Kinetic analysis of mineral formation during in vitro modeling of matrix vesicle mineralization: effect of annexin A5, phosphatidylserine, and type II collagen

Matrix vesicles (MVs) are involved in de novo mineral formation by nearly all vertebrate tissues. The driving force for MV mineralization is a nucleational core composed of three principal constituents: (i) amorphous calcium phosphate (ACP), complexed in part with phosphatidylserine (PS) to form (ii) calcium-phosphate-lipid complexes (CPLX), and (iii) annexin A5 (AnxA5), the principal lipid-dependent Ca(2+)-binding protein in MVs. We describe methods for reconstituting the nucleational core using a biomimetic approach and for analyzing the kinetics of its induction of mineral formation. The method is based on light scattering by the nascent crystallites at 340 nm and monitors mineral formation at regular intervals without disturbing the system using an automated plate reader. It yields precise replicate values that typically agree within less than 5%. As with MVs, mineral formation by the synthetic complex follows a sigmoidal pattern; following a quiescent induction period, rapid formation ensues for a limited time, followed by a distinct decline in rate that continues to slow, ultimately reaching a maximal asymptotic value. Key to quantization of mineral formation is the use of first-derivative analysis, which defines the induction time, the rate and the amount of initial mineral formation. Furthermore, using a five-parameter logistic curve-fitting algorithm, the maximal amount of mineral formation can be predicted accurately. Using these methods, we document the dramatic finding that AnxA5 synergistically activates PS-CPLX, transforming it from a very weak nucleator of mineral formation to a potent one. The methods presented should enable systematic study of the effects of numerous other factors thought to contribute to mineral formation.     

Anoxybacillus amylolyticus sp. nov., a thermophilic amylase producing bacterium isolated from Mount Rittmann (Antarctica)

A new thermophilic spore-forming strain MR3CT was isolated from geothermal soil located on Mount Rittmann in Antarctica. Strain MR3CT was Gram-positive, rod-shaped, occurring in pairs or filamentous. Growth was observed between 45 and 65 degrees C (optimum 61 degrees C) and at pH 5.0-6.5 (optimum pH 5.6). It was capable of utilizing galactose, trehalose, maltose and sucrose. The microorganism produced an exopolysaccharide and synthesized an extracellular constitutive amylolytic activity. The G + C content of DNA was 43.5 mol%. On the basis of 16S rRNA gene sequence similarity, strain MR3CT was shown to be related most closely to Anoxybacillus species. Chemotaxonomic data (major isoprenoid quinone-menaquinone-7; major fatty acid-iso-C15:0 and iso-C17:0) supported the affiliation of strain MR3C1T to the genus Anoxybacillus. The results of DNA-DNA hybridization, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain MR3CT from the validly published Anoxybacillus species. MR3CT therefore represents a new species, for which the name Anoxybacillus amylolyticus sp. nov., is proposed, with the type strain MR3CT (= ATCC BAA-872T = DSM 15939T = CIP 108338T).     

Retinoic acid stimulates matrix calcification and initiates type I collagen synthesis in primary cultures of avian weight-bearing growth plate chondrocytes

The effect of retinoic acid (RA) on primary cultures of growth plate chondrocytes obtained from weight-bearing joints was examined. Chondrocytes were isolated from the tibial epiphysis of 6- to 8-week-old broiler-strain chickens and cultured in either serum-containing or serum-free media. RA was administered at low levels either transiently or continuously after the cells had become established in culture. Effects of RA on cellular protein levels, alkaline phosphatase (AP) activity, synthesis of proteoglycan (PG), matrix calcification, cellular morphology, synthesis of tissue-specific types of collagen, and level of matrix metalloproteinase (MMP) activity were explored. RA treatment generally increased AP activity, and stimulated mineral deposition, especially if present continuously. RA also caused a shift in cell morphology from spherical/polygonal to spindle-like. This occurred in conjunction with a change in the type of collagen synthesized: type X and II collagens were decreased, while synthesis of type I collagen was increased. There was also a marked increase in the activity of MMP. Contrasting effects of continuous RA treatment on cellular protein levels were seen: they were enhanced in serum-containing media, but decreased in serum-free HL-1 media. Levels of RA as low as 10 nM significantly inhibited PG synthesis and caused depletion in the levels of PG in the medium and cell-matrix layer. Thus, in these appendicular chondrocytes, RA suppressed chondrocytic (PG, cartilage-specific collagens) and enhanced osteoblastic phenotype (cell morphology, type I collagen, alkaline phosphatase, and mineralization). J. Cell. Biochem. 65:209–230. © 1997 Wiley-Liss, Inc.     

