Complete mitochondrial genome sequence of the spinyhead croaker Collichthys lucidus (Perciformes, Sciaenidae) with phylogenetic considerations.

The complete mitochondrial genome of the spinyhead croaker Collichthys lucidus was determined using long-PCR and primer walking methods. It is a circular molecule of 16,451 bp in length with a standard set of 22 tRNAs, 2 rRNAs, 13 protein-coding genes as well as a non-coding control region in the same order as those of the other bony fishes. C. lucidus mitogenome exhibited a clear strand-specific bias in nucleotide composition, as evidence by a GC- skew of the H-strand of -0.319. The strand-specific bias was also reflected in the codon usage of genes oriented in opposite directions. All tRNA genes except for tRNA( Ser(AGY)) harbored the typical cloverleaf secondary structures and possessed anticodons that matched the vertebrate mitochondrial genetic code. Contrary to the typical structure of control region consistig of TAS, central, and CSB domains, there were no central conserved blocks available in C. lucidus mitogenome. Despite extensive studies based on both morphology and molecules, phylogenetic position of C. lucidus with Sciaenidae is still controversial. Our phylogenetic results provided more evidence to support previous morphological studies and consistently placed C. lucidus as a sister taxon to Collichthys niveatus, with both of these taxa forming the monophyletic Collichthys.

Complete mitochondrial genome sequence of bighead croaker Collichthys niveatus (Perciformes, Sciaenidae): a mitogenomic perspective on the phylogenetic relationships of Pseudosciaeniae.

The monophyly and phylogenetic relationships of Pseudosciaeniae have long been controversial. Here we describe the mitochondrial genome (mitogenome) sequence of Collichthys niveatus. It is a circular double-stranded DNA molecule of 16,450 base pairs (bp) in length with a standard set of 22 transfer RNA genes (tRNAs), 2 ribosomal RNA genes (rRNAs), 13 protein-coding genes as well as a non-coding control region. The mitogenome of C. niveatus shared common features with those of other bony fishes in terms of gene arrangement, base composition, and tRNA structures. The C. niveatus mitogenome exhibited pronounced strand-specific asymmetry in nucleotide composition, which was also reflected in the codon usage of genes oriented in opposite directions. Contrary to the typical structure of the control region, the central conserved blocks (CSB-D, -E, and -F) could not be detected in C. niveatus mitogenome. Phylogenetic analysis based on whole mitogenome sequences provided strong support for the monophyly of Pseudosciaeniae, and sister-group relationships of C. niveatus+Collichthys lucidus and Larimichthys crocea+Larimichthys polyactis, which was consistent with the traditional taxonomy. Unexpected divergence was found in two C. niveatus mitogenomes and several hypotheses were proposed to explain this observation including misidentification and introgressive hybridization between C. niveatus and L. polyactis, and polyphyletic origin of C. niveatus. We considered species misidentification to be the main hypothesis. However, additional data is essential to test these proposed hypotheses.

Sorting of neuropeptides and neuropeptide receptors into secretory pathways.

There are two major secretory pathways in neurons, the regulated pathway and the constitutive pathway. Neuropeptides and other regulated secretory proteins are known to be sorted into large dense-core vesicles of the regulated pathway in the trans-Golgi network and are secreted upon stimulus-induced increases in intracellular Ca(2+). The newly synthesized cell surface receptors are usually sorted into microvesicles of the constitutive pathway and inserted into the plasma membrane by spontaneous exocytosis. Small-diameter sensory neurons in dorsal root ganglia and pheochromocytoma cells express neuropeptides (e.g., substance P) and several neuropeptide receptors including opioid receptors. The mu-opioid receptors are delivered to the cell surface through the constitutive pathway, whereas another type of opioid receptor, the delta-opioid receptor, is often found in the membrane of large dense-core vesicles and can be inserted into the plasma membrane when exocytosis occurs. Recent studies show that sequences with opposite electrical polarity within the prohormones of substance P are essential for their sorting into large dense-core vesicles. Moreover, the delta-opioid receptor is sorted into large dense-core vesicles by its interaction with protachykinin, a prohormone of substance P. These findings provide insight into the molecular mechanisms that determine the sorting and trafficking of neuropeptides and neuropeptide receptors in neurons.

Short elements with charged amino acids form clusters to sort protachykinin into large dense-core vesicles.

The sorting of neuropeptide tachykinins into large dense-core vesicles (LDCVs) is a key step in their regulated secretion from neurons. However, the sorting mechanism for protachykinin has not yet to be clearly resolved. In this study, we report that the clustered short elements with charged amino acids regulate the efficiency of protachykinin sorting into LDCVs. A truncation experiment showed that the propeptide and the mature peptide-containing sequence of protachykinin were sorted into LDCVs. These two regions exhibit a polarized distribution of charged amino acids. The LDCV localization of the propeptide was gradually decreased with an increasing number of neutral amino acids. Furthermore, the short element with four to five amino acids containing two charged residues was found to be a basic unit for LDCV sorting that enables regulated secretion. In the native propeptide sequence, these charged short elements were clustered to enhance the intermolecular aggregation by electrostatic interaction and produce a gradual and additive effect on LDCV sorting. The optimal conditions for intermolecular aggregation of protachykinin were at millimolar Ca(2+) concentrations and pH 5.5-6.0. These results demonstrate that the charged short elements are clustered such that they serve as aggregative signals and regulate the efficiency of protachykinin sorting into LDCVs. These findings reveal a novel mechanism for the sorting of neuropeptides into a regulated secretory pathway.

Interaction with vesicle luminal protachykinin regulates surface expression of delta-opioid receptors and opioid analgesia.

Opioid and tachykinin systems are involved in modulation of pain transmission in the spinal cord. Regulation of surface opioid receptors on nociceptive afferents is critical for opioid analgesia. Plasma-membrane insertion of delta-opioid receptors (DORs) is induced by stimulus-triggered exocytosis of DOR-containing large dense-core vesicles (LDCVs), but how DORs become sorted into the regulated secretory pathway is unknown. Here we report that direct interaction between protachykinin and DOR is responsible for sorting of DORs into LDCVs, allowing stimulus-induced surface insertion of DORs and DOR-mediated spinal analgesia. This interaction is mediated by the substance P domain of protachykinin and the third luminal domain of DOR. Furthermore, deletion of the preprotachykinin A gene reduced stimulus-induced surface insertion of DORs and abolished DOR-mediated spinal analgesia and morphine tolerance. Thus, protachykinin is essential for modulation of the sensitivity of nociceptive afferents to opioids, and the opioid and tachykinin systems are directly linked by protachykinin/DOR interaction.