A unique feature of plant aspartic proteinase precursors is the presence of an internal domain, known as plant-specific insert, whose function is not completely understood. The three-dimensional structure of the plant-specific insert resembles that of saposin-like proteins, a group of lipid-binding proteins involved in a variety of physiological processes. Here we show that recombinant plant-specific insert is able to interact with phospholipid vesicles and to induce leakage of their contents in a pH-and lipid-dependent manner. The leakage activity is higher at pH 4.5 and requires the presence of acidic phospholipids such as phosphatidylserine. To determine whether the same effect could be observed when the plant-specific insert is part of the precursor form, procardosin A and a mutant form lacking this specific domain were produced and characterized. Procardosin A displays a similar activity profile, whereas the mutant without the plant-specific insert shows only residual activity. These findings indicate that the plant-specific insert domain of plant aspartic proteinases mediates an interaction of their precursors with phospholipid membranes and induces membrane permeabilization. It is therefore possible that the plantspecific insert, alone or in conjunction with the proteolytic activity of plant aspartic proteinases, may function either as a defensive weapon against pathogens or in late autolysis of plant cells.Plant aspartic proteinases have been purified and characterized from several species. They share high sequence and structure homology, as well as general biochemical properties, with the animal and microbial aspartic proteinases (1). A unique feature present in most aspartic proteinases of plant origin is an extra protein domain of about 100 amino acids, generally named plant-specific insert (PSI) 1 , that has no homology to any other aspartic protease sequence (2). The PSI is present in the precursors of plant aspartic proteinases but is absent from the mature form of the enzymes (2-6). The function of this plantspecific insert is still unclear. An important role in the vacuolar targeting of aspartic proteinase precursors has been proposed, either by its direct interaction with the membrane, where it would act as a targeting signal (4), or by the interaction of a putative membrane binding region, formed by a cluster of the mature protein and the PSI in prophytepsin, with the membrane itself or with a receptor in the Golgi apparatus (7). It has been suggested (8) that the PSI would interact with the membrane probably in the endoplasmic reticulum as a prerequisite for signaling plant aspartic proteases into the vacuole, because previous work identified a putative vacuolar targeting sequence on the C terminal end of the aspartic protease of cardoon (5). On the other hand, the PSI could be implicated in the correct folding of the enzyme, as suggested by the lack of activation of a mutant of recombinant procyprosin bearing a deletion of the PSI sequence (2).The plant-specific insert bears high homology wit...

BackgroundArray-based comparative genomic hybridization has been assumed to be the first genetic test offered to detect genomic imbalances in patients with unexplained intellectual disability with or without dysmorphisms, multiple congenital anomalies, learning difficulties and autism spectrum disorders.Our study contributes to the genotype/phenotype correlation with the delineation of laboratory criteria which help to classify the different copy number variants (CNVs) detected. We clustered our findings into five classes ranging from an imbalance detected in a microdeletion/duplication syndrome region (class I) to imbalances that had previously been reported in normal subjects in the Database of Genomic Variants (DGV) and thus considered common variants (class IV).ResultsAll the analyzed 1000 patients had at least one CNV independently of its clinical significance. Most of them, as expected, were alterations already reported in the DGV for normal individuals (class IV) or without known coding genes (class III-B). In approximately 14 % of the patients an imbalance involving known coding genes, but with partially overlapping or low frequency of CNVs described in the DGV was identified (class IIIA). In 10.4 % of the patients a pathogenic CNV that explained the phenotype was identified consisting of: 40 class I imbalances, 44 class II de novo imbalances and 21 class II X-chromosome imbalances in male patients. In 20 % of the patients a familial pathogenic or potentially pathogenic CNV, consisting of inherited class II imbalances, was identified that implied a family evaluation by the clinical geneticists.ConclusionsAs this interpretation can be sometimes difficult, particularly if it is not possible to study the parents, using the proposed classification we were able to prioritize the multiple imbalances that are identified in each patient without immediately having to classify them as pathogenic or benign.

BackgroundAutism is a global neurodevelopmental disorder which generally manifests during the first 2 years and continues throughout life, with a range of symptomatic variations. Epidemiological studies show an important role of genetic factors in autism and several susceptible regions and genes have been identified. The aim of our study was to validate a cost-effective set of commercial Multiplex Ligation dependent Probe Amplification (MLPA) and methylation specific multiplex ligation dependent probe amplification (MS-MLPA) test in autistic children refered by the neurodevelopmental center and autism unit of a Paediatric Hospital.ResultsIn this study 150 unrelated children with autism spectrum disorders were analysed for copy number variation in specific regions of chromosomes 15, 16 and 22, using MLPA. All the patients had been previously studied by conventional karyotype and fluorescence in situ hybridization (FISH) analysis for 15(q11.2q13) and, with these techniques, four alterations were identified. The MLPA technique confirmed these four and identified further six alterations by the combined application of the two different panels.ConclusionsOur data show that MLPA is a cost effective straightforward and rapid method for detection of imbalances in a clinically characterized population with autism. It contributes to strengthen the relationship between genotype and phenotype of children with autism, showing the clinical difference between deletions and duplications.