Cloning, expression, and nutritional regulation of the glutamine synthetase gene in Ctenopharyngodon idellus.

Glutamine synthetase (GS) is considered a master enzyme that catalyzes ATP-dependent biosynthesis of glutamine from glutamate. In the present study, the GS gene was cloned from the intestine of grass carp (Ctenopharyngodon idellus). The full-length cDNA sequence of GS encodes a 371-amino-acid polypetide. Phylogenetic analysis of the C. idellus GS sequence reveals common carp (Cyprinus carpio) as its closest neighbor. GS mRNA was differentially expressed in different tissues, with high to low gradient expression the intestine, brain, muscle, heart, gill, liver, pituitary gland, and spleen. Additionally, GS exhibited a dynamic pattern of expression during embryonic development, reaching maximal and minimal levels in the organ and hatching stages, respectively, and constant low levels from 7 to 28days post-hatching. We also assessed dietary protein levels and feed sources in diet-regulated fish, and the results suggested that low crude protein (CP) and fish meal stimulate GS gene expression. Furthermore, intestinal GS mRNA expression was significantly increased by 0.2, 0.4, 0.6, 0.8mM concentrations of glutamine dipeptide in vitro. This study provides valuable knowledge about the regulation of GS expression in teleosts.

Jingzhaotoxin-II, a novel tarantula toxin preferentially targets rat cardiac sodium channel.

Naturally occurring toxins are invaluable tools for exploration of the structure and function relationships of voltage-gated sodium channels (VGSCs). In this study, we isolated and characterized a novel VGSC toxin named jingzhaotoxin-II (JZTX-II) from the tarantula Chilobrachys jingzhao venom. JZTX-II consists of 32 amino acid residues including two acidic and two basic residues. Cloned and sequenced using 3'- and 5'-rapid amplification of the cDNA ends, the full-length cDNA for JZTX-II was found to encode a 63-residue precursor which contained a signal peptide of 21 residues, a propeptide of 10 residues and a mature peptide of 32 residues. Under whole-cell voltage-clamp conditions, JZTX-II significantly slowed rapid inactivation of TTX-resistant (TTX-R) VGSC on cardiac myocytes with the IC50=0.26+/-0.09 microM. In addition, JZTX-II had no effect on TTX-R VGSCs on rat dorsal root ganglion neurons but exerted a concentration-dependent reduction in tetrodotoxin-sensitive (TTX-S) VGSCs accompanied by a slowing of sodium current inactivation similar to delta-ACTXs. It is notable that TTX-S VGSCs on cultured rat hippocampal neurons were resistant to JZTX-II at high dose. Based on its high selectivity for mammalian VGSC subtypes, JZTX-II might be an important ligand for discrimination of VGSC subtypes and for exploration of the distribution and modulation mechanisms of VGSCs.

Jingzhaotoxin-I, a novel spider neurotoxin preferentially inhibiting cardiac sodium channel inactivation.

Jingzhaotoxin-I (JZTX-I), a 33-residue polypeptide, is derived from the Chinese tarantula Chilobrachys jing-zhao venom based on its ability to evidently increase the strength and the rate of vertebrate heartbeats. The toxin has three disulfide bonds with the linkage of I-IV, II-V, and III-VI that is a typical pattern found in inhibitor cystine knot molecules. Its cDNA determined by rapid amplification of 3'- and 5'-cDNA ends encoded a 62-residue precursor with a small proregion of eight residues. Whole-cell configuration indicated that JZTX-I was a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. Although JZTX-I also exhibits the interaction with channel isoforms expressing in mammalian and insect sensory neurons, its affinity for tetrodotoxin-resistant subtype in mammalian cardiac myocytes (IC50 = 31.6 nm) is approximately 30-fold higher than that for tetrodotoxin-sensitive subtypes in latter tissues. Not affecting outward delay-rectified potassium channels expressed in Xenopus laevis oocytes and tetrodotoxin-resistant sodium channels in mammal sensory neurons, JZTX-I hopefully represents a potent ligand to discriminate cardiac sodium channels from neuronal tetrodotoxin-resistant isoforms. Furthermore, different from any reported spider toxins, the toxin neither modifies the current-voltage relationships nor shifts the steady-state inactivation of sodium channels. Therefore, JZTX-I defines a new subclass of spider sodium channel toxins. JZTX-I is an alpha-like toxin first reported from spider venoms. The result provides an important witness for a convergent functional evolution between spider and other animal venoms.

Jingzhaotoxin-III, a novel spider toxin inhibiting activation of voltage-gated sodium channel in rat cardiac myocytes.

We have isolated a cardiotoxin, denoted jingzhaotoxin-III (JZTX-III), from the venom of the Chinese spider Chilobrachys jingzhao. The toxin contains 36 residues stabilized by three intracellular disulfide bridges (I-IV, II-V, and III-VI), assigned by a chemical strategy of partial reduction and sequence analysis. Cloned and sequenced using 3'-rapid amplification of cDNA ends and 5'-rapid amplification of cDNA ends, the full-length cDNA encoded a 63-residue precursor of JZTX-III. Different from other spider peptides, it contains an uncommon endoproteolytic site (-X-Ser-) anterior to mature protein and the intervening regions of 5 residues, which is the smallest in spider toxin cDNAs identified to date. Under whole cell recording, JZTX-III showed no effects on voltage-gated sodium channels (VGSCs) or calcium channels in dorsal root ganglion neurons, whereas it significantly inhibited tetrodotoxin-resistant VGSCs with an IC(50) value of 0.38 microm in rat cardiac myocytes. Different from scorpion beta-toxins, it caused a 10-mV depolarizing shift in the channel activation threshold. The binding site for JZTX-III on VGSCs is further suggested to be site 4 with a simple competitive assay, which at 10 microm eliminated the slowing currents induced by Buthus martensi Karsch I (BMK-I, scorpion alpha-like toxin) completely. JZTX-III shows higher selectivity for VGSC isoforms than other spider toxins affecting VGSCs, and the toxin hopefully represents an important ligand for discriminating cardiac VGSC subtype.