Reversal of MYB-dependent suppression of MAFB expression overrides leukaemia phenotype in MLL-rearranged AML.

The transcription factor MYB plays a pivotal role in haematopoietic homoeostasis and its aberrant expression is involved in the genesis and maintenance of acute myeloid leukaemia (AML). We have previously demonstrated that not all AML subtypes display the same dependency on MYB expression and that such variability is dictated by the nature of the driver mutation. However, whether this difference in MYB dependency is a general trend in AML remains to be further elucidated. Here, we investigate the role of MYB in human leukaemia by performing siRNA-mediated knock-down in cell line models of AML with different driver lesions. We show that the characteristic reduction in proliferation and the concomitant induction of myeloid differentiation that is observed in MLL-rearranged and t(8;21) leukaemias upon MYB suppression is not seen in AML cells with a complex karyotype. Transcriptome analyses revealed that MYB ablation produces consensual increase of MAFB expression in MYB-dependent cells and, interestingly, the ectopic expression of MAFB could phenocopy the effect of MYB suppression. Accordingly, in silico stratification analyses of molecular data from AML patients revealed a reciprocal relationship between MYB and MAFB expression, highlighting a novel biological interconnection between these two factors in AML and supporting new rationales of MAFB targeting in MLL-rearranged leukaemias.

Dynamic changes in thalamic microstructure of migraine without aura patients: a diffusion tensor magnetic resonance imaging study.

BACKGROUND AND PURPOSE: The thalamus seems to be profoundly involved in the cyclical recurrence of migraine clinical and neurophysiological features. Here possible structural changes in the thalamus of migraineurs were searched for by means of diffusion tensor (DT) magnetic resonance imaging (MRI). This MRI technique provides quantitative data on water molecule motion as a marker of tissue microstructure. METHODS: Twenty-four untreated migraine without aura (MO) patients underwent DT-MRI scans (3-T Siemens Gyroscan) during (n = 10) and between attacks (n = 14) and were compared with a group of 15 healthy volunteers (HVs). Fractional anisotropy (FA) and mean diffusivity (MD) were examined. RESULTS: During the interictal phase MO patients had a significantly higher FA and slightly lower MD values in bilateral thalami compared with HVs. During attacks, all MRI quantitative measurements in migraineurs were similar to those found in HVs. Right thalamic FA was positively correlated with the number of days since the last migraine attack in pooled patient data (r = 0.626, P = 0.003). CONCLUSIONS: These higher thalamic FA values noted during the interictal period which normalized during an attack are probably related to plastic peri-ictal modifications in regional branching and crossing of fibres. Whether these changes could be considered as the anatomical counterpart of the cyclical functional fluctuations previously observed in the neurophysiology of migraine remains to be determined.

Mutations in the mitochondrial glutamate carrier SLC25A22 in neonatal epileptic encephalopathy with suppression bursts.

Neonatal epileptic encephalopathies with suppression bursts (SBs) are very severe and relatively rare diseases characterized by neonatal onset of seizures, interictal electroencephalogram (EEG) with SB pattern and very poor neurological outcome or death. Their etiology remains elusive but they are occasionally caused by metabolic diseases or malformations. Studying an Arab Muslim Israeli consanguineous family, with four affected children presenting a severe neonatal epileptic encephalopathy, we have previously identified a mutation in the SLC25A22 gene encoding a mitochondrial glutamate transporter. In this report, we describe a novel SLC25A22 mutation in an unrelated patient born from first cousin Algerian parents and presenting severe epileptic encephalopathy characterized by an EEG with SB, hypotonia, microcephaly and abnormal electroretinogram. We showed that this patient carried a homozygous p.G236W SLC25A22 mutation which alters a highly conserved amino acid and completely abolishes the glutamate carrier's activity in vitro. Comparison of the clinical features of patients from both families suggests that SLC25A22 mutations are responsible for a novel clinically recognizable epileptic encephalopathy with SB.

