Global gene expression profiling and cluster analysis in Xenopus laevis

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.     

Effects of melatonin on behavioral dopaminergic supersensitivity

This study examines the effects of melatonin on dopaminergic supersensitivity induced by long-term treatment with haloperidol in rats. Enhancements of spontaneous general activity in an open-field and of stereotyped behavior induced by apomorphine after abrupt withdrawal from long-term treatment with haloperidol were used as experimental parameters for dopaminergic supersensitivity. Experiment 1 was conducted to investigate the effects of melatonin on the development of dopaminergic supersensitivity, and experiment 2 was conducted to investigate the effects of melatonin on the development as well as on expression of dopaminergic supersensitivity. Rats of both experiments were long-term treated with saline or haloperidol concomitant to saline or melatonin. In experiment 1 behavioral observations were performed after abrupt withdrawal from long-term treatment. In experiment 2 behavioral observations were performed 1 hour after an acute injection of saline or melatonin, administered after the abrupt withdrawal from long-term treatment. Both behavioral parameters used showed the development of central dopaminergic supersensitivity in rats treated with haloperidol since 24 hours after abrupt withdrawal. Concomitant treatment with melatonin intensified haloperidol-induced dopaminergic supersensitivity, observed 72 hours after withdrawal. Melatonin treatment per se also induced behavioral supersensitivity evaluated by both open-field and stereotyped behaviors, although it was more fugacious than that presented by haloperidol. Acute treatment with melatonin reverted the enhancement of the haloperidol-induced dopaminergic supersensitivity produced by concomitant long-term treatment with melatonin, as well as melatonin-induced dopaminergic supersensitivity per se. Our results support previous evidence of antidopaminergic effects of melatonin and demonstrate that repeated administration of this hormone modifies the plasticity of behaviors mediated by central dopaminergic systems.     

Functional avidity of tumor antigen-specific CTL recognition directly correlates with the stability of MHC/peptide multimer binding to TCR.

Avidity of Ag recognition by tumor-specific T cells is one of the main parameters that determines the potency of a tumor rejection Ag. In this study we show that the relative efficiency of staining of tumor Ag-specific T lymphocytes with the corresponding fluorescent MHC class I/peptide multimeric complexes can considerably vary with staining conditions and does not necessarily correlate with avidity of Ag recognition. Instead, we found a clear correlation between avidity of Ag recognition and the stability of MHC class I/peptide multimeric complexes interaction with TCR as measured in dissociation kinetic experiments. These findings are relevant for both identification and isolation of tumor-reactive CTL.     

New broad-host-range promoter probe vectors based on the plasmid RK2 replicon

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.     

Lewis acid catalysed methylation of N‐(9H‐fluoren‐9‐yl)methanesulfonyl (Fms) protected lipophilic α‐amino acid methyl esters

This work reports an efficient Lewis acid catalysed N‐methylation procedure of lipophilic α‐amino acid methyl esters in solution phase. The developed methodology involves the use of the reagent system AlCl₃/diazomethane as methylating agent and α‐amino acid methyl esters protected on the amino function with the (9H‐fluoren‐9‐yl)methanesulfonyl (Fms) group. The removal of Fms protecting group is achieved under the same conditions to those used for Fmoc removal. Thus the Fms group can be interchangeable with the Fmoc group in the synthesis of N‐methylated peptides using standard Fmoc‐based strategies. Finally, the absence of racemization during the methylation reaction and the removal of Fms group were demonstrated by synthesising a pair of diastereomeric dipeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

On the first record of the genus Octopoteuthis (Cephalopoda: Octopoteuthidae) in the Sardinian Channel (central western Mediterranean Sea)

A specimen of the rare squid genus Octopoteuthis (Cephalopoda: Octopoteuthidae) from 1600 m in the Sardinian Channel (central western Mediterranean) is described. Meristic and morphological data are provided. The specimen was a spent female and details of the reproductive system and implanted spermatangia are reported. Genetic analysis of this specimen and comparison with those available for species in this genus were carried out. Due to issues associated with the taxonomy of the genus Octopoteuthis, it was not possible to identify the specimen to the species level. The taxonomy of the genus is discussed.     

NY-ESO-1-specific circulating CD4+ T cells in ovarian cancer patients are prevalently T(H)1 type cells undetectable in the CD25+ FOXP3+ Treg compartment

Spontaneous CD4(+) T-cell responses to the tumor-specific antigen NY-ESO-1 (ESO) are frequently found in patients with epithelial ovarian cancer (EOC). If these responses are of effector or/and Treg type, however, has remained unclear. Here, we have used functional approaches together with recently developed MHC class II/ESO tetramers to assess the frequency, phenotype and function of ESO-specific cells in circulating lymphocytes from EOC patients. We found that circulating ESO-specific CD4(+) T cells in EOC patients with spontaneous immune responses to the antigen are prevalently T(H)1 type cells secreting IFN-γ but no IL-17 or IL-10 and are not suppressive. We detected tetramer(+) cells ex vivo, at an average frequency of 1:25,000 memory cells, that is, significantly lower than in patients immunized with an ESO vaccine. ESO tetramer(+) cells were mostly effector memory cells at advanced stages of differentiation and were not detected in circulating CD25(+)FOXP3(+)Treg. Thus, spontaneous CD4(+) T-cell responses to ESO in cancer patients are prevalently of T(H)1 type and not Treg. Their relatively low frequency and advanced differentiation stage, however, may limit their efficacy, that may be boosted by immunogenic ESO vaccines.     

Identification and sequencing of beta-myrcene catabolism genes from Pseudomonas sp. strain M1.

The M1 strain, able to grow on beta-myrcene as the sole carbon and energy source, was isolated by an enrichment culture and identified as a Pseudomonas sp. One beta-myrcene-negative mutant, called N22, obtained by transposon mutagenesis, accumulated (E)-2-methyl-6-methylen-2,7-octadien-1-ol (or myrcen-8-ol) as a unique beta-myrcene biotransformation product. This compound was identified by gas chromatography-mass spectrometry. We cloned and sequenced the DNA regions flanking the transposon and used these fragments to identify the M1 genomic library clones containing the wild-type copy of the interrupted gene. One of the selected cosmids, containing a 22-kb genomic insert, was able to complement the N22 mutant for growth on beta-myrcene. A 5,370-bp-long sequence spanning the region interrupted by the transposon in the mutant was determined. We identified four open reading frames, named myrA, myrB, myrC, and myrD, which can potentially code for an aldehyde dehydrogenase, an alcohol dehydrogenase, an acyl-coenzyme A (CoA) synthetase, and an enoyl-CoA hydratase, respectively. myrA, myrB, and myrC are likely organized in an operon, since they are separated by only 19 and 36 nucleotides (nt), respectively, and no promoter-like sequences have been found in these regions. The myrD gene starts 224 nt upstream of myrA and is divergently transcribed. The myrB sequence was found to be completely identical to the one flanking the transposon in the mutant. Therefore, we could ascertain that the transposon had been inserted inside the myrB gene, in complete agreement with the accumulation of (E)-2-methyl-6-methylen-2,7-octadien-1-ol by the mutant. Based on sequence and biotransformation data, we propose a pathway for beta-myrcene catabolism in Pseudomonas sp. strain M1.