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At least 19 records

Detergent-Specific Membrane Protein Crystallization Screens

A suite of reagents has been developed for three-dimensional crystallization of integral membranes present in solution as protein-detergent complexes (PDCs). The compositions of these reagents have been determined in part by proximity to the phase boundaries (lower consolute boundaries) of the detergents present in the PDCs. The acquisition of some of the requisite phase-boundary data and the preliminary design of several of the detergent- specific screens was supported by a NASA contract. At the time of expiration of the contract, a partial set of preliminary screens had been developed. This work has since been extended under non-NASA sponsorship, leading to near completion of a set of 20 to 30 different and unique detergent- specific 96-condition screens.

Wiener, Michael↗

Detergent solubilization of the EGF receptor from A431 cells

Functional reconstitution of purified preparations of human epidermal growth factor receptor (EGFR) requires dissociation of the protein from its plasma membrane lipid environment. Solubilization of membrane proteins in this manner requires the use of detergents, which are known to disrupt plasma membrane lipid/protein interactions. We have investigated the ability of three nonionic detergents to solubilize the human EGFR selectively, and have also analyzed the effect of these various treatments on the intrinsic tyrosyl kinase activity of the receptor. The nonionic detergent known as n-octyl glucoside (n-octyl beta-D-glucopyranoside) was found to give the best combination of selectivity, yield, and maintenance of enzymatic activity of the human EGFR.

Non-NASA Center↗

Effect of some detergents, humate, and composition of seedbed on crop of tomato plants in a hydroponic culture

It is established that single detergent doses distinctly stimulate vegetative development of plants in the initial stage when humates are available. When detergents are applied every four weeks in a hydroponic culture, in which the seedbed does not contain active humates, the crop is reduced by 50%. This adverse effect does not occur when the seedbed is a mixture of brown coal and peat.

Guminka, A. Z.↗

Cleanser, Detergent, Personal Care Product Pretreatment Evaluation

The purpose of the Cleanser, Detergent, Personal Care Product, and Pretreatment Evaluation & Selection task is to identify the optimal combination of personal hygiene products, crew activities, and pretreatment strategies to provide the crew with sustainable life support practices and a comfortable habitat. Minimal energy, mass, and crew time inputs are desired to recycle wastewater during long duration missions. This document will provide a brief background on the work this past year supporting the ELS Distillation Comparison Test, issues regarding use of the hygiene products originally chosen for the test, methods and results used to select alternative products, and lessons learned from testing.

Adam, Niklas↗

Cleanser, Detergent, Personal Care Product, and Pretreatment Evaluation

The purpose of the Cleanser, Detergent, Personal Care Product, and Pretreatment Evaluation & Selection task is to identify the optimal combination of personal hygiene products, crew activities, and pretreatment strategies to provide the crew with sustainable life support practices and a comfortable habitat. Minimal energy, mass, and crew time inputs are desired to recycle wastewater during long duration missions. This document will provide a brief background on the work this past year supporting the ELS Distillation Comparison Test, issues regarding use of the hygiene products originally chosen for the test, methods and results used to select alternative products, and lessons learned from testing.

Adam, Niklas↗

Self-contained, single-use hose and tubing cleaning module

A self contained, single use hose and tubing cleaning module which utilizes available water supplies without requiring access to precision cleaning facilities is presented. The module is attached to the water source at the inlet side and to the hose or tubing to be cleaned at the outlet side. The water flows through a water purification zone, a detergent dispensing zone, a filtration zone before the detergent laden water flows into the tubing to clean the tubing walls. The module contains an embedded pad which is impregnated with a pH indicator to indicate to the user when the detergent has dissolved and rinsing of the tubing begins.

Rollins, Fred P.↗

Formation and vesiculation of biomembranes during spaceflight

Shuttle flight, sounding rocket flight, and parabolic flight experiments demonstrate the formation of bilayer membrane vesicles (liposomes) in reduced gravity, following the dilution of detergent from detergent-phospholipid mixed micelles. The reduction in detergent concentration initiates assembly of bilayer membrane sheets, which are sensitive to solution disturbances. An increase in disturbances by forced dilution results in small diameter liposomes (less than 150 nm), in both ground and flight samples. In the absence of forced dilution, liposomes remain small at 1-g, but exhibit much larger diameters at 0-g (1000-2000 nm). Our spaceflight data reveal that membrane assembly and vesiculation are strongly influenced by gravity-induced solution disturbances (e.g., convection currents), which limit vesicle diameter.

