Qr: switch:"iLID"
Showing 226 - 240 of 240 results
226.
Investigating neuronal function with optically controllable proteins.
Abstract:
In the nervous system, protein activities are highly regulated in space and time. This regulation allows for fine modulation of neuronal structure and function during development and adaptive responses. For example, neurite extension and synaptogenesis both involve localized and transient activation of cytoskeletal and signaling proteins, allowing changes in microarchitecture to occur rapidly and in a localized manner. To investigate the role of specific protein regulation events in these processes, methods to optically control the activity of specific proteins have been developed. In this review, we focus on how photosensory domains enable optical control over protein activity and have been used in neuroscience applications. These tools have demonstrated versatility in controlling various proteins and thereby cellular functions, and possess enormous potential for future applications in nervous systems. Just as optogenetic control of neuronal firing using opsins has changed how we investigate the function of cellular circuits in vivo, optical control may yet yield another revolution in how we study the circuitry of intracellular signaling in the brain.
227.
Optimizing optogenetic constructs for control over signaling and cell behaviours.
Abstract:
Optogenetic tools have recently been developed that enable dynamic control over the activities of select signaling proteins. They provide the unique ability to rapidly turn signaling events on or off with subcellular control in living cells and organisms. This capability is leading to new insights into how the spatial and temporal coordination of signaling events governs dynamic cell behaviours such as migration and neurite outgrowth. These tools can also be used to dissect a protein's signaling functions at different organelles. Here we review the properties of photoreceptors from diverse organisms that have been leveraged to control signaling in mammalian cells. We emphasize recent engineering approaches that have been used to create optogenetic constructs with optimized spectral, kinetic, and signaling properties for controlling cell behaviours.
228.
Engineering an improved light-induced dimer (iLID) for controlling the localization and activity of signaling proteins.
Abstract:
The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling.
229.
Synthetic protein switches: design principles and applications.
Abstract:
Protein switches are ubiquitous in biological signal transduction systems, enabling cells to sense and respond to a variety of molecular queues in a rapid, specific, and integrated fashion. Analogously, tailor-engineered protein switches with custom input and output functions have become invaluable research tools for reporting on distinct physiological states and actuating molecular functions in real time and in situ. Here, we analyze recent progress in constructing protein-based switches while assessing their potential in the assembly of defined signaling motifs. We anticipate such systems will ultimately pave the way towards a new generation of molecular diagnostics and facilitate the construction of artificial signaling systems that operate in parallel to the signaling machinery of a host cell for applications in synthetic biology.
230.
Natural photoreceptors and their application to synthetic biology.
Abstract:
The ability to perturb living systems is essential to understand how cells sense, integrate, and exchange information, to comprehend how pathologic changes in these processes relate to disease, and to provide insights into therapeutic points of intervention. Several molecular technologies based on natural photoreceptor systems have been pioneered that allow distinct cellular signaling pathways to be modulated with light in a temporally and spatially precise manner. In this review, we describe and discuss the underlying design principles of natural photoreceptors that have emerged as fundamental for the rational design and implementation of synthetic light-controlled signaling systems. Furthermore, we examine the unique challenges that synthetic protein technologies face when applied to the study of neural dynamics at the cellular and network level.
231.
Optical control of protein function through unnatural amino acid mutagenesis and other optogenetic approaches.
Abstract:
Biological processes are naturally regulated with high spatial and temporal resolution at the molecular, cellular, and systems level. To control and study processes with the same resolution, light-sensitive groups and domains have been employed to optically activate and deactivate protein function. Optical control is a noninvasive technique in which the amplitude, wavelength, spatial location, and timing of the light illumination can be easily controlled. This review focuses on applications of genetically encoded unnatural amino acids containing light-removable protecting groups to optically trigger protein function, while also discussing select optogenetic approaches using natural light-sensitive domains to engineer optical control of biological processes.
232.
Sensitivity and precision of activated partial thromboplastin time (APTT) methods. A multicenter study.
Abstract:
The Activated Partial Thromboplastin Time (APTT) test, Cephotest, was compared to other APTT methods in current use in 4 specialized coagulation laboratories. In 3 of 4 laboratories, the sensitivity of Cephotest was superior (P less than 0.001) to that of the local APTT method. There was no statistically significant difference between the APTT methods with regard to precision of repetitive testing. In each laboratory, the normal range of Cephotest was estimated on freshly collected plasma samples from healthy subjects. A mean value between 28.8 and 35.8 s, with a standard deviation of 1.1-3.3 s, was obtained. It is concluded that the composition of the APTT method if of importance for the sensitivity of this test, but does not influence the precision of repetitive testing to a significant degree. The use of a standardized reagent facilitates comparison of the results obtained with the APTT method from one laboratory to another.
233.
Rhodopsin in model membranes: charge displacements in interfacial layers.
Abstract:
A model membrane was developed in which interfacial layers of rhodopsin were reoriented onto one side of a thin Teflon film separating two aqueous compartments. Flashes evoked fast photoelectric signals (1 ms) that originated from capacitative charge displacements of oriented rhodopsin upon bleaching. The photoelectric responses of rhodopsin in the model membrane are compared with the early receptor potential of photoreceptor cells; it is concluded that the signals in both systems originate from the same mechanism.
234.
Arrhythmogenic properties of thiamylal sodium in the dog.
Abstract:
Abstract not available.
235.
The specificity of heterophil antibodies in patients and healthy donors with no or minimal signs of infectious mononucleosis.
Abstract:
Over several years sera were collected from 14 heterophil-positive students or patients who did not fulfill minimal hematologic criteria for infectious mononucleosis (I.M.) The specificity of these heterophil reactions for I.M. was investigated by determining antibodies to Epstein-Barr virus-determined antigens, i.e., to viral capsid antigens (VCA), early antigens (EA), and EBV-associated nuclear antigens (EBNA). On the basis of detectable anti-EA and/or the early absence and late emergence of anti-EBNA, four of these 14 individuals showed evidence of a current or very recent primary Epstein-Barr virus infection. The other ten patients showed antibody patterns indicative of Epstein-Barr virus infections in the past, and no firm conclusions could be drawn with regard to the specificity of their heterophil reactions. It was assumed, however, that some represented atypical clinical forms of EBV infection and that timing of specimen collection was a factor in explaining the paucity of Downey cells. In three patients, the absorbed heterophil-positive reactions persisted with little change in titer for at least 22 mo and thus might represent false-positive tests.
236.
Citrate synthase.
Abstract:
Abstract not available.
237.
Beef liver L-Glutamate dehydrogenase mechanism: presteady state study of the catalytic reduction of 2.oxoglutarate by NADPH.
Abstract:
Abstract not available.
238.
A comparison of the substrate specificities of endo-beta-N-acetylglucosaminidases from Streptomyces griseus and Diplococcus Pneumoniae.
Abstract:
Abstract not available.
239.
[Elements of scientific work organization in ambulance feldshers' functions].
Abstract:
Abstract not available.
240.
Formate assay in body fluids: application in methanol poisoning.
Abstract:
Abstract not available.
