Endogenous Fluorescent Proteins in the Mucus of an Intertidal Polychaeta: Clues for Biotechnology.

The vast ocean holds many unexplored organisms with unique adaptive features that enable them to thrive in their environment. The secretion of fluorescent proteins is one of them, with reports on the presence of such compounds in marine annelids being scarce. The intertidal Eulalia sp. is an example. The worm secretes copious amounts of mucus, that when purified and concentrated extracts, yield strong fluorescence under UV light. Emission has two main maxima, at 400 nm and at 500 nm, with the latter responsible for the blue-greenish fluorescence. Combining proteomics and transcriptomics techniques, we identified ubiquitin, peroxiredoxin, and 14-3-3 protein as key elements in the mucus. Fluorescence was found to be mainly modulated by redox status and pH, being consistently upheld in extracts prepared in Tris-HCl buffer with reducing agent at pH 7 and excited at 330 nm. One of the proteins associated with the fluorescent signal was localized in secretory cells in the pharynx. The results indicate that the secretion of fluorescent proteinaceous complexes can be an important defense against UV for this dweller. Additionally, the internalization of fluorescent complexes by ovarian cancer cells and modulation of fluorescence of redox status bears important considerations for biotechnological application of mucus components as markers.

Efficacy of Precocene I from Desmosstachya bipinnata as an Effective Bioactive Molecules against the Spodoptera litura Fab. and Its Impact on Eisenia fetida Savigny.

The sustainability of agroecosystems are maintained with agro-chemicals. However, after more than 80 years of intensive use, many pests and pathogens have developed resistance to the currently used chemistries. Thus, we explored the isolation and bioactivity of a chemical compound, Precocene I, isolated from the perennial grass, Desmosstachya bipinnata (L.) Stapf. Fractions produced from chloroform extractions showed suppressive activity on larvae of Spodoptera litura (Lepidoptera: Noctuidae), the Oriental armyworm. Column chromatography analyses identified Precocene I confirmed using FTIR, HPLC and NMR techniques. The bioactivity of the plant-extracted Dp-Precocene I was compared to a commercially produced Precocene I standard. The percentage of mortality observed in insects fed on plant tissue treated with 60 ppm Db-Precocene I was 97, 87 and 81, respectively, for the second, third and fourth instar larvae. The LC50 value of third instars was 23.2 ppm. The percentages of survival, pupation, fecundity and egg hatch were altered at sub-lethal concentrations of Db-Precocene I (2, 4, 6 and 8 ppm, sprays on castor leaves). The observed effects were negatively correlated with concentration, with a decrease in effects as concentrations increased. Distinct changes in feeding activity and damage to gut tissues were observed upon histological examination of S. litura larvae after the ingestion of Db-Precocene I treatments. Comparative analyses of mortality on a non-target organism, the earthworm, Eisenia fetida, at equal concentrations of Precocene I and two chemical pesticides (cypermethrin and monocrotophos) produced mortality only with the chemical pesticide treatments. These results of Db-Precocene I as a highly active bioactive compound support further research to develop production from the grass D. bipinnata as an affordable resource for Precocene-I-based insecticides.

The origin identification method for crude drugs derived from arthropods and annelids using molecular biological techniques.

We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.

Analysis of petroleum biodegradation by a bacterial consortium isolated from worms of the polychaeta class (Annelida): Implications for NPK fertilizer supplementation.

