Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation

A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.     

Atypical presentation of the Prader-Willi syndrome. Mosaic trisomy 15?

We report a female with Prader-Willi syndrome and hemihypertrophy. We discuss the possibility of an undetected mosaicism for trisomy 15 explaining this unusual feature.     

Procathepsin D interacts with prosaposin in cancer cells but its internalization is not mediated by LDL receptor-related protein

The cell surface binding, endocytosis, and lysosomal routing of procathepsin D (procath-D) in cancer cells are mostly independent of the mannose-6-phosphate (M6P) receptors. In an attempt to define the receptor involved, we intracellularly cross-linked procath-D with a 68-kDa protein that we identified with specific antibodies as prosaposin in human breast and ovarian cancer cell lines. In cancer cells, this protein-protein interaction was resistant to ammonium chloride or M6P treatment, indicating that it was independent of the M6P receptors. A similar interaction also occurred in the breast cancer cell culture medium between the secreted prosaposin and procath-D. Since these two precursors can be endocytosed, we then determined whether they were interacting with the same cell surface receptor. In fibroblasts, we confirmed that the endocytosis of these two proteins was different since it was generally mediated by the M6P receptors for procath-D and mostly by LRP (LDL receptor-related protein) for prosaposin. In breast cancer cells, prosaposin endocytosis was not detected, in contrast to procath-D endocytosis, suggesting that the majority of procath-D is not internalized as a complex with prosaposin. Moreover, RAP (receptor-associated protein), a ligand inhibiting LRP-mediated endocytosis, prevented internalization of prosaposin in 49-F rat fibroblasts, but did not affect procath-D M6P-independent internalization in MDA-MB231 cells. We conclude that in breast cancer cells, even though procath-D interacts intracellularly and extracellarly with prosaposin, it is endocytosed independent of prosaposin by a receptor different from the M6P receptors and the LRP.     

Biochemical, molecular and structural analysis of multiple thaumatin-like proteins from the elderberry tree (Sambucus nigra L.)

Thaumatin-like proteins (TLPs) were isolated and characterized from fruits and leaves of elderberry (Sambucus nigra) and their corresponding genes cloned. In addition, the developmental regulation and induction of the different TLPs was followed in some detail. Ripening berries accumulated a fruit-specific TLP during the final stages of maturation. This fruit-specific TLP had no antifungal activity and was devoid of beta-glucanase activity. Leaves constitutively expressed a TLP that closely resembled the fruit-specific homologue. Treatment with jasmonate methyl ester induced two additional TLPs in leaves but did not induce or enhance the expression of TLPs in immature berries. In contrast to jasmonate methyl ester, both ethephon and garlic extract induced the expression of a TLP in unripe berries that normally do not express any TLP. Sequence analysis and molecular modeling indicated that all elderberry thaumatin-like proteins share a high sequence similarity with group-5 pathogenesis-related proteins. However, the proteins encoded by the different sequences differed from each other in isoelectric point and the distribution of the charges on the surface of the molecule.     

New in vivo and ex vivo models for the experimental study of sheep scrapie: development and perspectives

Sheep scrapie is a prototypical transmissible spongiform encephalopathy (TSE), and the most widespread of these diseases. Experimental study of TSE infectious agents from sheep and other species essentially depends on bioassays in rodents. Transmission of natural sheep scrapie to conventional mice commonly requires one or two years. In an effort to develop laboratory models in which investigations on the sheep TSE agent would be facilitated, we have established mice and cell lines that were genetically engineered to express ovine PrP protein and examined their susceptibility to the infection. A series of transgenic mice lines (tgOv) expressing the high susceptibility allele (VRQ) of the ovine PrP gene from different constructs was expanded. Following intracerebral inoculation with natural scrapie isolates, all animals developed typical TSE neurological signs and accumulated abnormal PrP in their brain. The survival time in the highest expressing tgOv lines ranged from 2 to 7 months, depending on the isolate. It was inversely related to the brain PrP content, and essentially unchanged on further passaging. Ovine PrP transgene expression thus enhanced scrapie disease transmission from sheep to mice. Such tgOv mice may bring new opportunities for analysing the natural variation of scrapie strains and measuring infectivity. As no relevant cell culture models for agents of naturally-occurring TSE exist, we have explored various strategies in order to obtain stable cell lines that would propagate the sheep agent ex vivo without prior adaptation to rodent. In one otherwise refractory rabbit epithelial cell line, a regulable expression of ovine PrP was achieved and found to enable an efficient replication of the scrapie agent in inoculated cultures. Cells derived from sheep embryos or from tgOv mice were also used in an attempt to establish permissive cell lines derived from the nervous system. Cells engineered to express PrP proteins of a specified sequence may thus represent a promising strategy to further explore, at the cellular level, various aspects of TSE diseases.     

A knowledge-driven agent-centred framework for data mining in EMG

In this paper, we present a multi-agent framework for data mining in electromyography. This application, based on a web interface, provides a set of functionalities allowing to manipulate 1000 medical cases and more than 25,000 neurological tests stored in a medical database. The aim is to extract medical information using data mining algorithms and to supply a knowledge base with pertinent information. The multi-agent platform gives the possibility to distribute the data management process between several autonomous entities. This framework provides a parallel and flexible data manipulation.     

