Do NOE distances contain enough information to assess the relative populations of multi-conformer structures?

Summary The feasibility of determining the relative populations of multi-conformer structures from NOE-derived distances alone is assessed. Without cross-validation of the NOE restraints, any population ratio can be refined to a similar quality of the fit. Complete cross-validation provides a less biased measure of fit and allows the estimation of the correct population ratio when used in conjunction with very tight distance restraints. With the qualitative distance restraints most commonly used in NMR structure determination, cross-validation is unsuccessful in providing the correct answer. Other experimental sources are therefore needed to determine relative populations of multi-conformer structures.To whom correspondence should be addressed.     

Analysis of the protein kinase activity of moss phytochrome expressed in fibroblast cell culture

In the moss Ceratodon purpureus a phytochrome gene encodes a phytochrome type (PhyCer) which has a C-terminal domain homologous to the catalytic domain of eukaryotic protein kinases (PKs). PhyCer exhibits sequence conservation to serine/ threonine as well to tyrosine kinases. Since PhyCer is expressed very weakly in moss cells, to investigate the proposed PK activity of PhyCer, we overexpressed PhyCer transiently in fibroblast cells. For this purpose we made a chimeric receptor, EC-R, which consists of the extracellular, the membrane-spanning and the juxtamembrane domains of the human epidermal growth-factor receptor (EGF-R) linked to the PK catalytic domain of PhyCer (CerKin). The expression of EC-R in transiently transfected cells was confirmed with antibodies directed against the extracellular domain of EGF-R or against CerKin. Both EGF-R and EC-R were immunoprecipitated from lysates of overexpressing cells with antibodies against the extracellular domain of EGF-R. Phosphorylation experiments were performed with the immunoprecipitates and the phosphorylation products were subjected to phosphoamino acid analysis. Phosphorylation products specifically obtained with EC-R-transfected cells exhibit phosphorylation on serine and threonine residues. In EC-R transfected cells the endogenous EGF-R showed enhanced phosphorylation of serine and threonine residues compared to EGF-R immuno-precipitated from control cells. Although CerKin is closest to the catalytic domain of a protein tyrosine kinase from Dictyostelium discoideum, EC-R does not appear to phosphorylate tyrosine residues in vitro. From our data we conclude that PhyCer carries an active PK domain capable of phosphorylating serine and threonine residues.Abbreviations CerKin protein kinase catalytic domain of PhyCer - EC-R chimeric receptor consisting of the extracellular, the membrane spanning and the juxtamembrane domains of the human epidermal growth factor receptor (EGF-R) linked to the protein kinase catalytic domain of PhyCer - EGF-R epidermal growth factor receptor - mAb monoclonal antibody - PhyCer phytochrome gene in Ceratodon encoding a phytochrome type which has a C-terminal domain homologous to the catalytic domain of eucaryotic protein kinases - PK protein kinase - PVDF polyvinyl difluoride - Ser serine - Thr threonine - Tyr tyrosine Dr. Patricia Algarra was supported by the Alexander von Humboldt Foundation, Germany. This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Bonn, Germany.     

Population structure and mycelial phenotypic variability of the ectomycorrhizal basidiomycete Laccaria bicolor (Maire) Orton

Mating type allele distribution and phenotypic variability were investigated in field populations of Laccaria bicolor. Sporophores associated with Norway spruce (Picea abies), colonized by natural sources of inoculum and growing in a seed orchard, were sampled to obtain dikaryotic strains and assay their phenotypic variability for traits important to the symbiosis. Basid-iospores were also collected for mating type analysis of different mycelia. Four sporophore mating types were identified containing seven A and five B factors. Out-breeding efficiency was estimated at 73.8% and the population was slightly inbred. Crosses with previously characterized L. bicolor strains from two nearby populations identified in total six sporophore mating types and ten A and nine B factors, for an estimated outbreeding efficiency (85.7%) similar to previous studies of more spatially disparate Laccaria spp. populations. Dikaryotic strains were tested for mycelial growth rate, as a measure of their competitive ability, on agar media containing a soluble (NaH₂PO₄), or an insoluble (CaHPO₄) phosphate source. Their ability to solubilize the latter was also tested to assess their relative capacity to access insoluble, inorganic phosphate. In most cases, significant variation was detected among strains from the same site for all variables. On three sites (VC4, VC5 and VC7), each determined previously to possess a uniform mycelial genotype, phenotypic variability was likely due to epigenetic variation among different strains of the same genotype. Possible evidence for dikaryon-monokaryon crosses was observed in vivo on one sample site (VC2) where adjacent mycelia shared two mating factors. The phenotypic variability of different mycelial genotypes reflected their genetic variability observed as mating type allele diversity and underlined the importance of basidiospore dispersal in introducing new genotypes into the population. The reproductive strategies of L. bicolor are discussed and compared to those of other basidiomycete species.     

The three polymorphic chains of human Hb-F studied in normal Italian newborns

Both the known polymorphisms of human Hb-F have been investigated here. Two similar groups of full-term newborns have been considered: pure F-globin has been studied by suitable fingerprinting techniques in the first group, while isoelectrofocusing techniques have been employed in the second. The results clearly indicate that G/A gamma polymorphism can be correctly determined with both the procedures, while the T/I gamma polymorphism cannot, because IEF is not sensitive enough towards low quantities of A gamma T chains. Moreover, in the first group of newborns, the A gamma T gene frequency (P) is 0.23, while the total G/A gamma ratio of both groups is about 7/3, the two data being in full agreement with those of the literature. Furthermore, additional reports show that the main functional parameters of the whole neonatal blood are not significantly different either in the presence or in the absence of A gamma T chains.     

Molecular and functional anomalies in two new mutant glucose-phosphate-isomerase variants with enzyme deficiency and chronic hemolysis

Summary Two new deficient glucose-phosphate-isomerase (GPI) variants have been described in patients suffering from severe chronic hemolytic anemias. The patients' parents were consanguineous, such that the patients were true homozygotes for the mutated GPI genes. In both cases the main cause of the defect in enzyme activity was molecular instability of the mutated GPI molecules, their catalytic activity being nearly normal.GPI Paris was characterized by a slow electrophoretic migration and, above all, a drastically altered affinity for the substrates glucose-6-phosphate (decreased) and fructose-6-phosphate (increased). GPI Enfants malades exhibited a slightly reduced electrophoretic mobility, an abnormal curve of the activity in function of pH, and an abnormal ratio of maximal velocity in the backward direction (fructose-6-phosphate»glucose-6-phosphate) to that in the forward direction (glucose-6-phosphate»fructose-6-phosphate).No clear relation could be proved between the kinetic abnormalities of the mutant GPI variants on the one hand and the metabolic changes of the GPI-deficient red cells and the severity of hemolysis on the other.Finally we emphasized the possible role of the impairment of hexosemonophosphate pathway in the reduction of viability of the GPI-deficient red cells.