Degradation of petroleum hydrocarbons using bacterial consortia may be a means of optimizing bioremediation techniques. In this study, bacterial strains were isolated from the digestive tract of polychaete worms and evaluated concerning the potential of the bacteria to degrade petroleum compounds (Acinetobacter sp., Bacillus sp., Pantoea sp. and Enterobacter sp.). The strains were separately screened regarding their potential to degrade oil after 24 h. The main experiment was carried out for 30 days with the addition of nitrogen, phosphorus and potassium (NPK) after 15 days (Bact-NPK15) and 28 days (Bact-NPK28) and without NPK (Bact). The Bact treatment biomass remained stable until the 20th day of the experiment. C13-C40 n-alkanes were degraded with all treatments in the following order: Bact>Bact-NPK28>Bact-NPK15. Significant differences were observed between the controls and all treatments (p = 0.00031). Measurement of polycyclic aromatic hydrocarbons (PAHs) indicates a lower contribution of these compounds in the Bact-NPK28 treatment, although no significant difference between groups was observed. Bact-NPK28 was able to remove 40% of naphthalene, while Bact-NPK15 removed 20%; this effect was not observed in Bact. Higher hopane degradation levels were observed in Bact and, to a lesser extent, in Bact-NPK28. NPK application for 28 days mainly favored PAH degradation. The evaluated consortium thus exhibits potential in the bioremediation of petroleum-contaminated areas.

Evaluation of Aristolochia indica L. and Piper nigrum L. methanol extract against centipede Scolopendra moristans L. using Wistar albino rats and screening of bioactive compounds by high pressure liquid chromatography: a polyherbal formulation.

The present study was aimed to explore the anti-venom activity of Aristolochia indica and Piper nigrum plants against the centipede (Scolopendra moristans) envenomation in animal model. In vtiro phytochemical, antioxidant and blocking of proteolysis were carried out by using standard spectrophotometric methods. In vivo anti-venom activity of methanol extracts was determined using Wistar albino rats after fixing lethal and effective doses. The electrolytes, lipid, liver, kidney, hematological parameters were analyzed and histopathology of skin and liver were also examined. Anti-skin cancer by MTT method and HPLC analysis were also carried out. The CAIPN extract showed higher total phenolics (150.65 ± 0.08 mg GAE/g extract) and flavonoids (158.97 ± 0.93 mg RE/g extract) content. Further, the same extract revealed the higher molybdenum reducing, inhibition of lipid peroxidation (80.08 ± 0.22%), DPPH radical scavenging (3.05 µg/mL), and blocking of proteolysis activities (96.45 ± 0.04%). The parameters like hypersensitivity, electrolytes, lipids, blood components, liver and kidney marker of the CAIPN methanol extract (200 mg/kg) treated envenomated rats was remarkable and same as in the normal animals. Such status was also achieved by RBAI and SPN at 600 mg/kg. The histopathological scoring of skin and liver confirmed the venom neutralizing activity of CAIPN. Also, the CAIPN methanol extract was notable in anti-skin cancer activity (208 µg/mL). The presence of the ferulic acid (04 ± 0.09 µg/mg) and quercetin (35.30 ± 0.30 µg/mg) like compounds was confirmed by HPLC analysis. Hence, the present investigation results conclude that the CAIPN was significant in their action and this polyherbal formulation could be considered as a new source for the pharmaceutical industries to develop a new effective, ecofriendly anti-venom drug.

Neuro-immune lessons from an annelid: The medicinal leech.

An important question that remains unanswered is how the vertebrate neuroimmune system can be both friend and foe to the damaged nervous tissue. Some of the difficulty in obtaining responses in mammals probably lies in the conflation in the central nervous system (CNS), of the innate and adaptive immune responses, which makes the vertebrate neuroimmune response quite complex and difficult to dissect. An alternative strategy for understanding the relation between neural immunity and neural repair is to study an animal devoid of adaptive immunity and whose CNS is well described and regeneration competent. The medicinal leech offers such opportunity. If the nerve cord of this annelid is crushed or partially cut, axons grow across the lesion and conduction of signals through the damaged region is restored within a few days, even when the nerve cord is removed from the animal and maintained in culture. When the mammalian spinal cord is injured, regeneration of normal connections is more or less successful and implies multiple events that still remain difficult to resolve. Interestingly, the regenerative process of the leech lesioned nerve cord is even more successful under septic than under sterile conditions suggesting that a controlled initiation of an infectious response may be a critical event for the regeneration of normal CNS functions in the leech. Here are reviewed and discussed data explaining how the leech nerve cord sensu stricto (i.e. excluding microglia and infiltrated blood cells) recognizes and responds to microbes and mechanical damages.

