Oxygen uptake rates in cultured rat hepatocytes

One potential treatment of acute liver failure involves the use of an extracorporeal device composed of functional hepatocytes. A major issue in the design of such a large-scale device is providing the hepatocytes with a sufficient supply of oxygen and other nutrients. In this study, we have designed and characterized a simple perfusion system hepatocytes using this system. The OUR of hepatocytes was determined during the first day after seeding on a single collagen gel and during the long-term stable culture after the addition of a top layer of collagen. The OUR increased to 20.7 +/- 0.57 pmol/sec/mug DNA during the first 13 hours of culture on a single collagen gel, while during the next 11 hours, the OUR declined to 10.6 +/- 1.5 pmol/sec/mug DNA. In parallel with the increase in OUR during the first 10 hours, we observed significant cell spreading, suggesting that the oxygen supply to the cells may be critical for the spreading and adaptation of the anchorage-dependent hepatocytes following isolation. Addition of a top layer of collagen to hepatocyte cultures for 24 hours of culture on a single collagen layer resulted in a stable OUR for 15 days. These results indicate that OUR of hepatocytes in culture may vary depending on the phase of culture (i.e., early vs. late) and on the extracellular environment. (c) 1992 John Wiley & Sons, Inc.     

微流控分析芯片在医学领域的应用

微全分析系统(μ_TAS)又称为芯片实验室,自从Manz等于20世纪90年代首次提出这一概念以来,经过十余年的发展μ_TAS已成为生物分析的一个独立领域并被学术界所认可。微流控分析芯片作为μ_TAS发展的主要方向以其快速、高效分析,低消耗和微型化等特点发展非常迅速。在此结合微流控分析芯片在医学领域的应用状况,着重从基因检测、蛋白质分析和细胞分析等方面,对该技术在医学领域里的应用及其未来发展趋势作一综述。     

Freeze-fracture autoradiography. Progress towards a routine technique

Freeze-fracture autoradiography was introduced in 1976 as a new technique for the autoradiography of diffusible compounds at the electron microscope level. With the original approach coating of the frozen replicated specimens was performed in a cryostat at atmospheric pressure. Ice contamination of the specimen surface acting as an outstanding source of artifacts was thereby not excluded. With the use of a specially designed coating device and volatile spreading substances it was made possible to coat the frozen replicated specimens in the maintained vacuum of the freeze-fracture plant. In this complicated technique we have recently extended the freeze-fracture autoradiography to labeled frozen-dried "half" membranes of red blood cells.     

An automated method for rapid determination of diffusion coefficients via measurements of boundary spreading

The use of a simple device by which a layer of solvent may be deposited onto a solution of an optically absorbing solute in a cylindrical quartz tube, without substantial mixing of solution and solvent, is described. The spreading of the boundary thus formed may be monitored as a function of time using an automated absorbance scanning device previously described [A. K. Attri and A. P. Minton (1983) Anal. Biochem. 133, 142-152]. A semiautomatic procedure for determining the diffusion coefficient from the time dependence of the shape of the boundary is described and is particularly well-suited for real-time data analysis with a laboratory microcomputer. The diffusion coefficients of several proteins have been measured using the technique reported, and the results are generally in good agreement with values reported in the literature. The feasibility of using this technique in combination with a previously described method for measuring the sedimentation coefficient [A. K. Attri and A. P. Minton (1984) Anal. Biochem. 136, 407-415] to rapidly determine the molecular weight of a protein is established.     

Selective Infection of Antigen-Specific B Lymphocytes by Salmonella Mediates Bacterial Survival and Systemic Spreading of Infection

Background

The bacterial pathogen Salmonella causes worldwide disease. A major route of intestinal entry involves M cells, providing access to B cell-rich Peyer’s Patches. Primary human B cells phagocytose Salmonella typhimurium upon recognition by the specific surface Ig receptor (BCR). As it is unclear how Salmonella disseminates systemically, we studied whether Salmonella can use B cells as a transport device for spreading.

