Photoproximity labeling of endogenous receptors in the live mouse brain in minutes.

Understanding how protein-protein interaction networks in the brain give rise to cognitive functions necessitates their characterization in live animals. However, tools available for this purpose require potentially disruptive genetic modifications and lack the temporal resolution necessary to track rapid changes in vivo. Here we leverage affinity-based targeting and photocatalyzed singlet oxygen generation to identify neurotransmitter receptor-proximal proteins in the live mouse brain using only small-molecule reagents and minutes of photoirradiation.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains.

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro.

Revisiting PFA-mediated tissue fixation chemistry: enables trapping of small molecules in the brain to visualize their distribution changes.

Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/excretion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited.

Protocol to visualize the distribution of exogenously administered small molecules in the mouse brain.

Here, we present fixation-driven chemical crosslinking of exogenous ligands, a protocol to visualize the distribution of exogenously administered small molecules in the mouse brain. We first describe the probe design of the small molecules of interest and the probe microinjection into a live mouse brain in detail. We then detail procedures for paraformaldehyde-perfusion fixation.

Coronaridine congeners induce sedative and anxiolytic-like activity in naïve and stressed/anxious mice by allosteric mechanisms involving increased GABA receptor affinity for GABA.

The sedative and anxiolytic-like activity of two coronaridine congeners, (+)-catharanthine and (-)-18-methoxycoronaridine (18-MC), was studied in male and female mice. The underlying molecular mechanism was subsequently determined by fluorescence imaging and radioligand binding experiments. The loss of righting reflex and locomotor activity results showed that both (+)-catharanthine and (-)-18-MC induce sedative effects at doses of 63 and 72 mg/kg in a sex-independent manner.

Chemical, Pharmacological, and Structural Characterization of Novel Acrylamide-Derived Modulators of the GABA Receptor.

Acrylamide-derived compounds have been previously shown to act as modulators of members of the Cys-loop transmitter-gated ion channel family, including the mammalian GABA receptor. Here we have synthesized and functionally characterized the GABAergic effects of a series of novel compounds (termed "DM compounds") derived from the previously characterized GABA and the nicotinic 7 receptor modulator (E)-3-furan-2-yl--p-tolyl-acrylamide (PAM-2). Fluorescence imaging studies indicated that the DM compounds increase apparent affinity to the transmitter by up to 80-fold in the ternary GABA receptor.

Recent applications of N-acyl imidazole chemistry in chemical biology.

N-Acyl imidazoles are unique electrophiles that exhibit moderate reactivity, relatively long-half life, and high solubility in water. Thanks to their tunable reactivity and chemical selectivity, the application of N-acyl imidazole derivatives has launched to a number of chemical biology researches, which include chemical synthesis of peptide/protein, chemical labeling of native proteins of interest (POIs), and structural analysis and functional manipulation of RNAs. Since proteins and RNAs play pivotal roles in numerous biological events in all living organisms, the methods that enable the chemical modification of endogenously existing POIs and RNAs in live cells may offer a variety of opportunities not only for fundamental scientific study but also for biotechnology and drug development.

Screening of protein-ligand interactions under crude conditions by native mass spectrometry.

A convenient analytical system for protein-ligand interactions under crude conditions was developed using native mass spectrometry (MS). As a model protein, Escherichia coli (E. coli) dihydrofolate reductase (DHFR) with and without a histidine tag was used for the study.

Peripartum myocardial infarction associated with coronary spasm and acquired protein S deficiency: A case report.

Rationale: Coronary angiography (CAG) findings of acute myocardial infarction (AMI) in pregnant women are characterized by a high incidence of normal coronary arteries. This is the first report of AMI with normal coronary arteries during pregnancy, showing coronary spasm and pregnancy-related acquired protein S (PS) deficiency.

Patient Concerns: A 30-year-old Japanese woman was admitted to an emergency department.

