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PubMed1993

Clinical experience with antitachycardia pacing and improved detection algorithms in a new implantable cardioverter-defibrillator.

OBJECTIVES: This study was conducted to assess the effectiveness of antitachycardia pacing modes and detection algorithms in patients with a new third-generation implantable cardioverter-defibrillator. METHODS: Twenty-three of 42 consecutive patients had coronary artery disease, 14 had dilated cardiomyopathy, 2 had prior valve replacement and 3 had arrhythmogenic right ventricular dysplasia. The mean ejection fraction was 41 +/- 14%; there were 31 men (74%) and 11 women, with a mean age of 53 years. On the basis of preoperative and postoperative electrophysiologic studies, in 28 patients antitachycardia pacing was postoperatively programmed randomly as "burst" (66%) or autodecremental "ramp" (34%) stimulation with a first coupling interval of 81% of tachycardia cycle length and up to 8 sequences with 3 to 10 stimuli. RESULTS: During a follow-up interval of 6.3 +/- 2.2 months, 15 patients were treated by antitachycardia pacing for a median of 6 (range 1 to 59) hemodynamically stable ventricular tachycardias (175 +/- 12 beats/min). In 5 patients, 22 ventricular tachycardias (9%) were not terminated by antitachycardia pacing but by cardioversion. Seven (3%) of these episodes accelerated (> 50 ms) during antitachycardia pacing. Syncope did not occur during these episodes. In seven patients initial antitachycardia pacing in cases of supraventricular tachycardias delayed charging and redetection prevented inappropriate discharges. Additional detection algorithms were programmed only after inappropriate therapy. The sudden "onset" and "sustained rate duration" criteria were programmed in three patients and the cycle length "stability" criteria in six patients, respectively. After activation of these detection algorithms only two of the seven patients had further inappropriate device discharges. CONCLUSIONS: Thus, antitachycardia pacing by this implantable cardioverter-defibrillator effectively and appropriately terminated 91% of hemodynamically stable ventricular tachycardias. Inappropriate device discharges were prevented in some patients by antitachycardia pacing and additional detection algorithms.

Adult↗
PubMed2003

Two EPR-detectable [4Fe-4S] clusters, N2a and N2b, are bound to the NuoI (TYKY) subunit of NADH:ubiquinone oxidoreductase (Complex I) from Rhodobacter capsulatus.

NADH:ubiquinone oxidoreductases (Complex I) contain a subunit, TYKY in the bovine enzyme and NuoI in the enzyme from Rhodobacter capsulatus, which is assumed to bind two [4Fe-4S] clusters because it contains two sets of conserved cysteine motifs similar to those found in the 2[4Fe-4S] ferredoxins. It was recently shown that the TYKY subunit is not an ordinary 2[4Fe-4S] ferredoxin, but has a unique amino acid sequence, which is only found in NAD(P)H:quinone oxidoreductases and certain membrane-bound [NiFe]-hydrogenases expected to be involved in redox-linked proton translocation [FEBS Lett. 485 (2000) 1]. We have generated a set of R. capsulatus mutants in which five out of the eight conserved cysteine residues in NuoI were replaced by other amino acids. The resulting mutants fell into three categories with virtually no, intermediate or quite normal Complex I activities. EPR-spectroscopic analysis of the membranes of the C67S and C106S mutants, two mutants belonging to the second and third group, respectively, showed a specific 50% decrease of the EPR signal attributed to cluster N2. It is concluded that the NuoI (TYKY) subunit binds two clusters N2, called N2a and N2b, which exhibit very similar spectral features when analyzed by X-band EPR spectroscopy.

Anti-Bacterial Agents↗
PubMed1997

Tc7, a Tc1-hitch hiking transposon in Caenorhabditis elegans.