Molecular dynamics studies on Mdm2 complexes: an analysis of the inhibitor influence

p53 is a powerful anti-tumoral molecule frequently inactivated by mutations or deletions in cancer. However, half of all human tumors expresses wild-type p53, and its activation, by antagonizing its negative regulator Mdm2, might offer a new strategy for therapeutic protocol. In this work, we present a molecular dynamics study on Mdm2 structure bound to two different known inhibitors with the aim to investigate the structural transitions between apo-Mdm2 and Mdm2-inhibitor complexes. We tried to gain information about conformational changes binding a benzodiazepine derivative inhibitor with respect the known nutlin and the apo form. The conformational changes alter the size of the cleft and were mainly in the linker regions, suggesting that the overall dynamic nature of Mdm2 is related to dynamic movements in these regions.     

Human plasmacytoid dendritic cells interact with gp96 via CD91 and regulate inflammatory responses

Glucose-regulated stress protein gp96 is known to be involved in the host response to pathogens and to cancer. Our study explored the relationships between gp96 and human blood plasmacytoid dendritic cells (pDC) and proved that gp96 directly targets pDC by a receptor-dependent interaction. Competition studies identified CD91 as a gp96 receptor on pDC, and laser confocal imaging indicated that CD91 triggering was followed by gp96 endocytosis and trafficking into early endosomes and later into the endoplasmic reticulum compartment. Using two alternative Abs, we showed that human blood pDC reproducibly expressed CD91, although different levels of expression were detectable among the analyzed donors. Moreover, CpG-matured pDC displayed CD91 receptor up-regulation that correlated with an increased gp96 binding. Functionally, gp96-pDC interaction activated the NF-kappaB pathway, leading to the nuclear translocation of the NF-kappaB complex. gp96-treated pDC maintained an immature phenotype, while they down-modulated the release of IL-8, suggesting an anti-inflammatory role of this pathway, and they strongly up-regulated the cell surface expression of the gp96 receptor CD91. CpG-matured or gp96-treated pDC, expressing high levels of the gp96 receptor CD91, antagonized the gp96-induced activation of monocyte-derived dendritic cells in terms of cell surface phenotype and cytokine production. Altogether, these results suggest that gp96-pDC interaction might represent an active mechanism controlling the strength of the immune response to free, extracellular available gp96; this mechanism could be particularly relevant in wounds and chronic inflammation.     

Analysis and molecular modeling of the formation, structure, and activity of the phosphatidylserine-calcium-phosphate complex associated with biomineralization

The nucleational core of matrix vesicles contains a complex (CPLX) of phosphatidylserine (PS), Ca(2+), and inorganic phosphate (P(i)) that is important to both normal and pathological calcification. Factors required for PS-CPLX formation and nucleational activity were studied using in vitro model systems and molecular dynamic simulations. Ca(2+) levels required for and rates of PS-CPLX formation were monitored by light scattering at 340 nm, assessing changes in amount and particle size. Fourier transform infrared spectroscopy was used to explore changes in chemical structure and composition. Washing with pH 5 buffer was used to examine the role of amorphous calcium phosphate in CPLX nucleational activity, which was assessed by incubation in synthetic cartilage lymph with varied pH values. Addition of 4 Ca(2+)/PS was minimally required to form viable complexes. During the critical first 10-min reaction period, rapid reduction in particle size signaled changes in PS-CPLX structure. Fourier transform infrared spectroscopy revealed increasing mineral phosphate that became progressively deprotonated to PO(4)(3-). This Ca(2+)-mediated effect was mimicked in part by increasing the Ca(2+)/PS reaction ratio. Molecular dynamic simulations provided key insight into initial interactions between Ca(2+) and P(i) and the carboxyl, amino, and phosphodiester groups of PS. Deduced interatomic distances agreed closely with previous radial distribution function x-ray-absorption fine structure measurements, except for an elongated Ca(2+)-N distance, suggesting additional changes in atomic structure during the critical 10-min ripening period. These findings clarify the process of PS-CPLX formation, reveal details of its structure, and provide insight into its role as a nucleator of crystalline calcium phosphate mineral formation.     

