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At least 145 records · Page 8

Permeation of membranes by the neutral form of amino acids and peptides: relevance to the origin of peptide translocation

The flux of amino acids and other nutrient solutes such as phosphate across lipid bilayers (liposomes) is 10(5) slower than facilitated inward transport across biological membranes. This suggest that primitive cells lacking highly evolved transport systems would have difficulty transporting sufficient nutrients for cell growth to occur. There are two possible ways by which early life may have overcome this difficulty: (1) The membranes of the earliest cellular life-forms may have been intrinsically more permeable to solutes; or (2) some transport mechanism may have been available to facilitate transbilayer movement of solutes essential for cell survival and growth prior to the evolution of membrane transport proteins. Translocation of neutral species represents one such mechanism. The neutral forms of amino acids modified by methylation (creating protonated weak bases) permeate membranes up to 10(10) times faster than charged forms. This increased permeability when coupled to a transmembrane pH gradient can result in significantly increased rates of net unidirectional transport. Such pH gradients can be generated in vesicles used to model protocells that preceded and were presumably ancestral to early forms of life. This transport mechanism may still play a role in some protein translocation processes (e.g. for certain signal sequences, toxins and thylakoid proteins) in vivo.

NASA Discipline Exobiology↗

Solar breeder: Energy payback time for silicon photovoltaic systems

The energy expenditures of the prevailing manufacturing technology of terrestrial photovoltaic cells and panels were evaluated, including silicon reduction, silicon refinement, crystal growth, cell processing and panel building. Energy expenditures include direct energy, indirect energy, and energy in the form of equipment and overhead expenses. Payback times were development using a conventional solar cell as a test vehicle which allows for the comparison of its energy generating capability with the energies expended during the production process. It was found that the energy payback time for a typical solar panel produced by the prevailing technology is 6.4 years. Furthermore, this value drops to 3.8 years under more favorable conditions. Moreover, since the major energy use reductions in terrestrial manufacturing have occurred in cell processing, this payback time directly illustrates the areas where major future energy reductions can be made -- silicon refinement, crystal growth, and panel building.

Lindmayer, J.↗

Effects of microgravity on osteoblast growth

Studies from space flights over the past two decades have demonstrated that basic physiological changes occur in humans during space flight. These changes include cephalic fluid shifts, loss of fluid and electrolytes, loss of muscle mass, space motion sickness, anemia, reduced immune response, and loss of calcium and mineralized bone. The cause of most of these manifestations is not known and until recently, the general approach was to investigate general systemic changes, not basic cellular responses to microgravity. Recently analyzed data from the 1973-1974 Skylabs disclose that there is a rise in the systemic hormone, cortisol, which may play a role in bone loss in flight. In two flights where bone growth was measured (Skylabs 3 and 4), the crew members had a significant loss of calcium accompanied by a rise in 24 hour urinary cortisol during the entire flight period. In ground-based work on osteoblasts, we have demonstrated that equivalent amounts of glucocorticoids can inhibit osteoblast cell growth. In addition, this laboratory has recently studied gene growth and activation of mouse osteoblasts (MC3T3-E1) during spaceflight. Osteoblast cells were grown on glass coverslips, loaded in the Biorack plunger boxes 18 hours before launch and activated 19 hours after launch in the Biorack incubator under microgravity conditions. The osteoblasts were launched in a serum deprived state, activated and collected in microgravity. Samples were collected at 29 hours after sera activation (0-g, n=4; 1-g, n=4). The osteoblasts were examined for changes in gene expression and cell morphology. Approximately one day after growth activation, remarkable differences were observed in gene expression in 0-g and 1-g flight samples. The 0-g activated cells had increased c-fos mRNA when compared to flight 1-g controls. The message of immediate early growth gene, cox-2 was decreased in the microgravity activated cells when compared to ground or 1-g flight controls. Cox-1 was not detected in any of the samples. There were no significant differences in the expression of actin mRNA between the 0-g and 1-g samples. These data indicate that quiescent osteoblasts are slower to enter the cell cycle in microgravity, suggesting that the force of gravity itself may be a significant factor in bone loss in spaceflight. Preliminary data from our STS 76 flight experiment support our hypothesis that a basic biological response occurs at the tissue, cellular, and molecular level in 0-g. Here we examine ground-based and space flown data on osteoblast growth in ground-based experiments mimicking space flight conditions and in microgravity to simulate lack of gravity stress to help us understand the mechanism of bone loss by experiments.

