Type XVII collagen (BP180) can function as a cell−matrix adhesion molecule via binding to laminin 332

Collagen XVII (COL17) is a transmembrane glycoprotein that is expressed on the basal surface of basal epidermal keratinocytes. Previous observations have led to the hypothesis that an interaction between COL17 and laminin 332, an extracellular matrix protein, contributes to the attachment of the basal keratinocyte to the basement membrane. In order to isolate and manipulate COL17 interactions with ECM components, we induced COL17 expression in two cells lines, SK-MEL1 and K562, that exhibit little or no capacity to attach to our test substrates, including laminin 332, types I and IV collagen, and fibronectin. Cells expressing high levels of COL17 preferentially adhered to a laminin 332 matrix, and, to a lesser extent, type IV collagen, while showing little or no binding to type I collagen or fibronectin. A quantitative analysis of cell adhesive forces revealed that, compared with COL17-negative cells, COL17-positive cells required over 7-fold greater force to achieve 50% detachment from a laminin 332 substrate. When a cell preparation (either K562 or SK-MEL1) with heterogeneous COL17 expression levels was allowed to attach to a laminin 332 matrix, the COL17-positive and COL17-negative cells differentially sorted to the bound and unbound cell fractions, respectively. COL17-dependent attachment to laminin 332 could be reduced or abolished by siRNA-mediated knock-down of COL17 expression or by adding to the assay wells specific antibodies against COL17 or laminin 332. These findings provide strong support for the hypothesis that cell surface COL17 can interact with laminin 332 and, together, participate in the adherence of a cell to the extracellular matrix.     

Expression analysis of the nucleocytoplasmic lectin 'Orysata' from rice in Pichia pastoris

The Oryza sativa lectin, abbreviated Orysata, is a mannose-specific, jacalin-related lectin expressed in rice plants after exposure to certain stress conditions. Expression of a fusion construct containing the rice lectin sequence linked to enhanced green fluorescent protein in Bright Yellow 2 tobacco cells revealed that Orysata is located in the nucleus and the cytoplasm of the plant cell, indicating that it belongs to the class of nucleocytoplasmic jacalin-related lectins. Since the expression level of Orysata in rice tissues is very low the lectin was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway and express the protein into the medium. Approximately 12 mg of recombinant lectin was purified per liter medium. SDS/PAGE and western blot analysis showed that the recombinant lectin exists in two molecular forms. Far western blot analysis revealed that the 23 kDa lectin polypeptide contains an N-glycan which is absent in the 18.5 kDa polypeptide. Characterization of the glycans present in the recombinant Orysata revealed high-mannose structures, Man9-11 glycans being the most abundant. Glycan array analysis showed that Orysata interacts with high-mannose as well as with more complex N-glycan structures. Orysata has potent anti-human immunodeficiency virus and anti-respiratory syncytial virus activity in cell culture compared with other jacalin-related lectins.     

Unusual N-glycan structures required for trafficking Toxoplasma gondii GAP50 to the inner membrane complex regulate host cell entry through parasite motility

Toxoplasma gondii motility, which is essential for host cell entry, migration through host tissues, and invasion, is a unique form of actin-dependent gliding. It is powered by a motor complex mainly composed of myosin heavy chain A, myosin light chain 1, gliding associated proteins GAP45, and GAP50, the only integral membrane anchor so far described. In the present study, we have combined glycomic and proteomic approaches to demonstrate that all three potential N-glycosylated sites of GAP50 are occupied by unusual N-glycan structures that are rarely found on mature mammalian glycoproteins. Using site-directed mutagenesis, we show that N-glycosylation is a prerequisite for GAP50 transport from the endoplasmic reticulum to the Golgi apparatus and for its subsequent delivery into the inner complex membrane. Assembly of key partners into the gliding complex, and parasite motility are severely impaired in the unglycosylated GAP50 mutants. Furthermore, comparative affinity purification using N-glycosylated and unglycosylated GAP50 as bait identified three novel hypothetical proteins including the recently described gliding associated protein GAP40, and we demonstrate that N-glycans are required for efficient binding to gliding partners. Collectively, these results provide the first detailed analyses of T. gondii N-glycosylation functions that are vital for parasite motility and host cell entry.     

SLIDER: a generic metaheuristic for the discovery of correlated motifs in protein-protein interaction networks

Correlated motif mining (cmm) is the problem of finding overrepresented pairs of patterns, called motifs, in sequences of interacting proteins. Algorithmic solutions for cmm thereby provide a computational method for predicting binding sites for protein interaction. In this paper, we adopt a motif-driven approach where the support of candidate motif pairs is evaluated in the network. We experimentally establish the superiority of the Chi-square-based support measure over other support measures. Furthermore, we obtain that cmm is an np-hard problem for a large class of support measures (including Chi-square) and reformulate the search for correlated motifs as a combinatorial optimization problem. We then present the generic metaheuristic slider which uses steepest ascent with a neighborhood function based on sliding motifs and employs the Chi-square-based support measure. We show that slider outperforms existing motif-driven cmm methods and scales to large protein-protein interaction networks. The slider-implementation and the data used in the experiments are available on http://bioinformatics.uhasselt.be.     

