Molecular cloning of precursors for TEP-1 and TEP-2: The GGNG peptide-related peptides of a prosobranch gastropod,Thais clavigera

TEP (Thais excitatory peptide)-1 and TEP-2 are molluscan counterparts of annelidan GGNG-peptides, identified in a neogastropod, Thais clavigera (Morishita et al., 2006). We have cloned two cDNAs encoding TEP-1 and TEP-2 precursor protein, respectively, by the standard molecular cloning techniques. Predicted TEP-1 precursor protein consists of 161 amino acids, while predicted TEP-2 precursor protein has 118 amino acids. Only a single copy of TEP was found on the respective precursor. The semi-quantitative RT-PCR showed that expression of TEP-1 was high in sub-esophageal, pleural, pedal and visceral ganglia, while it was low in supra-esophageal ganglion. By contrast, expression level of TEP-2 was high in pedal and visceral ganglia. In situ hybridization visualized different subsets of TEP-1 and TEP-2 expressing neurons in Thais ganglia. For example, supra-esophageal ganglion contained many TEP-2 expressing neuron, but not TEP-1 expressing ones. These results suggest that expression of TEP-1 and TEP-2 is differently regulated in the Thais ganglia.     

A novel GGNG-related neuropeptide from the polychaete Perinereis vancaurica

The GGNG peptides are myoactive peptides so far identified from earthworms and leeches, which are the earthworm excitatory peptides (EEP) and the leech excitatory peptide (LEP), respectively. A novel GGNG peptide was isolated and structurally determined from a marine polychaete, Perinereis vancaurica, using a combination of immunological assay and high performance liquid chromatography (HPLC). The peptide was a pentadecapeptide whose amino acid sequence was similar to that of EEP and LEP, and showed myoactivity on isolated esophagus of P. vancaurica with a threshold concentration of 10(-10)M. The peptide was designated as polychaete excitatory peptide (PEP). Amidation of the alpha-carboxyl group of C-terminal residue occurred in PEP. This is the case for LEP, but not for EEP. The cDNA cloning revealed that the structure of the PEP precursor is more similar to the EEP precursor than to the LEP precursor. Immunohistochemical staining showed the presence of PEP in several neurons of central nervous system (CNS) as somata and neuropile structure, epithelial cells of the pharynx and epidermal cells throughout the body wall. Altogether these results support the physiological significance of PEP in regulation of the CNS neural activity and the peripheral myoactivity.     

The leech excitatory peptide, a member of the GGNG peptide family: isolation and comparison with the earthworm GGNG peptides

A member of the GGNG peptide family was isolated from Hirudo nipponia (leech). GGNG peptides had only been isolated previously from earthworms. The C-terminus structure of the leech peptide, LEP (leech excitatory peptide), was –Gly–Gly–Asn–amide, while that of the earthworm peptides, EEP (earthworm excitatory peptide), was –Gly–Gly–Asn–Gly. LEP exerted 1000-fold more potent activities on leech gut than did EEP-2. On the other hand, EEP-2 was 1000-fold more potent than LEP on the crop-gizzard of the earthworm. Analog peptides of LEP and EEP-2 were synthesized, and the myoactive potency of each analog on the leech and earthworm tissues was compared.     

Comparison of precursor structures of the GGNG peptides derived from the earthworm Eisenia foetida and the leech Hirudo nipponia

Earthworm and leech cDNAs encoding the GGNG peptides, a family of myotropic peptides, were cloned and examined in this study. Both of the predicted precursor proteins are of polyprotein structure and contain several putative peptides distinct from the GGNG peptides. However, the precursors show organizations distinct from each other and no sequence similarity except for the GGNG peptides.     