Construction of a Fluorescent Screening System of Allosteric Modulators for the GABA Receptor Using a Turn-On Probe.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The fast inhibitory actions of GABA are mainly mediated by GABA receptors (GABARs), which are widely recognized as clinically relevant drug targets. However, it remains difficult to create screening systems for drug candidates that act on GABARs because of the existence of multiple ligand-binding sites and the delicate pentameric structures of GABARs.

Construction of ligand assay systems by protein-based semisynthetic biosensors.

Proteins as causative agents of diseases such as cancers, diabetes and neurological disorders are attractive drug targets. For developing chemicals selectively acting on key disease-causing proteins, one useful concept is the direct conversion of such target proteins into biosensors. This approach provides ligand-binding assay systems based on protein-based biosensors, which can quantitatively evaluate interactions between the protein and a specific ligand in many environments.

Recent Progress in Chemical Modification of Proteins.

Chemical modification of proteins is important for creating a myriad of engineered proteins and for elucidating the function and dynamics of proteins in live cells. A wide variety of chemical protein modification methods have been developed and can be categorized into three classes: (i) modification of proteins using the reactivity of naturally occurring amino acids; (ii) modification by bioorthogonal reactions using unnatural amino acids, most of which can be site-selectively incorporated into proteins-of-interest using genetic codon expansion techniques; and (iii) recognition driven chemical modification, which is the only approach that allows modification of endogenous proteins without any genetic manipulation even under heavily crowded and multi-molecular conditions, as in live cells and organisms. All of these approaches have merits and limitations.

Ligand-Directed Chemistry of AMPA Receptors Confers Live-Cell Fluorescent Biosensors.

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission in the central nervous system. Dysregulation of AMPAR function is associated with many kinds of neurological, neurodegenerative, and psychiatric disorders. As a result, molecules capable of controlling AMPAR functions are potential therapeutic agents.

Desflurane inhalation before ischemia increases ischemia-reperfusion-induced vascular leakage in isolated rabbit lungs.

Background: Isoflurane and sevoflurane protect lungs with ischemia-reperfusion (IR) injury. We examined the influence of desflurane on IR lung injury using isolated rabbit lungs perfused with a physiological salt solution.

Methods: The isolated lungs were divided into three groups: IR, desflurane-treated ischemia-reperfusion (DES-IR), and ventilation/perfusion-continued control (Cont) groups (n = 6 per group).

Activation of p53 Facilitates the Target Search in DNA by Enhancing the Target Recognition Probability.

Tumor suppressor p53 binds to the target in a genome and regulates the expression of downstream genes. p53 searches for the target by combining three-dimensional diffusion and one-dimensional sliding along the DNA. To examine the regulation mechanism of the target binding, we constructed the pseudo-wild type (pseudo-WT), activated (S392E), and inactive (R248Q) mutants of p53 and observed their target binding in long DNA using single-molecule fluorescence imaging.

Usefulness of 3D hybrid profile order technique with 3T magnetic resonance cholangiography: Comparison of image quality and acquisition time.

Purpose: To evaluate the image quality and acquisition time of magnetic resonance cholangiopancreatography (MRCP) with and without the 3D hybrid profile order technique.

Materials And Methods: We studied 32 consecutive patients at 3T. They underwent MRCP with and without the 3D hybrid profile order imaging technique during free breathing and MRCP with the 3D hybrid profile order technique during a single breath-hold.

Phosphoenolpyruvate administration protects ischemia-reperfusion injury in isolated rabbit lungs.

Phosphoenolpyruvate (PEP) is an intermediate metabolite of the glycolytic pathway and an in vivo high-energy phosphate compound. We have examined the protective effects of PEP on ischemia-reperfusion lung injury in isolated rabbits lungs perfused with a physiological salt solution. The lungs were divided into three treatment groups: (1) ischemia-reperfusion (IR), (2) ischemia-reperfusion with PEP treatment (PEP-IR), in which 1 mM PEP was pre-administered into the perfusate during the stable period, and (3) ventilation-perfusion continued without interruption (Cont).

