The benefits of fortification of poor quality food proteins such as wheat gluten with limiting amino acids depend on the biological availability of the added amino acids and their stability with respect to processing and storage. Although simple addition of amino acids in free form is convenient, the potential improvement in nutritional quality by this method of fortification may not materialize due to possible losses during processing steps such as washing, susceptibility to degradative reactions, and different rates of absorption and utilization compared to protein-bound amino acids. In this study, covalent attachment of lysine and threonine to wheat gluten was investigated using the chemical carbodiimide reaction and the enzymatic plastein reaction. The nutritional quality of enriched products was evaluated by in vitro and microbiological tests, and susceptibility to destruction by heating in the presence of a reducing sugar was investigated. Covalent attachment of lysine ethyl ester or threonine to gluten by the plastein reaction utilizing the enzyme papain was not successful. Although lysine and threonine contents in the products were increased, these results were attributed to selective enzymatic release of other amino acids. The amino acid compositions of undialyzable "plastein" products were markedly different from the original gluten substrate, and product yields were low. The results suggest the formation of many dialyzable peptides and free amino acids, and inability for protein re-synthesis from these low molecular weight compounds. Covalent lysine and threonine contents were increased using the carbodiimide reaction. In general, the reaction was most influenced by pH, reactant concentration and type of reactant. Products enriched via primarily peptide bonds as well as products enriched via peptide and isopeptide bonds could be prepared using various starting materials. Lysine, N -acetyl lysine, N -benzylidene lysine and threonine were coupled through amide bond formation to gluten, sodium stearate-solubilized gluten, acid-solubilized gluten or pepsin-solubilized gluten. Sodium stearate solubilization did not improve extent of amino acid incorporation. Pepsin hydrolysis of gluten enhanced amino acid attachment but decreased product yields. 4.0-fold and 6.5-fold increases in lysine content resulted by reaction of pepsin-solubilized gluten with N e-benzylidene lysine and N-acetyl lysine, respectively. However, yields of these products were low (47% and 58%). 1.6-fold, 2.0-fold and 2.5-fold increases were obtained by reaction of gluten with Ne-benzylidene lysine, lysine and N£-acetyl lysine, respectively, with product yields of 90 to 95%. At least 20-fold and 5-fold increases could be achieved by reaction of 0.5N HCI and 0.05N HCI solubilized glutens respectively with lysine, with product yields of 80 to 90%. 4-fold and 2-fold increases in threonine content resulted from reaction of threonine with pepsin-solubilized gluten and gluten, respectively. Simultaneous attachment of lysine or lysine derivative and threonine was not effective. In vitro evaluation of availability and digestibility of covalently enriched products was carried out by the DNBS reaction and pepsin pancreatin digestion tests. The results indicate the formation of isopeptide bonds involving the £-amino group of lysine unless N -substituents of lysine were used. Isopeptide bonds involving the Y_carboxyl groups were indicated when gluten had been solubilized by acid treatment. Peptide bond formation predominated when N -benzylidene or N -acetyl lysine was attached to pepsin-solubilized gluten or gluten. The high hi vitro availability and digestibility values for N £-benzylidene lysine enriched products suggest lability of the Schiff's base linkage of this N £-substituent, in contrast to the stability of the amide linkage in Ne -acetyl lysine. Microbiological evaluation by a Tetrahymena bioassay confirmed the nutritional improvement of gluten by covalent attachment of lysine, Ne-acetyl lysine or Ne-benzylidene lysine. Relative nutritive value of gluten was 54, whereas covalently and freely enriched glutens had relative nutritive values similar to that of the reference casein, assigned a value of 100. Covalently and freely lysine enriched glutens were compared for color and extent of lysine destruction after baking. In general, covalently enriched products had lighter color and higher percentages of total, DNBS-available and pepsin-pancreatin-digestible lysine contents than freely enriched products. N e-Benzylidene lysine enriched gluten was particularly stable, with relative nutritive value of 88 compared to 44 for baked gluten. It is concluded that covalently attached lysine is more stable than free lysine for enrichment of food proteins susceptible to Maillard reaction. / Land and Food Systems, Faculty of / Graduate