Fine-scale bioturbation effects of tubificid worm (Limnodrilus hoffmeisteri) on the lability of phosphorus in sediments.

This study investigated the effects of tubificid worm bioturbation on the lability of phosphorus (P) in microcosm sediments. High-resolution dialysis (HR-Peeper) and two types of diffusive gradients in thin films (DGT) (Zr-oxide DGT and ZrO-Chelex DGT) were used to measure soluble P and Fe, and labile P and Fe at a millimeter spatial scale. The worm bioturbation promoted P release (up to 511% of the control) to the overlying water on the 6th day, but it was reduced compared to the control (up to 171% of the control) from the 22nd day to the 102nd day because of the adsorption by Fe(III) oxyhydroxides. The worm bioturbation reduced the pore water soluble P concentration up to 48% and the DGT-labile P concentration up to 29% of the control from a sediment depth of -10 mm to approximately -130 mm before the 22nd day of incubation due to worm ingestion of sediment particles. Two-dimensional measurements of DGT-labile P also showed a much lower concentration of labile P around the worm burrow. This effect disappeared on the 53rd and 102nd day. However, the soluble P and DGT-labile P decreased again up to 41% and 38%, compared to the control from the sediment depth of -20 mm and -10 mm to approximately -130 mm, respectively, on the 152nd day of incubation due to the adsorption by Fe(III) oxyhydroxides. Soluble Fe(II) and DGT-labile Fe did not show significant changes from the worm bioturbation on the 6th day, but decreased up to 31% and 47% of the control after the 6th day. The results that worm ingestion of sediment particles is a significant driver of soluble and labile P reduction in the sediments before the 22nd day. After that, soluble and labile P reduction was attributed to P adsorption by Fe(III) oxyhydroxides.

[Effect of water temperature, stocking density and feeding cycle on growth of juvenile Poecilobdella manillensis].

The effect of water temperature, stocking density and feeding cycle on the growth of Poecilobdella manillensis juvenile was conducted P. manillensis was conducted respectively under different conditions: water temperatures(18, 22, 26, 30,34, 38 degrees C and CT), stocking density (75, 125, 200, 275, 350 individual/L) and feeding cycle(2, 4, 6, 8, 12, 16 d). After 30 days, survival rate, weight gain rate, specific growth rate were measured. There was a significant correlation between water temperature and specific growth rate (γ = -0.066x2 + 3.543 1x -38.09, R2 = 0.837 9). Based on the regression equation, the specific growth rate of P. manillensis achieved the maximum (9.461 4) at 26.84 degrees C. And the most optimal water temperature was 26-30 degrees C. Meanwhile, the survival rates of P. manillensis was 0 at 38 degrees C in 3 d. There was significant negative correlation between density and specific growth rate (γ = -0.005 7x + 9.197 3, R2 = 0.998 3) and between feeding cycle and specific growth rate (γ = -0.468 2x + 10.574, R2 = 0.998 8).

Ultrastructural study of spermatogenesis and sperm in the African medicinal leech Hirudo troctina Johnson, 1816 (Annelida, Hirudinida).

This paper presents the process of spermatogenesis in the leech Hirudo troctina Johnson, 1816 using light, fluorescent and transmission electron microscopy. At the onset of spermatogenesis in testes, the pear-shaped spermatogonia divide mitotically without full cytokinesis and as a result isogenic groups are formed (clusters, clones) with 2, 4, 8, 16, 32, 64, 128 spermatogonia and, finally, 256 primary spermatocytes occur. The final meiotic divisions of spermatocytes give rise to clones with 1024 spermatids. There are hundreds of developing germ-line clones in each testis. In each clone, the male germ cells divide in full synchrony and they are in the same phase of spermatogenesis. During complex spermiogenesis each spermatid becomes a filiform spermatozoon with a helicoid nucleus, which is characterized by the presence of a long acrosome with two regions - anterior and posterior, which are followed by a helicoid nucleus, a midpiece with only one mitochondrion and a long flagellum. Our results were compared to those on other clitellate annelids that have been studied to date, especially to sperm formation in Hirudo medicinalis Linnaeus, 1785. Only minor differences were found in the length and the diameter of different organelles and the number of spermatids in germ-line clones.

