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Contains fulltext : 48728.pdf (Publisher’s version ) (Closed access) The injection of 1.0 M glutamate into the masseter (jaw-closer) muscle results in a short period of muscle pain (5-10 min) and a prolonged period of mechanical sensitization (> 30 min). It is unclear, however, whether there is a temporal relationship between intramuscular glutamate concentration and either muscle pain or mechanical sensitization. In the present study, (1)H MRS and electrophysiological recording of masticatory muscle nerve fibers were performed in order to monitor glutamate clearance and nerve fiber activity, respectively, after injection of glutamate into rat masticatory muscles. Glutamate signal amplitude was found to decay rapidly (half-life t 1/2 = 108 +/- 42 s), and became indistinguishable from the baseline 10 min after the injection. Glutamate-evoked nerve fiber activity was also found to decay rapidly (t 1/2 = 76 +/- 28 s). These results suggest that glutamate clearance correlates well with the time course of glutamate-evoked muscle pain fiber discharge.

The availability of genome sequences and functional genomics data from multiple species enables us to compare the composition of biomolecular systems like biochemical pathways and protein complexes between species. Here, we review small- and large-scale, “genomics-based” approaches to biomolecular systems variation. In general, caution is required when comparing the results of bioinformatics analyses of genomes or of functional genomics data between species. Limitations to the sensitivity of sequence analysis tools and the noisy nature of genomics data tend to lead to systematic overestimates of the amount of variation. Nevertheless, the results from detailed manual analyses, and of large-scale analyses that filter out systematic biases, point to a large amount of variation in the composition of biomolecular systems. Such observations challenge our understanding of the function of the systems and their individual components and can potentially facilitate the identification and functional characterization of sub-systems within a system. Mapping the inter-species variation of complex biomolecular systems on a phylogenetic species tree allows one to reconstruct their evolution.

Contains fulltext : 32314.pdf (Publisher’s version ) (Open Access) Post-translational histone modifications abound and regulate multiple nuclear processes. Most modifications are targeted to the amino-terminal domains of histones. Here we report the identification and characterization of acetylation of lysine 56 within the core domain of histone H3. In the crystal structure of the nucleosome, lysine 56 contacts DNA. Phenotypic analysis suggests that lysine 56 is critical for histone function and that it modulates formamide resistance, ultraviolet radiation sensitivity, and sensitivity to hydroxyurea. We show that the acetylated form of histone H3 lysine 56 (H3-K56) is present during interphase, metaphase, and S phase. Finally, reverse genetic analysis indicates that none of the known histone acetyltransferases is solely responsible for H3-K56 acetylation in Saccharomyces cerevisiae.

Hydrogenosomes are organelles that produce ATP and hydrogen(1), and are found in various unrelated eukaryotes, such as anaerobic flagellates, chytridiomycete fungi and ciliates(2). Although all of these organelles generate hydrogen, the hydrogenosomes from these organisms are structurally and metabolically quite different, just like mitochondria where large differences also exist(3). These differences have led to a continuing debate about the evolutionary origin of hydrogenosomes(4,5). Here we show that the hydrogenosomes of the anaerobic ciliate Nyctotherus ovalis, which thrives in the hindgut of cockroaches, have retained a rudimentary genome encoding components of a mitochondrial electron transport chain. Phylogenetic analyses reveal that those proteins cluster with their homologues from aerobic ciliates. In addition, several nucleus-encoded components of the mitochondrial proteome, such as pyruvate dehydrogenase and complex II, were identified. The N. ovalis hydrogenosome is sensitive to inhibitors of mitochondrial complex I and produces succinate as a major metabolic end product - biochemical traits typical of anaerobic mitochondria(3). The production of hydrogen, together with the presence of a genome encoding respiratory chain components, and biochemical features characteristic of anaerobic mitochondria, identify the N. ovalis organelle as a missing link between mitochondria and hydrogenosomes.

Contains fulltext : 48708.pdf (Publisher’s version ) (Closed access) In clinical MR spectroscopy, double spin-echo point resolved spectroscopy (PRESS) sequences are routinely used for volume selection. For strongly coupled AB spin systems under PRESS excitation, the dependence of the signal on the echo time TE has been thoroughly investigated, whereas less attention has been paid to the signal modulation which occurs at constant TE with varying interpulse delays. A substantial TE-independent J modulation is here predicted from analytical solutions of the Liouville equation and density matrix simulations, and verified with experiments on citrate at 1.5 and 3T. It is also shown that this modulation effect could be exploited for editing of strongly coupled AB resonances or for removal of singlets in spectra-by means of difference spectroscopy-just using a standard PRESS sequence. The applicability in vivo of this new spectral editing approach is also demonstrated, with selective detection of citrate resonances in the human prostate. This novel approach has the advantages of being simple, and directly applicable on standard clinical MR scanners, provided that the exact behavior of the resonance is known.

