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Objective: To study the lipid soluble constituents of wild Russula in Fangcheng-Henan province. Methods: The lipid soluble chemical constituents of wild Russula in henan province were analyzed by GC-MS. Results: The Fatty acids was the highest content of lipid soluble components in wild Russula, 27 compounds were identified, accounting for 99.44% of the total detected components, the main compounds were (E)- 9-octadecenoic acid (44.93%), linoleic acid (28.18%), Palmitic acid (12.07%) and (E)- 12,15-octadecenoic acid (4.17%). Conclusion: The study provides experimental datas for the further development and utilization of wild Russula.

gamma-Secretase is a proteolytic membrane complex that processes a variety of substrates including the amyloid precursor protein and the Notch receptor. Earlier we showed that one of the components of this complex, nicastrin (NCT), functions as a receptor for gamma-secretase substrates. A recent report challenged this, arguing instead that the Glu-333 residue of NCT predicted to participate in substrate recognition only participates in gamma-secretase complex maturation and not in activity per se. Here, we present evidence that Glu-333 directly participates in gamma-secretase activity. By normalizing to the active pool of gamma-secretase with two separate methods, we establish that gamma-secretase complexes containing NCT-E333A are indeed deficient in intrinsic activity. We also demonstrate that the NCT-E333A mutant is deficient in its binding to substrates. Moreover, we find that the cleavage of substrates by gamma-secretase activity requires a free N-terminal amine but no minimal length of the extracellular N-terminal stub. Taken together, these studies provide further evidence supporting the role of NCT in substrate recognition. Finally, because gamma-secretase cleaves itself during its maturation and because NCT-E333A also shows defects in gamma-secretase complex maturation, we present a model whereby Glu-333 can serve a dual role via similar mechanisms in the recruitment of both Type 1 membrane proteins for activity and the presenilin intracellular loop during complex maturation.

As part of continuing studies of the identification of gene organization and cloning of novel α-conotoxins, the first α4/4-conotoxin identified in a vermivorous Conus species, designated Qc1.2, was originally obtained by cDNA and genomic DNA cloning from Conus quercinus collected in the South China Sea. The predicted mature toxin of Qc1.2 contains 14 amino acid residues with two disulfide bonds (I-III, II-IV connectivity) in a native globular configuration. The mature peptide of Qc1.2 is supposed to contain an N-terminal post-translationally processed pyroglutamate residue and a free carboxyl C-terminus. This peptide was chemically synthesized and refolded for further characterization of its functional properties. The synthetic Qc1.2 has two interconvertible conformations in aqueous solution, which may be due to the cis-trans isomerization of the two successive Pro residues in its first Cys loop. Using the Xenopus oocyte heterologous expression system, Qc1.2 was shown to selectively inhibit both rat neuronal α3β2 and α3β4 subtypes of nicotinic acetylcholine receptors with low potency. A block of ∼63% and 37% of the ACh-evoked currents was observed, respectively, and the toxin dissociated rapidly from the receptors. Compared with other characterized α-conotoxin members, the unusual structural features in Qc1.2 that confer to its receptor recognition profile are addressed.

Cone snails comprise approximately 700 species of venomous molluscs which have evolved the ability to generate multiple toxins with varied and exquisite selectivity. alpha-Conotoxin is a powerful tool for defining the composition and function of nicotinic acetylcholine receptors which play a crucial role in excitatory neurotransmission and are important targets for drugs and insecticides. An alpha4/7 conotoxin, Lp1.1, originally identified by cDNA and genomic DNA cloning from Conus leopardus, was found devoid of the highly conserved Pro residue in the first intercysteine loop. To further study this toxin, alpha-Lp1.1 was chemically synthesized and refolded into its globular disulfide isomer. The synthetic Lp1.1 induced seizure and paralysis on freshwater goldfish and selectively reversibly inhibited ACh-evoked currents in Xenopus oocytes expressing rat alpha3beta2 and alpha6alpha3beta2 nAChRs. Comparing the distinct primary structure with other functionally related alpha-conotoxins could indicate structural features in Lp1.1 that may be associated with its unique receptor recognition profile.

