Protoplasts from Neurospora crassa

Protoplasts from Neurospora crass

Characterization of antigen-presenting properties of tumour cells using virus-specific cytotoxic T lymphocytes

Immunotherapy of tumours by induction of tumour-specific cytotoxic T-lymphocytes (CTLs) will only be effective for tumours with a functional antigen processing and presentation machinery. However, many tumours are known to down-regulate expression of major histocompatibility complex (MHC) class I molecules and/or to impair antigen processing. It is therefore desirable to evaluate the ability of a given tumour to present antigenic epitopes before developing an immunotherapy protocol. In this study we have used influenza virus as a tool to determine the antigen-presenting capacities of the murine neuroblastoma C1300 cell line NB41A3, a frequently used model for human neuroblastoma. Immunofluorescence analyses revealed low and moderate expression of MHC class I molecules Ddand Kkrespectively. Nevertheless, infected NB41A3 cells were lysed efficiently by influenza-specific CTLs. These results demonstrate that all steps of the antigen-processing pathway function properly in the NB tumour cells, and that the limited MHC class I expression suffices for efficient recognition by CTLs. In addition, lysis of the NB tumour cells shows that the cells are susceptible to CTL-induced apoptosis, a pathway that is often impaired in tumour cells. These characteristics make neuroblastoma a suitable target for immunotherapy. The presented assay allows evaluation of various immunological properties of tumour cells and, thus, represents a valuable tool to assess whether a given tumour will be susceptible to immunotherapy or not. Copyright 2000 Cancer Research Campaign. © 2000 Cancer Research Campaig

HC4 The Potential Impact Of Recommendations Made Through The Common Drug Review Program At The Canadian Agency For Drugs And Technologies In Health

A full-length cDNA clone specifying the nuclear-encoded subunit VIb of human cytochrome c oxidase (COX) was isolated from a human skeletal muscle cDNA expression library. This was done with antiserum directed against the group of subunits VIa, b and c of bovine heart COX. A potential ribosome-binding site was located immediately upstream from the initiation codon. The predicted amino acid sequence revealed 85% similarity with the corresponding subunit of bovine heart COX. Subunit VIb lacks a cleavable presequence for mitochondrial addressing. We assume that there are no tissue-specific isoforms of subunit VIb, since (i) in a Northern blot experiment a single hybridizing band of approx. 500 nucleotides was demonstrated in RNA from liver, skeletal muscle, MOLT-4 cells and fibroblasts and (ii) a full-length cDNA clone with an identical sequence was isolated from a human liver cDNA library. Steady-state levels of the coxVIb transcript were different in the tissues examined

Oxidative phosphorylation in human muscle in patients with ocular myopathy and after general anaesthesia

Abstract The fuel preference of human muscle mitochondria has been given. Substrates which are oxidized with low velocity cannot be used to detect defects in oxidative phosphorylation. After general anaesthesia, the oxygen uptake with the different substrates is much lower than after local analgesia. The latter was therefore used in the subsequent study. In 15 out of 18 patients with ocular myopathy, defects in oxidative phosporylation could be detected in isolated muscle mitochondria prepared from freshly biopsied tissue. Measurement of the activity of segments of the respiratory chain in homogenate from frozen muscle showed no, or minor defects. In two of these patients showing exercise intolerance, decreased oxidation of NAD+-linked substrates and apparently normal mitochondrial DNA, further study revealed deficiency of pyruvate dehydrogenase in a girl with ptosis and a high Km of complex I for NADH in a man. Both patients responded to vitamin therapy

Analysis of the mitochondrial and nuclear genomes of two basidiomycetes, Coprinus cinereus and Coprinus stercorarius