Asymmetric distribution of visual evoked potentials in patients with migraine with aura during the interictal phase.

PURPOSE: One of the most commonly described electrocortical phenomena in patients with migraine is an increased interhemispheric asymmetry, in response to different sensory stimuli. This study aims to evaluate the bioelectrical activity of both occipital cortices in patients with migraine with visual aura (MA) during the interictal period, and its possible relationship with visual symptoms. METHODS: The authors recorded visual evoked potentials (VEPs) simultaneously from the left (O1) and right (O2) occipital cortices (80% contrast 60', 30', 15', and 7.5' checkerboard stimuli reversed at 2 Hz) in 22 patients with MA and 20 control subjects. The main outcome measure was interhemispheric asymmetry (IA) for both implicit time and amplitude, defined as the difference between the left and right scalp derivation (in absolute values). RESULTS: IA was significantly different in patients with MA with respect to controls when employing 60' (p<0.001) and 15' (p<0.05) checkerboard stimuli for implicit times, and 60' (p<0.05) checkerboard stimuli for amplitudes. On the other hand, IA was not statistically different (p>0.05) in patients with MA with respect to controls when employing 30' and 7.5' checkerboards for both implicit times and amplitudes, and 15' checkerboards for amplitudes. No correlations were found between IA and age, onset of disease, attack frequency, or side of headache/aura. CONCLUSIONS: Patients with MA presented asymmetries in VEP responses not related to visual aura or to headache side during the pain-free phase. These abnormalities may be ascribed to abnormal visual information processing, resulting in a different cortical activation when both foveal and parafoveal stimuli are used.

A novel ATP1A2 mutation in a family with FHM type II.

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura with an autosomal dominant pattern of inheritance. Six FHM families underwent extensive clinical and genetic investigation. The authors identified a novel ATP1A2 mutation (E700K) in three patients from one family. In the patients, attacks were triggered by several factors including minor head trauma. In one subject a 3-day coma developed after a cerebral angiography. Overall, the phenotype of the patients closely resembles that of previously reported cases of FHM type II. The E700K variant might be regarded as the cause of the disease in this family, but this was not tested functionally.

Identification of the human mitochondrial S-adenosylmethionine transporter: bacterial expression, reconstitution, functional characterization and tissue distribution.

The mitochondrial carriers are a family of transport proteins that, with a few exceptions, are found in the inner membranes of mitochondria. They shuttle metabolites and cofactors through this membrane, and connect cytoplasmic functions with others in the matrix. SAM (S-adenosylmethionine) has to be transported into the mitochondria where it is converted into S-adenosylhomocysteine in methylation reactions of DNA, RNA and proteins. The transport of SAM has been investigated in rat liver mitochondria, but no protein has ever been associated with this activity. By using information derived from the phylogenetically distant yeast mitochondrial carrier for SAM and from related human expressed sequence tags, a human cDNA sequence was completed. This sequence was overexpressed in bacteria, and its product was purified, reconstituted into phospholipid vesicles and identified from its transport properties as the human mitochondrial SAM carrier (SAMC). Unlike the yeast orthologue, SAMC catalysed virtually only countertransport, exhibited a higher transport affinity for SAM and was strongly inhibited by tannic acid and Bromocresol Purple. SAMC was found to be expressed in all human tissues examined and was localized to the mitochondria. The physiological role of SAMC is probably to exchange cytosolic SAM for mitochondrial S-adenosylhomocysteine. This is the first report describing the identification and characterization of the human SAMC and its gene.

Genomic organization and mapping of the gene (SLC25A19) encoding the human mitochondrial deoxynucleotide carrier (DNC).

The deoxynucleotide carrier (DNC) transports deoxynucleotides into mitochondria and is therefore essential for mtDNA synthesis. The human DNC gene (SLC25A19) spans about 16.5 kb and consists of nine exons with the translation start site in exon 4. It is located on chromosome 17q25.3. Three transcripts, which differ in their 5' ends and are generated by alternative splicing, have been identified.

