Precision of intracellular calcium spike timing in primary rat hepatocytes

Extracellular stimuli are often encoded in the frequency, amplitude and duration of spikes in the intracellular concentration of calcium ([Ca2+]i). However, the timing of individual [Ca2+]i-spikes in relation to the dynamics of an extracellular stimulus is still an open question. To address this question, we use a systems biology approach combining experimental and theoretical methods. Using computer simulations, we predict that more naturalistic pulsed stimuli generate precisely-timed [Ca2+]i-spikes in contrast to the application of constant stimuli of the same dose. These computational results are confirmed experimentally in single primary rat hepatocytes upon alpha1-adrenergic stimulation. Hormonal signalling in analogy to neuronal signalling thus has the potential to make use of temporal coding on the level of single cells. The [Ca2+]i-signalling cascade provides a first example for increasing the information capacity of an intracellular regulatory signal beyond the known coding mechanisms of amplitude (AM) and frequency modulation (FM).     

Mechanical and failure properties of single attached cells under compression

Eukaryotic cells are continuously subjected to mechanical forces under normal physiological conditions. These forces and associated cellular deformations induce a variety of biological processes. The degree of deformation depends on the mechanical properties of the cell. As most cells are anchorage dependent for normal functioning, it is important to study the mechanical properties of cells in their attached configuration. The goal of the present study was to obtain the mechanical and failure properties of attached cells. Individual, attached C2C12 mouse myoblasts were subjected to unconfined compression experiments using a recently developed loading device. The device allows global compression of the cell until cell rupture and simultaneously measures the associated forces. Cell bursting was characterized by a typical reduction in the force, referred to as the bursting force. Mean bursting forces were calculated as 8.7+/-2.5 microN at an axial strain of 72+/-4%. Visualization of the cell using confocal microscopy revealed that cell bursting was preceded by the formation of bulges at the cell membrane, which eventually led to rupturing of the cell membrane. Finite element calculations were performed to simulate the obtained force-deformation curves. A finite element mesh was built for each cell to account for its specific geometrical features. Using an axisymmetric approximation of the cell geometry, and a Neo-Hookean constitutive model, excellent agreement between predicted and measured force-deformation curves was obtained, yielding an average Young's modulus of 1.14+/-0.32 kPa.     

Pathway information for systems biology

Pathway information is vital for successful quantitative modeling of biological systems. The almost 170 online pathway databases vary widely in coverage and representation of biological processes, making their use extremely difficult. Future pathway information systems for querying, visualization and analysis must support standard exchange formats to successfully integrate data on a large scale. Such integrated systems will greatly facilitate the constructive cycle of computational model building and experimental verification that lies at the heart of systems biology.     

Cyclic tensile strain upregulates collagen synthesis in isolated tendon fascicles

Mechanical stimulation has been implicated as an important regulatory factor in tendon homeostasis. In this study, a custom-designed tensile loading system was used to apply controlled mechanical stimulation to isolated tendon fascicles, in order to examine the effects of 5% cyclic tensile strain at 1 Hz on cell proliferation and matrix synthesis. Sample viability and gross structural composition were maintained over a 24 h loading period. Data demonstrated no statistically significant differences in cell proliferation or glycosaminoglycan production, however, collagen synthesis was upregulated with the application of cyclic tensile strain over the 24 h period. Moreover, a greater proportion of the newly synthesised matrix was retained within the sample after loading. These data provide evidence of altered anabolic activity within tendon in response to mechanical stimuli, and suggest the importance of cyclic tensile loading for the maintenance of the collagen hierarchy within tendon.     

Determinants of antileukemia effects of allogeneic NK cells

In HLA-nonidentical bone marrow transplantation, we studied the characteristics of donor NK cells, recipient leukemia cells, and the cytokine environment that predict the antileukemia effects of allogeneic NK cells. We found that the risk of relapse in pediatric patients with hematologic malignancies was best predicted by a model taking into consideration the presence of inhibitory killer cell Ig-like receptors (KIRs) on the donor's NK cells and the absence of corresponding KIR ligand in the recipient's HLA repertoire (a receptor-ligand model). The risk of relapse was prognosticated less precisely by the Perugia donor-recipient KIR ligand-ligand mismatch model or by a natural cytotoxicity model. In contrast to the ligand-ligand model, we found that the new receptor-ligand model was accurate when analysis was applied to patients with lymphoid malignancy. These findings corroborate our observations that the recipient's KIR repertoire, which was derived from highly purified, HLA-disparate CD34+ cells, resumed a donor-specific pattern within 3 mo of transplantation, but did not correlate evidently with the donor or recipient ligand repertoire. In an in vitro assay and an in vivo mouse model, human NK cell cytotoxicity toward human leukemia cells with 11q23 chromosomal rearrangement increased with the number of receptor-ligand mismatch pairs or prestimulation with IL-12 and IL-18. These findings provide new insights into the determinants of antileukemia effects of allogeneic NK cells and therapeutic strategies.     

RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics

Legionella pneumophila translocates multiple bacterial effector proteins into host cells to direct formation of a replication vacuole for the bacterium. The emerging consensus is that formation of this compartment involves recruitment of membrane material that traffics between the endoplasmic reticulum (ER) and Golgi. To investigate this model, a targeted approach was used to knock down expression of proteins involved in membrane trafficking, using RNA interference in Drosophila cells. Surprisingly, few single knockdowns of ER-Golgi transport proteins decreased L. pneumophila replication. By analyzing double-stranded RNAs in pairs, combinations were identified that together caused defects in intracellular replication, consistent with the model that membrane traffic funnels into the replication vacuole from multiple sources. In particular, simultaneous depletion of the intermediate compartment and Golgi-tethering factor transport protein particle together with the ER SNARE protein Sec22 reduced replication efficiency, indicating that introduction of lesions at distinct sites in the secretory system reduces replication efficiency. In contrast to knockdowns in secretory traffic, which required multiple simultaneous hits, knockdown of single cytosolic components of ER-associated degradation, including Cdc48/p97 and associated cofactors, was sufficient to inhibit intracellular replication. The requirement for the Cdc48/p97 complex was conserved in mammalian cells, in which replication vacuoles showed intense recruitment of ubiquitinated proteins, the preferred substrates of Cdc48/p97. This complex promoted dislocation of both ubiquitinated proteins and bacterial effectors from the replication vacuole, consistent with the model that maintenance of high-level replication requires surveillance of the vacuole surface. This work demonstrates that L. pneumophila has the ability to gain access to multiple sites in the secretory system and provides the first evidence for a role of the Cdc48/p97 complex in promoting intracellular replication of pathogens and maintenance of replication vacuoles.     

Computational Modeling to Predict the Temporal Regulation of Chondrocyte Metabolism in Response to Various Dynamic Compression Regimens

Based on previously published experimental work, computational models were developed to simulate the effect of different dynamic compression regimens on the activity of chondrocytes seeded in agarose constructs. In particular, the balance between proliferation and matrix synthesis can be adjusted by applying different intervals of continuous or intermittent mechanical compression. A phenomenological compartment based-modeling approach was used as first model. A more mechanistic cell cycle model was used as the second model. The compartment-based modeling approach was found to be useful in representing a balance between proliferation and proteoglycan synthesis, when the effect of a certain stimulation protocol is known. In order to predict the response to different intervals of mechanical stimulation, however, a more mechanistic cell cycle-based approach is required. The cell cycle model supports an important role of the onset of loading. In addition, an inhibitory effect of further loading is required, which is more likely to be related to cell cycle progression velocity than to a decreased probability of commitment to the cell cycle. The mechanisms behind this inhibitory effect and the computational implementation, however, require further investigation.     

Intracellular nucleotide pools and ratios as tools for monitoring dedifferentiation of primary porcine hepatocytes in culture

The effect of two culture configurations (single collagen gel and double collagen gel) and of two hormones (insulin and glucagon) on the differentiated status and the intracellular nucleotide pools of primary porcine hepatocytes was investigated. The objective was to analyze and monitor the current state of differentiation supported by the two culture modes using intracellular nucleotide analysis. Specific intracellular nucleotide ratios, namely the nucleoside triphosphate (NTP) and the uridine (U) ratio were shown to consistently reflect the state of dedifferentiation status of the primary cells in culture affected by the presence of the two hormones insulin and glucagon. Continuous dedifferentiation of the cells was monitored in parallel by the reduction of the secretion of albumin, and changes in UDP-activated hexoses and UDP-glucuronate. The presence of insulin maintained the differentiated status of hepatocytes for more than 12 days when cultivated under double gel conditions whereas glucagon was less effective. In contrast, cells cultivated in a single gel matrix immediately started to dedifferentiate upon seeding. NTP and U ratios were shown to be more sensitive for monitoring dedifferentiation in culture than the albumin secretion. Their use allowed the generation of an easily applicable NTP–U plot in order to give a direct graphical representation of the current differentiation status of the cultured cells. Moreover, the transition from functional and differentiated hepatocytes to dedifferentiated fibroblasts could be determined earlier by the nucleotide ratios compared to the conventional method of monitoring the albumin secretion rate.