Positive Feedback Promotes Oscillations in Negative Feedback Loops

A simple three-component negative feedback loop is a recurring motif in biochemical oscillators. This motif oscillates as it has the three necessary ingredients for oscillations: a three-step delay, negative feedback, and nonlinearity in the loop. However, to oscillate, this motif under the common Goodwin formulation requires a high degree of cooperativity (a measure of nonlinearity) in the feedback that is biologically “unlikely.” Moreover, this recurring negative feedback motif is commonly observed augmented by positive feedback interactions. Here we show that these positive feedback interactions promote oscillation at lower degrees of cooperativity, and we can thus unify several common kinetic mechanisms that facilitate oscillations, such as self-activation and Michaelis-Menten degradation. The positive feedback loops are most beneficial when acting on the shortest lived component, where they function by balancing the lifetimes of the different components. The benefits of multiple positive feedback interactions are cumulative for a majority of situations considered, when benefits are measured by the reduction in the cooperativity required to oscillate. These positive feedback motifs also allow oscillations with longer periods than that determined by the lifetimes of the components alone. We can therefore conjecture that these positive feedback loops have evolved to facilitate oscillations at lower, kinetically achievable, degrees of cooperativity. Finally, we discuss the implications of our conclusions on the mammalian molecular clock, a system modeled extensively based on the three-component negative feedback loop.     

Identification of a Nuclear Retinoic Acid-binding Activity Representative of Endogeneous Retinoic Acid Receptors in the Nuclei of Rat Liver and Testis

We found a nuclear RA-binding activity by using a sucrose-density-gradient assay from rat liver and testis. From the sedimentation analysis, and the comparison with cloned RARs, it is likely that these binding activities represent endogenous RARs. Furthermore we showed that these binding activities were constant irrespective of the retinoid status in the rat.     

Retinoic Acid Increases Hydroxyindole-O-Methyltransferase Activity and mRNA in Human Y-79 Retinoblastoma Cells

Abstract: Hydroxyindole- O -methyltransferase (HIOMT) plays an important role as the final enzyme in the synthesis of melatonin. Here we present the first evidence that retinoic acid (RA) stereoisomers are potent regulators of HIOMT in the human retinoblastoma-derived Y-79 cell line. Treatment with all- trans -, 13- cis -, and 9- cis -RA induced a gradual 10-fold increase in HIOMT activity and mRNA, without changing the levels of mRNA encoding glyceraldehyde-3-phosphate dehydrogenase, actin, S-antigen, and interphotoreceptor retinoid-binding protein. These findings point to the possibility that RA may play a physiological role in the regulation of human HIOMT.     

孤生受体的相互作用对视黄酸应答元件的影响

孤生受体ＣＯＵＰ－ＴＦ和ｎｕｒ７７的功能及其作用机理仍未阐明．以ＤＮＡ瞬时转染和测定氯霉素乙酰转移酶（ＣＡＴ）活性,以及凝胶阻抑测定,分析ＣＯＵＰ－ＴＦ和ｎｕｒ７７的相互作用对视黄酸应答元件（ＲＡＲＥｓ）的影响．实验表明,ＣＯＵＰ－ＴＦ通过降低ＲＡＲＥｓ的基础活性,来增强ＲＡＲＥ对视黄酸（ＲＡ）的敏感性,而ｎｕｒ７７则拮抗ＣＯＵＰ－ＴＦ的作用．ｎｕｒ７７能够加强不同ＲＡＲＥｓ的转录活性,并且与ＲＡ的诱导无关．结果证实,ｎｕｒ７７通过与ＣＯＵＰ－ＴＦ的直接作用而对ＲＡＲＥｓ产生影响,从而抑制ＣＯＵＰ－ＴＦ与ＲＡＲＥｓ结合和ＣＯＵＰ－ＴＦ的转录活性     

Combination of Zinc and All-Trans Retinoic Acid Promotes Protection against Listeria monocytogenes Infection

Zinc (Zn) is the second most abundant transition metal after iron. It plays a vital role in living organisms and affects multiple aspects of the immune system. All-trans retinoic acid (atRA) is an isomeric form of the vitamin A or retinol. It possesses the greatest biological activity of Vitamin A. Vitamin A and related retinoids influence many aspects of immunity. In this study, we demonstrated that treatment with a combination of Zn and atRA contributes to host resistance against infection by Listeria monocytogenes. Pretreatment with Zn and atRA enhanced resistance against L. monocytogenes infection in mice and treatment with both Zn and atRA showed a higher protective effect than treatment with either alone. Supplementation with Zn, atRA or their combination decreased the number of L. monocytogenes present in target organs. In vitro, supplementation increased the bacterial uptake by macrophage cells and reduced the replication of L. monocytogenes. Our results suggest that the combination of Zn and atRA has a great bacteriostatic impact on L. monocytogenes and its infection.     

