Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.

Assembly of a Novel Cartilage Matrix Protein Filamentous Network: Molecular Basis of Differential Requirement of von Willebrand Factor A Domains

Cartilage matrix protein (CMP) is the prototype of the newly discovered matrilin family, all of which contain von Willebrand factor A domains. Although the function of matrilins remain unclear, we have shown that, in primary chondrocyte cultures, CMP (matrilin-1) forms a filamentous network, which is made up of two types of filaments, a collagen-dependent one and a collagen-independent one. In this study, we demonstrate that the collagen-independent CMP filaments are enriched in pericellular compartments, extending directly from chondrocyte membranes. Their morphology can be distinguished from that of collagen filaments by immunogold electron microscopy, and mimicked by that of self-assembled purified CMP. The assembly of CMP filaments can occur from transfection of a wild-type CMP transgene alone in skin fibroblasts, which do not produce endogenous CMP. Conversely, assembly of endogenous CMP filaments by chondrocytes can be inhibited specifically by dominant negative CMP transgenes. The two A domains within CMP serve essential but different functions during network formation. Deletion of the A2 domain converts the trimeric CMP into a mixture of monomers, dimers, and trimers, whereas deletion of the A1 domain does not affect the trimeric configuration. This suggests that the A2 domain modulates multimerization of CMP. Absence of either A domain from CMP abolishes its ability to form collagen-independent filaments. In particular, Asp22 in A1 and Asp255 in A2 are essential; double point mutation of these residues disrupts CMP network formation. These residues are part of the metal ion–dependent adhesion sites, thus a metal ion–dependent adhesion site–mediated adhesion mechanism may be applicable to matrilin assembly. Taken together, our data suggest that CMP is a bridging molecule that connects matrix components in cartilage to form an integrated matrix network.

Dynamics and elasticity of the fibronectin matrix in living cell culture visualized by fibronectin–green fluorescent protein

Fibronectin (FN) forms the primitive fibrillar matrix in both embryos and healing wounds. To study the matrix in living cell cultures, we have constructed a cell line that secretes FN molecules chimeric with green fluorescent protein. These FN–green fluorescent protein molecules were assembled into a typical matrix that was easily visualized by fluorescence over periods of several hours. FN fibrils remained mostly straight, and they were seen to extend and contract to accommodate movements of the cells, indicating that they are elastic. When fibrils were broken or detached from cells, they contracted to less than one-fourth of their extended length, demonstrating that they are highly stretched in the living culture. Previous work from other laboratories has suggested that cryptic sites for FN assembly may be exposed by tension on FN. Our results show directly that FN matrix fibrils are not only under tension but are also highly stretched. This stretched state of FN is an obvious candidate for exposing the cryptic assembly sites.

Preferential localization of self-stimulation sites in striosomes/patches in the rat striatum

Histological sections of the mammalian striatum reveal a “matrix” that is histochemically distinguishable from patches, or “striosomes”. The latter are cross sections of a compartment that consists primarily of tube-shaped structures radiating through the matrix. As a test of the hypothesis that the function of the striosome/patch compartment includes the mediation of behaviors related to reward, the present study examined electrical self-stimulation of the caudoputamen in rats with electrodes in either of the two compartments. Rats acquired and maintained bar-pressing responses that were contingent on stimulation through electrodes making contact with striosomes/patches more reliably than animals with electrodes terminating exclusively in the matrix. The results provide in vivo evidence that the striosome/patch compartment is functionally differentiated from the matrix compartment: Stimulation centered in or around the striosome/patch compartment but not in the matrix led to rapid acquisition of a new behavior.