We have found a novel transposon in the genome of Caenorhabditis elegans. Tc7 is a 921 bp element, made up of two 345 bp inverted repeats separated by a unique, internal sequence. Tc7 does not contain an open reading frame. The outer 38 bp of the inverted repeat show 36 matches with the outer 38 bp of Tc1. This region of Tc1 contains the Tc1-transposase binding site. Furthermore, Tc7 is flanked by TA dinucleotides, just like Tc1, which presumably correspond to the target duplication generated upon integration. Since Tc7 does not encode its own transposase but contains the Tc1-transposase binding site at its extremities, we tested the ability of Tc7 to jump upon forced expression of Tc1 transposase in somatic cells. Under these conditions Tc7 jumps at a frequency similar to Tc1. The target site choice of Tc7 is identical to that of Tc1. These data suggest that Tc7 shares with Tc1 all the sequences minimally required to parasitize upon the Tc1 transposition machinery. The genomic distribution of Tc7 shows a striking clustering on the X chromosome where two thirds of the elements (20 out of 33) are located. Related transposons in C. elegans do not show this asymmetric distribution.

Animals↗
PubMed1996

Molecular biological evidence for the occurrence of uncultured members of the actinomycete line of descent in different environments and geographical locations.

A 16S rDNA based molecular ecological study was performed on a sample taken from a peat bog in Germany. Total DNA was extracted by directly lysing micro-organisms in the peat matrix. The 5' 1400 nucleotides of the bacterial 16S rDNA were amplified using conserved bacterial PCR primers. A clone library was generated by blunt-end cloning and 262 16S rDNA clones were analysed. Of these, 37 were located in the Gram-positive phylum, as determined by hybridization to an oligonucleotide probe specific for Gram-positive bacteria. Analysis of 17 of these clones by sequence analysis and their comparison with published sequences representing all of the main bacterial phyla indicated their membership of the actinomycete line of descent. These peat clones were found to represent three novel lineages, two of which appear to be related to the species Acidimicrobium ferrooxidans, and 'Candidatus Microthrix parvicella'. Clone sequences of the third group are phylogenetically related to Rubrobacter radiotolerans. Comparison with short 16S rDNA clone sequences obtained from DNA isolated from a geothermally heated soil in New Zealand, and from DNA isolated from soil in Australia, Japan and Finland and marine environments from the Atlantic and the Pacific Oceans, suggests that members of these three groups occur in very different environments across the world.

Actinomycetales↗
PubMed1993

Expression of the Xenopus D2 dopamine receptor. Tissue-specific regulation and two transcriptionally active genes but no evidence for alternative splicing.

In the amphibian Xenopus laevis the D2 dopamine receptor is involved in the regulation of the melanotrope cells of the intermediate pituitary during background adaptation of the animal. The Xenopus D2 receptor has been found to be pharmacologically different from the mammalian D2 receptor. In a number of mammalian species alternative splicing generates two molecular forms of the D2 receptor. These isoforms differ by the presence or absence of 29 amino acids in the third cytoplasmic loop which is thought to be involved in guanine-nucleotide-binding-regulatory-protein (G-protein) binding of the receptor. We previously described a cDNA encoding the large isoform of the Xenopus D2 receptor. Here we report on the isolation of a brain cDNA encoding a second, structurally different Xenopus D2 dopamine receptor. Both Xenopus receptors correspond to the large isoform of the D2 receptor and they display a high degree of sequence identity with their mammalian counterparts. Their occurrence reflects the expression of two Xenopus D2 receptor genes and they are expressed to approximately the same level. In contrast to mammals, PCR analysis gave no evidence for alternative splicing during D2 receptor expression in Xenopus brain and pituitary. Tissue-specific expression of the Xenopus D2 receptor was observed in the pituitary during background adaptation. The low level of receptor mRNA in melanotrope cells of white animals compared to that of black animals may be caused by chronic dopamine stimulation of melanotrope cells in white animals with consequent cellular desensitization and down regulation of the D2 receptor gene.

Alternative Splicing↗
PubMed2002

Redundant cooperative interactions for assembly of a human U6 transcription initiation complex.

