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At least 145 records · Page 8

Collagen in the spicule organic matrix of the gorgonian Leptogorgia virgulata

Decalcification of the calcareous spicules from the gorgonian Leptogorgia virgulata reveals an organic matrix that may be divided into water insoluble and soluble fractions. The insoluble fraction displays characteristics typical of collagen, which is an unusual component of an invertebrate calcium carbonate structure. This matrix fraction exhibits a collagenous amino acid profile and behavior upon SDS-PAGE. Furthermore, the reducible crosslink, dihydroxylysinonorleucine (DHLNL), is detected in this fraction. The composition of the matrix varies seasonally; i.e., the collagenous composition is most prevalent in the summer. These results indicate that the insoluble matrix is a dynamic structure. Potential roles of this matrix in spicule calcification are discussed.

Non-NASA Center↗

Decorin modulates matrix mineralization in vitro

Decorin (DCN), a member of small leucine-rich proteoglycans, is known to modulate collagen fibrillogenesis. In order to investigate the potential roles of DCN in collagen matrix mineralization, several stable osteoblastic cell clones expressing higher (sense-DCN, S-DCN) and lower (antisense-DCN, As-DCN) levels of DCN were generated and the mineralized nodules formed by these clones were characterized. In comparison with control cells, the onset of mineralization by S-DCN clones was significantly delayed; whereas it was markedly accelerated and the number of mineralized nodules was significantly increased in As-DCN clones. The timing of mineralization was inversely correlated with the level of DCN synthesis. In these clones, the patterns of cell proliferation and differentiation appeared unaffected. These results suggest that DCN may act as an inhibitor of collagen matrix mineralization, thus modulating the timing of matrix mineralization.

NASA Discipline Musculoskeletal↗

Expression of spicule matrix protein gene SM30 in embryonic and adult mineralized tissues of sea urchin Hemicentrotus pulcherrimus

We have isolated a cDNA clone for spicule matrix protein, SM30, from sea urchin Hemicentrotus pulcherrimus and have studied the expression of this gene in comparison with that of another spicule matrix protein gene, SM50. In cultured micromeres as well as in intact embryos transcripts of SM30 were first detectable around the onset of spicule formation and rapidly increased with the growth of spicules, which accompanied accumulation of glycosylated SM30 protein(s). When micromeres were cultured in the presence of Zn2+, spicule formation and SM30 expression were suppressed, while both events resumed concurrently after the removal of Zn2+ from the culture medium. Expression of SM50, in contrast, started before the appearance of spicules and was not sensitive to Zn2+. Differences were also observed in adult tissues; SM30 mRNA was detected in spines and tube feet but not in the test, while SM50 mRNA was apparent in all of these mineralized tissues at similar levels. These results strongly suggest that the SM30 gene is regulated by a different mechanism to that of the SM50 gene and that the products of these two genes are differently involved in sea urchin biomineralization. A possible role of SM30 protein in skeleton formation is discussed.

NASA Discipline Musculoskeletal↗

Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions

We have described a powerful tool for the study of mechanical interactions between cells and their physical environment. Although the approach has already been used in a variety of ways to measure traction forces and to characterize active and passive responses of cultured cells to mechanical stimulation, it can be extended easily and combined with other microscopic approaches, including fluorescent analog imaging (Beningo et al., 2001), photobleaching, calcium imaging, micromanipulation, and electrophysiology. This method will be particularly useful for studying the functions of various components at focal adhesions, and the effects of mechanical forces on focal adhesion-mediated signal transduction. In addition, the method can be extended to a 3D setting, e.g., by sandwiching cultured cells between two layers of polyacrylamide to create an environment mimicking that in the tissue of a multicellular organism. Whereas chemical interactions between cells and the environment have been investigated extensively, many important questions remain as to the role of physical forces in cellular functions and the interplay between chemical and physical mechanisms of communication. The present approach, as well as other approaches capable of probing physical interactions, should fill in this important gap in the near future.