Claassen, Dale E.↗

Advanced Exploration Systems (AES) Logistics Reduction Project: Crew Clothing Care Process Development

This report covers the use of ozone and mist with detergent to develop zero-gravity laundering technology. Different concentrations of detergent with and without ozone were applied to remove odor from t-shirts used during exercise. The fabrics of these shirts were categorized into two groups: hydrophobic (polyester and modacrylic) and hydrophilic (cotton and Merino wool). It was found that applying detergent solution as a mist sufficient to achieve saturation can be effective in eliminating odors from athletically soiled shirts either with or without exposure to ozone.

Demmler, Morgan↗

Reverse osmosis for wash water recovery in space vehicles.

Tests were carried out on both synthetic and real wash water derived from clothes laundry to determine the utility of reverse osmosis in recovering the water for recycle use. A blend membrane made from cellulose di- and triacetates, and a cross-linked cellulose acetate/methacrylate were evaluated. Both were found acceptable. A number of detergents were evaluated, including a cationic detergent, sodium dodecyl sulfate, potassium palmitate, and sodium dodecylbenzenesulfonate. The tests were all made at a temperature of 165 F to minimize microbial growth. Long-term (15 to 30 day) runs were made at 600 and 400 psi on laundry water which was pretreated either by alum addition and sand filtration or by filtration only through 0.5 micron filters. A 30-day run was made using a 2-in. diameter by 22-in. long spiral module at 400 psig with filtering as the pretreatment. The membrane fouling by colloidal matter was found to be controllable. The unit produced initially 55 gal/day and 27 gal/day after 30 days.

Lawrence, R. W.↗

Evaluation of cleaners for photovoltaic modules exposed in an outdoor environment

Power recovery of silicone encapsulated and glass covered photovoltaic modules, exposed for two years to a suburban environment, was measured after washing with a variety of cleaners including detergents, abrasive soap, and hydrocarbon solvents. Silicone encapsulated modules in operating environments may experience significant power losses or require extensive periodic cleaning. Glass front-faced modules in similar situations are much less affected. Organic hydrocarbon solvents or abrasives were found to be about five times more effective than mild detergents in cleaning encapsulated modules.

Knapp, W. D.↗

Performance of high-recovery recycling reverse osmosis with wash water

Inclusion of a recycling loop for partially-desalted water from second-stage reverse-osmosis permeate has been shown useful for achieving high-recovery at moderate applied pressures. This approach has now been applied to simulated wash waters, to obtain data on retention by the membranes of solutes in a mixture comparable to anticipated spacecraft hygiene wastewaters, and to generate an estimate of the maximum concentration that can be expected without causing membrane fouling. A first experiment set provides selectivity information from a single membrane and an Igepon detergent, as a function of final concentration. A reject concentration of 3.1% Total Organic Carbon has been reached, at a pressure of 1.4 Mega Pascals, without membrane fouling. Further experiments have generated selectivity values for the recycle configuration from two washwater simulations, as a function of applied pump pressure. Reverse osmosis removal has also been tested for washwater containing detergent formulated for plant growth compatibility (containing nitrogen, phosphorous and potassium functional groups.)

Herrmann, Cal C.↗

Ozone as a laundry agent on orbit and on the ground

Ozone (03), is the strongest commercial oxidizing agent for aqueous systems and may be ideal for space station laundering operations. It can be generated electronically from air in situ. It kills virtually all microorganisms, attacks many organics and inorganics, and breaks down stable ring structures of benzene and related oils when coupled with ultra violet radiation. It cleans and disinfects in cold water without the need for detergent. It leaves no residues. Ozone permits up to 90% wash water recycling and it eliminates wash time, water volume, and recycling problems of a detergent rinse. Ozone is self purging and converts spontaneously to oxygen. It can be rapidly purged by well established catalytic and thermal processes. Scaling of an ozone laundering system for space station may have commercial applications in a consumer model for home use.

