A dense core vesicle protein is restricted to the cortex of granules in the exocrine atrial gland of Aplysia california

We have generated a monoclonal antibody (mAb) 5E10 which recognizes an antigen localized to dense core vesicles (DCVs) in the atrial gland of Aplysia californica. mAb5E10 immunoprecipitates an abundant 57-kDa glycoprotein (atrial gland granule-specific antigen, AGSA) which is a soluble component of atrial gland DCVs. Electron microscopy reveals that AGSA immunoreactivity is restricted to the region between the dense core, which contains neuropeptide immunoreactivity, and the membrane of atrial gland DCVs. AGSA was purified by immunoaffinity chromatography, and the amino acid sequences of both N-terminal and internal cyanogen bromide fragments were determined. This information was used to isolate a 2.8-kilobase cDNA which encodes a 47-kDa protein. The predicted amino acid sequence contains the micro-sequenced peptides, an N-terminal hydrophobic signal sequence, and four N-linked glycosylation sites, but does not contain any significant homologies to database sequences. Northern blots and light level immunocytochemistry demonstrate that the AGSA gene is specifically expressed in the atrial gland. The identification of a protein localized to the cortex of DCVs suggests that this region has a specialized role in the function of these vesicles.     

Ultrastructural localization of egg-laying prohormone-related peptides in the atrial gland of Aplysia californica

The atrial gland is an exocrine organ that secretes into the oviduct of Aplysia californica and expresses three homologous genes belonging to the egglaying hormone gene family. Although post-translational processing of the egg-laying hormone precursor in the neuroendocrine bag cells has been examined in detail, relatively little is known about the post-translational processing of egg-laying hormone-related gene products in the atrial gland. A combination of morphologic techniques that included light-microscopic histology and immunocytochemistry, transmission electron microscopy, and immuno-electron microscopy were used to localize egg-laying hormone-related peptides in the atrial gland and to evaluate the characteristic morphology of their secretory cells. Results of these studies showed that there were at least three major types of secretory cells in the atrial gland (types 1–3). Significantly, of these three cell types, only type 1 was immunoreactive to antisera against egg-laying hormone-related precursor peptides. The immunoreactivity studies established that all three egg-laying hormone-related precursor genes are expressed in type-1 cells and indicated that the processing of these precursors also occurs within the secretory granules of this cell type. Evidence was also obtained that proteolytic processing of the egg-laying hormone-related precursors differed significantly from that observed in the bag cells. In contrast to the bag cells, the NH₂-terminal and COOH-terminal products of the egg-laying hormone-related precursors of the atrial gland were not sorted into different types of vesicles.     

Localization of Aplysia neurosecretory peptides to multiple populations of dense core vesicles

Many neurons in the mollusc Aplysia are identifiable and provide a useful model system for investigating the cellular mechanisms used by the neuroendocrine system to mediate simple behaviors. In this study we determined the subcellular localization of eight Aplysia neuropeptides using immunogold labeling techniques, and analyzed the size distribution of dense core and granular vesicles in peptidergic neurons. Recent observations demonstrate that many neurons use multiple chemical messengers. Thus, an understanding of the functional significance of cotransmitters requires an analysis of their relative subcellular distributions. The peptides are expressed in a subset of neurons, or the exocrine atrial gland, and are primarily localized to dense core vesicles. Multiple regions of precursors which are cleaved into several components are co-localized. Each neuron has a distinct size distribution of peptide-containing dense core vesicles ranging in size from 65 to 600 nm. The atrial gland contains very large (up to 2 micron) peptide-containing granules. Single neurons have multiple populations of granules whose quantal sizes agree with predictions based on physical constraints. Some cells contain very large peptide-containing granules which are found in the cell soma and not in processes. Thus, the genetic determination of neuronal cell type includes not only transmitter choices but also multiple modes of packaging the intercellular messengers.     

Calsyntenins are secretory granule proteins in anterior pituitary gland and pancreatic islet alpha cells.

Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function.     

