Expression, purification, and structural characterization of human histone H4

Recombinant human histone H4 (hH4) was produced in milligrams quantities in Escherichia coli, without altering the codons of the original cDNA sequence. The hH4 cDNA was subcloned into the pQE30 expression vector, in frame with a sequence encoding an N-terminal stretch of six histidine residues. Purification to electrophoretic homogeneity was obtained by nickel-chelating chromatography, followed by gel filtration. The final yield of the entire expression and purification process was about 1 mg of pure histone H4 per liter of bacterial culture. SDS-PAGE analysis showed for the recombinant H4 a molecular weight corresponding to the expected one (12,535 Da). Circular dichroism spectroscopy was used to estimate the secondary structural composition of recombinant histone, when it is isolated from the physiological core particle. It was observed that under these conditions histone H4 exhibits an altered secondary conformation. In order to induce the recombinant histone to assume a conformation more similar to the one measured when it is organized inside the nucleosome, we resuspended it in buffers at increasing ionic strengths and in the presence of different concentrations of trifluoroethanol. We tried also to mimic the physiological situation of histone H4 by adding an equimolar amount of a commercial DNA to the protein solution. Finally, an estimation of protein thermal stability was evaluated by spectropolarimetry.     

Peroxisome senescence in human fibroblasts

The molecular mechanisms of peroxisome biogenesis have begun to emerge; in contrast, relatively little is known about how the organelle functions as cells age. In this report, we characterize age-related changes in peroxisomes of human cells. We show that aging compromises peroxisomal targeting signal 1 (PTS1) protein import, affecting in particular the critical antioxidant enzyme catalase. The number and appearance of peroxisomes are altered in these cells, and the organelles accumulate the PTS1-import receptor, Pex5p, on their membranes. Concomitantly, cells produce increasing amounts of the toxic metabolite hydrogen peroxide, and we present evidence that this increased load of reactive oxygen species may further reduce peroxisomal protein import and exacerbate the effects of aging.     

Myxobolus desaequalis n. sp. (Myxozoa,Myxosporea), parasite of the Amazonian freshwater fish,Apteronotus albifrons (Teleostei,Apteronotidae)

Myxobolus desaequalis n. sp. is described from the gill lamellae of the freshwater fish Apteronotus albifrons, collected in the Amazon River, near the city of Salvaterra, Brazil. Large spherical plasmodia filled with disporic pansporoblasts and spores were observed. Ellipsoidal to pyriform spores are 18.3 microm length x 11.2 microm width x 4.4 microm thickness. The anterior end of the spores contain two extremely unequal pyriform polar capsules measuring: (larger): 11.2 microm length, 4.9 microm width, and an isofilar polar filament with 11 to 12 turns obliquely to the longitudinal axis; (smaller): 4.6 microm length, 2.8 microm width, and an isofilar polar filament with 4 to 5 turns, obliquely to the longitudinal axis.     

An european pupil project linked to the scientific aims of the experiment AQUARIUS-XENOPUS on the taxi Soyuz flight Andromede to ISS

The German-French biological experiment AQUARIUS-XENOPUS which flew on the Soyuz flight Andromede to the International Space Station ISS (launched October 21, 2001 in Baikonour/Kazakhstan) was extended by an outreach project. Pupils of class 10 to 12 from Ulm/D and Nancy-Tomblaine/F studied swimming behavior of Xenopus tadpoles on ground. They were instructed to perform all experimental steps following the protocol of similar video recordings on ISS. After the flight, they evaluated the kinetics of swimming of both ground controls and space animals. The pupil project included theoretical components to introduce them to the field of gravitational biology. One feature of the project was the exchange of ideas between pupils by meetings which took place in Ulm (June 2001), Nancy (February 2002) and Paris (May 2002). We consider our approach as a successful way to include young people in space experiments on a cheap cost level and to bring ideas of gravitational biology into the curricula of European schools.     

Genotype to phenotype: associations,errors and complexity

The Keystone Symposium on Genotype to Phenotype: Focus on Disease was held in Santa Fe, New Mexico, USA, from 19 to 24 February 2002.     

Interaction of linear and cyclic peptide antagonists at the human B(2) kinin receptor

The ligand receptor interactions involving the C-terminal moiety of kinin B(2) receptor antagonists Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7gamma-10alpha)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser(111)Ala and Ser(111)Lys mutant human kinin B(2) receptors. Icatibant and [Lys(10)]-Icatibant maintained the same high affinity towards the three receptors. For Icatibant-NH(2), [Ala(10)]-Icatibant, MEN 11270 and [Glu(10)]-MEN 11270, the changes in affinity at the WT and Ser(111)Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser(111) residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg(10)]-Icatibant and [desArg(10)]-Icatibant-NH(2), moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg(10) side chain and the C-terminal free carboxylic function favours a high affinity interaction.     

