Sinus Gland Research Articles

Distinct or shared actions of peptide family isoforms: I. Peptide-specific actions of pyrokinins in the lobster cardiac neuromuscular system.

Although the crustacean heart is modulated by a large number of peptides and amines, few of these molecules have been localized to the cardiac ganglion itself; most appear to reach the cardiac ganglion only by hormonal routes. Immunohistochemistry in the American lobster Homarus americanus indicates that pyrokinins are present not only in neuroendocrine organs (pericardial organ and sinus gland), but also in the cardiac ganglion itself, where pyrokinin-positive terminals were found in the pacemaker cell region, as well as surrounding the motor neurons. Surprisingly, the single pyrokinin peptide identified from H. americanus, FSPRLamide, which consists solely of the conserved FXPRLamide residues that characterize pyrokinins, did not alter the activity of the cardiac neuromuscular system. However, a pyrokinin from the shrimp Litopenaeus vannamei [ADFAFNPRLamide, also known as Penaeus vannamei pyrokinin 2 (PevPK2)] increased both the frequency and amplitude of heart contractions when perfused through the isolated whole heart. None of the other crustacean pyrokinins tested (another from L. vannamei and two from the crab Cancer borealis) had any effect on the lobster heart. Similarly, altering the PevPK2 sequence either by truncation or by the substitution of single amino acids resulted in much lower or no activity in all cases; only the conservative substitution of serine for alanine at position 1 resulted in any activity on the heart. Thus, in contrast to other systems (cockroach and crab) in which all tested pyrokinins elicit similar bioactivities, activation of the pyrokinin receptor in the lobster heart appears to be highly isoform specific.

Bilateral Carotico-Clinoid Foramen

The carotico-clinoid foramen is the result of ossification either of the carotico-clinoid ligament or of a dural fold extending between the anterior and middle clinoid processes of the sphenoid bone. It is anatomically important due to its relations with the cavernous sinus and its contents, sphenoid sinus and pituitary gland. A case of bilateral foramen caroticoclinoid and interclinoid bar has been reported while teaching the cranial cavity to MBBS students. This carotico-clinoid foramen is seen as a consequence of fusion of anterior and middle clinoid processes. The existence of a bony caroticoclinoid foramen may cause compression, tightening or stretching of the internal carotid artery. Further, removing the anterior clinoid process is an important step in regional surgery. The presence of a bony carotico-clinoid foramen may have high risk. Therefore, detail knowledge of type of ossification between the anterior and middle clinoid processes is necessary to increase the success of regional surgery.

In vivo and in vitro inhibitory action of 17β-estradiol and environmental estrogen 4-nonylphenol on gonad-inhibiting hormone (GIH) expression in the eyestalks of Litopenaeus vannamei.

The gonad-inhibiting hormone (GIH) belongs to a neuropeptide family synthesized and released in an X-organ sinus gland complex of crustacean eyestalks. GIH inhibits crustacean ovarian maturation by suppressing vitellogenin (Vtg) synthesis, whereas estrogen is responsible for the stimulation of vitellogenesis (not established). In this study, the effects of 17β-estradiol (E2, 10(-6) M), estrogen receptor antagonist tamoxifen (TAM, 10(-6), 10(-7), and 10(-8) M), and the environmental estrogen nonylphenol (NP, 1 μg/L and 100 μg/L) on LvGIH expression in the eyestalks of shrimp were determined by quantitative real-time PCR. Results showed that LvGIH expression decreased significantly during the L. vannamei ovarian maturation cycle. E2 and NP significantly reduced LvGIH transcripts in vivo, but TAM neutralized the inhibitory action of E2 in a dose-dependent manner (P < 0.05). In addition, the LvGIH expression levels decreased significantly in a time-dependent manner (P < 0.05) when ovary fragments were cultured in vitro with E2. The results of this study suggested that estrogen regulates GIH expression in L. vannamei eyestalks. E2 promoted ovarian development not only by directly upregulating vitellogenesis in the hepatopancreas, but it was also capable of downregulating LvGIH expression, which indirectly resulted in the stimulation of L. vannamei vitellogenesis.

