Application of ultrafiltration method to measurement of catecholamines in plasma of human and rodents by high-performance liquid chromatography

In order to develop a reliable, simple and routine method using small sample volume to determine norepinephrine (NE) and epinephrine (E) concentrations in plasma of humans and rodents, we utilize the ultrafiltration (UF) method by Ultrafree-MC filter device and a high-performance liquid chromatography equipped with electrochemical detector (HPLC-ECD) to detect NE and E. Optimum UF and HPLC conditions were as follows: the filter nominal molecular weight limit size is 30,000, the pH of added phosphate buffer to each plasma sample for UF is 3.0, and the mobile phase is 0.1M phosphate buffer (pH 3)/acetonitrile (98:2) containing 0.05% sodium disulfite and 0.001% EDTA 2Na. The plasma samples and 1.0M phosphate buffer (pH 3) containing 3,4-dihydroxybenzylamine (DHBA), as an internal standard, was mixed and poured into the UF units. After the centrifugation for 60 min at 13,000 x g at 4 degrees C, the filtrate was directly injected into HPLC. The calibration curve of NE and E was linear for the concentrations studied (20-400 pg) with a correlation coefficient of >0.999. Intra-assay coefficients of variation for NE and E using this method were less than 3%. The method also correlated well with the well-established alumina method (r=0.954). The present findings suggest that a newly-developed UF method with HPLC-ECD would apply successfully to measure plasma NE and E concentrations in humans and rodents.     

Identification of 2,3-disubstituted pyridines as potent,orally active PDE4 inhibitors

A series of 2,3-disubstituted pyridines were synthesized and evaluated for their PDE4 inhibitory activity. We successfully modified undesirable cyano group of initial lead compound 2 to 4-pyridyl group with improvement of in vitro efficacy and optimized the position of nitrogen atoms in pyridine moiety and alkylene linker. The most potent compound showed significant efficacy in animal models of asthma and inflammation.     

Vesicular neurotransmitter transporter: bioenergetics and regulation of glutamate transport

Glutamate plays essential roles in chemical transmission as a major excitatory neurotransmitter. The accumulation of glutamate in secretory vesicles is mediated by vesicular glutamate transporters (VGLUTs) that together with the driving electrochemical gradient of proteins influence the subsequent quantum release of glutamate and the function of higher-order neurons. The vesicular content of glutamate is well correlated with membrane potential (Δψ), which suggests that Δψ determines the vesicular glutamate concentration. The transport of glutamate into secretory vesicles is highly dependent on Cl(-). This anion stimulates glutamate transport but is inhibitory at higher concentrations. Accumulating evidence indicates that Cl(-) regulates glutamate transport through control of VGLUT activity and the H(+) electrochemical gradient. Recently, a comprehensive study demonstrated that Cl(-) regulation of VGLUT is competitively inhibited by metabolic intermediates such as ketone bodies. It also showed that ketone bodies are effective in controlling epilepsy. These results suggest a correlation between metabolic state and higher-order brain function. We propose a novel function for Cl(-) as a fundamental regulator for signal transmission.     

Characterization of inhibitor binding sites of mitochondrial complex I using fluorescent inhibitor

Recent progress in complex I research suggests that a wide variety of complex I inhibitors share a common large binding domain with partially overlapping sites. To verify this concept, we carried out real-time displacement tests of a fluorescent ligand with various competitors using a novel quinazoline-type inhibitor (aminoquinazoline, AQ). In the presence of an excess amount of the competitors, the binding of AQ to the enzyme was completely suppressed, being in line with the concept mentioned above. However, AQ bound to the enzyme was not displaced by subsequent addition of an increasing amount of competitors in the concentration range expected from the relative magnitude of the K(d) values of AQ and competitors, rather, much higher concentrations of the competitors were needed to displace bound AQ. These results cannot be explained merely by the premise of a common or partially overlapping binding site(s) between AQ and competitors. On the other hand, double-inhibitor titration of steady state complex I activity suggested that additivity of inhibition is not necessarily observed for all pairs of complex I inhibitors. Our results are discussed in light of the cooperativity of the inhibitor binding sites.     

TG1 integrase-based system for site-specific gene integration into bacterial genomes

Serine-type phage integrases catalyze unidirectional site-specific recombination between the attachment sites, attP and attB, in the phage and host bacterial genomes, respectively; these integrases and DNA target sites function efficiently when transferred into heterologous cells. We previously developed an in vivo site-specific genomic integration system based on actinophage TG1 integrase that introduces ～2-kbp DNA into an att site inserted into a heterologous Escherichia coli genome. Here, we analyzed the TG1 integrase-mediated integrations of att site-containing ～10-kbp DNA into the corresponding att site pre-inserted into various genomic locations; moreover, we developed a system that introduces ～10-kbp DNA into the genome with an efficiency of ～10⁴ transformants/μg DNA. Integrations of attB-containing DNA into an attP-containing genome were more efficient than integrations of attP-containing DNA into an attB-containing genome, and integrations targeting attP inserted near the replication origin, oriC, and the E. coli “centromere” analogue, migS, were more efficient than those targeting attP within other regions of the genome. Because the genomic region proximal to the oriC and migS sites is located at the extreme poles of the cell during chromosomal segregation, the oriC–migS region may be more exposed to the cytosol than are other regions of the E. coli chromosome. Thus, accessibility of pre-inserted attP to attB-containing incoming DNA may be crucial for the integration efficiency by serine-type integrases in heterologous cells. These results may be beneficial to the development of serine-type integrases-based genomic integration systems for various bacterial species.     

Quantitative metabolome analysis using capillary electrophoresis mass spectrometry

A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.     

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.     

Protein S-guanylation by the biological signal 8-nitroguanosine 3',5'-cyclic monophosphate

The signaling pathway of nitric oxide (NO) depends mainly on guanosine 3',5'-cyclic monophosphate (cGMP). Here we report the formation and chemical biology of a nitrated derivative of cGMP, 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), in NO-mediated signal transduction. Immunocytochemistry demonstrated marked 8-nitro-cGMP production in various cultured cells in an NO-dependent manner. This finding was confirmed by HPLC plus electrochemical detection and tandem mass spectrometry. 8-Nitro-cGMP activated cGMP-dependent protein kinase and showed unique redox-active properties independent of cGMP activity. Formation of protein Cys-cGMP adducts by 8-nitro-cGMP was identified as a new post-translational modification, which we call protein S-guanylation. 8-Nitro-cGMP seems to regulate the redox-sensor signaling protein Keap1, via S-guanylation of the highly nucleophilic cysteine sulfhydryls of Keap1. This study reveals 8-nitro-cGMP to be a second messenger of NO and sheds light on new areas of the physiology and chemical biology of signal transduction by NO.     

Design and synthesis of novel hydrophilic spacers for the reduction of nonspecific binding proteins on affinity resins

Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets. Reduction of nonspecific binding proteins is important for success in finding such targets. We herein disclose the design, synthesis, and effectiveness in reduction of nonspecific binding proteins, of novel hydrophilic spacers (2-5), which were introduced between matrices and a ligand. Among them, tartaric acid derivative (5) exhibited the most effective reduction of nonspecific binding proteins, whilst maintaining binding of the target protein. Introduction of 5 on TOYOPEARL reduced tubulin and actin by almost 65% and 90% compared to that without the hydrophilic spacer, respectively, with effective binding to the target protein, FKBP12.