The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals.

The synthesis of DNA in mitochondria requires the uptake of deoxynucleotides into the matrix of the organelle. We have characterized a human cDNA encoding a member of the family of mitochondrial carriers. The protein has been overexpressed in bacteria and reconstituted into phospholipid vesicles where it catalyzed the transport of all four deoxy (d) NDPs, and, less efficiently, the corresponding dNTPs, in exchange for dNDPs, ADP, or ATP. It did not transport dNMPs, NMPs, deoxynucleosides, nucleosides, purines, or pyrimidines. The physiological role of this deoxynucleotide carrier is probably to supply deoxynucleotides to the mitochondrial matrix for conversion to triphosphates and incorporation into mitochondrial DNA. The protein is expressed in all human tissues that were examined except for placenta, in accord with such a central role. The deoxynucleotide carrier also transports dideoxynucleotides efficiently. It is likely to be medically important by providing the means of uptake into mitochondria of nucleoside analogs, leading to the mitochondrial impairment that underlies the toxic side effects of such drugs in the treatment of viral illnesses, including AIDS, and in cancer therapy.

Identification of the human mitochondrial oxodicarboxylate carrier. Bacterial expression, reconstitution, functional characterization, tissue distribution, and chromosomal location.

In Saccharomyces cerevisiae, the genes ODC1 and ODC2 encode isoforms of the oxodicarboxylate carrier. They both transport C5-C7 oxodicarboxylates across the inner membranes of mitochondria and are members of the family of mitochondrial carrier proteins. Orthologs are encoded in the genomes of Caenorhabditis elegans and Drosophila melanogaster, and a human expressed sequence tag (EST) encodes part of a closely related protein. Information from the EST has been used to complete the human cDNA sequence. This sequence has been used to map the gene to chromosome 14q11.2 and to show that the gene is expressed in all tissues that were examined. The human protein was produced by overexpression in Escherichia coli, purified, and reconstituted into phospholipid vesicles. It has similar transport characteristics to the yeast oxodicarboxylate carrier proteins (ODCs). Both the human and yeast ODCs catalyzed the transport of the oxodicarboxylates 2-oxoadipate and 2-oxoglutarate by a counter-exchange mechanism. Adipate, glutarate, and to a lesser extent, pimelate, 2-oxopimelate, 2-aminoadipate, oxaloacetate, and citrate were also transported by the human ODC. The main differences between the human and yeast ODCs are that 2-aminoadipate is transported by the former but not by the latter, whereas malate is transported by the yeast ODCs but not by the human ortholog. In mammals, 2-oxoadipate is a common intermediate in the catabolism of lysine, tryptophan, and hydroxylysine. It is transported from the cytoplasm into mitochondria where it is converted into acetyl-CoA. Defects in human ODC are likely to be a cause of 2-oxoadipate acidemia, an inborn error of metabolism of lysine, tryptophan, and hydroxylysine.

Identification and functions of new transporters in yeast mitochondria.

The genome of Saccharomyces cerevisiae encodes 35 putative members of the mitochondrial carrier family. Known members of this family transport substrates and products across the inner membranes of mitochondria. We are attempting to identify the functions of the yeast mitochondrial transporters via high-yield expression in Escherichia coli and/or S. cerevisiae, purification and reconstitution of their protein products into liposomes, where their transport properties are investigated. With this strategy, we have already identified the functions of seven S. cerevisiae gene products, whose structural and functional properties assigned them to the mitochondrial carrier family. The functional information obtained in the reconstituted system and the use of knock-out yeast strains can be usefully exploited for the investigation of the physiological role of individual transporters. Furthermore, the yeast carrier sequences can be used to identify the orthologous proteins in other organisms, including man.

Yeast mitochondrial carriers: bacterial expression, biochemical identification and metabolic significance.

