Functional Complementation in Yeast Allows Molecular Characterization of Missense Argininosuccinate Lyase Mutations

Deficiency of argininosuccinate lyase (ASL) causes argininosuccinic aciduria, an urea cycle defect that may present with a severe neonatal onset form or with a late onset phenotype. To date phenotype-genotype correlations are still not clear because biochemical assays of ASL activity correlate poorly with clinical severity in patients. We employed a yeast-based functional complementation assay to assess the pathogenicity of 12 missense ASL mutations, to establish genotype-phenotype correlations, and to screen for intragenic complementation. Rather than determining ASL enzyme activity directly, we have measured the growth rate in arginine-free medium of a yeast ASL^null strain transformed with individual mutant ASL alleles. Individual haploid strains were also mated to obtain diploid, “compound heterozygous” yeast. We show that the late onset phenotypes arise in patients because they harbor individual alleles retaining high residual enzymatic activity or because of intragenic complementation among different mutated alleles. In these cases complementation occurs because in the hybrid tetrameric enzyme at least one active site without mutations can be formed or because the differently mutated alleles can stabilize each other, resulting in partial recovery of enzymatic activity. Functional complementation in yeast is simple and reproducible and allows the analysis of large numbers of mutant alleles. Moreover, it can be easily adapted for the analysis of mutations in other genes involved in urea cycle disorders.Argininosuccinic aciduria (ASAuria, MIM 207900)³ is an autosomal recessive disorder of the urea cycle caused by mutations of the ASL gene (hASL, MIM 608310), encoding argininosuccinate lyase (ASL; EC 4.3.2.1.) (1). This enzyme is ubiquitously expressed and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. ASL belongs to a superfamily of hydrolases that includes adenylosuccinate lyase and fumarase, which share a homotetrameric structure and a similar catalytic mechanism. The tetrameric structure of ASL accounts for the phenomenon of intragenic complementation. This particular situation occurs when a multimeric protein is formed from subunits produced by differently mutated alleles of the same gene. On complementation, a partially functional hybrid protein is produced from the two distinct types of mutant subunits, neither of which individually has appreciable enzymatic activity (2).ASL participates to the urea cycle, and in humans it is essential for ammonia detoxification, whereas in lower organisms it is required for the biosynthesis of arginine. Saccharomyces cerevisiae strains harboring a deletion of the homolog of human ASL (ARG4) cannot grow on media lacking arginine (3).ASAuria is characterized by accumulation of argininosuccinic acid (ASA) in body fluids, and severe hyperammonaemia. The disease displays clinical heterogeneity with two main clinical phenotypes: the acute/neonatal onset form, with symptoms rapidly progressing to deep coma, apnea, and death (1), and the subacute/late onset type, which is diagnosed in infancy or childhood (4). Such patients may present simply with mental retardation or an epileptic disorder. In both types the diagnosis is established unambiguously by measuring plasma levels of ammonia (not always elevated in the late onset form), ASA, and its anhydrides by plasma amino acids assay (1). Over 40 mutations of the ASL gene have been reported, both amino acid substitutions and truncating variants, which are scattered throughout the gene (5, 6).We have previously reported the identification of novel mutations of the ASL gene in a cohort of Italian patients (7). In this study we employed a yeast model to validate the pathogenicity of missense ASL mutations found in our cohort, to study the effects of different allelic combinations, and to establish possible genotype-phenotype correlations.     

Structure and Function of Bacterial Communities Emerging from Different Sources under Identical Conditions

The aim of this study was to compare two major hypotheses concerning the formation of bacterial community composition (BCC) at the local scale, i.e., whether BCC is determined by the prevailing local environmental conditions or by “metacommunity processes.” A batch culture experiment where bacteria from eight distinctly different aquatic habitats were regrown under identical conditions was performed to test to what extent similar communities develop under similar selective pressure. Differently composed communities emerged from different inoculum communities, as determined by terminal restriction fragment length polymorphism analysis of the 16S rRNA gene. There was no indication that similarity increased between communities upon growth under identical conditions compared to that for growth at the ambient sampling sites. This suggests that the history and distribution of taxa within the source communities were stronger regulating factors of BCC than the environmental conditions. Moreover, differently composed communities were different with regard to specific functions, such as enzyme activities, but maintained similar broad-scale functions, such as biomass production and respiration.     

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K⁺ and Ca²⁺ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca²⁺ channels. Respective K⁺ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl^– efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 M), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 M), which inhibits K⁺ fluxes and part of the initial Ca²⁺ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.Abbreviations [Ca²⁺]_c cytosolic free Ca²⁺ - I-V current-voltage - E equilibrium potential - Pr red-light-absorbing phytochrome form - Pr far-red-light-absorbing phytochrome form - SPQ 6-methoxy-l-(3-sulphonatopropyl)quinolinium - TEA tetraethylammonium     

Effects of plant leachates from four boreal understorey species on soil N mineralization, and white spruce (Picea glauca) germination and seedling growth

BACKGROUND AND AIMS: Natural regeneration of white spruce (Picea glauca) after disturbance has been reported to be very poor. Here a study was made to determine whether C compounds released from understorey species growing together with white spruce could be involved in this regeneration failure, either by (1) changing soil nutrient dynamics, (2) inhibiting germination, and/or (3) delaying seedling growth. METHODS: Foliage leachates were obtained from two shrubs (Ledum palustre and Empetrum hermaphroditum) and one bryophyte (Sphagnum sp.) with high phenolic compound concentrations that have been reported to depress growth of conifers in boreal forests, and, as a comparison, one bryophyte (Hylocomium splendens) with negligible phenolic compounds. Mineral soil from a white spruce forest was amended with plant leachates to examine the effect of each species on net N mineralization. Additionally, white spruce seeds and seedlings were watered with plant leachates to determine their effects on germination and growth. KEY RESULTS: Leachates from the shrubs L. palustre and E. hermaphroditum contained high phenolic compound concentrations and dissolved organic carbon (DOC), while no detectable levels of C compounds were released from the bryophytes Sphagnum sp. or H. splendens. A decrease in net N mineralization was determined in soils amended with L. palustre or E. hermaphroditum leachates, and this effect was inversely proportional to the phenolic concentrations, DOC and leachate C/N ratio. The total percentage of white spruce germination and the growth of white spruce seedlings were similar among treatments. CONCLUSIONS: These results suggest that the shrubs L. palustre and E. hermaphroditum could negatively affect the performance of white spruce due to a decrease in soil N availability, but not by direct effects on plant physiology.     