Localization of leech excitatory peptide, a member of the GGNG peptides, in the central nervous system of a leech (Whitmania pigra) by immunohistochemistry and in situ hybridization

We have recently isolated a myoactive peptide, called leech excitatory peptide, belonging to the GGNG peptide family from two species of leeches, Hirudo nipponia and Whitmania pigra. Immunohistochemistry and in situ hybridization were employed to localize leech excitatory peptide-like peptide(s) and its gene expression in the central nervous system of W. pigra. A pair of neuronal somata were stained by both immunohistochemistry and in situ hybridization in the supraesophageal, subesophageal, and segmental ganglia. In addition, several other neurons showed positive signals by either immunohistochemistry or in situ hybridization in these ganglia. An immunoreactive fiber was observed to run in the anterior root of segmental ganglion 6, which is known to send axons to the sexual organs, though we failed to detect immunoreactivity in possible target tissues. Antiserum specificity was established by enzyme-linked immunosorbent assay using different leech excitatory peptide-related peptides. Leech excitatory peptide elicited muscular contraction of isolated preparations of penis and intestine at concentrations of 10(-8 )M. These results suggest that leech excitatory peptide is a neuropeptide modulating neuromuscular transmission in multiple systems, including regulation of reproductive behavior.     

Peptidomics analysis of neuropeptides involved in copulatory behavior of the mollusk Lymnaea stagnalis

Male copulation behavior in mollusks is controlled by an array of peptide messengers. In the present study, we have used a peptidomics approach employing liquid chromatography in conjunction with electrospray mass spectrometry to characterize peptides contained in the penial complex of the freshwater snail, Lymnaea stagnalis. In addition to the previously described peptides, we have identified a group of novel peptides that share the carboxyl termini of -FVRIamide. A cDNA cloning study revealed the organization of the precursor, which contains 20 peptide domains with the carboxyl termini of -F(X)RIamide which are flanked by many putative proteolytic sites including the KR and the less commonly occurring (G)K and (G)R sites. In addition, there are several monobasic R and dibasic RR and KK sites that may be used for processing. We then used MALDI-TOF/TOF-MS in a data-dependent mode, which selected all the molecular ion species with the predicted masses of the mature -F(X)RIamide peptides, and performed MS/MS analysis on these peptides. This approach allowed us to identify all the predicted -F(X)RIamide peptides. Immunocytochemistry showed the localization of -FVRIamide immunoreactive neurons in several central ganglia, and immunoreactive axons in the penial complex. Finally, application of synthetic -FVRIamide peptides to an in vitro posterior vas deferens preparation showed inhibitory effect on the spontaneous contraction/relaxation cycle of the vas deferens.     

Structural and functional characterization of neuropeptides involved in the control of male mating behavior of Lymnaea stagnalis.

Mating as a male in the simultaneous hermaphrodite freshwater snail, Lymnaea stagnalis, comprises a series of complex behaviors that are a prelude to copulation. Copulatory behavior itself is assumed to be controlled by various types of peptidergic neurons as well as serotonergic cells. Here we report the primary structure of two peptides that were extracted from a cluster of neurons that innervates the penial complex and that is located in the anterior lobe of the right cerebral ganglion. The sequences of the peptides were determined as: Ala-Pro-Gly-Trp-amide and Ser-Gly-Ser-Asp-Tyr-Cys-Glu-Thr-Leu-Lys-Glu-Val-Ala-Asp-Glu-Tyr-Ile-Leu- Leu- Ser-Tyr-Lys-Ile-Glu-Glu-Gln-Arg-Ala-Ala-Asp-Cys-Gly-Gly-Glu-Pro-Pro-Asn- Ser- Gln(amide), respectively. The longer peptide is a homodimer. Both peptides are processed from the recently identified Ala-Pro-Gly-Trp-amide prohormone, which is expressed in the neurons of the anterior lobe of the right cerebral ganglion. Ala-Pro-Gly-Trp-amide could also be recovered from the penial complex. This peptide, when applied in vitro, inhibits the contractions of the penis retractor muscles evoked by serotonin in a dose-dependent fashion.     

New insights into the functional morphology of the male copulatory apparatus of bullid gastropods