Peptide products of the atrial gland are not water-borne reproductive pheromones during egg laying in Aplysia

Mate attraction in Aplysia involves long-distance water-borne signaling via the secretion of the peptide pheromone attractin from the exocrine albumen gland during egg laying. Previous studies have shown that a second exocrine organ, the atrial gland, produces abundant egg-laying hormone (ELH) precursor-related peptides and mollusk-derived growth factor (MDGF), and crude extracts of the atrial gland are attractive in T-maze attraction assays. However, it is not known whether these peptides and proteins are secreted during egg laying. In this report, seawater eluates of freshly laid egg cordons were concentrated and fractionated by C18 RP-HPLC, and the resulting major peaks were examined by amino acid compositional analysis, microsequence analysis, and electrospray mass spectrometry. Concentrated egg cordon eluates were also examined by immunoblot analysis using anti-MDGF antisera as probe. The combined data demonstrated that the atrial gland of Aplysia californica does not secrete detectable levels of either ELH precursor-related peptides or MDGF during egg laying. Although the atrial gland is the last major exocrine organ to make contact with eggs before they are laid, the gland does not appear to secrete water-borne peptide pheromones during egg laying.     

1-Deoxy-1-phosphatidylethanolamino-lactitol-type neoglycolipids serve as acceptors for sialyltransferases from rat liver golgi vesicles.

1-Deoxy-1-phosphatidylethanolamino-lactitols (LacPtdEtns), 1-deoxy-1-phosphatidylethanolamino-sialyllactitols (NeuAcLacPtdEtns) and their corresponding N-acetylated derivatives were synthesized and characterized by fast-atom-bombardment mass spectrometry (FAB MS). The neoglycolipids were used as acceptors for sialyltransferases from rat liver Golgi vesicles. Sialylation rates were as good as or even better than those obtained with the corresponding authentic acceptors lactosylceramide (LacCer) and ganglioside GM3. The sialylation of LacPtdEtns and NeuAcLacPtdEtns yielded sialyl and disialyl compounds, respectively, as shown by FAB MS analysis of the reaction products. The results of competition experiments indicate that the neoglycolipids and the authentic acceptors are sialylated by the same sialyltransferases.     

Ontogeny of atrial natriuretic peptide receptors in fetal rat kidney and adrenal gland

Summary With in vitro autoradiography, specific receptors for atrial natriuretic peptide (ANP) were localized in fetal rat kidney and adrenal glands. Receptors were present over renal vesicles, in the primitive renal medulla and throughout the adrenal gland as early as 16 days gestation. By 20 days gestation, several layers of developing renal corpuscles were present and ANP receptors were localized over developing glomeruli in each layer. Larger accumulations occurred over the juxtamedullary glomeruli. In the medulla, the receptors were localized in a reticular pattern near the pelivis. With emulsion coated sections, ANP receptors in developing renal corpuscles were seen primarily over the lower curve of S-shaped vesicles and around the periphery of the more mature corpuscles. In the renal medulla, receptors were localized over the interstitial cells. In the 16-day-old adrenal gland, ANP receptors were present throughout the cortical area but at 20 days gestation and 1 day postpartum receptors appeared more numerous in the peripheral region. these data suggest that ANP has important developmental effects in the kidney and adrenal gland and may be involved in regulation of body fluid homeostasis in the late gestation rat fetus.     

Ontogeny of atrial natriuretic peptide receptors in fetal rat kidney and adrenal gland

With in vitro autoradiography, specific receptors for atrial natriuretic peptide (ANP) were localized in fetal rat kidney and adrenal glands. Receptors were present over renal vesicles, in the primitive renal medulla and throughout the adrenal gland as early as 16 days gestation. By 20 days gestation, several layers of developing renal corpuscles were present and ANP receptors were localized over developing glomeruli in each layer. Larger accumulations occurred over the juxtamedullary glomeruli. In the medulla, the receptors were localized in a reticular pattern near the pelvis. With emulsion coated sections, ANP receptors in developing renal corpuscles were seen primarily over the lower curve of S-shaped vesicles and around the periphery of the more mature corpuscles. In the renal medulla, receptors were localized over the interstitial cells. In the 16-day-old adrenal gland, ANP receptors were present throughout the cortical area but at 20 days gestation and 1 day postpartum receptors appeared more numerous in the peripheral region. These data suggest that ANP has important developmental effects in the kidney and adrenal gland and may be involved in regulation of body fluid homeostasis in the late gestation rat fetus.     