A regulação dependente de Ca2+ da atividade ATPásica da acto-miosina em concentrações fisiológicas de actina, tropomiosina e troponina ocorre exclusivamente na presença de troponina T (TnT). Nosso grupo demonstrou que um polipeptídeo correspondente aos primeiros 191 aminoácidos da TnT ativa a atividade ATPásica da acto-miosina na presença de tropomiosina e na ausência das outras duas subunidades do complexo troponina (TnI/TnC). Com o objetivo de mapear e caracterizar esse domínio ativatório da TnT, construímos fragmentos de TnT correspondentes às regiões compreendidas entre os resíduos de aminoácidos: 1-157 (TnTl-157), 1-76 (TnTl-76), 77-157 (TnT77-57), 77-191 (TnT77-191) e 158-191 (TnT158-191). Estudos das interações desses fragmentos com actina e tropomiosina demonstraram que: i) o fragmento TnTl-76 não se liga à tropomiosina ou a actina; ii) a região da TnT correspondente aos resíduos 158-191 liga-se à actina cooperativamente, mas não se liga à tropomiosina; iii) a região correspondente seqüência de aminoácidos 77-157 é necessária para a interação da TnT com o resíduo de aminoácido 263 da tropomiosina; iv) TnT77-191 ativa a atividade ATPásica da acto-miosina com a mesma intensidade que TnTl-191. Também observamos que TnTl-157, TnTl-76, TnT77-157, TnT158-91 e combinações de TnT158-191 com TnTl-157 e TnT77-157 não afetam a atividade ATPásica da acto-miosina. Concluímos que a região da TnT delimitada pelos aminoácidos 77 e 191 é essencial para a ativação da atividade ATPásica da actomiosina e que essa ativação é mediada pelas interações dessa região da TnT com a tropomiosina e a actina. / The Ca2+-regulation of the actomyosin ATPase activity at physiological ratios of actin, tropomyosin and troponin occurs only in the presence of troponin T. Our group has previously demonstrated that a recombinant polypeptide corresponding to the first 191 amino acids of TnT (TnTl-191) activates the aetomyosin Mg2+-ATPase activity in the presence of tropomyosin and in the absence of TnI/TnC. In order to further map and characterize this activation domain, we constructed a set of recombinant or synthetic TnT fragments, corresponding to amino acids 1-157 (TnTl-157), 1-76 (TnTl-76), 77-57 (TnT77-157), 77-191 (TnT77-191) and 158-191 (TnT158-191). Binding assays using these fragments demonstrated that: i) amino acids 1-76 of TnT do not bind to tropomyosin or actin; ii) amino acids 158-191 bind to actin cooperatively, but not to tropomyosin; iii) the sequence 77-157 is necessary for TnT\'s interaction with residue 263 of tropomyosin; iv) TnT77-191 on its own activates de actomyosin ATPase activity to the same extent as previously described for TnTl-191. TnT1-157; TnTl-76; TnT77-157; TnT158-191 and combinations of TnT158-191 with TnTl-157 or TnT77-157 showed no effect on the ATPase activity. We conclude that interactions of amino acids 77-191 of TnT with tropomyosin and actin are essential for the activation of actomyosin ATPase activity, and that this activation may be mediated in part by a direct interaction between TnT residues 158-191 and actin.

Murine Zfp106 is an 1,888 amino acid protein with two N-terminal and two C-terminal zinc finger domains with a beta-propeller upstream of the C-terminal zinc fingers. The transcription of the protein was found to be controlled through two promoter regions, leading to two isoform families, P1 and P2 Zfp106. The splice variants from each promoter are thought to have distinct starting exons and n-terminal regions. However, the isoforms are not well studied. Since its identification, Zfp106 has been implicated in RNA metabolism, transcription control, immune response, and muscle and testis development. It has been found to be capable of binding C9ORF72 repeats as well as being associated with TDP43 and FUS. However, its function is unknown. The aim of this study is to understand the role of Zfp106 in vivo through the use and development of various mouse models targeting exons specific to either the P1 or P2 family of isoforms. To begin with, we studied the Zfp106LacZ mouse model whose homozygous mice showed severe muscle atrophy beginning at 4 weeks leading to a premature death by 16 weeks. Research has supported the theory that the muscle atrophy is due to a motor neuron dysfunction potentially stemming from perturbed mitochondrial and spliceosome function. We, along with other researchers, found that this mouse model is not a complete disruption of Zfp106 through qPCR and RNAseq. We then found that this mouse model is an effective depletion of Zfp106 exon 2 and 3 which are exclusive to the P1 Zfp106 isoform family. Additionally, the Zfp106LacZ mouse model has an increased amount of the 1b exon associated with P2-Zfp106 in the skeletal muscle. Next, we established a CRISPR mouse line (ΔZfp106) targeting an exon common to the full-length splice variants of both the P1 and P2 family of isoforms, exon 5. This was in an attempt to dissect whether or not the muscle atrophy in the Zfp106LacZ mice was due to the interruption of exons 2 and 3 or from the increase in the P2 Zfp106 isoforms. Motor neurons derived from homozygous ΔZfp106 mouse embryonic stem cells, were found to be susceptible to CPA-induced endoplasmic reticulum stress and rotenone induced mitochondrial stress. Interestingly, the in vivo penetration rate of the muscle atrophy phenotype of homozygous ΔZfp106 mice is 60% for male and 12.5% for female mice. This is in stark contrast to the 100% penetration rate of the Zfp106LacZ mice. The reason behind this is currently unclear but may be due to either the incomplete backcrossing of the mouse model, a difference in the splice variants affected by the Zfp106 targeting, or because the muscle atrophy in the Zfp106LacZ mouse model is caused in part by the increase in the expression the P2 Zfp106 family of isoforms. These two mouse models show that affecting the expression of the full-length isoforms of P1-Zfp106 can lead to muscle atrophy. In an attempt to see if the Zfp106LacZ muscle atrophy was due to a lack of Zfp106 in the skeletal muscle, spinal cord, or necessitated its depletion in both, we derived a mouse line from the Zfp106LacZ that conditionally depletes exon 3 which impairs the expression of full length P1 Zfp106. This was used to target exon 3 removal to the skeletal muscle (Myf5), cholinergic neurons (ChAT), simultaneously (Myf5/ChAT), or a whole body depletion (Ella2). Surprisingly, the whole body depletion of Zfp106 exon 3 did not lead to muscle atrophy even though its removal leads to a frame shift and premature stop codon. The lack of a muscle atrophy phenotype may be because of the expression of a splice variant without exon 3, thereby rescuing the neuromuscular pathology. Lastly, to better understand the role of the P2 Zfp106 in vivo, we created a mouse line with a CRISPR mediated knockout of exon 1b (ΔP2). Exon 1b is the start exon of the P2 Zfp106 isoform family and the introduction of a destructive INDEL should independently affect the P2 isoform family. Interestingly, this mouse model showed no observed neuromuscular dysfunction or metabolic disorder, responding to a glucose bolus similarly with controls. The lack of a phenotype may be due to compensation by other Zfp106 isoforms or that the P2 isoform family is important in other biological roles.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 1977 / Vita. / Includes bibliographical references. / Ph. D. / Ph. D. Massachusetts Institute of Technology, Department of Chemistry

