The localisation of B lymphocyte precursor cells in mouse bone marrow and their associations with stromal reticular cells and macrophages have been investigated by in vivo radioimmunolabeling combined with light and electron microscope radioautography. Many early B lineage cells expressing the B220 glycoprotein prior to the expression of surface immunoglobulin and those regenerating after sub-lethal $ gamma$-irradiation, were located in bone-associated regions of femoral marrow. Labeled B220$ sp*$ lymphoid cells of undifferentiated morphology were closely associated with complex cytoplasmic processes of stromal reticular cells. The binding of a monoclonal antibody raised against a B lymphocyte-supportive stromal cell line (mAb KMI6), was highly restricted to the interface between stromal cells and undifferentiated lymphoid cells. The VCAM-1 specific mAb M/K-2, labeled electron-dense stromal cells that contacted lymphoid cells and a variety of other hemopoietic cell lineages. Within the bone marrow of normal mice there was evidence of death of B220$ sp+$ B lineage cells by apoptosis and their removal by macrophages. Cell loss, apoptosis and macrophage deletion of B precursor cells were greatly enhanced in E$ mu$-myc transgenic mice. The results reveal a complex in vivo microenvironmental organisation of B lymphocytopoiesis characterised by intimate interactions with stromal reticular cells and macrophages which regulate the development of precursor B cells and determine the ultimate output of B lymphocytes from the bone marrow.

No description available.

The nonciliated epithelial cells of the efferent ducts are specialized in internalizing luminal fluid. They possess an extensive endocytic apparatus which provides an ideal system to study the kinetics of endocytosis. The nonciliated cells actively endocytose sulfated glycoprotein-1 (SGP-1, 70 kDa), a major secretory protein of Sertoli cells. A second form of SGP-1 (65 kDa) present in the secondary lysosomes of Sertoli and nonciliated cells of the efferent ducts is believed to be the equivalent of human prosaposin, the precursor of four small heat stable proteins (saposins A, B, C and D) required for the hydrolysis of sphingolipids by specific lysosomal hydrolases. This study was undertaken to investigate the contribution of Sertoli-derived SGP-1 as well as a putative endogenous SGP-1 within the secondary lysosomes of nonciliated cells. The hormonal control of endogenous lysosomal SGP-1, specifically the influence of testosterone withdrawal and its subsequent replacement was also examined. The results provide evidence that the nonciliated cells of the efferent ducts are involved in the clearance of testicular SGP-1 and reveal the presence of 15 kDa saposins and their 65 kDa precursor in secondary lysosomes of these cells. In addition, the production of an endogenous lysosomal SGP-1 targeted from the Golgi apparatus to the lysosomes after its glycosylation has also been demonstrated. The results further reveal that the endocytosis and lysosomal targeting of SGP-1 in nonciliated cells of the efferent ducts appear to be pituitary controlled.

No description available.

<p> Chondrichthyans (sharks, rays, skates, and chimaeras) are a diverse taxonomic clade inhabiting bodies of water all over the world. As a lineage, chondrichthyans split from the other jawed vertebrates 450 million years ago, the most basal split in the gnathostome vertebrate tree. Although they have been studied for centuries, knowledge about these animals lags behind that of many other vertebrate groups. This work uses Computed Tomography (CT) to explore morphological variation across phylogenetically diverse species of chondrichthyans. CT imaging is a nondestructive method for viewing internal structures of extant and fossilized specimens. After CT scan data acquisition, reconstruction software was used to manually segment the skeletal anatomical into constituent structures, creating 3-Dimensional representations of the structures. In most groups of vertebrate organisms, skeletal structures are made of calcified bone which has high radiopacity, leading to greater contrast between the skeleton and soft tissues. Chondrichthyans, by comparison, have skeletons composed of cartilage which is much less radiopaque than bone, resulting in lower contrast with surrounding tissues. Variations in the skeletal structures are discussed along with notes on calcification within the chondrichthyan orders. This work is presented as a summary of the variation observed in the skeletal anatomy, building upon previous works in chondrichthyan anatomy, expanding the current state of knowledge of the diversity in chondrichthyan fish skeletons. This project is part of a collaborative effort to develop a phylogenetic tree of life for modern chondrichthyans.</p>

