New SUCLG1 patients expanding the phenotypic spectrum of this rare cause of mild methylmalonic aciduria

Deficiencies in two subunits of the succinyl-coenzyme A synthetase (SCS) have been involved in patients with encephalomyopathy and mild methylmalonic aciduria (MMA). In this study, we described three new SUCLG1 patients and performed a meta-analysis of the literature. Our report enlarges the phenotypic spectrum of SUCLG1 mutations and confirms that a characteristic metabolic profile (presence of MMA and C4-DC carnitine in urines) and basal ganglia MRI lesions are the hallmarks of SCS defects. As mitochondrial DNA depletion in muscle is not a constant finding in SUCLG1 patients, this may suggest that diagnosis should not be based on it, but also that alternative physiopathological mechanisms may be considered to explain the combined respiratory chain deficiency observed in SCS patients.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

Radiological innovations in the screening and diagnosis of the inborn errors of metabolism

New metabolic diseases are regularly identified by a genetic or biochemical approach. Indeed, the metabolic diseases result from an enzymatic block with accumulation of a metabolite upstream to the block and deficit of a metabolite downstream. The characterization of these abnormal metabolites by MRI spectroscopy permitted to identify the deficient enzyme in two new groups of diseases, creatine deficiencies and polyol anomalies. Creatine deficiency is implicated in unspecific mental retardation. A low peak of creatine at MRI spectroscopy is evocating of creatine deficiency which is treatable by creatine administration. Deficiency of synthesis of polyols, metabolites on the pentose pathway, represent new described metabolic diseases with variable symptoms including a neurological distress, liver disease, splenomegaly, cutis laxa and renal insufficiency. The deficit of ribose-5-phosphate isomerase, one of the enzymes whose diagnosis is evoked in front of the accumulation of ribitol, arabitol and xylitol leads to a leucodystrophy in adults. This new deficit was highlighted by the identification of an abnormal peak in cerebral MRI-spectroscopy corresponding to the abnormal accumulation of polyols in brain. Congenital hyperinsulinism (HI) is characterized by profound hypoglycaemia related to inappropriate insulin secretion. Focal and diffuse forms of hyperinsulinism share a similar clinical presentation but their treatment is dramatically different. Until recently, preoperative differential diagnosis was based on pancreatic venous sampling, an invasive and technically demanding technique. Positron emission tomography (PET) after injection of [18F]Fluoro-L-DOPA has been evaluated for the preoperative differentiation between focal and diffuse HI, by imaging uptake of radiotracer and the conversion of [18F]Fluoro-L-DOPA into dopamine by DOPA decarboxylase. PET with [18F]Fluoro-L-DOPA has been validated as a reliable test to differentiate diffuse and focal HI and is now a major differential diagnosis tool in infantile hyperinsulinemic hypoglycaemia.     

X-linked Sideroblastic Anemia Due to Carboxyl-terminal ALAS2 Mutations That Cause Loss of Binding to the β-Subunit of Succinyl-CoA Synthetase (SUCLA2)

Mutations in the erythroid-specific aminolevulinic acid synthase gene (ALAS2) cause X-linked sideroblastic anemia (XLSA) by reducing mitochondrial enzymatic activity. Surprisingly, a patient with the classic XLSA phenotype had a novel exon 11 mutation encoding a recombinant enzyme (p.Met567Val) with normal activity, kinetics, and stability. Similarly, both an expressed adjacent XLSA mutation, p.Ser568Gly, and a mutation (p.Phe557Ter) lacking the 31 carboxyl-terminal residues also had normal or enhanced activity, kinetics, and stability. Because ALAS2 binds to the β subunit of succinyl-CoA synthetase (SUCLA2), the mutant proteins were tested for their ability to bind to this protein. Wild type ALAS2 bound strongly to a SUCLA2 affinity column, but the adjacent XLSA mutant enzymes and the truncated mutant did not bind. In contrast, vitamin B6-responsive XLSA mutations p.Arg452Cys and p.Arg452His, with normal in vitro enzyme activity and stability, did not interfere with binding to SUCLA2 but instead had loss of positive cooperativity for succinyl-CoA binding, an increased K(m) for succinyl-CoA, and reduced vitamin B6 affinity. Consistent with the association of SUCLA2 binding with in vivo ALAS2 activity, the p.Met567GlufsX2 mutant protein that causes X-linked protoporphyria bound strongly to SUCLA2, highlighting the probable role of an ALAS2-succinyl-CoA synthetase complex in the regulation of erythroid heme biosynthesis.     

