Stereoisomeric flavour compounds XXXV: Chiral aroma compounds of sherry I. Optically pure stereoisomers of solerone and solerole

The enantioseparation of the sherry aroma components 5-oxo-4-hydroxyhexanoic acid γ-lactone (solerone) and 4,5-dihydroxyhexanoic acid γ-lactone (solerole) is achieved, using Chiraspher (Merck) as the chiral HPLC phase and the optical purity ascertained directly by HRGC with heptakis(3-O-acetyl-2,6-di-O-pentyl)-β-cyclodextrin (Lipodex D) as the chiral stationary phase. The absolute configurations of 4,5-dihydroxyhexanoic acid γ-lactones are assigned by ¹H-NMR spectral data of diastereomeric α-methoxy-α-trifluoromethylphenylacetic acid (MTPA) esters, according to Mosher's model. Sensory qualities of the isomers are given.     

Cu(I)-thionein release from copper-loaded yeast cells

Summary The release of intact CU(I)₈-thionein from copper-resistant copper-loaded yeast cells, strain X 2180-1Aa, has been shown. This copper(I)-thiolate-rich protein was characterized and compared with the chemical and physicochemical properties of intracellular yeast Cu-thionein. The same molecular mass and stoichiometry of 8 mol copper atoms/mol protein was found. No detectable difference between the Cu-thioneins was seen in luminescence emission, electronic absorption in the ultraviolet region, chiroptical data or amino acid composition. The importance of stable Cu(I)-thiolates in Cu-thionein as a safe vehicle for transporting copper in a non-reactive manner is confirmed.     

Rapid quantification of planktonic ciliates: comparison of improved live counting with other methods

THE FOLLOWING EFFICIENT AND QUANTITATIVELY VALID METHOD TO FILTER CONCENTRATE AND COUNT LIVE PLANKTONIC CILIATES WAS DEVELOPED AND COMPARED WITH OTHER TREATMENTS: unconcentrated (raw) samples and centrifuged samples were counted live, and the effects of five different fixatives (HgCl(2), Lugol's iodine, formaldehyde, glutaraldehyde, and Champy-DaFano) on the counts were monitored. Samples originated from a eutrophic mountain lake (Lake Aydat, near Clermont-Ferrand, France). Overall, live filtered counts were similar to counts of raw samples, but they were significantly higher (2 to 2.3 fold, P < 0.05) by analysis of variance than counts from centrifuged samples. Nevertheless, some taxa, i.e., Halteria and Loxodes spp., were sensitive to filtration. The live filtered counts were also comparable to counts of raw HgCl(2)-fixed and settled samples. HgCl(2) and Lugol fixation consistently gave the highest total counts, while significantly lower counts were always obtained with Champy-DaFano-fixed samples. Losses due to fixation were insignificant for raw samples but were substantial and statistically significant in concentrated samples (15% after filtration and 71% after centrifugation, compared with counts from the corresponding live samples). Live counting of passively filter-concentrated ciliates has many advantages over other methods. It is two to four times quicker and more efficient. Ciliates are recognized with certainty, more species are identified, and enumeration of dead organisms (e.g., tintinnid loricas) is avoided. It should be recommended as a quantitatively valid alternative to classical methods for assessing planktonic ciliate populations.     

Delineation of structural domains in eukaryotic 5S rRNA with a rhodium probe.

The three-dimensional folding of Xenopus oocyte 5S rRNA has been examined using the coordination complex Rh(phen)2phi3+ (phen = phenanthroline; phi = phenanthrenequinone diimine) as a structural probe. Rh(phen)2phi3+ binds neither double-helical RNA nor unstructured single-stranded regions of RNA. Instead, the complex targets through photoactivated cleavage sites of tertiary interaction which are open in the major groove and accessible to stacking. The sites targeted by the rhodium complex have been mapped on the wild-type Xenopus oocyte RNA, on a truncated RNA representing the arm of the molecule comprised of helix IV-loop E-helix V, and on several single-nucleotide mutants of the 5S rRNA. On the wild-type 5S rRNA, strong cleavage is found at residues U73, A74, A101, and U102 in the E loop and U80 and G81 in helix IV; additional sites are evident at A22 and A56 in the B loop, C29 and A32 in helix III, and C34, C39, A42, and C44 in the C loop. Given the similarity observed in cleavage between the full 5S RNA and the truncated fragment as well as the absence of any long-range effects on cleavage in mutant RNAs, the results do not support models which involve long-range tertiary interactions. Cleavage results with Rh(phen)2phi3+ do, however, indicate that the apposition of several noncanonical bases as well as stem--loop junctions may result in intimately stacked structures with opened major grooves. In particular, on the basis of cleavage results on mutant RNAs, both loops C and E represent structures where the strands constituting each loop are not independent of one another but are intrinsically structured.(ABSTRACT TRUNCATED AT 250 WORDS)     

The beta-amyloid domain is essential for axonal sorting of amyloid precursor protein.

