The pyloric neural circuit of the herbivorous crab Pugettia producta shows limited sensitivity to several neuromodulators that elicit robust effects in more opportunistically feeding decapods

Modulation of neural circuits in the crustacean stomatogastric nervous system (STNS) allows flexibility in the movements of the foregut musculature. The extensive repertoire of such resulting motor patterns in dietary generalists is hypothesized to permit these animals to process varied foods. The foregut and STNS of Pugettia producta are similar to those of other decapods, but its diet is more uniform, consisting primarily of kelp. We investigated the distribution of highly conserved neuromodulators in the stomatogastric ganglion (STG) and neuroendocrine organs of Pugettia, and documented their effects on its pyloric rhythm. Using immunohistochemistry, we found that the distributions of Cancer borealis tachykinin-related peptide I (CabTRP I), crustacean cardioactive peptide (CCAP), proctolin, red pigment concentrating hormone (RPCH) and tyrosine hydroxylase (dopamine) were similar to those of other decapods. For all peptides except proctolin, the isoforms responsible for the immunoreactivity were confirmed by mass spectrometry to be the authentic peptides. Only two modulators had physiological effects on the pyloric circuit similar to those seen in other species. In non-rhythmic preparations, proctolin and the muscarinic acetylcholine agonist oxotremorine consistently initiated a full pyloric rhythm. Dopamine usually activated a pyloric rhythm, but this pattern was highly variable. In only about 25% of preparations, RPCH activated a pyloric rhythm similar to that seen in other species. CCAP and CabTRP I had no effect on the pyloric rhythm. Thus, whereas Pugettia possesses all the neuromodulators investigated, its pyloric rhythm, when compared with other decapods, appears less sensitive to many of them, perhaps because of its limited diet

SIFamide peptides modulate cardiac activity differently in two species of Cancer crab

The SIFamides are a broadly conserved arthropod peptide family characterized by the C-terminal motif –SIFamide. In decapod crustaceans, two isoforms of SIFamide are known, GYRKPPFNGSIFamide (Gly1-SIFamide), which is nearly ubiquitously conserved in the order, and VYRKPPFNGSIFamide (Val1-SIFamide), known only from members of the astacidean genus Homarus. While much work has focused on the identification of SIFamide isoforms in decapods, there are few direct demonstrations of physiological function for members of the peptide family in this taxon. Here, we assessed the effects of Gly1- and Val1-SIFamide on the cardiac neuromuscular system of two closely related species of Cancer crab, Cancer borealis and Cancer irroratus. In each species, both peptides were cardioactive, with identical, dose-dependent effects elicited by both isoforms in a given species. Threshold concentrations for bioactivity are in the range typically associated with hormonal delivery, i.e., 10−9 to 10−8 M. Interestingly, and quite surprisingly, while the predicted effects of SIFamide on cardiac output are similar in both C. borealis and C. irroratus, frequency effects predominate in C. borealis, while amplitude effects predominate in C. irroratus. These findings suggest that, while SIFamide is likely to increase cardiac output in both crabs, the mechanism through which this is achieved is different in the two species. Immunohistochemical/mass spectrometric data suggest that SIFamide is delivered to the heart hormonally rather than locally, with the source of hormonal release being midgut epithelial endocrine cells in both Cancer species. If so, midgut-derived SIFamide may function as a regulator of cardiac output during the process of digestion

Identification, physiological actions, and distribution of TPSGFLGMRamide: A novel tachykinin-related peptide from the midgut and stomatogastric nervous system of Cancer crabs

In most invertebrates, multiple species-specific isoforms of tachykinin-related peptide (TRP) are common. In contrast, only a single conserved TRP isoform, APSGFLGMRamide, has been documented in decapod crustaceans, leading to the hypothesis that it is the sole TRP present in this arthropod order. Previous studies of crustacean TRPs have focused on neuronal tissue, but the recent demonstration of TRPs in midgut epithelial cells in Cancer species led us to question whether other TRPs are present in the gut, as is the case in insects. Using direct tissue matrix assisted laser desorption/ionization Fourier transform mass spectrometry, in combination with sustained off-resonance irradiation collision-induced dissociation, we found that at least one additional TRP is present in Cancer irroratus, Cancer borealis, Cancer magister, and Cancer productus. The novel TRP isoform, TPSGFLGMRamide, was present not only in the midgut, but also in the stomatogastric nervous system (STNS). In addition, we identified an unprocessed TRP precursor APSGFLGMRG, which was detected in midgut tissues only. TRP immunohistochemistry, in combination with preadsorption studies, suggests that APSGFLGMRamide and TPSGFLGMRamide are co-localized in the stomatogastric ganglion (STG), which is contained within the STNS. Exogenous application of TPSGFLGMRamide to the STG elicited a pyloric motor pattern that was identical to that elicited by APSGFLGMRamide, whereas APSGFLGMRG did not alter the pyloric motor pattern. © 2007 The Authors