Aromatic side-chain interactions in proteins. II. Near- and far-sequence Phe-X pairs

We have collected all aromatic pairs (3152) involving an N-phenyl partner in a dataset of 593 proteins of the PDB: 728 of these pairs involve a partner residue less than 6 apart in the sequence. These near-sequence Phe-X pairs correspond to specific conformations that stabilize secondary structures, mainly alpha-helices when the residues are 1, 3, and 4 apart, and beta-strands when they are 2 apart in the sequence. These conformations are not spatially random and have been examined in detail. The remaining phenylalanine pairs (2424) are between partners more than 5 apart in the sequence. Of these far-sequence pairs, 34% of occurrences are in sheets. Next in frequencies are pairs that bridge a beta-strand to a helix (24%), followed by pairs that bridge a beta-strand to a random coiled structure (15%). Helix to helix pairs only constitute 12% of these far-sequence pairs. Analysis of the pairing frequency supports the hypothesis that aromatic interactions are late events of protein folding.     

Aromatic side-chain interactions in proteins. I. Main structural features

In a data set of 593 nonhomologous proteins from the PDB, we have analyzed the pairing of phenylalanine, tyrosine, tryptophan, and histidine residues with their closest aromatic partner. The frequency distribution of the shortest interatomic distance of partners is bimodal with a sharp peak at approximately 3.8 A and a wider one at a longer distance. Only the 3.8 A peak corresponds to direct ring-ring interactions thus aromatic pairs. The aromatic pairs were separated into two classes, near-sequence pairs and far-sequence pairs. Near sequence pairs stabilize local structure, and far-sequence pairs stabilize tertiary structure. Far-sequence pairs (74% of all pairs) mainly bridge two beta-strands, followed by pairs that bridge a beta-strand and a helix, and pairs that bridge a beta-strand and a random coil structure. Pairs that bridge helices are rare. The secondary structure of the near-sequence pairs depends on the partner distance in the sequence. When the partners are 1, 3, or 4 residues apart in the sequence, pairs are mostly found in helical structures. When the partners are two apart, pairs are mostly found in the same beta-strand. Analysis of the frequency of near sequence pairs supports the hypothesis that aromatic pairing occurs after, rather than before, the formation of secondary structures.     

Major proteins of bovine seminal plasma bind to the low-density lipoprotein fraction of hen's egg yolk

Over the past 60 years, egg yolk (EY) has been routinely used in both liquid semen extenders and those used to cryopreserve sperm. However, the mechanism by which EY protects sperm during liquid storage or from freezing damage is unknown. Bovine seminal plasma contains a family of proteins designated BSP-A1/-A2, BSP-A3, and BSP-30-kDa (collectively called BSP proteins). These proteins are secretory products of seminal vesicles that are acquired by sperm at ejaculation, modifying the sperm membrane by inducing cholesterol efflux. Because cholesterol efflux is time and concentration dependent, continuous exposure to seminal plasma (SP) that contains BSP proteins may be detrimental to the sperm membrane, which may adversely affect the ability of sperm to be preserved. In this article, we show that the BSP proteins bind to the low-density fraction (LDF), a lipoprotein component of the EY extender. The binding is rapid, specific, saturable, and stable even after freeze-thawing of semen. Furthermore, LDF has a very high capacity for BSP protein binding. The binding of BSP proteins to LDF may prevent their detrimental effect on sperm membrane, and this may be crucial for sperm storage. Thus, we propose that the sequestration of BSP proteins of SP by LDF may represent the major mechanism of sperm protection by EY.     

Functional expression of murine LRP1 requires correction of Lrp1 cDNA sequences

Here, we describe the reconstruction of a functional 14 kbp full-length murine Lrp1 cDNA from overlapping partial cDNAs, which were described before [Biochim. Biophys. Acta 1173 (1993) 71]. The reconstructed full-length cDNA needed sequence correction (by mutagenesis) due to nucleotide errors present in the underlying partial cDNAs. These mistakes compromised the proteolytical maturation of the LRP precursor (4545 aa) into its alpha- and beta-subunits. To identify these mistakes initially, detailed sequence analyses and comparison of genomic and cDNA sequences of different murine strains proved to be necessary to obtain correct wild-type sequences. Comparison of Lrp1 cDNA sequences of CBA mice with Lrp1 genomic exon sequences of 129P3/J mice (like in man 89 exons) revealed only 24 nucleotide differences within about 14.8 kbp. Only 1 out of 23 nucleotide differences in the protein coding region affected an amino acid residue: Thr versus Ala at amino acid residue position 2642 in 129P3/J and CBA, respectively. After correction by mutagenesis, both a 129P3/J and a CBA-based version of a full-length wild-type Lrp1 cDNA were functionally expressed in an LRP-deficient mutant CHO cell line. Transient expression showed the expected maturation of the LRP precursor into its two subunits. Furthermore, stable transfection restored the sensitivity to exposure to Pseudomonas aeruginosa toxin A (PEA). Since LRP is the unique receptor for this toxin, this indicates that the toxin could enter the cells after binding to and endocytosis by its genuine receptor. This murine LRP expression system will be instrumental in future experimental dissection of this multifunctional receptor.