Daily magnesium supplementation on serum and urinary magnesium changes in rats during prolonged restriction of motor activity

The objective of this investigation was to determine whether a plentiful magnesium (Mg²⁺) supplementation might be used to normalize or prevent Mg deficiency. This is manifested by increased rather than decreased serum Mg²⁺ concentration as is observed during prolonged hospitalization, which is developed during prolonged hypokinesia (HK) (decreased motor activity). Eighty male Wistar rats with an initial body weight of 370–390 g were used to perform the studies: They were equally divided into four groups:

1. Unsupplemented control animals (UCA);
2. Supplemented control animals (SCA);
3. Unsupplemented hypokinetic animals (UHA); and
4. Supplemented hypokinetic animals (SHA).

For the simulation of the hypokinetic effect, the hypokinetic animals were kept in small individual cages made of wood, which restricted their movements in all directions without hindering food and water intake. The control and hypokinetic supplemental animals receive 0.9 mg/mL Mg sulfate daily with their drinking water. Prior to and during the experimental period, urinary excretions of Mg, calcium, and phosphate along with their concentrations in serum, water intake, and urine excretion, and body weight were determined in the control and hypokinetic animals. In the supplemental and unsupplemental hypokinetic rats, urinary excretions and serum concentrations of electrolytes increased significantly, whereas serum concentration and urinary excretion thereof remained unchanged in the supplemented and unsupplemented control animals. It was concluded that a daily intake of large amounts of Mg supplementation cannot be used to prevent or normalize Mg deficiency in rats during prolonged exposure to HK.     

How environmental factors regulate mutagenesis and gene transfer in microorganisms

This review is focused on the physiological and evolutionary strategies of the processes occurring during the entry of microbial cells into stationary phase and the subsequent period of stasis. The molecular mechanisms adapting microorganisms from exponential growth to a static state involve activation and complex regulation of the stationary factor Sigma-S, which directs RNA polymerase to the specific promoters. As a result the static cells acquire general resistance (simultaneous tolerances) to different environmental stresses. In parallel with the physiological adaptation to stasis, diverse genetical processes are aimed towards resuming the growth of the static cells. Different types of mutagenesis occur: (i) in cells entering stasis and (ii) in static cells (adaptive mutagenesis). Cessation of growth induces the transient hypermutator state resulting in the accumulation of random mutations in the subpopulation of the static cells. If by chance, one or a few of such mutations lead to resumption of division, the growing cell will return to a normal mechanism of spontaneous mutagenesis. Another mechanism for generating genetical variability in stressed cells involves transposons and conjugative plasmids. Stresses can stimulate the excision of some transposons, which, in turn, can generate chromosomal mutations and activate intracellular mechanisms of mutagenesis. Under stress some conjugative plasmids activate genes encoding antirestriction proteins that repress restriction-modification systems of the recipient cells. Moreover, under stress special cellular mechanisms decrease (alleviate) the activity of restriction-modification systems which, in turn, enhance the probability of gene transfer into the stressed cells. Under stress, the efficiency of inter-species genetical barriers also decreases. This, stimulates inter-species gene transfer and may lead to a burst of incipient speciation in the population of non-growing cells. After resumption of growth the genetical barriers leading to isolation will be restored. In general, the cessation of growth “switches on”, and resumption of growth “switches off”, a set of special processes that are responsible for generating bursts of genetical variability in populations of microorganisms. This article is dedicated to the memory of Nikolai V Timofeev-Ressovsky (1900–1981).     

Impact of Emission Anisotropy on Fluorescence Spectroscopy and FRET Distance Measurements

The objective of this report is to provide a practical and improved method for estimating Förster resonance energy transfer distance measurement error due to unknown angles in the dipole orientation factor based on emission anisotropy measurements. We improve on the method of Dale et al. (1979), which has minor mistakes and is frequently interpreted in overly optimistic ways in the literature. To facilitate proper fluorescence intensity measurements, we also evaluated instrument parameters that could impact the measurement. The apparent fluorescence intensity of isotropic samples depends on the sample emission anisotropy, fluorometer geometry, and optical apertures. We separate parameters of the sample, and those of the cylindrically symmetric illumination source and detector in the equations describing results of unpolarized and polarized fluorescence intensity measurements. This approach greatly simplifies calculations compared with the more universal method of Axelrod (1989). We provide a full computational method for calculating the Förster resonance energy transfer distance error and present a graph describing distance error in the simplest case.