The use of flow perfusion culture and subcutaneous implantation with fibroblast-seeded PLLA-collagen 3D scaffolds for abdominal wall repair.

Highly cellularised 3D-tissue constructs designed to repair large, complex abdominal wall defects were prepared using poly (lactic acid) (PLLA)-collagen scaffolds in vitro using a flow perfusion bioreactor. The PLLA-collagen scaffolds had a unique structure consisting of a collagen sponge formed within the pores of a mechanically stable knitted mesh of PLLA. The effect of the flow perfusion bioreactor culturing conditions was investigated in vitro for 0, 7, 14 and 28 days on scaffolds seeded with dermal fibroblasts.

Molecular misreading: the frequency of dinucleotide deletions in neuronal mRNAs for beta-amyloid precursor protein and ubiquitin B.

Human neuronal cells contain mutant beta-amyloid precursor protein (APP) and ubiquitin B (UBB) mRNAs, in which dinucleotide deletions ('Delta') are generated in/around GAGAG-motifs by an unknown mechanism referred to as 'Molecular Misreading.' The encoded frameshifted (+1) proteins accumulate in the neuropathological hallmarks of Alzheimer's disease (AD) and in other neurodegenerative and age-related diseases. To measure the concentration of Delta mRNAs, we developed a highly sensitive and specific assay, utilizing peptide nucleic acid-mediated PCR clamping, followed by cloning and colony hybridization with sequence-specific oligonucleotide probes.

RNA interference analyses suggest a transcript-specific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei.

Mitochondrial RNA-binding proteins MRP1 and MRP2 occur in a heteromeric complex that appears to play a role in U-insertion/deletion editing in trypanosomes. Reduction in the levels of MRP1 (gBP21) and/or MRP2 (gBP25) mRNA by RNA interference in procyclic Trypanosoma brucei resulted in severe growth inhibition. It also resulted in the loss of both proteins, even when only one of the MRP mRNAs was reduced, indicating a mutual dependence for stability.

Frameshifted beta-amyloid precursor protein (APP+1) is a secretory protein, and the level of APP+1 in cerebrospinal fluid is linked to Alzheimer pathology.

Molecular misreading of the beta-amyloid precursor protein (APP) gene generates mRNA with dinucleotide deletions in GAGAG motifs. The resulting truncated and partly frameshifted APP protein (APP+1) accumulates in the dystrophic neurites and the neurofibrillary tangles in the cortex and hippocampus of Alzheimer patients. In contrast, we show here that neuronal cells transfected with APP+1 proficiently secreted APP+1.

+1 Proteins and aging.

Molecular misreading is an expression used to describe errors in RNA that lead to the translation of mutated proteins. We have shown that dinucleotide deletions (delta GA, delta GU) are introduced in simple sequence repeats (e.g.

Mutational spectrum in the PEX7 gene and functional analysis of mutant alleles in 78 patients with rhizomelic chondrodysplasia punctata type 1.

Rhizomelic chondrodysplasia punctata (RCDP) is a genetically heterogeneous, autosomal recessive disorder of peroxisomal metabolism that is clinically characterized by symmetrical shortening of the proximal long bones, cataracts, periarticular calcifications, multiple joint contractures, and psychomotor retardation. Most patients with RCDP have mutations in the PEX7 gene encoding peroxin 7, the cytosolic PTS2-receptor protein required for targeting a subset of enzymes to peroxisomes. These enzymes are deficient in cells of patients with RCDP, because of their mislocalization to the cytoplasm.

Cloning and characterization of two guide RNA-binding proteins from mitochondria of Crithidia fasciculata: gBP27, a novel protein, and gBP29, the orthologue of Trypanosoma brucei gBP21.

In kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g)RNAs. Currently popular mechanisms for the editing process envisage a series of consecutive 'cut-and-paste' reactions, carried out by a complex RNP machinery. Here we report on the purification, cloning and functional analysis of two gRNA-binding proteins of 28.

Molecular misreading: a new type of transcript mutation expressed during aging.

Dinucleotide deletions (e.g. DeltaGA, DeltaGU) are created by molecular misreading in or adjacent to GAGAG motifs of neuronal mRNAs.

Molecular misreading. A new type of transcript mutation in gerontology.

Molecular misreading is a novel process that causes mutations in neuronal transcripts. It is defined as the inaccurate conversion of genomic information from DNA into nonsense transcripts and the subsequent translation into mutant proteins. As a result of dinucleotide deletions (delta GA, delta GU, delta CU) in and around GAGAG motifs in mRNA the reading frame shifts to the +1 frame, and subsequently the so-called +1 proteins are synthetized.

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks.

In trypanosomatids, the majority of the guide (g) RNAs that provide the information for U-insertion/deletion RNA editing are encoded by minicircles that are catenated into large networks. In contrast, in the distantly related cryptobiid Trypanoplasma borreli, gRNA genes appear to reside in large 180-kb noncatenated DNA circles. To shed light on the evolutionary history and function of the minicircle network, we have analyzed minicircle organization in the free-living bodonid Bodo saltans, which is more closely related to trypanosomatids than T.

MitBASE : a comprehensive and integrated mitochondrial DNA database. The present status.

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae. MitBASE reports all available information from different organisms and from intraspecies variants and mutants. Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.

