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.

Nuclear Pore Complex Number and Distribution throughout the Saccharomyces cerevisiae Cell Cycle by Three-Dimensional Reconstruction from Electron Micrographs of Nuclear Envelopes

The number of nuclear pore complexes (NPCs) in individual nuclei of the yeast Saccharomyces cerevisiae was determined by computer-aided reconstruction of entire nuclei from electron micrographs of serially sectioned cells. Nuclei of 32 haploid cells at various points in the cell cycle were modeled and found to contain between 65 and 182 NPCs. Morphological markers, such as cell shape and nuclear shape, were used to determine the cell cycle stage of the cell being examined. NPC number was correlated with cell cycle stage to reveal that the number of NPCs increases steadily, beginning in G1-phase, suggesting that NPC assembly occurs continuously throughout the cell cycle. However, the accumulation of nuclear envelope observed during the cell cycle, indicated by nuclear surface area, is not continuous at the same rate, such that the density of NPCs per unit area of nuclear envelope peaks in apparent S-phase cells. Analysis of the nuclear envelope reconstructions also revealed no preferred NPC-to-NPC distance. However, NPCs were found in large clusters over regions of the nuclear envelope. Interestingly, clusters of NPCs were most pronounced in early mitotic nuclei and were found to be associated with the spindle pole bodies, but the functional significance of this association is unknown.

The nucleotide changes governing cuticular hydrocarbon variation and their evolution in Drosophila melanogaster

The cuticular hydrocarbon (CH) pheromones in Drosophila melanogaster exhibit strong geographic variation. African and Caribbean populations have a high ratio of 5,9 heptacosadiene/7,11 heptacosadiene (the “High” CH type), whereas populations from all other areas have a low ratio (“Low” CH type). Based on previous genetic mapping, DNA markers were developed that localized the genetic basis of this CH polymorphism to within a 13-kb region. We then carried out a hierarchical search for diagnostic nucleotide sites starting with four lines, and increasing to 24 and 43 lines from a worldwide collection. Within the 13-kb region, only one variable site shows a complete concordance with the CH phenotype. This is a 16-bp deletion in the 5′ region of a desaturase gene (desat2) that was recently suggested to be responsible for the CH polymorphism on the basis of its expression [Dallerac, R., Labeur, C., Jallon, J.-M., Knipple, D. C., Roelofs, W. L. & Wicker-Thomas, C. (2000) Proc. Natl. Acad. Sci. 97, 9449–9454]. The cosmopolitan Low type is derived from the ancestral High type, and DNA sequence variations suggest that the former spread worldwide with the aid of positive selection. Whether this CH variation could be a component of the sexual isolation between Zimbabwe and other cosmopolitan populations remains an interesting and unresolved question.

Phenotypic variation and intracellular parasitism by Histoplasma capsulatum

The success of Histoplasma capsulatum as an intracellular pathogen depends completely on successful conversion of the saprophytic mycelial (mold) form of this fungus to a parasitic yeast form. It is therefore not surprising that yeast phase-specific genes and gene products are proving to be important for survival and proliferation of H. capsulatum within macrophages. In this study, we have focused on the role and regulation of two yeast-specific characteristics: α-(1,3)-glucan, a cell wall polysaccharide modulated by cell-density (quorum) sensing, and a secreted calcium-binding protein (CBP) that is essential for pathogenicity.

Protein Changes in Response to Progressive Water Deficit in Maize1 : Quantitative Variation and Polypeptide Identification

Three-week-old plants of two unrelated lines of maize (Zea mays L.) and their hybrid were submitted to progressive water stress for 10 d. Changes induced in leaf proteins were studied by two-dimensional electrophoresis and quantitatively analyzed using image analysis. Seventy-eight proteins out of a total of 413 showed a significant quantitative variation (increase or decrease), with 38 of them exhibiting a different expression in the two genotypes. Eleven proteins that increased by a factor of 1.3 to 5 in stressed plants and 8 proteins detected only in stressed plants were selected for internal amino acid microsequencing, and by similarity search 16 were found to be closely related to previously reported proteins. In addition to proteins already known to be involved in the response to water stress (e.g. RAB17 [Responsive to ABA]), several enzymes involved in basic metabolic cellular pathways such as glycolysis and the Krebs cycle (e.g. enolase and triose phosphate isomerase) were identified, as well as several others, including caffeate O-methyltransferase, the induction of which could be related to lignification.