Creation of a caspase-3 sensing system using a combination of split-GFP and split-intein.

A genetically encodable caspase-3 sensing system has been created using self-assembling split-GFP, in which a C-terminal fragment is "covalently" cyclized via a caspase-3 substrate sequence mediated by split-intein. The specific cleavage of the cyclic C-terminal fragment by caspase-3 induces the GFP reassembly and fluorescence recovery.

Design and semisynthesis of photoactivable split-GFP by incorporation of photocleavable functionality.

The design of proteins whose structure and function can be manipulated by the external stimuli has been of great interest in the field of protein engineering. In particular, caged proteins which can be activated by photo-irradiation become powerful tools for investigating a variety of biological events. Although protein caging is straightforward to render light-responsive protein functions, this approach mostly have difficulties based on the preparation of caged proteins in which amino acid residues required for biological activities must be specifically modified with synthetic photolabile groups.

One-pot sequential alcohol oxidation and asymmetric α-oxyamination in aqueous media using recyclable resin-supported peptide catalyst.

An efficient tandem reaction system was developed, in which primary alcohols were used for the oxidation to the corresponding aldehydes followed by an asymmetric α-oxyamination with a resin-supported peptide catalyst.

Effect of intraoperative acetated Ringer's solution with 1% glucose on glucose and protein metabolism.

Purpose: To investigate the effects of the intraoperative administration of Ringer's solution with 1% glucose on the metabolism of glucose, lipid and muscle protein during surgery.

Methods: Thirty-one adult patients, American Society of Anesthesiologists physical status I or II, undergoing elective otorhinolaryngeal, head and neck surgeries were randomly assigned to one of two patient groups: those receiving acetated Ringer's solution with 1% glucose (Group G) or those receiving acetated Ringer's solution without glucose (Group R) throughout the surgical procedure. Plasma glucose was measured at anesthetic induction (T0), artery 1 h (T1), 2 h (T2), 3 h after anesthetic induction (T3) and at the end of surgery (T4).

Efficient asymmetric alpha-oxyamination of aldehydes by resin-supported peptide catalyst in aqueous media.

The resin-supported peptide catalyst having the terminal five-residue Pro-d-Pro-Aib-Trp-Trp combined with polyleucine successfully catalyzed the asymmetric alpha-oxyamination of aldehydes in aqueous media. The secondary structure and the chirality sense of the hydrophobic polyleucine chain significantly affected both reactivity and enantioselectivity.

Studies on the effects of arginine residues introduced to peptide ribonucleic acids (PRNA) on the complex stability with RNA.

In this study, a series of novel alpha-Peptide ribonucleic acid (alpha-PRNA) oligomers, possessing alternative alpha-PRNA/arginine or serine sequences, were newly designed, synthesized, and evaluated as the third-generation PRNA. As expected, these alpha-PRNAs formed highly stable sequence-specific complexes with the complementary RNAs, for which both the conventional hydrogen-bonding interactions between the complementary nucleobase pairs and the electrostatic interactions between the arginine's guanidinium cation and the RNA's phosphate anion on the backbone are jointly responsible. Moreover, in the cases of alpha-PRNA and single point mismatched DNA mixing systems, appreciable T(m) could not be observed, thus alpha-PRNAs containing Arg were expected to have high nucleobase sequence discrimination abilities.

Supramolecular control of split-GFP reassembly by conjugation of beta-cyclodextrin and coumarin units.

The design of proteins whose structure and function can be manipulated by binding with specific ligands has been of great interest in the field of protein engineering. Some successful examples of small-molecule-dependent proteins have been reported, but their ligand-binding domains have mainly been limited to those derived from natural proteins. The introduction of synthetic components for ligand responsiveness may expand the versatility of small-molecule-dependent proteins.