Effect of humidity on nitric acid uptake to mineral dust aerosol particles

International audienceThis study presents the first laboratory observation of HNO3 uptake by airborne mineral dust particles. The model aerosols were generated by dry dispersion of Arizona Test Dust (ATD), SiO2, and by nebulizing a saturated solution of calcium carbonate. The uptake of 13N-labeled gaseous nitric acid was observed in a flow reactor on the 0.2?2 s reaction time scale at room temperature and atmospheric pressure. The amount of nitric acid appearing in the aerosol phase at the end of the flow tube was found to be a linear function of the aerosol surface area. SiO2 particles did not show any significant uptake, while the CaCO3 aerosol was found to be more reactive than ATD. Due to the smaller uncertainty associated with the reactive surface area in the case of suspended particles as compared to bulk powder samples, we believe that we provide an improved estimate of the rate of uptake of HNO3 to mineral dust. The fact that the rate of uptake was smaller at a concentration of 1012 than at 1011 was indicative of a complex uptake mechanism. The uptake coefficient averaged over the first 2 s of reaction time at a concentration of 1012 molecules cm-3 was found to increase with increasing relative humidity, from 0.022±0.007 at 12% RH to 0.113±0.017 at 73% RH , which was attributed to an increasing degree of solvation of the more basic minerals. The extended processing of the dust by higher concentrations of HNO3 at 85% RH led to a water soluble coating on the particles and enhanced their hygroscopicity

Identification, physiological actions, and distribution of TPSGFLGMRamide: A novel tachykinin-related peptide from the midgut and stomatogastric nervous system of Cancer crabs

In most invertebrates, multiple species-specific isoforms of tachykinin-related peptide (TRP) are common. In contrast, only a single conserved TRP isoform, APSGFLGMRamide, has been documented in decapod crustaceans, leading to the hypothesis that it is the sole TRP present in this arthropod order. Previous studies of crustacean TRPs have focused on neuronal tissue, but the recent demonstration of TRPs in midgut epithelial cells in Cancer species led us to question whether other TRPs are present in the gut, as is the case in insects. Using direct tissue matrix assisted laser desorption/ionization Fourier transform mass spectrometry, in combination with sustained off-resonance irradiation collision-induced dissociation, we found that at least one additional TRP is present in Cancer irroratus, Cancer borealis, Cancer magister, and Cancer productus. The novel TRP isoform, TPSGFLGMRamide, was present not only in the midgut, but also in the stomatogastric nervous system (STNS). In addition, we identified an unprocessed TRP precursor APSGFLGMRG, which was detected in midgut tissues only. TRP immunohistochemistry, in combination with preadsorption studies, suggests that APSGFLGMRamide and TPSGFLGMRamide are co-localized in the stomatogastric ganglion (STG), which is contained within the STNS. Exogenous application of TPSGFLGMRamide to the STG elicited a pyloric motor pattern that was identical to that elicited by APSGFLGMRamide, whereas APSGFLGMRG did not alter the pyloric motor pattern. © 2007 The Authors

Mass spectrometric identification of pEGFYSQRYamide: A crustacean peptide hormone possessing a vertebrate neuropeptide Y (NPY)-like carboxy-terminus

