Preparation of O-phosphotyrosine-containing peptides by Fmoc solid-phase synthesis: Evaluation of several Fmoc-Tyr(PO3R2)-OH derivatives

Summary The synthesis of two model Tyr(P)-containing peptides using Fmoc-Tyr(PO₃ tBu₂)-OH, Fmoc-Tyr(PO₃Bzl₂)-OH and Fmoc-Tyr(PO₃H₂)-OH established that the t-butylphosphate-protected derivative was the preferred derivative for use in Fmoc solid-phase peptide synthesis, since it afforded phosphopeptides in high purity and with the lowest amount of Tyr-peptide contamination. In addition, this study confirmed that commercially available Fmoc-Tyr(PO₃H₂)-OH is also suitable for use in Fmoc solid-phase synthesis but gives less pure phosphopeptides, along with the generation of 1–4% of the tyrosine-containing peptide for the model sequences studied. In view of the good performance of Fmoc-Tyr(PO₃ tBu₂)-OH, a large-scale three-step synthetic procedure was developed which involved phenacyl protection of the carboxyl group, phosphite-triester phosphorylation of the tyrosyl hydroxyl using di-t-butyl N,N-diethylphosphoramidite, and final removal of the phenacyl group by zinc reduction in acetic acid.Abbreviations BOP benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate - tBu t-butyl - Bzl benzyl - DBU 1,8-diazabicyclo[5,4,0]undec-7-ene - DMF N,N-dimethylformamide - EDT ethanedithiol - Fmoc 9-fluorenylmethoxycarbonyl - HOBt N-hydroxybenzotriazole - HPLC high performance liquid chromatography - NMM N-methylmorpholine - Pac phenacyl - TFA trifluoroacetic acid - THF tetrahydrofuran - Tyr(P) O-phosphotyrosine     

Evaluation of genes involved in Norway lobster (<Emphasis Type="Italic">Nephrops norvegicus</Emphasis>) female sexual maturation using transcriptomic analysis

In marine ecosystems, the most significant migration observed in terms of biomass distribution is the one connected with the vertical movements in the water column. In the present study, the vertical profiles of the mesopelagic shrimps Gennadas elegans, Eusergestes arcticus, Sergia robusta, and the epipelagic Parasergestes vigilax in the Balearic Sea (western Mediterranean), during the stratified (summer) and non-stratified (autumn) hydrographic conditions, were investigated through their ontogeny, from the larval to adult stages. The mesopelagic adults were observed to move down to the deeper layers during the night more than during the daylight hours. Most larvae aggregated within the limits of the upper water column. The P. vigilax larvae were collected only during the stratified period. The first two larval stages vertical distribution indicates that the mesopelagic crustacean spawning could occur at greater depths. During the non-stratified period, the larvae of the mesopelagic species tended to remain at about 500 m depth at night, rising towards the upper layers at sunrise. Vertical patterns are discussed, as strategies associated with predator–prey trade-offs. To our knowledge, the present study is the first such attempt to jointly analyze the vertical migrations of the developmental stages of the pelagic shrimps in the Mediterranean Sea.     

Dual nature of translational control by regulatory BC RNAs

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3' stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems.     

A phylogeny of howler monkeys (Cebidae: Alouatta) based on mitochondrial, chromosomal and morphological data

The current taxonomic status of the species and subspecies belonging to the genus Alouatta is addressed by combined phylogenetic analysis using morphological, kariotipyc and molecular data (mitochondrial genes cytocrome oxidase II and cytochrome B). Our result demonstrated that Alouatta palliata is the most basal taxon for the genus in concordance with previous studies, as well as showing the validity of the taxon Alouatta sara as a species. Also our analysis shows that the sex chromosome has evolved from a XY/XX system to a X1X2Y1Y2/X1X1X2X2 system within the genus, as well as an increase in the size and complexity of the hioideal bone.     

Activity of the adenosine deaminase promoter in transgenic mice.

The promoter of the human gene for adenosine deaminase (ADA) is extremely G/C-rich, contains several G/C-box motifs (GGGCGGG) and lacks any apparent TATA or CAAT boxes. These features are commonly found in promoters of genes that lack a strong tissue specificity, and are referred to as "housekeeping genes". Like other housekeeping genes, the ADA gene is expressed in all tissues. However, there is a considerable variation in the levels of expression of the ADA protein in different tissues. In order to study the activity of the ADA promoter, transgenic mice were generated that harbor a chimeric gene composed of the ADA promoter linked to a reporter gene encoding the bacterial enzyme Chloramphenicol Acetyl Transferase (CAT). These mice reproducibly showed CAT expression in all tissues examined, including the hemopoietic organs (spleen, thymus and bone marrow). However, examination of the actual cell types expressing the CAT gene revealed the ADA promoter to be inactive in the hemopoietic cells. This was substantiated by a transplantation experiment in which bone marrow from ADA-CAT transgenic mice was used to reconstitute the hemopoietic compartment of lethally irradiated mice. The engrafted recipients revealed strongly reduced CAT activity in their hemopoietic organs. The lack of expression in hemopoietic cells was further shown to be correlated with a hypermethylated state of the transgene. Combined, our data suggest that the ADA promoter sequences tested can direct expression in a wide variety of tissues as expected for a regular housekeeping gene promoter. However, the activity of the ADA promoter fragment did not reflect the tissue-specific variations in expression levels of the endogenous ADA gene. Additionally, regulatory elements are needed for expression in the hemopoietic cells.     

Ovarian mesothelial and extramesothelial cells in interactive culture

Summary The ovarian mesothelium (OM) represents the tissue of origin of ovarian epithelial cancer. To gain insight into the regulation of this tissue, OM organoids and submesothelial ovarian stromal cells (SC) were isolated from New Zealand White rabbits by a stepwise tissue dispersal technique, while granulosa cells (GC) were aspirated from mature follicles (14±4 groups/animal). OM and SC dispersal were sequentially accomplished by: a) 1-h incubation in collagenase type I (300 U/ml), gentle scraping of the ovarian surface, and 1 g sedimentation of OM organoids (equivalent to 0.93±0.40 × 10⁶ cells/animal) on 5% bovine serum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%–300 U/ml) under periodical resuspension and gentle scraping of SC (1.40±0.25 × 10⁶/animal) from OM-denuded ovaries. After a week-long in vitro expansion, OM cells (OMC) were cultured alone and with SC or GC within monocameral vessels or bicameral transfilter vessels in serumless, fibronectinrich (4μg/ml) HL-1 medium. After 7 d of contact cell-cell interaction, cytokeratin-positive OMC became surrounded by fibroblastoid, vimentin-positive SC or by cytokeratin and vimentin-weakly positive GC. Filter-bound OMC humorally interacting with underlying SC or GC displayed a biphasic, epithelioid and spindle, morphology with universal cytokeratin expression. Bromo-2′-deoxyuridine (BrdU) immunoperoxidase revealed mean cell proliferation indices of 14.88% for OMC cultured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monocameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vessels over GC or SC, respectively. This model provides an experimental tool for investigating the unexplored role of stromal-mesothelial interaction in OM pathobiology.