Macrolide use in the previous years is associated with failure to eradicate Helicobacter pylori with clarithromycin‐containing regimens

Background

There is some evidence that prior use of macrolide antibiotics is a useful predictor of the likelihood of standard triple therapy failure in Helicobacter pylori eradication. In this study, we have evaluated whether previous intake of macrolides correlates with failure to eradicate H. pylori using two different first‐line clarithromycin‐containing regimens.

Materials and Methods

Retrospective study of 212 patients with H. pylori infection treated with one of two first‐line clarithromycin‐containing regimens: 108 patients treated with triple therapy for 10 days and 104 patients treated with concomitant therapy for 10 days. The intake of macrolides (clarithromycin, azithromycin, and other macrolides) prior to the eradication therapy was obtained from the electronic medical record, which contains information regarding all the medication prescribed to the patients since the year 2004.

Results

One hundred of 212 patients (47.2%) had received at least one treatment with macrolides during the years prior to the eradication therapy. H. pylori eradication rates were significantly lower in patients with previous use compared to patients without previous use of macrolides, both with triple therapy (60.8% vs 92.9%; P < .0001) and with concomitant therapy (85.7% vs 98.2%; P = .024).

Conclusions

Previous use of macrolides correlates with a low H. pylori eradication rate with triple and concomitant clarithromycin‐containing regimens. In addition, our study shows that in patients without previous use of macrolides, triple therapy achieves per‐protocol eradication rates over 90%.     

Changes in the biological activity of agropyrene on combined application with humus substances in the cultivation ofScenedesmus obliquus (TURP.) krüger

The work is concerned with the question of the effect of humic acids on the biological action of agropyrene, the effective substance from the rootstock of Agropyron repens (L.) P. BEAUV. The test organism was the alga Scenedesmus obliquus (TURP.) KRÜGER, cultivated in mineral solution. The basic biological test was the determination of the number and size of the cells of this alga. It was found that the way in which agropyrene acts on combined application with humus acids depends on which humus fraction is used. It can display a synergic action if humic acid is used, since this substance probably facilitates the penetration of agropyrene into the cells. On the other hand, fulvic acid counteracts the activity of agropyrene and this most probably by mutual binding and blocking of active groups and links.     

Exercise-induced lipid mobilization in subcutaneous adipose tissue is mainly related to natriuretic peptides in overweight men

Involvement of sympathetic nervous system and natriuretic peptides in the control of exercise-induced lipid mobilization was compared in overweight and lean men. Lipid mobilization was determined using local microdialysis during exercise. Subjects performed 35-min exercise bouts at 60% of their maximal oxygen consumption under placebo or after oral tertatolol [a beta-adrenergic receptor (AR) antagonist]. Under placebo, exercise increased dialysate glycerol concentration (DGC) in both groups. Phentolamine (alpha-AR antagonist) potentiated exercise-induced lipolysis in overweight but not in lean subjects; the alpha(2)-antilipolytic effect was only functional in overweight men. After tertatolol administration, the DGC increased similarly during exercise no matter which was used probe in both groups. Compared with the control probe under placebo, lipolysis was reduced in lean but not in overweight men treated with the beta-AR blocker. Tertatolol reduced plasma nonesterified fatty acids and insulin concentration in both groups at rest. Under placebo or tertatolol, the exercise-induced changes in plasma nonesterified fatty acids, glycerol, and insulin concentrations were similar in both groups. Exercise promoted a higher increase in catecholamine and ANP plasma levels after tertatolol administration. In conclusion, the major finding of our study is that in overweight men, in addition to an increased alpha(2)-antilipolytic effect, the lipid mobilization in subcutaneous adipose tissue that persists during exercise under beta-blockade is not dependent on catecholamine action. On the basis of correlation findings, it seems to be related to a concomitant exercise-induced rise in plasma ANP when exercise is performed under tertatolol intake and a decrease in plasma insulin.     

Isocitrate dehydrogenase: A NADPH-generating enzyme in the lumen of the endoplasmic reticulum

The aim of the present study was the investigation of the occurrence of NADPH-generating pathways in the endoplasmic reticulum others then hexose-6-phosphate dehydrogenase. A significant isocitrate and a moderate malate-dependent NADP⁺ reduction were observed in endoplasmic reticulum-derived rat liver microsomes. The isocitrate-dependent activity was very likely attributable to the appearance of the cytosolic isocitrate dehydrogenase isozyme in the lumen. The isocitrate dehydrogenase activity of microsomes was present in the luminal fraction; it showed a strong preference towards NADP⁺versus NAD⁺, and it was almost completely latent. Antibodies against the cytosolic isoform of isocitrate dehydrogenase immunorevealed a microsomal protein of identical molecular weight; the microsomal enzyme showed similar kinetic parameters and oxalomalate inhibition as the cytosolic one. Measurable luminal isocitrate dehydrogenase activity was also present in microsomes from rat epididymal fat. The results suggest that isocitrate dehydrogenase is an important NADPH-generating enzyme in the endoplasmic reticulum.