In invertebrates, peptides possessing the carboxy (C)-terminal motif -RXRFamide have been proposed as the homologs of vertebrate neuropeptide Y (NPY). Using matrix assisted laser desorption/ionization mass spectrometry, in combination with sustained off-resonance irradiation collision-induced dissociation and chemical and enzymatic reactions, we have identified the peptide pEGFYSQRYamide from the neuroendocrine pericardial organ (PO) of the crab Pugettia producta. This peptide is likely the same as that previously reported, but misidentified, as PAFYSQRYamide in several earlier reports (e.g. [Li, L., Kelley, W.P., Billimoria, C.P., Christie, A.E., Pulver, S.R., Sweedler, J.V., Marder, E. 2003. Mass spectrometric investigation of the neuropeptide complement and release in the pericardial organs of the crab, Cancer borealis. J. Neurochem. 87, 642-656; Fu, Q., Kutz, K.K., Schmidt, J.J., Hsu, Y.W., Messinger, D.I., Cain, S.D., de la Iglesia, H.O., Christie, A.E., Li, L. 2005. Hormone complement of the Cancer productus sinus gland and pericardial organ: an anatomical and mass spectrometric investigation. J. Comp. Neurol. 493, 607-626.]). The -QRYamide motif contained in pEGFYSQRYamide is identical to that present in many vertebrate members of the NPY superfamily. Mass spectrometric analysis conducted on the POs of several other decapods showed that pEGFYSQRYamide is present in three other brachyurans (Cancer borealis, Cancer irroratus and Cancer productus) as well as in one species from another decapod infraorder (Lithodes maja, an anomuran). Thus, our findings show that at least some invertebrates possess NPY-like peptides in addition to those exhibiting an -RXRFamide C-terminus, and raise the question as to whether the invertebrate -QRYamides are functionally and/or evolutionarily related to the NPY superfamily. © 2007 Elsevier Inc. All rights reserved

Molecular and mass spectral identification of the broadly conserved decapod crustacean neuropeptide pQIRYHQCYFNPISCF: The first PISCF-allatostatin (Manduca sexta- or C-type allatostatin) from a non-insect

The PISCF-allatostatins (Manduca sexta- or C-type allatostatins) are a family of pentadecapeptides characterized by a pyroglutamine blocked N-terminus, an unamidated-PISCF C-terminus, and a disulfide bridge between two internal Cys residues. Several isoforms of PISCF-AST are known, all from holometabolous insects. Using a combination of transcriptomics and mass spectrometry, we have identified the first PISCF-type peptides from a non-insect species. In silico analysis of crustacean ESTs identified several Litopenaeus vannamei (infraorder Penaeidea) transcripts encoding putative PISCF-AST precursors. Translation of these ESTs, with subsequent prediction of their putative post-translational processing, revealed the existence of as many as three PISCF-type peptides, including pQIRYHQCYFNPISCF (disulfide bridging between Cys7 and Cys14). Although none of the predicted isoforms was detected by mass spectrometry in L. vannamei, MALDI-FTMS mass profiling identified an m/z signal corresponding to pQIRYHQCYFNPISCF (disulfide bridge present) in neural tissue from 28 other decapods, which included members of six infraorders (Stenopodidea, Astacidea, Thalassinidea, Achelata, Anomura and Brachyura). Further characterization of the peptide using SORI-CID and chemical derivatization/enzymatic digestion supported the theorized structure. In both the crab Cancer borealis and the lobster Homarus americanus, MALDI-based tissue surveys suggest that pQIRYHQCYFNPISCF is broadly distributed in the nervous system; it was also detected in the posterior midgut caecum. Collectively, our data show that members of the PISCF-AST family are not restricted to the holometabolous insects, but instead may be broadly conserved within the Pancrustacea. Moreover, our data suggest that one highly conserved PISCF-type peptide, pQIRYHQCYFN-PISCF, is present in decapod crustaceans, functioning as a brain-gut paracrine/hormone. © 2009 Elsevier Inc. All rights reserved

Arginine methylation of yeast mRNA-binding protein Npl3 directly affects its function, nuclear export, and intranuclear protein interactions

Arginine methylation can affect both nucleocytoplasmic transport and protein-protein interactions of RNA-binding proteins. These effects are seen in cells that lack the yeast hnRNP methyltransferase (HMT1), raising the question of whether effects on specific proteins are direct or indirect. The presence of multiple arginines in individual methylated proteins also raises the question of whether overall methylation or methylation of a subset of arginines affects protein function. We have used the yeast mRNA-binding protein Npl3 to address these questions in vivo. Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry was used to identify 17 methylated arginines in Npl3 purified from yeast: whereas 10 Arg-Gly-Gly (RGG) tripeptides were exclusively dimethylated, variable levels off methylation were found for 5 RGG and 2 RG motif arginines. We constructed a set of Npl3 proteins in which subsets of the RGG arginines were mutated to lysine. Expression of these mutant proteins as the sole form of Npl3 specifically affected growth of a strain that requires Hmtl. Although decreased growth generally correlated with increased numbers of Arg-to-Lys mutations, lysine substitutions in the N terminus of the RGG domain showed more severe effects. Npl3 with all 15 RGG arginines mutated to lysine exited the nucleus independent of Hmtl, indicating a direct effect of methylation on Npl3 transport. These mutations also resulted in a decreased, methylation-independent interaction of Npl3 with transcription elongation factor Tho2 and inhibited Npl3 self-association. These results support a model in which arginine methylation facilitates Npl3 export directly by weakening contacts with nuclear proteins. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc

Characterization of the mature form of a β-defensin-like peptide, Hoa-D1, in the lobster Homarus americanus

We report on the characterization of the native form of an American lobster, Homarus americanus, β-defensin-like putative antimicrobial peptide, H. americanus defensin 1 (Hoa-D1), sequenced employing top-down and bottom-up peptidomic strategies using a sensitive, chip-based nanoLC-QTOF-MS/MS instrument. The sequence of Hoa-D1 was determined by mass spectrometry; it was found to contain three disulfide bonds and an amidated C-terminus. The sequence was further validated by searching publicly-accessible H. americanus expressed sequence tag (EST) and transcriptome shotgun assembly (TSA) datasets. Hoa-D1, SYVRScSSNGGDcVYRcYGNIINGAcSGSRVccRSGGGYamide (with c representing a cysteine participating in a disulfide bond), was shown to be related to β-defensin-like peptides previously reported from Panulirus japonicas and Panulirus argus. We found Hoa-D1 in H. americanus hemolymph, hemocytes, the supraoesophageal ganglion (brain), eyestalk ganglia, and pericardial organ extracts, as well as in the plasma of some hemolymph samples. Using discontinuous density gradient separations, we fractionatated hemocytes and localized Hoa-D1 to hemocyte sub-populations. While Hoa-D1 was detected in semigranulocytes and granulocytes using conventional proteomic strategies for analysis, the direct analysis of cell lysates exposed evidence of Hoa-D1 processing, including truncation of the C-terminal tyrosine residue, in the granulocytes, but not semigranulocytes. These measurements demonstrate the insights regarding post-translational modifications and peptide processing that can be revealed through the MS analysis of intact peptides. The identification of Hoa-D1 as a widely-distributed peptide in the lobster suggests the possibility that it may be pleiotropic, with functions in addition to its proposed role as an antimicrobial molecule in the innate immune system

Multiple transcriptome mining coupled with tissue specific molecular cloning and mass spectrometry provide insights into agatoxin-like peptide conservation in decapod crustaceans

Over the past decade, in silico genome and transcriptome mining has led to the identification of many new crustacean peptide families, including the agatoxin-like peptides (ALPs), a group named for their structural similarity to agatoxin, a spider venom component. Here, analysis of publicly accessible transcriptomes was used to expand our understanding of crustacean ALPs. Specifically, transcriptome mining was used to investigate the phylogenetic/structural conservation, tissue localization, and putative functions of ALPs in decapod species. Transcripts encoding putative ALP precursors were identified from one or more members of the Penaeoidea (penaeid shrimp), Sergestoidea (sergestid shrimps), Caridea (caridean shrimp), Astacidea (clawed lobsters and freshwater crayfish), Achelata (spiny/slipper lobsters), and Brachyura (true crabs), suggesting a broad, and perhaps ubiquitous, conservation of ALPs in decapods. Comparison of the predicted mature structures of decapod ALPs revealed high levels of amino acid conservation, including eight identically conserved cysteine residues that presumably allow for the formation of four identically positioned disulfide bridges. All decapod ALPs are predicted to have amidated carboxyl-terminals. Two isoforms of ALP appear to be present in most decapod species, one 44 amino acids long and the other 42 amino acids in length, both likely generated by alternative splicing of a single gene. In carideans, a gene or terminal exon duplication appears to have occurred, with alternative splicing producing four ALPs, two 44 and two 42 amino acid isoforms. The identification of ALP precursor-encoding transcripts in nervous system-specific transcriptomes (e.g., Homarus americanus brain, eyestalk ganglia, and cardiac ganglion assemblies, finding confirmed using RT-PCR) suggests that members of this peptide family may serve as locally-released and/or hormonally-delivered neuromodulators in decapods. Their detection in testis- and hepatopancreas-specific transcriptomes suggests that members of the ALP family may also play roles in male reproduction and innate immunity/detoxification