Synthesis Of Vesicles Research Articles

Water-Dispersible Oil-Filled ABC Triblock Copolymer Vesicles and Nanocapsules

Customer-engineered block copolymers can self-assemble under appropriate conditions in block-selective solvents into vesicles. Such vesicles have been of enormous academic interest over the past decade for their potential applications in enzyme encapsulation as well as in controlled release of drugs, fragrance, fertilizer, etc. A drawback with the self-assembled vesicles is that the solvent external to and in the internal cavity of a vesicle is essentially the same. This makes loading of reagents into the vesicle cavities difficult. Reported in this paper is a methodology for the one-pot synthesis of water-dispersible oil-filled ABC triblock copolymer vesicles or nanocapsules. Such a methodology achieves reagent (oil) filling and vesicle preparation simultaneously and should help commercial acceptance of block copolymer vesicles.

A Singular Bacteriophytochrome Acquired by Lateral Gene Transfer

Bacteriophytochromes are phytochrome-like proteins that mediate photosensory responses in various bacteria according to their light environment. The genome of the photosynthetic and plant-symbiotic Bradyrhizobium sp. strain ORS278 revealed the presence of a genomic island acquired by lateral transfer harboring a bacteriophytochrome gene, BrBphP3.ORS278, and genes involved in the synthesis of phycocyanobilin and gas vesicles. The corresponding protein BrBphP3.ORS278 is phylogenetically distant from the other (bacterio)phytochromes described thus far and displays a series of unusual properties. It binds phycocyanobilin as a chromophore, a unique feature for a bacteriophytochrome. Moreover, its C-terminal region is short and displays no homology with any known functional domain. Its dark-adapted state absorbs maximally around 610 nm, an unusually short wavelength for (bacterio)phytochromes. This form is designated as Po for orange-absorbing form. Upon illumination, a photo-reversible switch occurs between the Po form and a red (670 nm)-absorbing form (Pr), which rapidly backreacts in the dark. Because of this instability, illumination results in a mixture of the Po and Pr states in proportions that depend on the intensity. These uncommon features suggest that BrBphP3.ORS278 could be fitted to measure light intensity rather than color.

Frankia alni proteome under nitrogen‐fixing and nitrogen‐replete conditions

Frankia alni induces root nodules on Alnus, in which the bacterium differentiates into nitrogen (N)‐fixing cells called vesicles. In culture, F. alni also undergoes major morphological changes as it alternates between N‐replete and N‐fixing conditions. Lack of biologically available N induces the synthesis of vesicles in which nitrogenase is protected from molecular oxygen by a thick lipid hopanoid envelope. Very little is known about the molecular basis of Frankia–host interaction as well as Frankia cell differentiation. The recent determination of the complete genome sequence of F. alni strain ACN14a has permitted us to characterize its proteome, particularly in the extracellular compartment, which could be involved in Frankia–host interaction, and in the switch from N‐replete to N‐fixing conditions. To that end, 126 bacterial proteins were analyzed by two‐dimensional protein gel electrophoresis and identified by matrix‐assisted laser desorption/ionization time of flight fingerprinting using a F. alni proteome database. Interestingly, the extracellular fraction contains some glycolytic enzymes lacking secretion signals, already reported to be extracellularly localized in some streptococci, as well as some abundant stress‐resistance proteins. As expected, several proteins involved in N assimilation and oxidative defense system were upregulated in F. alni grown under N‐fixing vs N‐replete conditions. Furthermore, two Raf kinase inhibitor protein homologs that could play a role in cellular signaling, and a hemoglobin‐like protein HbN that could be involved in detoxification of nitric oxide were also upregulated. More surprising, a succinate dehydrogenase was strongly downregulated, which could be linked to the need of pyruvate for the biosynthesis of hopanoids or to reduced oxygen diffusion in vesicles.