Mapping of contact sites in complex formation between transducin and light-activated rhodopsin by covalent crosslinking: Use of a photoactivatable reagent

Interaction of light-activated rhodopsin with transducin (T) is the first event in visual signal transduction. We use covalent crosslinking approaches to map the contact sites in interaction between the two proteins. Here we use a photoactivatable reagent, N-[(2-pyridyldithio)-ethyl], 4-azido salicylamide. The reagent is attached to the SH group of cytoplasmic monocysteine rhodopsin mutants by a disulfide-exchange reaction with the pyridylthio group, and the derivatized rhodopsin then is complexed with T by illumination at λ >495 nm. Subsequent irradiation of the complex at λ310 nm generates covalent crosslinks between the two proteins. Crosslinking was demonstrated between T and a number of single cysteine rhodopsin mutants. However, sites of crosslinks were investigated in detail only between T and the rhodopsin mutant S240C (cytoplasmic loop V-VI). Crosslinking occurred predominantly with Tα. For identification of the sites of crosslinks in Tα, the strategy used involved: (i) derivatization of all of the free cysteines in the crosslinked proteins with N-ethylmaleimide; (ii) reduction of the disulfide bond linking the two proteins and isolation of all of the Tα species carrying the crosslinked moiety with a free SH group; (iii) adduct formation of the latter with the N-maleimide moiety of the reagent, maleimido-butyryl-biocytin, containing a biotinyl group; (iv) trypsin degradation of the resulting Tα derivatives and isolation of Tα peptides carrying maleimido-butyryl-biocytin by avidin-agarose chromatography; and (v) identification of the isolated peptides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. We found that crosslinking occurred mainly to two C-terminal peptides in Tα containing the amino acid sequences 310–313 and 342–345.

Mapping of contact sites in complex formation between light-activated rhodopsin and transducin by covalent crosslinking: Use of a chemically preactivated reagent

Contact sites in interaction between light-activated rhodopsin and transducin (T) have been investigated by using a chemically preactivated crosslinking reagent, N-succinimidyl 3-(2-pyridyldithio)propionate. The 3 propionyl-N-succinimidyl group in the reagent was attached by a disulfide exchange reaction to rhodopsin mutants containing single reactive cysteine groups in the cytoplasmic loops. Complex formation between the derivatized rhodopsin mutants and T was carried out by illumination at λ > 495 nm. Subsequent increase in pH (from 6 to 7.5 or higher) of the complex resulted in crosslinking of rhodopsin to the Tα subunit. Crosslinking to Tα was demonstrated for the rhodopsin mutants K141C, S240C, and K248C, and the crosslinked sites in Tα were identified for the rhodopsin mutant S240C. The peptides carrying the crosslinking moiety were isolated from the trypsin-digested peptide mixture, and their identification was carried out by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The main site of crosslinking is within the peptide sequence, Leu-19–Arg-28 at the N-terminal region of Tα. The total results show that both the N and the C termini of Tα are in close vicinity to the third cytoplasmic loop of rhodopsin in the complex between rhodopsin and T.

Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme.

Certain matrix metalloproteinases (MMP) are expressed within the fibrous areas surrounding acellular lipid cores of atherosclerotic plaques, suggesting that these proteinases degrade matrix proteins within these areas and weaken the structural integrity of the lesion. We report that matrilysin and macrophage metalloelastase, two broad-acting MMPs, were expressed in human atherosclerotic lesions in carotid endarterectomy samples (n = 18) but were not expressed in normal arteries (n = 7). In situ hybridization and immunohistochemistry revealed prominent expression of matrilysin in cells confined to the border between acellular lipid cores and overlying fibrous areas, a distribution distinct from other MMPs found in similar lesions. Metalloelastase was expressed in these same border areas. Matrilysin was present in lipid-laden macrophages, identified by staining with anti-CD-68 antibody. Furthermore, endarterectomy tissue in organ culture released matrilysin. Staining for versican demonstrated that this vascular proteoglycan was present at sites of matrilysin expression. Biochemical studies showed that matrilysin degraded versican much more efficiently than other MMPs present in atherosclerotic lesions. Our findings suggest that matrilysin, specifically expressed in atherosclerotic lesions, could cleave structural proteoglycans and other matrix components, potentially leading to separation of caps and shoulders from lipid cores.

A general additive distance with time-reversibility and rate variation among nucleotide sites.

