Protein trafficking events in the secretory and endosomal apparatus were evaluated in rat liver hepatocytes by EM immunogold cytochemistry. The cellular distribution of apolipoprotein E and other abundant hepatic secretory and endosomal proteins was quantitatively examined in ultrathin cryosections of liver hepatocytes. Under steady-state conditions, apoE was concentrated within the Golgi apparatus, along sinusoidal plasma membrane microvilli and within all components of the endosomal apparatus, as evaluated immunocytochemically and confirmed by quantitative immunoblotting of organelles isolated from liver homogenates. / Within the secretory pathway, the hepatic Golgi apparatus was a site of protein concentration as evaluated by the gold labeling density of another major secretory protein of liver hepatocytes, albumin, which was concentrated $ sim$10-fold in the Golgi apparatus relative to the ER. Sorting of this secretory protein within pre-Golgi compartments was not observed. Within the Golgi apparatus, apoE was concentrated within Golgi saccular distensions while being predominantly absent from flattened saccular components; apoB was similarly segregated within peripheral distensions. In contrast albumin, as well as two other monomeric proteins, transferrin (Tf) and the polymeric immunoglobulin receptor (pIg-R) were distributed homogeneously throughout Golgi stacks. In an attempt to assess a key prediction of the vesicular transport hypothesis, small 60-90 nm vesicles in the immediate vicinity of Golgi apparatus, postulated to mediate intersaccular transport were examined for their content of cargo secretory or plasma membrane proteins. Lack of immunoreactive apoE, apoB, albumin, Tf, or pIgR, within small vasicular profiles suggests limits to current models of vesicle-mediated intra-Golgi transport. / Along the endocytic pathway, at the cell surface, apoE and pIgR were dispersely distributed along the sinusoidal microvilli. Quantitative analysis of the immunolabeling distribution of these proteins did not reveal concentration within plasma membrane pits. These findings which were confirmed by observations of cell surface labeling of two other ligands, Tf and apoB, are consistent with receptors and ligands gaining access to the endocytic machinery likely without receptor/ligand preclustering or prolonged clustering events within plasma membrane pits. Intracellularly, apoE was concentrated within endocytic structures which were double-labeled for apoE and internalized HRP. Large endocytic vesicles closely juxtaposed to Golgi stacks also revealed a high content of apoE. Together the endosomal labeling distribution of apoE as well as morphological features of endosomal components are consistent with the maturation model for endosomes.

Butyrate is a short chained fatty acid that induces histone hyperacetylation by inhibiting histone deacetylases. This hyperacetylation of histones then leads to a change in chromatin conformation and transcription of genes. Histone deacetylases have recently been found to directly affect gene expression by associating with transcriptional repressor complexes. The present thesis describes the initial characterization of histone deacetylase activity in variants of HeLa cells that are capable of growth in cytotoxic concentrations of butyrate. Analysis of acetylation levels of total histones by triton-acid-urea gels indicated that the histone deacetylases in the variants are less sensitive to the effects of histone deacetylase inhibitors than the parental HeLa cells. Control experiments showed that this resistance was not due to the transport of butyrate into the cell nor from the overexpression of HDAC1 or HDAC2, further suggesting that the resistance to butyrate is due to an alteration in a gene encoding HDAC.

The aim of our investigation was to gain insight on the regulation of flagellar movement, at the axonemal level. In our laboratory a panel of monoclonal antibodies (MoAbs) has been produced against the axoneme of the biflagellated algae, Chlamydomonas reinhardtii, a well-characterized model for the study of flagellar movement. Of these MoAbs, L2H12 has been selected, because it has a potent inhibitory effect on the motility of de membranated-reactivated flagella of Chlamydomonas cells. Using video micrography, we demonstrated that low concentrations of L2H12 cause a progressive decrease in the wave amplitude and beat frequency of the flagella. Results of Western blotting of the axonemal proteins indicates that L2H12 recognizes a 105 kDa protein. Analysis of Chlamydomonas radial spoke mutants deficient in one or more radial spoke proteins (RSPs) suggests that this protein is RSP2. Immunoprecipitation of this protein was performed to further characterize it.

