It has been shown that a non-synonymous polymorphism Asp299Gly of Toll-like receptor-4 (TLR-4) gene could be associated with lipopolysaccharide hyporesponsiveness and decreased risk of atherosclerosis in Caucasians. However, this polymorphism is not detected in Asians including Japanese and hence considered to be a population-specific one. To investigate if there are common non-synonymous SNPs of TLR-4 gene in Japanese, 100 DNA samples randomly selected from 613 healthy volunteers were sequenced for exons 2 and 3. As a result, Thr175Ala (A690G) was found in 6 out of 100 samples. Genotyping of Thr175Ala was then performed for 842 DNA samples from 613 healthy volunteers and 229 patients with ulcerative colitis. Frequency of heterozygote (Thr/Ala) or homozygote (Ala/Ala) in healthy subjects was 2.3% (14/613) or 0% (0/613), respectively. There was no significant difference of the genotype distribution between ulcerative colitis patients and age- and gender-matched controls. Furthermore, Thr¹⁷⁵ is known to be involved in a potential N-linked glycosylation site and hence the polymorphism might affect the function of TLR-4. These data suggest that Thr175Ala is a common non-synonymous polymorphism of TLR-4 gene in a Japanese population.

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Protein chips are useful tools for profiling proteins and analyzing protein-protein interactions and post-translational modifications. In previous work, we developed a diamond-like carbon-coated stainless steel plate (DLC plate) as a novel protein chip plate. Gel-resolved proteins can be covalently immobilized on the surface of the DLC plate by electroblotting to produce a high-density protein chip. The proteins can then be identified by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS) using the plates. The interactions of the immobilized proteins with other proteins, and their post-translational modifications, can also be analyzed. However, to improve the efficiency and reproducibility of analyses using DLC plates, it is important to automate these analytical processes. Therefore, we developed a system for automatically transferring gels from the gel electrophoresis glass plates to the DLC plates for electroblotting. This is an essential first step toward complete automation of the production of high-density protein chips for immobilizing gel-resolved proteins.

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