NASA Discipline Cell Biology↗

Compositional and toxicological evaluation of the diazotrophic cyanobacterium, Cyanothece sp. strain ATCC 51142

Compositional analyses of Cyanothece sp. strain ATCC 51142 showed high protein (50-60%) and low fat (0.4-1%) content, and the ability to synthesize vitamin B12. The amino acid profile indicated that Cyanothece sp. was a balanced protein source. Fatty acids of the 18:3n-3 type were also present. Mineral analyses indicated that the cellular biomass may be a good source of Fe, Zn and Na. Caloric content was 4.5 to 5.1 kcal g dry weight-1 and the carbon content was approximately 40% on a dry weight basis. Nitrogen content was 8 to 9% on a dry weight basis and total nucleic acids were 1.3% on a dry weight basis. Short-term feeding studies in rats followed by histopathology found no toxicity or dietary incompatibility problems. The level of uric acid and allantoin in urine and tissues was low, suggesting no excess of nucleic acids, as sometimes reported in the past for a cyanobacteria-containing diet. The current work discusses the potential implications of these results for human nutrition applications.

NASA Discipline Life Support Systems↗

How do tissues respond to damage at the cellular level? The role of cytokines in irradiated tissues

The capacity of ionizing radiation to affect tissue function, control tumor growth and elicit pathological sequelae has been attributed in great part to its effects on cellular DNA, which, as the transmitter of genetic information, can both register damage and perpetuate it. Nonetheless, multicellular organisms function as the result of the cooperation of many cell types. What then occurs when individual cells are damaged by ionizing radiation? Is tissue response a sum of cellular effects such as cell death and DNA damage? Or does the tissue respond as a coherent unit to the damage of its parts? In this paper, data in support of the latter model that indicate a role for cytokines, in particular transforming growth factor beta1, as critical components of extracellular signaling pathways that mediate tissue response to radiation will be reviewed. The key to manipulating the consequences of radiation exposure lies in understanding the complex interplay of events initiated at the cellular level, but acting on the tissue.

Review↗

Differences in elasticity of vinculin-deficient F9 cells measured by magnetometry and atomic force microscopy

We have investigated a mouse F9 embryonic carcinoma cell line, in which both vinculin genes were inactivated by homologous recombination, that exhibits defective adhesion and spreading [Coll et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9161-9165]. Using a magnetometer and RGD-coated magnetic microbeads, we measured the local effect of loss and replacement of vinculin on mechanical force transfer across integrins. Vinculin-deficient F9Vin(-/-) cells showed a 21% difference in relative stiffness compared to wild-type cells. This was restored to near wild-type levels after transfection and constitutive expression of increasing amounts of vinculin into F9Vin(-/-) cells. In contrast, the transfection of vinculin constructs deficient in amino acids 1-288 (containing the talin- and alpha-actinin-binding site) or substituting tyrosine for phenylalanine (phosphorylation site, amino acid 822) in F9Vin(-/-) cells resulted in partial restoration of stiffness. Using atomic force microscopy to map the relative elasticity of entire F9 cells by 128 x 128 (n = 16,384) force scans, we observed a correlation with magnetometer measurements. These findings suggest that vinculin may promote cell adhesions and spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, thereby affecting the elastic properties of the cell.