The neuron-specific Rai (ShcC) adaptor protein inhibits apoptosis by coupling Ret to the phosphatidylinositol 3-kinase/Akt signaling pathway

Rai is a recently identified member of the family of Shc-like proteins, which are cytoplasmic signal transducers characterized by the unique PTB-CH1-SH2 modular organization. Rai expression is restricted to neuronal cells and regulates in vivo the number of postmitotic sympathetic neurons. We report here that Rai is not a common substrate of receptor tyrosine kinases under physiological conditions and that among the analyzed receptors (Ret, epidermal growth factor receptor, and TrkA) it is activated specifically by Ret. Overexpression of Rai in neuronal cell lines promoted survival by reducing apoptosis both under conditions of limited availability of the Ret ligand glial cell line-derived neurotrophic factor (GDNF) and in the absence of Ret activation. Overexpressed Rai resulted in the potentiation of the Ret-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt. Notably, increased Akt phosphorylation and PI3K activity were also found under basal conditions, e.g., in serum-starved neuronal cells. Phosphorylated and hypophosphorylated Rai proteins form a constitutive complex with the p85 subunit of PI3K: upon Ret triggering, the Rai-PI3K complex is recruited to the tyrosine-phosphorylated Ret receptor through the binding of the Rai PTB domain to tyrosine 1062 of Ret. In neurons treated with low concentrations of GDNF, the prosurvival effect of Rai depends on Rai phosphorylation and Ret activation. In the absence of Ret activation, the prosurvival effect of Rai is, instead, phosphorylation independent. Finally, we showed that overexpression of Rai, at variance with Shc, had no effects on the early peak of mitogen-activated protein kinase (MAPK) activation, whereas it increased its activation at later time points. Phosphorylated Rai, however, was not found in complexes with Grb2. We propose that Rai potentiates the MAPK and PI3K signaling pathways and regulates Ret-dependent and -independent survival signals.     

A novel locus for Meckel-Gruber syndrome,MKS3, maps to chromosome 8q24

Meckel-Gruber syndrome (MKS), the most common monogenic cause of neural tube defects, is an autosomal recessive disorder characterised by a combination of renal cysts and variably associated features, including developmental anomalies of the central nervous system (typically encephalcoele), hepatic ductal dysplasia and cysts, and polydactyly. Locus heterogeneity has been demonstrated by the mapping of the MKS1locus to 17q21-24 in Finnish kindreds, and of MKS2 to 11q13 in North African-Middle Eastern cohorts. In the present study, we have investigated the genetic basis of MKS in eight consanguineous kindreds, originating from the Indian sub-continent, that do not show linkage to either MKS1 or MKS2. We report the localisation of a third MKS locus ( MKS3) to chromosome 8q24 in this cohort by a genome-wide linkage search using autozygosity mapping. We identified a 26-cM region of autozygosity between D8S586 and D8S1108 with a maximum cumulative two-point LOD score at D8S1179 ( Z(max)=3.04 at theta=0.06). A heterogeneity test provided evidence of one unlinked family. Exclusion of this family from multipoint analysis maximised the cumulative multipoint LOD score at locus D8S1128 ( Z(max)=5.65). Furthermore, a heterozygous SNP in DDEF1, a putative candidate gene, suggested that MKS3 mapped within a 15-cM interval. Comparison of the clinical features of MKS3-linked cases with reports of MKS1- and MKS2-linked kindreds suggests that polydactyly (and possibly encephalocele) appear less common in MKS3-linked families.     

Development of Genus/Species-Specific PCR Analysis for Identification of <Emphasis Type="Italic">Carnobacterium</Emphasis> Strains

Heterofermentative lactic acid bacteria belonging to the genus Carnobacterium are currently divided into seven different species, C. piscicola, C. mobile, C. gallinarum, C. inhibens, C. divergens, C. funditum, and C. alterfunditum. 16S rDNA-targeted PCR assay was carried out for the identification of the genus Carnobacterium. In addition, type strains of all Carnobacterium species were analyzed by 16S–23S rDNA intergenic spacer analysis in comparison with type strains of phylogenetically related lactic acid bacteria. These methods enabled the identification and the discrimination among Carnobacterium species and the other phylogenetically related lactic acid bacteria. Likewise, analogous results were obtained by restriction analysis of amplified 16S rDNA performed with HaeIII and HinfI as restriction enzymes. Received: 25 July 2001 / Accepted: 19 October 2001     

Pseudopodium dynamics and rapid cell movement in Dictyostelium Ras pathway mutants

Loss of either of the Ras pathway members RasS or GefB causes growing Dictyostelium cells to move aberrantly rapidly. In this study, we describe the changes in motility that underlie these phenotypes using computer-assisted 3D dynamic image analysis. Unexpectedly, the two mutants use different mechanisms to achieve rapid migration. The rasS(-) cells' motility is characterised by highly dynamic cell morphology, with rapidly extending and retracting pseudopodia. The gefB(-) cells do not have an unusually dynamic morphology, and achieve their efficient translocation by the continual remodelling of an existing dominant anterior pseudopodium. In spite of these dramatic changes in pseudopodium behaviour, the underlying motility cycle of both mutants remains normal. The levels of F-actin in both mutant cell lines are significantly elevated with respect to the wild-type parental cells, suggesting a possible biochemical basis for these emphatic phenotypes.