Genomes, functional genomics data and 3D structure reflect different aspects of protein function. Here, we combine these data to predict that BolA, a widely distributed protein family with unknown function, is a reductase that interacts with a glutaredoxin. Comparisons at the 3D structure level as well as at the sequence profile level indicate homology between BolA and OsmC, an enzyme that reduces organic peroxides. Complementary to this, comparative analyses of genomes and genomics data provide strong evidence of an interaction between BolA and the mono-thiol glutaredoxin family. The interaction between BolA and a mono-thiol glutaredoxin is of particular interest because BolA does not, in contrast to its homolog OsmC, have evolutionarily conserved cysteines to provide it with reducing equivalents. We propose that BolA uses the mono-thiol glutaredoxin as the source for these.

A full description of a protein's function requires knowledge of all partner proteins with which it specifically associates. From a functional perspective, ‘association’ can mean direct physical binding, but can also mean indirect interaction such as participation in the same metabolic pathway or cellular process. Currently, information about protein association is scattered over a wide variety of resources and model organisms. STRING aims to simplify access to this information by providing a comprehensive, yet quality-controlled collection of protein–protein associations for a large number of organisms. The associations are derived from high-throughput experimental data, from the mining of databases and literature, and from predictions based on genomic context analysis. STRING integrates and ranks these associations by benchmarking them against a common reference set, and presents evidence in a consistent and intuitive web interface. Importantly, the associations are extended beyond the organism in which they were originally described, by automatic transfer to orthologous protein pairs in other organisms, where applicable. STRING currently holds 730 000 proteins in 180 fully sequenced organisms, and is available at http://string.embl.de/.

Force characteristics of skeletal muscle of knockout mice lacking creatine (Cr) due to a deletion of guanidinoacetate methyltransferase (GAMT) were studied in situ. Medial gastrocnemius muscles of anesthetized GAMT-deficient (GAMT−/−) and control (Con) littermates were stimulated at optimum length via the sciatic nerve at different stimulation frequencies (60–250 Hz). GAMT−/− mice showed reduced maximal tetanic and twitch force, reduced relative force at 60 Hz, and increased relaxation times. High-intensity fatigue protocols consisting of 30 successive isometric or dynamic contractions showed a strong reduction in force at the beginning of the series in GAMT−/− mice, followed by a smaller reduction compared with Con littermates toward the end of the series. Cr supplementation for 2 days in GAMT−/− animals (GAMT[Formula: see text]) resulted in normalization to Con values for relaxation times, relative force at lower stimulation frequencies, and relative force during 30 isometric contractions. Force per muscle mass, however, remained decreased. Furthermore,GAMT[Formula: see text] mice showed differences compared with both Con and unsupplemented animals in maximal rates of force rise and relaxation times during the isometric protocol as well as in force during the dynamic protocol. Our results show that the absence of Cr plays a direct role in relaxation times, maximal rate of force rise, and force production during high-intensity fatigue protocols. The lower force per muscle mass, however, is probably caused by other factors; i.e., high intracellular guanidinoacetate concentrations.

Item does not contain fulltext Congenital adrenal hyperplasia (CAH) is a disorder of adrenal steroid synthesis. The symptoms and signs of CAH depend on the degree of enzyme deficiency; severe salt-wasting (SW) form, less severe simple virilizing (SV) form and mild nonclassic (NC) form. In this paper, puberty and fertility in CAH are discussed. The time of onset of puberty and progress of pubertal development is quite normal, except in NC patients (earlier). Also the age of menarche in CAH girls is normal, but it can depend on the level of therapeutic control. In prepuberty, bone age is advanced. In puberty, peak height velocity is normal but occurs at a younger age and can therefore be considered to be low (compared to healthy early maturers). In puberty there seems to be an increased sensitivity for glucocorticoids leading to growth inhibition. All three above factors can play a role in reducing adult height. Subfertility is frequently found in both female and male CAH patients. In females, the pregnancy rate depends on the severity of 21-hydroxylase deficiency (SW

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Contains fulltext : 32331.pdf (Publisher’s version ) (Closed access) The purpose of this paper is to evaluate the effect of the combination of magnetic resonance spectroscopic imaging (MRSI) data and magnetic resonance imaging (MRI) data on the classification result of four brain tumor classes. Suppressed and unsuppressed short echo time MRSI and MRI were performed on 24 patients with a brain tumor and four volunteers. Four different feature reduction procedures were applied to the MRSI data: simple quantitation, principal component analysis, independent component analysis and LCModel. Water intensities were calculated from the unsuppressed MRSI data. Features were extracted from the MR images which were acquired with four different contrasts to comply with the spatial resolution of the MRSI. Evaluation was performed by investigating different combinations of the MRSI features, the MRI features and the water intensities. For each data set, the isolation in feature space of the tumor classes, healthy brain tissue and cerebrospinal fluid was calculated and visualized. A test set was used to calculate classification results for each data set. Finally, the effect of the selected feature reduction procedures on the MRSI data was investigated to ascertain whether it was more important than the addition of MRI information. Conclusions are that the combination of features from MRSI data and MRI data improves the classification result considerably when compared with features obtained from MRSI data alone. This effect is larger than the effect of specific feature reduction procedures on the MRSI data. The addition of water intensities to the data set also increases the classification result, although not significantly. We show that the combination of data from different MR investigations can be very important for brain tumor classification, particularly if a large number of tumors are to be classified simultaneously. Copyright (C) 2005 John Wiley Sons, Ltd.