Detection of mutagens in water-distribution systems after disinfection

This research examined the quality of water-before and after distribution-of four drinking-water production plants located in Northern Italy, two of which collected water from local aquifers and two from the River Po. A battery of genotoxicity assays for monitoring drinking-water was performed to assess the quality of the water produced by the treatment plants under study. Three different sampling stations were selected at each plant, one right at the outlet of the treatment plant and two along with the distribution pipelines. Raw river water was also sampled and analysed as a control. The water samples (500 l) were concentrated on silica C18 cartridges and the extracts were tested in in vitro mutagenicity assays (Salmonella/microsome assay with strains TA 98 and TA 100; SOS Chromotest with Escherichia coli strain PQ37); gene conversion, point mutation and mitochondrial DNA mutability assays with the diploid Saccharomyces cerevisiae strain D7 and a toxicity test using the bioluminescent bacterium Vibrio fischeri (Microtox). The Microtox test and the mitochondrial DNA mutability assay showed the greatest sensitivity towards toxic or mutagenic substances in the water extracts considered. The results show that this battery of short-term tests is applicable in the routine monitoring of drinking-water quality before and after distribution.     

Novel pathogenic mechanisms of congenital insensitivity to pain with anhidrosis genetic disorder unveiled by functional analysis of neurotrophic tyrosine receptor kinase type 1/nerve growth factor receptor mutations.

Congenital insensitivity to pain with anhidrosis (CIPA) is a rare genetic disease characterized by absence of reaction to noxious stimuli and anhidrosis. The genetic bases of CIPA have remained long unknown. A few years ago, point mutations affecting both coding and noncoding regions of the neurotrophic tyrosine receptor kinase type 1 (NTRK1)/nerve growth factor receptor gene have been detected in CIPA patients, demonstrating the implication of the nerve growth factor/NTRK1 pathway in the pathogenesis of the disease. We have previously shown that two CIPA mutations, the G571R and the R774P, inactivate the NTRK1 receptor by interfering with the autophosphorylation process. We have extended our functional analysis to seven additional NTRK1 mutations associated with CIPA recently reported by others. Through a combination of biochemical and biological assays, we have identified polymorphisms and pathogenic mutations. In addition to the identification of residues important for NTRK1 activity, our analysis suggests the existence of two novel pathogenic mechanisms in CIPA: one based on the NTRK1 receptor processing and the other acting through the reduction of the receptor activity.     

Inhibition of terminal differentiation and matrix calcification in cultured avian growth plate chondrocytes by Rous sarcoma virus transformation

Endochondral bone formation involves the progression of epiphyseal growth plate chondrocytes through a sequence of developmental stages which include proliferation, differentiation, hypertrophy, and matrix calcification. To study this highly coordinated process, we infected growth plate chondrocytes with Rous sarcoma virus (RSV) and studied the effects of RSV transformation on cell proliferation, differentiation, matrix synthesis, and mineralization. The RSV-transformed chondrocytes exhibited a distinct bipolar, fibroblast-like morphology, while the mock-infected chondrocytes had a typical polygonal morphology. The RSV-transformed chondrocytes actively synthesized extracellular matrix proteins consisting mainly of type I collagen and fibronectin. RSV-transformed cells produced much less type X collagen than was produced by mock-transformed cells. There also was a significant reduction of proteoglycan levels secreted in both the cell-matrix layer and culture media from RSV-transformed chondrocytes. RSV-transformed chondrocytes expressed two- to- threefold more matrix metalloproteinase, while expressing only one-half to one-third of the alkaline phosphatase activity of mock infected cells. Finally, RSV-transformed chondrocytes failed to calcify the extracellular matrix, while mock-transformed cells deposited high levels of calcium and phosphate into their extracellular matrix. These results collectively indicate that RSV transformation disrupts the preprogrammed differentiation pattern of growth plate chondrocytes and inhibit chondrocyte terminal differentiation and mineralization. They also suggest that the expression of extracellular matrix proteins, type II and type X collagens, and the cartilage proteoglycans are important for chondrocyte terminal differentiation and matrix calcification. J. Cell. Biochem. 69:453–462, 1998. © 1998 Wiley-Liss, Inc.