Delay model of circadian gene expression with two negative feedback loops

In this theoretical paper we propose a quantitative minimal model for circadian gene expression based on two negative feedback loops. We perform numerical simulations to analyse its dynamics and parameter sensitivities in free-running conditions, and verify the entrainability by a single periodic driver. We furthermore apply two simultaneously acting external drivers, leading to aperiodic oscillations in the case of a single-loop system. These can be turned into regular periodic oscillations by introduction of a second loop. Our studies confirm the increasing evidence that multiple feedback loops increase the robustness of regulatory systems, and stress the particular situation of systems that are close to transition from free-running oscillation to steady-state behaviour. We discuss possible molecular realisations of the featured feedback loops and suggest the application of complex patterns of external stimulation as a generally useful approach to assess the functionality of models of circadian systems.     

Sprouty fine-tunes EGF signaling through interlinked positive and negative feedback loops

BACKGROUND: Growth factors and their receptor tyrosine kinases play pivotal roles in development, normal physiology, and pathology. Signal transduction is regulated primarily by receptor endocytosis and degradation in lysosomes ("receptor downregulation"). c-Cbl is an adaptor that modulates this process by recruiting binding partners, such as ubiquitin-conjugating enzymes. The role of another group of adaptors, Sprouty proteins, is less understood; although, studies in insects implicated the founder protein in the negative regulation of several receptor tyrosine kinases. RESULTS: By utilizing transfection of living cells, as well as reconstituted in vitro systems, we identified a dual regulatory mechanism that combines human Sprouty2 and c-Cbl. Upon activation of the receptor for the epidermal growth factor (EGFR), Sprouty2 undergoes phosphorylation at a conserved tyrosine that recruits the Src homology 2 domain of c-Cbl. Subsequently, the flanking RING finger of c-Cbl mediates poly-ubiquitination of Sprouty2, which is followed by proteasomal degradation. Because phosphorylated Sprouty2 sequesters active c-Cbl molecules, it impedes receptor ubiquitination, downregulation, and degradation in lysosomes. This competitive interplay occurs in endosomes, and it regulates the amplitude and longevity of intracellular signals. CONCLUSIONS: Sprouty2 emerges as an inducible antagonist of c-Cbl, and together they set a time window for receptor activation. When incorporated in signaling networks, the coupling of positive (Sprouty) to negative (Cbl) feedback loops can greatly enhance output diversification.     

The effect of negative feedback loops on the dynamics of boolean networks

Feedback loops play an important role in determining the dynamics of biological networks. To study the role of negative feedback loops, this article introduces the notion of distance-to-positive-feedback which, in essence, captures the number of independent negative feedback loops in the network, a property inherent in the network topology. Through a computational study using Boolean networks, it is shown that distance-to-positive-feedback has a strong influence on network dynamics and correlates very well with the number and length of limit cycles in the phase space of the network. To be precise, it is shown that, as the number of independent negative feedback loops increases, the number (length) of limit cycles tends to decrease (increase). These conclusions are consistent with the fact that certain natural biological networks exhibit generally regular behavior and have fewer negative feedback loops than randomized networks with the same number of nodes and same connectivity.     

Overlaid positive and negative feedback loops shape dynamical properties of PhoPQ two-component system

Bacteria use two-component systems (TCSs) to sense environmental conditions and change gene expression in response to those conditions. To amplify cellular responses, many bacterial TCSs are under positive feedback control, i.e. increase their expression when activated. Escherichia coli Mg²⁺ -sensing TCS, PhoPQ, in addition to the positive feedback, includes a negative feedback loop via the upregulation of the MgrB protein that inhibits PhoQ. How the interplay of these feedback loops shapes steady-state and dynamical responses of PhoPQ TCS to change in Mg²⁺ remains poorly understood. In particular, how the presence of MgrB feedback affects the robustness of PhoPQ response to overexpression of TCS is unclear. It is also unclear why the steady-state response to decreasing Mg²⁺ is biphasic, i.e. plateaus over a range of Mg²⁺ concentrations, and then increases again at growth-limiting Mg²⁺. In this study, we use mathematical modeling to identify potential mechanisms behind these experimentally observed dynamical properties. The results make experimentally testable predictions for the regime with response robustness and propose a novel explanation of biphasic response constraining the mechanisms for modulation of PhoQ activity by Mg²⁺ and MgrB. Finally, we show how the interplay of positive and negative feedback loops affects the network’s steady-state sensitivity and response dynamics. In the absence of MgrB feedback, the model predicts oscillations thereby suggesting a general mechanism of oscillatory or pulsatile dynamics in autoregulated TCSs. These results improve the understanding of TCS signaling and other networks with overlaid positive and negative feedback.     