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The increasing availability of sequenced genomes enables the reconstruction of the evolutionary history of large protein complexes. Here, we trace the evolution of NADH:ubiquinone oxidoreductase (Complex I), which has increased in size, by so-called supernumary subunits, from 14 subunits in the bacteria to 30 in the plants and algae, 37 in the fungi and 46 in the mammals. Using a combination of pair-wise and profile-based sequence comparisons at the levels of proteins and the DNA of the sequenced eukaryotic genomes, combined with phylogenetic analyses to establish orthology relationships, we were able to (1) trace the origin of six of the supernumerary subunits to the alpha-proteobacterial ancestor of the mitochondria, (2) detect previously unidentified homology relations between subunits from fungi and mammals, (3) detect previously unidentified subunits in the genomes of several species and (4) document several cases of gene duplications among supernumerary subunits in the eukaryotes. One of these, a duplication of N7BM (B17.2), is particularly interesting as it has been lost from genomes that have also lost Complex I proteins, making it a candidate for a Complex I interacting protein. A parsimonious reconstruction of eukaryotic Complex I evolution shows an initial increase in size that predates the separation of plants, fungi and metazoa, followed by a gradual adding and incidental losses of subunits in the various evolutionary lineages. This evolutionary scenario is in contrast to that for Complex I in the prokaryotes, for which the combination of several separate, and previously independently functioning modules into a single complex has been proposed.

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Contains fulltext : 48742.pdf (Publisher’s version ) (Closed access) BACKGROUND: In adult patients with a slowly progressive demyelinating neuropathy, it may be difficult to distinguish between a hereditary neuropathy and chronic inflammatory demyelinating polyneuropathy (CIDP). The authors previously observed clustering of macrophages around endoneurial blood vessels in sural nerve biopsies from patients with CIDP. OBJECTIVES: To quantitate macrophage clustering around endoneurial blood vessels in CIDP vs hereditary neuropathies. METHODS: The authors studied 21 patients with CIDP, 18 patients with hereditary neuropathies, and 5 normal sural nerves. Numbers of macrophages, T-cells, and blood vessels were counted after immunohistochemical staining. The presence of three or more macrophages around one blood vessel was defined as a cluster. In a subsequent validation analysis, 65 stored biopsy specimens obtained from patients with a chronic neuropathy were re-evaluated for perivascular macrophage clustering according to criteria derived from the quantitative analysis of the first 221 biopsies in a blinded fashion. RESULTS: The percentage of endoneurial vessels with macrophage clusters was higher in CIDP than in hereditary neuropathies (CIDP median = 9.4, range 0 to 48; hereditary NP median = 0, range 0 to 7.7; p < 0.001). The evaluation of the 65 further biopsies showed that the presence of one perivascular macrophage cluster per fascicle proved to be a valid criterion to differentiate between inflammatory and other forms of neuropathy (chi2 test p = 0.0000025, sensitivity 75%, specificity 72%). CONCLUSION: The presence of clusters of macrophages around endoneurial vessels in sural nerve biopsies may serve as a useful additional marker for establishing the pathologic diagnosis of chronic inflammatory demyelinating polyneuropathy (CIDP).

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Video-rate confocal microscopy of Indo-1-loaded human skeletal myotubes was used to assess the relationship between the changes in sarcoplasmic ([Ca(2+)](S)) and nuclear ([Ca(2+)](N)) Ca(2+) concentration during low- and high-frequency electrostimulation. A single stimulus of 10 ms duration transiently increased [Ca(2+)] in both compartments with the same time of onset. Rate and amplitude of the [Ca(2+)] rise were significantly lower in the nucleus (4.0- and 2.5-fold, respectively). Similarly, [Ca(2+)](N) decayed more slowly than [Ca(2+)](S) (mono-exponential time constants of 6.1 and 2.5 s, respectively). After return of [Ca(2+)] to the prestimulatory level, a train of 10 stimuli was applied at a frequency of 1 Hz. The amplitude of the first [Ca(2+)](S) transient was 25% lower than that of the preceding single transient. Thereafter, [Ca(2+)](S) increased stepwise to a maximum that equalled that of the single transient. Similarly, the amplitude of the first [Ca(2+)](N) transient was 20% lower than that of the preceding single transient. In contrast to [Ca(2+)](S), [Ca(2+)](N) then increased to a maximum that was 2.3-fold higher than that of the single transient and equalled that of [Ca(2+)](S). In the nucleus, and to a lesser extent in the sarcoplasm, [Ca(2+)] decreased faster at the end of the stimulus train than after the preceding single stimulus (time constants of 3.3 and 2.1 s, respectively). To gain insight into the molecular principles underlying the shaping of the nuclear Ca(2+) signal, a 3-D mathematical model was constructed. Intriguingly, quantitative modelling required the inclusion of a satiable nuclear Ca(2+) buffer. Alterations in the concentration of this putative buffer had dramatic effects on the kinetics of the nuclear Ca(2+) signal. This finding unveils a possible mechanism by which the skeletal muscle can adapt to changes in physiological demand.