The core human U6 promoter consists of a proximal sequence element (PSE) located upstream of a TATA box. The PSE is recognized by the snRNA-activating protein complex (SNAP(c)), which consists of five types of subunits, SNAP190, SNAP50, SNAP45, SNAP43, and SNAP19. The TATA box is recognized by TATA box binding protein (TBP). In addition, basal U6 transcription requires the SANT domain protein Bdp1 and the transcription factor IIB-related factor Brf2. SNAP(c) and mini-SNAP(c), which consists of just SNAP43, SNAP50, and the N-terminal third of SNAP190, bind cooperatively with TBP to the core U6 promoter. By generating complexes smaller than mini-SNAP(c), we have identified a 50-amino-acid region within SNAP190 that is (i) required for cooperative binding with TBP in the context of mini-SNAP(c) and (ii) sufficient for cooperative binding with TBP when fused to a heterologous DNA binding domain. We show that derivatives of mini-SNAP(c) lacking this region are active for transcription and that with such complexes, TBP can still be recruited to the U6 promoter through cooperative interactions with Brf2. Our results identify complexes smaller than mini-SNAP(c) that are transcriptionally active and show that there are at least two redundant mechanisms to stably recruit TBP to the U6 transcription initiation complex.

DNA↗
PubMed1990

On the mechanism of non-allelically excluded V alpha-J alpha T cell receptor secondary rearrangements in a murine T cell lymphoma.

We had previously demonstrated that several subclones derived from a CD3+, CD4-/CD8-, TCR-alpha beta+ murine T cell line have undergone secondary V alpha-J alpha rearrangements at the TCR-alpha locus (1). In an effort to examine the molecular mechanism responsible for these V alpha-J alpha replacements, the structures of TCR-alpha cDNA prepared from both the parental and subcloned T cell lines have been determined. Here we report that: 1) the mechanism whereby the secondary rearrangements occur is a precise deletion event that involves germ-line V alpha genes 5' to the preexisting V alpha-J alpha complex joining to J alpha segments 3' of the preexisting complex deleting the region in between, 2) preexisting productive V alpha-J alpha rearrangements of the parental line do not allelically exclude productive and nonproductive secondary rearrangements, 3) both productively rearranged TCR-alpha alleles of the parental cell line can undergo secondary rearrangements, 4) the presence of unrearranged germline V alpha transcripts in the parental line support an "accessibility" model of regulated lymphocyte receptor gene rearrangement. In addition, we present data which suggests that one of the subcloned lines has undergone a third rearrangement of one of its TCR-alpha alleles. One interpretation of these results is that T cells may have the ability to circumvent allelic exclusion at the TCR-alpha locus early in their ontogeny. This could provide T cells with an additional mechanism for generating an Ag receptor repertoire which is not found in B cells.

Alleles↗
PubMed2006

Genetic characterization of the natural hybrid species Phytophthora alni as inferred from nuclear and mitochondrial DNA analyses.

The different subspecies of Phytophthora alni, P. alni subsp. alni (Paa), P. alni subsp. uniformis (Pau), and P. alni subsp. multiformis (Pam), are recent and widespread pathogens of alder in Europe. They are believed to be a group of emergent heteroploid hybrids between two phylogenetically close Phytophthora species. Nuclear and mitochondrial DNA analyses were performed, using a broad collection of P. alni and two closely related species, P. cambivora and P. fragariae. Paa possesses three different alleles for each of the nuclear genes we studied, two of which are present in Pam as well, whereas the third matches the single allele present in Pau. Moreover, Paa displays common mtDNA patterns with both Pam and Pau. A combination of the data suggests that Paa may have been generated on several occasions by hybridization between Pam and Pau, or their respective ancestors. Pau might have P. cambivora as a species ancestor, whereas Pam seems to have either been generated itself by an ancient reticulation or by autopolyploidization.

Alleles↗
PubMed1993

Site-directed mutagenesis of beta-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes.