Agosto, William N.↗

Characterization of auxin-binding proteins from zucchini plasma membrane

We have previously identified two auxin-binding polypeptides in plasma membrane (PM) preparations from zucchini (Cucurbita pepo L.) (Hicks et al. 1989, Proc. Natl. Acad. Sci. USA 86, 4948-4952). These polypeptides have molecular weights of 40 kDa and 42 kDa and label specifically with the photoaffinity auxin analog 5-N3-7-3H-IAA (azido-IAA). Azido-IAA permits both the covalent and radioactive tagging of auxin-binding proteins and has allowed us to characterize further the 40-kDa and 42-kDa polypeptides, including the nature of their attachment to the PM, their relationship to each other, and their potential function. The azido-IAA-labeled polypeptides remain in the pelleted membrane fraction following high-salt and detergent washes, which indicates a tight and possibly integral association with the PM. Two-dimensional electrophoresis of partially purified azido-IAA-labeled protein demonstrates that, in addition to the major isoforms of the 40-kDa and 42-kDa polypeptides, which possess isoelectric points (pIs) of 8.2 and 7.2, respectively, several less abundant isoforms that display unique pIs are apparent at both molecular masses. Tryptic and chymotryptic digestion of the auxin-binding proteins indicates that the 40-kDa and 42-kDa polypeptides are closely related or are modifications of the same polypeptide. Phase extraction with the nonionic detergent Triton X-114 results in partitioning of the azido-IAA-labeled polypeptides into the aqueous (hydrophilic) phase. This apparently paradoxical behavior is also exhibited by certain integral membrane proteins that aggregate to form channels. The results of gel filtration indicate that the auxin-binding proteins do indeed aggregate strongly and that the polypeptides associate to form a dimer or multimeric complex in vivo. These characteristics are consistent with the hypothesis that the 40-kDa and 42-kDa polypeptides are subunits of a multimeric integral membrane protein which has an auxin-binding site, and which may possess transporter or channel function.

NASA Program Space Biology↗

Stoichiometry of the large conductance bacterial mechanosensitive channel of E. coli. A biochemical study

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M. tuberculosis reveals a pentameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches. Detergent-purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels. Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product. DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker. It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species. Thus the longer spacer arm could permit both intra- and intercomplex linkages. Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers. The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization. Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor. The channel is thus a pentameric core with the 'extra' subunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress.

NASA Discipline Cell Biology↗

Helicity, membrane incorporation, orientation and thermal stability of the large conductance mechanosensitive ion channel from E. coli

In this report, we present structural studies on the large conductance mechanosensitive ion channel (MscL) from E. coli in detergent micelles and lipid vesicles. Both transmission Fourier transform infrared spectroscopy and circular dichroism (CD) spectra indicate that the protein is highly helical in detergents as well as liposomes. The secondary structure of the proteins was shown to be highly resistant towards denaturation (25-95 degrees C) based on an ellipticity thermal profile. Amide H+/D+ exchange was shown to be extensive (ca. 66%), implying that two thirds of the protein are water accessible. MscL, reconstituted in oriented lipid bilayers, was shown to possess a net bilayer orientation using dichroic ratios measured by attenuated total-reflection Fourier transform infrared spectroscopy. Here, we present and discuss this initial set of structural data on this new family of ion-channel proteins.

NASA Discipline Cell Biology↗

Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton

Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two vinculin-transfected clones (5.51Vin3 and Vin4; Samuels et al., 1993, J. Cell Biol. 121, 909-921). In the present study, we used this model system to determine how the presence of vinculin promotes cytoskeletal alterations and associated changes in cell shape. Microscopic analysis of cell spreading at early times, revealed that 5.51 cells retained the ability to form filopodia; however, they could not form lamellipodia, assemble stress fibers, or efficiently spread over the culture substrate. Detergent (Triton X-100) studies revealed that these major differences in cell morphology and cytoskeletal organization did not result from differences in levels of total polymerized or cross-linked actin. Biochemical studies showed that 5.51 cells, in addition to lacking vinculin, exhibited slightly reduced levels of alpha-actinin and paxillin in their detergent-insoluble cytoskeleton. The absence of vinculin correlated with a decrease in the mechanical stiffness of the integrin-cytoskeleton linkage, as measured using cell magnetometry. Furthermore, when vinculin was replaced by transfection in 5.51Vin3 and 5.51Vin4 cells, the levels of cytoskeletal-associated alpha-actinin and paxillin, the efficiency of transmembrane mechanical coupling, and the formation of actin stress fibers were all restored to near wild-type levels. These findings suggest that vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, rather than by altering the total level of actin polymerization or cross-linking.

NASA Program Space Biology↗