Female sex pheromone of Cameraria ohridella Desch. and Dim. (Lepidoptera: Gracillariidae): structure confirmation,synthesis and biological activity of (8E,10Z)-8,10-tetradecadienal and some analogues

Mass spectrometric investigations confirmed the structure of the female produced sex pheromone of the horse-chestnut leafminer Cameraria ohridella Desch. and Dim. to be (8E,IOZ)-8,10-tetradecadienal. Pure samples, prepared in a straightforward synthesis, were highly attractive in field tests and proved to be suitable for monitoring of flight activities and population dynamics. In mixtures with the synthetic pheromone, analogues like 9-tridecynal and 7-dodecynyl formate were shown to reduce trap catches. In electroantennographic experiments, pheromone analogues were less active than the pheromone. 9-Tridecynal was the most EAG active analogue tested, followed by 7-dodecyn-1-yl formate and 7-undecyn-1-yl formate.     

The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism

The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants; however, no conditions suitable for the unambiguous assay of the enzyme are known. As a result, all attempts to purify the protein and clone its corresponding gene have failed. By screening for plants that accumulate reduced levels of soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. Using radiotracer-feeding experiments, we have determined that the ref8 mutant is unable to synthesize caffeic acid, suggesting that the mutant is defective in a gene required for the activity or expression of C3H. We have isolated the REF8 gene using positional cloning methods, and have verified that it encodes C3H by expression of the wild-type gene in yeast. Although many previous reports in the literature have suggested that C3H is a phenolase, the isolation of the REF8 gene demonstrates that the enzyme is actually a cytochrome P450-dependent monooxygenase. Although the enzyme accepts p-coumarate as a substrate, it also exhibits significant activity towards other p-hydroxylated substrates. These data may explain the previous difficulties in identifying C3H activity in plant extracts and they indicate that the currently accepted version of the lignin biosynthetic pathway is likely to be incorrect.     

Conservation of the gene structure and membrane-targeting signals of germ cell-specific lamin LIII in amphibians and fish

Targeting of nuclear lamins to the inner nuclear membrane requires CaaX motif-dependent posttranslational isoprenylation and carboxyl methylation. We previously have shown that two variants of lamin LIII (i.e., LIII and LIIIb) in amphibian oocytes are generated by alternative splicing and differ greatly in their membrane association. An extra cysteine residue (as a potential palmitoylation site) and a basic cluster in conjunction with the CaaX motif function as secondary targeting signals responsible for stable membrane association of lamin LIIIb. cDNA sequencing and genomic analysis of the zebrafish Danio rerio lamin LIII uncovers a remarkable conservation of the genomic organization and of the two secondary membrane-targeting signals in amphibians and fish. The expression pattern of lamin LIII genes is also conserved between amphibians and fish. Danio lamin LIII is expressed in diplotene oocytes. It is absent from male germ cells but is expressed in Sertoli cells of the testis. In addition, we provide sequence information of the entire coding sequence of zebrafish lamin A, which allows comparison of all major lamins from representatives of the four classes of vertebrates.     

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Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids and formation of extracellular matrix

In plants, extracellular matrix polymers built from polysaccharides and cuticular lipids have structural and protective functions. The cuticle is found to be ten times thinner in Arabidopsis thaliana (L.) Heynh than in many other plants, and there is evidence that it is unusual in having a high content of α-,ω-dicarboxylic fatty acids (FAs) in its polyesters. We designated the new organ fusion mutant hth-12 after it appeared to be allelic to adhesion of calyx edges (ace) and hothead (hth), upon molecular cloning of the gene by transposon tagging. This mutant is deficient in its ability to oxidize long-chain ω-hydroxy FAs to ω-oxo FAs, which results in leaf polyesters in decreased α-,ω-dicarboxylic FAs and increased ω-hydroxy FAs. These chemical phenotypes lead to disorder of the cuticle membrane structure in hth-12. ACE/HTH is a single-domain protein showing sequence similarity to long-chain FA ω-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA ω-hydroxylases in the ω-oxidation pathway. We show that ACE/HTH is specifically expressed in epidermal cells. It appears very likely therefore that the changes in the amount of α-,ω-dicarboxylic FAs in hth-12 reflect the different composition of cuticular polyesters. The ACE/HTH gene is also expressed in root epidermal cells which do not form a polyester membrane on the exterior surface, thereby making it possible that the end products of the pathway, α-,ω-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.     