Methodology/Principal Findings

Human primary B cells or Ramos cell line were incubated with GFP-expressing Salmonella. Intracellular survival and escape was studied in vitro by live cell imaging, flow cytometry and flow imaging. HEL-specific B cells were transferred into C57BL/6 mice and HEL-expressing Salmonella spreading in vivo was analyzed investigating mesenteric lymph nodes, spleen and blood. After phagocytosis by B cells, Salmonella survives intracellularly in a non-replicative state which is actively maintained by the B cell. Salmonella is later excreted followed by reproductive infection of other cell types. Salmonella-specific B cells thus act both as a survival niche and a reservoir for reinfection. Adoptive transfer of antigen-specific B cells before oral infection of mice showed that these B cells mediate in vivo systemic spreading of Salmonella to spleen and blood.

Conclusions/Significance

This is a first example of a pathogenic bacterium that abuses the antigen-specific cells of the adaptive immune system for systemic spreading for dissemination of infection.     

In situ determination of slurry nutrient content by electrical conductivity

Land application of animal slurries has both agronomic and environmental implications. It can be supported by the quantification of available nutrients in the field. A prototype device for indirect measurement of the nutrient content of slurry based on electrical conductivity (EC) was calibrated on manure samples collected from farms with different livestock typologies. The resulting correlations between EC and nutrient contents of slurries from laboratory analyses have shown good agreement (r(2) from 0.73 to 0.95) with total and ammoniacal nitrogen and, with some exception, Potassium, but failed to demonstrate any significant relationship with total phosphorous. The mean errors obtained using the device in field conditions for nitrogen content were always lower or equal to 10%, while the standard deviations were 12-13% for pig and calf slurries, and 20-21% for dairy cow slurry. The results obtained suggest that the equipment, provided the regression line used to convert EC readings to nutrient contents is related to the livestock typology under observation, can provide good support to practical slurry spreading, even though it does not reach an accuracy comparable to laboratory methods and does not give reliable information on phosphorus.     

Characterization of Endothelial Progenitor Cell Interactions with Human Tropoelastin

The deployment of endovascular implants such as stents in the treatment of cardiovascular disease damages the vascular endothelium, increasing the risk of thrombosis and promoting neointimal hyperplasia. The rapid restoration of a functional endothelium is known to reduce these complications. Circulating endothelial progenitor cells (EPCs) are increasingly recognized as important contributors to device re-endothelialization. Extracellular matrix proteins prominent in the vessel wall may enhance EPC-directed re-endothelialization. We examined attachment, spreading and proliferation on recombinant human tropoelastin (rhTE) and investigated the mechanism and site of interaction. EPCs attached and spread on rhTE in a dose dependent manner, reaching a maximal level of 56±3% and 54±3%, respectively. EPC proliferation on rhTE was comparable to vitronectin, fibronectin and collagen. EDTA, but not heparan sulfate or lactose, reduced EPC attachment by 81±3%, while full attachment was recovered after add-back of manganese, inferring a classical integrin-mediated interaction. Integrin α_Vβ₃ blocking antibodies decreased EPC adhesion and spreading on rhTE by 39±3% and 56±10% respectively, demonstrating a large contribution from this specific integrin. Attachment of EPCs on N-terminal rhTE constructs N25 and N18 accounted for most of this interaction, accompanied by comparable spreading. In contrast, attachment and spreading on N10 was negligible. α_Vβ₃ blocking antibodies reduced EPC spreading on both N25 and N18 by 45±4% and 42±14%, respectively. In conclusion, rhTE supports EPC binding via an integrin mechanism involving α_Vβ₃. N25 and N18, but not N10 constructs of rhTE contribute to EPC binding. The regulation of EPC activity by rhTE may have implications for modulation of the vascular biocompatibility of endovascular implants.     