From sequence alignments, two groups can be defined for the carbenicillin-hydrolysing beta-lactamases (CARB enzymes). One group includes the Pseudomonas-specific enzymes PSE-1, PSE-4, CARB-3, CARB-4 and also the Proteus mirabilis GN79, for which the well-conserved residue Lys 234 in all class-A beta-lactamases is changed to an arginine residue. The second group includes the enzymes PSE-3 and AER-1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM-1 beta-lactamase to study their potential role in defining the substrate profile of the CARB enzymes. The mutations K234R and E240G in TEM-1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second-generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to alpha-carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third-generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM-1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three-dimensional structures of the active site of the TEM-1 enzyme and PSE-3, PSE-4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM-1 protein variants.

Base Sequence↗
PubMed2003

Genomic structure and promoter analysis of PKC-delta.

Protein kinase C-delta (PKC-delta) is a ubiquitously expressed kinase involved in a variety of cellular signaling pathways including cell growth, differentiation, apoptosis, tumor promotion, and carcinogenesis. While signaling pathways downstream of PKC-delta are well studied, the regulation of the gene has not been extensively analyzed. A mouse genomic DNA fragment containing the PKC-delta gene was sequenced by the primer-walking method, and the subsequent DNA sequence data were used as a query to clone Caenorhabditis elegans and human genomic homologs from the publicly available genomic databases. The genomic structures of C. elegans, mouse, rat, and human PKC-delta were analyzed, and the result revealed that PKC-delta genes comprise 12, 18, 19, and 18 exons for C. elegans, mouse, rat, and human, respectively. The translation start methionine resides in the second exon in mouse and human and in the third exon in rat. The first intron between the first exon and the exon with the translation start methionine in mammalian genes represents a very large gap, as long as 17 kb in human, indicating a complexity involved in gene splicing. Overall exon-intron genomic structure is highly conserved among mammals, while significantly diverged in C. elegans. Putative transcription factor binding sites on the 1.7-kb promoter region of the mouse gene suggest that PKC-delta might be involved in spermatogenesis, embryogenesis, development, brain generation, immune response, oxidative environment, and oncogenesis. Studies on the promoter and subsequent biological testing on mouse keratinocytes indicate that tumor necrosis factor (TNF)-alpha increases the expression of PKC-delta, and this correlates with the time of NFkappaB nuclear translocation and activation. This TNF-alpha-mediated upregulation of PKC-delta is repressed in keratinocytes that are preinfected with IkappaB superrepressor adenovirus, suggesting that NFkappaB is involved directly in PKC-delta expression.

Animals↗
PubMed1992

The molecular defect in a family with mild atypical osteogenesis imperfecta and extreme joint hypermobility: exon skipping caused by an 11-bp deletion from an intron in one COL1A2 allele.

We have investigated a family with an autosomal dominantly inherited connective-tissue defect causing extreme joint hypermobility, premature osteoporosis and late-onset fractures. Analysis of collagenous proteins from affected individuals showed a deletion in some alpha 2(I) chains. Peptide mapping localized this to the CB peptide alpha 2CB4, which covers the N-terminal one-third of the protein chain. Polymerase chain reaction amplification and sequencing of cDNA derived from this region of the mRNA identified a heterozygous deletion of the 54 bp comprising exon 9. Similar analysis of the genomic DNA revealed an 11-bp deletion from bp3 to bp13 of IVS-9. This disrupts the consensus 5' splice signal (GTAAGT) and leads to exon skipping. In a family study of 13 affected and unaffected family members using both heteroduplex formation and direct analysis for the deletion, all of the affected, but no unaffected individuals, were found to carry the deletion. This generated a positive Lod score of 2.6 with the Liped programme.

Alleles↗
PubMed2005

Two novel transposable elements in a cytochrome P450 gene govern anthocyanin biosynthesis of commercial petunias.