The genome of Saccharomyces cerevisiae encodes 35 members of a family proteins that transport metabolites and substrates across the inner membranes of mitochondria. They include three isoforms of the ADP/ATP translocase and the phosphate and citrate carriers. At the start of our work, the functions of the remaining 30 members of the family were unknown. We are attempting to identify these 30 proteins by overexpression of the proteins in specially selected host strains of Escherichia coli that allow the carriers to accumulate at high levels in the form of inclusion bodies. The purified proteins are then reconstituted into proteoliposomes where their transport properties are studied. Thus far, we have identified the dicarboxylate, succinate-fumarate and ornithine carriers. Bacterial overexpression and functional identification, together with characterization of yeast knockout strains, has brought insight into the physiological significance of these transporters. The yeast dicarboxylate carrier sequence has been used to identify the orthologous protein in Caenorhabditis elegans and, in turn, this latter sequence has been used to establish the sequence of the human ortholog.

Organization and sequence of the gene for the human mitochondrial dicarboxylate carrier: evolution of the carrier family.

The dicarboxylate carrier (DIC) is a nuclear-encoded protein located in the mitochondrial inner membrane. It catalyses the transport of dicarboxylates such as malate and succinate across the mitochondrial membrane in exchange for phosphate, sulphate and thiosulphate. We have determined the sequences of the human cDNA and gene for the DIC. The gene sequence was established from overlapping genomic clones generated by PCRs by use of primers and probes based upon the human cDNA sequence. It is spread over 8.6 kb of human DNA and is divided into 11 exons. Five short interspersed repetitive Alu sequences are found in intron I. The protein encoded by the gene is 287 amino acids long. In common with the rat protein, it does not have a processed presequence to help to target it into mitochondria. It has been demonstrated by Northern- and Western-blot analyses that the DIC is present in high amounts in liver and kidney, and at lower levels in all the other tissues analysed. The positions of introns contribute towards an understanding of the processes involved in the evolution of human genes for carrier proteins.

Transcranial Doppler evaluation of cerebral hemodynamics in migraineurs during prophylactic treatment with flunarizine.

Transcranial Doppler (TCD) recording was used to evaluate the mean flow velocity (MFV) and cerebrovascular reactivity to CO2 in 21 migraineurs during the interictal phase. Nine were affected by migraine with aura (MwA) and 12 by migraine without aura (MwoA). During each session the middle cerebral artery (MCA) flow velocity was examined in basal conditions, in hypocapnia after a 3-min period of hyperventilation, in basal conditions a second time, and in hypercapnia after breath-holding. The same procedure was followed in a group of 21 age- and sex-matched volunteers. Recordings were performed before (T1), during (T2), and after (T3) prophylactic treatment with flunarizine (10 mg/day for 2 months) to assess the possible effect of this drug on cerebral hemodynamics. In basal condition, increased MFV values were found in both MwA and MwoA patients. In MwA patients the reactivity index (RI) to hypocapnia was significantly increased in T1 (p < 0.05). This abnormal cerebrovascular reactivity disappeared during flunarizine treatment (T2) and in the post-therapy period (T3).

The sequence, bacterial expression, and functional reconstitution of the rat mitochondrial dicarboxylate transporter cloned via distant homologs in yeast and Caenorhabditis elegans.

The dicarboxylate carrier (DIC) belongs to a family of transport proteins found in the inner mitochondrial membranes. The biochemical properties of the mammalian protein have been characterized, but the protein is not abundant. It is difficult to purify and had not been sequenced. We have used the sequence of the distantly related yeast DIC to identify a related protein encoded in the genome of Caenorhabditis elegans. Then, related murine expressed sequence tags were identified with the worm sequence, and the murine sequence was used to isolate the cDNA for the rat homolog. The sequences of the worm and rat proteins have features characteristic of the family of mitochondrial transport proteins. Both proteins were expressed in bacteria and reconstituted into phospholipid vesicles where their transport characteristics closely resembled those of whole rat mitochondria and of the rat DIC reconstituted into vesicles. As expected from the role of the DIC in gluconeogenesis and ureogenesis, its transcripts were detected in rat liver and kidney, but unexpectedly, they were also detected in rat heart and brain tissues where the protein may fulfill other roles, possibly in supplying substrates to the Krebs cycle.

Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria.

The two isoforms of the mammalian mitochondrial phosphate carrier (PiC), A and B, differing in the sequence near the N terminus, arise from alternative splicing of a primary transcript of the PiC gene (Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994) J. Biol. Chem. 269, 10451-10460). To date, the PiC isoforms A and B have not been studied at the protein level. To explore the tissue-distribution and the potential functional differences between the two isoforms, polyclonal site-directed antibodies specific for PiC-A and PiC-B were raised, and the two bovine isoforms were obtained by expression in Escherichia coli and reconstituted into phospholipid vesicles. Western blot analysis demonstrated that isoform A is present in high amounts in heart, skeletal muscle, and diaphragm mitochondria, whereas isoform B is present in the mitochondria of all tissues examined. Heart and liver bovine mitochondria contained 69 and 0 pmol of PiC-A/mg of protein, and 10 and 8 pmol of PiC-B/mg of protein, respectively. In the reconstituted system the pure recombinant isoforms A and B both catalyzed the two known modes of transport (Pi/Pi antiport and Pi/H+ symport) and exhibited similar properties of substrate specificity and inhibitor sensitivity. However, they strongly differed in their kinetic parameters. The transport affinities of isoform B for phosphate and arsenate were found to be 3-fold lower than those of isoform A. Furthermore, the maximum transport rate of isoform B is about 3-fold higher than that of isoform A. These results support the hypothesis that the sequence divergence between PiC-A and PiC-B may have functional significance in determining the affinity and the translocation rate of the substrate through the PiC molecule.

Tissue-specific expression of the two isoforms of the mitochondrial phosphate carrier in bovine tissues.

Comparison of the sequence of the human mitochondrial phosphate carrier (PiC) gene with cDNA clones characterised from a human heart cDNA library suggested the existence of two isoforms of the PiC, which were generated by alternative splicing of exon IIIA or exon IIIB and which differed in 13 amino acids [Dolce et al. (1994) J. Biol. Chem. 269, 10451]. In this work the expression of isoforms A and B of the PiC was investigated in different bovine tissues by Northern blot analysis using two probes that are specific for bovine exon IIIA and exon IIIB, respectively. Isoform A is highly expressed in heart and skeletal muscle. Isoform B is ubiquitously expressed in all tissues that were examined, although at different levels. The tissue-specific expression pattern of the two PiC isoforms is similar to that reported for the isoforms of several mitochondrial proteins required for energy production.

Cerebrovascular CO2 reactivity in migraine with aura and without aura. A transcranial Doppler study.

INTRODUCTION: We studied by means of Transcranial Doppler (TCD) recordings the CO2 cerebrovascular reactivity in migraine patients during the headache-free period. MATERIAL & METHODS: In three groups of subjects (15 controls, 15 suffering from migraine with aura and 15 from migraine without aura) the middle cerebral artery (MCA) mean flow velocity (MFV) was recorded under basal condition and hypocapnia induced by hyperventilation. Relative MFV, PI (Pulsatility Index) changes and Reactivity Index (RI) were calculated. RESULTS: Reactivity Index values were: 0.019 +/- 0.007 (mean +/- SD) in control subjects: 0.029 +/- 0.008 in migraine with aura; 0.022 +/- 0.008 in migraine without aura. Statistical analysis showed a significantly (P < 0.05) increased RI in migraine with aura group. CONCLUSION: Cerebrosvascular CO2 reactivity is increased during the interictal period in migraine with aura patients.