Non-specific Cross-reacting Antigen or NCA was isolated from colonic tumor (T-NCA), normal lung (L-NCA) and normal spleen (S-NCA) tissues. In each case, NCA consisted of a 50,000 dalton glycoprotein. Tumor NCA and carcinoembryonic antigen (CEA) had a similar amino acid composition but differed in carbohydrate content and composition. Enzymatically and chemically derived fragments of T-NCA, L-NCA and S-NCA were identical by SDS-gel but demonstrated only 25% homology by two-dimensional peptide mapping. Normal tissue NCAs demonstrated 62% peptide homology. These differences are thought to result from glycosylation differences. / Antigenic determinants involved in CEA/T-NCA cross-reactivity were localized on purified fragments of CEA and T-NCA which ranged in size between 20,000 and 30,000 daltons. Two-dimensional peptide mapping revealed 30-50% homology of CEA, T-NCA and fragments of these molecules, even after partial deglycosylation. Deglycosylation of CEA and T-NCA with trifluoromethanesulfonic acid (TFMS) generated from each antigen a soluble immunochemically identical 40,000 dalton polypeptide demonstrating 45% homology by peptide mapping. In the case of CEA, the 40,000 dalton polypeptide may represent an acid cleavage product. Deglycosylation of CEA by TFMS treatment also generated an insoluble 85,000 dalton polypeptide which may represent the intact deglycosylated CEA polypeptide. These data demonstrate that a structural basis exists for CEA/T-NCA cross-reactivity.

Formiminotransferase (EC 2.1.2.5)-cyclodeaminase (EC 4.3.1.4) catalyzes two sequential H(,4)folate-dependent reactions. With derivatives of the H(,4)folate substrate having 4,5, or 6 glutamyl residues, the formiminoH(,4)pteroylpolyglutamate formed by the transferase activity is preferentially transferred (channeled) to the deaminase site rather than released into the solution. This channeling is essentially complete with the pentaglutamate derivative. The enzyme has highest affinity for the hexaglutamate as measured by K(,d) and K(,m), but does not show specificity for a given polyglutamate as measured by V(,m)/K(,m). The results indicate that steric length of the polyglutamate chain, not simply affinity, is critical for optimal channeling. Binding studies demonstrate four sites for the binding of H(,4)pteroylpolyglutamates to the native octamer, suggesting the formation of sites between subunits. The transferase and deaminase sites are kinetically independent, but share a common polyglutamate subsite. The results support the concept that the polyglutamate chain anchors the H(,4)folate molecule during its transfer between active sites.

A family of four low molecular weight peptides, that are rich in arginine and cysteine and which have the ability to inhibit ACTH stimulated adrenocortical steroidogenesis have been purified from rabbit lung and neutrophil extracts and sequenced. They are called corticostatin I (CSI), II (CSII), III (CSIII), and IV (CSIV) respectively. Among them CSI is the most potent with ED$ sb{50}$ of 25 nM against a concentration of 150 pg/ml ACTH stimulation. The mechanism of action of CSI has been studied extensively. CSI shows a high degree of specificity in that it does not inhibit the action of angiotensin II in the adrenal cortex. CSI does not inhibit dbcAMP induced steroidogenesis, however it does inhibit the accumulation of cAMP in response to ACTH in the rat adrenal cell suspension. CSI acts by competing with the basic--Gly$ sb{14}$-Lys$ sb{15}$-Lys$ sb{16}$-Arg$ sb{17}$-Arg$ sb {18}$--sequence of ACTH for its binding site.