Contains fulltext : 48204.pdf (Publisher’s version ) (Closed access) Recently, we described the molecular identification of dendritic cell-specific TrAnsMembrane protein (DC-STAMP), a multimembrane-spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC-STAMP (mDC-STAMP) as well as the characterization of the DC-STAMP protein. The results demonstrate that mDC-STAMP is over 90% homologous to hDC-STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC-STAMP expression is enhanced by interleukin-4 and down-regulated upon DC maturation. Analysis of differently tagged DC-STAMP proteins further demonstrates that hDC-STAMP and mDC-STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC-STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC-STAMP-green fluorescent protein fusion protein. These data imply that DC-STAMP may exert its effect in the ER.

Contains fulltext : 33148.pdf (Publisher’s version ) (Closed access) This study investigated the value of information from both magnetic resonance imaging and magnetic resonance spectroscopic imaging (MRSI) to automated discrimination of brain tumours. The influence of imaging intensities and metabolic data was tested by comparing the use of MR spectra from MRSI, MR imaging intensities, peak integration values obtained from the MR spectra and a combination of the latter two. Three classification techniques were objectively compared: linear discriminant analysis, least squares support vector machines (LS-SVM) with a linear kernel as linear techniques and LS-SVM with radial basis function kernel as a nonlinear technique. Classifiers were evaluated over 100 stratified random splittings of the dataset into training and test sets. The area under the receiver operating characteristic (ROC) curve (AUC) was used as a global performance measure on test data. In general, all techniques obtained a high performance when using peak integration values with or without MR imaging intensities. For example for low- versus high-grade tumours, low- versus high-grade gliomas and gliomas versus meningiomas, the mean test AUC was higher than 0.91, 0.94, and 0.99, respectively, when both MR imaging intensities and peak integration values were used. The use of metabolic data from MRSI significantly improved automated classification of brain tumour types compared to the use of MR imaging intensities solely. (c) 2004 Elsevier Inc. All rights reserved.

Item does not contain fulltext Here, we report the first isolation of Wautersiella falsenii from the urine of an infant with a complicated urinary tract infection. W. falsenii was correctly identified by matrix-assisted laser desorption ionisation time of flight mass spectrometry. The identification was confirmed by 16S polymerase chain reaction. Susceptibility test results of this isolate are reported. Ciprofloxacin treatment resulted in clinical and microbiological improvement.

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Contains fulltext : 51010.pdf (Publisher’s version ) (Closed access) Mutations in SURF1, an assembly gene for cytochrome c oxidase (COX), the fourth complex of the oxidative phosphorylation system, are most frequently encountered in patients with COX deficiency. We describe a patient with Leigh syndrome harbouring a mutation in SURF1 who was reported decades ago with a tissue-specific cytochrome c oxidase deficiency.

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Mitochondrial oxidative phosphorylation (OXPHOS) is responsible for generating the majority of cellular ATP. Complex III (ubiquinol-cytochrome c oxidoreductase) is the third of five OXPHOS complexes. Complex III assembly relies on the coordinated expression of the mitochondrial and nuclear genomes, with 10 subunits encoded by nuclear DNA and one by mitochondrial DNA (mtDNA). Complex III deficiency is a debilitating and often fatal disorder that can arise from mutations in complex III subunit genes or one of three known complex III assembly factors. The molecular cause for complex III deficiency in about half of cases, however, is unknown and there are likely many complex III assembly factors yet to be identified. Here, we used Massively Parallel Sequencing to identify a homozygous splicing mutation in the gene encoding Ubiquinol-Cytochrome c Reductase Complex Assembly Factor 2 (UQCC2) in a consanguineous Lebanese patient displaying complex III deficiency, severe intrauterine growth retardation, neonatal lactic acidosis and renal tubular dysfunction. We prove causality of the mutation via lentiviral correction studies in patient fibroblasts. Sequence-profile based orthology prediction shows UQCC2 is an ortholog of the Saccharomyces cerevisiae complex III assembly factor, Cbp6p, although its sequence has diverged substantially. Co-purification studies show that UQCC2 interacts with UQCC1, the predicted ortholog of the Cbp6p binding partner, Cbp3p. Fibroblasts from the patient with UQCC2 mutations have deficiency of UQCC1, while UQCC1-depleted cells have reduced levels of UQCC2 and complex III. We show that UQCC1 binds the newly synthesized mtDNA-encoded cytochrome b subunit of complex III and that UQCC2 patient fibroblasts have specific defects in the synthesis or stability of cytochrome b. This work reveals a new cause for complex III deficiency that can assist future patient diagnosis, and provides insight into human complex III assembly by establishing that UQCC1 and UQCC2 are complex III assembly factors participating in cytochrome b biogenesis., Author Summary Mitochondrial complex III deficiency is a devastating disorder that impairs energy generation, and leads to variable symptoms such as developmental regression, seizures, kidney dysfunction and frequently death. The genetic basis of complex III deficiency is not fully understood, with around half of cases having no known cause. This lack of genetic diagnosis is partly due to an incomplete understanding of the genes required for complex III assembly and function. We have identified two key proteins required for complex III, UQCC1 and UQCC2, and have elucidated the role of these inter-dependent proteins in the biogenesis of cytochrome b, the only complex III subunit that is encoded by mitochondrial DNA. We have shown that mutations in UQCC2 cause human complex III deficiency in a patient with neonatal lactic acidosis and renal tubulopathy. This work contributes to an improved understanding of complex III biogenesis, and will aid future molecular diagnoses of complex III deficiency.

