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.     

Fatal Chytridiomycosis in the Tyrrhenian Painted Frog

Batrachochytrium dendrobatidis (Bd), the causative agent of the amphibian disease chytridiomycosis, is an important factor in the global decline of amphibians. Within Europe, animals that exhibit clinical signs of the disease have only been reported in Spain despite the pathogen’s wide, but patchy, distribution on the continent. Recently, another occurrence of chytridiomycosis was reported in Euproctus platycephalus, the Sardinian brook newt, on the Mediterranean island of Sardinia, but without any evidence of fatal disease. We report further evidence of the emergence of Bd on Sardinia and the first evidence of lethal chytridiomycosis outside of Spain. Unusual mortalities of the Tyrrhenian painted frog (Discoglossus sardus) were found at three sites in the Limbara mountains of northern Sardinia. Molecular and histological screens of corpses, frogs, and tadpoles from these sites revealed infection with Bd. Infection and mortality occurred at locations that are unusual in terms of the published habitat requirements of the pathogen. Given the endemicity, the IUCN Red List status of the amphibian species on Sardinia, and the occurrence of infection and mortality caused by chytridiomycosis, there is serious reason for concern for the impact that disease emergence may have on the conservation of the amphibians of the island.     

CXC chemokine receptor 4 is expressed in uveal malignant melanoma and correlates with the epithelioid-mixed cell type

PURPOSE: Although relatively rare, uveal melanoma is the most common ocular tumor of adults. Up to half of uveal melanoma patients die of metastatic disease. CXCR4, a chemokine receptor, is a prognostic factor in cutaneous melanoma involved in angiogenesis and metastasis formation. The aim of this study was to evaluate the expression of CXCR4 in uveal melanoma. METHODS: CXCR4 was detected by immunohistochemistry in 44 samples of uveal melanoma. Staining was categorized into three semiquantitative classes based on the rate of stained (positive) tumor cells: absence of staining, <50% of cell (+) and >50% (++). Correlations between CXCR4 expression, data on patient and tumor features were studied by contingency tables and the chi2 test. Time-to-event curves were studied using the Kaplan-Meier method. Univariate analysis was performed using the log-rank test. Ninety-five percent confidence intervals (95% CI) of hazard ratios were also reported. RESULTS: Staining for CXCR4 protein was absent in 18 tumors (40.9%), present in <50% of cells in 19 (43.2%) and in >50% of cells in 7 (15.9%) tumors. CXCR4 expression correlated to the epithelioid-mixed cell type (P=0.030). No statistically significant relation emerged between CXCR4 expression, largest tumor diameter (LTD) and extracellular matrix patterns as evaluated through histological patterns stained with periodic acid-Schiff (PAS). Events occurred in 2 out of 18 patients (11.1%) with negative tumors (2 deaths), in 3 out of 19 patients (15.8%) with <50% of positive tumor cells (2 deaths and 1 occurrence of metastases) and in 1 out of 7 patients (14.3%) with >50% of positive tumor cells (1 occurrence of metastases). The cell type (P=0.0457) but not CXCR4 showed prognostic value at univariate analysis. CONCLUSION: This study shows that CXCR4 is commonly expressed in uveal melanoma and correlates with cell type a well-established prognostic factor.     

The autonomic innervation of rat jugular vein

Summary The autonomic innervation of rat jugular vein was studied using glyoxylic acid fluorescence and acetylcholinesterase histochemical methods. The rat jugular vein is provided with both adrenergic and cholinergic nerve fibers organized in plexuses located at the adventitial-medial border. The existence of these nerve plexuses does not seem to support biochemical findings that suggest a lack of innervation in the rat jugular vein and which propose this blood vessel as a model for the analysis of drug-smooth muscle cell interaction without the interference of neuronal uptake mechanisms.     

Manipulation of Tissues for Improved Aceto-Carmine Staining

The importance of the study of the structural-mechanic properties of any particular material in order to improve the technic for aceto-carmine smears and to obtain better preparations of that material has not been, perhaps, sufficiently emphasized in the large number of papers on such cytological technic. The usefulness of such a study will be shown here in two cases met by the writer.     

Aerobic biodegradation of propylene glycol by soil bacteria

Propylene glycol (PG) is a main component of aircraft deicing fluids and its extensive use in Northern airports is a source of soil and groundwater contamination. Bacterial consortia able to grow on PG as sole carbon and energy source were selected from soil samples taken along the runways of Oslo Airport Gardermoen site (Norway). DGGE analysis of enrichment cultures showed that PG-degrading populations were mainly composed by Pseudomonas species, although Bacteroidetes were found, as well. Nineteen bacterial strains, able to grow on PG as sole carbon and energy source, were isolated and identified as different Pseudomonas species. Maximum specific growth rate of mixed cultures in the absence of nutrient limitation was 0.014 h^?1 at 4 °C. Substrate C:N:P molar ratios calculated on the basis of measured growth yields are in good agreement with the suggested values for biostimulation reported in literature. Therefore, the addition of nutrients is suggested as a suitable technique to sustain PG aerobic degradation at the maximum rate by autochthonous microorganisms of unsaturated soil profile.     

Copper(II) complexes with chicken prion repeats: influence of proline and tyrosine residues on the coordination features

The prion protein (PrP^c) is a copper-binding glycoprotein that can misfold into a β-sheet-rich and pathogenic isoform (PrP^sc) leading to prion diseases. The first non-mammalian PrP^c was identified in chicken and it was found to keep many structural motifs present in mammalian PrP^c, despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY , HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV–vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species.Electronic Supplementary Material Supplementary material is available for this article at .This revised version was published online in June 2005 with corrections to the text. The author name LaMendola has been corrected to La Mendola.     

Vascular-homing peptides for targeted drug delivery and molecular imaging: Meeting the clinical challenges

The vasculature of each organ expresses distinct molecular signatures critically influenced by the pathological status. The heterogeneous profile of the vascular beds has been successfully unveiled by the in vivo phage display, a high-throughput tool for mapping normal, diseased, and tumor vasculature. Specific challenges of this growing field are targeted therapies against cancer and cardiovascular diseases, as well as novel bioimaging diagnostic tools. Tumor vasculature-homing peptides have been extensively evaluated in several preclinical and clinical studies both as targeted-therapy and diagnosis. To date, results from several Phase I and II trials have been reported and many other trials are currently ongoing or recruiting patients. In this review, advances in the identification of novel peptide ligands and their corresponding receptors on tumor endothelium through the in vivo phage display technology are discussed. Emphasis is given to recent findings in the clinical setting of vascular-homing peptides selected by in vivo phage display for the treatment of advanced malignancies and their altered vascular beds.