Gel-based proteomics of liver cancer progression in rat

A significant challenge in proteomics biomarker research is to identify the changes that are of highest diagnostic interest, among the many unspecific aberrations associated with disease burden and inflammation. In the present study liver tissue specimens (n=18) from six experimental stages were collected from the resistant hepatocyte (RH) rat model of liver cancer and analyzed by 2D DIGE. The study included triplicates of regenerating liver, control "sham-operated" liver, three distinct premalignant stages and hepatomas. Out of 81 identified proteins two-thirds were differentially abundant in rat hepatomas compared to control rat liver and, secondly, the majority of proteins were also changed in precursor stages. This underscores the importance of adequate control samples in explorative cancer biomarker research. We confirm several proteomic changes previously identified in human hepatocellular carcinoma (HCC) and we identify novel candidate proteomic aberrations for further analysis in human HCC. In particular, increased levels of HSP70, HSP90, AKR1B1, AKR7A3, GCLM, ANXA5, VDBP, RGN and SULT1E1 were associated specifically with rat hepatomas, or with liver cancer progression in rat. In addition, we examine an integrated gel-based workflow for analysis of protein post-translational modifications (PTMs) and microtubule-association. We highlight differential PTM and localization of HSP60 as an interesting target for further analysis in liver cancer.     

The role of extrahepatic retinol binding protein in the mobilization of retinoid stores

Although the major tissue site of retinol binding protein (RBP) synthesis in the body is the liver, other sites of synthesis have been reported. The physiological role(s) of circulating RBP that is produced and secreted extrahepatically has not been systematically investigated. To address this question, we used as a model a mouse strain (hRBP(-/-)) that expresses human RBP (hRBP) cDNA under the control of the mouse muscle creatine kinase promoter in an rbp-null background (RBP(-/-)). By comparing hRBP(-/-), RBP(-/-), and wild-type mice, we asked whether extrahepatic RBP can perform all of the physiological functions of RBP synthesized in the liver. We demonstrate that extrahepatically synthesized hRBP, unlike RBP expressed in liver, cannot mobilize liver retinoid stores. Consistent with this conclusion, we find that circulating hRBP is not taken up by hepatocytes. RBP has been proposed to play an essential role in distributing hepatic retinoids between hepatocytes and hepatic stellate cells. We find, however, that the distribution of retinoid in the livers of the three mouse strains described above is identical. Thus, RBP is not required for intrahepatic transport and storage of retinoid. These and other observations are discussed.     

Adenosinetriphosphatase Activity in the Cell Membranes of Kidney Tubule Cells

This cytochemical study demonstrates high levels of apparent ATPase activity in the infolded cell membranes of the proximal tubules (dog, rat, human, mouse, monkey, and opossum) and ascending loops of Henle (dog, rat, human and, to a variable degree, mouse). Electron microscopy has shown (see Rhodin (1)) that these membranes separate adjacent cells where they interlock with one another by multiple cytoplasmic lamellae containing oriented mitochondria. The significance of the high ATPase activity is considered in relation to possible movements of the membranes and to "active transport" believed to occur there. In the rat, enzyme activity in the proximal tubule membranes does not survive formol-calcium fixation, and it is therefore necessary to use unfixed sections for its demonstration. However, in edematous rats with experimental nephrosis (induced by the injection of aminonucleoside or with antikidney serum) marked ATPase activity is exhibited in these membranes even after formol-calcium fixation. When proximal tubule or Henle loop cells of the dog acquire an altered metabolism, as indicated by accumulated lipide spheres or by "droplets," the infolded ATPase-rich membranes are no longer demonstrable.     

GOLGI APPARATUS, GERL, AND LYSOSOMES OF NEURONS IN RAT DORSAL ROOT GANGLIA, STUDIED BY THICK SECTION AND THIN SECTION CYTOCHEMISTRY

New insights into the ultrastructure and phosphatase localizations of Golgi apparatus and GERL, and into the probable origin of lysosomes in the neurons of fetal dorsal root ganglia and the small neurons of adult ganglia have come from studying thick (0.5–1.0 µ) as well as thin (up to 500 A) sections by conventional electron microscopy. Tilting the thick specimens, by a goniometer stage, has helped to increase our understanding of the three-dimensional aspects of the Golgi apparatus and GERL. One Golgi element, situated at the inner aspect of the Golgi stack, displays thiamine pyrophosphatase and nucleoside diphosphatase activities. This element exhibits regular geometric arrays (hexagons) of interconnected tubules without evidence of a flattened portion (saccule or cisterna). In contrast, GERL shows acid phosphatase activity and possesses small cisternal portions and anastomosing tubules. Lysosomes appear to bud from GERL. Osmium deposits, following prolonged osmication, are found in the outer Golgi element. Serial 0.5-µ and thin sections of thiamine pyrophosphatase-incubated material demonstrate that, in the neurons studied, the Golgi apparatus is a continuous network coursing through the cytoplasm. Serial thick sections of acid phosphatase-incubated tissue suggest that GERL is also a continuous structure throughout the cytoplasm. Tubules of smooth endoplasmic reticulum, possibly part of GERL, extend into the polygonal compartments of the inner Golgi element. The possible physiological significance of a polygonal arrangement of a phosphatase-rich Golgi element in proximity to smooth ER is considered. A tentative diagram of the Golgi stack and associated endoplasmic reticulum in these neurons has been drawn.     