Ligand Induction of Retinoic Acid Receptors Alters an Acute Infection by Murine Cytomegalovirus

Here we report that administration of retinoids can alter the outcome of an acute murine cytomegalovirus (MCMV) infection. We show that a crucial viral control element, the major immediate-early enhancer, can be activated by retinoic acid (RA) via multiple RA-responsive elements (DR2) that bind retinoid X receptor-retinoic acid receptor (RAR) heterodimers with apparent dissociation constants ranging from 15 to 33 nM. Viral growth is dramatically increased upon RA treatment of infected tissue culture cells. Using synthetic retinoid receptor-specific agonists and antagonists, we provide evidence that RAR activation in cells is required for mediating the response of MCMV to RA. Oral administration of RA to infected immunocompetent mice selectively exacerbates an infection by MCMV, while cotreatment with an RAR antagonist protects against the adverse effects of RA on MCMV infection. In conclusion, these chemical genetic experiments provide evidence that an RAR-mediated pathway can modulate in vitro and in vivo infections by MCMV.     

Tumor Necrosis Factor Receptors in Neuroblastoma SKNBE Cells and Their Regulation by Retinoic Acid

Abstract: The human neuroblastoma cell line SKNBE can be differentiated either by serum removal or by adding to the culture medium different morphogens, for instance, retinoic acid (RA), cyclic AMP derivatives, and phorbol esters. Both the differentiated and undifferentiated cells express the two types of membrane tumor necrosis factor (TNF) receptors (TNFRs) of 55 and 75 kDa (p55 and p75 TNFR, respectively) and also their soluble forms. After RA addition the number of the surface TNFRs per cell is increased approximately twofold, but the kinetics of expression are different, depending on the receptor type. The level of the mRNAs of 2.4 and 4.2 kb, which, respectively, encode the p55 and p75 TNFRs, is also increased during the time course of differentiation, and the kinetics of their expression are biphasic. In contrast, the number of TNFRs and the level of their encoding mRNAs remain unchanged after exposure of the cells to both a phorbol and a cyclic AMP derivative.     

Retinoic Acid Improves a Hybridoma Culture in a Fructose-Based Medium by Up-Regulation of Fructose Incorporation via Retinoid Nuclear Receptors

Fructose was focused on as an alternative sugar source to glucose in a hybridoma culture medium because it decreases lactate production during cultivation, leading to cell and product stability. But, not all human hybridoma cell lines grew well in a fructose-based serum-free medium. We found that the addition of all-trans-retinoic acid to the fructose-based medium improved the growth and monoclonal antibody production of hybridoma cell lines by up-regulation of fructose incorporation that represented increased expression of the fructose transporter, GLUT5. Selective activation of retinoid nuclear receptor by synthetic ligands showed that both retinoic acid receptors and retinoid X receptors might be related to the improvement of the fructose-based hybridoma culture. This study might be applicable to cell cultures susceptible to lactate and pH changes as well as hybridoma cultures.     

A Positive Feedback Loop of TET3 and TGF-β1 Promotes Liver Fibrosis

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胃癌细胞中视黄酸受体抑制AP-1活性的不同方式

 研究胃癌细胞中视黄酸受体RARα和RARβ抑制活化蛋白 1(activatorprotein 1,AP 1)活性的不同方式及其与全反式视黄酸 (ATRA)作用的相关性 .瞬时转染RARβ表达载体到MKN 4 5细胞后 ,佛波脂 (TPA)诱导的AP 1活性受到明显抑制 ,且与RARβ浓度正相关 ,与ATRA存在与否无关 ;相反 ,RARα转染细胞后 ,对TPA诱导的AP 1活性的抑制不仅与RARα的浓度相关 ,而且依赖于AT RA .凝胶阻抑测定表明 ,TPA可以显著加强AP 1结合活性 ,当ATRA处理不表达RARβ和低表达RARα的MKN 4 5细胞后 ,AP 1结合活性不受影响 ;然而 ,表达RARα和RARβ的BGC 82 3细胞经AT RA处理后 ,TPA诱导的AP 1结合活性则受到抑制 .另外 ,分析与抗AP 1活性相关的RARβ功能区表明 ,DNA结合区的缺失导致RARβ抑制AP 1活性作用的丧失 ,而配体结合区对于RARβ抑制AP 1活性则是非必需的 .以上结果证实 ,有胃癌细胞中 ,RARβ可能是AP 1活性的抑制因子 ,RARα则可能是ATRA作用的靶向 .尽管它们的作用方式有所不同 ,但最终都可以通过抑制AP 1活性来抑制胃癌细胞生长     