The process of islet isolation destroys extracellular matrix and eliminates potentially important inter-cellular relationships. We have previously shown that isolated islets embedded in a type I collagen gel, in the presence of a defined medium, undergo a phenotypic switch to duct-like epithelial structures through a process known as transdifferentiation. The aim of this study was to characterize the specific effectors implicated in islet cell transdifferentiation in order to better understand the factors that confer morphogenetic stability on cells in the isolated islets. / We demonstrated cytoplasmic immunoreactivity for TGF-beta isoforms over 8 days post isolation using canine islets. Islet-to-duct epithelial transdifferentiation was correlated with the total amount of TGF-beta and was maximal at 48 h of culture. Up regulation of TGF-betaRI and TGF-betaRII expression on day 2 post-isolation was demonstrated by immunohistochemistry and Western blot analysis, and correlated temporally with the induction of cell proliferation. The presence of TGF-beta1 in culture supernatants was detected using the PAI/L assay. The peak TGF-beta1 level was 10.94 +/- 2.27 pM (active form) and 52.23 +/- 1.57 pM (total TGF-beta) at 48h. Addition of exogenous TGF-beta1 at different concentrations caused an accelerated and more pronounced epithelial transformation at 5--10 ng/mL compared to lower concentrations (0.5--1 ng/mL). / These studies confirm the biological potential of islets of Langerhans to transdifferentiate to duct epithelial structures. TGF-beta signal transduction appears to play an important role in this process.

Mechanical loading inhibits bone resorption by osteoclasts and stimulates bone formation by osteoblasts. We examined the mechanotransduction among bone cells. We generated osteoclasts and osteoblasts from mouse bone marrow, RAW 264.7 and MC3T3-E1 cells. Single cell was mechanically stimulated and changes in cytosolic free calcium concentration ([Ca2+]i) were recorded and analyzed. Mechanical stimulation of an osteoclast or an osteoblast induced a transient increase in its [Ca2+]i. Stimulated bone marrow osteoblasts had significantly higher basal [Ca2+]i, significant higher amplitude and significant faster [Ca2+]i rate of rise. Mechanical stimulation of a single osteoclast or osteoblast induced delayed transient elevations of [Ca2+]i in neighboring non-connected cells, consistent with propagation of an intercellular signal. Inhibitors studies suggest that the mediator is likely ATP degradation product, such as ADP, acting through Suramin-sensitive P2 receptor, potentially P2Y1. Our data demonstrated that mechanically stimulated bone cells release soluble mediator, which communicates this signal to neighboring non-connected cells. / Chargement mécanique inhibe la résorption osseuse par les ostéoclastes et stimule la formation osseuse par les ostéoblastes. Nous avons examiné la transduction mécanique entre les cellules osseuses. Nous avons généré des ostéoclastes et des ostéoblastes à partir de la moelle osseuse de souris, RAW 264.7 et MC3T3-E1 cellules. Une cellule a été stimulé mécaniquement et les modifications de la concentration de calcium cytosolique ([Ca2+]i) ont été enregistrées et analysées. Une stimulation mécanique d'un ostéoclaste ou un ostéoblaste induit une augmentation transitoire de son [Ca2+]i. Les ostéoblastes stimulés de moelle osseuse ont un [Ca2+]i basale significativement plus élevé, un amplitude significativement plus élevé et un taux d'augmentation de [Ca2+]i significativement plus rapide. D'une stimulation mécanique de un ostéoblaste ou un ostéoclaste, il ya eu des élévations retardés et transitoires de [Ca2+]i dans les cellules voisines pas connectés, compatible avec la propagation d'un signal intercellulaire. Les études des inhibiteurs suggèrent que le médiateur est probablement un produit de dégradation d’ATP, comme ADP, et est agit par l'intermédiaire des récepteurs sensibles de Suramine P2, potentiellement P2Y1. Nos données ont démontré que les cellules osseuses mécaniquement stimulé la libération de médiateur soluble, qui communique ce signal aux cellules voisins pas connectés.

Amyloid enhancing factor (AEF), which has recently been shown to have identity with ubiquitin (Ub), is believed to play a causative role in experimentally induced AA amyloidosis in mice. Here, the profile of Ub is examined in activated leukocytes and splenic reticulo-endothelial (RE) cells and its relationship with serum amyloid A protein (SAA) and AA amyloid (AA) deposits in an alveolar hydatid cyst (AHC)-infected mouse model of AA amyloidosis. In response to AHC infection, the diffuse Ub immunoreactivity in normal mouse leukocytes and RE cells promptly changed to a discrete granular pattern suggesting an increase in the intracellular concentration of Ub and the formation of Ub-protein conjugates. This corresponded to elevation in SAA levels, SAA uptake by Ub-positive phagocytic cells, co-localization of Ub and SAA in the RE, cells and deposition of Ub-bound AA amyploid in the splenic and hepatic tissues. Ultrastructural immunogold studies have revealed SAA/AA and Ub enrichment in endosome-lysosomes (EL) in activated monocytoid cells, thus implicating EL in AA formation. Aspects of AA amyloidogenesis are discussed in relation to other experimental models in which stress-induced Ub-protein conjugate formation and its transport to lysosomal (EL) vesicles have been studied. Whether plasma membrane bound or EL enzymes from activated murine splenic/hepatic RE cells degrade SAA$ sb2$ to generate amyloidogenic AA-like peptides is not clearly understood. These results suggest that Ub (or AEF)-loaded monocytoid cells may play an important role in the physiological processing of the sequestered SAA into AA amyloid.