Freeze-fracture autoradiography: The in-vacuo coating technique

Summary Freeze-fracture autoradiography (FFA) was introduced in 1976 as a new method for electron microscopic autoradiography of diffusible compounds (Fisher and Branton, Rix et al.). With the original technique, the film monolayer was applied to the cold specimen in a cryostat at atmospheric pressure. Coating under these conditions did not exclude the risk of artifacts, mainly due to uncontrolled ice contamination of the cold specimen surface.A new method has been developed for coating the frozen specimen, immediately after replication, in the maintained vacuum of the freeze-fracture unit. Two main components of the new technique are described in detail, a specially designed coating device, and the use of spreading substances promoting adhesion of the film in vacuo. Using this technique artifacts so far inherent in the FFA method can be eliminated.Supported by the Deutsche Forschungsgemeinschaft     

Cell spreading factor. Occurrence and specificity of action.

A factor required for spreading of substratum-attached baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, HeLa cells, and L cells has been isolated and purified from fetal calf serum. A similar factor has also been found in calf, porcine, human, rabbit, and chicken sera. The spreading factor was active when adsorbed to the substratum and prior adsorption of other proteins prevented cell spreading, regardless of the addition of spreading factor or unfractionated serum to the incubation medium. Antibody against the fetal calf spreading factor inhibited the spreading activity associated with unfractionated fetal calf serum and also the spreading activity associated with calf serum and porcine serum. In model system studies it was found that antibody against BHK cell surfaces induced cell spreading when the antibody was adsorbed to the substratum; when it was present in the incubation medium as well as on the substratum, cell spreading was not observed. The data are discussed in terms of the hypothesis that there is a specific serum factor which adsorbs to the substratum surface and is thereby activated, and which then forms the target for certain cell surface receptors. Interaction between adsorbed-activated factor and cell surface receptors leads to cell spreading.     

Rapid nested-PCR for tyrosinase gene detection on chip

The availability of non-invasive, fast and sensitive technologies for detection of circulating cancer cells is still a critical need of clinical oncology, particularly for diagnosis of aggressive and highly metastatic tumors, like malignant melanoma. Here we present the first nested polymerase chain reaction process carried out by a microfabricated, hybrid plastic-glass microfluidic chip on the tyrosinase gene, a predictive marker for melanoma diagnosis. The device is a hybrid system consisting of a glass microchannel embedded in an elastomeric matrix, and operating in flow-oscillating modality on a droplet of biological sample. The convection heat transfer and the temperature distribution inside the carrier fluid in the device are investigated. The oil responds to temperature changes with a characteristic time around 53 s, and exhibits three different thermal gradients along the capillary, with temperature variations below 4°C in correspondence of heater electrodes. The sample heating/cooling rates in the chip are as high as 16°C/s, allowing rapid processes. The nested polymerase chain reaction process is performed in less than 50 min, namely more than four times faster than in a standard thermocycler. The rapidity of the analysis method, combined with the simple and low-cost fabrication, reduced sample evaporation, and flexibility of the overall microfluidic platform, make it promising for the detection of events of tumor spreading.     

Activated R-ras, Rac1, PI 3-kinase and PKCepsilon can each restore cell spreading inhibited by isolated integrin beta1 cytoplasmic domains

