call number: LD2668 .T4 1978 F58 / Master of Science

Histone lysine demethylases comprise an important family of epigenetic regulatory enzymes. They catalyse the demethylation of tri-, di- and monomethylated lysine residues on histone H3, thus contributing to either silencing or activation of chromatin. Their biological roles are widespread and have just begun to be elucidated. Among other functions, they contribute to establishment and maintenance of pluripotent states in embryonic stem cells, and also to cellular differentiation during development. Abnormal expression or mutation of some demethylases has been linked to diverse diseases, from prostate and oesophageal cancers to X-linked mental retardation. The development of small molecule inhibitors of histone demethylases is therefore of interest, both from the therapeutic perspective, and with the aim of developing chemical probes to understand the diverse functions of the demethylases in vivo. Most histone lysine demethylases belong to the 2-oxoglutarate and ferrous iron dependent dioxygenase superfamily. This family utilises molecular oxygen to catalyse hydroxylation of substrates, together with oxidation/decarboxylation of the 2-oxoglutarate cofactor. In the work outlined in this thesis, the JMJD2 family of histone demethylases was characterised biochemically, with attention given to mechanism, substrate selectivity and the role of eo factors. JMJD2E was identified herein as a novel histone demethylase in H. sapiens, and was shown to be selective for the demethylation of tri-, di- and monomethylated lysine 9 in histone H3. JMJD2E was also found to be particularly amenable to mechanistic and inhibition studies in vitro. A variety of mechanistic investigations established details of the catalytic cycle, its substrate selectivity and the role of iron and ascorbic acid as cofactors. Crystallographic analyses were also employed to compare its substrate selectivity to other JMJD2 family members. Assays suitable for the evaluation of inhibitors of the JMJD2 demethylases were then developed. These included a coupled enzyme assay suitable for kinetic measurements, and two mass spectrometric assays for observing inhibitor binding and catalytic activity. A critical review of the 20G oxygenase inhibitor literature carried out, and was then used as a basis for the identification of inhibitor scaffolds for the JMJD2 demethylases. These were characterised both in vitro (using kinetic assays, mass spectrometry and crystallography), and in cell culture. Some were further developed to achieve selective inhibition of the JMJD2 demethylases over the related prolyl hydroxylase PHD2; crystallography was again employed to understand the mode of inhibition of these potent inhibitors. The kinetic assays developed were optimised for use in a high-throughput screen, and a library of 240 000 compounds was screened against JMJD2E. This was the first instance of high-throughput screening against these promising therapeutic targets. Several hit compounds were identified and characterised further in vitro. Finally, alternative means of inhibiting the JMJD2 demethylases were investigated. Compounds were identified that inhibited JMJD2A by ejection of its unique structural zinc ion, thus demonstrating that selective inhibition of the JMJD2 demethylase family is possible. In summary, this work contains the first detailed investigation of a histone demethylase subfamily, and also the first steps towards identifying potent, selective inhibitors of these epigenetic regulatory enzymes.