Metallothioneins (MTs) have a major role in metal metabolism and may also protect DNA against oxidants. MT protein has been found localized in the nucleus during S-phase. The mRNA encoding for the MT-1 isoform is found localized around the nucleus and associated with the cytoskeleton; this is due to targeting signals within the 3'untranslated region (3'UTR). Using cells transfected with gene constructs differing in their 3'UTRs, the role of perinuclear mRNA localization in facilitating MT synthesis close to its site of function and subsequent import of protein into the nucleus has been investigated, as well as the role of MT protein in the nucleus. We transfected CHO cells, which have a low constitutive level of MT expression, with either the full MT-gene (MTMT) or with MR 5'UTR and coding region linked to the 3'UTR of glutathione peroxidase (MTGSH). Immunocytochemistry showed that MT protein was localized in the perinuclear cytoplasm in the MTMT cells whereas no distinct localization was found in the MTGSH cells. The cells were then synchronised in S-phase by serum depletion/repletion. After serum repletion, MT was found in the nucleus of MTMT cells but not in the MTGSH cells. This suggests that perinuclear localization of MT-1 mRNA and its association with the cytoskeleton is necessary for MT protein localization, particularly for the shuttling of MT protein into the nucleus during S-phase. Functional studies demonstrated that the extent of oxidative stress and DNA damage was lower in the MTMT than the MTGSH, showing that a loss of MT protein localization led to a reduced protection of the cell. Therefore, it seems that perinuclear localization of mRNAs coding for MT is necessary for subsequent transport and targeting of proteins into the nucleus and that the localization of the protein within the cell is important for its function.

Both the endometrium and the trophoblast secrete a number of proteins. Some of these, e.g. human chorionic gonadotrophin (hCG) from the trophoblast and pregnancy-associated endometrial α2-globulin (α2-PEG) from the endometrium, are secreted into the bloodstream and may act as endocrines. Moreover, they may also function as paracrines, signalling to adjacent tissues. Several models have been developed, by which the paracrine activity of the endometrium and the trophoblast might be examined <i>in vitro</i>. These include, cultures of endometrium and trophoblast, dual chamber superfusion of membranes and dual perfusion of individual placental cotyledons. The purpose of the present study was to examine some aspects of experimental design on the results from these models and consider the factors that might govern the rate of release. A good many trophoblast proteins have been characterised and methods devised for their quantitative measurement, however, the study of endometrial proteins is less advanced. As a first step, a method was developed for the measurement of α2-PEG, the major secretory protein of the secretory endometrium during the first trimester of pregnancy. This thesis describes the development of a competitive ELISA for measuring α2-PEG. This assay was employed for measuring α2-PEG in the medium from decidual cultures. In this thesis, release of protein <i>in vitro</i> has been examined as a mode of basal unstimulated secretion <i>in vivo</i>. The release is compounded of at least two simultaneous processes, biosynthesis and the transport of formed material to the outside of the cell. The concept is put forward that protein release is determined by a dynamic balance between biosynthesis of new protein and diffusion of protein down the concentration gradient between cell cytoplasm and the surrounding medium.

The aim of this study was to assess changes in the amino-acid composition of the body and in the fractional protein synthesis rates of individual organs or of haemoglobin as a result of long-term amino-acid deficiency in both growing and adult animals. The body weight loss of adults and the growth rates of growing animals were controlled by adjusting the degree of amino-acid deficiency according to individual body weight changes. Adult rats given amino acid-deficient diets had lower absolute weights of muscle (p = 0.052), liver (p = 0.002) and gastrointestinal tract (p < 0.001) than adequately-fed animals but chronic marginal amino-acid deficiency did not result in any measurable effect on whole-body amino-acid composition in comparison with well-nourished rats. In growing rats, liver weight, liver protein mass, fractional (% per day) and absolute (g / liver per day) synthesis rates of total liver protein, liver RNA activity and absolute albumin synthesis rate (g / liver per day) were depressed by long-term dietary deficiency of leucine, isoleucine or histidine. There were no significant differences in liver RNA capacities or relative albumin synthesis rates, however, between the adequate and the deficient groups. In growing pigs, deficiency of isoleucine, histidine or tryptophan depressed growth rate and the weights of liver, gastrointestinal tract, and muscle (<I>biceps femoris</I>). The fractional rates of total protein synthesis, both in individual tissues (muscle, skin and liver) and of haemoglobin, were also reduced. Pigs given histidine- or tryptophan- deficient diets had higher body hydroxyproline concentrations, suggesting changes in the deposition of collagen relative to other body proteins. The histidine-deficient pigs had lower whole-body histidine concentrations, due to absolute decreases in both haemoglobin and in free peptides rich in histidine. The histidine-deficient pigs also had low histidine concentrations in skin.