In this study, semi-thin sections stained with histochemical techniques and transmission electron microscopy were used to obtain new data about the morphology and function of the male copulatory apparatus of the cephalaspidean gastropod Bulla striata. The apparatus comprises a vestibule, a penial papilla and a prostate consisting of a coiled unbranched tube ending in a blind caecum. The penial papilla and the coiled tubular prostate are enclosed by a muscular sheath, which is continuous with the muscular tissue of the vestibule. The epithelium lining the lumen of the vestibule is formed by ciliated and mucus-secreting cells. Two new types of subepithelial secretory cells were discovered in this region. The penial papilla is a muscular structure without secretory cells in the epithelium lining the narrow lumen. The tubule that constitutes the prostate possesses a muscular wall and can be divided in three distinct regions: a non-secretory duct connected to the penial papilla, a glandular region rich in large secretory cells and the terminal caecum containing just a few small secretory cells. In the terminal blind caecum, the muscular sheath is fused with the muscular wall of the tubular prostate. Large numbers of spermatozoa were found in the glandular region and in the terminal caecum of the prostate. A new functional mechanism is proposed to explain penial eversion during copulation. This differs from a previous hypothesis in two main aspects: (1) existence of a permanent penial papilla in mature animals acting as a functional penis and (2) functional role of vestibule during copulation, which everts and surrounds the penial papilla, while the latter protrudes outwards.     

Molecular Basis for Genetic Resistance of Anopheles gambiae to Plasmodium: Structural Analysis of TEP1 Susceptible and Resistant Alleles

Thioester-containing protein 1 (TEP1) is a central component in the innate immune response of Anopheles gambiae to Plasmodium infection. Two classes of TEP1 alleles, TEP1*S and TEP1*R, are found in both laboratory strains and wild isolates, related by a greater or lesser susceptibility, respectively to both P. berghei and P. falciparum infection. We report the crystal structure of the full-length TEP1*S1 allele which, while similar to the previously determined structure of full-length TEP1*R1, displays flexibility in the N-terminal fragment comprising domains MG1-MG6. Amino acid differences between TEP1*R1 and TEP1*S1 are localized to the TED-MG8 domain interface that protects the thioester bond from hydrolysis and structural changes are apparent at this interface. As a consequence cleaved TEP1*S1 (TEP1*S1_cut) is significantly more susceptible to hydrolysis of its intramolecular thioester bond than TEP1*R1_cut. TEP1*S1_cut is stabilized in solution by the heterodimeric LRIM1/APL1C complex, which preserves the thioester bond within TEP1*S1_cut. These results suggest a mechanism by which selective pressure on the TEP1 gene results in functional variation that may influence the vector competence of A. gambiae towards Plasmodium infection.     

Introduction to Physidae (Gastropoda: Hygrophila); biogeography, classification, morphology

Physidae, a world-wide family of freshwater snails with about 80 species, are reclassified by progressive characters of the penial complex (the terminal male reproductive system): form and composition of penial sheath and preputium, proportions and structure of penis, presence or absence of penial stylet, site of pore of penial canal, and number and insertions of penial retractor muscles. Observation of these characters, many not recognized previously, has been possible only by the technique used in anesthetizing, fixing, and preserving. These progressive characters are the principal basis of 23 genera, four grades and four clades within the family. The two established subfamilies are divided into seven new tribes including 11 new genera, with diagnoses and lists of species referred to each. Proposed as new are: in Aplexinae, Austrinautini, with Austrinauta g.n. and Caribnautu harryi g.n., nom.nov.; Aplexini; Amecanautini with Amecanauta jaliscoensis g.n., sp.n., Mexinauta g.n., and Mayabina g.n., with M. petenensis, polita, sanctijohannis, tempisquensis spp.nn., Tropinauta sinusdulcensis g.n., sp.n.; and Stenophysini, with Stenophysa spathidophallus sp.n.; in Physinae, Haitiini, with Haitia moreleti sp.n.; Physini, with Laurentiphysa chippevarum g.n., sp.n., Physa mirollii nom.nou.; and Physellini, with Chiapaphysa g.n., and C. grijalvae, C. pacifica spp.nn., Utahphysa g.n., Archiphysa g.n., with A. ashmuni, A. sonomae spp.nn., Physella hemphilli sp.n., and Ultraphysella sinaloae g.n., sp.n. The simplest reproductive system is found in Austrinauta of the Aplexinae; its penial complex approaches that in the related family Lymnaeidae. Within Physinae a close approximation is found in Haitia. By these two genera the two subfamilies are drawn close together. Four grades of progressive complexity are recognized: (I) penial sheath entirely muscular; (II) penial sheath with both glandular and muscular tissue; (III) penis with penial stylet or other specialization of the tip of the penis; and (IV) pore of penial canal lateral rather than terminal as in the lower grades. In both subfamilies there are clades with glandular tissue in the penial sheath, a penial sheath subdivided into two parts, and tip of penis specialized in various ways. These clades are formalized as new tribes. Of 23 genera of Physidae, 17 occur in Pacific drainages of North and Central America, eight of these restricted to the region. Concentration of primitive genera along the Pacific coast from Mexico to Costa Rica conforms to previous observations that primitive pulmonate families are concentrated within, or along the continental margins of, the Pacific Ocean. An ancestral origin of Physidae along an ancient eastern Pacific coast is probable. From this region the several lineages have spread north, south and east in the Americas, and through Siberia to Europe. Although Physinae have fewer genera than Aplexinae (11 v. 12), they have more species (47 v. 34). Greater land area in the temperate zone has provided more opportunity for speciation of Physinae, in contrast to the generally tropical and warm-temperate range of Aplexinae. Furthermore, 10 species of Physinae are localized in individual lakes, whereas Aplexinae are not lake-dwellers. Both well-developed egg strings and capsular strings are found in the spawn of Sibirenauta elongatus. These structures have been known in Lymnaeidae, but not hitherto in Physidae; they are a link with some marine groups, such as Siphonariidae. Spiral color bands and white streaks in the shell of Mexinauta recall those in Lancidae (Lymnaeacea), whereas the radula of Physidae is like that of Chilinidae. Physidae thus show affinities to various basal stocks of aquatic pulmonates; no clear-cut sister-group can be recognized. Most species have a restricted range; out of 55 with sufficiently detailed information for analysis, 25 are limited to a single 1 degrees x 1 degrees quadrangle. Only a few species are widespread, on one or even two continents. Accordingly, more species of Physidae are threatened by habitat destruction than in other families of Hygrophila with generally wider distributions. Other features are a key to genera; catalog of more than 430 names applied to living Physidae, with original reference, type locality, and location of type specimens; summary of museums with types; and glossary.     