The gene Hf1 plays a key role in the expression of floral color in petunias. Hf1 encodes a flavonoid-3',5'-hydroxylase (F3'5'H). The recessive allele (hf1) in an inbred line of petunia is known to be generated by the insertion of a transposable element (Psl). We isolated a novel Mutator-like transposable element (named dTph9) from the hf1 allele of a commercial petunia with red flowers. Another novel transposable element (named rTph1) was found in hf1 of another red petunia. rTph1 shared features with the copia-like retrotransposable element family. These novel elements were inserted independently in the third exon of the Hf1 gene, at different positions. The hf1 allele harboring dTph9 or rTph1 cannot produce an active F3'5'H enzyme because there are stop codons in the dTph9 and rTph1 sequences. Southern analysis showed that these elements were present in relatively low copy numbers and that mutation of the Hf1 locus was associated with the transposition of both elements. We conclude that a loss-of-function mutation of the petunia Hf1 gene is caused by the insertion of at least two different transposable elements, other than Psl, within the Hf1 gene.

Alleles↗
PubMed2006

CTX-M-1- and CTX-M-15-type beta-lactamases in clinical Escherichia coli isolates recovered from food-producing animals in France.

Clinical Escherichia coli strains with resistance or variable susceptibility to third-generation cephalosporins were detected in cattle, swine and poultry in France. These strains were shown to produce extended-spectrum beta-lactamases (ESBLs), with CTX-M-1- and CTX-M-15-type beta-lactamases being responsible for this phenotype. The bla(CTX-M-1) gene was encountered most commonly and was characterised in seven E. coli strains isolated from cattle, swine and poultry, whereas bla(CTX-M-15) was identified in one E. coli isolated from cattle. These genes were located on a conjugative plasmid and were linked to the insertion sequence ISEcp1, which could have contributed to dissemination of the resistance gene. No epidemiological link between the strains was determined by pulsed-field gel electrophoresis, although two plasmids were identical in two strains isolated from swine and in two strains isolated from cattle and poultry. Thus, this study describes the emergence of ESBLs in animals in France, with a probable similar prevalence rate to that observed in humans. This is a major concern because of the possibility of transfer of these genes between animal species as well as to humans, leading to treatment failures in veterinary and human medicine.

Animals↗
PubMed2005

Developmental progression of immunoglobulin heavy chain diversity in sheep.

In order to assess the respective impacts of combinatorial rearrangement, junctional diversification, somatic hypermutation and gene conversion in the generation of immunoglobulin heavy chain variable regions diversity, the sequences of 42 variable regions from late fetal, newborn and young sheep were determined and compared to those of adult animals. At earlier stages of development, the use of germline diversity segments appears restricted, junctional variability is already established, and somatic hypermutations are scarce. The sequence diversity in adults is much higher, which we suggest results from a higher hymermutation activity and possibly from the use of a variety of diversity segments. Altogether, this pattern is very reminiscent of the situation observed in cattle, except for the length of the third complementarity determining regions (CDR3) which are shorter in sheep than in bovine. Unlike the chicken and rabbit systems, it seems that new rearrangements continue to occur in sheep for at least several months after birth.

Amino Acid Sequence↗
PubMed1998

Molecular characterization of six variant Fcgamma receptor class I (CD64) transcripts.

In humans, three distinct but closely related classes of receptors that bind the Fc portion of IgG (FcgammaRI, II and III) have been identified. FcgammaRI can bind monomeric IgG with high affinity and has a unique third extracellular domain (EC3). Three very similar genes have been characterized for FcgammaRI (A, B, C). Although the sequences are remarkably similar, a number of coding-region differences discriminate between the genes and amongst their transcripts. Six distinct FcgammaRI transcripts were analysed. Three transcripts, one from each gene, contain all six exons. Only the gene A transcript appears to encode a bona fide high affinity receptor, a three Ig-domain membrane spanning receptor that can bind monomeric IgG. Stop codons in the EC3 domains of the gene B and gene C isoforms would be predicted to generate secreted receptors. Three transcripts are alternatively spliced isoforms, one from gene A and two from gene B. One gene B transcript encodes a two Ig-domain transmembrane receptor which has structural characteristics of a low affinity FcgammaR.