MitBASE: a comprehensive and integrated mitochondrial DNA database.

MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects all available information from different organisms and from intraspecie variants and mutants. Research institutions from different countries are involved, each in charge of developing, collecting and annotating data for the organisms they are specialised in. The design of the actual structure of the database and its implementation in a user-friendly format are the care of the European Bioinformatics Institute.

Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity.

We report the characterization of a Trypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of the TBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite.

RNA editing in the free-living bodonid Bodo saltans.

In parasitic kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g) RNAs. In order to further explore the role and evolutionary history of this process, we searched for editing in mt RNAs of the free-living bodonid Bodo saltans. We found extensive editing in the transcript for NADH dehydrogenase (ND) subunit 5, which is unedited in trypanosomatids.

Further evidence for the presence of mitochondrially encoded subunits in cytochrome c oxidase of the trypanosomatid Crithidia fasciculata.

Mitochondrial mRNAs in trypanosomatids are edited by uridylate insertion and deletion. The respiratory chain complexes cytochrome c reductase, cytochrome c oxidase and F0F1-ATPase of the insect trypanosomatid Crithidia fasciculata have been isolated and analysed by peptide microsequencing, but so far, proteins encoded by edited (and unedited) mitochondrial mRNAs have not been found. In this paper, we provide evidence that the mitochondrial mRNAs encoding the three large subunits of cytochrome c oxidase are indeed translated.

RNA editing in kinetoplastid parasites: what to do with U.

The editing of the mitochondrial RNAs of kinetoplastid protozoa is a bizarre form of transcript maturation that involves insertion and deletion of uridylate residues. Editing leads to the formation of translational initiation and termination codons, the correction of gene-encoded reading frame shifts and the creation of complete reading frames in mRNAs. It is therefore an essential step in mitochondrial gene expression.

Characterization of the respiratory chain from cultured Crithidia fasciculata.

Mitochondrial mRNAs encoding subunits of respiratory-chain complexes in kinetoplastids are post-transcriptionally edited by uridine insertion and deletion. In order to identify the proteins encoded by these mRNAs, we have analyzed respiratory-chain complexes from cultured cells of Crithidia fasciculata with the aid of 2D polyacrylamide gel electrophoresis (PAGE). The subunit composition of F0F1-ATPase (complex V), identified on the basis of its activity as an oligomycin-sensitive ATPase, is similar to that of bovine mitochondrial F0F1-ATPase.

An intronic mutation in a lariat branchpoint sequence is a direct cause of an inherited human disorder (fish-eye disease).

The first step in the splicing of an intron from nuclear precursors of mRNA results in the formation of a lariat structure. A distinct intronic nucleotide sequence, known as the branchpoint region, plays a central role in this process. We here describe a point mutation in such a sequence.

Purification and characterization of cytochrome c oxidase from the insect trypanosomatid Crithidia fasciculata.

Cytochrome c oxidase was purified from the mitochondrial lysate of the insect trypanosomatid Crithidia fasciculata with the aid of a methyl hydrophobic interaction column in a rapid one-step procedure. The purified complex displayed all characteristics expected from a eukaryotic cytochrome c oxidase: the presence of CuA in electron paramagnetic resonance analysis, a characteristic 605 nm peak in reduced-minus-oxidized optical spectroscopy, and the capacity to efficiently oxidize homologous, but not heterologous, cytochrome c. Two-dimensional PAGE showed that C.

RNA editing: how a message is changed.

Considerable progress has been made in unraveling the mechanistic features of RNA editing processes in a number of genetic systems. Recent highlights include the identification of the catalytic subunit of the mammalian apolipoprotein B mRNA editing enzyme as a zinc-dependent cytidine deaminase that binds to RNA, the demonstration that adenosines in brain glutamate receptor pre-mRNAs are converted into inosines and that double-stranded RNA A deaminase (dsRAD), the candidate enzyme, is another zinc-dependent RNA nucleotide deaminase, and a mounting body of evidence for a cleavage-ligation mechanism for U insertion/deletion editing in kinetoplastid protozoa.

The sequence of a small subunit of cytochrome c oxidase from Crithidia fasciculata which is homologous to mammalian subunit IV.

The sequence of subunit 8 of cytochrome c oxidase from Crithidia fasciculata was determined by sequencing cDNA and N-terminus of the mature protein (Mr = 15.7 kDA). The (inferred) protein is homologous to mammalian cox IV and the corresponding cox subunits from yeast, Neurospora crassa and Dictyostelium discoideum, which is reflected in a very similar hydropathy profile.

A possible role for the guide RNA U-tail as a specificity determinant in formation of guide RNA-messenger RNA chimeras in mitochondrial extracts of Crithidia fasciculata.

Chimeric g(uide) RNA:pre-mRNA molecules are potential intermediates of the RNA editing process in kinetoplastid mitochondria. We have studied the characteristics of chimeric molecules formed in mitochondrial extracts of the insect trypanosomatid Crithidia fasciculata which had been supplied with synthetic NADH dehydrogenase (ND) subunit-7 gRNA and pre-mRNA variants. The ability of a gRNA to participate in chimera formation in this system depends on the possibility of base pairing with the pre-mRNA via the anchor sequence, but not on the presence of a U-tail or a full-length informational part.