NASA Experiment Number 9403056↗

Extracellular signaling through the microenvironment: a hypothesis relating carcinogenesis, bystander effects, and genomic instability

Cell growth, differentiation and death are directed in large part by extracellular signaling through the interactions of cells with other cells and with the extracellular matrix; these interactions are in turn modulated by cytokines and growth factors, i.e. the microenvironment. Here we discuss the idea that extracellular signaling integrates multicellular damage responses that are important deterrents to the development of cancer through mechanisms that eliminate abnormal cells and inhibit neoplastic behavior. As an example, we discuss the action of transforming growth factor beta (TGFB1) as an extracellular sensor of damage. We propose that radiation-induced bystander effects and genomic instability are, respectively, positive and negative manifestations of this homeostatic process. Bystander effects exhibited predominantly after a low-dose or a nonhomogeneous radiation exposure are extracellular signaling pathways that modulate cellular repair and death programs. Persistent disruption of extracellular signaling after exposure to relatively high doses of ionizing radiation may lead to the accumulation of aberrant cells that are genomically unstable. Understanding radiation effects in terms of coordinated multicellular responses that affect decisions regarding the fate of a cell may necessitate re-evaluation of radiation dose and risk concepts and provide avenues for intervention.

NASA Discipline Radiation Health↗

Targeted inhibition of MASTL kinase activity induces apoptosis in breast cancer

Microtubule-associated serine/threonine kinase-like (MASTL) (or Greatwall kinase (GWL)) is an important cell cycle regulating kinase that regulates the G2-M transition. Uncontrolled MASTL activity is implicated in breast cancer progression. To date, very few inhibitors have been reported against this protein. Here, structure-based computational modeling indicates that the natural product flavopiridol (FLV) binds strongly to MASTL and these results are validated using molecular dynamics simulation studies. Further, an in vitro kinase assay reveals an EC 50 (effective concentration) value of FLV to be 82.1 nM and a better IC 50 compared to the positive reference compound, staurosporine. FLV is found to inhibit MASTL kinase activity, arresting the cell growth in the G1 phase and inducing apoptosis in breast cancer cells. Consistent with these results differential gene expression obtained using RNA sequencing studies, and validated by RT PCR and immunoblot analysis, indicate that MASTL inhibition induces cell cycle arrest and apoptotic-related genes. Furthermore, metastasis- and inflammation- related genes are downregulated. Thus, the deregulation of MASTL signaling pathways on targeted inhibition of its kinase activity is revealed. This study lays a strong foundation for investigating FLV as a lead compound in breast cancer therapeutics.

60 APPLIED LIFE SCIENCES↗

The evolution of convective storms from their footprints on the sea as viewed by synthetic aperture radar from space

SEASAT synthetic aperture radar (SAR) echoes from the sea have previously been shown to be the result of rain and winds produced by convective stroms; rain damps the surface waves and causes ech-free holes, while the diverging winds associated with downdraft generate waves and associated echoes surrounding the holes. Gust fronts are also evident. Such a snapshot from 8 July 1978 has been examined in conjunction with ground-based radar. This leads to the conclusion that the SAR storm footprints resulted from storm processes that occurred up to an hour or more prior to the snapshot. A sequence of events is discerned from the SAR imagery in which new cell growth is triggered in between the converging outflows of two preexisting cells. In turn, the new cell generates a mini-squall line along its expanding gust front. While such phenomena are well known over land, the spaceborne SAR now allows important inferences to be made about the nature and frequency of convective storms over the oceans. The storm effects on the sea have significant implications for spaceborne wind scatterometry and rainfall measurements. Some of the findings herein remain speculative because of the great distance to the Miami weather radar-the only source of corroborative data.