The effect of phosphorylation on the in vitro assembly of NF subunits was investigated using alkaline phosphatase treated subunits. The assembly properties of exhaustively dephosphorylated NF subunits were found to be similar to those of untreated subunits, as determined by SDS-PAGE and electron microscopy. Moreover, in vitro dephosphorylation removed only about half of the phosphate moieties suggesting the possible presence of inaccessible phosphorylation site(s). / The anomalous migration of NF subunits on SDS-PAGE was abolished when the glutamyl carboxyl groups, localized largely in the carboxyl terminal domain, were chemically modified by glycinamidation. The apparent molecular weights of NF subunits on SDS-PAGE after glycinamidation, and dephosphorylation were $ sim60$ Kd, $ sim112$ Kd, and $ sim138$ Kd for NF-L, NF-M, and NF-H, respectively. / The expression of all three NF subunits in cultured adrenal chromaffin cells was demonstrated by immunocytochemical staining, and by immunoprecipitation techniques. The NFs in chromaffin cells, which are largely restricted to the perikaryon, were found to be hypophosphorylated, as they comigrated with in vitro dephosphorylated bovine NF subunits on SDS-PAGE. Triton-soluble pools of NF-M, and NF-H were shown to be present in chromaffin cells. These were found to be distinct from the cytoskeleton-associated NF subunits based on differences in their relative amounts, and in the rate of $ sp{32}$P turnover. In addition, differences in the phosphopeptide maps of Triton-soluble and cytoskeletal NF-M suggested that certain phosphorylation sites may be important in regulating NF subunit assembly. / Treatment of chromaffin cells with phorbol esters markedly increased the phosphorylation of NF subunits in both the cytoskeletal and Triton-soluble fractions. This protein kinase C-mediated phosphorylation was accompanied by a decrease in the electrophoretic mobilities of NF-M and NF-H on SDS-PAGE, indicating the attainment of higher phosphorylation levels. Phosphopeptide mapping analysis indicated that protein kinase C phosphorylates NF-M at a new site(s). / Incubation of chromaffin cells in growth medium containing 8-Br cAMP and NGF also resulted in the increased $ sp{32}$P labelling of NF subunits. The in vitro phosphorylation of cytoskeleton preparations from chromaffin cells resulted in the hyperphosphorylation of NF subunits to levels seen for axonal NFs.

Malaria is a worldwide disaster that is still killing two million lives annually. The recent rise of the epidemic is mainly attributed to the emergence of resistance to the most effective, affordable and safe antimalarial, chloroquine. Unfortunately, neither the mechanism of action of these 4-aminoquinolines nor the basis of chloroquine resistance is well understood. It has been noted that a known channel blocker, verapamil, is able to modulate chloroquine resistance. / In this study, a chemical biology approach was taken to gain some light into these mechanisms. The design, synthesis and characterization of fluorescent and photoreactive probes, analogues of chloroquine and verapamil, are presented. These can be used in fluorescent microscopy and photoaffinity labeling experiments to identify a putative chloroquine export protein, Plasmodium falciparum chloroquine resistance transporter (PfCRT). / As a result of the hemoglobin degradation pathway, the liberated, toxic heme is sequestered by P. falciparum into an insoluble inert crystal, hemozoin. Hemoglobin proteases knockout parasites were generated. Their hemozoin was isolated, analyzed by synchrotron powder X-ray diffraction, and imaged using scanning electron microscopy.

Initiation of DNA replication is a cellular process whose regulation is not well understood yet. It certainly depends on cis-acting elements, or origins of DNA replication sequences, and trans-acting factors, or initiator proteins. Here, we have characterized the Cruciform Binding Protein (CBP) as an origin binding protein and its role in initiation of DNA replication. / CBP was previously shown to contain the β, ɣ, ε, and ζ isoforms of the 14-3-3 family, which is composed of seven mammalian isoforms (β, ɣ, ε, η, σ, τ, and ζ) that can form homo- and heterodimers, and plays a variety of roles in different cellular processes. In this thesis, I showed by Western blot analysis with anti-14-3-3σ antibody, which partially interfered with the CBP-cruciform DNA complex formation, that the isolated CBP-cruciform DNA complex contained also the σ isoform. The same antibody reduced the in vitro DNA replication efficiency of HeLa cell total extracts, in an assay that used p186, a plasmid bearing the 186-bp minimal origin of ors8, as template DNA. Similarly, I found that antibodies against 14-3-3 isoforms β, ɣ, ε, and ζ also interfered with the CBP-cruciform DNA complex formation, and reduced the in vitro p186 replication efficiency of HeLa cell total extracts. The five isoforms of 14-3-3 (β, ɣ, ε, σ, and ζ) were found to associate with the monkey cell (CV-1) origins of DNA replication ors8 and ors12 in a cell cycle-dependent manner, the association being higher at the G1/S phase. Furthermore, we found that 14-3-3 yeast homologues, Bmh1p and Bmh2p, were able to bind cruciform DNA in vitro, and to associate in vivo with the autonomous replication sequence 307 (ARS307) in a cell cycle-dependent manner, again the association being higher at G1/S. Finally, I showed that recombinant 14-3-3ζ, tagged with maltose-binding protein (r14-3-3ζ-MBP), could only bind cruciform DNA after pre-incubation with a CBP-enriched HeLa cell extract (FTH), in which it heterodimerized with endogenous 14-3-3 isoforms β and ε. Addition of r14-3-3ζ-MBP to HeLa cell total extracts increased the in vitro replication of p186, suggesting that increased CBP activity could lead to multiple rounds of initiation of DNA replication.