Isolation of crustacean hyperglycemic hormone-family peptides with vitellogenesis-inhibiting activity from the shiba shrimp Metapenaeus joyneri

Vitellogenesis-inhibiting hormone (VIH) in Crustacea belongs to the crustacean hyperglycemic hormone (CHH)-family. In order to characterize multiple VIH molecules in the shiba shrimp Metapenaeus joyneri, four CHH-family peptides designated as Mej-SGP-I, -II, -III, and -IV were purified by reversed-phase HPLC and identified by N-terminal amino acid sequencing. The vitellogenesis-inhibiting activities of these peptides were examined using ex vivo incubation of ovarian fragments of the kuruma prawn Marsupenaeus japonicus. All of the four peptides significantly reduced vitellogenin mRNA levels in the ovarian fragments. Among them, the inhibitory activity of Mej-SGP-III which was the most abundant CHH-family peptide in the sinus gland was apparent. Therefore, a cDNA encoding the Mej-SGP-III precursor was cloned by RT-PCR coupled with 5′- and 3′-RACE. The mature Mej-SGP-III was found to consist of 72 amino acid residues containing six conserved cysteine residues and possess an amidated C-terminus. The mature Mej-SGP-III showed the highest sequence identity to Pej-SGP-V (one of six CHH/VIHs in M. japonicus) and Liv-SGP-G (one of six VIHs in Litopenaeus vannamei). These results indicate that Mej-SGP-III is one of the hormones regulating vitellogenesis in M. joyneri.

Functional characterization of neuroendocrine regulation of branchial carbonic anhydrase induction in the euryhaline crab Callinectes sapidus.

Carbonic anhydrase (CA) plays an essential role as a provider of counterions for Na(+)/H(+) and Cl(-)/HCO3 (-) exchange in branchial ionic uptake processes in euryhaline crustaceans. CA activity and gene expression are low in crabs acclimated to full-strength seawater, with transfer to low salinity resulting in large-scale inductions of mRNA and subsequent enzyme activity in the posterior ion-regulating gills (e.g., G7). In the green crab Carcinus maenas, CA has been shown to be under inhibitory neuroendocrine control by a putative hormone in the x-organ-sinus gland complex (XOSG), located in the eyestalk. This study characterizes the neuroendocrine regulation of CA induction in the blue crab Callinectes sapidus, a commonly used experimental organism for crustacean osmoregulation. In crabs acclimated to full-strength seawater, eyestalk ligation (ESL) triggered a 1.8- and 100-fold increase in CA activity and mRNA, respectively. Re-injection with eyestalk homogenates abolished increases in CA activity and fractionally reduced CA gene expression. ESL also enhanced CA induction by 33% after 96 h in crabs transferred to 15 ppt salinity. Injection of eyestalk homogenates into intact crabs transferred from 35 to 15 ppt diminished by 43% the CA induction stimulated by low salinity. These results point to the presence of a repressor hormone in the eyestalk. Separate injections of medullary tissue (MT) and sinus gland (SG), two components of the eyestalk, reduced salinity-stimulated CA activity by 22% and 49%, suggesting that the putative repressor is localized to the SG. Crabs injected with SG extract harvested from crabs acclimated to 5 ppt showed no decrease in CA activity, demonstrating that the hormone is down-regulated at low salinity. Our results show the presence in the XOSG of an inhibitory compound that regulates salinity-stimulated CA induction.

Quantitative neuropeptidomics study of the effects of temperature change in the crab Cancer borealis.