pK_n values for step-wise dissociation of protons from the protonated ligands (glycine, α-amino isobutyric acid, sarcosine and theonine) were determined potentiometrically with a Beckman Model GS pH meter in aqueous solution at 10, 20, 30 and 40° C. Log K values for step-wise interaction of glycine, α-amino isobutyric acid, sarcosine and threonine with copper(II) ion were also determined potentiometrically with a pH meter at 10, 20, 30 and 40° C. The standard free energy of formation for the step-wise interaction of copper(II) with each of the above ligands was calculated from the values of log K. Enthalpy and entropy changes were calculated for each of the above reactions at 20° C. Measurements of pH were obtained at several ionic strengths for each metal io-ligand system at each temperature. It was assumed that the zwitterion has no charge. The hydrolysis of copper(II) ion and the liquid junction potential were assumed to be negligible. Log K_1 and log K_2 values for the copper(II) chelate of α-amino isobutyric acid are lower than those of glycine at 10 20° C. but higher at 30 and 40° C. This may be related to the positive inductive effect and the steric effect of methyl groups. The large decrease in the log K values of the copper(II) chelate of sarcosine is probably due to two effects, namely, steric and hydration. These two effects counteract the positive inductive effect introduced by the methyl group on the nitrogen and actually decrease its basic strength and thus decrease the bond strength between nitrogen and copper. THe decrease in the log K values of the copper (II) chelate of threonine is explained by the presence of the hydroxyl group which increases the acid strength of the NH_3^+ group and decreases its electron donating power. This is probably due to the hydrogen bonding between the hydrogen of the hydroxyl group and nitrogen of the amino group. Values of log K for the copper(II) chelate of each ligand decrease with the increase in the temperature, indicating that the entropy effect is greater at a lower temperature than at a higher temperature. Calculations of n, [A^-] and log K were carried out on an IBM 650 Computer.

The carbohydrates play a vital role in the chemistry of the life processes. As man’s study of these processes, both fundamentally and in applied areas such as medicine, becomes ever more intensive, the limitations of our analytical procedures become ever more serious. While there are innumerable quantitative analytic procedures for the monosaccharides, there is a severe dearth of good micro colorimetric procedures. A particularly useful and widely used micro and semimicro procedure, based upon periodic acid oxidation, was originally introduced by Malaprade in France in 1928. In general, the consumption of the periodic acid in estimated by a back titration procedure involved the liberation of iodine by the excess periodic acid and subsequent estimation of the iodine by titration with thiosulfate. We conceived of the idea of an appropriately chosen reagent that would react with periodic acid to yield a compound that was stable under the working conditions and would have a spectra significantly different from that of the parent compound.

No description available.

Four casein-derived peptides fractions of varying hydrophobicity were obtained from á-chymotryptic digest of acid casein using hydrophobic interaction chromatography, termed fractions one through four (abbreviated, F1, F2, F3, and F4). Four standard methods involving alkoxyl, peroxyl, 2, 2-diphenyl-1-picrylhydrazl (DPPH), and 2, 2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) ABTS•+ radicals, were used to measure antioxidative properties. While significantly superior efficacy was exhibited by F2 for all tests except against DPPH, no correlation between antioxidant efficacy and surface hydrophobicity was found. By using capillary electrophoresis and high performance liquid chromatography, the detection of aromatic chromophores by ultraviolet at 280 nm in the fractions revealed that F2 contained the highest concentration of aromatic amino acids and a unique peptide. Result from circular dichroism exhibited remaining residual structure in F2 compared with undigested casein. The F2 possesses a high potential to be used in food industry as a natural source of antioxidant with pronounced antioxidant capacity.

No description available.

No description available.