Engineering hepatocyte functional fate through growth factor dynamics: the role of cell morphologic priming.

We have reported previously that cellular stimulation induced by variable mechanochemical properties of the extracellular microenvironment can significantly alter liver-specific function in cultured hepatocytes (Semler et al., Biotech Bioeng 69:359-369, 2000). Cell activation via time-invariant presentation of biochemical growth factors was found to either enhance or repress cellular differentiation of cultured hepatocytes depending on the mechanical properties of the underlying substrate. In this work, we investigated the effects of dynamic growth factor stimulation on the cell growth and differentiation behavior of hepatocytes cultured on either compliant or rigid substrates. Specifically, hepatotrophic growth factors (epidermal and hepatocyte) were either temporally added or withdrawn from hepatocyte cultures on Matrigel that was crosslinked to yield differential degrees of mechanical compliance. We determined that the functional responsiveness of hepatocytes to fluctuations in GF stimulation is substrate specific but only in conditions in which the initial mechanochemical environment induced significant cell morphogenesis. Our studies indicate that in conditions under which hepatocytes adopted a "rounded" phenotype, they exhibited increased levels of differentiated function upon soluble stimulation and markedly decreased function upon the depletion of GF stimulation. In contrast, hepatocytes that assumed a "spread" phenotype exhibited slightly increased function upon the depletion of GF stimulation. By examining the functional responsiveness of hepatocytes of differential morphology to varied fluctuations in GF activation, insights into the ability of cell shape to "prime" hepatocyte behavior in dynamic microenvironments were elucidated. We report on the possibility of uncoupling and, thus, selectively manipulating, the concerted contributions of GF-induced cellular activation and substrate- and GF-induced cell morphogenesis toward induction of cell function.     

Nanoscale amphiphilic macromolecules as lipoprotein inhibitors: the role of charge and architecture

A series of novel amphiphilic macromolecules composed of alkyl chains as the hydrophobic block and poly(ethylene glycol) as the hydrophilic block were designed to inhibit highly oxidized low density lipoprotein (hoxLDL) uptake by synthesizing macromolecules with negatively charged moieties (ie, carboxylic acids) located in the two different blocks. The macromolecules have molecular weights around 5,500 g/mol, form micelles in aqueous solution with an average size of 20-35 nm, and display critical micelle concentration values as low as 10(-7) M. Their charge densities and hydrodynamic size in physiological buffer solutions correlated with the hydrophobic/ hydrophilic block location and quantity of the carboxylate groups. Generally, carboxylate groups located in the hydrophobic block destabilize micelle formation more than carboxylate groups in the hydrophilic block. Although all amphiphilic macromolecules inhibited unregulated uptake of hoxLDL by macrophages, inhibition efficiency was influenced by the quantity and location of the negatively charged-carboxylate on the macromolecules. Notably, negative charge is not the sole factor in reducing hoxLDL uptake. The combination of smaller size, micellar stability and charge density is critical for inhibiting hoxLDL uptake by macrophages.     

What's Nu(SAP) in mitosis and cancer?

Unperturbed mitosis is a prerequisite for the generation of two genetically identical daughter cells. Nucleolar-spindle associated protein (NuSAP) is an important mitotic regulator. The activity of NuSAP is essential for a variety of cellular events that occur during mitosis starting from spindle assembly to cytokinesis. In addition to playing crucial roles during mitosis, NuSAP has been in the spotlight recently due to different studies exhibiting its importance in embryogenesis and cancer. In this review, we have extensively mined the current literature and made connections between different studies involving NuSAP. Importantly, we have assembled data pertaining to NuSAP from several proteomic studies and analyzed it thoroughly. Our review focuses on the role of NuSAP in mitosis and cancer, and brings to light several unanswered questions regarding the regulation of NuSAP in mitosis and its role in carcinogenesis.     

Functional engineering of hepatocytes via heterocellular presentation of a homoadhesive molecule, E-cadherin.