Temperature changes influence the reaction rates of all biological processes, which can pose dramatic challenges to cold-blooded organisms, and the capability to adapt to temperature fluctuations is crucial for the survival of these animals. In order to understand the roles that neuropeptides play in the temperature stress response, we employed a mass spectrometry-based approach to investigate the neuropeptide changes associated with acute temperature elevation in three neural tissues from the Jonah crab Cancer borealis. At high temperature, members from two neuropeptide families, including RFamide and RYamide, were observed to be significantly reduced in one of the neuroendocrine structures, the pericardial organ, while several orcokinin peptides were detected to be decreased in another major neuroendocrine organ, the sinus gland. These results implicate that the observed neuropeptides may be involved with temperature perturbation response via hormonal regulation. Furthermore, a temperature stress marker peptide with the primary sequence of SFRRMGGKAQ (m/z 1137.7) was detected and de novo sequenced in the circulating fluid (hemolymph) from animals under thermal perturbation.

Crustacean hyperglycemic hormones of two cold water crab species, Chionoecetes opilio and C. japonicus: Isolation of cDNA sequences and localization of CHH neuropeptide in eyestalk ganglia

Crustacean hyperglycemic hormone (CHH) is primarily known for its prototypical function in hyperglycemia which is induced by the release of CHH. The CHH release takes place as an adaptive response to the energy demands of the animals experiencing stressful environmental, physiological or behavioral conditions. Although >63 decapod CHH nucleotide sequences are known (GenBank), the majority of them is garnered from the species inhabiting shallow and warm water. In order to understand the adaptive role of CHH in Chionoecetes opilio and Chionoecetes japonicus inhabiting deep water environments, we first aimed for the isolation of the full-length cDNA sequence of CHH from the eyestalk ganglia of C. opilio (ChoCHH) and C. japonicus (ChjCHH) using degenerate PCR and 5′ and 3′ RACE. Cho- and ChjCHH cDNA sequences are identical in 5′ UTR and ORF with 100% sequence identity of the putative 138aa of preproCHHs. The length of 3′ UTR ChjCHH cDNA sequence is 39 nucleotides shorter than that of ChoCHH. This is the first report in decapod crustaceans that two different species have the identical sequence of CHH. ChoCHH expression increases during embryogenesis of C. opilio and is significantly higher in adult males and females. C. japonicus males have slightly higher ChjCHH expression than C. opilio males, but no statistical difference. In both species, the immunostaining intensity of CHH is stronger in the sinus gland than that of X-organ cells. Future studies will enable us to gain better understanding of the comparative metabolic physiology and endocrinology of cold, deep water species of Chionoecetes spp.

Effects of l-tryptophan-supplemented dietary on growth performance and 5-HT and GABA levels in juvenile Litopenaeus vannamei

A 60-day feeding trial was conducted to evaluate the effects of dietary l-tryptophan level on the growth performance and neurotransmitter, including 5-hydroxytryptamine (5-HT) and γ-aminobutyric acid (GABA) of juvenile Litopenaeus vannamei. Six experimental diets were formulated to supplement with graded levels of tryptophan, resulting in different dietary tryptophan levels at 0 (control), 0.12, 0.24, 0.36, 0.48 and 0.60 %, respectively. A total of 960 shrimps (0.63 ± 0.01 g) were divided into six treatments and fed with the respective tryptophan diets in an indoor recirculating system. The results showed that the body weight, body length, live weight gain, specific growth rate, protein efficiency ratio and survival rate were significantly increased by dietary tryptophan levels, and the maximum were all obtained at 0.60 % tryptophan-supplemented diet. Feed conversion ratio was significantly decreased as dietary tryptophan level increased. No significant difference was found in hepatopancreas index and body proximate composition among all the treatments. High-performance liquid chromatography analysis showed that 5-HT level increased first and then decreased in all examined tissues except in thoracic ganglia with dietary tryptophan level increased, while GABA level first elevated and then decreased in X organ–sinus gland, brain, stomach and intestinal tract, and on the contrary, in thoracic ganglia, hepatopancreas, but declined in hemolymph with higher dietary tryptophan level. qRT-PCR results exhibited 5-HT1 mRNA expression was similar to 5-HT level variation. Based on these results, the dietary tryptophan supplemented 0.36 % dry diet or 1.72 % protein diet is found to be optimum to improve L. vannamei growth performance. In conclusion, it suggested that diet supplemented with tryptophan was beneficial to improve growth performance possibly by mediating 5-HT as well as GABA pathway in L. vannamei. These data provide new insight into the physiological functions of tryptophan in shrimp.