Structure-function analysis of the Anopheles gambiae LRIM1/APL1C complex and its interaction with complement C3-like protein TEP1

Malaria threatens half the world's population and exacts a devastating human toll. The principal malaria vector in Africa, the mosquito Anopheles gambiae, encodes 24 members of a recently identified family of leucine-rich repeat proteins named LRIMs. Two members of this family, LRIM1 and APL1C, are crucial components of the mosquito complement-like pathway that is important for immune defense against Plasmodium parasites. LRIM1 and APL1C circulate in the hemolymph exclusively as a disulfide-bonded complex that specifically interacts with the mature form of the complement C3-like protein, TEP1. We have investigated the specificity of LRIM1/APL1C complex formation and which regions of these proteins are required for interactions with TEP1. To address these questions, we have generated a set of LRIM1 and APL1C alleles altering key conserved structural elements and assayed them in cell culture for complex formation and interaction with TEP1. Our data indicate that heterocomplex formation is an intrinsic ability of LRIM1 and APL1C and identify key homologous cysteine residues forming the intermolecular disulfide bond. We also demonstrate that the coiled-coil domain is the binding site for TEP1 but also contributes to the specificity of LRIM1/APL1C complex formation. In addition, we show that the LRIM1/APL1C complex interacts with the mature forms of three other TEP proteins, one of which, TEP3, we have characterized as a Plasmodium antagonist. We conclude that LRIM1 and APL1C contain three distinct modules: a C-terminal coiled-coil domain that can carry different TEP protein cargoes, potentially with distinct functions, a central cysteine-rich region that controls complex formation and an N-terminal leucine-rich repeat with a putative role in pathogen recognition.     

