Congruence of fatty acid methyl ester profiles and morphological characters of arbuscular mycorrhizal fungi in Gigasporaceae.

Arbuscular mycorrhizal (AM) fungi (Order Glomales, Class Zygomycetes) are a diverse group of soil fungi that form mutualistic associations with the roots of most species of higher plants. Despite intensive study over the past 25 years, the phylogenetic relationships among AM fungi, and thus many details of evolution of the symbiosis, remain unclear. Cladistic analysis was performed on fatty acid methyl ester (FAME) profiles of 15 species in Gigaspora and Scutellospora (family Gigasporaceae) by using a restricted maximum likelihood approach of continuous character data. Results were compared to a parsimony analysis of spore morphological characters of the same species. Only one tree was generated from each character set. Morphological and developmental data suggest that species with the simplest spore types are ancestral whereas those with complicated inner wall structures are derived. Spores of those species having a complex wall structure pass through stages of development identical to the mature stages of simpler spores, suggesting a pattern of classical Haeckelian recapitulation in evolution of spore characters. Analysis of FAME profiles supported this hypothesis when Glomus leptotichum was used as the outgroup. However, when Glomus etunicatum was chosen as the outgroup, the polarity of the entire tree was reversed. Our results suggest that FAME profiles contain useful information and provide independent criteria for generating phylogenetic hypotheses in AM fungi. The maximum likelihood approach to analyzing FAME profiles also may prove useful for many other groups of organisms in which profiles are empirically shown to be stable and heritable.

Distinct pharmacological properties and distribution in neurons and endocrine cells of two isoforms of the human vesicular monoamine transporter.

A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1-mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2-mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.

Visualization of the vesicular acetylcholine transporter in cholinergic nerve terminals and its targeting to a specific population of small synaptic vesicles.

Immunohistochemical visualization of the rat vesicular acetylcholine transporter (VAChT) in cholinergic neurons and nerve terminals has been compared to that for choline acetyltransferase (ChAT), heretofore the most specific marker for cholinergic neurons. VAChT-positive cell bodies were visualized in cerebral cortex, basal forebrain, medial habenula, striatum, brain stem, and spinal cord by using a polyclonal anti-VAChT antiserum. VAChT-immuno-reactive fibers and terminals were also visualized in these regions and in hippocampus, at neuromuscular junctions within skeletal muscle, and in sympathetic and parasympathetic autonomic ganglia and target tissues. Cholinergic nerve terminals contain more VAChT than ChAT immunoreactivity after routine fixation, consistent with a concentration of VAChT within terminal neuronal arborizations in which secretory vesicles are clustered. These include VAChT-positive terminals of the median eminence or the hypothalamus, not observed with ChAT antiserum after routine fixation. Subcellular localization of VAChT in specific organelles in neuronal cells was examined by immunoelectron microscopy in a rat neuronal cell line (PC 12-c4) expressing VAChT as well as the endocrine and neuronal forms of the vesicular monoamine transporters (VMAT1 and VMAT2). VAChT is targeted to small synaptic vesicles, while VMAT1 is found mainly but not exclusively on large dense-core vesicles. VMAT2 is found on large dense-core vesicles but not on the small synaptic vesicles that contain VAChT in PC12-c4 cells, despite the presence of VMAT2 immunoreactivity in central and peripheral nerve terminals known to contain monoamines in small synaptic vesicles. Thus, VAChT and VMAT2 may be specific markers for "cholinergic" and "adrenergic" small synaptic vesicles, with the latter not expressed in nonstimulated neuronally differentiated PC12-c4 cells.

14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae.

Deletion of the clathrin heavy-chain gene, CHC1, in the budding yeast Saccharomyces cerevisiae results in growth, morphological, and membrane trafficking defects, and in some strains chc1-delta is lethal. A previous study identified five genes which, in multicopy, rescue inviable strains of Chc- yeast. Now we report that one of the suppressor loci, BMH2/SCD3, encodes a protein of the 14-3-3 family. The 14-3-3 proteins are abundant acidic proteins of approximately 30 kDa with numerous isoforms and a diverse array of reported functions. The Bmh2 protein is > 70% identical to the mammalian epsilon-isoform and > 90% identical to a previously reported yeast 14-3-3 protein encoded by BMH1. Single deletions of BMH1 or BMH2 have no discernable phenotypes, but deletion of both BMH1 and BMH2 is lethal. High-copy BMH1 also rescues inviable strains of Chc- yeast, although not as well as BMH2. In addition, the slow growth of viable strains of Chc- yeast is further impaired when combined with single bmh mutations, often resulting in lethality. Overexpression of BMH genes also partially suppresses the temperature sensitivity of the cdc25-1 mutant, and high-copy TPK1, encoding a cAMP-dependent protein kinase, restores Bmh- yeast to viability. High-copy TPK1 did not rescue Chc- yeast. These genetic interactions suggest that budding-yeast 14-3-3 proteins are multifunctional and may play a role in both vesicular transport and Ras signaling pathways.

Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal.