Atlas, David↗

Convective flow effects on protein crystal growth

A high-resolution microscopic interferometric setup for the monitoring of protein morphologies has been developed. Growth or dissolution of a crystal can be resolved with a long-term depth resolution of 200 A and a lateral resolution of 2 microns. This capability of simultaneously monitoring the interfacial displacement with high local depth resolution has yielded several novel results. We have found with lysozyme that (1) the normal growth rate is oscillatory, and (2) depending on the impurity content of the solution, the growth step density is either greater or lower at the periphery of a facet than in its center. The repartitioning of Na plus and Cl minus ions between lysozyme solutions and crystals was studied for a wide range of crystallization conditions. A nucleation-growth-repartitioning model was developed, to interpret the large body of data in unified way. The results strongly suggest that (1) the ion to lysozyne ratio in the crystal depends mostly on kinetic rather than crystallographic parameters, and (2) lysozyme crystals possess a salt-rich core with a diameter electron microscopy results appear to confirm this finding, which could have far-reaching consequences for x-ray diffraction studies. A computational model for diffusive-convective transport in protein crystallization has been applied to a realistic growth cell geometry, taking into account the findings of the above repartitioning studies and our kinetics data for the growth of lysozyme. The results show that even in the small cell employed, protein concentration nonuniformities and gravity-driven solutal convection can be significant. The calculated convection velocities are of the same order to magnitude as those found in earlier experiments. As expected, convective transport, i.e., at Og, lysozyme crystal growth remains kinetically limited. The salt distribution in the crystal is predicted to be non-uniform at both 1g and 0g, as a consequence of protein depletion in the solution. Static and dynamic light scattering studies in undersaturated and supersaturated solutions have been performed. Diffusivities in undersaturated solutions, were found to vary with lysozyme concentrations. Depending on the salt concentration, the diffusivities either increase or decrease. Interestingly, the corresponding static scattering intensities behave oppositely, Our current analysis indicates that these changes are inconsistent with aggregation in undersaturated solutions. However, the data are compatible with concentration-dependent changes of the interactions between protein and salt.

Rosenberger, Franz↗

Determining protein polarization proteome-wide using physical dissection of individual Stentor coeruleus cells

Cellular components are non-randomly arranged with respect to the shape and polarity of the whole cell. Patterning within cells can extend down to the level of individual proteins and mRNA. But how much of the proteome is actually localized with respect to cell polarity axes? Proteomics combined with cellular fractionation has shown that most proteins localize to one or more organelles but does not tell us how many proteins have a polarized localization with respect to the large-scale polarity axes of the intact cell. Genome-wide localization studies in yeast found that only a few percent of proteins have a localized position relative to the cell polarity axis defined by sites of polarized cell growth. Here, we describe an approach for analyzing protein distribution within a cell with a visibly obvious global patterning - the giant ciliate Stentor coeruleus. Ciliates, including Stentor, have highly polarized cell shapes with visible surface patterning. A Stentor cell is roughly 2mmlong, allowing a ‘‘proteomic dissection’’ in which microsurgery is used to separate cellular fragments along the anterior-posterior axis, followed by comparative proteomic analysis. In our analysis, 25% of the proteome, including signaling proteins, centrin/SFI proteins, and GAS2 orthologs, shows a polarized location along the cell’s anterior-posterior axis. We conclude that a large proportion of all proteins are polarized with respect to global cell polarity axes and that proteomic dissection provides a simple and effective approach for spatial proteomics.