Non-NASA Center↗

Mice Drawer System

The Mice Drawer System (MDS) is an Italian Space Agency (ASI) facility which is able to support mice onboard the International Space Station during long-duration exploration missions (from 100 to 150-days) by living space, food, water, ventilation and lighting. Mice can be accommodated either individually (maximum 6) or in groups (4 pairs). MDS is integrated in the Space Shuttle middeck during transportation (uploading and downloading) to the ISS and in an EXPRESS Rack in Destiny, the US Laboratory during experiment execution. Osteoporosis is a debilitating disease that afflicts millions of people worldwide. One of the physiological changes experienced by astronauts during space flight is the accelerated loss of bone mass due to the lack of gravitational loading on the skeleton. This bone loss experienced by astronauts is similar to osteoporosis in the elderly population. MDS will help investigate the effects of unloading on transgenic (foreign gene that has been inserted into its genome to exhibit a particular trait) mice with the Osteoblast Stimulating Factor-1, OSF-1, a growth and differentiation factor, and to study the genetic mechanisms underlying the bone mass pathophysiology. MDS will test the hypothesis that mice with an increased bone density are likely to be more protected from osteoporosis, when the increased bone mass is a direct effect of a gene involved in skeletogenesis (skeleton formation). Osteoporosis is a debilitating disease that afflicts millions worldwide. One of the physiological changes experienced by astronauts during space flight is the accelerated loss of bone mass due to the lack of gravitational loading on the skeleton, a loss that is similar to osteoporosis in the elderly population on Earth. Osteoblast Stimulating Factor-1 (OSF-1), also known as pleiotrophin (PTN) or Heparin-Binding Growth- Associated Molecule (HB-GAM) belongs to a family of secreted heparin binding proteins..OSF-1 is an extracellular matrix-associated growth and differentiation factor that is normally expressed in cartilage; it can stimulate the proliferation and differentiation of human osteoprogenitor cells (cell that differentiate into an osteoblast) in vitro. The Mice Drawer System will study the effects of microgravity on transgenic mouse bones in order to identify genetic mechanisms playing a role in the reduction of the bone mass observed in humans and animals as a consequence of long-duration (greater than 100 days) microgravity exposure. Onboard the ISS, MDS is relatively self-sufficient; a crewmember will check the health status of the rodents on a daily basis, by assessing them through the viewing window. Water levels will be assessed by the crew daily and refilled as needed. Replacement of the food bars and replacement of the waste filters will be conducted inflight by crewmembers every 20-days.

Cancedda, Ranieri↗

Biofilm Mitigation Testing Phase III

Biofilm is produced from bacterial or fungal organisms that can survive in extreme conditions. The microorganisms attach themselves to surfaces and produce an extracellular polymeric matrix that develops into a community structure and ecosystem. Biofilm has been known to cause blockage and clogging inside the Water Processor Assembly (WPA) and Water Recovery System (WRS) piping. The WPA and WRS on the International Space Station (ISS) is responsible for recycling humidity condensate and urine distillate to produce potable water for the astronauts to survive. The Environmental Control and Life Support System (ECLSS) department is developing a way to mitigate biofilm accumulation inside the wastewater tank.

Biofilm↗

A contribution with review to the description of mineralization of bone and other calcified tissues in vivo

This manuscript considers certain aspects of mineral deposition in bone and other vertebrate calcifying tissues in order to examine physical, chemical, and biological factors important in the mineralization process. The paper in a discussion format principally presents a new data and the formulation of concepts based on such data as well as a summary of background material as necessary review. Mineralization is found to occur at spatially independent sites throughout the organic extracellular tissue matrices. Matrix vesicles and collagen fibrils each may serve as independent nucleation centers for mineral with vesicle mineralization being local and collagen mineralization dominating the tissues as a whole. Collagen fibril organization is suggested to be such that hole zones are aligned in three dimensions, creating extensive channels for mineral accommodation. Nucleation occurs initially in hole zones and crystal growth leads to the development of plate-like mineral particles whose orientation, disposition, and sizes within fibrils are detailed. Effects of diffusion, crystallinity, and critical nucleation and growth events are described with respect to their influence on mineral deposition in bulk and local regions of tissue matrices.

Review↗

Determination of the Three-Dimensional Morphology of ALH84001 and Biogenic MV-1 Magnetite: Comparison of Results from Electron Tomography and Classical Transmission Electron Microscopy

Dated at approximately 3.9 billion years of age, carbonate disks, found within fractures of the host rock of Martian meteorite ALH84001, have been interpreted as secondary minerals that formed at low temperature in an aqueous medium. Heterogeneously distributed within these disks are magnetite nanocrystals that are of Martian origin. Approximately one quarter of these magnetites have morphological and chemical similarities to magnetite particles produced by magnetotactic bacteria strain MV-1, which are ubiquitous in aquatic habitats on Earth. Moreover, these types of magnetite particles are not known or expected to be produced by abiotic means either through geological processes or synthetically in the laboratory. The remaining three quarters of the ALH84001 magnetites are likely products of multiple processes including, but not limited to, precipitation from a hydrothermal fluid, thermal decomposition of the carbonate matrix in which they are embedded, and extracellular formation by dissimilatory Fe-reducing bacteria. We have proposed that the origins of magnetites in ALH84001 can be best explained as the products of multiple processes, one of which is biological. Recently the three-dimensional (3-D) external morphology of the purported biogenic fraction of the ALH84001 magnetites has been the subject of considerable debate. We report here the 3-D geometry of biogenic magnetite crystals extracted from MV-1 and of those extracted from ALH84001 carbonate disks using a combination of high resolution classical and tomographic transmission electron microscopy (TEM). We focus on answering the following questions: (1) which technique provides adequate information to deduce the 3-D external crystal morphology?; and, (2) what is the precise 3-D geometry of the ALH84001 and MV-1 magnetites?