Engineering functional activity of liver cell cultures requires the modulation of specific cell-cell interactions. We have investigated the quantitative role of systematically varied presentation of the cell-cell adhesion molecule, E-cadherin, on the differentiated function of cocultured parenchymal liver cells, hepatocytes. Specifically, we incorporated different proportions of E-cadherin transfected L-929 chaperone cells and untransfected chaperone cells, within cultures of primary rat hepatocytes on a collagen substrate. By using a strongly adhesive substrate that restricted cadherin-induced variations in cell spreading and growth-arresting chaperone cells, we could carefully isolate the potential role of cell-cell adhesion on cell differentiation. Using immunofluorescence microscopy, we confirmed that cadherins expressed at hepatocyte-hepatocyte contacts as well as hepatocyte-chaperone contacts were crossreactive. However, hepatocytes cocultured with cadherin-presenting chaperone cells had a 55-65% increase in longterm function over hepatocytes cocultured with control, nonpresenting chaperone cells. Notably, the cadherin-induced increase in function occurred over and above the basal, coculture-induced functional elevation. Further, we quantified the stoichiometric importance of cadherin contacts by comparing established markers of hepatocyte functional activity across a graded range of E-cadherin presentation. At low levels of cadherin-mediated contacts, the induction of differentiated function was weak, while high levels of contacts elicited a marked increase in function. Thus, hepatocyte biochemical functions (albumin and urea secretion) were biphasically governed by the degree of cadherin-based contacts presented during culture. Overall, our results demonstrate the unequivocal role of cell-cell adhesion molecules in hepatocyte functional engineering, through the graded use of cadherin presentation from functionally incompetent, heterotypic chaperone cells.     

Glutathione preconditioning attenuates Ac-LDL-induced macrophage apoptosis via protein kinase C-dependent Ac-LDL trafficking

Oxidized low-density lipoprotein (ox-LDL) incorporation into intimally resident vascular cells via scavenger receptors marks one of the early steps in atherosclerosis. Cellular apoptotic damage results from two major serial intracellular events: the binding and scavenger receptor-mediated uptake of oxidizable lipoproteins and the intracellular oxidative responses of accumulated lipoproteins. Most molecular approaches to prevent apoptotic damage have focused on singular events within the cascade of lipoprotein trafficking. To identify a multifocal strategy against LDL-induced apoptosis, we evaluated the role of cellular preconditioning by glutathione-ethyl ester (GSH-Et), a native redox regulator, in the prevention of the uptake and apoptotic effects of an oxidizable scavenger receptor-specific ligand, acetylated low-density lipoprotein (Ac-LDL). Our results indicate that GSH-Et-mediated protein kinase C (PKC) pathway modulation regulates Ac-LDL binding and incorporation into GSH-Et preconditioned cells and subsequently delays reactive oxygen intermediate generation and apoptotic conversion. The GSH-Et protective effects on apoptosis and Ac-LDL binding were reversed by calphostin C, a PKC inhibitor, and were accompanied by an increase in PKC phosphorylation. However, the rate of reactive oxygen intermediate accumulation was not increased following calphostin C treatment, suggesting that GSH-Et may play an important nonreactive oxygen-intermediate-based protective role in regulating apoptotic dynamics. Overall, we report on the novel role for GSH-Et preconditioning as a molecular strategy to limit lipoprotein entry into the cells, which presents a proactive modality to prevent cellular apoptosis in contrast with the prevalent antioxidant approaches that treat damage retroactively.     

A microsatellite-based consensus linkage map for species of Eucalyptus and a novel set of 230 microsatellite markers for the genus

Background  

Eucalypts are the most widely planted hardwood trees in the world occupying globally more than 18 million hectares as an important source of carbon neutral renewable energy and raw material for pulp, paper and solid wood. Quantitative Trait Loci (QTLs) in Eucalyptus have been localized on pedigree-specific RAPD or AFLP maps seriously limiting the value of such QTL mapping efforts for molecular breeding. The availability of a genus-wide genetic map with transferable microsatellite markers has become a must for the effective advancement of genomic undertakings. This report describes the development of a novel set of 230 EMBRA microsatellites, the construction of the first comprehensive microsatellite-based consensus linkage map for Eucalyptus and the consolidation of existing linkage information for other microsatellites and candidate genes mapped in other species of the genus.