Rapid Communication: cis-Methyldioxolane Specifically Recognizes the m2 Muscarinic Receptor

Abstract: cis -Methyldioxolane (CD) is a muscarinic receptor agonist. [³H] CD has been used to label a subpopulation of muscarinic receptors described as exhibiting high agonist affinity. Pharmacological evidence suggests that the population of receptors labeled by [³H] CD consists of m2 and/or m4 subtypes; however, no studies have directly addressed the subtype selectivity of [³H] CD. The present study characterizes binding of this ligand to individual human receptor subtypes expressed in transfected Chinese hamster ovary cells. Results indicate that [³H] CD binds with high affinity only to Hm2 receptors but not to all Hm2 receptors. Twenty-eight percent of Hm2 receptors bound [³H] CD with a K _D of 3.5 ± 0.5 nM. Binding was eliminated in the presence of guanosine 5'- O -(3-thiotriphosphate), indicating that the Hm2 receptors labeled by [³H] CD are those that are associated with GDP-bound G protein. Binding of [³H] CD by only a subpopulation of Hm2 receptors is in agreement with data generated from studies of [³H] CD binding in mammalian brain. Because muscarinic receptors have been implicated to play a role in the pathogenesis of both Alzheimer's and Parkinson's disease, as well as the neurotoxicity of organophosphorus compounds, knowledge of the binding specificity of the muscarinic agonist [³H] CD should aid research in these areas.     

Acclimation of Arabidopsis thaliana to the light environment: Changes in composition of the photosynthetic apparatus

Acclimation to changes in the light environment was investigated in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Plants grown under four light regimes showed differences in their development, morphology, photosynthetic performance and in the composition of the photosynthetic apparatus. Plants grown under high light showed higher maximum rates of oxygen evolution and lower levels of light-harvesting complexes than their low light-grown counterparts; plants transferred to low light showed rapid changes in maximum photosynthetic rate and chlorophyll-a/b ratio as they became acclimated to the new environment. In contrast, plants grown under lights of differing spectral quality showed significant differences in the ratio of photosystem II to photosystem I. These changes are consistent with a model in which photosynthetic metabolism provides signals which regulate the composition of the thylakoid membrane.Abbreviations Aac1 gene encoding actin - Chl chlorophyll - F far-red-enriched light (R:FR = 0.72) - FR far-red light - H high light (400 mol · m^–2 · s^–1) - L low light (100 ml · m^–2 · s^–1) - LHCII light-harvesting complex of PSII - Lhcb genes encoding the proteins of LHCII - R red light - Rbcs genes encoding the small subunit of Rubisco - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - W white light (R:FR = 1.40) This work was supported by Natural Environment Research Council Grant No. GR3/7571A. We would like to thank H. Smith (Botany Department, University of Leicester) and E. Murchie (University of Sheffield) for helpful discussions.     

Genetic mapping of 14 short tandem repeat polymorphisms on human chromosome 22

We have constructed a linkage map of 14 short tandem repeat polymorphisms (11 with heterozygosity > 70%) on the long arm of human chromosome 22 using 23 non-CEPH pedigrees. Twelve of the markers could be positioned uniquely with a likelihood of at least 1,000:1, and distributed at an average distance of 6.62 cM (range 1.5–16.1 cM). The sex-combined map covers a total of 79.6 cM, the female map 93.2 cM and the male map 64.6 cM. Based on comparisons between physical maps and other genetic maps, we estimate that our map covers 70%–80% of the chromosome. The map integrates markers from previous genetic maps and uniquely positions one marker (D22S307). Data from physical mapping on the location of four genetic markers correlates well with our linkage map, and provides information on an additional marker (D22S315). This map will facilitate high resolution mapping of additional polymorphic loci and disease genes on chromosome 22, and act as a reference for building and verifying physical maps.     

Molecular cytogenetic analysis of a duplication Xp in a male: further delineation of a possible sex influencing region on the X chromosome

We describe a male infant with severe mental retardation and autism with a duplication of the short arm of the X chromosome. Chromosome painting confirmed the origin of this X duplication. Molecular cytogenetic analysis with fluorescence in situ hybridization (FISH) identified one copy of the zinc finger protein on the X chromosome (ZFX) and two copies of the steroid sulfatase gene (STS), further delineating the breakpoints. Based on cytogenetic and molecular comparisons of cases from the literature of sex-reversal in dup(X),Y patients and our patient, we suggest that a possible secondary sexinfluencing gene involved in the regulation of sex determination or testis morphogenesis is present at the distal Xp21.1 to p21.2 region.     