The anatomy and functional morphology of the large hermaphroditic duct of three species of Aplysia, with special reference to the atrial gland

The anatomy and functional morphology of the large hermaphroditic duct of three species of gastropod mollusc (Aplysia californica, A. dactylomela, and A. brasiliana) were examined. Each duct is composed of two parallel compartments, the red hemiduct (RHD) and the white hemiduct (WHD), which are distinguishable from the outside of the duct. Four secretory regions, all exocrine in morphology, are recognizable: the RHD secretory epithelium, the atrial gland (or atrial gland-like epithelium), the WHD secretory epithelium, and the accessory gland of the copulatory duct (AGCD). Of these regions, only the atrial gland (or atrial gland-like epithelium) contains egglaying activity and only the atrial gland (or atrial gland-like epithelium) is immunocytochemically labeled by serum antibodies generated against low molecular weight A. californica atrial gland peptides. The RHD is the functional oviduct: the egg cordon passes through a channel lined by the RHD secretory epithelium and bordered by the atrial gland (or atrial gland-like epithelium); the eggs are separated from both the WHD secretory epithelium and the AGCD by internal folds of the duct. The WHD is the functional copulatory duct: the penis, exogenous sperm, and endogenous sperm pass directly by the AGCD and in close proximity to the WHD secretory epithelium; they are separated from both the RHD secretory epithelium and the atrial gland (or atrial gland-like epithelium) by internal folds. The atrial gland (or atrial gland-like epithelium) is thus not likely to have a prostatic function or to be directly stimulated by the penis during copulation; it may play a role in oviductal function.     

Osteoprotegerin in Exosome-Like Vesicles from Human Cultured Tubular Cells and Urine

Urinary exosomes have been proposed as potential diagnostic tools. TNF superfamily cytokines and receptors may be present in exosomes and are expressed by proximal tubular cells. We have now studied the expression of selected TNF superfamily proteins in exosome-like vesicles from cultured human proximal tubular cells and human urine and have identified additional proteins in these vesicles by LC-MS/MS proteomics. Human proximal tubular cells constitutively released exosome-like vesicles that did not contain the TNF superfamily cytokines TRAIL or TWEAK. However, exosome-like vesicles contained osteoprotegerin (OPG), a TNF receptor superfamily protein, as assessed by Western blot, ELISA or selected reaction monitoring by nLC-(QQQ)MS/MS. Twenty-one additional proteins were identified in tubular cell exosome-like vesicles, including one (vitamin D binding protein) that had not been previously reported in exosome-like vesicles. Twelve were extracellular matrix proteins, including the basement membrane proteins type IV collagen, nidogen-1, agrin and fibulin-1. Urine from chronic kidney disease patients contained a higher amount of exosomal protein and exosomal OPG than urine from healthy volunteers. Specifically OPG was increased in autosomal dominant polycystic kidney disease urinary exosome-like vesicles and expressed by cystic epithelium in vivo. In conclusion, OPG is present in exosome-like vesicles secreted by proximal tubular epithelial cells and isolated from Chronic Kidney Disease urine.     

Size and Surface Charge Properties of Myelin Vesicles from Normal and Diseased (Multiple Sclerosis) Brain