Fibrillin proteins are the major components of microfibrils in elastic and non-elastic extracellular matrices. Microfibrils exert critical functions in the development and homeostasis of tissues and organs. Mutations in fibrillin-1 lead to heritable diseases such as Marfan syndrome, Weill-Marchesani syndrome and others. While different studies have been performed to characterize human fibrillins in details, mouse fibrillins have not yet been studied in depth. In this thesis, recombinant expression plasmids for the C-terminal halves of mouse fibrillin-1 and -2 were generated. The recombinant proteins were expressed in human embryonic kidney cells, and subsequently purified by immobilized metal ion affinity chromatography. N-terminal sequencing validated the correct sequence of the recombinant proteins. Both purified mouse fibrillin fragments consistently presented in monomeric, intermediate and multimeric forms. Similar to human fibrillins, the mouse fibrillin-1 and -2 fragments showed strong interaction with heparin in solid phase binding assays. Addition of the recombinant mouse fibrillin-1 C-terminal fragment to fibroblasts inhibited mouse microfibril assembly. The recombinant mouse fibrillin fragments were used to generate rabbit antisera, which were extensively characterized. Both antisera had very high titers against the mouse fibrillin fragments as determined by ELISA. As expected, based on the >95% homology on the amino acid level, the antisera showed high cross-reactivity between the mouse and human fibrillin-1 and -2 fragments. Surprisingly, however, the antiserum against mouse fibrillin-1 reacted much more strongly with microfibrils produced from mouse cells and with mouse tissue compared to the antiserum against human fibrillin-1. / Les fibrillines sont les principaux composants des microfibrilles dans les matrices extracellulaires élastiques et non élastiques. Les microfibrilles jouent un rôle majeur dans le développement et l'homéostasie des tissus et des organes. Des mutations dans la fibrilline-1 causent des maladies héréditaires telles que le syndrome de Marfan, le syndrome de Weill-Marchesani et autres.. Alors que de nombreuses études ont été menées afin de caractériser les fibrillines humaines, les fibrillines murines n'ont été que partiellement étudiées.Dans cette thèse, des plasmides permettant l'expression des portions C-terminales des fibrillines 1 et 2 de souris ont été générés. Les protéines recombinantes ont été exprimées dans les cellules de rein embryonnaire humain, et ensuite purifiées par chromatographie d'échange ionique. La séquence des protéines recombinantes a été validée par séquençage N-terminal . Les deux fragments de fibrilline de souris purifiés se présentaient sous des formes monomériques, intermédiaires et multimèriques. Tout comme les fibrillines humaines, les fragments de fibrilline1 et 2 ont montré une forte interaction avec l'héparine dans les essais de liaison protéine-protéine en phase solide. L'addition du fragment C-terminal de fibrillin 1 de souris à des fibroblastes induit l'inhibition de l'assemblage des microfibrilles. Les fragments de fibrillines recombinantes de souris ont également été utilisés pour générer des antisérums de lapin, qui ont été largement caractérisés. Des tests ELISA ont montré une forte réactivité des deux antisérums contre les fragments de fibrilline de souris. Comme la forte homologie au niveau peptidique (95%) le laissait supposer, les antisérums ont montré une forte réactivité croisée entre les fragments de fibrillines 1 et 2 murins et humains. Cependant, l'antisérum contre la fibrilline-1 de souris réagissait plus fortement à des microfibrilles produites à partir de cellules de souris et avec des tissus murins comparé à l'antisérum dirigé contre la fibrilline-1 humaine.

Cytochrome b(,5) is an integral membrane protein of the endoplasmic reticulum of the hepatocyte. Previous work by the groups of Ozols and Strittmatter has determined the primary sequence and the orientation of the molecule. It is embedded on the cytoplasmic face of microsomal membranes via a hydrophobic 40 amino acid long "tail" at the carboxy terminus. The synthesis of this protein is assigned to the free polyribosome population of rat hepatocytes, and the experimental documentation of this assignment is reported. / Free and membrane-bound polyribosomes were separated by a modification of the method of Ramsey and Steele (1976) and characterized by electron microscopy. Poly A('+) RNA isolated from the total free (poly A('+) RNA(,f)) or total membrane-bound polyribosomes (poly A('+) RNA(,mb)) was translated using the heterologous wheat germ system. Fluorographic SDS-PAGE analysis demonstrated that the translation products of poly A('+) RNA(,f) had electrophoretic mobilities similar to those of liver cytosol proteins. In contrast, poly A('+) RNA(,mb) was translated into polypeptides with mobilities similar to serum proteins (the major secretory product of the hepatocyte). Antiserum to the complete cytochrome b(,5) molecule immunoprecipitated from the translation products of poly A('+) RNA(,f) a single polypeptide with electrophoretic mobility identical to that of native cytochrome b(,5) (17,500 daltons). Quantitative analysis indicated 85% synthesized from poly A('+) RNA(,f) compared to only 15% from poly A('+) RNA(,mb). Control experiments with antiserum to serum albumin demonstrated that following immunoprecipitation, a single polypeptide of M(,r) 68,000 was synthesized by poly A('+) RNA(,mb). This M(,r) was shown to be larger by 3000 daltons than that of native serum albumin; the difference being accounted by the known "signal sequence". Quantitation indicated 90% synthesized from poly A('+) RNA(,mb) and 10% from poly A('+) RNA(,f). / In conclusion, an exclusive location of the site of synthesis of microsomal cytochrome b(,5) is assigned to the free polyribosome population. This conclusion provides a ready explanation (i) for the ubiquitous distribution of the protein to most intracellular organelles; (ii) for the topographic orientation on the cytoplasmic face of membranes and (iii) for the hyrophobic tail (membrane insertion sequence) of amino acids at the carboxy terminal end of the molecule.

Mouse L cell mutants defective in receptor mediated endocytosis were isolated using selection with modeccin followed by screening for loss of mannose 6-phosphate (Man 6-P) dependent uptake or for resistance to Pseudomonas exotoxin. Mutants L146 and L15D were resistant to modeccin, lacked 215 kD Man 6-P receptor, were defective in Man 6-P dependent uptake and binding, and oversecreted several lysosomal enzymes. Mutant LEFIC was resistant to modeccin, Pseudomonas exotoxin and ricin. The mutant exhibited delayed (1) oligosaccharide processing of VSV G protein, (2) transport of VSV G to the cell surface and (3) release of radiolabeled VSV G into virions. LEFIC was blocked in proteolytic processing of the Sindbis glycoprotein pE2 and showed intracellular accumulation of Sindbis nucleocapsid. The mutant showed a delay in secretion of fibronectin. An intersection of the secretory and endocytic pathways is suggested by the isolation of this cross-toxin resistant mutant defective in intracellular transport of membrane and secretory proteins.

No description available.

No description available.