Attachment of many cell types to extracellular matrix proteins triggers cell spreading, a process that strengthens cell adhesion and is a prerequisite for many adhesion-dependent processes including cell migration, survival, and proliferation. Cell spreading requires integrins with intact beta cytoplasmic domains, presumably to connect integrins with the actin cytoskeleton and to activate signaling pathways that promote cell spreading. Several signaling proteins are known to regulate cell spreading, including R-Ras, PI 3-kinase, PKCepsilon and Rac1; however, it is not known whether they do so through a mechanism involving integrin beta cytoplasmic domains. To study the mechanisms whereby cell spreading is regulated by integrin beta cytoplasmic domains, we inhibited cell spreading on collagen I or fibrinogen by expressing tac-beta1, a dominant-negative inhibitor of integrin function, and examined whether cell spreading could be restored by the coexpression of either V38R-Ras, p110alpha-CAAX, myr-PKCepsilon, or L61Rac1. Each of these activated signaling proteins was able to restore cell spreading as assayed by an increase in the area of cells expressing tac-beta1. R-Ras and Rac1 rescued cell spreading in a GTP-dependent manner, whereas PKCstraightepsilon required an intact kinase domain. Importantly, each of these signaling proteins required intact beta cytoplasmic domains on the integrins mediating adhesion in order to restore cell spreading. In addition, the rescue of cell spreading by V38R-Ras was inhibited by LY294002, suggesting that PI 3-kinase activity is required for V38R-Ras to restore cell spreading. In contrast, L61Rac1 and myr-PKCstraightepsilon each increased cell spreading independent of PI 3-kinase activity. Additionally, the dominant-negative mutant of Rac1, N17Rac1, abrogated cell spreading and inhibited the ability of p110alpha-CAAX and myr-PKCstraightepsilon to increase cell spreading. These studies suggest that R-Ras, PI 3-kinase, Rac1 and PKCepsilon require the function of integrin beta cytoplasmic domains to regulate cell spreading and that Rac1 is downstream of PI 3-kinase and PKCepsilon in a pathway involving integrin beta cytoplasmic domain function in cell spreading.     

On the synthesis of DNA error correcting codes

Regeneration of damaged central nervous systems (CNS) is an important topic in neuroscience and neuroengineering. Grafting new neurons derived from pluripotent stem cells into damaged regions can be done to restore functions after injury. Little is known, however, about network-wide interactions between stem-cell-derived neurons and CNS neurons. In this study, we developed a co-culture method of stem cell-derived neuronal networks and CNS networks and observed spontaneous activity in the co-culture samples. By using a microfabricated poly(dimethylsiloxane) device having two culture compartments and 20 connecting microconduits, we are able to compartmentalize P19-derived neurons and mouse cortical neurons and connect them via the microconduits. Furthermore, we combined the co-culture device and a microelectrode array (MEA)-based recording system and recorded spontaneous activity in the co-cultured networks. We found that periodic synchronized bursting spreading over both neuronal networks occurred during the second week in vitro and that P19-derived neurons in the co-cultured networks had different developmental processes compared with those grown in monoculture. These findings suggest that functional interactions form between P19-dervived neurons and mouse cortical neurons and that the co-culture method is useful for exploring the network-wide integrations between stem cell-derived neurons and CNS neurons.     

Macrophage spreading in vitro : II. Manganese and other metals as inducers or as co-factors for induced spreading

The cation requirement for the spreading of macrophages on glass and in simple media was examined. Several divalents allowed spreading induced by glass-bound immune-complexes. Their rank was MnCo > Ni, Zn > Cd > Mg > Fe in bicarbonate-buffered saline (SBK). Concentrations of Mn of 3μM allowed for extensive spreading in SBK while 10 μM were required for full spreading in Tris- or imidazole-buffered saline. Similar concentrations of Mn permitted spreading induced by dithiothreitol. Ca²⁺, Al³⁺ or Th⁴⁺, among other metals, were ineffective in immune-complex triggered spreading. Spreading in the presence of Mg was inhibited by EDTA but not by EGTA, giving further support for lack of a Ca requirement. At higher concentrations, Mn and several other metals induced macrophage spreading in the absence of other inducers. Features of the spreading promoted by 1 mM Mn were similar to those previously found for other inducers [23], and cells induced to spread by Mn were fully capable of phagocytosis. The specificity and the low concentrations needed for co-factor activity are incompatible with a charge reduction role of divalent cations and are consistent with a metal activated enzymatic reaction step. The similarity between macrophage spreading and cell to substrate adhesion in regard to Mg requirements and other features, suggests that cell ‘adhesion’, as usually measured, may be dependent on the extent of cell spreading.     

Myosin is involved in postmitotic cell spreading

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time- lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.     