Molecular identification of a new androgenic gland-specific insulin-like gene from the mud crab, Scylla paramamosain

It is well known that the androgenic gland is a specific endocrine organ of the male crustacean, controlling its primary and secondary sexual characteristics. The androgenic gland hormone is a key peptide hormone involved in sexual differentiation in crustaceans. In this study, a full length cDNA encoding insulin-like gland hormone from Scylla paramamosain, named Sp-IAG, was identified. The predicted protein had similar molecular organization to other insulin-like androgenic gland factors reported in the Decapoda, encoding a signal peptide, B chain, C peptide and A chain. There were two disulfide bridges between the B chain and A chain, and one disulfide in the B chain. In particular, a full genomic DNA sequence of Sp-IAG with a 5′ flanking region of 2988 bp was revealed from which many potential regulatory binding sites were first identified in S. paramamosain. The Sp-IAG gene was highly expressed in the androgenic gland and also lowly expressed in the seminal vesicle and ejaculatory duct, which was not reported in previous studies. It is noteworthy that Sp-IAG was highly expressed both in the androgenic gland and in the seminal vesicle using western blot analysis, suggesting that Sp-IAG might play a major role in these two organs. In addition, it was quantitatively detected that Sp-IAG mRNA expression was up-regulated after mating, which suggested that this protein might have an activity related to mating behavior. Eyestalk ablation can induce a high level of Sp-IAG expression, demonstrating that the Sp-IAG of S. paramamosain was negatively controlled by the X-organ–sinus gland in the eyestalk. These findings provided the basis for elucidating the mechanism of sex differentiation in crustaceans.

Endoscopic endonasal transcavernous posterior clinoidectomy with interdural pituitary transposition.

OBJECT.: The object of this paper was to describe the surgical anatomy and technical nuances of the endonasal transcavernous posterior clinoidectomy approach with interdural pituitary transposition and to report the clinical outcome of this technical modification. The surgical anatomy of the proposed approach was studied in 10 colored silicon-injected anatomical specimens. The medical records of 12 patients that underwent removal of the posterior clinoid(s) with this technique were reviewed. The natural anatomical corridor provided by the cavernous sinus is used to get access to the posterior clinoid by mobilizing the pituitary gland in an interdural fashion. The medial wall of the cavernous sinus is preserved intact and attached to the gland during its medial and superior mobilization. This provides protection to the gland, allowing for preservation of its venous drainage pathways. The inferior hypophyseal artery is transected to facilitate the manipulation of the medial wall of the cavernous sinus and pituitary gland. This approach was successfully performed in all patients, including 6 with chordomas, 5 with petroclival meningiomas, and 1 with an epidermoid tumor. No patient in this series had neurovascular injury related to the posterior clinoidectomy. There were no instances of permanent hypopituitarism or diabetes insipidus. The authors introduce a surgical variant of the endoscopic endonasal posterior clinoidectomy approach that does not require intradural pituitary transposition and is more effective than the purely extradural approach. The endoscopic endonasal transcavernous approach facilitates the removal of prominent posterior clinoids increasing the working space at the lateral recess of the interpeduncular cistern, while preserving the pituitary function.