Contains fulltext : 109893.pdf (Publisher’s version ) (Open Access) Recurrent submicroscopic deletions in genes affecting key cellular pathways are a hallmark of pediatric acute lymphoblastic leukemia (ALL). To gain more insight into the mechanism underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B-cell translocation gene 1 (BTG1), in a large cohort of pediatric ALL cases. BTG1 was found to be exclusively affected by genomic deletions, which were detected in 65 out of 722 B-cell precursor ALL (BCP-ALL) patient samples (9%), but not in 109 T-ALL cases. Eight different deletion sizes were identified, which all clustered at the telomeric site in a hotspot region within the second (and last) exon of the BTG1 gene, resulting in the expression of truncated BTG1 read-through transcripts. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests illegitimate RAG1/RAG2-mediated recombination as the responsible mechanism. Moreover, high levels of histone H3 lysine 4 trimethylation (H3K4me3), which is known to tether the RAG enzyme complex to DNA, were found within the BTG1 gene body in BCP-ALL cells, but not T-ALL cells. BTG1 deletions were rarely found in hyperdiploid BCP-ALLs, but were predominant in other cytogenetic subgroups, including the ETV6-RUNX1 and BCR-ABL1 positive BCP-ALL subgroups. Through sensitive PCR-based screening, we identified multiple additional BTG1 deletions at the subclonal level in BCP-ALL, with equal cytogenetic distribution which, in some cases, grew out into the major clone at relapse. Taken together, our results indicate that BTG1 deletions may act as "drivers" of leukemogenesis in specific BCP-ALL subgroups, in which they can arise independently in multiple subclones at sites that are prone to aberrant RAG1/RAG2-mediated recombination events. These findings provide further evidence for a complex and multiclonal evolution of ALL. 01 februari 2012

Contains fulltext : 108461.pdf (Publisher’s version ) (Open Access) BACKGROUND: Orthology is a central tenet of comparative genomics and ortholog identification is instrumental to protein function prediction. Major advances have been made to determine orthology relations among a set of homologous proteins. However, they depend on the comparison of individual sequences and do not take into account divergent orthologs. RESULTS: We have developed an iterative orthology prediction method, Ortho-Profile, that uses reciprocal best hits at the level of sequence profiles to infer orthology. It increases ortholog detection by 20% compared to sequence-to-sequence comparisons. Ortho-Profile predicts 598 human orthologs of mitochondrial proteins from Saccharomyces cerevisiae and Schizosaccharomyces pombe with 94% accuracy. Of these, 181 were not known to localize to mitochondria in mammals. Among the predictions of the Ortho-Profile method are 11 human cytochrome c oxidase (COX) assembly proteins that are implicated in mitochondrial function and disease. Their co-expression patterns, experimentally verified subcellular localization, and co-purification with human COX-associated proteins support these predictions. For the human gene C12orf62, the ortholog of S. cerevisiae COX14, we specifically confirm its role in negative regulation of the translation of cytochrome c oxidase. CONCLUSIONS: Divergent homologs can often only be detected by comparing sequence profiles and profile-based hidden Markov models. The Ortho-Profile method takes advantage of these techniques in the quest for orthologs.

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Contains fulltext : 88978.pdf (Publisher’s version ) (Open Access) Mitochondrial disorders are a heterogeneous group of often multisystemic and early fatal diseases, which are amongst the most common inherited human diseases. These disorders are caused by defects in the oxidative phosphorylation (OXPHOS) system, which comprises five multisubunit enzyme complexes encoded by both the nuclear and the mitochondrial genomes. Due to the multitude of proteins and intricacy of the processes required for a properly functioning OXPHOS system, identifying the genetic defect that underlies an OXPHOS deficiency is not an easy task, especially in the case of combined OXPHOS defects. In the present communication we give an extensive overview of the proteins and processes (in)directly involved in mitochondrial translation and the biogenesis of the OXPHOS system and their roles in combined OXPHOS deficiencies. This knowledge is important for further research into the genetic causes, with the ultimate goal to effectively prevent and cure these complex and often devastating disorders.