59 BASIC BIOLOGICAL SCIENCES↗

Biohydrogen Generation from Un-Pretreated Spent Coffee Grounds with Clostridium Thermocellum

Coffee is among the world's most widely consumed beverages, generating 18 million wet tonnes of waste spent coffee grounds annually. Disposal of SCGs is complicated by their high moisture content, their recalcitrant chemical composition, and the presence of caffeine and other bioactive compounds. Dark fermentation is a promising technology for conversion of waste cellulosic biomass into renewable hydrogen, carbon dioxide, and volatile fatty acids. Typical SCGs have high moisture content, are pre-milled to fine particle sizes, and contain 8%-19% cellulose and up to 40% hemicellulose, making them an attractive feedstock for dark fermentation. In this study, we investigate biohydrogen production from unpretreated SCGs using Clostridium thermocellum strain KJC19-9, a cellulolytic bacterium engineered to co-utilize xylose, specifically examining the inhibitory effects of residual caffeine and high solids loadings on microbial growth and hydrogen production efficiency. While SCGs were initially resistant to both cell growth and biohydrogen production, a strategy to co-ferment with low concentrations of cellobiose dramatically enhanced process performance, reducing lag phase duration and enabling cellulosome production for efficient cellulose hydrolysis. This co-substrate approach generated up to 944 mL hydrogen per L reaction from 50 g/L of spent coffee grounds over 120 h (1.34 mol H2/mol carbohydrate), validating SCGs as a promising dark fermentation feedstock and underscoring the value of mixed substrate approaches in consolidated bioprocessing.

08 HYDROGEN↗

Some results from studies on the effects of weightlessness on the growth of epiphytic orchids

Epidendrum orchids were placed in a Malakhit-2 micro-greenhouse aboard the Soyuz-36-Salyut-6 space station to test their growth under weightless conditions. Growth occurred but was less than in control plants left on Earth; cells were smaller and parenchymal development slowed in all tissues. Stems, roots, and leaves were smaller. The number of stomas on the leaves was about the same as in the controls, but, because of the smaller leaf size, there were more per unit area. A modeling experiment using a clinostat revealed a large decrease in gibberellin activity and auxin activity. It was assumed that weightlessness primarily affects gibberellin biosynthesis, inhibiting cell growth. Reestablishment of growth compound activity upon return of the plants to Earth was indicated by the fact that the orchids resumed growth thereafter.

Cherevchenko, T. M.↗

Traction in smooth muscle cells varies with cell spreading

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

Non-NASA Center↗

Protein Kinases Possibly Mediate Hypergravity-Induced Changes in F-Actin Expression by Endothelial Cells

Basic cellular functions such as electrolyte concentration, cell growth rate, glucose utilization, bone formation, response to growth stimulation, and exocytosis are modified in microgravity. These studies indicate that microgravity affects a number of physiological systems and included in this are cell signaling mechanisms. Rijken and coworkers performed growth factor studies that showed PKC signaling and actin microfilament organization appears to be sensitive to microgravity, suggesting that the inhibition of signal transduction by microgravity may be related to alterations in actin microfilament organization. However, similar studies have not been done for vascular cells. Vascular endothelial cells play critical roles in providing nutrients to organ and tissues and in wound repair. The major deterrent to ground-based microgravity studies is that it is impossible to achieved true microgravity for longer than a few minutes on earth. Hence, it has not been possible to conduct prolonged microgravity studies except for two models that simulate certain aspects of microgravity. However, hypergravity is quite easily achieved. Several researchers have shown that hypergravity will increase the proliferation of several different cell lines while decreasing cell motility and slowing liver regeneration following partial hepatectomy, These studies indicate the hypergravity also alters the behavior of most cells. Several investigators have shown that hypergravity affects the activation of several protein kinases (PKs) in cells. In this study, we investigated whether hypergravity alters the expression of f-actin by bovine aortic endothelial cells (BAECs) and the role of PK's (calmodulin 11 dependent, PKA and PKC) as mediators of these effects.

