Evaluation of progesterone receptor expression in eosinophils using real-time quantitative PCR

Progesterone has been shown in many instances to have immune-suppressant activities. Most of these activities have been investigated in the light of general immune suppression or with a focus on lymphocytes. However, many clinical and in vitro studies have shown that progesterone also has a suppressive effect on eosinophilia. This effect so far has not been thoroughly investigated. The purpose of this study was to evaluate whether the effect is mediated via the classical progesterone receptor (PR). We developed a new real-time quantitative PCR (RQ-PCR) for the analysis and quantification of expression of the classical PR. The test was first validated both on breast cancer cell lines and on breast cancer biopsies. Subsequently, when using eosinophils isolated from peripheral blood of healthy volunteers, we could not find evidence for the expression of PR. These data suggest that the effects of progesterone on eosinophils are not mediated by the classical PR.     

A two-protein strategy for the functional loading of a cellular replicative DNA helicase

The delivery of a ring-shaped hexameric helicase onto DNA is a fundamental step of DNA replication, conserved in all cellular organisms. We report the biochemical characterization of the bacterial hexameric replicative helicase DnaC of Bacillus subtilis with that of the two replication initiation proteins DnaI and DnaB. We show that DnaI and DnaB interact physically and functionally with the DnaC helicase and mediate its functional delivery onto DNA. Thus, DnaB and DnaI form a pair of helicase loaders, revealing a two-protein strategy for the loading of a replicative helicase. We also present evidence that the DnaC helicase loading mechanism appears to be of the ring-assembly type, proceeding through the recruitment of DnaC monomers and their hexamerization around single-stranded DNA by the coordinated action of DnaI and DnaB.     

Rescue of the neural tube defect of loop-tail mice by a BAC clone containing the Ltap gene

The mouse mutant loop-tail (Lp) is an accepted model for the study of neural tube defects (NTDs) in humans. Whereas Lp/+ heterozygotes show a mild tail defect (looped), homozygous Lp/Lp embryos show a very severe form of NTD, with a completely open neural tube from the hindbrain region to the caudal portion of the spinal cord (craniorachischisis). We have recently identified a positional candidate for Lp on chromosome 1, designated as Ltap. Here, we have used an in vivo complementation approach in transgenic mice to attempt to correct the looped-tail phenotype with a bacterial artificial chromosome clone (BAC280A23) that harbors a full-length copy of the Ltap gene. Genotype:phenotype correlations in Lp/+ heterozygotes carrying BAC280A23 show that this clone can rescue the looped-tail phenotype in two independent founder lines (P < 0.05 and P < 0.0001). Importantly, BAC280A23 is also observed to rescue the lethal NTD of Lp/Lp homozygotes, because several viable transgenic Lp/Lp mice could be identified and appeared normal (P < 0.05). Results from these gain-of-function transgenic animals strongly suggest that the positional candidate Ltap present in this BAC is indeed the gene that is defective in loop-tail.     

Prenylated isoflavonoids from Millettia pervilleana

From the root bark of Millettia pervilleana, which had shown significant cytotoxic activity, a 3-phenylcoumarin, named pervilleanine, two new pterocarpans, pervilline and pervillinine, and one known, emoroidocarpan, were isolated in addition to rotenone and 3alpha-hydroxyrotenone. The anticancer activity of two previously isolated isoflavanones, pervilleanone and 3'-O-demethylpervilleanone is reported.     

Treatment of complex interphalangeal joint fractures with dynamic external traction: a series of 20 cases

Data are reported for a series of 20 patients who were treated with the pins and rubbers traction system for fractures of the proximal interphalangeal joints of the long fingers. This technique allows fracture reduction with external dynamic traction and immediate active mobilization. Two patients in the series were lost to follow-up monitoring. For two others, the pins and rubbers traction system needed to be removed early (during the first week) because of intolerance or infection. Sixteen patients who were reexamined after minimal follow-up periods of 1 year demonstrated a mean active range of motion of 85.9 degrees for the injured joint; only one patient experienced intermittent pain.     

Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant

We examined anoxic and aerobic basins and an anaerobic digestor of a municipal wastewater treatment plant for the presence of novel planctomycete-like diversity. Three 16S rRNA gene libraries were constructed by using a 16S rRNA-targeted universal reverse primer and a forward PCR primer specific for Planctomyces: Phylogenetic analysis of 234 16S rRNA gene sequences defined 110 operational taxonomic units. The majority of these sequences clustered with the four known genera, Pirellula (32%), Planctomyces (18.4%), Gemmata (3.8%), and Isosphaera (0.4%). More interestingly, 42.3% of the sequences appeared to define two distantly separated monophyletic groups. The first group, represented by 35.5% of the sequences, was related to the Planctomyces group and branched as a monophyletic cluster. It exhibited between 11.9 and 20.3% 16S rRNA gene sequence dissimilarity in comparisons with cultivated planctomycetes. The second group, represented by 6.8% of the sequences, was deeply rooted within the Planctomycetales tree. It was distantly related to the anammox sequences (level of dissimilarity, 20.3 to 24.4%) and was a monophyletic cluster. The retrieved sequences extended the intralineage phylogenetic depth of the Plantomycetales from 23 to 30.6%. The lineages described here may have a broad diversity of undiscovered biochemical and metabolic novelty. We developed a new 16S rRNA-targeted oligonucleotide probe and localized members of one of the phylogenetic groups using the fluorescent in situ hybridization technique. Our results indicate that activated sludge contains very diverse representatives of this group, which grow under aerobic and anoxic conditions and even under anaerobic conditions. The majority of species in this group remain poorly characterized.     

Proteomics application exercise of the Swiss Proteomics Society: report of the SPS'02 session

After the success of the mass spectrometry (MS) round table that was held at the first Swiss Proteomics Society congress (SPS'01) in Geneva, the SPS has organized a proteomics application exercise and allocated a full session at the SPS'02 congress. The main objective was to encourage the exchange of expertise in protein identification, with a focus on the use of mass spectrometry, and to create a bridge between the users' questions and the instrument providers' solutions. Two samples were sent to fifteen interested labs, including academic groups and MS hardware providers. Participants were asked to identify and partially characterize the samples. They consisted of a complex mixture of peptide/proteins (sample A) and an almost pure recombinant peptide carrying post-translational modifications (sample B). Sample A was an extract of snake venom from the species Bothrops jararaca. Sample B was a recombinant and modified peptide derived from the shrimp Penaeus vannamei penaeidin 3a. The eight labs that returned results reported the use of a wide range of MS instrumentation and techniques. They mentioned a variety of time and manpower allocations. The origin of sample A was generally identified together with a number of database protein entries. The difficulty of the sample identification lay in the incomplete knowledge of the Bothrops species genome sequence and is discussed. Sample B was generally and correctly identified as penaeidin. However, only one group reported the full primary structure. Interestingly, the approaches were again varied and are discussed in the text.     

Synthesis of 5-substituted-2,3-dihydro-1,4-benzoxathiins: biological evaluation as melatonin receptors ligands

The synthesis of benzoxathiins bearing a retroamide function is described from 8-hydroxythiochroman, the key step involving the synthesis of the benzoxathiin ring through a sulfonium salt. These new melatonin analogues were evaluated on human receptors MT1 and MT2 and have a similar affinity to that of melatonin itself.     

Novel roles for the flagellum in cell morphogenesis and cytokinesis of trypanosomes

Flagella and cilia are elaborate cytoskeletal structures conserved from protists to mammals, where they fulfil functions related to motility or sensitivity. Here we demonstrate novel roles for the flagellum in the control of cell size, shape, polarity and division of the protozoan Trypanosoma brucei. To investigate the function of the flagellum, its formation was perturbed by inducible RNA interference silencing of com ponents required for intraflagellar transport, a dynamic process necessary for flagellum assembly. First, we show that down-regulation of intraflagellar transport leads to assembly of a shorter flagellum. Strikingly, cells with a shorter flagellum are smaller, with a direct correlation between flagellum length and cell size. Detailed morphogenetic analysis reveals that the tip of the new flagellum defines the point where cytokinesis is initiated. Secondly, when new flagellum formation is completely blocked, non-flagellated cells are very short, lose their normal shape and polarity, and fail to undergo cytokinesis. We show that flagellum elongation controls formation of cytoskeletal structures (present in the cell body) that act as molecular organizers of the cell.