Contains fulltext : 80701.pdf (Publisher’s version ) (Open Access) BACKGROUND: Mammalian ATAD3 is a mitochondrial protein, which is thought to play an important role in nucleoid organization. However, its exact function is still unresolved. RESULTS: Here, we characterize the Caenorhabditis elegans (C. elegans) ATAD3 homologue (ATAD-3) and investigate its importance for mitochondrial function and development. We show that ATAD-3 is highly conserved among different species and RNA mediated interference against atad-3 causes severe defects, characterized by early larval arrest, gonadal dysfunction and embryonic lethality. Investigation of mitochondrial physiology revealed a disturbance in organellar structure while biogenesis and function, as indicated by complex I and citrate synthase activities, appeared to be unaltered according to the developmental stage. Nevertheless, we observed very low complex I and citrate synthase activities in L1 larvae populations in comparison to higher larval and adult stages. Our findings indicate that atad-3(RNAi) animals arrest at developmental stages with low mitochondrial activity. In addition, a reduced intestinal fat storage and low lysosomal content after depletion of ATAD-3 suggests a central role of this protein for metabolic activity. CONCLUSIONS: In summary, our data clearly indicate that ATAD-3 is essential for C. elegans development in vivo. Moreover, our results suggest that the protein is important for the upregulation of mitochondrial activity during the transition to higher larval stages.

Background Neurons require an elaborate system of intracellular transport to distribute cargo throughout axonal and dendritic projections. Active anterograde and retrograde transport of mitochondria serves in local energy distribution, but at the same time also requires input of ATP. Here we studied whether brain-type creatine kinase (CK-B), a key enzyme for high-energy phosphoryl transfer between ATP and CrP in brain, has an intermediary role in the reciprocal coordination between mitochondrial motility and energy distribution. Therefore, we analysed the impact of brain-type creatine kinase (CK-B) deficiency on transport activity and velocity of mitochondria in primary murine neurons and made a comparison to the fate of amyloid precursor protein (APP) cargo in these cells, using live cell imaging. Results Comparison of average and maximum transport velocities and global transport activity showed that CK-B deficiency had no effect on speed of movement of mitochondria or APP cargo, but that the fraction of motile mitochondria was significantly increased by 36% in neurons derived from CK-B knockout mice. The percentage of motile APP vesicles was not altered. Conclusion CK-B activity does not directly couple to motor protein activity but cells without the enzyme increase the number of motile mitochondria, possibly as an adaptational strategy aimed to enhance mitochondrial distribution versatility in order to compensate for loss of efficiency in the cellular network for ATP distribution.

We read with interest the paper by Bianchi et al,[1][1] in which the authors report on the treatment monitoring of 2 patients with guanidinoacetate methyltransferase (GAMT) deficiency and 3 patients with an arginine:glycine amidinotransferase defect (AGAT-d). Repetitive MR measurements of these

Contains fulltext : 52286.pdf (Publisher’s version ) (Closed access) Cobalamin is an essential cofactor for two mammalian enzymes: methionine synthase and methylmalonyl-CoA mutase. Patients with the cobalamin C (CblC) defect have combined methylmalonic aciduria and homocystinuria. Recently, the gene responsible for the CblC type, MMACHC, was identified, which enables molecular diagnostics. In this study, we describe two siblings, a 16-year-old girl and her 11-year-old brother, of a consanguineous family who presented with a very distinct clinical manifestation. The girl presented at the age of 13 years with macrocytic anaemia, cognitive regression and Marfanoid features such as increased arm-span, arachnodactyly, joint hyperlaxity and scoliosis. Her brother presented at the age of 10 months with developmental delay and behavioural abnormalities. Biochemical analysis showed severely increased homocysteine and methylmalonic acid levels in plasma of both siblings. In addition, plasma cysteine levels were decreased in the girl but not in her brother. The diagnosis of CblC defect was confirmed by genomic sequencing of the coding exons of the MMACHC gene. Two heterozygous mutations were identified in both siblings; the common c.271dupA p.Arg91LysfsX14 and a novel mutation, c.1A > G p.Met1?. Therapy consisting of folic acid, vitamin B6, l-carnitine and intramuscular vitamin B12 resulted in a clear improvement of biochemical parameters and, importantly, resulted in amelioration of the Marfanoid features in the girl. These data might suggest that low cysteine levels account for the Marfanoid features observed in the girl and indicate that the CblC type of combined methylmalonic aciduria and homocystinuria should be considered in the differential diagnosis of patients with Marfanoid features.

Syntheticrather than biological building blocks are used because of theproceduresemployed. For biomedical applications it would beadvantageous if capsules could be prepared from the vast re-pertoireof(large) naturalmacromolecules, preferablywithoutthe need for amphiphilicity. One such example encompasseshollow microcapsules prepared by using cavitation methods,for example, spray-drying of aqueous albumin solutions.