Qualitative and quantitative top‐down mass spectral analysis of crustacean hyperglycemic hormones in response to feeding

An efficient pipeline for peptide discovery accelerates peptidomic analysis and facilitates a better understanding of the functional roles of neuropeptides. However, qualitative and quantitative analysis of large neuropeptides is challenging due to the bigger molecular sizes, multiple PTMs, and interference by homologous isoforms. Herein, we refined two methodologies in the pipeline for highly confident and efficient MS-based peptide discovery. For the qualitative analysis, the so-called "high resolution/accurate mass" measurement on Orbitrap mass spectrometers was integrated with computer-assisted homology search, which was successfully applied to decipher the substituted amino acid residues in large neuropeptides by referring to homologous sequences. For the quantitative analysis, a new isotopic labeling-assisted top-down MS strategy was developed, which enabled direct monitoring of the abundance changes of endogenous large neuropeptides. By using the refined peptide discovery pipeline, one novel crustacean hyperglycemic hormone (CHH) from the Dungeness crab sinus glands was confidently identified and de novo sequenced, and its relative abundance was quantified. Comparative analysis of CHHs in unfed and fed crabs revealed that the peptide abundance in the sinus glands was significantly increased after food intake, suggesting that the release of CHHs might be altered by feeding behavior.

An insulin-like growth factor found in hepatopancreas implicates carbohydrate metabolism of the blue crab Callinectes sapidus

Hyperglycemia that is caused by the release of crustacean hyperglycemic hormone (CHH) from the sinus gland to hemolymph is one of the hallmark physiological phenomena, occurring in decapod crustaceans experiencing stressful conditions. However, the mechanism(s) by which such elevated glucose levels return to resting levels is still unknown. Interestingly, noted is a difference in the clearance rate of hemolymph glucose between adult females and adult males of the blue crab, Callinectes sapidus: the former with more rapid clearance than the latter. The presence of an endogenous-insulin-like molecule is suggested in C. sapidus because an injection of bovine insulin, significantly reduces the levels of hemolymph glucose that were previously elevated by emersion stress or the glucose injection. Using 5′ and 3′ RACE, the full-length cDNA of an insulin-like molecule is isolated from the hepatopancreas of an adult female C. sapidus and shows the same putative sequence of an insulin-like androgenic gland factor (IAG) but differs in 5′ and 3′ UTR sequences. A knock-down study using five injections of double-stranded RNA of CasIAG-hep (dsRNA-CasIAG-hep, 10μg/injection) over a 10-day period reduces CasIAG-hep expression by ∼50%. The levels of hemolymph glucose are also kept higher in dsRNA-CasIAG-hep injected group than those treated with dsRNA-green fluorescent protein (dsRNA-IAG-hep) or saline. Most importantly, the hepatopancreas of dsRNA-CasIAG-hep injected animals contains amounts of carbohydrate (glucose, trehalose, and glycogen) significantly lower than those of control groups, indicating that the function of CasIAG-hep in carbohydrate metabolism in crustaceans is similar to carbohydrate metabolism in vertebrates.

Nitric oxide production and sequestration in the sinus gland of the green shore crab Carcinus maenas.

Molting in decapod crustaceans is regulated by molt-inhibiting hormone (MIH), a neuropeptide produced in the X-organ (XO)/sinus gland (SG) complex of the eyestalk ganglia (ESG). Pulsatile release of MIH from the SG suppresses ecdysteroidogenesis by the molting gland or Y-organ (YO). The hypothesis is that nitric oxide (NO), a neuromodulator that controls neurotransmitter release at presynaptic membranes, depresses the frequency and/or amount of MIH pulses to induce molting. NO synthase (NOS) mRNA was present in Carcinus maenas ESG and other tissues and NOS protein was present in the SG. A copper based ligand (CuFL), which reacts with NO to form a highly fluorescent product (NO-FL), was used to image NO in the ESG and SG and quantify the effects of NO scavenger (cPTIO), NOS inhibitor (l-NAME), and sodium azide (NaN3) on NO production in the SG. Pre-incubation with cPTIO prior to CuFL loading decreased NO-FL fluorescence ~30%; including l-NAME had no additional effect. Incubating SG with l-NAME during pre-incubation and loading decreased NO-FL fluorescence ~40%, indicating that over half of the NO release was not directly dependent on NOS activity. Azide, which reacts with NO-binding metal groups in proteins, reduced NO-FL fluorescence to near background levels without extensive cell death. Spectral shift analysis showed that azide displaced NO from a soluble protein in SG extract. These data suggest that the SG contains NO-binding protein(s) that sequester NO and releases it over a prolonged period. This NO release may modulate neuropeptide secretion from the axon termini in the SG.