Folate metabolism is compartmentalized between the cytoplasm and the mitochondria of mammalian cells. Certain folate-dependent enzymes are present in both of these compartments, such as methylenetetrahydrofolate dehydrogenases, which are required to interconvert one-carbon substituted tetra hydrofolates. In the cytoplasm, there is a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase (DCS). Its mitochondrial counterpart is a bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC), expressed during embryogenesis and in immortalized cells. A comparison of the 3' untranslated region of the NMDMC cDNA with the synthetase domain of the DCS cDNA and gene among different species have revealed significant regions of homology. This suggests that the mammalian mitochondrial NAD-dependent DC evolved from an NADP-dependent DCS precursor through a change in cofactor specificity of the dehydrogenase from NADP to NAD. / Although the folate pathways are compartmentalized, the mitochondrial folate pathway makes an important contribution to total cellular folate metabolism. Mouse fibroblasts that have a completely inactivated NMDMC gene are glycine auxotrophs. Furthermore, growth of these Nmdmc-/- cell lines is stimulated by supplementation with formate or hypoxanthine. These cell lines also show enhanced incorporation of radioactivity into DNA from formate as compared to serine, demonstrating that formate is a preferred one-carbon donor for the Nmdmc-/- cell lines. This indicates that NMDMC is required for optimal purine biosynthesis during periods of rapid cellular proliferation such as embryogenesis and tumorigenesis. The rescue of these Nmdmc-/- cell lines with NMDMC expression reversed the glycine auxotrophy and the formate to serine incorporation ratio reverted toward the wild type ratio. The rescue of these Nmdmc-/- cell lines with the NAD-dependent monofunctional dehydrogenase activity also reversed the glycine auxotrophy but these cell lines did not grow as well as the NMDMC-rescued cell lines. This indicates that although the cyclohydrolase activity is not required in the mitochondria, the rate of 10-formyl-THF production is not optimal in its absence. Furthermore, when these Nmdmc-/- cell lines were rescued with the expression of the NADP-dependent DCS in the mitochondria there was reversal of the glycine auxotrophy as well, indicating that the NAD cofactor specificity of the mitochondrial methylenetetrahydrofolate dehydrogenase is not absolutely essential to maintain the flux of one-carbon metabolites.

Nuclear receptors play essential roles in many aspects of cellular regulation such as cell development, differentiation, cell death, energy homeostasis, and metabolism of the organism. ERRα and COUP-TFII are both orphan nuclear receptors known to recognize similar DNA response elements. ERRα often binds to an extended half site TNAAGGTCA in the promoter regions of target genes, whereas COUP-TFII prefers to bind to a number of variably spaced imperfect AGGTCA direct or inverted repeats. To better understand the cross talk between these two proteins, ChIP-on-chip experiments were performed on mouse liver in order to identify the targets of ERRα and COUP-TFII on a genome-wide scale. 299 genes with a large variety of biological processes were found to be common targets of ERRα and COUP-TFII, meaning that these genes recruit the two nuclear receptors to the same binding sites. In these cases, ERRα and COUP-TFII are likely to compete for the common DNA binding site in order to regulate their target genes. / Les récepteurs nucléaires jouent un rôle critique dans plusieurs aspects de la physiologie cellulaire, incluant le développement, la différentiation, l'apoptose, l'homéostasie et le métabolisme énergétique. ERRα et COUP-TFII sont tous deux des récepteurs nucléaires orphelins connus pour se lier à des éléments de réponse similaires au niveau de l'ADN. ERRα se lie généralement à un demi-site allongé TNAAGGTCA situé dans la région du promoteur de ses gènes cibles. COUP-TFII se lie quant à lui à des motifs AGGTCA, en répétition directe ou en palindrome, séparés par un nombre variable de nucléotides. Afin de mieux comprendre les interactions entre ces deux protéines, une analyse génomique ChIP-sur-chip (pour Chromatin ImmunoPrécipitation sur Chip) a été faite dans des tissus de foie murin. Cette analyse a permis d'établir la liste des gènes cibles d'ERRα et COUP-TFII dans cet organe. 299 gènes communs aux deux récepteurs nucléaires ont été identifiés. Ces gènes, impliqués dans une grande variété de processus biologiques, sont liés au même élément de réponse de l'ADN par les deux récepteurs. Nos résultats suggèrent qu'ERRα et COUP-TFII exercent une liaison compétitive à l'ADN, sur les éléments de réponse commun, pour la régulation de leurs gènes cibles.