Cloning, expression, and distribution of a Ca(2+)-activated K+ channel beta-subunit from human brain.

We have cloned and expressed a Ca(2+)-activated K+ channel beta-subunit from human brain. The open reading frame encodes a 191-amino acid protein possessing significant homology to a previously described subunit cloned from bovine muscle. The gene for this subunit is located on chromosome 5 at band q34 (hslo-beta). There is no evidence for alternative RNA splicing of this gene product. hslo-beta mRNA is abundantly expressed in smooth muscle, but expression levels are low in most other tissues, including brain. Brain subregions in which beta-subunit mRNA expression is relatively high are the hippocampus and corpus callosum. The coexpression of hslo-beta mRNA together with hslo-alpha subunits in either Xenopus oocytes or stably transfected HEK 293 cells give rise to Ca(2+)-activated potassium currents with a much increased calcium and/or voltage sensitivity. These data indicate that the beta-subunit shows a tissue distribution different to that of the alpha-subunit, and in many tissues there may be no association of alpha-subunits with beta-subunits. These beta-subunits can play a functional role in the regulation of neuronal excitability by tuning the Ca2+ and/or the voltage dependence of alpha-subunits.

Antigenic variation and the within-host dynamics of parasites.

Many parasites exhibit antigenic variation within their hosts. We use mathematical models to investigate the dynamical interaction between an antigenically varying parasite and the host's immune system. The models incorporate antigenic variation in the parasite population and the generation of immune responses directed against (i) antigens specific to individual parasite variants and (ii) antigens common to all the parasite variants. Analysis of the models allows us to evaluate the relative importance of variant-specific and cross-reactive immune responses in controlling the parasite. Early in the course of infection within the host, when parasite diversity is below a defined threshold value (the value is determined by the biological properties of the parasite and of the host's immune response), the variant-specific immune responses are predominant. Later, when the parasite diversity is high, the cross-reactive immune response is largely responsible for controlling the parasitemia. It is argued that increasing antigenic diversity leads to a switch from variant-specific to cross-reactive immune responses. These simple models mimic various features of observed infections recorded in the experimental literature, including an initial peak in parasitemia, a long and variable duration of infection with fluctuating parasitemia that ends with either the clearance of the parasite or persistent infection.

Chloroplast DNA variation and the recent radiation of the giant senecios (Asteraceae) on the tall mountains of eastern Africa.

Chloroplast DNA restriction-site variation was surveyed among 40 accessions representing all 11 species of giant senecios (Dendrosenecio, Asteraceae) at all but one known location, plus three outgroup species. Remarkably little variation (only 9 variable sites out of roughly 1000 sites examined) was found among the 40 giant senecio accessions, yet as a group they differ significantly (at 18 sites) from Cineraria deltoidea, the closest known relative. This pattern indicates that the giant senecios underwent a recent dramatic radiation in eastern Africa and evolved from a relatively isolated lineage within the Senecioneae. Biogeographic interpretation of the molecular phylogeny suggests that the giant senecios originated high on Mt. Kilimanjaro, with subsequent dispersion to the Aberdares, Mt. Kenya, and the Cherangani Hills, followed by dispersion westward to the Ruwenzori Mountains, and then south to the Virunga Mountains, Mt. Kahuzi, and Mt. Muhi, but with dispersion back to Mt. Elgon. Geographic radiation was an important antecedent to the diversification in eastern Africa, which primarily involved repeated altitudinal radiation, both up and down the mountains, leading to morphological parallelism in both directions. In general, the plants on a given mountain are more closely related to each other than they are to plants on other mountains, and plants on nearby mountains are more closely related to each other than they are to plants on more distant mountains. The individual steps of the geographic radiation have occurred at various altitudes, some clearly the result of intermountain dispersal. The molecular evidence suggests that two species are extant ancestors to other species on the same or nearby mountains.