Hypoglycemic effects of glycosaminoglycan from Urechis unicinctus in diabetic mice.

To further utilize glycosaminoglycan from Urechis unicinctus, the hypoglycemic effect and possible mechanism of glycosaminoglycan on diabetic mice were evaluated. Diabetes was induced in mice by intraperitoneal injections of streptozotocin for 3 consecutive days and fed with high-sugar and high-lipid fodder. After diabetes was confirmed, the hypoglycemic effect of glycosaminoglycan from U. unicinctus was investigated in the diabetic mice. Results demonstrated that glycosaminoglycan could significantly decrease blood glucose concentrations, HOMA-IR, AUG, and liver MDA content in diabetic mice. In addition, it significantly enhanced liver SOD and GSH-Px activity, as well as liver GCK activity and hepatic glycogen levels. Glycosaminoglycan from U. unicinctus exhibited efficacy against diabetes, suggesting its potential use as a natural intervention against diabetes.

[Reasonable and safe application of Shuxuetong injection and intravenous medication's combined application in acute cerebral infarction's therapy].

Shuxuetong injection is a kind of compound injection which is made from traditional Chinese medicine Hirudo and Pheretime, which has a clear anticoagulant, fibrinolytic promoting, blood rheology improving, blood lipids regulating and cell protecting effect, and the injection has been widely used in clinical. Especially, the injection has often been combined with other Chinese and modern medicine in the treatment of cerebral infarction disease. However, there are still many non-standard and irrational aspects in clinical practice so as to make a more reasonable and safer use of Shuxuetong injection. In order to avoid the occurrence of adverse reactions to provide a reference for regulating the use of the injection,the paper systematically expounds the Shuxuetong injection's main clinical problems and the reasonable combination.

Reasonable and safe application of Shuxuetong injection and intravenous medication's combined application in acute cerebral infarction's therapy / 中国中药杂志

Shuxuetong injection is a kind of compound injection which is made from traditional Chinese medicine Hirudo and Pheretime, which has a clear anticoagulant, fibrinolytic promoting, blood rheology improving, blood lipids regulating and cell protecting effect, and the injection has been widely used in clinical. Especially, the injection has often been combined with other Chinese and modern medicine in the treatment of cerebral infarction disease. However, there are still many non-standard and irrational aspects in clinical practice so as to make a more reasonable and safer use of Shuxuetong injection. In order to avoid the occurrence of adverse reactions to provide a reference for regulating the use of the injection,the paper systematically expounds the Shuxuetong injection's main clinical problems and the reasonable combination.

Effects of uranium-contaminated sediments on the bioturbation activity of Chironomus riparius larvae (Insecta, Diptera) and Tubifex tubifex worms (Annelida, Tubificidae).