The Cut homeobox 1 (CUX1) gene codes for several isoforms that display distinct biochemical and biological activities. To compare the oncogenic potential of various CUX1 isoforms in the mammary gland, transgenes comprising the Mouse Mammary Tumor Virus long terminal repeat (MMTV-LTR) and CUX1 coding sequences were inserted into the hprt locus by specific transgenesis into embryonic stem cells and transgenic mice were obtained following injection of these cells into mouse blastocysts. In this study, I characterized the phenotype of transgenic mice of the FVB strain that carry the MMTV-p200 CUX1 transgene. These mice developed mammary tumors with a penetrance of 26% and a mean latency of 20.1 months. Mammary tumors were of various histological types the most frequent being adenosquamous carcinoma, solid carcinoma and carcinoma cribriform. Immunohistochemical analysis revealed variable expression of cytokeratins (CK) 6, 8/18, and 14. From reverse-transcription polymerase chain reaction (RT-PCR) assays, the p200 CUX1 transgene was expressed in the mammary glands but not in the mammary tumors. Instead, in mammary tumors we detected elevated CUX1 expression from the endogenous CUX1 gene. These results suggest that transgene expression was shut-off in mammary tumors, whereas the endogenous CUX1 was over-expressed. / Le gène Cut homeobox 1 (CUX1) code pour plusieurs isoformes avec des activités bicohimiques et biologiques distinctes. Afin the comparer le potentiel oncogénique de certaines isoformes dans la glande mammaire, nous avons utilisé la méthode de transgénèse spécifique pour insérer les transgènes dans le locus hprt. Chaque transgène comprenait les séquences codantes d'une isoforme particulière sous le contrôle transcriptionnel de séquences régulatrices provenant du " Mouse Mammary Tumor Virus (MMTV). Dans cette étude, j'ai caractérisé le phénotype des souris de la souche FVB qui portait le transgène MMTV-p200 CUX1. Ces souris ont développé des tumeurs mammaires avec une pénétrance de 26% et un temps de latence moyen de 20.1 mois. L'analyse des tumeurs mammaires par notre pathologiste a identifié plusieurs types histologiques dont les plus fréquents étaient les carcinomes adénosqameux, les carcinomes solides et les carcinome cribiformes. De fait, j'ai détecté par immunohistochimie l'expression des cytokératines 6, 8/18 et 14. L'analyse de l'ARN par "reverse-transcription polymerase chain reaction" (RT-PCR) a montré que le transgène p200 CUX1 était exprimé dans les glandes mammaires mais pas dans les tumeurs mammaires. Par contre, le gène CUX1 endogène était surexprimé dans plusieurs tumeurs mammaires. Ces résultats suggèrent que le transgène est éventuellement réprimé dans les tumeurs alors que le gène CUX1 endogène est activé.

Translation is the conversion of one language into another: from the language of nucleotides to the language of amino acids; from our genetic code to functional proteins. This process is fundamental to cellular activities such as growth, differentiation, proliferation and even cell death. Translation is the final step in the flow of the genetic information. Therefore, regulation at this level allows for an immediate and rapid response to changes in physiological conditions. Translation is a complex multi-stage process, which is highly regulated, especially at the level of initiation. It requires several players: initiator transfer RNA (tRNA), 40S and 60S ribosomal subunits, as well as several eukaryotic initiation factors (eIFs), culminating in the positioning of the 80S ribosome at the initiation codon of a messenger RNA (mRNA). Recognition of the cap structure of the mRNA by eIF4F (composed of the cap binding protein eIF4E, the RNA helicase eIF4A and the scaffolding protein eIF4G), is a key step in this process and also the rate-limiting step of translation initiation. / The human eIF4E-Homologous protein (h4EHP) was originally cloned in our laboratory in 1998. This cytoplasmic protein bares 30% identity and 60% similarity to the human eIF4E and is also a cap-binding protein. However, the function of 4EHP in translation has remained elusive: it does not interact with any known initiation factor and has no effect on overall translation rate. Recently, isoforms of human 4EHP in D. melanogaster and C. elegans were found to play a role in repression or activation of specific mRNAs. In order to assess the role of human 4EHP as a translational modulator, novel interacting partners were investigated using various methods. h4EHP was shown to specifically interact with eIF4E-transporter (4ET), elongation factor 2 (eEF2), eIF4A and certain ribosomal proteins (L4, L13, L27a). In addition, specific assays were performed to examine the post-translational modifications of h4EHP, such as phosphorylation. Lastly, the possible roles of h4EHP in metastasis and in microRNA-mediated repression, were investigated.