Dental microwear of a basal ankylosaurine dinosaur, Jinyunpelta and its implication on evolution of chewing mechanism in ankylosaurs.

Jinyunpelta sinensis is a basal ankylosaurine dinosaur excavated from the mid Cretaceous Liangtoutang Formation of Jinyun County, Zhejiang Province, China. In the present study, its dental microwear was observed using a confocal laser microscope. Jinyunpelta had steep wear facets that covered most of buccal surfaces of posterior dentary teeth. Observation of dental microwear on the wear facet revealed that scratch orientation varied according to its location within the wear facet: vertically (i.e. apicobasally) oriented scratches were dominant in the upper half of the wear facet, and horizontally (i.e. mesiolaterally) oriented ones were in the bottom of the facet. These findings indicated that Jinyunpelta adopted precise tooth occlusion and biphasal jaw movement (orthal closure and palinal lower jaw movement). The biphasal jaw movement was widely observed among nodosaurids, among ankylosaurids, it was previously only known from the Late Cretaceous North American taxa, and not known among Asian ankylosaurids. The finding of biphasal jaw movement in Jinyunpelta showed sophisticate feeding adaptations emerged among ankylosaurids much earlier (during Albian or Cenomanian) than previously thought (during Campanian). The Evolution of the biphasal jaw mechanism that contemporaneously occurred among two lineages of ankylosaurs, ankylosaurids and nodosaurids, showed high evolutionary plasticity of ankylosaur jaw mechanics.

Characterization of voltage-dependent calcium channel blocking peptides from the venom of the tarantula Grammostola rosea.

Voltage-dependent calcium channel blocking peptides were purified and sequenced from the venom of the tarantula, Grammostola rosea. cDNAs encoding the peptide sequences were cloned from the venom gland cDNA library. The electrophysiological effects of the peptides on several types of voltage-dependent calcium channels were evaluated using a Xenopus laevis oocyte expression system. A peptide contained in one of the HPLC peak fractions inhibited P/Q type voltage-dependent calcium channels (Ca(v)2.1). The amino acid sequence of this peptide is identical to that of ω-grammotoxin SIA. A peptide from another discrete peak, which is identical to GsAFII except for one tryptophan residue in the C-terminus, inhibited L-type voltage-dependent calcium channels (Ca(v)1.2). A novel peptide, named GTx1-15 (Accession number, AB201016), shows 76.5% sequence homology with the sodium channel blocker phrixotoxin 3, however, GTx1-15 preferentially inhibited T-type voltage-dependent calcium channels (Ca(v)3.1). In silico secondary and tertiary structure prediction revealed that GTx1-15 and sodium channel blockers such as hainantoxin-IV, phrixotoxin 3, and ceratotoxin 2 show very similar ß-strand composition, distribution of Optimal Docking Areas (continuous surface patches likely to be involved in protein-protein interactions), and surface electrostatic potential. These findings suggest that these peptide toxins evolved from common ancestors by gene duplication to maintain surface atmospheres appropriate for interaction with low-voltage-dependent ion channels.

cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions.

We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA-protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a 'ligation site' for T4 RNA ligase, a 'biotin site' for solid-phase handling, a 'reverse transcription primer site' for the efficient and rapid conversion from an unstable mRNA-protein fusion (mRNA display) to a stable mRNA/cDNA-protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a 'restriction enzyme site' for the release of a complex from the solid support. This enables not only stabilizing mRNA-protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.

Modulation of a feeding neural circuit by microinjection of K+ channel expression genes into a single identified neuron in Aplysia kurodai.

In Aplysia buccal ganglion expression genes for voltage-dependent K(+) channels (AKv1.1a) were injected into one of four electrically coupled multi-action (MA) neurons that directly inhibit jaw-closing (JC) motor neurons and may cooperatively generate their firing pattern during the feeding response. Following the DNA injection, the firing threshold increased and the spike frequency at the same current decreased in the current-induced excitation of the MA neuron; indicating a decrease in excitability of the MA neuron. This procedure also reduced the firing activity of MA neurons during the feeding-like rhythmic responses induced by the electrical nerve stimulation. Moreover, the firing pattern in JC motor neurons was remarkably changed, suggesting the effective contribution of a single MA neuron or electrically coupled MA neurons to the generation of the firing pattern in the JC motor neurons. This method appears useful for exploring the functional roles of specific neurons in complex neural circuits.

Cloning and functional characterization of squid voltage-dependent Ca2+ channel beta 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)