Modeling genetic switches with positive feedback loops

In this paper, we develop a new methodology to design synthetic genetic switch networks with multiple genes and time delays, by using monotone dynamical systems. We show that the networks with only positive feedback loops have no stable oscillation but stable equilibria whose stability is independent of the time delays. In other words, such systems have ideal properties for switch networks and can be designed without consideration of time delays, because the systems can be reduced from functional spaces to Euclidian spaces. Therefore, we can ensure that the designed switches function correctly even with uncertain delays. We first prove the basic properties of the genetic networks composed of only positive feedback loops, and then propose a procedure to design the switches, which drastically simplifies analysis of the switches and makes theoretical analysis and design tractable even for large-scaled systems. Finally, to demonstrate our theoretical results, we show biologically plausible examples by designing a synthetic genetic switch with experimentally well investigated lacI, tetR, and cI genes for numerical simulation.     

Histochemical identification of human T lymphocytes from paraffin sections

The alpha-naphthyl acetate esterase (ANEA) is a histochemical marker for human T lymphocytes in cell smears and frozen tissue sections. We have now applied the ANAE method to paraffin-embedded tissue sections. We first demonstrated with cytocentrifuged cell smears of blood leukocytes that the ANAE activity is preserved upon prolonged storage in formol calcium, Holt's buffer, acetone, xylene, and heat. When the tissue sections were similarly processed and embedded in paraffin, the ANAE positive (T) lymphocytes were identified by their distinct display of one or more reddish-brown reaction dots in the cell cytoplasm. ANAE positive mononuclear phagocytes were easioy distinguished from the T lymphocytes by their diffuse, sodium fluoride-sensitive pancytoplasmic reaction. The extension of the ANAE method to paraffin-embedded tissue sections with superior morphological integrity, makes it possible to apply it in practical biopsy pathology.     

Prolactin induces calcium influx and release from intracellular pools in human T lymphocytes by activation of tyrosine kinases

The early events related to intracellular signals after prolactin (PRL) activation in T lymphocytes are not clearly established. The aim of this work was to study the effect of PRL in cytosolic calcium levels in human T lymphocytes. By using the dye FURA-2 AM, the variations in cytosolic Ca(2+) were studied in peripheral human T lymphocytes isolated from extracted blood from healthy donors. Fifty nanograms per milliliter PRL induces a small increase in cytosolic calcium. When the cells are preincubated overnight (16-20 h) in the presence of PRL, the increase in calcium is higher. This high increase is due to the release from intracellular pools and to the influx from the extracellular media. That is, after overnight incubation with PRL, calcium influx in T cells follows the capacitative model. Since PRL receptor (PRL-R) activation involves the tyrosine kinase pathway, we check calcium effect in the presence of genistein, a known inhibitor of tyrosine kinases. When cells are preincubated in the presence of 10 microM genistein, and PRL is immediately added, no increase in cytosolic calcium is observed. The presence of genistein also completely blocks the increase in cytosolic calcium stimulated by PRL after overnight incubation with PRL. In the presence of PRL and N,N-dimethyl-D-erythro-sphingosine (DMS), a stimulus that increases cytosolic calcium in T cells by tyrosine kinase stimulation, a high, even insignificant, calcium influx is induced. However, when the cells are incubated overnight in the presence of PRL, and then DMS is added, a significant increase in cytosolic calcium levels takes place. This increase is associated with an increase in calcium release from intracellular pools and an increase in calcium uptake. Genistein reduces the influx of external calcium induced by DMS after short incubation with PRL and significantly inhibits both, calcium pools empty, and calcium influx is induced by DMS after overnight incubation with PRL. In summary, PRL induces calcium influx in normal T lymphocytes. The influx is magnified after long PRL exposures, intracellular Ca(2+) pool-dependent, and activated through tyrosine kinases.     

Absence of negative feedback by oxytocin on release from magnocellular neurones in conscious rats.

Peripheral administration of vasopressin (VP) was previously shown to exert a negative feedback influence on its own release and on the release of oxytocin (OT). In this study we examined the possible influence that OT has on the function of hypothalamic magnocellular neurones. Oxytocin was administered intraperitoneally and its effects on release from VP neurones and from OT neurones were determined as indexed by plasma concentrations of vasopressin-associated neurophysin ([VP-RNP]) and oxytocin-associated neurophysin ([OT-RNP]) under basal conditions and conditions of high plasma osmolality (Posm) induced by acute salt loading. Studies were performed on conscious, chronically instrumented Long-Evans rats. Oxytocin (1 nmol or 10 nmol) dissolved in 1 mL of 0.9% saline was administered intraperitoneally to animals 1 h before they received an intravenous infusion of hypertonic saline over 60 min at a rate designed to raise Posm by approximately 0.75 mosmol.min-1. Intraperitoneal injection of vehicle or 1 nmol of OT did not significantly alter [VP-RNP], [OT-RNP], or basal Posm. Administration of 10 nmol OT also had no effect on [VP-RNP] or [OT-RNP], but this dose of peptide significantly lowered basal Posm (299 +/- 2 to 290 +/- 2 mosmol/kg H2O, p less than 0.001). Both doses of OT did not significantly alter the responsiveness of VP neurones to hyperosmotic stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation requirements for antigen- and mitogen-induced interferon-gamma release from cytotoxic T lymphocytes

