The structural basis for methylmalonic aciduria. The crystal structure of archaeal ATP:cobalamin adenosyltransferase

ATP:cobalamin adenosyltransferase MMAB was recently identified as the gene responsible for a disorder of cobalamin metabolism in humans (cblB complementation group). The crystal structure of the MMAB sequence homologue from Thermoplasma acidophilum (TA1434; GenBank identification number gi|16082403) was determined to a resolution of 1.5 A. TA1434 was confirmed to be an ATP:cobalamin adenosyltransferase, which depended absolutely on divalent metal ions (Mg2+ > Mn2+ > Co2+) and only used ATP or dATP as adenosyl donors. The apparent Km of TA1434 was 110 microM (kcat = 0.23 s(-1)) for ATP, 140 microM (kcat = 0.11 s(-1)) for dATP, and 3 microM (kcat = 0.18 s(-1)) for cobalamin. TA1434 is a trimer in solution and in the crystal structure, with each subunit composed of a five-helix bundle. The location of disease-related point mutations and other residues conserved among the homologues of TA1434 suggest that the active site lies at the junctions between the subunits. Mutations in TA1434 that correspond to the disease-related mutations resulted in proteins that were inactive for ATP:cobalamin adenosyltransferase activity in vitro, confirming that these mutations define the molecular basis of the human disease.     

Separable roles of UFO during floral development revealed by conditional restoration of gene function

The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.     

A missense mutation (R565W) in cirhin (FLJ14728) in North American Indian childhood cirrhosis

North American Indian childhood cirrhosis (CIRH1A, or NAIC), a severe autosomal recessive intrahepatic cholestasis described in Ojibway-Cree children from northwestern Quebec, is one of several familial cholestases with unknown molecular etiology. It typically presents with transient neonatal jaundice, in a child who is otherwise healthy, and progresses to biliary cirrhosis and portal hypertension. Clinical and physiological investigations have not revealed the underlying cause of the disease. Currently, liver transplantation is the only effective therapy for patients with advanced disease. We previously identified the NAIC locus by homozygosity mapping to chromosome 16q22. Here we report that an exon 15 mutation in gene FLJ14728 (alias Cirhin) causes NAIC: c.1741C-->T in GenBank cDNA sequence NM_032830, found in all NAIC chromosomes, changes the conserved arginine 565 codon to a tryptophan, altering the predicted secondary structure of the protein. Cirhin is preferentially expressed in embryonic liver, is predicted to localize to mitochondria, and contains WD repeats, which are structural motifs frequently associated with molecular scaffolds.     

Archaeal Binding Protein-Dependent ABC Transporter: Molecular and Biochemical Analysis of the Trehalose/Maltose Transport System of the Hyperthermophilic Archaeon Thermococcus litoralis

We report the cloning and sequencing of a gene cluster encoding a maltose/trehalose transport system of the hyperthermophilic archaeon Thermococcus litoralis that is homologous to the malEFG cluster encoding the Escherichia coli maltose transport system. The deduced amino acid sequence of the malE product, the trehalose/maltose-binding protein (TMBP), shows at its N terminus a signal sequence typical for bacterial secreted proteins containing a glyceride lipid modification at the N-terminal cysteine. The T. litoralis malE gene was expressed in E. coli under control of an inducible promoter with and without its natural signal sequence. In addition, in one construct the endogenous signal sequence was replaced by the E. coli MalE signal sequence. The secreted, soluble recombinant protein was analyzed for its binding activity towards trehalose and maltose. The protein bound both sugars at 85°C with a K_d of 0.16 μM. Antibodies raised against the recombinant soluble TMBP recognized the detergent-soluble TMBP isolated from T. litoralis membranes as well as the products from all other DNA constructs expressed in E. coli. Transmembrane segments 1 and 2 as well as the N-terminal portion of the large periplasmic loop of the E. coli MalF protein are missing in the T. litoralis MalF. MalG is homologous throughout the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins. The strong homology found between the components of this archaeal transport system and the bacterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogenetic branches.     

Increase in epidermal growth factor receptor and its messenger ribonucleic acid levels with differentiation of human trophoblast cells in culture

Epidermal growth factor receptor (EGFR) expression was studied during the differentiation of human trophoblast cells in culture. In vitro, intravillous mononuclear cytotrophoblasts aggregate and fuse within 24 h to form a syncytium. This morphological differentiation was associated with a significant twofold increase in specific ¹²⁵I-EGF binding capacity (P < 0.01). Scatchard analyses showed an apparent rise in the number of high-affinity binding sites (0.33 ± 0.04 and 0.63 ± 0.07 pmol/mg protein at 24 and 48 h, respectively), with no change in their affinity (1.34 and 1.42 × 10^?10 mol/L). Affinity labeling of ¹²⁵I-EGF in cultured trophoblast cells followed by SDS-PAGE and autoradiography revealed a band of 175 KDa corresponding to EGFR, the intensity of which increased with the time in culture. EGF-dependent phosphorylation of membrane proteins from cultured trophoblast cells revealed major phosphorylated proteins of 170 KDa (EGFR) and 35 KDa, which were both increased at 48 h, indicating a rise in EGFR-kinase activity during syncytium formation. Northern blot analysis of EGFR-mRNA, followed by hybridization with a ³²P-cDNA probe for EGFR, revealed an increase in EGFR gene expression in syncytiotrophoblasts, as compared to cytotrophoblasts. Thus, the increase in bioactive EGFR observed during the differentiation of trophoblast cells was due to an increase in their synthesis. Cultured trophoblast cells are therefore a good model of spontaneous up-regulation of EGFR expression with cell differentiation. © 1993 Wiley-Liss, Inc.     