As additivity is a very useful property for a distance measure, a general additive distance is proposed under the stationary time-reversible (SR) model of nucleotide substitution or, more generally, under the stationary, time-reversible, and rate variable (SRV) model, which allows rate variation among nucleotide sites. A method for estimating the mean distance and the sampling variance is developed. In addition, a method is developed for estimating the variance-covariance matrix of distances, which is useful for the statistical test of phylogenies and molecular clocks. Computer simulation shows (i) if the sequences are longer than, say, 1000 bp, the SR method is preferable to simpler methods; (ii) the SR method is robust against deviations from time-reversibility; (iii) when the rate varies among sites, the SRV method is much better than the SR method because the distance is seriously underestimated by the SR method; and (iv) our method for estimating the sampling variance is accurate for sequences longer than 500 bp. Finally, a test is constructed for testing whether DNA evolution follows a general Markovian model.

X-ray absorption fine structure as a monitor of zinc coordination sites during oogenesis of Xenopus laevis.

The x-ray absorption fine structure (XAFS) zinc K-edge steps for intact stages I,II and V,VI Xenopus laevis oocytes demonstrate that the zinc concentration is about 3 and 1 mM, respectively. However, the chi(k) function for the early stage oocytes differs markedly from that for the late one. Analysis of the XAFS data for stage I,II oocytes indicates that zinc is bound to 2.0 +/- 0.5 sulfur atoms at an average coordination distance of 2.29 +/- 0.02 angstroms and 2.0 +/- 0.5 nitrogen or oxygen (N/O) atoms at 2.02 +/- 0.02 angstroms. In marked contrast, in stage V,VI oocytes, zinc is bound to 4.1 +/- 0.4 N/O atoms at an average distance of 1.98 +/- 0.01 angstroms. Our previous studies demonstrated that 90% of the zinc in stage VI oocytes is sequestered within yolk platelets, associated with a single molecule, lipovitellin, the proteolytically processed product of vitellogenin. XAFS analysis of yolk platelets, lipovitellin, and vitellogenin demonstrates that zinc is bound to 4.0 +/- 0.5 N/O ligands at an average distance of 1.98 +/- 0.01 angstroms in each case, identical to that of stage V,VI oocytes. The higher shell contributions in the Fourier transforms indicate that two of the N/O zinc ligands are His in both stage V,VI and I,II oocytes. The results show that in stage I,II oocytes, there is a high concentration of a zinc protein whose zinc coordination site likely is composed of (His)2(Cys)2, such as, e.g., TFIIIA. As the oocytes develop, the predominant zinc species becomes one that exhibits the (His)2(N/0)2 zinc site found in lipovitellin. Hence, the ligands to the zinc atoms in intact oocytes and the changes that take place as a function of oogenesis and after their fertilization, during embryogenesis, now can be examined and explored.

Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly.

The human immunodeficiency virus type 1 (HIV-1) matrix protein forms a structural shell associated with the inner viral membrane and performs other essential functions throughout the viral life cycle. The crystal structure of the HIV-1 matrix protein, determined at 2.3 angstrom resolution, reveals that individual matrix molecules are composed of five major helices capped by a three-stranded mixed beta-sheet. Unexpectedly, the protein assembles into a trimer in three different crystal lattices, burying 1880 angstrom2 of accessible surface area at the trimer interfaces. Trimerization appears to create a large, bipartite membrane binding surface in which exposed basic residues could cooperate with the N-terminal myristoyl groups to anchor the protein on the acidic inner membrane of the virus.

Age determinations for ODP Sites in the Norwegian-Greenland Sea

Records of biogenic and terrigenous components have been obtained from the interval corresponding to the last 2.6 m.y. of ODP Sites 643 and 644 in order to reconstruct surface and deep water regimes in the Norwegian Sea. Surface water regimes record long lasting moderate glacial conditions during the interval 2.6 1.0 Ma. Small intrusions of Atlantic water episodically penetrated into the Norwegian Sea forming a narrow tongue along the eastern margin, which is documented at Site 644. The polar front was most probably situated between the Site 644 and 643 locations on the outer Voring Plateau during these time intervals. Deep water regimes reflect long-term persistent corrosive bottom waters, most probably due to a weakly undersaturated water column and a low rate of carbonate shell production in surface waters. Deep water production in the Norwegian-Greenland Sea may have operated in a different way, e.g. brine formation during winter sea ice growth. Bottom waters were oxygenated throughout the entire period, and deep water was exchanged persistently with the North Atlantic. Increased glacial/interglacial enviromental contrasts are documented, reflecting a strengthening of the Norwegian Current and intensified glaciations on the surrounding land masses during the interval 1.0 0.6 Ma. During this time a major shift in the mode of deep water production occurred. Tile onset of large amplitudes in glacial/interglacial environmental conditions with maximum contrasts in surface water regimes, different modes of deep water production, and intensified exchange with the North Atlantic marks the last 0.6 Ma. A broad development of the Norwegian Current is observed during peak interglacials, while during glacials seasonally variable sea ice cover and iceberg drift dominate surface water conditions.

