Development of dual receptor biosensors: an analysis of FRET pairs

The development of a dual receptor detection method for enhanced biosensor monitoring was investigated by analyzing potential fluorescent resonance energy transfer (FRET) pairs. The dual receptor scheme requires the integration of a chemical transducer system with two unique protein receptors that bind to a single biological agent. The two receptors are tagged with special molecular groups (donors and acceptors fluorophores) while the chemical transduction system relies on the well-known mechanisms of FRET. During the binding event, the two FRET labeled receptors dock at the binding sites on the surface of the biological agent. The resulting close proximity of the two fluorophores upon binding will initiate the energy transfer resulting in fluorescence. The paper focuses on the analysis and optimization of the chemical transduction system. A variety of FRET fluorophore pairs were tested in a spectrofluorimeter and promising FRET pairs were then tagged to the protein, avidin and its ligand, biotin. Due to their affinities, the FRET-tagged biomolecules combine in solution, resulting in a stable, fluorescent signal from the acceptor FRET dye with a simultaneous decrease in fluorescent signal from the donor FRET dye. The results indicate that the selected FRET pairs can be utilized in the development of dual receptor sensors.     

The Nicotinic Acetylcholine Receptor: The Founding Father of the Pentameric Ligand-gated Ion Channel Superfamily

A critical event in the history of biological chemistry was the chemical identification of the first neurotransmitter receptor, the nicotinic acetylcholine receptor. Disciplines as diverse as electrophysiology, pharmacology, and biochemistry joined together in a unified and rational manner with the common goal of successfully identifying the molecular device that converts a chemical signal into an electrical one in the nervous system. The nicotinic receptor has become the founding father of a broad family of pentameric membrane receptors, paving the way for their identification, including that of the GABA_A receptors.     

人血小板生成素受体c—MPL膜内部分的聚合作用

血小板生成素（ＴＰＯ）是调节血小板生成最主要的细胞因子,其生物学效应由其受体ｃ－ＭＰＬ介导。利用酵母双杂合系统研究ｃ－ＭＰＬ膜内部分在ＴＰＯ信号转导途径中的功能。首先用反转录ＰＣＲ（ＲＴ－ＰＣＲ）方法从人红白血病ＨＥＬ细胞系总ＲＮＡ中扩增并克隆Ｐ型ｃ－ＭＰＬ（ＭＰＬＰ）膜内部分ｃＤＮＡ,经测序验证后克隆至双杂合载体ｐＡＳ２和ｐＧＡＤ４２４中,重组质粒命名为ｐＡＳＭＭ和ｐＧＡＤＭＭ。将ｐＡＳＭＭ与ｐ     

Antennal morphology as a physical filter of olfaction: temporal tuning of the antennae of the honeybee,Apis mellifera

There are many different antennal morphologies for insects, yet they all have the same functional role in olfaction. Chemical signals are dispersed through two physical forces; diffusion and fluid flow. The interaction between antennal morphology and fluid flow generates a region of changing flow velocity called the boundary layer. The boundary layer determines signal dispersion dynamics and therefore influences the signal structure and information that arrives at the receptor cells. To investigate how the boundary layer changes the information in the signals arriving at receptor cells, we measured chemical dynamics within the boundary layer around the bee antennae using microelectrodes. We used two types of chemical signals: pulsed and continuous. The results showed that the boundary layer increased the decay time of the chemical signal for the pulsatile stimuli and increased the peak height for the continuous data. Spectral analysis of continuous signals showed that the temporal aspects of the chemical signal are changed by the boundary layer. Particularly the temporal dynamics of the signal are dampened at the slowest flow speed and amplified at the intermediate and fast flow speeds. By altering the structure of the chemical signal, the morphology will function as a sensory filter.     

Approaches to define antigen receptor-induced serine kinase signal transduction pathways

In the present report we describe the properties of a novel phospho-specific antiserum that has opened a route to the characterization of antigen receptor-activated serine kinase pathways in lymphocytes. The basis for the present work was that Ser-21 in glycogen synthase kinase 3alpha is robustly phosphorylated following antigen receptor triggering. We predicted accordingly that antigen receptors would also stimulate phosphorylation of other proteins with a similar sequence. To test this idea we raised an antibody against the phospho-peptide RARTSpSFAEP, where pS is a phospho-serine corresponding to the glycogen synthase kinase 3alpha Ser-21 sequence. The resulting antiserum was called phospho antibody for proteomics-1 (PAP-1). The present study describes the properties of PAP-1 and shows that it can reveal quite striking differences in the phospho-proteome of different cell types and is able to pinpoint new targets in important signal transduction pathways. PAP-1 was used to map protein phosphorylations regulated by the antigen receptor in T cells. One of these PAP-1-reactive proteins was purified and revealed to be a previously unrecognized target for antigen receptor signal transduction, namely an "orphan" adapter SLY (Src homology 3 (SH3) domain-containing protein expressed in lymphocytes). The use of sera detecting specific phosphorylation sites is thus proved as a powerful method for the discovery of novel downstream components of antigen receptor signals in T cells.     