Freshwater sediments represent a compartment for accumulation of toxic substances, notably of metallic pollutants such as uranium. However, they also constitute a privileged habitat for many benthic macro-invertebrate species with important roles in the functioning of these ecosystems, particularly through their bioturbation activities. Uranium accumulation in sediments can thus have harmful effects on these organisms (e.g., developmental delay, malformations, mortality). The present study aimed to evaluate the consequences of these effects on the bioturbation activity of Chironomus riparius larvae and Tubifex tubifex worms. These two species, which are widespread in freshwater ecosystems, are characteristic of two different modes of bioturbation: bioirrigation and upward bioconveying, respectively. By quantifying the burial and redistribution of fluorescent particulate tracers (microspheres), sediment reworking induced by these macro-invertebrates was measured after 12d of exposure. Biodiffusion D(b) and bioadvection W rates, as well as several other parameters, were estimated to assess and compare the bioturbation activity of the two species, separately and in combination, between uncontaminated and uranium-spiked sediments. The results reveal that C. riparius larvae were more sensitive to uranium, but their bioturbation activity, even under uncontaminated conditions, had little effect on sediment reworking. Particle mixing was mainly induced by T. tubifex worms, which were only affected by uranium at high concentrations in the sediment. Finally, bioturbation by T. tubifex led to a high degree of uranium release from sediment to the overlying water, which highlights the crucial role of this mostly dominant species on uranium biogeochemical cycles at concentrations existing in naturally contaminated sites.

Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution.

Neurosecretory control centers form part of the forebrain in many animal phyla, including vertebrates, insects, and annelids. The evolutionary origin of these centers is largely unknown. To identify conserved, and thus phylogenetically ancient, components of neurosecretory brain centers, we characterize and compare neurons that express the prohormone vasotocin (vasopressin/oxytocin)-neurophysin in the developing forebrain of the annelid Platynereis dumerilii and of the zebrafish. These neurons express the same tissue-restricted microRNA, miR-7, and conserved, cell-type-specific combinations of transcription factors (nk2.1, rx, and otp) that specify their identity, as evidenced by the specific requirement of zebrafish rx3 for vasotocin-neurophysin expression. MiR-7 also labels another shared population of neurons containing RFamides. Since the vasotocinergic and RFamidergic neurons appear to be directly sensory in annelid and fish, we propose that cell types with dual sensory-neurosecretory properties were the starting point for the evolution of neurosecretory brain centers in Bilateria.

Phosphagen kinase of the giant tubeworm Riftia pachyptila. Cloning and expression of cytoplasmic and mitochondrial isoforms of taurocyamine kinase.

The giant tubeworm Riftia pachyptila lives at deep-sea hydrothermal vents along the East Pacific Rise and the Galapagos Rift. The large size and high growth rate of R. pachyptila is supported by an endosymbiotic relationship with a chemosynthetic bacterium. Elucidation of the regulation of energy metabolism of the giant tubeworm remains an interesting problem. The purpose of this study is to determine the cDNA sequence of phosphagen kinase, one of the most important enzymes in energy metabolism, and to characterize its function. Two phosphagen kinase cDNA sequences amplified from the cDNA library of R. pachyptila showed high derived amino acid sequence identity (74%) with those of cytoplasmic taurocyamine kinase (TK) and mitochondrial TK from an annelid Arenicola brasiliensis. The cytoplasmic form of the Riftia recombinant enzyme showed stronger activity for the substrates taurocyamine and also considerable activity for lombricine (21% that of taurocyamine). The mitochondrial form, which was structurally similar to mitochondrial creatine kinase, showed stronger activity for taurocyamine, and a broader activity for various guanidine compounds: glycocyamine (35% that of taurocyamine), lombricine (31%) and arginine (3%). Both forms showed no activity for creatine. The difference in substrate specificities between the cytoplasmic and mitochondrial forms might be attributable to the large difference in the amino acid sequence of the GS region and/or several key amino acid residues for establishing guanidine substrate specificity. Based on these results, we conclude that Riftia contains at least two forms of TK as phosphagen kinase. We also report the kinetic parameters, Km and kcat, of Arenicola and Riftia TKs for the first time. The Km values for taurocyamine of Arenicola and Riftia TKs ranged from 0.9 to 4.0 mM and appear to be comparable to those of other annelid-specific enzymes, lombricine kinase and glycocyamine kinase, but are significantly lower than those of Neanthes cytoplasmic and mitochondrial creatine kinases. Comparison of kcat/Km value in various annelid phosphagen kinases indicates that Arenicola mitochondrial TK has the highest catalytic efficiency (16.2 s-1 mM-1). In Arenicola TKs, the mitochondrial form has seven-fold higher efficiency than the cytoplasmic form.