Differences have been observed between myelin vesicles prepared from normal human central nervous system and from white matter of patients who died with multiple sclerosis (MS). The mean cross-sectional area of the vesicles was 5.69 +/- 0.17 micron 2 from normal myelin and 3.71 +/- 0.28 micron 2 for diseased myelin. Vesicle size was reduced to 4.08 +/- 0.21 micron 2 when normal myelin vesicles were prepared in the presence of 0.1 mM EDTA. The presence of Ca2+ during the preparation of the vesicles had no effect on the mean cross-sectional area. In the case of MS myelin vesicles, 0.1 mM EDTA had no effect on vesicle size, whereas the presence of Ca2+ increased the vesicle size from 3.71 +/- 0.28 to 5.40 +/- 0.31 micron 2. Electrokinetic analysis revealed that the electrophoretic mobility of normal myelin vesicles was -5.169 +/- 0.193 X 10(-8) compared with -6.093 +/- 0.202 X 10(-8) m2 s-1 V-1 for the MS myelin vesicles. The presence of 0.1 mM EDTA increased the electrophoretic mobility of the normal vesicles to -6.483 +/- 0.151 X 10(-8) m2 s-1 V-1 but did not significantly affect that of the MS vesicles. Addition of 0.1 mM Ca2+ decreased the electrophoretic mobility of both normal and MS vesicles to similar mobilities. From these data, the surface charge densities were calculated for both normal and MS myelin vesicles and found to be -2.93 and -5.39 mV m-1, respectively. The phase transition temperature determined by wide-angle x-ray diffraction studies was 63 degrees C for normal myelin vesicles and 43 degrees C for MS myelin vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opaline gland ultrastructure in Aplysia californica (Gastropoda: Anaspidea)

Secretions released from the ink and opaline glands of Aplysiacalifornica protect this shell-less mollusc from predators inseveral ways; the most recently discovered, phagomimicry, stimulatesthe feeding behaviours of the predator, distracting it fromthe sea hare. The structure of the ink gland has been reported,but little is known about the opaline gland. This paper comparesthe structure of the opaline gland of A. californica with thatof its ink gland, as well as two additional vesicle types foundin the epidermis. The opaline gland consists of single largecells, the vesicle cells, each with an enlarged nucleus, themaximum size of both exceeding that of respective structuresin the ink gland. Opaline vesicles, like ink vesicles, are enclosedby an external layer of muscle. Opaline vesicles, unlike inkvesicles, are not immersed in additional cells, but are freewithin the haemolymph and are, therefore, the probable sitefor the synthesis of their protein contents. The necks of individualopaline vesicles are fused into a central canal, but short necksconnecting each vesicle to the central canal remain; these arefilled with epithelial cells, but lack a muscular release valvelike that in the long necks of ink vesicles. Mucous cells containcircular arrays and are structurally distinct from opaline vesicles;mucous cells, though enlarged, are smaller than opaline or inkvesicle cells; they lack an external layer of muscle and a multicellularneck and, therefore, more closely match another vesicle typein the skin of A. californica, the white vesicle, which is involvedwith excess calcium excretion. (Received 22 September 2006; accepted 20 March 2007)     

Identification of novel Cry1Ac binding proteins in midgut membranes from Heliothis virescens using proteomic analyses

Proteins such as aminopeptidases and alkaline phosphatases, both glycosyl-phosphatidyl-inositol (GPI) anchored proteins, were previously identified as Cry1Ac binding proteins in the Heliothis virescens midgut. To identify additional toxin binding proteins, brush border membrane vesicles from H. virescens larvae were treated with phosphatidyl inositol phospholipase C, and released proteins were resolved by two-dimensional electrophoresis. Protein spots selected by their ability to bind Cry1Ac were identified by MALDI-TOF mass spectrometry coupled to peptide mass fingerprinting (PMF) and database searching. As in previous studies, H. virescens alkaline phosphatase was identified as a Cry1Ac binding protein. V-ATP synthase subunit A and actin were identified as novel Cry1Ac binding proteins in H. virescens. Additional toxin-binding proteins were predicted based on MS/MS fragmentation and de novo sequencing, providing amino acid sequences that were used in database searches to identify a phosphatase and a putative protein of the cadherin superfamily as additional Cry1Ac binding proteins.     

Comparative Cytology and Morphology of Seminal Vesicles in male gobiid fishes

Anatomical, histological and EM studies are presented on the male seminal vesicles of 111 species of gobies (Gobiidae, Teleostei). These vesicles, attached to the sperm-ducts, are lined with an excretory epithelium composed of three types of cells: 1) columnar cells with giant Golgi cisterns in the form of large rings apical to the nucleus; 2) excretory cells with remarkably interdigitative basal lamina, that resemble sperm-duct cells; and 3) groups of interstitial, Leydig-type cells that possibly form a part of the mesorchial gland. Morphologically the vesicles appear to be taxa specific, the simplest one in the form of a few tubules on the sperm duct, and the most elaborate one forming large, wing-like structures, with an elaborate mass of passages. In many species the final form of the vesicles develops during the fish ontogenesis; in others, the form of this organ does not change during the lifetime of the fish. With the onset of reproduction, secretion and sperm accumulate in the tubule of the seminal vesicle and are expelled from there onto the eggs during fertilization. The comparative morphology, as well as possible ecological and physiological role of this secretion is discussed.     