Multicompartment micelles and vesicles from π-shaped ABC block copolymers: a dissipative particle dynamics study

Dissipative particle dynamics simulations were performed on the morphology and structure of multicompartment micelles and vesicles formed from pi-shaped ABC block copolymers in water. By varying the chain architecture and the composition of copolymers, a rich variety of morphologies were observed, such as oblate vesicles, trumpet vesicles, layered ribbon-like micelles and Y-shaped micelles. The simulations show that the hydrophilic block length and the distance between the two grafts play important roles in the control of the morphology. Since pi-shaped ABC block copolymers can reduce to linear ABC and star ABC block copolymers, they are good model copolymers for studying the self-assembly of complex block copolymers into micelles or vesicles. Thus, the knowledge obtained in this work as well as the new morphologies identified provides useful information for future rational design and synthesis of novel multicompartment micelles and vesicles.

Clathrin-dependent pathways and the cytoskeleton network are involved in ceramide endocytosis by a parasitic protozoan, Giardia lamblia

Although identified as an early-diverged protozoan, Giardia lamblia shares many similarities with higher eukaryotic cells, including an internal membrane system and cytoskeleton, as well as secretory pathways. However, unlike many other eukaryotes, Giardia does not synthesize lipids de novo, but rather depends on exogenous sources for both energy production and organelle or membrane biogenesis. It is not known how lipid molecules are taken up by this parasite and if endocytic pathways are involved in this process. In this investigation, we tested the hypothesis that highly regulated and selective lipid transport machinery is present in Giardia and necessary for the efficient internalization and intracellular targeting of ceramide molecules, the major sphingolipid precursor. Using metabolic and pathway inhibitors, we demonstrate that ceramide is internalized through endocytic pathways and is primarily targeted into perinuclear/endoplasmic reticulum membranes. Further investigations suggested that Giardia uses both clathrin-dependent pathways and the actin cytoskeleton for ceramide uptake, as well as microtubule filaments for intracellular localization and targeting. We speculate that this parasitic protozoan has evolved cytoskeletal and clathrin-dependent endocytic mechanisms for importing ceramide molecules from the cell exterior for the synthesis of membranes and vesicles during growth and differentiation.

Synthesis of PEDOT Nanoparticles and Vesicles by Dispersion Polymerization in Alcoholic Media

AbstractSummary: The synthesis of PEDOT nanoparticles and vesicles by dispersion polymerization in a methanol/water mixture (3/2, v/v) is reported, using either ammonium persulfate or iron(III) p‐toluenesulfonate as oxidants and α‐EDOT‐PEO as a reactive stabilizer. The influence of the oxidant as well as the α‐EDOT‐PEO molar mass and concentration on the core‐shell particle morphology and conductivity properties have been investigated. PEDOT particles with conductivities up to 1.5 × 10−2 S · cm−1 have been obtained in high yield.TEM image of PEDOT vesicles prepared using PEO‐based stabilizers of 25 000 g · mol−1 in water/methanol mixture (2:3 v/v) at room temperature using ammonium persulfate as an oxidant.magnified imageTEM image of PEDOT vesicles prepared using PEO‐based stabilizers of 25 000 g · mol−1 in water/methanol mixture (2:3 v/v) at room temperature using ammonium persulfate as an oxidant.

Synthesis and characterization of nanoscale biomimetic polymer vesicles and polymermembranes for bioelectronic applications

An amphiphilic ABA triblock copolymer was synthesized using poly(2-ethyl-2-oxazoline)(PEtOz) as hydrophilic block [A] and poly(dimethylsiloxane) (PDMS) as hydrophobicblock [B]. The cationic ring-opening polymerization of 2-ethyl-2-oxazoline was initiated bybenzyl chloride in the presence of NaI. PEtOz–PDMS–PEtOz was characterized in aqueoussolution using transmission electron microscopy (TEM). The block copolymers formedvesicles with a [B] block hydrophobic component thickness of 4 nm, which is thinenough for successful reconstitution of proteins. The mean diameters of the vesicleswere measured to be in the range of 150–250 nm, with a narrow distribution. Theelectrochemical properties of planar PEtOz–PDMS–PEtOz membrane films spread across aTeflon aperture were investigated by electrochemical impedance spectroscopy(EIS). The impedance data showed an increase of planar membrane capacitance(CMEM) from2.58 × 10−7 to2.71 × 10−7 F cm−2 and a decrease ofmembrane resistance (RMEM) from 12 to 10.8 Ω cm2.The increase of CMEM over time in buffer solution can be explained by an increase of the dielectric constant as aresult of membrane electrolyte incorporation and/or by the formation and growth of defectin the free-standing films. In contrast to the formation of thin-walled vesicles, thespread triblock copolymer formed a free-standing membrane with a 9 nm thickness.Based on the two possible conformations (bridge midblock conformation and loopmidblock conformation) that the triblock copolymer can have, we can conclude thatPEtOz–PDMS–PEtOz formed 4 nm thick polymer vesicles with intercalated loop midblockstructure in aqueous solution, while 9 nm thick free-standing polymer films with bilayerloop midblock conformation or with bridge midblock conformation were formed by aperturespreading.