We have analysed the axonal sorting signals of amyloid precursor protein (APP). Wild-type and mutant versions of human APP were expressed in hippocampal neurons using the Semliki forest virus system. We show that wild-type APP and mutations implicated in Alzheimer's disease and another brain beta-amyloidosis are sorted to the axon. By analysis of deletion mutants we found that the membrane-inserted APP ectodomain but not the cytoplasmic tail is required for axonal sorting. Systematic deletions of the APP ectodomain identified two regions required for axonal delivery: one encoded by exons 11-15 in the carbohydrate domain, the other encoded by exons 16-17 in the juxtamembraneous beta-amyloid domain. Treatment of the cells with the N-glycosylation inhibitor tunicamycin induced missorting of wild-type APP, supporting the importance of glycosylation in axonal sorting of APP. The data revealed a hierarchy of sorting signals on APP: the beta-amyloid-dependent membrane proximal signal was the major contributor to axonal sorting, while N-glycosylation had a weaker effect. Furthermore, recessive somatodendritic signals, most likely in the cytoplasmic tail, directed the protein to the dendrites when the ectodomain was deleted. Analysis of detergent solubility of APP and another axonally delivered protein, hemagglutinin, demonstrated that only hemagglutinin formed CHAPS-insoluble complexes, suggesting distinct mechanisms of axonal sorting for these two proteins. This study is the first delineation of sorting requirements of an axonally targeted protein in polarized neurons and indicates that the beta-amyloid domain plays a major role in axonal delivery of APP.     

Structural basis for the red light induced repolarization of tip growth in caulonema cells ofCeratodon purpureus

Summary Two dynamic changes are associated with the phytochrome-regulated phototropic response in tip cells of the mossCeratodon purpureus: a tip-located gradient shift of chlortetracy-cline (CTC)-stained calcium and a structural reorganization of apical microfilaments (MFs). We examined the interdependence of these processes. Cells were treated with the antimicrotubule drug oryzalin, the antimicrofilament drug cytochalasin-D, and the calcium channel blocker nifedipine. respectively. The effects on phototropic growth, on the structural alignment of the cytoskeleton (microtubules, MTs; microfilaments) and on the distribution of CTC-stained calcium were studied under each of these conditions. In gravitropically growing tip cells the apical MFs form a cortical collar-like structure, consisting of actin bundles with a parallel axial alignment. These MFs point towards the presumptive growing point, a weakly stained region in the tip of the cell from which bundles are absent. MTs are present in the cortex and in the endoplasm of the tip, predominantly oriented longitudinally. The MTs converge within the central apex. The cells show a steep tip-to-base CTC-calcium gradient with its highest signal in the central apex. Destruction of MTs by 1 M oryzalin induces several translocational effects: (i) the growing zone and phototropic outgrowth shift from the apex to subapical parts of the cell; (ii) the structural integrity of the apical MFs and the tip-to-base alignment of the CTC-calcium gradient are disturbed; and (iii) the red light induced gradient shift and the reorientation of MFs proceed in an expanded area spanning from the tip to subapical parts of the cell. Cytochalasin-D (10 g/ml) destroys the MFs. Under these conditions tip growth stops and the phototropic outgrowth is suppressed. The apical MT-structure and the CTC-calcium gradient are not influenced by the agent. Unilateral red light still induces the light-directed translocation of the gradient. Tip cells memorize a unilateral irradiation applied during growth inhibition with cytochalasin-D. After recovery in darkness the cells start to grow in the former light direction. The restoration of the MFs precedes the outgrowth. The structural alignment of the rebuilt actin bundles indicates the future growth direction. The calcium channel blocker nifedipine (10 M) also inhibits tip growth and concurrently phototropic outgrowth. Nifedipine destroys the CTC-calcium gradient and apical MFs; MTs are not influenced by the channel blocker.Abbreviations CTC chlortetracycline - DIC differential interference contrast - DMSO dimethyl sulfoxide - EGTA ethyleneglycol-bis-(-aminoethylether) N,N,N-N-tetraacetic acid - MBS 3-maleimidobenzoic acid N-hydroxysuccimide ester - MF microfilament - MT microtubule - MTSB microtubule stabilizing buffer - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - RP rhodamine labeled phalloidin     

Genetic Analysis of the Epidermal Differentiation Complex (EDC) on Human Chromosome 1q21: Chromosomal Orientation, New Markers, and a 6-Mb YAC Contig

The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis. It is localized within 2.05 Mb of region q21 on human chromosome 1. We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619–D1S3628). Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC. Fluorescencein situhybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen–D1S442–D1S498–S100A10–THH–FLG–D1S1664–IVL–SPRR3–SPRR1–SPRR2–LOR–S100A9–S100A8–S100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A1–D1S305–1qtel. These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.     

Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function.

We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8. Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end. In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity). The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure. The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335. Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold. The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data. However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Insulin receptors in syncytitrophoblast and fetal endothelium of human placenta. Immunohistochemical evidence for developmental changes in distribution pattern

The localisation of insulin receptors (IR) was investigated on cryosections of human non-pathologic first trimester and full term placentae by indirect immunohistochemistry with three different monoclonal antibodies (MABS). In placentae from 6 to 10 weeks post-menstruation (p-m.), only syncytiotrophoblast was stained, predominantly that of mesenchymal villi and syncytial sprouts, which are areas of high proliferative activity. In placentae from 11 to 14 weeks p-m., endothelial cells commenced to react with the IR MABS and the syncytiotrophoblast was less intensely labelled than at weeks 6 to 10 p-m. In term placentae, the microvillous membrane of the syncytiotrophoblast showed only patches of weak immunoreactivity. In contrast, the endothelial cells in the placenta but not in the umbilical cord were strongly stained. The amniotic epithelium in the chorionic plate and fibroblasts in the stroma were conspicuously labelled. The data indicate: (1) the receptor density on villous syncytiotrophoblast decreases and that of fetal endothelium increases throughout gestation; (2) syncytiotrophoblast of human term placentae expresses a low level per unit area of surface IR; and (3) the majority of IR in human term placentae is located in fetal endothelium. Apart from yet unknown functional effects of maternal and fetal insulin at the placental barrier, the results suggest a growth promoting effect on the trophoblast of maternal insulin in first trimester as well as developmental effects of fetal insulin on the feto-placental vessels at term.