Plant signaling in stress: G-protein coupled receptors,heterotrimeric G-proteins and signal coupling via phospholipases

Plant growth and development are coordinalely controlled by several internal factors and environmental signals. To sense these environmental signals, the higher plants have evolved a complex signaling network, which may also cross talk with each other. Plants can respond to the signals as individual cells and as whole organisms. Various receptors including phytochromes, G-proteins coupled receptors (GPCR), kinase and hormone receptors play important role in signal transduction but very few have been characterized in plant system. The heterotrimeric G-proteins mediate the coupling of signal transduction from activated GPCR to appropriate downstream effectors and thereby play an important role in signaling. In this review we have focused on some of the recent work on G-proteins and two of the effectors, PLC and PLD, which have been shown to interact with Gα subunit and also discussed their role in abiotic stress tolerance.Key words: abiotic stress, G-protein couple receptor, heterotrimeric G-protein, phospholipases, plant receptors, signal transduction     

Stochastic resonance and signal detection in an energy detector – implications for biological receptor systems

Stochastic resonance is demonstrated in a simple energy detector model, as a non-monotonic relationship between signal-to-noise ratio and detection of a sinusoid signal in bandpass-limited Gaussian noise. The behaviour of the model detecting signals of various intensities and signal-to-noise ratios was investigated. Significant improvement in detection was obtained by adding noise for mean signal intensities below the detection criterion of the detector. The range of usable noise levels, however, may be too small to be biologically meaningful. It is demonstrated that improving detection in the analysed model by adding noise to an otherwise undetectable signal is only at best as efficient as what can be obtained by adjusting the criterion to the signal-to-noise ratio. Improving detection by means of stochastic resonance is thus a sub-optimal strategy. It is speculated whether a demonstration of stochastic resonance in a biological system indicates any adaptive significance. More than anything, it indicates the presence of a mismatch between receptor sensitivity and the signal-to-noise ratio of the experiment, not the cause of this mismatch. Received: 22 December 1999 / Accepted in revised form: 14 April 2000     

Engineering key components in a synthetic eukaryotic signal transduction pathway

Signal transduction underlies how living organisms detect and respond to stimuli. A goal of synthetic biology is to rewire natural signal transduction systems. Bacteria, yeast, and plants sense environmental aspects through conserved histidine kinase (HK) signal transduction systems. HK protein components are typically comprised of multiple, relatively modular, and conserved domains. Phosphate transfer between these components may exhibit considerable cross talk between the otherwise apparently linear pathways, thereby establishing networks that integrate multiple signals. We show that sequence conservation and cross talk can extend across kingdoms and can be exploited to produce a synthetic plant signal transduction system. In response to HK cross talk, heterologously expressed bacterial response regulators, PhoB and OmpR, translocate to the nucleus on HK activation. Using this discovery, combined with modification of PhoB (PhoB‐VP64), we produced a key component of a eukaryotic synthetic signal transduction pathway. In response to exogenous cytokinin, PhoB‐VP64 translocates to the nucleus, binds a synthetic PlantPho promoter, and activates gene expression. These results show that conserved‐signaling components can be used across kingdoms and adapted to produce synthetic eukaryotic signal transduction pathways.     

Long-term control of neuronal excitability by corticosteroid hormones

Hippocampal CA1 neurons express both mineralocorticoid and glucocorticoid receptors. Due to the difference in affinity of the two receptor types for corticosterone and variations in endogenous steroid levels, occupation of the receptors will range between a situation of predominant mineralocorticoid receptor activation and conditions where both receptor types are occupied. It was observed that local signal transduction is regulated by activation of the corticosteroid receptors. Particularly, transmission mediated by biogenic amines appears to be sensitive to steroid control. The data indicate that cholinergic and serotonergic responses are small with predominant mineralocorticoid receptor activation, while additional glucocorticoid receptor activation results in large responses; the reverse has been found for noradrenalin. The steroid-dependent control over transmission by biogenic amines will influence local excitability and therefore functional processes in which the hippocampal system is involved.     

光受体及光信号转导

植物在进化过程中形成了对环境信号反应的能力,光是植物生长发育中的一个重要的环境信号。植物为了更好地生长和发育形成了精密的光信号接收和转导系统。本文介绍近年来光信号接收即光受体和光信号的转导研究进展。     

Abnormal signal transduction: a hypothetical model for bipolar affective disorder

The molecular basis of bipolar affective disorder is poorly understood at this time. The episodic nature of the condition in which relatively euthymic periods of variable duration separate periods of mania and depression, and the specificity of lithium therapy suggests that a molecular target of the illness may be a system that bidirectionally influences neurotransmission and is affected by lithium. Signal transduction pathways, which are important mediators of neurotransmitter generated signals, may represent such a system because they: 1) generate second messenger molecules that stimulate neurotransmission and also mediate negative feedback mechanisms, and 2) appear to be a direct target of lithium's action on cells. In this paper, we present a model in which abnormal regulation of signal transduction could lead to the episodic accumulation of biologically active transducers or second messengers. These alterations may result in prolonged effector stimulation which may underlie mania, followed by excessive receptor desensitization, which may result in depression. Using our model we suggest a plausible hypothesis that can explain the clinical spectrum of the disorder and the therapeutic action of lithium.     