Sterols block binding of COPII proteins to SCAP, thereby controlling SCAP sorting in ER.

Sterols inhibit their own synthesis in mammalian cells by blocking the vesicular endoplasmic reticulum-to-Golgi transport of sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP), a sterol-sensing protein that escorts SREBPs. Unable to reach the Golgi, SREBPs are not processed by Golgi-resident proteases, and they fail to activate genes required for cholesterol synthesis. The current studies were designed to reveal whether sterols block SCAP movement by inhibiting synthesis of special vesicles dedicated to SCAP, or whether sterols block SCAP incorporation into common coat protein (COP)II-coated vesicles. Through immunoisolation, we show that SCAP-containing vesicles, formed in vitro, also contain vesicular stomatitis virus glycoprotein (VSVG) protein, a classic marker of COPII-coated vesicles. Sterols selectively block incorporation of SCAP into these vesicles without blocking incorporation of VSVG protein. We show that the mammalian vesicular budding reaction can be reconstituted by recombinant yeast COPII proteins that support incorporation of SCAP as well as VSVG into vesicles. Sterols block SCAP incorporation into vesicles by blocking Sar1-dependent binding of the COPII proteins Sec 23/24 to SCAP. These studies demonstrate feedback control of a biosynthetic pathway by the regulated binding of COPII proteins to an endoplasmic reticulum-to-Golgi transport protein.

The sequence of the major gas vesicle protein, GvpA, influences the width and strength of halobacterial gas vesicles.

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p-gvpA was used to complement a Vac(-) strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p-gvpA. Replacement of p-gvpA with mc-gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c-gvpA from Hbt. salinarum or mutated p-gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.

The sequence of the major gas vesicle protein, GvpA, influences the width and strength of halobacterial gas vesicles

Transformation experiments with Haloferax volcanii show that the amino acid sequence of the gas vesicle protein GvpA influences the morphology and strength of gas vesicles produced by halophilic archaea. A modified expression vector containing p- gvpA was used to complement a Vac − strain of Hfx. volcanii that harboured the entire p-vac region (from Halobacterium salinarum PHH1) except for p- gvpA. Replacement of p- gvpA with mc- gvpA (from Haloferax mediterranei) led to the synthesis of gas vesicles that were narrower and stronger. Other gene replacements (using c- gvpA from Hbt. salinarum or mutated p- gvpA sequences) led to a significant but smaller increase in gas vesicle strength, and less marked effects on gas vesicle morphology.

Potassium Current Development and its Linkage to Membrane Expansion During Growth of Cultured Embryonic Mouse Hippocampal Neurons: Sensitivity to Inhibitors of Phosphatidylinositol 3-Kinase and Other Protein Kinases

