CFC syndrome: a syndrome distinct from Noonan syndrome

We report two children with a common pattern of birth defects. Both have very sparse, curly hair, nystagmus and mental retardation. The first one has Noonan syndrome habitus associated with keratosis plantaris and nystagmus; the second one has a slightly Noonan-like face, macrocephaly, keratosis pilaris, and hypertrophic cardiomyopathy. They represent the extreme of a spectrum of congenital defects recently reported independently as CFC syndrome by Reynolds and as "Noonan-like short stature syndrome with sparse hair" by Baraitser and Patton. The clinical features are reviewed and the autonomy of the syndrome with regards to Noonan syndrome, is disputed, since every sign seems to occur independently in Noonan syndrome. The father of the second case probably has a minor syndrome expression, pointing to probable autosomal dominant inheritance.     

Duplication of HRAS1, INS, and IGF2 is not a common event in Beckwith-Wiedemann syndrome

A few cases of Beckwith-Wiedemann syndrome (BWS) have in common a duplication of 11p15. Among the genes located in 11p15, c-Ha-ras 1 (HRAS1), insulin (INS), and insulin-like growth factor II (IGF2) may account for the clinical features and the increased risk for malignancy. Using eight 11p15 markers including HRAS1, INS and IGF2 we have studied eight sporadic and hereditary cases of BWS whether or not associated with a nephroblastoma. By gene dosage determination and family studies, we have shown the following: the eight patients examined had an apparent diploid representation of all of the eight markers studied, thus indicating that a microduplication of these markers or of the region characterized by these markers is not a common event in BWS; in a family with three affected sibs the genes for HRAS1 and INS/IGF2 did not cosegregate with BWS and therefore may not participate in the pathogenic processes here observed.     

Quantitative Immunologic Analysis of the Methanogenic Flora of Digestors Reveals a Considerable Diversity

To determine which methanogens occur in digestors, we performed a quantitative immunologic analysis of a variety of samples. A comprehensive panel of calibrated polyclonal antibody probes of predefined specificity spectra was used. This allowed precise identification of bacteria by antigenic fingerprinting. A considerable diversity of methanogens was uncovered, much larger than previously reported, encompassing at least 14 strains of 11 species. Strategies were developed to measure the load of any given methanogen in a sample and to compare samples quantitatively. Two methanogens were found to predominate which were antigenically closely related with either Methanobacterium formicicum MF or Methanobrevibacter arboriphilus AZ. Fundamental data, probes, and methods are now available to monitor methanogenic subpopulations during digestor operation and thus learn about their respective roles and predictive significance.     

Density dependent mortality in the springtail species Orchesella cincta due to predation by the carabid beetle Notiophilus biguttatus

Mortality of the collembole species Orchesella cincta (L.), due to predation by the carabid Notiophilus biguttatus F., has been measured in the laboratory at three levels of prey density. Prey densities were chosen such that the predator's consumption at the lowest prey density was a little above the maintenance ration. Between the two lowest prey densities, mortality increased significantly with prey density. We presume that density-dependent search activity by the predator is responsible for the positive density-dependent mortality. A method is proposed to solve the problem of decreasing prey density during the observations.

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Relation positive entre la densité de Orchesella cincta et la mortalité due à la prédation part Nothiophilus biguttarus

Résumé Cet article décrit l'importance de la prédation par le carabe N. biguttatus en fonction de la densité de la proie, O. cincta. Les résultats montrent que, sous l'effet de la prédation, la mortalité de la proie augmente avec sa densité. La quantité minimale de proies qui doit être consommée pour permettre la reproduction du carabe, a été déterminée. La relation entre la fécondité du carabe et l'effectif consommé, montre l'effet stabilisant de la prédation sur les populations de proies.La relation de dépendance de la densité observée avec la mortalité est due à une modification du comportement du carabe en fonction de la densité de sa proie. Nous proposons un modèle permettant de prévoir la régression de la population de proies sous les effets de la prédation.

     

Molecular mechanics calculation of geometries of NAD+ derivatives, modified in the nicotinamide group, in a ternary complex with horse liver alcohol dehydrogenase

The geometry of seven NAD+ analogues bound to horse liver alcohol dehydrogenase (LADH) modified only in their nicotinamide group, have been studied using AMBER molecular mechanics energy-minimization procedures. Starting geometries were taken from X-ray crystallographic data for NAD+/Me2SO/LADH reported by Eklund and co-workers. In this study the NAD+ analogues were encaged by the constituent amino acids of the enzyme within a range of 0.6 nm from the initial NAD+/Me2SO/Zn2+ complex. The calculational method used is able to rationalize individual substituent effects and to evaluate the essential interactions between NAD+ analogue, enzyme, Me2SO and Zn2+ without the necessity of additional X-ray data. The results presented here demonstrate that the reactivity of NAD+ derivatives as reported in literature can be qualitatively related to the position of the pyridine moiety in the active site.     

Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.     

Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector

A stable transformed cell line constitutively expressing human factor IX has been established. Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX l-1 day-1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N-terminal sequencing shows tow differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX. Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX. The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1.     

Monoclonal antibodies reveal evolutionary conservation of alternative splicing of the alpha A-crystallin primary transcript

Because of their specificity and sensitivity, monoclonal antibodies are powerful tools in studies of protein structure and function. Therefore, we raised monoclonal antibodies against alpha A-crystallin and identified the antigenic determinant for two of these antibodies. Applying limited-digestion methods, we show that the region spanning residues 158-168 of alpha A-crystallin contains the epitope for the two monoclonal antibodies. These monoclonals were then used to study the occurrence in the lenses of different vertebrates of the elongated alpha Ains-crystallin chain, a product of alternative splicing. It appears that the mutational event resulting in the alternative splicing pattern of the alpha A-crystallin gene took place at least 70 million years ago. This alternative splicing phenomenon has been maintained in rodents and some other, unrelated mammals, but disappeared again in most mammalian lineages.     

Lipoamide dehydrogenase from Azotobacter vinelandii. Molecular cloning, organization and sequence analysis of the gene

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9-23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded form SDS gel electrophoresis of the cell-free extract, stained for protein or used for Western blotting. After subcloning of the 14.7-kb insert of this plasmid the structural gene could be located on a 3.2-kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined. The protein-coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome-binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.     

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.