Tadashi Kimura

Molecular Cloning and Sequence Analysis of the cDNAs Encoding Toxin-Like Peptides from the Venom Glands of Tarantula Grammostola rosea

Tarantula venom glands produce a large variety of bioactive peptides. Here we present the identification of venom components obtained by sequencing clones isolated from a cDNA library prepared from the venom glands of the Chilean common tarantula, Grammostola rosea. The cDNA sequences of about 1500 clones out of 4000 clones were analyzed after selection using several criteria. Forty-eight novel toxin-like peptides (GTx1 to GTx7, and GTx-TCTP and GTx-CRISP) were predicted from the nucleotide sequences. Among these peptides, twenty-four toxins are ICK motif peptides, eleven peptides are MIT1-like peptides, and seven are ESTX-like peptides. Peptides similar to JZTX-64, aptotoxin, CRISP, or TCTP are also obtained. GTx3 series possess a cysteine framework that is conserved among vertebrate MIT1, Bv8, prokineticins, and invertebrate astakines. GTx-CRISP is the first CRISP-like protein identified from the arthropod venom. Real-time PCR revealed that the transcripts for TCTP-like peptide are expressed in both the pereopodal muscle and the venom gland. Furthermore, a unique peptide GTx7-1, whose signal and prepro sequences are essentially identical to those of HaTx1, was obtained.

Cloning and functional characterization of squid voltage-dependent Ca2+ channel β subunits: involvement of N-terminal sequences in differential modulation of the current

cDNAs that encode beta subunits of voltage-dependent Ca(2+) channel were cloned from the optic lobe of the squid Loligo bleekeri. The subunits, LoCa(v)beta(1a) and LoCa(v)beta(1b) are 96% identical in amino acid sequence. The sole sequence differences are in the N-terminal region and in a five amino acid insertion in the central region of LoCa(v)beta(1b). RT-PCR revealed that LoCa(v)beta(1a) and LoCa(v)beta(1b) transcripts were expressed mainly in the optic lobe and stellate ganglion, and more weakly in mantle muscle, systemic heart, gill, branchial heart, stomach and liver. Coexpression of LoCa(v)beta(1a) or LoCa(v)beta(1b) with mammalian Ca(v)2.3 and alpha(2)/delta subunits in the Xenopus oocyte resulted in high-voltage-activated currents, and showed slow current inactivation and moderate steady-state inactivation. Comparison of the squid subunits with four mammalian beta subunits, beta(1b), beta(2a), beta(3) and beta(4), demonstrated that the modulatory effects of the beta subunits on steady-state inactivation kinetics were beta(3)<beta(4) approximately beta(1b)<LoCa(v)beta(1a) approximately LoCa(v)beta(1b)<beta(2a). LoCa(v)beta(1a)-induced current amplitude was about two to four times higher than that of LoCa(v)beta(1b). Experiments with point mutants and chimeras suggest that potential PKC and CK2 phosphorylation sites in the N-terminal region of LoCa(v)beta(1b) affect the current amplitude reciprocally, and may be responsible for regulating current amplitude.

High Proteolytic Resistance of Spider-Derived Inhibitor Cystine Knots.

Proteolytic stability in gastrointestinal tract and blood plasma is the major obstacle for oral peptide drug development. Inhibitor cystine knots (ICKs) are linear cystine knot peptides which have multifunctional properties and could become promising drug scaffolds. ProTx-I, ProTx-II, GTx1-15, and GsMTx-4 were spider-derived ICKs and incubated with pepsin, trypsin, chymotrypsin, and elastase in physiological conditions to find that all tested peptides were resistant to pepsin, and ProTx-II, GsMTx-4, and GTx1-15 showed resistance to all tested proteases. Also, no ProTx-II degradation was observed in rat blood plasma for 24 hours in vitro and ProTx-II concentration in circulation decreased to half in 40 min, indicating absolute stability in plasma and fast clearance from the system. So far, linear peptides are generally thought to be unsuitable in vivo, but all tested ICKs were not degraded by pepsin and stomach could be selected for the alternative site of drug absorption for fast onset of the drug action. Since spider ICKs are selective inhibitors of various ion channels which are related to the pathology of many diseases, engineered ICKs will make a novel class of peptide medicines which can treat variety of bothering symptoms.

The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system

Highlights • Human Kv2.1 was expressed in the inner membrane of E. coli using prokaryotic codon.• Bacterial spheroplasts large enough for the automated patch clamp were prepared by microfluidic chips.• Kv2.1 current was recorded from the giant spheroplast by the automated patch clamp.• E. coli spheroplasts were used for dose–response assay of potassium channel inhibitors.• Our system will become the simple and sensitive drug assay method anyone can use.

The constant threat from a non-native predator increases tail muscle and fast-start swimming performance in Xenopus tadpoles

ABSTRACT Predator-induced phenotypic plasticity is the ability of prey to adapt to their native predator. However, owing to environmental changes, encounters with unknown predators are inevitable. Therefore, study of prey and non-native predator interaction will reveal the primary stages of adaptive strategies in prey-predator interactions in the context of evolutionary processes. Here, Xenopus tadpoles exposed to a non-native predator, a larval salamander, showed a significant increase in body weight and tail length to body length ratio. The Tmax2 test indicated a significant enhancement of the tail muscle and decrease in the relative ventral fin height in tadpoles exposed to predation risk, leading to significantly higher average swimming speeds. The analysis of muscle-related metabolites revealed that sarcosine increased significantly in tadpoles exposed to non-native predators. Multiple linear regression analysis of the fast-start swimming pattern showed that the fast-start swimming speed was determined by the time required for a tadpole to bend its body away from the threat (C-start) and the angle at which it was bent. In conclusion, morphological changes in tadpoles were functionally adaptive and induced by survival behaviors of Xenopus tadpoles against non-native predators. Summary: Xenopus tadpoles exposed to a non-native predator increase their tail length, muscle tissue and swimming speed, all of which are functionally adaptive and induced by tadpole survival behavior.