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Portal fibroblasts are newly discovered liver cells that may be of particular importance in biliary fibrosis. Recent data indicate that portal fibroblasts express NTPDase2, an ecto-nucleoside triphosphate diphosphohydrolase. Portal fibroblasts exist within the peri-portal regions of rat livers and express NTPDase2 adjacent to the basolateral side of intrahepatic bile ducts. Because extracellular nucleotides regulate secretion via activation of P2Y purinergic receptors, extracellular nucleotide hydrolysis via NTPDase2 makes NTPDase2 a potential regulator of bile ductular secretion. We propose that NTPDase2 expression may be altered in biliary fibrosis, especially in conditions in which bile duct epithelia are the target of disease. To test this hypothesis we have contrasted the distribution of NTPDase2 in normal and diseased liver states. Using confocal immunofluorescence, we assessed differences in expression of NTPDase2 in liver biopsy specimens from normal liver, primary biliary cirrhosis (PBC), and hepatitis C (HepC). We found that NTPDase2 was down regulated in the peri-portal regions of patients with PBC when compared to normal patients. Hepatitis C, however, showed NTPDase2 staining equal to or nearly equal to that of normal liver. The intermediate filament vimentin was down regulated in both PBC and Hep C when compared to normal liver. We conclude that NTPDase2 expression is down regulated in PBC but not Hep C, while vimentin is down regulated in both disease states when compared to normal liver.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--Case Western Reserve University, 2005. / Author's name on approval form and OhioLink ETD database: Maria Isabel Rico-Salas. [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--Case Western Reserve University, 2006. / [School of Medicine] Department of Pathology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Cellular prion protein (PrPC ) is a membrane bound glycoprotein. The protein is expressed in all vertebrates, mainly in the nervous system, but it is present also in the cells of gastrointestinal tract, bone marrow, germ cells and heart. PrPC is necessary for pathogenesis of prion diseases, which are deadly and without the possibility of therapy. The pathogenic isoform of prion protein is formed by changing of secondary structure of PrPC and it's the main constituent of infectious prion particles. Pathological form of prion protein accumulates in brain of infected patients and this process is associated with neurodegradation. Physiological function of PrPC is poorly understood. Knock-out of the PrPC gene (PRNP) is not connected with any noticeable phenotype. Potential functions of PrPC are dispersed, protein may have antiapoptotic effect, it can be involved in ions metabolism or in protection against oxidative stress. Latest results show, that PrPC can play important role in cell differentiation. During the differentiation PrPC can influence the development of cells and their typing. It could affect cell cycle and have an influence on formation of nervous system. Aim of the present study was to elucidate, whether the down-regulation of PrPC or infection with prions has an impact on differentiation of...

Sorghum (Sorghum bicolor L. Moench) ranks fifth worldwide in production among cereals. It is a major staple food for millions in Africa and Asia, and a major livestock feed grain in developed countries. However, the sorghum grain is poor in lysine content, limiting its value as food and feed. In this study, I hypothesize that reduction of some of the major storage proteins that are inherently poor in lysine through in vitro manipulation will result in the enhanced expression of proteins with a better lysine profile and, thus, increased overall grain lysine content. Sorghum genotypes were screened for in vitro amenability and a sorghum genotype-tissue culture medium combination that yielded the highest somatic embryo callus formation and regeneration potential, was identified. This resulted in the establishment of a sorghum biolistic transformation method with a transformation efficiency of 3.36%, the highest reported to date. Using genetic engineering tools, the enhancement of the nutritional quality of grain sorghum was achieved by increasing the seed lysine content. An RNAi co-suppression strategy was employed and resulted in 45.23 and 77.55% increase in whole seed and endosperm lysine increase, respectively. The co-suppression RNAi constructs targeted the endosperm specific suppression of three lysine-poor storage proteins, namely ä-kaf-2, ã-kaf-1 and -2, and an enzyme that catalyzes seed lysine degradation, lysine keto-gluterate reductase (LKR). Seven independent transgenic events displayed successful transgene integration for both the selectable marker gene and the target constructs. However, the Southern blot hybridization analysis revealed two transgenic events that displayed transgene re-arrangement at the 5’promoter end, thus resulting in a lack of suppression of target proteins. Variations in target proteins co-suppression was observed with Western blot analysis and RT-PCR for both the target kafirins and LKR suppression, and no lysine improvement was observed where no kafirin suppression occurred. The transgenic co-suppression of the target kafirins resulted in the endosperm structural change from a hard, corneous endosperm to a soft, floury endosperm, consistent with ã-zein suppression in the Opaque-2 maize mutant. / Thesis (PhD)--University of Pretoria, 2010. / Plant Science / unrestricted

