Effect of 1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) on leukocytes I. Analysis of the in vitro migration of human neutrophils

The effect of synthetic 1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) and of 1-O-octadecyl-sn-glycero-3-phosphocholine (lyso-PAF-acether) on human neutrophil migration was studied in modified Boyden chambers, with the following results: (1) By checker-board analysis and deactivation experiments, the factors are chemokinetic at low (10^?8 M) and chemotactic at higher concentrations (10^?6 M), with lyso-PAF-acether being less potent at all concentrations. (2) Cross-deactivation occurs between the two PAF compounds, but not with two other chemotactic factors, suggesting a specific, common receptor for the PAFs on the neutrophil membrane. (3) Other chemotactic substances may act as potentiating or additive factors to the PAF compounds. (4) Inhibition of arachidonic acid turnover during chemotaxis by compound BW 755 C enhances leukocyte chemotaxis towards the PAF compounds and towards other chemotactic factors. The data suggest that PAF and its lyso-derivate may contribute in a unique and potent fashion to leukocyte accumulation at inflammatory sites.     

Selection and growth regulation of genetically modified cells with hapten‐specific antibody/receptor tyrosine kinase chimera

Although receptor tyrosine kinases (RTKs) play a pivotal role in the development and maintaining the homeostasis of the body, overexpression or mutation of RTKs often induces tumorigenesis or metastasis. To mimic the function of RTKs, we developed two fusion receptors consisting of anti‐fluorescein antibody single‐chain Fv, extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of epidermal growth factor receptor or c‐fms based on previously constructed antibody/cytokine receptor chimeras. The expression of these chimeric receptors in the hematopoietic cell line Ba/F3 and non‐hematopoietic cell line NIH/3T3 resulted in the activation of receptors themselves, downstream signaling molecules and cell proliferation in response to fluorescein‐conjugated BSA, leading to selective expansion of transduced cells up to almost 100%. These results indicate that the cognate antigen could activate the chimeric receptors even though the wild‐type extracellular domains were switched to the antibody fragment. This is the first study to show that our antigen‐mediated genetically modified cell amplification (AMEGA) system could be applied to non‐hematopoietic cells by utilizing antibody/RTK chimeras. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Construction of unnatural heterodimeric receptors based on IL‐2 and IL‐6 receptor subunits

Cells have various receptors on their surface for responding to extracellular signals that involve intercellular communication. Although the mechanism of signal transduction by such wild‐type receptors has been studied intensively, there has been minimal effort in investigating whether such receptors could signal when unnaturally coupled. In this study, we investigated whether unnatural receptor pairs comprising interleukin‐2 (IL‐2) and interleukin‐6 (IL‐6) receptor subunits could transduce a signal through forced dimerization. We replaced the extracellular domain of IL‐2R and IL‐6R signaling subunits (IL‐2Rβ, IL‐2Rγ, and gp130) with the FK506‐binding protein (FKBP) or the FKBP12‐rapamycin binding (FRB) domain, the protein pair known to be heterodimerized by rapamycin. When expressed in a hematopoietic cell line, unnatural heterodimers (IL‐2Rβ‐gp130 and IL‐2Rγ‐gp130 pairs) successfully transduced a signal. While the IL‐2Rγ‐gp130 pair maximally mimicked gp130 signaling, the IL‐2Rβ‐gp130 pair gave weaker gp130 signaling and no significant induction of IL‐2Rβ signaling, indicating a high potential of the IL‐2Rγ chain in terms of activating the coupled partners. This is the first report demonstrating that heterodimeric combinations of IL‐2R and IL‐6R subunits are functional for signaling. Further extension of this approach may attain a creative design of artificial receptor pairs that have distinct signaling properties when compared with natural receptors. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1512–1518, 2013     

Cell regulation by sphingosine and more complex sphingolipids

Sphingolipids have the potential to regulate cell behavior at essentially all levels of signal transduction. They serve as cell surface receptors for cytoskeletal proteins, immunoglobulins, and some bacteria; as modifiers of the properties of cell receptors for growth factors (and perhaps other agents); and as activators and inhibitors of protein kinases, ion transporters, and other proteins. Furthermore, the biological activity of these compounds resides not only in the more complex species (e.g., sphingomyelin, cerebrosides, gangliosides, and sulfatides), but also in their turnover products, such as the sphingosine backbone which inhibits protein kinase C and activates the EGF-receptor kinase,inter alia. Since sphingolipids change with cell growth, differentiation, and neoplastic transformation, they could be vital participants in the regulation of these processes.This review is dedicated to Professor Herbert E. Carter on the occasion of his 80th birthday.     