Fibronectin‐mediated cell spreading requires ABBA‐Rac1 signaling

ABBA was reported to be an actin dynamics regulator. However, the molecular mechanism of action of ABBA is still totally obscure. Here, we show that ABBA is ubiquitously expressed in all the examined cultured cells. We found that expression of ABBA in NIH3T3 cells promotes cell spreading. ABBA binds to and markedly promotes cell spreading‐induced Rac1 activation. Cell spreading stimulates ABBA activation probably by inducing it tyrosine phosphorylation, which endows ABBA much higher activity to activate Rac1, and attenuates the interaction between ABBA and Rac1. Loss of function suggests that deletion of ABBA in C6‐R cells markedly inhibits Rac1 activation and cell spreading; this suggests that and the interaction between ABBA and activated Rac1 is required for ABBA‐promoted cell spreading. Taken together, our results indicate that ABBA is activated in response to cell spreading, which markedly promotes cell spreading, and ABBA is required for Rac1 activation and cell spreading. J. Cell. Biochem. 114: 773–781, 2013. © 2012 Wiley Periodicals, Inc.     

Band 4.1 proteins regulate integrin-dependent cell spreading

Integrins link the extracellular matrix (ECM) to the cytoskeleton to control cell behaviors including adhesion, spreading and migration. Band 4.1 proteins contain 4.1, ezrin, radixin, moesin (FERM) domains that likely mediate signaling events and cytoskeletal reorganization via integrins. However, the mechanisms by which Band 4.1 proteins and integrins are functionally interconnected remain enigmatic. Here we have investigated roles for Band 4.1 proteins in integrin-mediated cell spreading using primary astrocytes as a model system. We demonstrate that Proteins 4.1B and 4.1G show dynamic patterns of sub-cellular localization in astrocytes spreading on fibronectin. During early stages of cell spreading Proteins 4.1B and 4.1G are enriched in ECM adhesion sites but become more diffusely localized at later stages of spreading. Combinatorial inactivation of Protein 4.1B and 4.1G expression leads to impaired astrocyte spreading. Furthermore, in exogenous expression systems we show that the isolated Protein 4.1 FERM domain significantly enhances integrin-mediated cell spreading. Protein 4.1B is dispensable for reactive astrogliosis in experimental models of cortical injury, likely due to functional compensation by related Protein 4.1 family members. Collectively, these findings reveal that Band 4.1 proteins are important intracellular components for integrin-mediated cell spreading.     

Cell adhesion and spreading factor: Similarity to cold insoluble globulin in human serum

Data are presented which indicate that the serum factor required for cell adhesion and spreading is very similar to cold insoluble globulin. Clotting of plasma under conditions that remove cold insoluble globulin also remove the adhesion and spreading factor. The activity of the adhesion and spreading factor co-chromatographs with cold insoluble globulin antigenicity on DEAE-cellulose and the mobilities of adhesion and spreading factor and cold insoluble globulin are the same in disc gel electrophoresis. Finally, antibody which is directed against cold insoluble globulin cross-reacts with a single component in the adhesion and spreading factor and inhibits its activity. The close similarity of the cell adhesion and spreading factor with cold insoluble globulin suggests that, in vivo, cold insoluble globulin which is adsorbed to collagen or part of a fibrin clot may constitute the normal substratum for fibroblast adhesion and migration.     

Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein

The mammalian growth plate is a dynamic structure rich in extracellular matrix (ECM). Interactions of growth plate chondrocytes with ECM proteins regulate cell behavior. In this study, we compared chondrocyte adhesion and spreading dynamics on fibronectin (FN) and bone sialoprotein (BSP). Chondrocyte adhesion and spreading were also compared with fibroblasts to analyze potential cell-type-specific effects. Chondrocyte adhesion to BSP is independent of posttranslational modifications but is dependent on the RGD sequence in BSP. Whereas chondrocytes and fibroblasts adhered at similar levels on FN and BSP, cells displayed more actin-dependent spread on FN despite a 16x molar excess of BSP adsorbed to plastic. To identify intracellular mediators responsible for this difference in spreading, we investigated focal adhesion kinase (FAK)-Src and Rho-Rho kinase (ROCK) signaling. Although activated FAK localized to the vertices of adhered chondrocytes, levels of FAK activation did not correlate with the extent of spreading. Furthermore, Src inhibition reduced chondrocyte spreading on both FN and BSP, suggesting that FAK-Src signaling is not responsible for less cell spreading on BSP. In contrast, inhibition of Rho and ROCK in chondrocytes increased cell spreading on BSP and membrane protrusiveness on FN but did not affect cell adhesion. In fibroblasts, Rho inhibition increased fibroblast spreading on BSP while ROCK inhibition changed membrane protrusiveness of FN and BSP. In summary, we identify a novel role for Rho-ROCK signaling in regulating chondrocyte spreading and demonstrate both cell- and matrix molecule-specific mechanisms controlling cell spreading.     

Pre-emptive targeting of the epitope spreading cascade with genetically modified regulatory T cells during autoimmune demyelinating disease.

Epitope spreading or endogenous self-priming has been implicated in mediating the progression of autoimmune disease. In the present study we created an immune-deviated, epitope spreading response in SWXJ mice after the onset of experimental autoimmune encephalomyelitis, a prototypic autoimmune animal model widely used in multiple sclerosis research. We established an immunoregulatory spreading repertoire by transferring T cells genetically modified to produce high levels of IL-10 in response to a dominant epitope spreading determinant. Installation of a Th2/Tr1-like spreading repertoire resulted in a marked and prolonged inhibition of disease progression and demyelination characterized by 1) bystander inhibition of the recall response to the priming immunogen, and 2) a Th1-->Tr1 immune-deviated spreading response involving a shift in the source of IL-10 production from the transferred regulatory population to the host-derived, endogenously primed repertoire. Thus, our data provide a rationale for cell-based therapeutic intervention in multiple sclerosis by showing that pre-emptive targeting of the epitope spreading cascade with regulatory T cells effectively induces an immune-deviated spreading response capable of inhibiting ongoing inflammatory autoreactivity and disease progression.     

The vasodilator-stimulated phosphoprotein is regulated by cyclic GMP-dependent protein kinase during neutrophil spreading

The expression and phosphorylation state of the vasodilator-stimulated phosphoprotein (VASP), a membrane-associated focal adhesion protein, was investigated in human neutrophils. Adhesion and spreading of neutrophils induced the rapid phosphorylation of VASP. The phosphorylation of VASP was dependent on cell spreading, as VASP was expressed as a dephosphorylated protein in round adherent cells and was phosphorylated at the onset of changes in cell shape from round to spread cells. Immunofluorescence microscopy demonstrated that VASP was localized at the cell cortex in round cells and redistributed to focal adhesions at the ventral surface of the cell body during cell spreading. Dual labeling of spread cells indicated that VASP was colocalized with F-actin in filopodia and in focal adhesions, suggesting that the phosphorylation of VASP during cell spreading may be involved in focal adhesion complex organization and actin dynamics. VASP is a prominent substrate for both cGMP-dependent protein kinase (cGK) and cAMP-dependent protein kinase. Evidence suggested that cGK regulated neutrophil spreading, as both VASP phosphorylation and neutrophil spreading were inhibited by Rp-8-pCPT-cGMPS (cGK inhibitor), but not KT5720 (cAMP-dependent protein kinase inhibitor). In contrast, neutrophil spreading was accelerated when cGMP levels were elevated with 8-Br-cGMP, a direct activator of cGK. Furthermore, the same conditions that lead to VASP phosphorylation during neutrophil adherence and spreading induced significant elevations of cGMP in neutrophils. These results indicate that cGMP/cGK signal transduction is required for neutrophil spreading, and that VASP is a target for cGK regulation.