The requirement of mitochondrial precursor proteins for cytosolic factors was investigated. The precursor to rat ornithine carbamyl transferase (pOCT) was imported into isolated mitochondria when synthesized in either animal or bacterial extracts. Neither it nor several engineered hybrid proteins could enter mitochondria in the absence of cytosol; import was reconstituted by adding back reticulocyte lysate. The use of one of the chimeras, pO-DHFR, showed that the requisite factor in lysate functions to maintain precursors in an import-competent conformation. pO-DHFR diluted from urea in the presence of mitochondria was imported, but this competence was lost when urea was removed in the absence of organelles, unless the cytosolic protein(s) was present. The factor was NEM-sensitive, did not need ATP to maintain precursor competence, and chromatographed with an apparent molecular weight in excess of 200 kDa when partially purified. The mg quantities of pO-DHFR purified from E. coli now available will facilitate purification of this novel mitochondrial competence factor.

The potential use of stem cells for therapy depends on our ability to optimize differentiation conditions to best take advantage of their multipotentiality. Skin derived precursor cells (SKPs) are recently described adult stem cells that have been shown to differentiate into both neuronal and mesodermal cell types in vitro. We asked specifically whether they could be induced to differentiate into skeletal muscle. An accurate assay for detecting skeletal muscle was first developed, using RT-PCR, immunocytochemistry and Western blotting. We then used traditional differentiation methods, and found that SKPs could differentiate into cells expressing an immature skeletal muscle phenotype, as determined by appropriate morphology and expression of various skeletal muscle markers. By modifying this protocol, we observed a small percentage of multinucleated, fused myotubes, suggesting that SKPs have the potential to differentiate into mature skeletal muscle given appropriate conditions. Future work could enhance this population, creating an accessible, new source of cells for autologous transplants.

Tissue engineering of the intervertebral disc using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. Since it is not known how to differentiate stem cells into disc cells, our rationale is to differentiate stem cells into chondrocyte-like cells. This proposal is based on the fact that cartilage and immature nucleus possess similar macromolecules in their matrix. Our hypothesis is that these cells can produce a matrix that mimics native nucleus pulposus with properties resembling that found in healthy intervertebral disc. We used a pellet culture system to promote in vitro chondrogenesis of MSCs, in which the cells were cultured in defined chondrogenic medium and supplemented with TGF-beta1 or TGF-beta3, IGF-1, with or without dexamethasone. Markers of chondrogenesis include collagen type II and aggrecan, with collagen type X being used as a marker of late stage chondrocyte hypertrophy. The purpose of this study was to investigate the above growth factors on the chondrogenic differentiation pathway using these markers to follow cell differentiation. Our results show evidence of constitutive expression of aggrecan message and early expression of type X collagen message, surprisingly before the appearance of that for type II collagen. This raises the question whether they are good markers of chondrogenesis and chondrocyte hypertrophy, respectively, during MSC differentiation.

Prosaposin is a 65 kDa glycoprotein that can either be targeted to the lysosomal compartment or further glycosylated to a 70 kDa form and secreted from the cell. In the lysosome, prosaposin is cleaved into four individual proteins known as saposins A, B, C and D that function as activators of sphingolipid degradation. Most soluble lysosomal proteins use the mannose 6-phophate receptor to traffic to the lysosome. However, several proteins including prosaposin have been shown not to use this mechanism. Since prosaposin binds several types of sphingolipids and that lipids are involved in the trafficking of proteins to various cellular destinations, we tested the hypothesis that these lipids mediate the trafficking of prosaposin to the lysosomes. Using inhibitors of sphingolipid synthesis in conjunction with mutagenic analysis and dominant negative competition with chimeric constructs, we demonstrated that the D functional domain of prosaposin interacts with sphingomyelin and that the carboxy-terminal region of prosaposin binds a sorting receptor. We also demonstrated that prosaposin binds to sortilin, a recently identified lysosomal sorting receptor. In conclusion, our research unfolded a novel mechanism of lysosomal transport involving a protein-lipid interaction and the sorting receptor sortilin.