Functional morphology of the mammiliform penial glands ofLittorina saxatilis (Gastropoda)

Summary The functional morphology of the mammiliform penial glands ofLittorina saxatilis has been investigated with both light and electron microscopy. These penial glands line the ventral edge of the penis and orient with the female mantle during copulation. Secretions are released from the penial glands to this interface where they probably function in adhesion. The penial gland secretions comprise heterogeneous granules as well as apocrine and mucous secretions. The heterogeneous granules are produced in separate multicellular glands arranged in a series of lobes that lie outside a thick smooth muscle layer enclosing the lumen. Each glandular lobe is surrounded by a thin layer of smooth muscle. Secretions are transported in individual cellular processes that pass through the thick smooth muscle layer and empty into the lumen. Surrounding the lumen is an epithelium containing apocrine secretory cells as well as occasional goblet-type, mucous cells. The combined action of the muscles forces secretions out of the lumen through the penial papilla, onto the external surface of the mammiliform penial gland. Longitudinal muscles extend into the penial papilla enabling its protrusion or retraction. Retraction of the penial papilla following secretion release is thought to create negative pressure beneath the penial gland producing suction adhesion. The visco-elastic properties of the penial gland secretion are qualitatively different from foot mucus and may represent specialization to an adhesive function.     

Structure-activity relationships of 18 endogenous neuropeptides on the motor nervous system of the nematode Ascaris suum

Neuropeptides play an important role in all nervous systems and structure-activity studies of related peptides is one approach to understanding this role. This study of the motor nervous system of the parasitic nematode Ascaris suum describes the physiological effects of a family of 18 endogenous Ascaris FMRFamide-like peptides (AF peptides) on the membrane potential and input resistance of the dorsal excitatory type 2 (DE2) and dorsal inhibitory (DI) motor neurons. These motor neurons are part of the final common output pathway from the motor nervous system to the somatic muscle cells responsible for locomotion. AF peptide effects on the frequency of excitatory postsynaptic potentials (EPSPs) in DE2 motor neurons were also measured to infer peptide effects on central presynaptic spiking neurons. AF peptide injections into intact worms were made to assess their qualitative effects on behavior, providing a context for interpreting motor neuron data. One category of AF peptides, N-terminally extended -FIRFa peptides (AF5, AF7 and AF1), has pronounced behavioral effects and qualitatively similar, but quantitatively different effects on DE2 and DI motor neurons. A second category of AF peptides (AF2, AF9, and AF8) also produces dramatic behavioral effects and strong electrophysiological effects on DE2 and/or DI motor neurons. A third category of AF peptides, consisting of six members of the -PGVLRFa group (which are encoded by the same gene and have closely related sequences) and peptide AF11, have pronounced behavioral effects, but relatively weak or negligible effects on DE2 and DI motor neurons. A fourth category of AF peptides, also consisting of structurally unrelated members, has pronounced behavioral effects and, as individual peptides, similar effects on both DE2 and DI motor neurons; AF15 is excitatory, while AF17 and AF19 are inhibitory, on both motor neuron types. Finally, two AF peptides (AF6, AF16) are relatively weak or inactive in producing behavioral or motor neuronal effects. Based on comparisons of the effects of AF peptides on DE2 and DI motor neurons, a tentative list of 5 major response-types is proposed as a working hypothesis to guide the search for AF peptide receptors. The findings attest to the potential complexity of neurosignaling in this comparatively simple nervous system.     

Contribution of transparent exopolymer particles to carbon sinking flux in an oligotrophic reservoir

Transparent exopolymer particles (TEP) compose an important pool of particulate organic matter (POM) in aquatic systems. However, no studies of TEP contribution to C export to sediment exist for freshwaters. We quantify the contribution of TEP to C sinking fluxes in an oligotrophic reservoir (Quéntar, Southern Spain) by monitoring TEP in the water column and TEP, particulate organic carbon (POC) and dry weight in sedimentation traps. TEP sinking fluxes ranged from 0.73 to 183.23 mg C m^?2 day^?1 and from 0.51 to 177.04 mg C m^?2 day^?1 at the surface and at the bottom layer, respectively. These values represent that, over an annual basis, 5.59 Ton TEP-C (over 61.32 Ton POC) are exported, on an average, from the water column to the sediment of Quentar reservoir. TEP concentrations (average = 48.0 μg XG eq l^?1) were lower than the scarce data reported for freshwaters. No significant relationships between TEP and Chl a concentrations or BA were observed. Average value for daily sedimentation flux (6.63 g Dry Weight m^?2 day^?1) in the study reservoir was higher than that documented for low productive natural aquatic ecosystems as a consequence of the high amount of allochthonous material input characterizing reservoirs. TEP contributed to C export to sediment with a value that range from 0.02 to 31%. Our results show that even in man-made systems, which are predominantly controlled by allochthonous inputs, TEP may be relevant for explaining POM settling fluxes.     