M-superfamily conotoxins can be divided into four branches (M-1, M-2, M-3 and M-4) according to the number of amino acid residues in the third Cys loop. In general, it is widely accepted that the conotoxin signal peptides of each superfamily are strictly conserved. Recently, we cloned six cDNAs of novel M-superfamily conotoxins from Conus leopardus, Conus marmoreus and Conus quercinus, belonging to either M-1 or M-3 branch. These conotoxins, judging from the putative peptide sequences deducted from cDNAs, are rich in acidic residues and share highly conserved signal and pro-peptide region. However, they are quite different from the reported conotoxins of M-2 and M-4 branches even in their signal peptides, which in general are considered highly conserved for each superfamily of conotoxins. The signal sequences of M-1 and M-3 conotoxins composed of 24 residues start with MLKMGVVL-, while those of M-2 and M-4 conotoxins composed of 25 residues start with MMSKLGVL-. It is another example that different types of signal peptides can exist within a superfamily besides the I-conotoxin superfamily. In addition to the different disulfide connectivity of M-1 conotoxins from that of M-4 or M-2 conotoxins, the sequence alignment, preferential Cys codon usage and phylogenetic tree analysis suggest that M-1 and M-3 conotoxins have much closer relationship, being different from the conotoxins of other two branches (M-4 and M-2) of M-superfamily.

Cone snails, a group of gastropod animals that inhabit tropical seas, are capable of producing a mixture of peptide neurotoxins, namely conotoxins, for defense and predation. Conotoxins are mainly disulfide-rich short peptides that act on different ion channels, neurotransmitter receptors, or transporters in the nervous system. They exhibit highly diverse compositions, structures, and biological functions. In this work, a novel Cys-free 15-residue conopeptide from Conus marmoreus was purified and designated as conomarphin. Conomarphin is unique because of its D-configuration Phe at the third residue from the C-terminus, which was identified using HPLC by comparing native conomarphin fragments and the corresponding synthetic peptides cleaved by different proteases. Surprisingly, the cDNA-encoded precursor of conomarphin was found to share the conserved signal peptide with other M-superfamily conotoxins, clearly indicating that conomarphin should belong to the M-superfamily, although conomarphin shares no homology with other six-Cys-containing M-superfamily conotoxins. Furthermore, NMR spectroscopy experiments established that conomarphin adopts a well-defined structure in solution, with a tight loop in the middle of the peptide and a short 3(10)-helix at the C-terminus. By contrast, no loop in L-Phe13-conomarphin was found, which suggests that D-Phe13 is essential for the structure of conomarphin. In conclusion, conomarphin may represent a new conotoxin family, whose biological activity remains to be identified.

The biological underpinnings and the pathological lesions of psychiatric disorders are centuries-old questions that have yet to be understood. Recent studies suggest that schizophrenia and related disorders likely have their origins in perturbed neurodevelopment and can result from a large number of common genetic variants or multiple, individually rare genetic alterations. It is thus conceivable that key neurodevelopmental pathways underline the various genetic changes and the still unknown pathological lesions in schizophrenia. Here, we report that mice defective of the nicastrin subunit of γ-secretase in oligodendrocytes have hypomyelination in the central nervous system. These mice have altered dopamine signaling and display profound abnormal phenotypes reminiscent of schizophrenia. In addition, we identify an association of the nicastrin gene with a human schizophrenia cohort. These observations implicate γ-secretase and its mediated neurodevelopmental pathways in schizophrenia and provide support for the "myelination hypothesis" of the disease. Moreover, by showing that schizophrenia and obsessive-compulsive symptoms could be modeled in animals wherein a single genetic factor is altered, our work provides a biological basis that schizophrenia with obsessive-compulsive disorder is a distinct subtype of schizophrenia. ispartof: Journal of Biological Chemistry vol:291 issue:22 pages:11647-56 ispartof: location:United States status: published

Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a 3' RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1-Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.

The oxidative folding of disulfide-rich conotoxins is essential for their biological functions. In vivo, disulfide bond formation is mainly catalyzed by protein disulfide isomerase. To elucidate the physiologic roles of protein disulfide isomerase in the folding of conotoxins, we have cloned a novel full-length protein disulfide isomerase from Conus marmoreus. Its ORF encodes a 500 amino acid protein that shares sequence homology with protein disulfide isomerases from other species, and 70% homology with human protein disulfide isomerase. Enzymatic analyses of recombinant C. marmoreus protein disulfide isomerase showed that it shared functional similarities with human protein disulfide isomerase. Using conotoxins tx3a and sTx3.1 as substrate, we analyzed the oxidase and isomerase activities of the C. marmoreus protein disulfide isomerase and found that it was much more efficient than glutathione in catalyzing oxidative folding and disulfide isomerization of conotoxins. We further demonstrated that macromolecular crowding had little effect on the protein disulfide isomerase-catalyzed oxidative folding and disulfide isomerization of conotoxins. On the basis of these data, we propose that the C. marmoreus protein disulfide isomerase plays a key role during in vivo folding of conotoxins.