Current advances on different kinases involved in tau phosphorylation, and implications in Alzheimer's disease and tauopathies.

Hyperphosphorylation and accumulation of tau in neurons (and glial cells) is one the main pathologic hallmarks in Alzheimer's disease (AD) and other tauopathies, including Pick's disease (PiD), progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease and familial frontotemporal dementia and parkinsonism linked to chromosome 17 due to mutations in the tau gene (FTDP-17-tau). Hyperphosphorylation of tau is regulated by several kinases that phosphorylate specific sites of tau in vitro. GSK-3-immunoprecipitated sarcosyl-insoluble fractions in AD have the capacity to phosphorylate recombinant tau. In addition, GSK-3 phosphorylated at Ser9, that inactivates GSK-3, is found in the majority of neurons with neurofibrillary tangles and dystrophic neurites of senile plaques in AD, and in Pick bodies and other phospho-tau-containing neurons and glial cells in other tauopathies. Increased expression of active kinases, including stress-activated kinase, c-Jun N-terminal kinase (SAPK/JNK) and kinase p38 has been found in brain homogenates in all the tauopathies. Strong active SAPK/JNK and p38 immunoreactivity has been observed restricted to neurons and glial cells containing hyperphosphorylated tau, as well as in dystrophic neurites of senile plaques in AD. Moreover, SAPK/JNK- and p38-immunoprecipitated sub-cellular fractions enriched in abnormal hyperphosphorylated tau have the capacity to phosphorylate recombinant tau and c-Jun and ATF-2 which are specific substrates of SAPK/JNK and p38 in AD and PiD. Interestingly, increased expression of phosphorylated (active) SAPK/JNK and p38 and hyperphosphorylated tau containing neurites have been observed around betaA4 amyloid deposits in the brain of transgenic mice (Tg 2576) carrying the double APP Swedish mutation. These findings suggest that betaA4 amyloid has the capacity to trigger the activation of stress kinases which, in turn, phosphorylate tau in neurites surrounding amyloid deposits. Complementary findings have been reported from the autopsy of two AD patients who participated in an amyloid-beta immunization trial and died during the course of immunization-induced encephalitis. The neuropathological examination of the brain showed massive focal reduction of amyloid plaques but not of neurofibrillary degeneration. Activation of SAPK/JNK and p38 were reduced together with decreased tau hyperphosphorylation of aberrant neurites in association with decreased amyloid plaques in both Tg2576 mice and human brains. These findings support the amyloid cascade hypothesis of tau phosphorylation mediated by stress kinases in dystrophic neurites of senile plaques but not that of neurofibrillary tangles and neuropil threads in AD.

Asphalt volcanism and chemosynthetic life in the Campeche Knolls, Gulf of Mexico.

In the Campeche Knolls, in the southern Gulf of Mexico, lava-like flows of solidified asphalt cover more than 1 square kilometer of the rim of a dissected salt dome at a depth of 3000 meters below sea level. Chemosynthetic tubeworms and bivalves colonize the sea floor near the asphalt, which chilled and contracted after discharge. The site also includes oil seeps, gas hydrate deposits, locally anoxic sediments, and slabs of authigenic carbonate. Asphalt volcanism creates a habitat for chemosynthetic life that may be widespread at great depth in the Gulf of Mexico.

Antibacterial properties of hemerythrin of the sand worm Nereis diversicolor.