Hippocampal pyramidal neurons express three major voltage-dependent potassium currents, IA, ID, and IK. During hippocampal development, IA, the rapidly activating and inactivating transient potassium current, is detected soon after pyramidal neurons can be morphologically identified. Appearance of IA in developing pyramidal neurons is dependent on contact with cocultured astroglial cells; cultured pyramidal neurons not in contact with astroglial cells have reduced membrane area and IA (Wu and Barish, 1994). We have examined intracellular signaling pathways that could contribute to the regulation of IA development by probing developing pyramidal neurons with kinase inhibitors. We observed that exposure to LY294002 or wortmannin, inhibitors of phosphatidylinositol (PI) 3-kinase, reduced somatic cross-sectional area, neurite outgrowth, whole-cell capacitance, IA amplitude and density (amplitude normalized to membrane area), and immunoreactivity for Kv4.2 and/or Kv4.3 (potassium channel subunits likely to be present in the channels carrying IA). In contrast, exposure to ML-9 or KN-62, inhibitors of myosin light chain kinase or Ca2+-calmodulin-dependent protein kinase II (CaMKII), reduced membrane area and IA amplitude but did not affect IA density or Kv4. 2/3 immunoreactivity to the same extent as inhibitors of PI 3-kinase. Unexpectedly, exposure to bisindolymaleimide I or calphostin C, inhibitors of protein kinase C (PKC), did not affect membrane area or potassium current development. Our data suggest that PI 3-kinases regulate both A-type potassium channel synthesis and plasmalemmal insertion of vesicles bearing these potassium channels. CaMKII appears to regulate fusion of channel-bearing vesicles with the plasmalemma and myosin light chain kinase to regulate centripetal transport of channel-bearing vesicles from the Golgi. We further suggest that astroglial cells exert their influence on pyramidal neuron development through activation of PI 3-kinases.

Brefeldin A inhibits cell-free, de novo synthesis of poliovirus.

Brefeldin A (BFA), an inhibitor of intracellular vesicle-dependent secretory transport, is a potent inhibitor of poliovirus RNA replication in infected cells. We have determined that the unknown mechanism of BFA inhibition of replication is reproduced in the cell-free poliovirus translation, replication, and encapsidation system. Furthermore, we provide evidence suggesting that the cellular mechanism targeted by BFA, the GTP-dependent synthesis of secretory transport vesicles, may be involved in viral RNA replication in the system via a soluble cellular GTP-binding and -hydrolyzing activity. This activity is related to the ARF (ADP-ribosylation factor) family of GTP-binding proteins. ARFs are required for the formation of several classes of secretory vesicles, and some family members are indirectly inactivated by BFA. Peptides that function as competitive inhibitors of ARF activity in cell-free transport systems also inhibit poliovirus RNA replication, and this inhibitory effect can be countered by the addition of exogenous ARF. We suggest that BFA inhibition of replication is diagnostic of a requirement for ARF activity in the cell-free system.

Effect of Ethanol on Intracellular Vesicular Transport from Golgi to the Apical Cell Membrane: Role of Phosphatidylinositol 3‐Kinase and Phospholipase A2 in Golgi Transport Vesicles Association and Fusion with the Apical Membrane

The study of ethanol effects on intracellular transport and membrane biogenesis in rat hepatocytes revealed that, during synthesis of transport vesicles, the cytosolic phosphatidylinositol 3-kinase incorporated into the membrane of Golgi transport vesicles and a portion of the vesicular phosphatidylinositol was phosphorylated to phosphatidylinositol 3-phosphate. Association of the enzyme with Golgi transport vesicles and the transport to the apical portion of the cell membrane was not affected by 0 to 120 mM ethanol, but was dependent on the presence of the p85 subunit of the phosphatidylinositol 3-kinase. In the presence of ATP-enriched cytosol and calcium ions, association of Golgi transport vesicles with the apical membrane was followed by phospholipase A2-specific hydrolysis of phosphatidylinositol 3-phosphate and incorporation of the transport vesicle membrane into the apical membrane. Association of Golgi transport vesicles with apical membranes was not affected by preincubation of the cell membrane or Golgi transport vesicles with 0 to 120 mM ethanol, but was inhibited when the p85 phosphatidylinositol 3-kinase was incorporated into the membrane before incubation with Golgi transport vesicles. The fusion of Golgi transport vesicles with the apical membrane and generation of lysophosphatidylinositol 3-phosphate and arachidonate was inhibited with EGTA or after depletion of ATP from cytosol. Results of these studies provide evidence that phosphatidylinositol 3-kinase and phospholipase A2 activities are crucial for the final step of exocytotic transport. The process consists of two stages. First, the p85 subunit of phosphatidylinositol 3-kinase is involved in the specific association of the vesicle with membrane receptor, and that is followed by phospholipase A2-specific lysophospholipid generation, perturbation of the membranes, and fusion of the transport vesicle membrane with the apical membrane. Addition of ethanol to the in vitro transport system decreased production of Golgi transport vesicles, but had no effect on their association with apical membrane or fusion with the membrane.