By combining different types of large datasets that give evidence for protein-interactions, qualitative labels on the predicted protein interaction network of S. cerevisiae could be inferred, providing guidance towards direct experimental verification of the predicted interactions., Background In the post-genomic era various functional genomics, proteomics and computational techniques have been developed to elucidate the protein interaction network. While some of these techniques are specific for a certain type of interaction, most predict a mixture of interactions. Qualitative labels are essential for the molecular biologist to experimentally verify predicted interactions. Results Of the individual protein-protein interaction prediction methods, some can predict physical interactions without producing other types of interactions. None of the methods can specifically predict metabolic interactions. We have constructed an 'omics evidence landscape' that combines all sources of evidence for protein interactions from various types of omics data for Saccharomyces cerevisiae. We explore this evidence landscape to identify areas with either only metabolic or only physical interactions, allowing us to specifically predict the nature of new interactions in these areas. We combine the datasets in ways that examine the whole evidence landscape and not only the highest scoring protein pairs in both datasets and find specific predictions. Conclusion The combination of evidence types in the form of the evidence landscape allows for qualitative labels to be inferred and placed on the predicted protein interaction network of S. cerevisiae. These qualitative labels will help in the biological interpretation of gene networks and will direct experimental verification of the predicted interactions.

Contains fulltext : 52444.pdf (Publisher’s version ) (Closed access) Hereditary fructose intolerance (HFI) is caused by a deficiency of aldolase B due to mutations of the ALDOB gene. The disease poses diagnostic problems because of unspecific clinical manifestations. We report three cases of HFI all of whom had a chronic disease with neurological, nephrological or gastroenterological symptoms, whereas nutritional fructose intolerance, the pathognomonic sign of HFI, was apparent only in retrospect. In all patients a hypoglycosylated pattern of transferrin isoforms was found but was misinterpreted as a sign of CDG Ix. The correct diagnosis was achieved with marked delay (26, 36 and 24 months, respectively) by sequencing of the ALDOB gene two common mutations were identified on both alleles or on one (A150P/A175D, A150P/-, and A150P/A175D). The diagnosis was further supported by normalization of transferrin isoforms on a fructose-free diet. Data available in two patients showed that following the fructose restriction the type I pattern of carbohydrate-deficient transferrin detectable on fructose-containing diet disappeared after 3-4 weeks. These cases illustrate that in the first years of life HFI may show misleading variability in clinical presentation and that protein glycosylation analysis such as transferrin isofocusing may give important diagnostic clues. However, care should be taken not to misinterpret the abnormal results as CDG Ix as well as to remember that a normal profile does not exclude HFI due to the possibility of spontaneous fructose restriction in the diet. The presented data also emphasize the usefulness of ALDOB mutation screening for diagnosis of HFI.

Contains fulltext : 36066.pdf ( ) (Open Access) BACKGROUND: The transfer of functional annotations from model organism proteins to human proteins is one of the main applications of comparative genomics. Various methods are used to analyze cross-species orthologous relationships according to an operational definition of orthology. Often the definition of orthology is incorrectly interpreted as a prediction of proteins that are functionally equivalent across species, while in fact it only defines the existence of a common ancestor for a gene in different species. However, it has been demonstrated that orthologs often reveal significant functional similarity. Therefore, the quality of the orthology prediction is an important factor in the transfer of functional annotations (and other related information). To identify protein pairs with the highest possible functional similarity, it is important to qualify ortholog identification methods. RESULTS: To measure the similarity in function of proteins from different species we used functional genomics data, such as expression data and protein interaction data. We tested several of the most popular ortholog identification methods. In general, we observed a sensitivity/selectivity trade-off: the functional similarity scores per orthologous pair of sequences become higher when the number of proteins included in the ortholog groups decreases. CONCLUSION: By combining the sensitivity and the selectivity into an overall score, we show that the InParanoid program is the best ortholog identification method in terms of identifying functionally equivalent proteins.

Contains fulltext : 35781.pdf ( ) (Open Access) BACKGROUND: The horizontal transfer of expressed genes from Bacteria into Ciliates which live in close contact with each other in the rumen (the foregut of ruminants) was studied using ciliate Expressed Sequence Tags (ESTs). More than 4000 ESTs were sequenced from representatives of the two major groups of rumen Cilates: the order Entodiniomorphida (Entodinium simplex, Entodinium caudatum, Eudiplodinium maggii, Metadinium medium, Diploplastron affine, Polyplastron multivesiculatum and Epidinium ecaudatum) and the order Vestibuliferida, previously called Holotricha (Isotricha prostoma, Isotricha intestinalis and Dasytricha ruminantium). RESULTS: A comparison of the sequences with the completely sequenced genomes of Eukaryotes and Prokaryotes, followed by large-scale construction and analysis of phylogenies, identified 148 ciliate genes that specifically cluster with genes from the Bacteria and Archaea. The phylogenetic clustering with bacterial genes, coupled with the absence of close relatives of these genes in the Ciliate Tetrahymena thermophila, indicates that they have been acquired via Horizontal Gene Transfer (HGT) after the colonization of the gut by the rumen Ciliates. CONCLUSION: Among the HGT candidates, we found an over-representation (>75%) of genes involved in metabolism, specifically in the catabolism of complex carbohydrates, a rich food source in the rumen. We propose that the acquisition of these genes has greatly facilitated the Ciliates' colonization of the rumen providing evidence for the role of HGT in the adaptation to new niches.