Comparison of three vegetation monitoring methods: Their relative utility for ecological assessment and monitoring

Vegetation cover and composition are two indicators commonly used to monitor terrestrial ecosystems. These indicators are currently quantified with a number of different methods. The interchangeability and relative benefits of different methods have been widely discussed in the literature, but there are few published comparisons that address multiple criteria across a broad range of grass- and shrub-dominated communities, while keeping sampling effort (time) approximately constant. This study compared the utility of three field sampling methods for ecological assessment and monitoring: line-point intercept, grid-point intercept, and ocular estimates. The criteria used include: (1) interchangeability of data, (2) precision, (3) cost, and (4) value of each method based on its potential to generate multiple indicators. Foliar cover by species was measured for each method in five plant communities in the Chihuahuan Desert. Line- and grid-point intercept provide similar estimates of species richness which were lower than those based on ocular estimates. There were no differences in the precision of the number of species detected. Estimates of foliar cover with line- and grid-point intercept were similar and significantly higher than those based on ocular estimates. Precision of cover estimates with line-point intercept was higher than for ocular estimates. Time requirements for the three methods were similar, despite the fact that the point-based methods included cover estimates for all canopy layers and the soil surface, while the ocular estimates included only the top canopy layer. Results suggest that point-based methods provide interchangeable data with higher precision than ocular estimates. Moreover these methods can be used to generate a much greater number of indicators that are more directly applicable to a variety of monitoring objectives, including soil erosion and wildlife habitat.     

Effect of three pollination methods on embryo development and seedset in intra- and interspecific crosses between seven Lilium species

Summary Interspecific crosses were made between seven Lilium species, viz. L. candidum, L. concolor, L. dauricum, L. henryi, L. longiflorum, L. nepalense and L. rubellum. A complete diallel cross was carried out between these seven species, including self- and intraspecific pollinations using three pollination methods: normal pollination on the stigma, pollination on the ovary after cutting the style, and pollination on the stigma with the aid of mentor (non-functional, compatible) pollen. Embryo rescue, starting 35 days after pollination, was applied to all interspecific combinations. The percentage of successful crosses was about 2.8% after normal pollination, 5.4% after cut-style pollination and 3.8% with the mentor pollen technique. Crosses with L. nepalense were exceptional in that embryos died during the embryo culture phase. Seventeen cross combinations (including 4 reciprocals) yielded 62 embryo plantlets from 839 interspecific pollinations.     

X-chromosome polysomy in the male

Summary A review of 569 male patients with X-chromosome polysomies (544 Klinefelter and 25 patients with other types of X-chromosome polysomy) is presented here. These patients were detected among the 77000 persons karyotyped in the Leuven cytogenetic center between the years 1966 and 1987. In the group of 544 Klinefelter patients special attention was paid to (1) the age at diagnosis, (2) social and marital status of the postpubertal males, (3) physical and intellectual abilities of the prepubertal boys, (4) delineation of the concurrence of Klinefelter syndrome and fragile X syndrome, and (5) the frequency of malignancies. In 25 patients with other X-chromosome polysomies (2 n48 chromosomes) genotype/phenotype correlation is reviewed, especially for the patients with 48,XXYY and 49,XXXXY karyotypes. Finally, double aneuploidy and rare structural X-chromosome aberrations are briefly discussed.     

Ligand-induced trafficking of the sphingosine-1-phosphate receptor EDG-1

The endothelial-derived G-protein-coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1-green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP-EDG-1 interaction. SPP binds to EDG-1-GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1-GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a tau1/2 of approximately 15 min. The EDG-1-GFP-containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1-GFP recycles back to the plasma membrane with a tau1/2 of approximately 30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.     

No direct linkage between proton pumping and photosynthate unloading from seed coats of Phaseolus vulgaris L.

The energization of the active sucrose release from bean seed-coat halves was investigated. For this purpose, seed coat tissues adjacent to the apoplastic space were exposed to a variety of treatments and proton and photosynthate release were measured. Fusicoccin (10^–5 moll^–1) stimulated proton pump activities. Orthovanadate (2×10^–4 moll^–1) and abscisic acid (10^–5 moll^–1) diminished the proton extrusion evoked by fusicoccin. Fusicoccin inhibited sucrose release, whereas orthovanadate and abscisic acid stimulated it. Addition of 100 mmoll^–1 K⁺ had a promotory effect on photosynthate unloading, fading away with time. This extra unloading was linearly related to an enhanced proton loss. It was concluded that the photosynthate unloading apparently is not a proton/sucrose antiport and that a pump-leak system for photosynthate release is unlikely. A tentative model for photosynthate/proton symport not directly linked to proton pumping is presented as the mechanism of unloading.Abbreviations ABA abscisic acid - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DTE diethioerythritol - FC fusicoccin - MES 2-(N-morpholino) ethanesulfonic acid monohydrate - NEM n-ethylmaleimide - PCMBS p-chloromercuriphenylsulfonic acid - TRIS 2-amino-2-(hydroxymethyl) propane-1,3 diol - VAN sodium orthovanadate