The SIE,SRE, CRE,and FAP‐1 four intracellular signal pathways between stimulus and the expression of c‐fos promoter

c‐fos gene has a close relationship with the osteoblasts. Mechanical signal effect on osteoblasts would change the expression level of c‐fos. Authors introduce the signal pathways of four cis‐response elements on the promoter of c‐fos, that is, CRE (cAMP responsive element), FAP‐1 (Fbs‐AP‐1 site), SRE (serum response element), and SIE (sis‐inducible element), as the regulatory mechanism for c‐fos gene expression following various stimuli. J. Cell. Biochem. 106: 764–768, 2009. © 2009 Wiley‐Liss, Inc.     

Structure‐based simulations reveal concerted dynamics of GPCR activation

G protein‐coupled receptors (GPCRs) are a vital class of proteins that transduce biological signals across the cell membrane. However, their allosteric activation mechanism is not fully understood; crystal structures of active and inactive receptors have been reported, but the functional pathway between these two states remains elusive. Here, we use structure‐based (Gō‐like) models to simulate activation of two GPCRs, rhodopsin and the β₂ adrenergic receptor (β₂AR). We used data‐derived reaction coordinates that capture the activation mechanism for both proteins, showing that activation proceeds through quantitatively different paths in the two systems. Both reaction coordinates are determined from the dominant concerted motions in the simulations so the technique is broadly applicable. There were two surprising results. First, the main structural changes in the simulations were distributed throughout the transmembrane bundle, and not localized to the obvious areas of interest, such as the intracellular portion of Helix 6. Second, the activation (and deactivation) paths were distinctly nonmonotonic, populating states that were not simply interpolations between the inactive and active structures. These transitions also suggest a functional explanation for β₂AR's basal activity: it can proceed through a more broadly defined path during the observed transitions. Proteins 2014; 82:2538–2551. © 2014 Wiley Periodicals, Inc.     

Association and dissociation kinetics for CheY interacting with the P2 domain of CheA

The chemotaxis system of Escherichia coli makes use of an extended two-component sensory response pathway in which CheA, an autophosphorylating protein histidine kinase (PHK) rapidly passes its phosphoryl group to CheY, a phospho-accepting response regulator protein (RR). The CheA-->CheY phospho-transfer reaction is 100-1000 times faster than the His-->Asp phospho-relays that operate in other (non-chemotaxis) two-component regulatory systems, suggesting that CheA and CheY have unique features that enhance His-->Asp phospho-transfer kinetics. One such feature could be the P2 domain of CheA. P2 encompasses a binding site for CheY, but an analogous RR-binding domain is not found in other PHKs. In previous work, we removed P2 from CheA, and this decreased the catalytic efficiency of CheA-->CheY phospho-transfer by a factor of 50-100. Here we examined the kinetics of the binding interactions between CheY and P2. The rapid association reaction (k(assn) approximately 10(8)M(-1)s(-1) at 25 degrees C and micro=0.03 M) exhibited a simple first-order dependence on P2 concentration and appeared to be largely diffusion-limited. Ionic strength (micro) had a moderate effect on k(assn) in a manner predictable based on the calculated electrostatic interaction energy of the protein binding surfaces and the expected Debye-Hückel shielding. The speed of binding reflects, in part, electrostatic interactions, but there is also an important contribution from the inherent plasticity of the complex and the resulting flexibility that this allows during the process of complex formation. Our results support the idea that the P2 domain of CheA contributes to the overall speed of phospho-transfer by promoting rapid association between CheY and CheA. However, this alone does not account for the ability of the chemotaxis system to operate much more rapidly than other two-component systems: k(cat) differences indicate that CheA and CheY also achieve the chemical events of phospho-transfer more rapidly than do PHK-RR pairs of slower systems.     