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Summary Objective Firstly, this paper aims to systematically review the mitochondrial disease literature to identify studies assessing the needs and problems in the daily life of children with a mitochondrial disease and of their parents and family. The second aim is to provide more insight into the need for information by the parents of these children during the diagnostic process while in hospital. Design A systematic review and a pilot study, using a qualitative (focus group interviews; n=7) and a quantitative (questionnaire; n=77) design. Results Mothers reported great socioeconomic and psychoaffective strain and showed psychopathological symptoms in the two studies published with respect to this topic. The pilot study showed that parents considered an honest and interested attitude of the person who is giving the information as most important. Furthermore they wanted oral and written information and a central point where they could go with their questions at any time they felt the need. The need for information increased during the four phases of the diagnostic process and was highest in the fourth phase. Conclusions The few studies found in the review, combined with expectations that having a mitochondrial disease must have a great impact on these children and their parents and family, call for more research in their needs and problems. Furthermore, there are gaps in the current information provision to parents of these children. A better understanding of the needs and problems of these children and their family is essential for effective care planning and might result in an improved quality of life. Electronic Supplementary Material The online version of this article (doi:10.1007/s10545-007-0426-0) contains supplementary material, which is available to authorized users.

Item does not contain fulltext The heterogeneous group of 3-methylglutaconic aciduria (3-MGA-uria) syndromes includes several inborn errors of metabolism biochemically characterized by increased urinary excretion of 3-methylglutaconic acid. Five distinct types have been recognized: 3-methylglutaconic aciduria type I is an inborn error of leucine catabolism; the additional four types all affect mitochondrial function through different pathomechanisms. We provide an overview of the expanding clinical spectrum of the 3-MGA-uria types and provide the newest insights into the underlying pathomechanisms. A diagnostic approach to the patient with 3-MGA-uria is presented, and we search for the connection between urinary 3-MGA excretion and mitochondrial dysfunction. 01 januari 2012

Contains fulltext : 89368.pdf (Publisher’s version ) (Closed access) We investigated the genetic basis for a global and uniform decrease in mitochondrial translation in fibroblasts from patients in two unrelated pedigrees who developed Leigh syndrome, optic atrophy, and ophthalmoplegia. Analysis of the assembly of the oxidative phosphorylation complexes showed severe decreases of complexes I, IV, and V and a smaller decrease in complex III. The steady-state levels of mitochondrial mRNAs, tRNAs, and rRNAs were not reduced, nor were those of the mitochondrial translation elongation factors or the protein components of the mitochondrial ribosome. Using homozygosity mapping, we identified a 1 bp deletion in C12orf65 in one patient, and DNA sequence analysis showed a different 1 bp deletion in the second patient. Both mutations predict the same premature stop codon. C12orf65 belongs to a family of four mitochondrial class I peptide release factors, which also includes mtRF1a, mtRF1, and Ict1, all characterized by the presence of a GGQ motif at the active site. However, C12orf65 does not exhibit peptidyl-tRNA hydrolase activity in an in vitro assay with bacterial ribosomes. We suggest that it might play a role in recycling abortive peptidyl-tRNA species, released from the ribosome during the elongation phase of translation.

Item does not contain fulltext Human mitochondrial (mt) ATP synthase, or complex V consists of two functional domains: F(1), situated in the mitochondrial matrix, and F(o), located in the inner mitochondrial membrane. Complex V uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP. This review covers the architecture, function and assembly of complex V. The role of complex V di-and oligomerization and its relation with mitochondrial morphology is discussed. Finally, pathology related to complex V deficiency and current therapeutic strategies are highlighted. Despite the huge progress in this research field over the past decades, questions remain to be answered regarding the structure of subunits, the function of the rotary nanomotor at a molecular level, and the human complex V assembly process. The elucidation of more nuclear genetic defects will guide physio(patho)logical studies, paving the way for future therapeutic interventions. 01 maart 2012

Contains fulltext : 51463.pdf (Publisher’s version ) (Closed access) The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the beta(1) subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH(4)(+) as activating cation. The enzyme was also stimulated by Na(+), but in contrast to the rat enzyme, hardly by K(+). SCH 28080 inhibited the NH(4)(+)-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K(+)], and [Na(+)] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E(1)P to E(2)P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H(+)/Na(+) and/or H(+)/NH(4)(+) transport but not in Na(+)/K(+) transport.

Contains fulltext : 88992.pdf (Publisher’s version ) (Closed access) The alpha-kinase family represents a class of atypical protein kinases that display little sequence similarity to conventional protein kinases. Early studies on myosin heavy chain kinases in Dictyostelium discoideum revealed their unusual propensity to phosphorylate serine and threonine residues in the context of an alpha-helix. Although recent studies show that some members of this family can also phosphorylate residues in non-helical regions, the name alpha-kinase has remained. During evolution, the alpha-kinase domains combined with many different functional subdomains such as von Willebrand factor-like motifs (vWKa) and even cation channels (TRPM6 and TRPM7). As a result, these kinases are implicated in a large variety of cellular processes such as protein translation, Mg(2+) homeostasis, intracellular transport, cell migration, adhesion, and proliferation. Here, we review the current state of knowledge on different members of this kinase family and discuss the potential use of alpha-kinases as drug targets in diseases such as cancer. 01 maart 2010