The expression of cecropin peptide in transgenic tobacco does not confer resistance to Pseudomonas syringae pv tabaci

Summary We used in vitro growth inhibition assays to demonstrate that synthetic cecropin protein has potent activity against a range of plant pathogenic bacteria. We then prepared transgenic tobacco plants which express cecropin mRNA and protein. We have used Pseudomonas syringae pv tabaci infection of these transgenic tobacco as a model system to evaluate whether the plants which express cecropin protein also have increased tolerance to infection. We found no dramatic difference in disease response between plants which are expressing cecropin protein and control plants which were derived from the transformation with a binary vector which did not carry the gene encoding cecropin protein.     

The effect of age on the recognition thresholds of three sweeteners: sucrose,saccharin and aspartame

It iS believed that people's sensitivity to taste declines with age but the evidence is inconclusive. This study was designed to test the hypothesis that taste recognition thresholds (TRTs) for sweetness are higher in older than in younger individuals, using groups of 16 younger subjects (18–30) and 16 older subjects (60–85). Three test substances were used: sucrose, aspartame and saccharin. A questionnaire recorded variables which might have affected TRTs, but data failed to show any trend that might have biased the principle variate-age. There was a significant alteration with age of recognition thresholds, at least for sucrose and saccharin. The differences between the groups for the three sweeteners were due to the fact that all the very sensitive subjects were young. None of the older subjects had particularly poor discrimination: all but one had TRTs within the range of younger subjects. Although there are age-related taste changes, they are much less dramatic than commonly occurs with other senses, such as sight and hearing. The findings of this study have implications for institutional catering and the dietary management of older people using non-sugar sweeteners.     

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac^–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g^–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity. Correspondence to: R. F. M.van Gorcom     

Iron uptake byBifidobacterium thermophilum protoplasts

Protoplasts ofBifidobacterium thermophilum were prepared by a combination of lysozyme and protease digestion, and ferrous iron uptake studies were carried out. Little, if any, iron was internalized by the protoplasts, although large amounts of iron were bound to the protoplast surface. This binding was much greater than that of intact cells, which prefer to internalize iron by an energy-dependent process. It was also found that the binding of iron by protoplasts of cells grown in an iron-deficient medium was much more extensive than that of cells grown in an iron-sufficient medium. Soluble and particulate fractions of protoplasts were prepared by grinding them in a glass homogenizer, and the particulate fraction was also subjected to iron binding studies. The amount of iron bound was the same as that in intact protoplasts, indicating that the particulate fraction membrane fragments bound iron on their outer surface only. Nevertheless, when iron-preloaded cells were protoplasted and their surface cleared of iron, their particulate fraction contained considerable amounts of iron, indicating that the inner surface of the membranes is capable of binding iron as long as the cell is intact. The amount of iron so bound was dose-dependent on the amount of iron entering the cell. The failure of the outer and inner surface iron pools to mix was confirmed by the fact that when iron-preloaded protoplasts were incubated with additional iron, only the latter (surface-bound) was elutable with nonradioactive 2 mM FeSO₄. It is concluded that increasing bifidobacterial iron load increases the amount of iron bound to the inner surface of the membrane; the procedure, which is effective in forming bifidobacterial protoplasts, destroys their iron transport mechanism while uncovering surface iron-binding sites; and that such iron-binding sites may be of significance in the cellular iron metabolism processes.     

Structure of the intergenic spacer region from the ribosomal RNA gene family of white spruce (Picea glauca)

Five genomic clones containing ribosomal DNA repeats from the gymnosperm white spruce (Picea glauca) have been isolated and characterized by restriction enzyme analysis. No nucleotide variation or length variation was detected within the region encoding the ribosomal RNAs. Four clones which contained the intergenic spacer (IGS) region from different rDNA repeats were further characterized to reveal the sub-repeat structure within the IGS. The sub-repeats were unusually long, ranging from 540 to 990 bp but in all other respects the structure of the IGS was very similar to the organization of the IGS from wheat, Drosophila and Xenopus.