OBJECTIVES: To investigate the immune defense of the annelid Nereis diversicolor and the key role of a oxygen-binding protein, the metalloprotein MPII animals were subjected to bacteria infection. METHODS AND RESULTS: Using RACE-PCR, we have cloned the complete cDNA coding for the MPII related to the hemerythrin family in the sand worm Hediste diversicolor. This cDNA (883 pb) codes for a polypeptide of 119 amino acid residues with no signal peptide. Previous works have identified this protein as a cadmium scavenger. We here clearly demonstrated that this protein is also involved in the worm defence towards bacteria growth by its iron scavenger ability. This protein is expressed and produced in a haematopoietic center that floats freely in the coelomic fluid before stored in a particular hemocyte type: the granulocyte type 1. During bacterial challenge, this protein contained in these cells is discharged into the blood stream 3-4 hours after the infection and remains active for approximately 10 hours. This time period blocks progression of the pathogen and its attachment to tissues. CONCLUSION: These results reflect that MPII in conjunction with others partners like lysozyme act as defence molecule for the sand worm.

Protein tyrosine phosphatases in Chaetopterus egg activation.

Changes in protein tyrosine phosphorylation are an essential aspect of egg activation after fertilization. Such changes result from the net contributions of both tyrosine kinases and phosphatases (PTP). This study was conducted to determine what role(s) PTP may have in egg activation. We identified four novel PTP in Chaetopterus pergamentaceus oocytes, cpPTPNT6, cpPTPNT7, cpPTPR2B, and cpPTPR2A, that have significant homology to, respectively, human PTPsigma, -rho, -D2 and -BAS. The first two are cytosolic and the latter two are transmembrane. Several PTP inhibitors were tested to see if they would affect Chaetopterus pergamentaceus fertilization. Eggs treated with beta-bromo-4-hydroxyacetophenone (PTP inhibitor 1) exhibited microvillar elongation, which is a sign of cortical changes resulting from activation. Those treated with Na3VO4 underwent full parthenogenetic activation, including polar body formation and pseudocleavage and did so independently of extracellular Ca2+, which is required for the Ca2+ oscillations that initiate development after fertilization. Fluorescence microscopy identified phosphotyrosine-containing proteins in the cortex and around the nucleus of vanadate-activated eggs, whereas in fertilized eggs they were concentrated only in the cortex. Immunoblots of vanadate-activated and fertilized eggs showed tyrosine hyperphosphorylation of approximately 140 kDa protein. These results suggest that PTP most likely maintain the egg in an inactive state by dephosphorylation of proteins independent of the Ca2+ oscillations in the activation process.

A novel tachykinin-related peptide receptor. Sequence, genomic organization, and functional analysis.

Structurally tachykinin-related peptides have been isolated from various invertebrate species and shown to exhibit their biological activities through a G-protein-coupled receptor (GPCR) for a tachykinin-related peptide. In this paper, we report the identification of a novel tachykinin-related peptide receptor, the urechistachykinin receptor (UTKR) from the echiuroid worm, Urechis unitinctus. The deduced UTKR precursor includes seven transmembrane domains and typical sites for mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors. A functional analysis of the UTKR expressed in Xenopus oocytes demonstrated that UTKR, like tachykinin receptors and tachykinin-related peptide receptors, activates calcium-dependent signal transduction upon binding to its endogenous ligands, urechistachykinins (Uru-TKs) I-V and VII, which were isolated as Urechis tachykinin-related peptides from the nervous tissue of the Urechis unitinctus in our previous study. UTKR responded to all Uru-TKs equivalently, showing that UTKR possesses no selective affinity with Uru-TKs. In contrast, UTKR was not activated by substance P or an Uru-TK analog containing a C-terminal Met-NH2 instead of Arg-NH2. Furthermore, the genomic analysis revealed that the UTKR gene, like mammalian tachykinin receptor genes, consists of five exons interrupted by four introns, and all the intron-inserted positions are completely compatible with those of mammalian tachykinin receptor genes. These results suggest that mammalian tachykinin receptors and invertebrate tachykinin-related peptide receptors were evolved from a common ancestral GPCR gene. This is the first identification of an invertebrate tachykinin-related peptide receptor from other species than insects and also of the genomic structure of a tachykinin-related peptide receptor gene.