In the venoms of cone snails, alpha conotoxins are competitive antagonists of nicotinic acetylcholine receptors. Eleven novel cDNA and eight partial gene sequences (including two pseudogenes) of alpha conotoxins were identified from five species of cone snail. As expected, every cDNA encodes a precursor of prepropeptide. In all the partial genes of a conotoxins identified, there is a long intron inserted at a fixed position in the pro-region, dividing the encoding region into two exons. The mutation rate in exon I (encoding the signal peptide and a part of pro-region) is much lower than that in exon II (encoding the other part of pro-region, the mature peptide and 3' untranslational region). Interestingly, the sequences at the 5' and 3' end of introns are highly conserved. In addition, in the identified introns exist long dinucleotide (e.g. "GT", "CA") or trinucleotide ("CAT") repeats. In the special case of Pu1.1, there are five almost identical repeats of a 150bp sequence in the long intron. Taking advantage of the conserved 3' end sequence of intron, 16 alpha. conotoxins, as well as a pseudogene and three kappa A conotoxins, were identified from their genomic DNAs. Based on the comparison of these cDNA and gene sequences, a hypothesis of the alpha conotoxin evolution was proposed. (c) 2007 Elsevier Ltd. All rights reserved.

The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.

The full-length cDNAs of two novel T-superfamily conotoxins, Lp5.1 and Lp5.2, were cloned from a vermivorous cone snail Conus leopardus using 3'/5'-rapid amplification of cDNA ends. The cDNA of Lp5.1 encodes a precursor of 65 residues, including a 22-residue signal peptide, a 28-residue propeptide and a 15-residue mature peptide. Lp5.1 is processed at the common signal site-X-Arg- immediately before the mature peptide sequences. In the case of Lp5.2, the precursor includes a 25-residue signal peptide and a 43-residue sequence comprising the propeptide and mature peptide, which is probably cleaved to yield a 29-residue propeptide and a 14-residue mature toxin. Although these two conotoxins share a similar signal sequence and a conserved disulfide pattern with the known T-superfamily, the pro-region and mature peptides are of low identity, especially Lp5.2 with an identity as low as 10.7% compared with the reference Mr5.1a. The elucidated cDNAs of these two toxins will facilitate a better understanding of the species distribution, the sequence diversity of T-superfamily conotoxins, the special gene structure and the evolution of these peptides.

Remarkable sequence diversity of T-superfamily conotoxins was found in a mollusk-hunting cone snail Conus marmoreus. The sequence of mr5a purified from the snail venom was determined, while six other sequences of Mr5.1a, Mr5.1b, Mr5.2, Mr5.3, Mr5.4a, and Mr5.4b were deduced from their corresponding cDNA cloned by RACE approach. mr5a of 10 amino acid residues is one of the shortest T-superfamily conotoxins ever found. They all share a typical (-CC-CC-) Cys pattern, a conserved signal peptide and a long 3'-untranslated region. A consensus Glu residue is preceded by the second two adjacent cysteines in all these toxins except in mr5a, whereas Mr5.1a, Mr5.1b, Mr5.4a and Mr5.4b are abundant in Trp residues. The identification of these highly divergent T-superfamily conotoxins will facilitate the understanding the relationship of their structure and function.

BmBKTx1 is a novel short chain toxin purified from the venom of the Asian scorpion Buthus martensi Karsch. It is composed of 31 residues and is structurally related to SK toxins. However, when tested on the cloned rat SK2 channel, it only partially inhibited rSK2 currents, even at a concentration of 1 microm. To screen for other possible targets, BmBKTx1 was then tested on isolated metathoracic dorsal unpaired median neurons of Locusta migratoria, in which a wide variety of ion channels are expressed. The results suggested that BmBKTx1 could specifically block voltage-gated Ca(2+)-activated K(+) currents (BK-type). This was confirmed by testing the BmBKTx1 effect on the alpha subunits of BK channels of the cockroach (pSlo), fruit fly (dSlo), and human (hSlo), heterologously expressed in HEK293 cells. The IC(50) for channel blocking by BmBKTx1 was 82 nm for pSlo and 194 nm for dSlo. Interestingly, BmBKTx1 hardly affected hSlo currents, even at concentrations as high as 10 microm, suggesting that the toxin might be insect specific. In contrast to most other scorpion BK blockers that also act on the Kv1.3 channel, BmBKTx1 did not affect this channel as well as other Kv channels. These results show that BmBKTx1 is a novel kind of blocker of BK-type Ca(2+)-activated K(+) channels. As the first reported toxin active on the Drosophila Slo channel dSlo, it will also greatly facilitate studying the physiological role of BK channels in this model organism.