No description available.

No description available.

No description available.

No description available.

Guanidine-stable chymoelastase (GSC-Elastase), an enzyme isolated from a commercial protease preparation (Pronase), has been shown to be stable and active in the presence of 6.0 M guanidinium chloride (Si egel , S . , et. al . , J . Biol. Chem. 247:4155, J . Pi of . Chem. 248:3233.) The amino acid sequence around the active site serine residue for the protease is similar to that found for bovine chymotrypsin (Awad, W. M. et.al., J. Biol. Chem. 247:4144). This investigation involved the qualitative comparison of the protein cleavage specificity of GSC-Elastase in the presence and absence of denaturant. This specificity was also compared to that found for a-chymotrypsin for the existence of possible similarities in their actionson protein substrates. S-Carboxymethylated lysozyme (CM-HEL) was hydrolyzed in separate trials which were catalyzed by GSC-Elastase in the presence and absence of 6.0 M guanidinium chloride and by a-chymotrypsin. Two-dimensional peptide maps were made from each of the hydrolysates by performing thin-layer chromatography in one direction followed by electrophoresis in a perpendicular direction. The results of the mapping procedure indicated that the cleavage of protein substrates by GSC-Elastase is reproducible and therefore specific under denaturing conditions as well as in the absence of denaturant. These results also suggested that the protein cleavage specificity of GSC-Elastase was found to be significantly different from that of bovine chymotrypsin.

Access to abstract permanently restricted to Ball State community only. / Access to thesis permanently restricted to Ball State community only.

Gap junctions are communicating junctions between cells that allow small molecules to pass from the cytoplasm of one cell to the cytoplasm of an adjacent cell. The pores of gap junctions are comprised of two adjacent connexons on neighboring cells, and each connexon is comprised of six connexin proteins. The eye lens of vertebrates is an avascular tissue that is dependent on gap junctions for the distribution of nutrients as well as the removal of waste products. In addition, as the lens cells develop into fibers, they lose their intracellular organelles including the membrane-bound organelles, and are highly dependent on connexons for movement of metabolites and waste materials. Only two connexins, in Bos Taurus Cx44 and Cx49, are highly expressed in lens fiber cells. Thus, the lens offers an excellent system for studying gap junctions. In this study, high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) techniques were used to isolate and characterize connexin proteins from the eye lens of the cow and mouse. Despite over 300 proteins being identified from bovine lens using MS techniques, it was still possible to identify the two connexin proteins following proteolytic digests and MS analysis of the resultant peptides. Several post- translational modifications (PTMs) were identified and characterized in lens fiber connexins, including phosphorylations, acetylations and deamidations and proteolytic cleavages. Changes in phosphorylation of several other lens proteins upon the activation of protein kinase C were also identified. Detection of the orthologous proteins in mouse lens proved more challenging, but peptides derived from both connexin proteins were also detected from this tissue and PTMs of mouse connexins were also observed. Glutathione-S-transferase fusions to mouse Cx44 and Cx50 were used to identify a number of proteins that may interact with the mouse connexins, and the relevance of those interactions was considered. The utility of mass spectrometry to the identification of specific proteins from complex mixtures was clearly demonstrated, and its application to understanding the functional relevance of PTMs was discussed.