The p80 homology region of TEP1 is sufficient for its association with the telomerase and vault RNAs, and the vault particle

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1–871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1–RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1–RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1–RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding.     

The CLIP-Domain Serine Protease Homolog SPCLIP1 Regulates Complement Recruitment to Microbial Surfaces in the Malaria Mosquito Anopheles gambiae

The complement C3-like protein TEP1 of the mosquito Anopheles gambiae is required for defense against malaria parasites and bacteria. Two forms of TEP1 are present in the mosquito hemolymph, the full-length TEP1-F and the proteolytically processed TEP1_cut that is part of a complex including the leucine-rich repeat proteins LRIM1 and APL1C. Here we show that the non-catalytic serine protease SPCLIP1 is a key regulator of the complement-like pathway. SPCLIP1 is required for accumulation of TEP1 on microbial surfaces, a reaction that leads to lysis of malaria parasites or triggers activation of a cascade culminating with melanization of malaria parasites and bacteria. We also demonstrate that the two forms of TEP1 have distinct roles in the complement-like pathway and provide the first evidence for a complement convertase-like cascade in insects analogous to that in vertebrates. Our findings establish that core principles of complement activation are conserved throughout the evolution of animals.     

Effects of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B26-34), and the 8-amino acid alpha-bag cell peptide (alpha-BCP1-8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both alpha-BCP1-8 and B26-34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells is mimicked by alpha-BCP1-8. The excitatory effect of the atrial gland extract cannot be duplicated with B26-34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B26-34 seems to mimick alpha-BCP1-8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.     

Spatial distribution of transparent exopolymer particles in the Bransfield Strait, Antarctica

Transparent exopolymer particles (TEP) are recognized to playan important role in the flux of exported carbon to the deepocean. However, there is little information on how TEP standingstocks are affected by different hydrographic conditions andother relevant ecological factors in situ. This lack of knowledgeis particularly serious for the Southern Ocean. During Australsummer 1999, the Strait of Bransfield presented high mesoscalevariability. Two fronts were present, the Bransfield hydrographicfront and a slope front along the South Shetland Islands andseveral mesoscale anticyclonic eddies and/or frontal meanders.The spatial distributions of biological properties were largelyaffected by this complex hydrography. Chlorophyll a (Chl a)(0.05–4.81 µg L^–1), TEP (from undetectableto 346 µg GXeq L^–1) and heterotrophic bacteria (HB)(1.7–9.4 x 10⁵ cells mL^–1) were positively correlateddespite the wide hydrographic heterogeneity of the BransfieldStrait. Higher abundances of autotrophic biomass, and correspondlyhigher TEP and heterotrophic bacteria (HB), were found in themore stratified waters. TEP spatial distribution was mostlyrelated to the abundance of autotrophic biomass although localhigh TEP concentrations were not matched by similarly high valuesof Chl a in some areas where diatoms were relatively abundant.     

The Telomerase/vault-associated protein TEP1 is required for vault RNA stability and its association with the vault particle

Vaults and telomerase are ribonucleoprotein (RNP) particles that share a common protein subunit, TEP1. Although its role in either complex has not yet been defined, TEP1 has been shown to interact with the mouse telomerase RNA and with several of the human vault RNAs in a yeast three-hybrid assay. An mTep1(-/-) mouse was previously generated which resulted in no apparent change in telomere length or telomerase activity in six generations of mTep1-deficient mice. Here we show that the levels of the telomerase RNA and its association with the telomerase RNP are also unaffected in mTep1(-/-) mice. Although vaults purified from the livers of mTep1(-/-) mice appear structurally intact by both negative stain and cryoelectron microscopy, three-dimensional reconstruction of the mTep1(-/-) vault revealed less density in the cap than previously observed for the intact rat vault. Furthermore, the absence of TEP1 completely disrupted the stable association of the vault RNA with the purified vault particle and also resulted in a decrease in the levels and stability of the vault RNA. Therefore, we have uncovered a novel role for TEP1 in vivo as an integral vault protein important for the stabilization and recruitment of the vault RNA to the vault particle.