Animals↗
PubMed2002

A P-insertion screen identifying novel X-linked essential genes in Drosophila.

The recent determination and annotation of the entire euchromatic sequence of the Drosophila melanogaster genome predicted the existence of about 13600 different genes (Science 287 (2000) 2185; http://www.fruitfly.org/annot/index.html). In parallel, the Berkeley Drosophila Genome Project (BDGP) has undertaken systematic P-insertion screens, to isolate new lethals and misexpressing lines. To date, however, the genes of the X chromosome have been under-represented in the screens performed. In order both to characterize several X-linked genes of prime interest to our laboratories and contribute to the collection of lethal P-insertions available to the community, we performed a P-insertion mutagenesis of the X chromosome. Using the PlacW and PGawB P-elements as mutagens, we generated two complementary sets of enhancer-trap lines, l(1)(T)PL and l(1)(T)PG, respectively, which both contain a reporter gene whose developmental expression can be monitored when driven by nearby enhancer sequences. We report here the characterization of 260 new insertions, mapping to 133 different genes or predicted CGs. Of these, 83 correspond to genes for which no lethal mutation had yet been reported. For 64 of those, we could confirm that lethality was solely due to the P-element insertion. The primary molecular data, reporter gene expression patterns (observed in embryos, third instar larvae and adult ovaries) and proposed CG assignment for each strain can be accessed and updated on our website at the following address: http://www-cbd.ups-tlse.fr:8080/screen.

Animals↗
PubMed1978

Amino acid sequence of two cyanogen bromide fragments of glycogen phosphorylase.

This communication presents the strategy and experimental details to prove the amino acid sequence of two large fragments of rabbit muscle glycogen phosphorylase generated by cleavage with cyanogen bromide. These fragments, CB18 and CB15, represent 241 of the 841 residues in the whole molecule. In addition to applying methods of automated liquid phase Edman degradation, techniques of selective immobilization and solid phase Edman degradation are used. One of the two cyanogen bromide fragments (CB15) contains two of the sites of cleavage with hydroxylamine which have proved to be important in the overall strategy of determining the complete sequence of this molecule. Together with the accompanying reports by Koide, A., et al., and Titani, K., et al. ((1978) Biochemistry 17 (first and third papers, respectively, in a series in this issue)), the present communication completes the proof of the amino acid sequence of phosphorylase and provides the basis for examining the relationship between its structure and function.

Amino Acid Sequence↗
PubMed2004

Sequences of flavivirus-related RNA viruses persist in DNA form integrated in the genome of Aedes spp. mosquitoes.

Flavivirus-related sequences have been discovered in the dsDNA genome of Aedes albopictus and Aedes aegypti mosquitoes, demonstrating for the first time an integration into a eukaryotic genome of a multigenic sequence from an RNA virus that replicates without a recognized DNA intermediate. In the Aedes albopictus C6/36 cell line, an open reading frame (ORF) of 1557 aa with protease/helicase and polyprotein processing domains characteristic of flaviviruses was identified. It is closely related to NS1-NS4A genes of the Cell Fusing Agent and Kamiti River virus and the corresponding mRNAs were detected. Integrated sequences homologous to the envelope, NS4B and polymerase genes of flaviviruses were identified. Overall, approximately two-thirds of a flavivirus-like genome were characterized. In the Aedes aegypti A20 cell line, a 492 aa ORF related to the polymerase of the Cell Fusing Agent and Kamiti River virus was identified. These flavivirus-related integrated DNA sequences were detected in laboratory-bred and wild Aedes albopictus and Aedes aegypti mosquitoes, demonstrating that their discovery is not an artefact resulting from the manipulation of mosquito cell lines, since they exist under natural conditions. This finding has major implications regarding evolution, as it represents an entirely different mechanism by which genetic diversity may be generated in eukaryotic cells distinct from accepted processes.

Aedes↗