Ubiquitin‐specific protease 19 blunts pathological cardiac hypertrophy via inhibition of the TAK1‐dependent pathway

Ubiquitin‐specific protease 19 (USP19) belongs to USP family and is involved in promoting skeletal muscle atrophy. Although USP19 is expressed in the heart, the role of USP19 in the heart disease remains unknown. The present study provides in vivo and in vitro data to reveal the role of USP19 in preventing pathological cardiac hypertrophy. We generated USP19‐knockout mice and isolated neonatal rat cardiomyocytes (NRCMs) that overexpressed or were deficient in USP19 to investigate the effect of USP19 on transverse aortic constriction (TAC) or phenylephrine (PE)‐mediated cardiac hypertrophy. Echocardiography, pathological and molecular analysis were used to determine the extent of cardiac hypertrophy, fibrosis, dysfunction and inflammation. USP19 expression was markedly increased in rodent hypertrophic heart or cardiomyocytes underwent TAC or PE culturing, the increase was mediated by the reduction of Seven In Absentia Homolog‐2. The extent of TAC‐induced cardiac hypertrophy, fibrosis, dysfunction and inflammation in USP19‐knockout mice was exacerbated. Consistently, gain‐of‐function and loss‐of‐function approaches that involved USP19 in cardiomyocytes suggested that the down‐regulation of USP19 promoted the hypertrophic phenotype, while the up‐regulation of USP19 improved the worsened phenotype. Mechanistically, the USP19‐elicited cardiac hypertrophy improvement was attributed to the abrogation of the transforming growth factor beta‐activated kinase 1 (TAK1)‐p38/JNK1/2 transduction. Furthermore, the inhibition of TAK1 abolished the aggravated hypertrophy induced by the loss of USP19. In conclusion, the present study revealed that USP19 and the downstream of TAK1‐p38/JNK1/2 signalling pathway might be a potential target to attenuate pathological cardiac hypertrophy.     

The endocrine regulation network of growth hormone synthesis and secretion in fish: Emphasis on the signal integration in somatotropes

In teleosts, growth hormone (GH) production is governed by multiple neuroendocrine factors from the hypothalamus and other regulators from the pituitary and peripheral organs. Exploring the principles followed by pituitary somatotropes when differentiating and integrating the signals from these regulators at the cellular and intracellular level is essential for understanding the endocrine regulation network of growth hormone synthesis and secretion in fish. This paper discusses recent advances in the action...     

Down-regulation of MicroRNAs (MiRs) 203, 887, 3619 and 182 Prevents Vimentin-triggered,Phospholipase D (PLD)-mediated Cancer Cell Invasion

Breast cancer is a leading cause of morbidity and mortality among women. Metastasis is initiated after epithelial-mesenchymal-transition (EMT). We have found a connection between EMT markers and the expression of four microRNAs (miRs) mediated by the signaling enzyme phospholipase D (PLD). Low aggressive MCF-7 breast cancer cells have low endogenous PLD enzymatic activity and cell invasion, concomitant with high expression of miR-203, -887, and -3619 (that decrease PLD2 translation and a luciferase reporter) and miR-182 (targeting PLD1) that are, therefore, “tumor-suppressor-like” miRs. The combination miR-887+miR-3619 abolished >90% of PLD enzymatic activity. Conversely, post-EMT MDA-MB-231 cells have low miR expression, high levels of PLD1/2, and high aggressiveness. The latter was reversed by ectopically transfecting the miRs, which was negated by silencing miRs with specific siRNAs. We determined that the molecular mechanism is that E-cadherin triggers expression of the miRs in pre-EMT cells, whereas vimentin dampens expression of the miRs in post-EMT invasive cells. This novel work identifies for the first time a set of miRs that are activated by a major pre-EMT marker and deactivated by a post-EMT marker, boosting the transition from low invasion to high invasion, as mediated by the key phospholipid metabolism enzyme PLD.     