Cystic fibrosis mutations in North American populations of French ancestry: Analysis of Quebec French-Canadian and Louisiana Acadian families

A 3-bp deletion (ΔF508) in the cystic fibrosis (CF) gene is the mutation on the majority of CF chromosomes. We studied 112 CF families from North American populations of French ancestry: French-Canadian families referred from hospitals in three cities in Quebec and from the Saguenay-Lac St. Jean region of northeastern Quebec and Acadian families living in Louisiana. ΔF508 was present on 71%, 55%, and 70% of the CF chromosomes from the major-urban Quebec, Saguenay-Lac St. Jean, and Louisiana Acadian families, respectively. A weighted estimate of the proportion of ΔF508 in the French-Canadian patient population of Quebec was 70%. We found that 95% of the CF chromosomes with ΔF508 had D7S23 haplotype B, the most frequent haplotype on CF chromosomes. In the Saguenay-Lac St. Jean families, 86% of the CF chromosomes without ΔF508 had the B haplotype, compared with 31% for the major-urban Quebec and Louisiana Acadian families. The incidence of CF in the Saguenay-Lac St. Jean population was 1/895 live-born infants.     

DNA relatedness among serovars ofListeria monocytogenes sensu lato

Sixty-six strains ofListeria monocytogenes (as defined in the eighth edition ofBergey's Manual of Determinative Bacteriology) were characterized by DNA relatedness. These strains formed five distinct DNA relatedness groups: (i)L. monocytogenes sensu stricto (30 strains) including the type spain ATCC 15313; (ii) serovar 5 strains (9 strains) corresponding to L. bulgarica; (iii) L. inocua (11 strains of serovars 6a, 6b, 4ab, and undesignated ones) including the reference strains; (iv) six strains of serovars 6a and 6b; (v) ten strains of various serovars. These five groups were clearly distinct fromL. grayi (4 strains) andL. murrayi (3 strains).     

The First Steps of Adaptation of Escherichia coli to the Gut Are Dominated by Soft Sweeps

The accumulation of adaptive mutations is essential for survival in novel environments. However, in clonal populations with a high mutational supply, the power of natural selection is expected to be limited. This is due to clonal interference - the competition of clones carrying different beneficial mutations - which leads to the loss of many small effect mutations and fixation of large effect ones. If interference is abundant, then mechanisms for horizontal transfer of genes, which allow the immediate combination of beneficial alleles in a single background, are expected to evolve. However, the relevance of interference in natural complex environments, such as the gut, is poorly known. To address this issue, we have developed an experimental system which allows to uncover the nature of the adaptive process as Escherichia coli adapts to the mouse gut. This system shows the invasion of beneficial mutations in the bacterial populations and demonstrates the pervasiveness of clonal interference. The observed dynamics of change in frequency of beneficial mutations are consistent with soft sweeps, where different adaptive mutations with similar phenotypes, arise repeatedly on different haplotypes without reaching fixation. Despite the complexity of this ecosystem, the genetic basis of the adaptive mutations revealed a striking parallelism in independently evolving populations. This was mainly characterized by the insertion of transposable elements in both coding and regulatory regions of a few genes. Interestingly, in most populations we observed a complete phenotypic sweep without loss of genetic variation. The intense clonal interference during adaptation to the gut environment, here demonstrated, may be important for our understanding of the levels of strain diversity of E. coli inhabiting the human gut microbiota and of its recombination rate.     

Comprehensive profiling of 8p11-12 amplification in breast cancer

In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer.     

Combinations of cytokines promote survival of mice and limit acute radiation damage in concert with amelioration of vascular damage

Recovery from hematopoietic aplasia is a predominant factor in the survival of total-body-irradiated mice within 30 days after exposure. However, other radiation-induced pathophysiological events have been shown to play a role, among which an inflammatory reaction must be considered. In the present study, we evaluated the therapeutic potential of a hematopoietic growth factor (thrombopoietin, Tpo) and pleiotropic cytokines (Il4 or Il11), used alone or in combination, on the survival of mice, hematopoietic reconstitution, inflammatory reaction and vascular changes. All treatments including Tpo induced a higher level of survival than did treatment with a placebo, with combinations being the most efficient. The increased survival could not be explained solely by an improved hematopoietic recovery. Treatments with Tpo also reduced the level of the chemokine KC in plasma and the level of expression of mRNA for inflammatory and coagulation proteins in the lungs of irradiated mice. In addition, radiation- induced vascular hyperpermeability was reduced with the use of Tpo. In summary, our results show that Tpo may improve survival by limiting vascular leakage, which in turn could limit inflammatory reactions and the ensuing tissue damage.