Uptake of l-Glutamate into Rat Brain Synaptic Vesicles: Effect of Inhibitors that Bind Specifically to the Glutamate Transporter

Abstract: In this study we have described a series of new and potent inhibitors of the vesicular uptake of glutamate. The two most efficient inhibitors were the dyes Evans blue and Chicago Skye Blue 6B, which are structurally related to glutamate and were competitive inhibitors in the nanomolar range. The anion channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (SITS) and the diuretics furosemide and bumetanide are inhibitors of chloride transport in other organs but were competitive inhibitors of glutamate and noncompetitive with respect to chloride ions. Evans blue, Chicago Skye Blue 6B, SITS, furosemide, and bumetanide are all large organic acids with two centers of negative charge and an electron-donating group at close vicinity of the negative charge at physiological pH. The inhibition of the glutamate uptake with these inhibitors was noncompetitive with respect to Cl⁻. The inhibitors, therefore, probably interact directly with the glutamate carrier. Bafilomycin A₁, which is a specific vacuolar ATPase inhibitor, was used as a control and inhibited the vesicular dopamine, glutamate, and GABA uptake to the same extent. None of the inhibitors had any effect on the plasma membrane carrier, which is therefore clearly different from the vesicular carrier.     

Influence of nitrate supply on concentrations and translocation of abscisic acid in barley (Hordeum vulgare)

Spring barley ( Hordeum vulgare L. cv. Golf) was grown at different nitrate supply rates, controlled by using the relative addition rate technique, in order to elucidate the relationship between nitrate-N supply and root and shoot levels of abscisic acid (ABA). The plants were maintained as (1) standard cultures where nitrate was supplied at relative addition rates (RAs) of 0.03, 0.09 and 0.18 day⁻¹, and (2) split-root cultures at RA 0.09 day⁻¹ but with the nitrate distributed between the two root parts in ratios of 100:0, 80:20 and 60:40. Time-dependent changes in root and shoot concentrations of ABA (determined by radioimmunoassay using a monoclonal antibody) were observed in both standard and split-root cultures during 12 days of acclimation to the different nitrate regimes. However, the ABA responses were similar at all nitrate supply rates. Further experiments were performed with split-root cultures where the distribution of nitrate between the two root parts was reversed from 80:20 to 20:80 so that short-term effects to local perturbations of nitrate supply could be studied without altering whole-plant N absorption. Transient increases in ABA concentrations (maximum of 25 to 40% after 3 to 4 h) were observed in both subroot parts, as well as in xylem sap and shoot tissue. By pruning the root system it was demonstrated that the change in ABA had its origin in the subroot part receiving the increased nitrate supply (i.e. switched from 20 to 80% of the total nitrate supply). The data indicate that ABA responses are easily transmitted between different organs, including transmission from one set of seminal roots to another via the shoot. The data do not provide any indication that long-term nitrate supplies or general nitrogen status of barley plants affect, or are otherwise related to, the average tissue ABA concentrations of roots and shoots.     

Studies on the import and processing of the alternative oxidase precursor by isolated soybean mitochondria

Import of the synthetic precursor of the alternative oxidase from soybean was shown to be dependent on a membrane potential and ATP. The membrane potential in soybean mitochondria may be formed either by respiration through the cytochrome pathway, or through the alternative oxidase pathway with NAD⁺-linked substrates. Import of the alternative oxidase precursor in the presence of succinate as respiratory substrate was inhibited by KCN. Import in the presence of malate was insensitive to KCN and SHAM added separately, but was inhibited by KCN and SHAM added together (inhibitors of the cytochrome and alternative oxidases respectively). Import of the alternative oxidase was accompanied by processing of the precursor to a single 32 kDa product in both cotyledon and root mitochondria. This product had a different mobility than the two alternative oxidase bands detected by immunological means (34 and 36 kDa), suggesting that the enzyme had been modified in situ. When the cDNA clone of the alternative oxidase was modified by a single mutation (–2 Arg changed to –2 Gly), the processing of the precursor was inhibited.     

Population genetic structure of a California endemic branchiopod,Branchinecta sandiegonensis

Branchinecta sandiegonensis (Crustacea: Anostraca) is a narrow range endemic fairy shrimp discontinuously distributed in ephemeral pools on coastal mesas in San Diego County, USA. Ten populations across the range of the species were subjected to allozyme analysis for eleven loci. The species exhibits low variability (P₉₅ =9.1–45.5) and one third of the loci tested did not conform to Hardy-Weinberg equilibrium expectations. The species also exhibited a high degree of genetic differentiation between populations. F _ST values (fixation index) for most pairs of populations were above 0.25 (0.036–0.889).Low genetic variability and high genetic structure may result from low gene flow and founder effects due to habitat fragmentation and the lack of potential vectors for cyst dispersal. The unpredictable rainfall of the region also creates potential for variable population sizes which could affect structure and variability. This revised version was published online in August 2006 with corrections to the Cover Date.     