Energy balance of spermatophores and sperm viability during the molt cycle in intact and bilaterally eyestalk ablated male Pacific white shrimp Litopenaeus vannamei

The energy levels of spermatophores and sperm viability during the molt cycle were investigated in intact and bilaterally eyestalk ablated male Pacific white shrimp Litopenaeus vannamei. Sexual organs and spermatophores, expressed either as absolute weight or as a somatic index, and sperm counts were significantly higher in the ablated group. A continuous increase in the weight of the vas deferens throughout the molt cycle was observed in the ablated group but not in the control one. This suggests formation of new spermatophores in the vas deferens from the early premolt stage, negatively controlled by hormones from the X organ–sinus gland complex. Spermatophores contained the highest ATP level and adenylate energy charge (AEC), once they were visible in the terminal ampoule after ecdysis at the late postmolt stage of the first molt cycle, where it reached a mean ATP value of 3.3±0.5nmol per spermatophore in the intact group and 5.85±1.4nmol per spermatophore in the ablated group. Both groups had an AEC of ~0.73. ATP and AEC progressively decreased in both groups during the first molt cycle. At the late premolt stage, the intact group had values of ~1.6nmol per spermatophore and ~0.5 respectively, whereas the ablated group had values of ~2.9nmol per spermatophore and ~0.6, respectively. However, after ecdysis at the early postmolt stage of the second molt cycle, ATP and AEC increased in the intact group to ~2.2nmol per spermatophore and ~0.6, respectively, whereas in the ablated group, they decreased to ~1.5nmol per spermatophore and ~0.48, respectively. This differential pattern was also found for arginine phosphate, where it increased in the intact group from 24.8±7.5 to 55.2±5.5nmol per spermatophore, while it decreased in the ablated group from 78.8±10.5 to 59.6±15.5nmol per spermatophore. This suggests a cyclic molt-regulated energy balance, where new spermatophores are energy “recharged” in intact shrimp, while this process is apparently disrupted in ablated shrimp.

A multi-scale strategy for discovery of novel endogenous neuropeptides in the crustacean nervous system

The conventional mass spectrometry (MS)-based strategy is often inadequate for the comprehensive characterization of various size neuropeptides without the assistance of genomic information. This study evaluated sequence coverage of different size neuropeptides in two crustacean species, blue crab Callinectes sapidus and Jonah crab Cancer borealis using conventional MS methodologies and revealed limitations to mid- and large-size peptide analysis. Herein we attempt to establish a multi-scale strategy for simultaneous and confident sequence elucidation of various sizes of peptides in the crustacean nervous system. Nine novel neuropeptides spanning a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ, the sinus gland of the spiny lobster Panulirus interruptus. These novel neuropeptides included seven allatostatin (A- and B-type) peptides, one crustacean hyperglycemic hormone precursor-related peptide, and one crustacean hyperglycemic hormone. Highly accurate multi-scale characterization of a collection of varied size neuropeptides was achieved by integrating traditional data-dependent tandem MS, improved bottom-up sequencing, multiple fragmentation technique-enabled top-down sequencing, chemical derivatization, and in silico homology search. Collectively, the ability to characterize a neuropeptidome with vastly differing molecule sizes from a neural tissue extract could find great utility in unraveling complex signaling peptide mixtures employed by other biological systems. Mass spectrometry (MS)-based neuropeptidomics aims to completely characterize the neuropeptides in a target organism as an important first step toward a better understanding of the structure and function of these complex signaling molecules. Although liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) with data-dependent acquisition is a powerful tool in peptidomic research, it often lacks the capability for de novo sequencing of mid-size and large peptides due to inefficient fragmentation of peptides larger than 4kDa. This study describes a multi-scale strategy for complete and confident sequence elucidation of various sizes of neuropeptides in the crustacean nervous system. The aim is to fill a technical gap where the conventional strategy is inefficient for comprehensive characterization of a complex neuropeptidome without assistance of genomic information. Nine novel neuropeptides in a wide range of molecular weights (0.9-8.2kDa) were fully sequenced from a major neuroendocrine organ of the spiny lobster, P. interruptus. The resulting molecular information extracted from such multi-scale peptidomic analysis will greatly accelerate functional studies of these novel neuropeptides.