全反式维甲酸通过γ-氨基丁酸途径促进小鼠胚胎腭板间充质细胞的凋亡

维甲酸(RA)是一种能够诱导腭裂发生的致畸物．研究显示γ-氨基丁酸(GABA)在腭板的发育过程中发挥重要作用．而GABA是否参与了RA诱导的腭裂发生还不清楚．本研究以小鼠胚胎腭板间充质细胞(MEPM)为研究对象,观察全反式维甲酸(atRA)(0.2、0.67、2.0和 6.7 μmol/L)对MEPM细胞增殖和凋亡的影响,并探讨GABA信号通路在其中的可能作用．结果显示,atRA(2.0 μmol/L和6.7 μmol/L)显著性抑制了MEPM的增殖,并促进了细胞凋亡．atRA(0.67、2.0和 6.7 μmol/L)显著性降低了GABA合成的关键酶谷氨酸脱羧酶(GAD67)mRNA和蛋白质的表达,但对γ-氨基丁酸A型受体-β3(GABAAR-β3)mRNA和蛋白质的表达没有影响．1.0 μmol/L的GABA逆转了atRA(6.7 μmol/L)对MEPM细胞增殖和凋亡的影响．以上结果表明,atRA通过下调GAD67的表达,减少GABA的产生,抑制MEPM的增殖和促进MEPM的凋亡,从而可能影响腭板的发育,诱导腭裂形成．     

Macrophage Death following Influenza Vaccination Initiates the Inflammatory Response that Promotes Dendritic Cell Function in the Draining Lymph Node

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Heterodimeric BMP-2/7 Antagonizes the Inhibition of All-Trans Retinoic Acid and Promotes the Osteoblastogenesis

Objectives

Hypervitaminosis A and alcoholism can result in a low mineral density and compromised regenerative capacity of bone, thus delaying implant osteointegration. The inhibitory effect of all-trans retinoic acid on osteoblastogenesis is considered to be one of the mechanisms. We hypothesized that heterodimeric bone morphogenetic protein-2/7 could antagonize all-trans retinoic acid and enhance osteoblastogenesis, with an aim to accelerate and enhance bone regeneration and implant osteointegration.

Materials and Methods

We applied 5 ng/ml or 50 ng/ml bone morphogenetic protein-2/7 to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line) that was inhibited by 1 µM all-trans retinoic acid. We evaluated the efficacy by assessing cell numbers (proliferation), alkaline phosphatase activity (a marker for early differentiation), osteocalcin (a marker for late differentiation), calcium deposition (a marker for final mineralization) and the expression of osteoblastogenic genes (such as Runx2, Collagen Ia, alkaline phosphatase and osteocalcin) at different time points.

Results

All-trans retinoic acid significantly inhibited the expression of all the tested osteoblastogenic genes and proteins except alkaline phosphatase activity. In the presence of ATRA, 50 ng/ml bone morphogenetic protein-2/7 not only completely restored but also significantly enhanced all the osteoblastogenic genes and proteins. On the 28^th day, mineralization was completely inhibited by all-trans retinoic acid. In contrast, 50 ng/ml BMP-2/7 could antagonize ATRA and significantly enhance the mineralization about 2.5 folds in comparison with the control treatment (no ATRA, no BMP2/7).

Conclusions

Heterodimeric bone morphogenetic protein-2/7 bears a promising application potential to significantly promote bone regeneration and implant osteointegration for the patients with hypervitaminosis A and alcoholism.     

Inhibition of Receptor Dimerization as a Novel Negative Feedback Mechanism of EGFR Signaling

Dimerization of the epidermal growth factor receptor (EGFR) is crucial for initiating signal transduction. We employed raster image correlation spectroscopy to continuously monitor the EGFR monomer-dimer equilibrium in living cells. EGFR dimer formation upon addition of EGF showed oscillatory behavior with a periodicity of about 2.5 min, suggesting the presence of a negative feedback loop to monomerize the receptor. We demonstrated that monomerization of EGFR relies on phospholipase Cγ, protein kinase C, and protein kinase D (PKD), while being independent of Ca²⁺ signaling and endocytosis. Phosphorylation of the juxtamembrane threonine residues of EGFR (T654/T669) by PKD was identified as the factor that shifts the monomer-dimer equilibrium of ligand bound EGFR towards the monomeric state. The dimerization state of the receptor correlated with the activity of an extracellular signal-regulated kinase, downstream of the EGFR. Based on these observations, we propose a novel, negative feedback mechanism that regulates EGFR signaling via receptor monomerization.