The mitochondrial and nuclear genomes of Coprinus stercorarius and C. cinereus were compared to assess their evolutionary relatedness and to characterize at the molecular level changes that have occurred since they diverged from a common ancestor. The mitochondrial genome of C. stercorarius (91.1 kb) is approximately twice as large as that of C. cinereus (43.3 kb). The pattern of restriction enzyme recognition sites shows both genomes to be circular, but reveals no clear homologies; furthermore, the order of structural genes is different in each species. The C. stercorarius mitochondrial genome contains a region homologous to a probe derived from the yeast mitochondrial var1 gene, whereas its nuclear genome does not. By contrast, the C. cinereus nuclear, but not mitochondrial, genome contains a region homologous to the var1 probe. Only a small fraction of either the nuclear or mitochondrial genomes, perhaps corresponding to the coding sequences, is capable of forming duplexes in interspecies solution reassociations, as measured by binding to hydroxylapatite. Those sequences capable of reassociating were found to have approximately 15% divergence for the mitochondrial genomes and 7%–15% divergence for the nuclear genomes, depending on the conditions of reassociation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46959/1/294_2004_Article_BF00447385.pd

Processing of precursor RNAs from mitochondria of Neurospora crassa.

Neurospora mitochondrial DNA is transcribed into long molecules containing the information of several genes. Processing leads to formation of functionally active RNAs. It has been shown previously that when tRNA sequences are present in these transcripts excision of mRNAs occurs at the acceptor stem of these tRNA sequences. We have investigated the processing of precursor RNAs transcribed from a region of the mitochondrial genome devoid of tRNA genes. This region comprises the genes encoding subunit 6 of the mitochondrial ATPase, subunit 2 of cytochrome aa3 and a mitochondrial ATPase proteolipid-like gene. We have proved that a common precursor of the putative mRNAs of these genes exists and we have determined the positions of the 5' and 3' ends of processing intermediates and of the mature mRNAs. We will discuss possible processing routes and secondary structures that substitute for tRNA sequences as processing sites

Decreased synthesis and inefficient mitochondrial import of hsp60 in a patient with a mitochondrial encephalomyopathy

In a recent paper (Agsteribbe et al. (1993) Biochem. Biophys. Res. Commun. 193, 146-154) we suggested deficiency of heat shock protein 60 (hsp60) as the possible cause of a systemic mitochondrial encephalomyopathy with multiple deficiency of mitochondrial enzymes. In this paper we present new data which strongly support this hypothesis. Hsp60 deficiency appeared to be not a common side effect of impaired mitochondrial metabolism as eight out of ten fibroblast cultures from patients with systemic mitochondrial myopathy contained normal quantities of the protein. The low steady state amount of hsp60 in the fibroblasts of our patient is caused by decreased synthesis of the protein and not by its enhanced degradation indicating that the hsp60 deficiency is indeed a primary defect. Processing of hsp60 but not of other mitochondrial proteins is markedly retarded in the patient cells. Other functional properties of the patient hsp60 like the assembly of hsp60 monomers to the native 14mer complex and the affinity of this complex to denatured protein are not impaired. Our results underline that a primary defect in hsp60 synthesis and/or processing causing a low steady state amount of hsp60 is the molecular basis of this mitochondrial disorder. The presented data provide for the first time substantial evidence that deficiency of a heat shock protein can give rise to pathological conditions in man

Duplication of the tRNA MetM and tRNACys genes and of fragments of a gene encoding a subunit of the NADH dehydrogenase complex in Neurospora grassa mitochondrial DNA

Neurospora crassa mitochondrial DNA (mtDNA) contains duplications of the tRNA MetMgene upstream of a gene (ND2) encoding a subunit of the NADH dehydrogenase complex and of the tRNACys gene which is found downstream of the apocytochrome b gene. Both duplicated genes are located upstream of the small rRNA gene. The duplications are extended to flanking sequences. In the case of the tRNA MetMduplication, two fragments of the ND2 gene are also duplicated. These two fragments, which are not contiguous in the ND2 gene, are connected to each other by a palindromic sequence of 37 by and together they constitute an open reading frame. The possible involvement of this palindromic sequence in the processes of gene duplication and transfer is discussed. Two overlapping reading frames are present between the tRNA MetMand tRNACys copies. All information of the ND2 duplication and the two overlapping reading frames are present on a polycistronic transcrip