EFFECTS OF ARGININE DEPRIVATION, ULTRAVIOLET RADIATION, AND X-RADIATION ON CULTURED KB CELLS : A Cytochemical and Ultrastructural Study

Cultured KB cells (derived from a human oral carcinoma) grown in monolayers were injured by one of three agents: starvation by arginine deprivation or treatment with high doses of either ultraviolet radiation or x-radiation. The different agents produced changes in nucleolar structure and varying accumulations of triglyceride and glycogen. All three agents produced an increase in number and size of lysosomes. These were studied in acid phosphatase preparations, viewed by both light and electron microscopy, and, occasionally, in vital dye, esterase, and aryl sulfatase preparations. Ultrastructurally, alterations in lysosomes suggested that "residual bodies" developed in a variety of ways, i.e., from the endoplasmic reticulum, multivesicular bodies, or autophagic vacuoles. Following all three agents the endoplasmic reticulum assumed the form of "rough" or "smooth" whorls, and, after two of the agents, arginine deprivation or ultraviolet radiation, it acquired cytochemically demonstrable acid phosphatase activity. Near connections between the endoplasmic reticulum and lysosomes raise the possibility that in KB cells, at least when injured, the endoplasmic reticulum is involved in the formation of lysosomes and the transport of acid phosphatase to them.     

Cyclin D1 determines mitochondrial function in vivo

The cyclin D1 gene encodes a regulatory subunit of the holoenzyme that phosphorylates and inactivates the pRb tumor suppressor to promote nuclear DNA synthesis. cyclin D1 is overexpressed in human breast cancers and is sufficient for the development of murine mammary tumors. Herein, cyclin D1 is shown to perform a novel function, inhibiting mitochondrial function and size. Mitochondrial activity was enhanced by genetic deletion or antisense or small interfering RNA to cyclin D1. Global gene expression profiling and functional analysis of mammary epithelial cell-targeted cyclin D1 antisense transgenics demonstrated that cyclin D1 inhibits mitochondrial activity and aerobic glycolysis in vivo. Reciprocal regulation of these genes was observed in cyclin D1-induced mammary tumors. Cyclin D1 thus integrates nuclear DNA synthesis and mitochondrial function.     

Collagen-induced arthritis in common marmosets: a new nonhuman primate model for chronic arthritis

Introduction  

There is an ever-increasing need for animal models to evaluate efficacy and safety of new therapeutics in the field of rheumatoid arthritis (RA). Particularly for the early preclinical evaluation of human-specific biologicals targeting the progressive phase of the disease, there is a need for relevant animal models. In response to this requirement we set out to develop a model of collagen-induced arthritis (CIA) in a small-sized nonhuman primate species (300 to 400 g at adult age); that is, the common marmoset (Callithrix jacchus).     

Aspects of early arthritis. What determines the evolution of early undifferentiated arthritis and rheumatoid arthritis? An update from the Norfolk Arthritis Register

Over 3500 patients with recent onset inflammatory polyarthritis (IP) have been recruited by the Norfolk Arthritis Register (NOAR) since 1990. Longitudinal data from this cohort have been used to examine the prevalence and predictors of remission, functional disability, radiological outcome, cardiovascular mortality and co-morbidity and the development of non-Hodgkin's lymphoma. Rheumatoid factor titre, high baseline C-reactive protein and high baseline HAQ score are all predictors of a poor outcome. There is a strong association between possession of the shared epitope and the development of erosions. Patients who satisfy the American College of Rheumatology criteria for rheumatoid arthritis (RA) have a worse prognosis than those who do not. However, it appears that these patients are a poorly defined subset of all those with IP rather than having an entirely separate disease entity. New statistical techniques offer exciting possibilities for using longitudinal datasets such as NOAR to explore the long-term effects of treatment in IP and RA.     

Liver repopulation with hepatocyte transplantation: new avenues for gene and cell therapy

Liver-directed gene therapy is appropriate for many conditions. Recent work established that liver repopulation with transplanted cells can be effective in treating genetic disorders. Although hepatocytes express therapeutic genes with considerable efficiency, correction of genetic disorders is constrained by limitations in permanent gene transfer into hepatocytes and repopulation of the liver with transplanted cells. Adenoviral vectors are highly efficient for hepatic gene transfer but the onset of deleterious host immune responses against adenoviral vectors, along with clearance of transduced hepatocytes have caused problems. Nonetheless, recent work concerning engraftment and proliferation of transplanted hepatocytes in the liver has provided significant new information, which should refocus interest in hepatocyte-based therapies. Moreover, hepatocyte transplantation systems offer creative tools for defining critical mechanisms in gene regulation and survival of transduced cells.