Introducing fluorescence resonance energy transfer‐based biosensors for the analysis of cAMP‐PKA signalling in the fungal pathogen Candida glabrata

The cyclic adenosine monophosphate‐protein kinase A (cAMP‐PKA) pathway is central to signal transduction in many organisms. In pathogenic fungi such as Candida albicans, this signalling cascade has proven to be involved in several processes, such as virulence, indicating its potential importance in antifungal drug discovery. Candida glabrata is an upcoming pathogen of the same species, yet information regarding the role of cAMP‐PKA signalling in virulence is largely lacking. To enable efficient monitoring of cAMP‐PKA activity in this pathogen, we here present the usage of two FRET‐based biosensors. Both variations in the activity of PKA and the quantity of cAMP can be detected in a time‐resolved manner, as we exemplify by glucose‐induced activation of the pathway. We also present information on how to adequately process and analyse the data in a mathematically correct and physiologically relevant manner. These sensors will be of great benefit for scientists interested in linking the cAMP‐PKA signalling cascade to downstream processes, such as virulence, possibly in a host environment.     

Differentiation potential of individual olfactory c‐Kit+ progenitors determined via multicolor lineage tracing

Olfactory tissue undergoes lifelong renewal, due to the presence of basal neural stem cells. Multiple categories of globose basal stem cells have been identified, expressing markers such as Lgr5, Ascl1, GBC‐2, and c‐Kit. The differentiation potential of individual globose cells has remained unclear. Here, we utilized Cre/loxP lineage tracing with a multicolor reporter system to define c‐Kit+ cell contributions at clonal resolution. We determined that reporter expression permitted identification of c‐Kit derived progeny with fine cellular detail, and that clones were found to be comprised by neurons only, microvillar cells only, microvillar cells and neurons, or gland/duct cells. Quantification of reporter‐labeled cells indicated that c‐Kit+ cells behave as transit amplifying or immediate precursors, although we also found evidence for longer‐term c‐Kit+ cell contributions. Our results from the application of multicolor fate mapping delineate the clonal contributions of c‐Kit+ cells to olfactory epithelial renewal, and provide novel insight into tissue maintenance of an adult neuroepithelium. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 241–251, 2016     

PI3K is involved in L‐selectin‐ and PSGL‐1‐mediated neutrophil rolling on E‐selectin via F‐actin redistribution and assembly

L‐selectin and P‐selectin glycoprotein ligand‐1 (PSGL‐1) are adhesion molecules that play critical roles in neutrophil rolling during inflammation and lymphocyte homing. On the other hand they also function as signaling receptors to induce cytoskeleton changes. The present study is to investigate the signaling kinases responsible for the F‐actin changes mediated by L‐selectin and PSGL‐1 during neutrophil rolling on E‐selectin. Western blot analysis demonstrated that PI3K activation, peaking within 5 min, was induced by ligation of L‐selectin and PSGL‐1 with E‐selectin, and that Vav1 (the pivotal downstream effector of PI3K signaling pathway involved in cytoskeleton regulation) was recruited to the membrane and tyrosine‐phosphorylated, depending on PI3K. Furthermore, the F‐actin redistribution and assembly mediated by ligation with E‐selectin were blocked by LY294002, a PI3K specific inhibitor. Additional experiments showed that PI3K activity was involved in neutrophil rolling on E‐selectin. However, Syk/Zap70, the well‐known upstream kinase of PI3K, was not involved in this event. These data suggest that PI3K is required for the F‐actin‐based cytoskeleton changes during neutrophil rolling on E‐selectin, which may consequently regulate the rolling event. J. Cell. Biochem. 110: 910–919, 2010. © 2010 Wiley‐Liss, Inc.     

High‐resolution imaging demonstrates dynein‐based vesicular transport of activated trk receptors

Target‐derived neurotrophins signal from nerve endings to the cell body to influence cellular and nuclear responses. The retrograde signal is conveyed by neurotrophin receptors (Trks) themselves. To accomplish this, activated Trks may physically relocalize from nerve endings to the cell bodies. However, alternative signaling mechanisms may also be used. To identify the vehicle wherein the activated Trks are located and transported, and to identify associated motor proteins that would facilitate transport, we use activation‐state specific antibodies in concert with immunoelectron microscopy and deconvolution microscopy. We show that the activated Trks within rat sciatic nerve axons are preferentially localized to coated and uncoated vesicles. These vesicles are moving in a retrograde direction and so accumulate distal to a ligation site. The P‐Trk containing vesicles, in turn, colocalize with dynein components, and not with kinesins. Collectively, these results indicate activated Trk within axons travel in vesicles and dynein is the motor that drives these vesicles towards the cell bodies. © 2002 Wiley Periodicals, Inc. J Neurobiol 51: 302–312, 2002