Clinical and Molecular Characterization of Patients with Distal 11q Deletions

Jacobsen syndrome is caused by segmental aneusomy for the distal end of the long arm of chromosome 11. Typical features include mild to moderate psychomotor retardation, trigonocephaly, facial dysmorphism, cardiac defects, and thrombocytopenia, though none of these features are invariably present. To define the critical regions responsible for these abnormalities, we studied 17 individuals with de novo terminal deletions of 11q. The patients were characterized in a loss-of-heterozygosity analysis using polymorphic dinucleotide repeats. The breakpoints in the complete two-generation families were localized with an average resolution of 3.9 cM. Eight patients with the largest deletions extending from 11q23.3 to 11qter have breakpoints, between D11S924 and D11S1341. This cytogenetic region accounts for the majority of 11q⁻ patients and may be related to the FRA11B fragile site in 11q23.3. One patient with a small terminal deletion distal to D11S1351 had facial dysmorphism, cardiac defects, and thrombocytopenia, suggesting that the genes responsible for these features may lie distal to D11S1351. Twelve of 15 patients with deletion breakpoints as far distal as D11S1345 had trigonocephaly, while patients with deletions distal to D11S912 did not, suggesting that, if hemizygosity for a single gene is responsible for this dysmorphic feature, the gene may lie distal to D11S1345 and proximal to D11S912.     

Appomattoxia ancistrophora gen. et sp. nov., a new Early Cretaceous plant with similarities to Circaeaster and extant Magnoliidae

A new genus and species of fossil angiosperm (Appomattoxia ancistrophora) is established based on well-preserved fruiting units and associated pollen from the Early Cretaceous (Early or Middle Albian) Puddledock locality in the Potomac Group sequence of Virginia, eastern North America. Fruiting units are small, unilocular, and with a single, pendulous, orthotropous seed. The fruit surface is characterized by densely spaced unicellular spines with hooklike tips, which probably functioned in biotic dispersal. Pollen grains adhering to the stigmatic area of many specimens are monocolpate and tectate with granular to columellate infratectal structure, and are similar to dispersed grains assigned to Tucanopollis and Transitoripollis. Comparison of fossil Appomattoxia ancistrophora with extant plants reveals an unusual combination of characters that includes similarities with some magnoliid taxa, particularly Piperales (Piperaceae, Saururaceae) and Laurales (Chloranthaceae), as well as the monotypic ranunculid family Circaeasteraceae. Appomattoxia ancistrophora differs from extant Piperales in having a pendulous rather than erect ovule, and differs from extant Circaeaster in details of the fruit wall, as well as the presence of monosulcate rather than tricolpate pollen.     

Seven New Mutations in hMSH2, an HNPCC Gene, Identified by Denaturing Gradient-Gel Electrophoresis

Hereditary nonpolyposis colorectal cancer (HNPCC) is a relatively common autosomal dominant cancer-susceptibility condition. The recent isolation of the DNA mismatch repair genes (hMSH2, hMLH1, hPMS1, and hPMS2) responsible for HNPCC has allowed the search for germ-line mutations in affected individuals. In this study we used denaturing gradient-gel electrophoresis to screen for mutations in the hMSH2 gene. Analysis of all the 16 exons of hMSH2, in 34 unrelated HNPCC kindreds, has revealed seven novel pathogenic germ-line mutations resulting in stop codons either directly or through frameshifts. Additionally, nucleotide substitutions giving rise to one missense, two silent, and one useful polymorphism have been identified. The proportion of families in which hMSH2 mutations were found is 21%. Although the spectrum of mutations spread at the hMSH2 gene among HNPCC patients appears extremely heterogeneous, we were not able to establish any correlation between the site of the individual mutations and the corresponding tumor spectrum. Our results indicate that, given the genomic size and organization of the hMSH2 gene and the heterogeneity of its mutation spectrum, a rapid and efficient mutation detection procedure is necessary for routine molecular diagnosis and presymptomatic detection of the disease in a clinical setup.     

Apoptosis and nutrition: Involvement of amino acid transport system in repression of hybridoma cell death

Mouse hybridoma cells cultured on the verge of starvation-induced apoptosis, i.e. in a medium diluted with saline, proved to serve as a sensitive screening system for apoptosis-suppressing activity of nutrient medium components. Conventional amino acid mixtures were found to suppress the starvation-induced apoptosis, whereas a vitamin mixture was ineffective. (Frank F (1995) Biotechnol. Bioeng. 45: 86–90). Recent experiments showed that suppression of apoptosis, and concurrent resumption of growth, could be achieved by addition of single substances at millimolar concentrations. The set of active substances included certain coded L-amino acids (glycine, alanine, serine, threonine, proline, asparagine, glutamine, histidine), non-coded amino acids (-alanine, taurine, 4-aminobutyric acid), and a non-metabolizable analogue (2-aminoisobutyric acid). This finding shows that some amino acids do not act solely as nutrients, but also as specific signal molecules. The specificity of the effect points to the involvement of adaptively regulated amino acid transport systems A and N in maintaining the balance between triggering and suppression of starvation-induced apoptosis.