Continuous enzyme-linked fluorometric detection of l-(+)-lactate released from rat brain vesicles under anoxic conditions

A method is described for the on-line detection of l-(+)-lactate released from brain vesicles under physiological conditions. The principle of l-lactate detection is based on the reversible oxidation of l-lactate catalysed by l-lactate dehydrogenase (LDH, EC 1.1.1.27) employing 3-acetylpyridine-adenine-dinucleotide (APAD) as analogue of NAD according to the reaction: l-lactate + APAD ⇌ pyruvate + APADH. In practical terms, l-lactate synthesis of vesicles incubated in the presence of LDH and APAD was continuously followed by the fluorescence (490 nm) of APADH excited at 410 nm. Addition of a l-lactate standard (10 μmol/l) enhanced APADH fluorescence with a half-life of 6.0 ± 0.6 s allowing us to uncover a short-term alteration of l-lactate synthesis. This method was applied to evaluate a prospective change of l-lactate generation caused by the anoxia-induced increase in intravesicular Na + and Ca 2+ concentration ([Na +] i, [Ca 2+] i), both fluorometrically determined by SBFI and Fura, respectively. Upon anoxia, [Na +] i and [Ca 2+] i increased continuously up to 40 mmol/l Na + and 900 nmol/l Ca 2+ within 400 s. Concurrently, intravesicular NADH ([NADH] i) and basal l-lactate synthesis were enhanced within a few seconds, the latter from 4.2 ± 1.5 to 15.8 ± 1.5 nmol l-lactate/min per mg protein. Incubation of vesicles in the presence of 10 μmol/l tetrodotoxin (TTX) suppressed the increase in [Na +] i and [Ca 2+] i but failed to influence l-lactate synthesis. The data indicate a continuous Na + influx via voltage-dependent Na + channels accompanied by an increase in [Ca 2+] i during anoxia which did not affect anaerobic l-lactate synthesis. The method of fluorometric l-lactate determination was confirmed to be suitable for the detection of l-lactate released under physiological conditions from brain vesicles and seems to be applicable to various cell models.

Synthesis of zoospore peripheral vesicles during sporulation of Phytophthora cinnamomi

Three types of spherical vesicles (dorsal, ventral and large peripheral vesicles) are found in the peripheral cytoplasm of Phytophthora cinnamomi zoospores. Each is believed to participate in the infection of host plants through formation of a cyst coat, deposition of an adhesive pad or supply of proteins during early germling growth. In the present study, the occurrence of these three vesicles was determined during the asexual life cycle of P. cinnamomi . The vesicles were found to be absent from rapidly growing, vegetative hyphae, but to appear in the hyphae under conditions that induced sporulation. Vesicle formation was correlated with sporulation via the production of sporangia or chlamydospores. In mycelia transferred to nutrient-poor medium, large peripheral and ventral vesicles first appeared about 5 h after transfer; dorsal vesicles appeared about 30 min later. The first sporangia were detected at 7·5 h. Large peripheral and ventral vesicles were also produced before dorsal vesicles in mycelia growing on nutrient agar and in the hyphae formed by germinating cysts. On nutrient agar, the three vesicle types appeared about 10 h in advance of the development of chlamydospores.

Synthesis of magnetic DDAB vesicles

The synthesis of magnetic vesicles is described. The vesicles are constituted by didodecyldimethylammonium bromide and have a diameter of about 1 μm. An aqueous magnetic fluid, constituted by charged magnetic nanoparticles dispersed in water without surfactant, is encapsulated in the vesicle with a volume fraction in particles that may range up to 10%. The first step of the encapsulation is the synthesis of a multiple emulsion the intermediate oily phase being evaporated to obtain the DDAB bilayer. The magnetic vesicles thus synthesized align and change shape when a magnetic field is applied.

COPII: A membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum

In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60–65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.