Synaptotagmin (Syt) is an inositol high-polyphosphate series [IHPS inositol 1,3,4,5-tetrakisphosphate (IP4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate] binding synaptic vesicle protein. A polyclonal antibody against the C2B domain (anti-Syt-C2B), an IHPS binding site, was produced. The specificity of this antibody to the C2B domain was determined by comparing its ability to inhibit IP4 binding to the C2B domain with that to inhibit the Ca2+/phospholipid binding to the C2A domain. Injection of the anti-Syt-C2B IgG into the squid giant presynapse did not block synaptic release. Coinjection of IP4 and anti-Syt-C2B IgG failed to block transmitter release, while IP4 itself was a powerful synpatic release blocker. Repetitive stimulation to presynaptic fiber injected with anti-Syt-C2B IgG demonstrated a rapid decline of the postsynaptic response amplitude probably due to its block of synaptic vesicle recycling. Electron microscopy of the anti-Syt-C2B-injected presynapse showed a 90% reduction of the numbers of synaptic vesicles. These results, taken together, indicate that the Syt molecule is central, in synaptic vesicle fusion by Ca2+ and its regulation by IHPS, as well as in the recycling of synaptic vesicles.

The vesicular monoamine transporter 2 is present in small synaptic vesicles and preferentially localizes to large dense core vesicles in rat solitary tract nuclei.

In central neurons, monamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.

Efficient recovery of infectious vesicular stomatitis virus entirely from cDNA clones.

Infectious vesicular stomatitis virus (VSV), the prototypic nonsegmented negative-strand RNA virus, was recovered from a full-length cDNA clone of the viral genome. Bacteriophage T7 RNA polymerase expressed from a recombinant vaccinia virus was used to drive the synthesis of a genome-length positive-sense transcript of VSV from a cDNA clone in baby hamster kidney cells that were simultaneously expressing the VSV nucleocapsid protein, phosphoprotein, and polymerase from separate plasmids. Up to 10(5) infectious virus particles were obtained from transfection of 10(6) cells, as determined by plaque assays. This virus was amplified on passage, neutralized by VSV-specific antiserum, and shown to possess specific nucleotide sequence markers characteristic of the cDNA. This achievement renders the biology of VSV fully accessible to genetic manipulation of the viral genome. In contrast to the success with positive-sense RNA, attempts to recover infectious virus from negative-sense T7 transcripts were uniformly unsuccessful, because T7 RNA polymerase terminated transcription at or near the VSV intergenic junctions.

Recombinant vesicular stomatitis viruses from DNA.

We assembled a DNA clone containing the 11,161-nt sequence of the prototype rhabdovirus, vesicular stomatitis virus (VSV), such that it could be transcribed by the bacteriophage T7 RNA polymerase to yield a full-length positive-strand RNA complementary to the VSV genome. Expression of this RNA in cells also expressing the VSV nucleocapsid protein and the two VSV polymerase subunits resulted in production of VSV with the growth characteristics of wild-type VSV. Recovery of virus from DNA was verified by (i) the presence of two genetic tags generating restriction sites in DNA derived from the genome, (ii) direct sequencing of the genomic RNA of the recovered virus, and (iii) production of a VSV recombinant in which the glycoprotein was derived from a second serotype. The ability to generate VSV from DNA opens numerous possibilities for the genetic analysis of VSV replication. In addition, because VSV can be grown to very high titers and in large quantities with relative ease, it may be possible to genetically engineer recombinant VSVs displaying foreign antigens. Such modified viruses could be useful as vaccines conferring protection against other viruses.

Regulación del ciclo vesicular sináptico por las quinasas dependientes de GMPc en neuronas grabulares de cerebelo

La comunicación entre las neuronas ocurre en regiones anatómicamente identificables denominadas sinapsis. Existen dos tipos de transmisión sináptica, las sinapsis químicas y las eléctricas, aunque predominan las sinapsis químicas. En este tipo de sinapsis, la comunicación neuronal ocurre en zonas especializadas de los axones, denominados terminales sinápticos, los cuales almacenan en su interior pequeñas vesículas que contienen un neurotransmisor. Ante la llegada de un potencial de acción al terminal presináptico, el flujo de calcio, generado a través de la apertura de canales de calcio voltaje-dependientes, provoca la fusión de las vesículas con la membrana del terminal presináptico, y la liberación del neurotransmisor a la hendidura sináptica. La fusión vesicular tiene lugar en regiones de membrana del terminal presináptico, molecularmente especializadas para dicho evento exocitótico, denominadas Zonas Activas. Este neurotransmisor liberado difunde por la hendidura sináptica y se une a receptores específicos ubicados en la membrana de la neurona postsináptica, propagándose así el impulso nervioso. Tras este evento exocitótico, que implica la fusión de multitud de vesículas, es necesario un proceso de endocitosis, que ocurre en las zonas perisinápticas, y que está encargado de recuperar las fracciones de membrana que formaban las vesículas sinápticas, con dos objetivos: 1. Impedir el aumento de la superficie de la zona activa, lo cual llevaría a su desestructuración, y 2. La formación de nuevas vesículas que se rellenen de neurotransmisor y puedan prepararse para una nueva ronda de exocitosis. La endocitosis que sigue a un estímulo moderado, está mediada por clatrina y recicla vesículas independientes preparadas para el rellenado con neurotransmisor. Tras estímulos intensos que provocan exocitosis de múltiples vesículas, la retirada de membrana ocurre a través de otro mecanismo más lento y menos eficaz, denominado endocitosis en masa, el cual recicla grandes fragmentos de membrana y acumula estructuras endosomales en el interior del terminal, los cuales no siempre rinden vesículas funcionales inmediatamente...

Special report of the Joint Legislative Committee on Agriculture and Livestock Problems, covering the committee's investigations and recommendations on problems involving: 1. Vesicular Exanthema, 2. Foot-and-Mouth Disease, 3. Federal Animal Research Laboratory, Plum Island, New York.

Mode of access: Internet.

Vesicular exanthema of swine, 1933-1963 : a bibliography.

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Swine vesicular disease, 1966-1973 : a bibliography /

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