Mineral an chemical composition of basalts from DSDP Sites 417 and 418, West Atlantic Ocean

Major element composition ranges of closely associated basalt glass-whole rock pairs from individual small cooling units approach the total known range of basalt glass and whole rock compositions at IPOD sites 417 and 418. The whole rock samples fall into two groups: one is depleted in MgO and distinctly enriched in plagioclase but has lost some olivine and/or pyroxene relative to its corresponding glass; and the other is enriched in MgO and in phenocrysts of olivine and pyroxene as well as plagioclase compared to its corresponding glass. By analogy with observed phenocryst distributions in lava pillows, tubes, and dikes, and with some theoretical studies, we infer that bulk rock compositions are strongly affected by phenocryst redistribution due to gravity settling, flotation, and dynamic sorting after eruption, although specific models are not well constrained by the one-dimensional geometry of drill core. Compositional trends or groupings in whole rock data resulting from such late-stage processes should not be confused with more fundamental compositional effects produced in deep chambers or during partial melting.

Development and transplantation of a mineralized matrix formed by osteoblasts in vitro for bone regeneration

The use of extracellular matrix materials as scaffolds for the repair and regeneration of tissues is receiving increased attention. The current study was undertaken to test whether extracellular matrix formed by osteoblasts in vitro could be used as a scaffold for osteoblast transplantation and induce new bone formation in critical size osseous defects in vivo. Human osteoblasts derived from alveolar bone were cultured in six-well plates until confluent and then in mineralization media for a further period of 3 weeks to form an osteoblast-mineralized matrix complex. Histologically, at this time point a tissue structure with a connective tissue-like morphology was formed. Type I collagen was the major extracellular component present and appeared to determine the matrix macrostructure. Other bone-related proteins such as alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and -4, bone sialoprotein (BSP), osteopontin (OPN), and osteocalcin (OCN) also accumulated in the matrix. The osteoblasts embedded in this matrix expressed mRNAs for these bone-related proteins very strongly. Nodules of calcification were detected in the matrix and there was a correlation between calcification and the distribution of BSP and OPN. When this matrix was transplanted into a critical size bone defect in skulls of inummodeficient mice (SCID), new bone formation occurred. Furthermore, the cells inside the matrix survived and proliferated in the recipient sites, and were traceable by the human-specific Alu gene sequence using in situ hybridization. It was found that bone-forming cells differentiated from both transplanted human osteoblasts and activated endogenous mesenchymal cells. This study indicates that a mineralized matrix, formed by human osteoblasts in vitro, can be used as a scaffold for osteoblast transplantation, which subsequently can induce new bone formation.

In vitro heterogeneity of osteogenic cell populations at various equine skeletal sites

Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted. The numbers of nodules produced from osteogenic cells harvested from different skeletal sites were compared with the use of a mixed linear model. On average, cortical bone sites yielded significantly greater numbers of nodules than did cancellous bone sites. Between cortical bone sites, there was no significant difference in nodule numbers. Among cancellous sites, the radial cancellous bone yielded significantly more nodules than did the tibial cancellous bone. Among appendicular skeletal sites, tibial metaphyseal bone yielded significantly fewer nodules than did all other long bone sites. This study detected evidence of heterogeneity of equine osteogenic cell populations at various skeletal sites. Further characterization of the dissimilarities is warranted to determine the potential role heterogeneity plays in differential rates of fracture healing between skeletal sites.