A DNA region of 9kbp contains all genes necessary for gas vesicle synthesis in halophilic archaebacteria

We determined the minimal size of the genomic region necessary for gas vesicle synthesis in halophilic archaebacteria by transformation experiments, comparative DNA sequence analysis and investigation of gas vesicle (Vac) mutants. The comparison of the three genomic regions encoding gas vesicles in Halobacterium halobium (p-vac- and c-vac-region) and Haloferax mediterranei (mc-vac-region) indicates high DNA sequence similarity throughout a contiguous sequence of 9 kbp. In each case, this area encompassed at least 13 open reading frames (ORFs). Ten of these ORFs (gvpD to gvpM) were located 5' to the vac gene encoding the major gas vesicle protein, but were transcribed from the opposite strand. At least two ORFs (gvpC, and gvpN) were located 3' to each vac gene and transcribed from the same strand as the respective vac gene. In the p-vac-region present on plasmid pHH1 these ORFs were transcribed as at least three units, one transcript encompassing gvpD-gvpE, the second encompassing ORFs gvpF to gvpM, and the third unit comprising the ORFs located 3' to the p-vac gene. In H. halobium Vac mutants copies of the insertion elements ISH2, ISH23, ISH26 or ISH27 were found to be integrated throughout the p-vac-region. The de novo synthesis of gas vesicles was tested by transformation of the Vac-negative species, Haloferax volcanii, with various subfragments of the mc-vac- or p-vac-region cloned into vector plasmids. In contrast to a fragment containing the entire 9 kbp region, none of the subfragments tested was sufficient to promote gas vesicle synthesis. However, gas vesicle synthesis could be restored in each Vac mutant containing an ISH element when the entire transcription unit encompassing the mutated gene on pHH1 was present in the wild-type form on the vector construct.

Structure of the gas vesicle plasmid in Halobacterium halobium: inversion isomers, inverted repeats, and insertion sequences

Halobacterium-halobium NRC-1 harbors a 200-kb plasmid, pNRC100, which contains a cluster of genes for synthesis of buoyant gas-filled vesicles. Physical mapping of pNRC100 by using pulsed-field gel electrophoresis showed the presence of a large (35 to 38-kb) inverted repeat (IR) sequence. Inversion isomers of pNRC100 were demonstrated by Southern hybridization analysis using two restriction enzymes, AflII and SfiI, that cut asymmetrically within the intervening small single-copy region and the large single-copy region, respectively, but not within the large IRs. No inversion isomers were observed for a deletion derivative of pNRC100 lacking one IR, which suggests that both copies are required for inversion to occur. Additionally, the identities and approximate positions of 17 insertion sequences (IS) in pNRC100 were determined by Southern hybridization and limited nucleotide sequence analysis across the IS element-target site junctions: ISH2, a 0.5-kb element, was found in four copies; ISH3, a 1.4-kb heterogeneous family of elements, was present in seven copies; ISH8, a 1.4-kb element, was found in five copies; and ISH50, a 1.0-kb element, was present in a single copy. The large IRs terminated at an ISH2 element at one end and an ISH3 element at the other end. pNRC100 is similar in structure to chloroplast and mitochondrial genomes, which contain large IRs and other large halobacterial and prokaryotic plasmids that are reservoirs of IS elements but lack the large IRs.

Synaptophysin. A widespread constituent of small neuroendocrine vesicles and a new tool in tumor diagnosis.

Synaptophysin, a vesicular integral membrane protein, is specifically expressed in neuroendocrine tissues. According to cDNA cloning studies, it has a molecular weight of 33,300 Dalton, one potential N-glycosylation site at the vesicle inside, four major hydrophobic domains as well as a C-terminus containing approximately 90 amino acids. The C- and N-termini of synaptophysin are located on the cytoplasmic side of the vesicle membrane. No signal sequence is found. Transfection of non-neuroendocrine cells with synaptophysin cDNA leads to the synthesis of synaptophysin-containing vesicles, which contain this protein in highly enriched form and have biophysical properties similar to presynaptic vesicles of neurons. So far, the vesicular content has only been determined in rat neurons, where classical neurotransmitters such as biogenic amines and transmitter-active amino acids were found. Reconstitution of the purified protein in liposomes suggests a possible channel function of synaptophysin. Using mono- and polyvalent antibodies against synaptophysin, a considerable number of studies in several laboratories have shown that this protein is a reliable marker molecule for neuroendocrine tumors of various degrees of differentiation.