We have studied the activation signals that regulate interferon-gamma (IFN-gamma) secretion from murine cytotoxic T lymphocytes (CTL) upon binding mitogen or antigen. CTL clones were found to require at least 1 hr of stimulation with concanavalin A (Con A) in order to produce detectable levels of IFN-gamma. Full activation of IFN-gamma synthesis in CTL clones occurred after stimulation for 2 hr or more, and in those cultures CTL continued to produce high levels of IFN-gamma even after the effects of Con A had been neutralized. Splenic T cells and uncloned long-term CTL lines required a longer period of stimulation than cloned CTL for Con A-induced IFN-gamma secretion. The relationship between IFN-gamma secretion and cytotoxic activity was studied in an antigen-specific system. These studies reveal marked differences in the types of effector responses generated by CTL upon contact with antigen, demonstrating that some antigen-bearing cells promote high levels of IFN-gamma secretion and are poorly lysed by CTL, whereas other cell lines are lysed with high efficiency by CTL but induce low levels of IFN-gamma secretion.     

Syntaxin7 is required for lytic granule release from cytotoxic T lymphocytes

SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant-negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTL-mediated killing of target cells. Real-time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7-deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high-resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE-dependent process and that Stx7-mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs.     

Interaction of synthetic decapeptide SLTCLVKGFY with human T lymphocytes

The synthetic peptide SLTCLVKGFY, corresponding to the 364-373 amino acid sequence of the human IgG heavy chain (Immunorphin), was found to compete with [125I] beta-endorphin for binding by high-affinity receptors on T lymphocytes isolated from the blood of healthy donors (Ki 0.6 nM). The fragments 3-10, 4-10, 5-10, and 6-10 of Immunorphin also inhibited the binding (Ki 2.2, 3.4, 8.0, and 15 nM, respectively). Specificity of these receptors was studied: they turned out to be insensitive to naloxone and, therefore, are not opioid. The Kd values of the specific binding of 125I-labeled Immunorphin and its 6-10 fragment to the receptor were found to be 7.4 and 36.3 nM, respectively.     

The interaction of positive and negative sensory feedback loops in dynamic regulation of a motor pattern

In many rhythmic behaviors, phasic sensory feedback modifies the motor pattern. This modification is assumed to depend on feedback sign (positive vs. negative). While on a phenomenological level feedback sign is well defined, many sensory pathways also process antagonistic, and possibly contradictory, sensory information. We here model the locust flight pattern generator and proprioceptive feedback provided by the tegula wing receptor to test the functional significance of sensory pathways processing antagonistic information. We demonstrate that the tegula provides delayed positive feedback via interneuron 301, while all other pathways provide negative feedback. Contradictory to previous assumptions, the increase of wing beat frequency when the tegula is activated during flight is due to the positive feedback. By use of an abstract model we reveal that the regulation of motor pattern frequency by sensory feedback critically depends on the interaction of positive and negative feedback, and thus on the weighting of antagonistic pathways.     

Hybrids from fusion of normal human T lymphocytes with immortal human cells exhibit limited life span

A number of normal human cell types have been shown to exhibit cellular senescence in vitro. We and others had found that fusion of normal human fibroblasts with immortal human cells yielded hybrids having limited lifespan. This indicated that the phenotype of cellular senescence is dominant and that immortality results from recessive changes in genes involved in growth control. They also supported the hypothesis that senescence results from genetic mechanisms rather than random damage. Since T lymphocytes are a highly differentiated cell type, in contrast to fibroblasts, it was of interest to determine whether similar mechanisms caused senescence in the T cells. We therefore fused normal human T lymphocytes with an immortal human cell line to determine whether they could restore the senescent, nondividing phenotype in hybrids, as do normal human fibroblasts. Eleven of fifteen hybrid clones studied exhibited limited proliferative potential after achieving a range of population doubling similar to that observed in the cell fusion studies involving normal fibroblasts. These results provide evidence that cellular senescence in T lymphocytes occurs via genetic mechanisms.