A high-speed atomic force microscope for studying biological macromolecules.

The atomic force microscope (AFM) is a powerful tool for imaging individual biological molecules attached to a substrate and placed in aqueous solution. At present, however, it is limited by the speed at which it can successively record highly resolved images. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behavior of biomolecules. For this purpose, we have developed a high-speed scanner, free of resonant vibrations up to 60 kHz, small cantilevers with high resonance frequencies (450-650 kHz) and small spring constants (150-280 pN/nm), an objective-lens type of deflection detection device, and several electronic devices of wide bandwidth. Integration of these various devices has produced an AFM that can capture a 100 x 100 pixel(2) image within 80 ms and therefore can generate a movie consisting of many successive images (80-ms intervals) of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica (see http://www.s.kanazawa-u.ac.jp/phys/biophys/bmv_movie.htm).

A trimmed viral cap-independent translation enhancing sequence for rapid in vitro gene expression.

We prepared a short (29 nucleotides) 5' UTR that enhanced cap-independent translation in a wheat germ translation system by trimming the tobacco etch virus 5' UTR. The trimmed sequence, designated as TE(37-65), was obtained from a conserved region among several potyviruses. The productivities of uncapped reporter mRNAs carrying the TE(37-65) sequence were comparable to those of capped counterparts, in that 5-20 microg of proteins were synthesized per 1 mL of translation reaction mixture. The ribosome that entered onto the TE(37-65) sequence precisely initiated polypeptide synthesis at the defined initiation codon, which ensures rapid and efficient protein truncation analyses. Moreover, the TE(37-65) sequence is short enough to be involved in a PCR primer, which allows a simple method for rapid gene expression, i.e., PCR amplification of a target gene and succeeding in vitro transcription and translation. As a demonstration, the rapid in vitro expression of rice cDNAs using the TE(37-65) sequence was also performed.

Anti-inflammatory, analgesic and anti-pyretic effects of d-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid (M-5011), a new non-steroidal anti-inflammatory drug, in rats and guinea pigs.

Anti-inflammatory, analgesic and anti-pyretic effects of d-2-[4-(3-methyl-2-thienyl)phenyl]propionic acid (M-5011), a new non-steroidal anti-inflammatory drug (NSAID), were compared with those of indomethacin, diclofenac sodium and ketoprofen in rats and guinea pigs. Anti-inflammatory effect of M-5011 on ultraviolet-induced erythema in guinea pigs was 11.7 and 1.8 times more potent than that of indomethacin and ketoprofen, respectively. Inhibitory effect of M-5011 on carrageenin-induced paw edema was 2 and 1.5 times more potent than that of indomethacin and diclofenac sodium, respectively. Analgesic effect of M-5011 on dry yeast-induced hyperalgesia or adjuvant-induced arthritic pain was equipotent to that of indomethacin, diclofenac sodium or ketoprofen. Anti-pyretic effect of M-5011 on yeast-induced pyrexia in rats was 4.2 and 4.6 times more potent than that of indomethacin and ketoprofen, respectively. Inhibitory effect of M-5011 on prostaglandin E2 production in the exudate of air-pouch inflammation induced by carrageenin was 1.75 times more potent than that in the non-inflamed site (stomach). As a result, gastric ulcerogenic activity of M-5011 was half that of indomethacin in rat. These results suggest that M-5011 shows more potent anti-inflammatory and anti-pyretic effects and equipotent analgesic effect with low gastro-ulcerogenic activity compared with classical NSAIDs.