The pyramidal lobe: Connecting a case study with lymphatic system topics in gross anatomical education

The pyramidal lobe is a variably present structure extending superiorly from the midline of the thyroid gland. A remnant of the thyroglossal duct, the pyramidal lobe may harbor precricoid (also known as Delphian) lymph nodes, which receive lymphatic drainage predominantly from the larynx, pyriform sinus, and thyroid gland. The grossly visible presence of precricoid lymph nodes can serve as an indication of metastasis of head and neck cancers. A sixty‐one year old male donor present in a human anatomy course displayed a shockingly pronounced pyramidal lobe, measuring nine centimeters in length. This individual also displayed several small swellings in the center of the pyramidal lobe where the precricoid lymph nodes frequently rest. This donor's cause of death was metastatic salivary gland carcinoma, and therefore involvement of the precricoid lymph nodes is an intriguing possibility. This case highlights the importance of the lymphatic system in gross anatomical education. Unfortunately, the lymphatic system may not be examined extensively in gross anatomy laboratory settings in part due to reduction in course hours and this system's relative inconspicuousness and fragility in dissection. However, this case study brings to light the clinical importance of seemingly insignificant segments of the lymphatic system as well as the necessity of comprehensive gross anatomical education on this topic.Grant Funding Source: none

Large-scale Identification of Endogenous Secretory Peptides Using Electron Transfer Dissociation Mass Spectrometry

Mass spectrometry-based unbiased analysis of the full complement of secretory peptides is expected to facilitate the identification of unknown biologically active peptides. However, tandem MS sequencing of endogenous peptides in their native form has proven difficult because they show size heterogeneity and contain multiple internal basic residues, the characteristics not found in peptide fragments produced by in vitro digestion. Endogenous peptides remain largely unexplored by electron transfer dissociation (ETD), despite its widespread use in bottom-up proteomics. We used ETD, in comparison to collision induced dissociation (CID), to identify endogenous peptides derived from secretory granules of a human endocrine cell line. For mass accuracy, both MS and tandem MS were analyzed on an Orbitrap. CID and ETD, performed in different LC-MS runs, resulted in the identification of 795 and 569 unique peptides (ranging from 1000 to 15000 Da), respectively, with an overlap of 397. Peptides larger than 3000 Da accounted for 54% in CID and 46% in ETD identifications. Although numerically outperformed by CID, ETD provided more extensive fragmentation, leading to the identification of peptides that are not reached by CID. This advantage was demonstrated in identifying a new antimicrobial peptide from neurosecretory protein VGF (non-acronymic), VGF[554-577]-NH2, or in differentiating nearly isobaric peptides (mass difference less than 2 ppm) that arise from alternatively spliced exons of the gastrin-releasing peptide gene. CID and ETD complemented each other to add to our knowledge of the proteolytic processing sites of proteins implicated in the regulated secretory pathway. An advantage of the use of both fragmentation methods was also noted in localization of phosphorylation sites. These findings point to the utility of ETD mass spectrometry in the global study of endogenous peptides, or peptidomics.