Distinct endocytic pathways regulate TGF-beta receptor signalling and turnover

Endocytosis of cell surface receptors is an important regulatory event in signal transduction. The transforming growth factor beta (TGF-beta) superfamily signals to the Smad pathway through heteromeric Ser-Thr kinase receptors that are rapidly internalized and then downregulated in a ubiquitin-dependent manner. Here we demonstrate that TGF-beta receptors internalize into both caveolin- and EEA1-positive vesicles and reside in both lipid raft and non-raft membrane domains. Clathrin-dependent internalization into the EEA1-positive endosome, where the Smad2 anchor SARA is enriched, promotes TGF-beta signalling. In contrast, the lipid raft-caveolar internalization pathway contains the Smad7-Smurf2 bound receptor and is required for rapid receptor turnover. Thus, segregation of TGF-beta receptors into distinct endocytic compartments regulates Smad activation and receptor turnover.     

Periplasmic binding proteins: a versatile superfamily for protein engineering

The diversity of biological function, ligand binding, conformational changes and structural adaptability of the periplasmic binding protein superfamily have been exploited to engineer biosensors, allosteric control elements, biologically active receptors and enzymes using a combination of techniques, including computational design. Extensively redesigned periplasmic binding proteins have been re-introduced into bacteria to function in synthetic signal transduction pathways that respond to extracellular ligands and as biologically active enzymes.     

Boundary-layer effect on chemical signal movement near the antennae of the sphinx moth, Manduca sexta : temporal filters for olfaction

For olfaction to occur, signal molecules must move through the environment from the source to the receptor cells. As molecules approach receptor structures they pass through a boundary layer surrounding those receptor structures. Within boundary layers the interaction between the forces causing chemical dispersion changes. To investigate how the boundary layer changes the dynamics of the chemical signals, we measured chemical dynamics within the boundary layer around the moth antennae using microelectrodes. The results showed that the boundary layer amplified three aspects of the chemical signal: peak height, peak onset, and decay time. Spectral analysis of turbulent signals showed that the temporal aspects of the chemical signal were altered. The boundary layers around the male and female antennae have different effects on the spectrum of chemical temporal fluctuations. Specifically, at a flow speed of 0.12 m s⁻¹, the analysis showed distinct amplification patterns for each sex. Thus, the fluid flow around the antennae functions as a filter, altering the structure of the chemical signal that is arriving at the receptors. The results illustrated in this study show that male and female moths have different physical filters that can alter the information that can be extracted from odor plumes. Accepted: 1 September 1997     

Mapping signal transduction pathways by phage display

Rapid identification of proteins that interact with a novel gene product is an important element of functional genomics. Here we describe a phage display-based technique for interaction screening of complex cDNA libraries using proteins or synthetic peptides as baits. Starting with the epidermal growth factor receptor (EGFR) cytoplasmic tail, we identified known protein interactions that link EGFR to the Ras/MAP kinase signal transduction cascade and several novel interactions. This approach can be used as a rapid and efficient tool for elucidating protein networks and mapping intracellular signal transduction pathways.     

A theoretical approach to G-protein modulation of cellular responsiveness

 Structure and function of cells often depend critically on molecular signals arriving at their surface. There are universal mechanisms of signal transduction and signal processing across cell membranes. In this paper the mechanisms involving guanine-nucleotide regulatory proteins (“G-proteins”) and certain receptor-kinases are considered. On the basis of recent findings in molecular biology a mathematical model is developed taking into account all essential components in the biochemical network between first and second messenger. There are two coupled feedback loops inherent in this process. The model finally consists of three nonlinear equations, which are obtained from a system of originally ten equations by using conservation laws and quasi-steady state conditions. The second part of the paper contains a mathematical analysis of the model. Solutions describing the temporal development of the involved biochemical species are shown to be bounded, more specifically to remain, independent of the size of the input signal, in a bounded domain of the state space. For the situation of stationary input signals existence, uniqueness and asymptotic stability of steady states are derived. We also demonstrate biologically relevant stimulus-response properties like monotonicity and saturation effects. For temporally non-constant input signals we show numerically that the model is able to produce phenomena of hypersensitivity and desensitization which are important characteristics of cellular responsiveness. Received 18 March 1996; received in revised form 15 April 1996     

寡糖诱导植物抗病性研究进展

寡糖在植物中可作为早期信息分子,激活植物防御系统,诱导植物产生抗病性。综述近年来对寡糖激发子受体、信号转导、基因表达等方面的研究进展。