Subtypes of melanocytes and melanoma cells distinguished by their intercellular contacts: heterotypic adherens junctions,adhesive associations,and dispersed desmoglein 2 glycoproteins

In the tissue integration of melanocytes and melanoma cells, an important role is attributed to cell adhesion molecules, notably the cadherins. In cultured melanoma cells, we have previously described a more heterogeneous repertoire of cadherins than normal, including some melanoma subtypes synthesizing the desmosomal cadherin, desmoglein 2, out of the desmosomal context. Using biochemical and immunological characterization of junctional molecules, confocal laser scanning, and electron and immunoelectron microscopy, we now demonstrate homo- and heterotypic cell-cell adhesions of normal epidermal melanocytes. In human epidermis, both in situ and in cell culture, melanocytes and keratinocytes are connected by closely aligned membranes that are interspersed by small puncta adhaerentia containing heterotypic complexes of E- and P-cadherin. Moreover, melanocytes growing in culture often begin to synthesize desmoglein 2, which is dispersed over extended areas of intimate adhesive cell-cell associations. As desmoglein 2 is not found in melanocytes in situ, we hypothesize that its synthesis is correlated with cell proliferation. Indeed, in tissue microarrays, desmoglein 2 has been demonstrated in a sizable subset of nevi and primary melanomas. The biological meanings of these cell-cell adhesion molecule arrangements, the possible diagnostic and prognostic significance of these findings, and the implications of the heterogeneity types of melanomas are discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported in parts by grants from the Deutsche Forschungsgemeinschaft to W. K. Peitsch (project PE 896/1) and the Deutsche Krebshilfe to W. W. Franke (project 10-2049).     

In vitro cultivation of Dipetalonema viteae third-stage larvae: evaluation of culture media, serum, and other supplements

Third-stage larvae of Dipetalonema viteae obtained from the tick vector developed to the fourth stage in several cell-free culture systems. Survival and development of larvae in a number of commercially available cell culture media, supplemented with serum and other defined and undefined components, were compared. All cultures were gassed with 5% carbon dioxide in nitrogen. Best survival, growth and development were obtained in stationary cultures containing 1:1 (v/v) mixtures of NCTC 135, either RPMI 1640 or Iscove's Modified Dulbecco's Medium, and a supplement of 20% non-heat-inactivated fetal bovine serum. The importance of the medium composition and physical environment of the culture system, for the survival, growth and development of D. viteae was demonstrated.     

High Affinity Peptide Inhibitors of the Hepatitis C Virus NS3-4A Protease Refractory to Common Resistant Mutants

Hepatitis C virus (HCV) NS3-4A protease is essential for viral replication. All current small molecular weight drugs against NS3-4A are substrate peptidomimetics that have a similar binding and resistance profile. We developed inhibitory peptides (IPs) capping the active site and binding via a novel “tyrosine” finger at an alternative NS3-4A site that is of particular interest for further HCV drug development. The peptides are not cleaved due to a combination of geometrical constraints and impairment of the oxyanion hole function. Selection and optimization through combinatorial phagemid display, protein crystallography, and further modifications resulted in a 32-amino acid peptide with a K_i of 0.53 nm. Inhibition of viral replication in cell culture was demonstrated by fusion to a cell-penetrating peptide. Negligible susceptibility to known (A156V and R155K) resistance mutations of the NS3-4A protease was observed. This work shows for the first time that antiviral peptides can target an intracellular site and reveals a novel druggable site on the HCV protease.     

Expression of simple epithelial type cytokeratins in stratified epithelia as detected by immunolocalization and hybridization in situ

Multi-layered ("stratified") epithelia differ from one-layered ("simple") polar epithelia by various architectural and functional properties as well as by their cytoskeletal complements, notably a set of cytokeratins characteristic of stratified tissue. The simple epithelial cytokeratins 8 and 18 have so far not been detected in any stratified epithelium. Using specific monoclonal antibodies we have noted, in several but not all samples of stratified epithelia, including esophagus, tongue, exocervix, and vagina, positive immunocytochemical reactions for cytokeratins 8, 18, and 19 which in some regions were selective for the basal cell layer(s) but extended into suprabasal layers in others. In situ hybridization with different probes (riboprobes, synthetic oligonucleotides) for mRNAs of cytokeratin 8 on esophageal epithelium has shown, in extended regions, relatively strong reactivity for cytokeratin 8 mRNA in the basal cell layer. In contrast, probes to cytokeratin 18 have shown much weaker hybridization which, however, was rather evenly spread over basal and suprabasal strata. These results, which emphasize the importance of in situ hybridization in studies of gene expression in complex tissues, show that the genes encoding simple epithelial cytokeratins can be expressed in stratified epithelia. This suggests that continual expression of genes coding for simple epithelial cytokeratins is compatible with the formation of squamous stratified tissues and can occur, at least in basal cell layers, simultaneously with the synthesis of certain stratification-related cytokeratins. We also emphasize differences of expression and immunoreactivity of these cytokeratins between different samples and in different regions of the same stratified epithelium and discuss the results in relation to changes of cytokeratin expression during fetal development of stratified epithelia, in response to environmental factors and during the formation of squamous cell carcinomas.