Molecular cloning of a cDNA encoding vitellogenesis-inhibiting hormone in the whiteleg shrimp Litopenaeus vannamei and preparation of its recombinant peptide using an E. coli expression system

The cDNA encoding a precursor of Liv-SGP-G, the most abundant crustacean hyperglycemic hormone-family peptide in the sinus gland of the whiteleg shrimp Litopenaeus vannamei, was cloned by RT-PCR coupled with 5′- and 3′-RACE. The determined cDNA sequence consisted of 621 nucleotides comprising a 69-bp 5′-untranslated region, a 357-bp open reading frame, and a 195-bp 3′-untranslated region. The deduced sequence of 72 amino acid residues corresponding to mature Liv-SGP-G was completely identical to that of natural Liv-SGP-G, determined in our previous study. The recombinant Liv-SGP-G was thereafter heterologously expressed in Escherichia coli, and its vitellogenesis-inhibiting activity in ex vivo cultured ovarian fragments was examined. The recombinant peptides inhibited vitellogenin gene expression with almost the same efficacy as that of natural Liv-SGP-G purified from sinus glands.

Mass spectrometric characterization of the neuropeptidome of the ghost crab Ocypode ceratophthalma (Brachyura, Ocypodidae)

The horn-eyed ghost crab Ocypode ceratophthalma is a terrestrial brachyuran native to the Indo-Pacific region, including the islands of Hawaii. Here, multiple mass spectrometric platforms, including matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS) and nanoflow liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-Q-TOF MS/MS), were used to characterize the neuropeptidome of this species. In total, 156 peptide paracrines/hormones, representing 15 peptide families, were identified from the O. ceratophthalma supraesophageal ganglion (brain), eyestalk ganglia, pericardial organ and/or sinus gland, including 59 neuropeptides de novo sequenced here for the first time. Among the de novo sequenced peptides were isoforms of A-type allatostatin, B-type allatostatin, FMRFamide-like peptide (FLP), orcokinin, orcomyotropin and RYamide. Of particular note, were several novel FLPs including DVRAPALRLRFamide, an isoform of short neuropeptide F, and NRSNLRFamide, the orcokinins NFDEIDRSGYGFV and DFDEIDRSSFGFH, which exhibit novel Y for F and D for N substitutions at positions 10 and 1, respectively, and FDAYTTGFGHS, a member of the orcomyotropin family exhibiting a novel Y for F substitution at position 4. Taken collectively, the set of peptides described here represents the largest number of neuropeptides thus far characterized via mass spectrometry from any single crustacean, and provides a framework for future investigations of the physiological roles played by these molecules in this species.

Incidence Of Ossified Interclinoid Bars In Dry Human Skulls Of Gujarat State

A group of intrinsic ligaments of the sphenoid bone which connect the anterior, middle and posterior clinoid processes occasionally get ossified and give rise to various types of ossified interclinoid bars. In present study the incidence of various types of ossified interclinoid bars were observed in 200 dry human skulls of unknown age and sex belonging to department of Anatomy of various Medical Colleges of Gujarat region. The interclinoid bars are classified on the basis of two classifications, proposed by Rani Archana et al and Keyers. Rani Archana et al, classified interclinoid bars into four types : Type I (caroticoclinoid foramen) bridge present between anterior and middle clinoid process, Type II bridge between anterior, middle and posterior clinoid process, Type III bridge between anterior and posterior clinoid process and Type IV bridge between the middle and posterior clinoid process. Each interclinoid bar was classified into contact, incomplete, complete types based on the classification of Keyers. In present study total incidence of interclinoid bars was 18%. Type I were present in 10.5%, out of which 8% were complete and 2.5% were incomplete. Types II in 3.5%, amongst them 2% were complete and 1.5% was mixed. Type III in 4%, out of which 2% were complete and 2% were incomplete. Total incidence of caroticoclinoid foramens was 14.5%, out of which 11.5% were complete and 3% were incomplete. Total incidence of sellar bridge was 7.5%, out of which 4.5% were complete and 3% were incomplete. Presence of osseous interclinoid bars may cause compression of surrounding structures like the cavernous sinus and its content, sphenoid sinus and pituitary gland. Therefore, detailed anatomical knowledge of various types of interclinoid bars can increase the success of diagnostic evaluation and surgical approaches to the region.