Characterization of the interaction between von Willebrand factor and osteoprotegerin.

BACKGROUND AND OBJECTIVE: Osteoprotegerin (OPG), a member of the tumor necrosis-factor receptor superfamily, plays an important role in bone remodeling and is also involved in vascular diseases. OPG is physically associated with von Willebrand factor (VWF), a glycoprotein involved in primary hemostasis, within the Weibel-Palade bodies (WPBs) of endothelial cells and in plasma. The present study aimed to elucidate the molecular mechanisms underlying the interaction between OPG and VWF. METHODS AND RESULTS: In a solid-phase binding assay, VWF was able to bind specifically to OPG in a calcium-dependent manner. This interaction displayed strong pH dependence with optimal binding occurring at pH 6.5 and was severely impaired by chloride-ion concentrations above 40 mm. Using a series of purified VWF derivatives the functional site that supports VWF interaction with OPG was localized on its Al domain. Fluorescence microscopy on human umbilical vein endothelial cells showed co-localization of VWF and OPG in WPBs. When secretion was induced, OPG remained associated with VWF in extracellular patches of release under biochemical conditions found in blood plasma. CONCLUSIONS: Our observations demonstrate the existence of an interactive site for OPG within the VWF A1-domain. This study established that the optimal biochemical parameters allowing a complex formation between VWF and OPG are those thought to prevail in the trans-Golgi network. These conditions would allow VWF to act as a cargo targeting OPG to WPBs. Finally, blood environments appear suitable to preserve the complex, which may participate in vascular injury, arterial calcification and inflammation.

Fifteen new mutations (-195C>T, L-12X, 298-2A>G, T117N, A159T, R229S, 997+2T>A, E274X, A331T, H364R, D389G, 1256delC, R433H, N461I, C472S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with hypophosphatasia.

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and liver/bone/kidney-type alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 12 families affected by severe or mild hypophosphatasia. Twenty distinct mutations were found, 5 of which were previously reported. Nine of the 15 new mutations were missense mutations (T117N, A159T, R229S, A331T, H364R, D389G, R433H, N461I, and C472S). The others were 2 nonsense mutations (L-12X and E274X), one single nucleotide deletion (1256delC), 2 mutations affecting splicing (298-2A>G, 997+2T>A), and a mutation in the major transcription start site (-195C>T). Hum Mutat 15:293, 2000.

Strains of BCMV and BCMNV characterized from lima bean plants affected by deforming mosaic disease in Peru.

Viruses of the species Bean common mosaic virus (BCMV) and Bean common mosaic necrosis virus (BCMNV) were simultaneously detected by the different size of PCR amplicons in lima bean plants (Phaseolus lunatus) displaying deforming mosaic symptoms in Peru. Phylogenetic analysis of partial deduced CP amino acid sequences indicated that the Peruvian BCMV isolates belong to new strains. One isolate differed from the other Peruvian isolates, and also from the ten previously described BCMV strains recognized by responses on differential bean varieties. The sequence of the 3'-proximal part (2547 nucleotides) of the genome confirmed that this isolate also belongs to BCMV.

Evaluation of various species demarcation criteria in attempts to classify ten new tombusvirus isolates.

The usefulness of various suggested species demarcation criteria was compared in attempts to determine the taxonomic status of ten new tombusvirus isolates. Five of them (Lim 1, 2, 3, 5 and 6) were obtained from different sources of commercially grown statice (Limonium sinuatum), two (Gyp 1 and 2) from different sources of commercially grown Gypsophila paniculata and three from water samples, i.e. from a small river (Schunter) in Northern Germany, from a brook (near Dossenheim) in Southern Germany and from the groundwater in a Limonium production glasshouse in the Netherlands (Lim 4). The immunoelectron microscopical decoration test allowed a quick preliminary assignment of various isolates to several known tombusviruses. A more precise analysis of the relationships was achieved by comparing the deduced amino acid sequences of the coat proteins. Sequence as well as serological data suggested that eight of the isolates should be classified as strains or variants of either Carnation Italian ringspot virus, Grapevine Algerian latent virus, Petunia asteroid mosaic virus or Sikte waterborne virus, respectively, whereas the 9th isolate (Lim 2) appears to represent a distinct new tombusvirus species. The case of the 10th isolate (Lim 5) illustrates the classification problems experienced when the properties of a virus place it close to the more or less arbitrary man-made borderline between virus species and virus strains. The coat protein gene sequences were also determined for some viruses for which these data had not yet been available, i.e. Neckar river virus, Sikte waterborne virus and Eggplant mottled crinkle virus. The sequences of the coat protein gene and also of ORF 1 of the latter virus proved to be almost identical to the corresponding genome regions of the recently described Pear latent virus, which for priority reasons should be renamed. Criteria which have been suggested in addition to serology and sequence comparisons for tombusvirus species demarcation, i.e. differences in natural and in experimental host ranges, in cytopathological features and in coat protein size, appear to be of little value for the classification of new tombusviruses.

Strains of Peru tomato virus infecting cocona (Solanum sessiliflorum), tomato and pepper in Peru with reference to genome evolution in genus Potyvirus.

Two isolates (SL1 and SL6) of Peru tomato virus (PTV, genus Potyvirus) were obtained from cocona plants (Solanum sessiliflorum) growing in Tingo María, the jungle of the Amazon basin in Peru. One PTV isolate (TM) was isolated from a tomato plant (Lycopersicon esculentum) growing in Huaral at the Peruvian coast. The three PTV isolates were readily transmissible by Myzus persicae. Isolate SL1, but not SL6, caused chlorotic lesions in inoculated leaves of Chenopodium amaranticolor and C. quinoa. Isolate TM differed from SL1 and SL6 in causing more severe mosaic symptoms in tomato, and vein necrosis in the leaves of cocona. Pepper cv. Avelar (Capsicum annuum) showed resistance to the PTV isolates SL1 and SL6 but not TM. The 5'- and 3'-proximal sequences of the three PTV isolates were cloned, sequenced and compared to the corresponding sequences of four PTV isolates from pepper, the only host from which PTV isolates have been previously characterised at the molecular level. Phylogenetic analyses on the P1 protein and coat protein amino acid sequences indicated, in accordance with the phenotypic data from indicator hosts, that the PTV isolates from cocona represented a distinguishable strain. In contrast, the PTV isolates from tomato and pepper were not grouped according to the host. Inclusion of the sequence data from the three PTV isolates of this study in a phylogenetic analysis with other PTV isolates and other potyviruses strengthen the membership of PTV in the so-called "PVY subgroup" of Potyvirus. This subgroup of closely related potyvirus species was also distinguishable from other potyviruses by their more uniform sizes of the protein-encoding regions within the polyprotein.