Thomas-Keprta, Kathie L.↗

Microscopic physical biomarkers in carbonate hot springs: implications in the search for life on Mars

Physical evidence of life (physical biomarkers) from the deposits of carbonate hot springs were documented at the scale of microorganisms--submillimeter to submicrometer. The four moderate-temperature (57 to 72 degrees C), neutral pH springs reported on in this study, support diverse communities of bacteria adapted to specific physical and chemical conditions. Some of the microbes coexist with travertine deposits in endolithic communities. In other cases, the microbes are rapidly coated and destroyed by precipitates but leave distinctive mineral fabrics. Some microbes adapted to carbonate hot springs produce an extracellular polymeric substance which forms a three-dimensional matrix with living cells and cell remains, known as a biofilm. Silicon and iron oxides often coat the biofilm, leading to long-term preservation. Submicrometer mineralized spheres composed of calcium fluoride or silica are common in carbonate hot spring deposits. Sphere formation is biologically mediated, but the spheres themselves are apparently not fossils or microbes. Additionally, some microbes selectively weather mineral surfaces in distinctive patterns. Hot spring deposits have been cited as prime locations for exobiological exploration of Mars. The presence of preserved microscopic physical biomarkers at all four sites supports a strategy of searching for evidence of life in hot spring deposits on Mars.

NASA Center JSC↗

Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

Non-NASA Center↗

The regulation and regulatory role of collagenase in bone

Interstitial collagenase plays an important role in both the normal and pathological remodeling of collagenous extracellular matrices, including skeletal tissues. The enzyme is a member of the family of matrix metalloproteinases. Only one rodent interstitial collagenase has been found but there are two human enzymes, human collagenase-1 and -3, the latter being the homologue of the rat enzyme. In developing rat and mouse bone, collagenase is expressed by hypertrophic chondrocytes, osteoblasts, and osteocytes, a situation that is replicated in a fracture callus. Cultured osteoblasts derived from neonatal rat calvariae show greater amounts of collagenase transcripts late in differentiation. These levels can be regulated by parathyroid hormone (PTH), retinoic acid, and insulin-like growth factors, as well as the degree of matrix mineralization. Much of the work on collagenase in bone has been derived from studies on the rat osteosarcoma cell line, UMR 106-01. All bone-resorbing agents stimulate these cells to produce collagenase mRNA and protein, with PTH being the most potent stimulator. Determination of secreted levels of collagenase has been difficult because UMR cells, normal rat osteoblasts, and rat fibroblasts possess a scavenger receptor that removes the enzyme from the extracellular space, internalizes and degrades it, thus imposing another level of control. PTH can also regulate the abundance of the receptor as well as the expression and synthesis of the enzyme. Regulation of the collagenase gene by PTH appears to involve the cAMP pathway as well as a primary response gene, possibly Fos, which then contributes to induction of the collagenase gene. The rat collagenase gene contains an activator protein-1 sequence that is necessary for basal expression, but other promoter regions may also participate in PTH regulation. Thus, there are many levels of regulation of collagenase in bone perhaps constraining what would otherwise be a rampant enzyme.