Influenza A virus (IAV) is a major threat to human health with seasonal epidemics, occasional pandemics and emergence of new highly pathogenic strains from the animal reservoir. Our laboratory has shown that the human Annexin A6 (AnxA6) interacts with the IAV M2 proton channel and limits production of progeny IAV from infected cells. We have found that overexpression of AnxA6 impairs morphogenesis and release of progeny viruses. These findings are supported by another study showing that AnxA6 has a critical role in the late endosomal cholesterol balance and affects IAV replication and propagation in AnxA6-overexpressing cells. However, the molecular mechanism responsible for restriction of IAV morphogenesis by AnxA6 is still unclear. AnxA6 is a calcium-dependent phospholipid-binding protein which plays a major role in cellular events such as regulation of cholesterol homeostasis and membrane organisation or repair. AnxA6 is also implicated in the regulation of intracellular signalling pathways required for IAV infection. In this study, we used a combination of virology, cellular biology and biochemistry approaches to decipher the restriction mechanism of IAV by human AnxA6. We found that AnxA6 down-regulates M2 viral protein expression and impairs viral morphogenesis and budding. We also found that AnxA6 regulates chemokines and cytokines expression during viral infection, suggesting that AnxA6 triggers an innate immune response to IAV by modulating signalling pathways required for viral replication. Finally, we observed that IAV down-regulates AnxA6 expression at mRNA level during early stages of infection and at protein level during late infection, suggesting that IAV has developed a strategy to respond to AnxA6 restriction mechanism during viral infection. We conclude that it is essential to better understand the interaction between human AnxA6 and IAV to elucidate the potential of AnxA6 as an antiviral candidate.

The muscarinic acetylcholine type 2 receptor (M2 mAChR), a member of the GPCR superfamily, is found throughout the parasympathetic nervous system where it controls pulmonary, urinary, and cardiac function, and neurotransmission. The molecular mechanisms that regulate M2 mAChR availability at the cell surface are an important component in controlling these physiological events. Since beta-arrestin proteins are known to regulate the activity of other GPCRs, we sought to identify their role in regulating M2 mAChR activity, a topic that remains contentious in the field. To achieve this goal we utilized mouse embryonic fibroblasts (MEFs) derived from beta-arrestin knockout mice lacking one or both isoforms (MEF KO1, KO2, or KO1/2 cells) in addition to exogenous expression of beta-arrestin mutants. This study demonstrates that agonist-induced internalization of M2 mAChR is beta-arrestin- and clathrin-dependent, and that the receptor stably co-localizes with beta-arrestin in early endosomal vesicles suggesting it behaves as a class B receptor. Next, we sought to identify beta-arrestin s function in regulating the post-endocytic trafficking (down-regulation) of the M2 mAChR. MEF KO1/2 cells were unable to down-regulate M2 mAChRs whereas MEF KO1 or KO2 cells retained the ability to do so. In MEFwt cells, both M2 mAChR and beta-arrestin exhibited basal ubiquitination that increased following agonist stimulation. Receptor degradation appeared to be regulated by the ubiquitination status of beta-arrestin 2, since expression of a chimeric â-arrestin 2 form fused to ubiquitin increased both constitutive and agonist-promoted down-regulation, whereas expression of a beta-arrestin 2 mutant lacking putative ubiquitination sites, beta-arrestin 2K18R, K107R, K108R, K207R, K296R, significantly blocked degradation while internalization and stable association remained intact. Upon further analysis, the beta-arrestin 2K18R, K107R, K108R, K207R, K296R mutant blocked delivery of M2 mAChR to the late endosome/lysosome, presumably where degradation occurs. Inhibition of proteasome-dependent recycling of ubiquitin blocked receptor down-regulation without affecting internalization or the ubiquitination state of the M2 mAChR while ubiquitination of beta-arrestin 2 diminished significantly. These results support a role for ubiquitinated beta-arrestin in mediating M2 mAChR sorting and degradation in the lysosome. Collectively, these studies give us new insight on the function of beta-arrestin in regulating the activity of the M2 mAChR.