Morphometric studies of the parotid gland of rats after bilateral gonadectomy and after gonadectomy and administration of mesterone

The influence of bilateral gonadectomy on the secretory segments of the partoid gland in male rats was investigated. The changes in the section area of cell nuclei, secretory cells, serous vesicles as well as in the mean number of cells in the vesicles were statistically analyzed. Eleven-day gonadectomy caused a statistically significant increase of the mean section area of cell nuclei, secretory vesicles and cells. Eight-day mesterone treatment in the dose of 2 mg/kg of body mass does not eliminate the changes caused by gonadectomy but results in the further increase of the mean section area of cell nuclei and secretory vesicles, as well as in the increase of cells in the vesicles.     

Absorption of ferritin by cells of the digestive gland in Nassarius tegula (Gastropoda : Prosobranchia)

Within 12 min after the prosobranch snail, Nassarius, begins feeding on ferritin-labeled food, ferritin reaches the lumen of the digestive gland and is absorbed by the digestive cells lining the gland. Within the digestive cells, the ferritin is present in coated pinocytotic vesicles, in microvesicles and in macrovesicles. It is probable that ferritin (and components of the food as well) progresses rapidly in order from the gland lumen to the pinocytotic vesicles to the microvesicles to the macrovesicles. The macro vesicles are presumably an important site of intracellular digestion.     

Source of the host marking pheromone in the egg parasitoid Trissolcus basalis (Hymenoptera: Scelionidae)

After oviposition, Trissolcus basalis females always mark the host's surface, depositing host marking substances for herself and to warn other ovipositing females. The perception of these host marking substances, probably through the antennae, can induce the female to leave and seek healthy hosts. Parasitoid females exposed to conspecific parasitized egg masses left the host egg masses significantly more often than when exposed to non-parasitized egg masses. More egg mass leaving behavior also was observed when the egg masses were treated with Dufour's gland secretion but not when treated with secretion from the common oviducts. The common oviduct has a secretory epithelium that produces electron-dense vesicles, probably containing proteinaceous substances. The secretory cells of the accessory gland, Dufour's gland, contain electron-lucid vesicles, whose secretion appears to be a lipid similarly to that found in pheromone secreting glands. Ultrastructural and behavioral evidence suggests that Dufour's gland is the host marking pheromone source.     

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.     

The synaptic vesicle proteome

Synaptic vesicles are key organelles in neurotransmission. Vesicle integral or membrane-associated proteins mediate the various functions the organelle fulfills during its life cycle. These include organelle transport, interaction with the nerve terminal cytoskeleton, uptake and storage of low molecular weight constituents, and the regulated interaction with the pre-synaptic plasma membrane during exo- and endocytosis. Within the past two decades, converging work from several laboratories resulted in the molecular and functional characterization of the proteinaceous inventory of the synaptic vesicle compartment. However, up until recently and due to technical difficulties, it was impossible to screen the entire organelle thoroughly. Recent advances in membrane protein identification and mass spectrometry (MS) have dramatically promoted this field. A comparison of different techniques for elucidating the proteinaceous composition of synaptic vesicles revealed numerous overlaps but also remarkable differences in the protein constituents of the synaptic vesicle compartment, indicating that several protein separation techniques in combination with differing MS approaches are required to identify and characterize the synaptic vesicle proteome. This review highlights the power of various gel separation techniques and MS analyses for the characterization of the proteome of highly purified synaptic vesicles. Furthermore, the newly detected protein assignments to synaptic vesicles, especially those proteins which are new to the inventory of the synaptic vesicle proteome, are critically discussed.