NASA Discipline Musculoskeletal↗

Mechanotransduction and the functional response of bone to mechanical strain

Mechanotransduction plays a crucial role in the physiology of many tissues including bone. Mechanical loading can inhibit bone resorption and increase bone formation in vivo. In bone, the process of mechanotransduction can be divided into four distinct steps: (1) mechanocoupling, (2) biochemical coupling, (3) transmission of signal, and (4) effector cell response. In mechanocoupling, mechanical loads in vivo cause deformations in bone that stretch bone cells within and lining the bone matrix and create fluid movement within the canaliculae of bone. Dynamic loading, which is associated with extracellular fluid flow and the creation of streaming potentials within bone, is most effective for stimulating new bone formation in vivo. Bone cells in vitro are stimulated to produce second messengers when exposed to fluid flow or mechanical stretch. In biochemical coupling, the possible mechanisms for the coupling of cell-level mechanical signals into intracellular biochemical signals include force transduction through the integrin-cytoskeleton-nuclear matrix structure, stretch-activated cation channels within the cell membrane, G protein-dependent pathways, and linkage between the cytoskeleton and the phospholipase C or phospholipase A pathways. The tight interaction of each of these pathways would suggest that the entire cell is a mechanosensor and there are many different pathways available for the transduction of a mechanical signal. In the transmission of signal, osteoblasts, osteocytes, and bone lining cells may act as sensors of mechanical signals and may communicate the signal through cell processes connected by gap junctions. These cells also produce paracrine factors that may signal osteoprogenitors to differentiate into osteoblasts and attach to the bone surface. Insulin-like growth factors and prostaglandins are possible candidates for intermediaries in signal transduction. In the effector cell response, the effects of mechanical loading are dependent upon the magnitude, duration, and rate of the applied load. Longer duration, lower amplitude loading has the same effect on bone formation as loads with short duration and high amplitude. Loading must be cyclic to stimulate new bone formation. Aging greatly reduces the osteogenic effects of mechanical loading in vivo. Also, some hormones may interact with local mechanical signals to change the sensitivity of the sensor or effector cells to mechanical load.

Non-NASA Center↗

Hypergravity Stimulates the Extracellular Matrix/Integrin-Signaling Axis and Proliferation in Primary Osteoblasts

We set out to determine the molecular mechanisms involved in the proliferative response of primary rat osteoblasts to mechanical stimulation using cell culture centrifugation as a model for hypergravity. We hypothesized that this proliferative response is mediated by specific integrin/Extracellular Matrix (ECM) interactions. To investigate this question we developed a cell culture centrifuge and an automated system that performs cell fixation during hypergravity loading. We generated expression vectors for various focal adhesion and cytoskeletal proteins fused to GFP or dsRed and visualized these structures in transfected (or infected) osteoblasts. The actin cytoskeleton was also visualized using rhodamine-phalloidin staining and Focal Adhesion Kinase (FAK) levels were assessed biochemically. We observed that a 24 hour exposure to 50-g stimulated proliferation compared to the 1-g control when cells were plated on fibronectin, collagen Type I , and collagen Type IV, but not on uncoated tissue culture plastic surfaces. This proliferative response was greatest for osteoblasts grown on fibronectin (2-fold increase over 1-g control) and collagen Type I (1.4 fold increase over 1-g control), suggesting that specific matrices and integrins are involved in the signaling pathways required for proliferation. Exposing osteoblasts grown on different matrices to 10-g or 25-g showed that effects on proliferation depended on both matrix type and loading level. We found that osteoblasts exposed to a short pulse of hypergravity during adhesion spread further and had more GFP-FAK containing focal adhesions compared to their 1-g controls. While overall levels of FAK did not change, more FAK was in the active (phosphorylated) form under hypergravity than in the 1-g controls. Cytoskeletal F-actin organization into filaments was also more prominent after brief exposures to hypergravity during the first five minutes of adhesion. These results suggest that specific integrins sense hypergravity and activate distinct matrix-dependent FAK signaling pathways that can enhance proliferation. Our results also imply that brief exposures to hypergravity accelerate cell adhesion and spreading processes via the focal adhesion-signaling axis. These results support the role of the ECM/integrin-signaling axis in osteoblast response to hypergravity loading.

Parra, M.↗

Know thy neighbor: stromal cells can contribute oncogenic signals

Although the stroma within carcinogenic lesions is known to be supportive and responsive to tumors, new data increasingly show that the stroma also has a more active, oncogenic role in tumorigenesis. Stromal cells and their products can transform adjacent tissues in the absence of pre-existing tumor cells by inciting phenotypic and genomic changes in the epithelial cells. The oncogenic action of distinctive stromal components has been demonstrated through a variety of approaches, which provide clues about the cellular pathways involved.

Review↗