S-(2-(acylamino)phenyl) 2,2-dimethylpropanethioates as CETP inhibitors

Studies on the relationship between the structure of the benzene moiety of S-(2-(acylamino)phenyl) 2,2-dimethylpropanethioates and CETP inhibitory activity were performed. Substituents on the benzene moiety influenced CETP inhibitory activity in a type and position dependent manner, and electron-withdrawing groups at the 4- or 5-position increased the activity. The most potent compound showed 50% inhibition of CETP activity in human plasma at a concentration of 2 microM.     

GroEL mediates protein folding with a two successive timer mechanism

GroEL encapsulates nonnative substrate proteins in a central cavity capped by GroES, providing a safe folding cage. Conventional models assume that a single timer lasting approximately 8 s governs the ATP hydrolysis-driven GroEL chaperonin cycle. We examine single molecule imaging of GFP folding within the cavity, binding release dynamics of GroEL-GroES, ensemble measurements of GroEL/substrate FRET, and the initial kinetics of GroEL ATPase activity. We conclude that the cycle consists of two successive timers of approximately 3 s and approximately 5 s duration. During the first timer, GroEL is bound to ATP, substrate protein, and GroES. When the first timer ends, the substrate protein is released into the central cavity and folding begins. ATP hydrolysis and phosphate release immediately follow this transition. ADP, GroES, and substrate depart GroEL after the second timer is complete. This mechanism explains how GroES binding to a GroEL-substrate complex encapsulates the substrate rather than allowing it to escape into solution.     

Prediction of the coding sequences of mouse homologues of FLJ genes: the complete nucleotide sequences of 110 mouse FLJ-homologous cDnas identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse KIAA-homologous genes since 2001. As an extension of this project, we also started to accumulate mouse cDNA clones homologous to the human FLJ cDNA clones which are another long cDNA resource produced in our institute. We have isolated the cDNA clones from size-fractionated cDNA libraries derived from five different mouse tissues and natural killer T-cells. Although the human FLJ cDNA clones were originally derived from human spleen libraries, one-third of their mouse homologues were obtained from the brain library. We designated these homologues "mFLJ" plus a 5-digit number and herein characterized 110 mFLJ cDNA clones. We assigned an integrity of the CDSs from the comparison of the 110 cDNA clones with the corresponding human FLJ cDNA clones. The average size of the 110 mouse cDNA sequences was 3.8 kb and that of the deduced amino acid sequences from their longest CDS in each cDNA was 663 amino acid residues. Homology and/or motif search against public databases revealed new domains and/or motifs in 26 mFLJ gene products which provide additional speculation regarding the function of FLJ genes.     

A comprehensive approach for establishment of the platform to analyze functions of KIAA proteins: generation and evaluation of anti-mKIAA antibodies

Since December 2001 we have been conducting a project to isolate and determine entire sequences of mouse KIAA cDNA clones which encode polypeptides corresponding to human KIAA proteins. The ultimate goal of this project is the elucidation of the functions of KIAA proteins. A critical step in this project is the generation of antibodies based on the cDNA sequence information. Although antibodies are the most optimal tools for biological analysis, the production and isolation of multiple recombinant proteins for an antigen is a rate-limiting step in antibody production. To address this problem, we established a system utilizing the in vitro recombination-assisted method and shotgun clones that were generated during the sequencing of mouse KIAA cDNAs (DNA Res. 2003, 10, 129-136). The authenticity of the expressed proteins was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Another critical step for antibody production is the evaluation of the antibodies. Thus, we also made efforts to develop a systematic approach for evaluation of the titer and the specificity of the antibodies. Using these systems, we have produced and evaluated more than 500 antibodies raised against mouse KIAA proteins to date. We are currently generating antibody arrays for analysis of protein expression profiles. We will verify protein-protein interactions using immunoprecipitation and tandem mass spectrometry analysis.     

Accelerated biodegradation of poly(vinyl alcohol) by glycosidations of the hydroxyl groups or addition of sugars

Biodegradabilities of N-acetyl-d-glucosamine (GlcNAc)- (1) and chitobiose-substituted (2) poly(vinyl alcohol)s (PVA)s in a soil suspension (pH 6.5) were investigated at 25 degrees C for 40 days. Biochemical oxygen demand of 1 with a degree of substitution of 0.2-0.3 (DP = 430-480) was higher than that of PVA under the degradation condition. Size exclusion chromatography, (1)H NMR, and Fourier-transform infrared measurements of the recovered sample indicated that biodegradation of the PVA main chain was accelerated by partial glycosidation of hydroxyl groups in PVA. Similar acceleration was observed in a PVA/GlcNAc (50:50, w/w) mixture. Microbes which relate with degradation of the glycosidated polymers were grown in a culture medium including the soil suspension and the polymer as the carbon source. Polyacrylamide gel electrophoresis (SDS-PAGE) and IR measurements indicated that a cell-free extract derived from GlcNAc-substituted PVA was different from that in the PVA/GlcNAc mixture. The results suggested that the PVA main chain in GlcNAc-substituted PVA was cleaved by a different microorganism or via a mechanism different from that in the mixture. Chitobiose-substituted PVA 2 showed more enhanced acceleration, indicating that the sugar length influenced the degradability.     

Identification of two novel mutations in adenine phosphoribosyltransferase gene in patients with 2,8-dihydroxyadenine urolithiasis

Five mutations in the adenine phosphoribosyltransferase (APRT) gene have been described in Japanese patients with APRT deficiency. We investigated the APRT gene from three patients with APRT deficiency and two novel mutations, G133D and V84M, were determined.     

Activation of a signaling cascade by cytoskeleton stretch

Cells sense and respond to mechanical force. However, the mechanisms of transduction of extracellular matrix (ECM) forces to biochemical signals are not known. After removing the cell membrane and soluble proteins by Triton X-100 extraction, we found that the remaining complex (Triton cytoskeletons) activated Rap1 upon stretch. Rap1 guanine nucleotide exchange factor, C3G, was required for this activation; C3G as well as the adaptor protein, CrkII, in cell extract bound to Triton cytoskeletons in a stretch-dependent manner. CrkII binding, which was Cas dependent, correlated with stretch-dependent tyrosine phosphorylation of proteins in Triton cytoskeletons including Cas at the contacts with ECM. These in vitro findings were compatible with in vivo observations of stretch-enhanced phosphotyrosine signals, accumulation of CrkII at cell-ECM contacts, and CrkII-Cas colocalization. We suggest that mechanical force on Triton cytoskeletons activates local tyrosine phosphorylation, which provides docking sites for cytosolic proteins, and initiates signaling to activate Rap1.     

Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operates in submucosal gland acinar cells

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.