Love, Felisha D.↗

Cellular mechanisms underlying growth asymmetry during stem gravitropism

Plant stems respond to gravitropic stimulation with a rapid, local and reversible change in cell growth rate (elongation), generally on both the upper and lower sides of the stem. The cellular and biochemical mechanisms for this differential growth are reviewed. Considerable evidence implicates an asymmetry in wall pH in the growth response. The strengths and weaknesses of the wall "loosening enzyme" concept are reviewed and the possibility of expansin involvement in the bending response of stems is considered. Also discussed is the possibility that wall stiffening processes, e.g. phenolic coupling driven by oxidative bursts or altered orientation of newly deposited cellulose, might mediate the growth responses during gravitropism.

NASA Discipline Plant Biology↗

Unveiling the Dynamics of KRAS4b on Lipid Model Membranes

Abstract Small GTPase proteins are ubiquitous and responsible for regulating several processes related to cell growth and differentiation. Mutations that stabilize their active state can lead to uncontrolled cell proliferation and cancer. Although these proteins are well characterized at the cellular scale, the molecular mechanisms governing their functions are still poorly understood. In addition, there is limited information about the regulatory function of the cell membrane which supports their activity. Thus, we have studied the dynamics and conformations of the farnesylated KRAS4b in various membrane model systems, ranging from binary fluid mixtures to heterogeneous raft mimics. Our approach combines long time-scale coarse-grained (CG) simulations and Markov state models to dissect the membrane-supported dynamics of KRAS4b. Our simulations reveal that protein dynamics is mainly modulated by the presence of anionic lipids and to some extent by the nucleotide state (activation) of the protein. In addition, our results suggest that both the farnesyl and the polybasic hypervariable region (HVR) are responsible for its preferential partitioning within the liquid-disordered (Ld) domains in membranes, potentially enhancing the formation of membrane-driven signaling platforms. Graphic Abstract

59 BASIC BIOLOGICAL SCIENCES↗

Cell-free styrene biosynthesis at high titers

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 ± 0.63 mM to 40.33 ± 1.03 mM, the highest amount achieved using biosynthesis without process modifications and product removal strategies. Finally, cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules.

59 BASIC BIOLOGICAL SCIENCES↗

Materials processing in a centrifuge - Numerical modeling of macrogravity effects

The fluid mechanics associated with crystal growth processes on a centrifuge is investigated. A simple scaling analysis is used to examine the relative magnitudes of the forces acting on the system and good agreement is obtained with previous studies. A two-dimensional model of crystal growth on a centrifuge is proposed and calculations are undertaken to help in understanding the fundamental transport processes within the crystal growth cell. Results from three-dimensional calculations of actual centrifuge-based crystal growth systems are presented both for the thermodynamically stable and unstable configurations. The calculations show the existence of flow bifurcations in certain configurations but not in all instances. The numerical simulations also show that the centrifugal force is the dominant stabilizing force on fluid convection in the stable configuration. The stabilizing influence of the Coriolis force is found to be only secondary in nature. No significant impact of gravity gradient is found in the calculations. Simulations of unstable configurations show that the Coriolis force has a stabilizing influence on fluid motion by delaying the onset of unsteady convection. Detailed flow and thermal field characteristics are presented for all the different cases that are simulated.

Ramachandran, N.↗

Medical Research System

Based on Johnson Space Flight Center's development of a rotating bioreactor cell culture apparatus for Space Shuttle medical research, Johnson Space Flight Center engineers who worked on the original project formed a company called Synthecon, with the intention of commercializing the bioreactor technology. Synthecon grows three dimensional tissues in the bioreactor. These are superior to previous two-dimensional tissue samples in the study of human cell growth. A refined version of the Johnson Space Center technology, Synthecon's Rotary Cell Culture System includes a cell culture chamber that rotates around a horizontal axis. The cells establish an orbit that approximates free fall through the liquid medium in the chamber. The technology has significant applications for cancer research and treatment as well as AIDS research.

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