Experimental formation of bio-like structures <eksperimental'noye formirovaniye biopodobnykh struktur<
Bio-like structures formed in distilled water and on agar gel in electric field
GEL↗
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Purification of the small mechanosensitive channel of Escherichia coli (MscS): the subunit structure, conduction, and gating characteristics in liposomes
The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of approximately 200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20-60 mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (gamma). Po(gamma) curves had the midpoint at 5.5 +/- 0.1 dyne/cm and gave estimates for the energy of opening DeltaG = 11.4 +/- 0.5 kT, and the transition-related area change DeltaA = 8.4 +/- 0.4 nm(2) when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed.
NASA Discipline Cell Biology↗
Organization of photosystem I polypeptides examined by chemical cross-linking
Photosystem I from the cyanobacterium Synechocystis sp. PCC 6803 was examined using the chemical cross-linkers glutaraldehyde and N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide to investigate the organization of the polypeptide subunits. Thylakoid membranes and photosystem I, which was isolated by Triton X-100 fractionation, were treated with cross-linking reagents and were resolved using a Tricine/urea low-molecular-weight resolution gel system. Subunit-specific antibodies and western blotting analysis were used to identify the components of cross-linked species. These analyses identified glutaraldehyde-dependent cross-linking products composed of small amounts of PsaD and PsaC, PsaC and PsaE, and PsaE and PsaF. The novel cross-link between PsaE and PsaF was also observed following treatment with N-ethyl-1-3-[3-(dimethylamino)propyl]carbodiimide. These cross-linking results suggest a structural interaction between PsaE and PsaF and predict a transmembrane topology for PsaF.
NASA Discipline Plant Biology↗
Nonenzymatic template-directed synthesis on oligodeoxycytidylate sequences in hairpin oligonucleotides
We have developed a novel method for studying template-directed synthesis in hairpin oligonucleotides. An unpaired segment at the 5'-terminus of the hairpin acts as an intramolecular template for the extension of the paired 3'-terminus. Products are analyzed by denaturing gel electrophoresis of [32P]-labeled hairpins. Using this system, we have studied the synthesis of oligoguanylates on an oligodeoxycytidylate template. We find that guanosine 5'-phosphoro(2-methyl)imidazolide adds efficiently to a terminal riboguanylate residue at temperatures in the range 0-37 degrees C but not at 50 degrees C. At 0 degree C, the half-time for addition of the first G residue is about 3 h, and the reaction rate is independent of pH in the range 6.5-8.0. The first addition reaction results in the formation of a predominantly 3'-5'-internucleotide bond. When the 3'-terminal riboguanylate residue is placed by a deoxyguanylate residue, the half-time for the first addition increases from about 3 to about 30 h.
NASA Discipline Exobiology↗
Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution
We have prepared a [32P]-labled oligonucleotide probe carrying a free primary amine at its 3'-terminus. This probe is used to initiate polymerization of aziridine (ethyleneimine) in aqueous solution. The nature of the oligomeric products and the kinetics of their formation are then monitored by gel electrophoresis. Our results are generally consistent with those obtained using conventional techniques. We have also investigated the effect of polyanionic templates on the rate of oligomerization of aziridine. We find that water-soluble polyanions generally accelerate the polymerization. The sodium salt of polymethacrylic acid is the most effective of the templates that we studied. The methods introduced in this paper should be applicable to a variety of polymerization reactions in aqueous solution. They should greatly simplify the screening of potentially prebiotic polymerization reactions.
NASA Discipline Exobiology↗
4-Coumarate:coenzyme A ligase and isoperoxidase expression in Zinnia mesophyll cells induced to differentiate into tracheary elements
When cultured in inductive medium containing adequate auxin and cytokinin, isolated mesophyll cells of Zinnia elegans L. cv Envy differentiate into tracheary elements with lignified secondary wall thickenings. Differentiation does not occur when cells are cultured in control medium, which has reduced levels of auxin and/or cytokinin. The activities of two enzymes involved in lignin synthesis, 4-coumarate:coenzyme A ligase and peroxidase, were examined. An induction-specific cationic isoperoxidase, visualized by low pH polyacrylamide gel electrophoresis, is detectable in soluble and wall fractions of cultured Zinnia cells long before tracheary elements visibly differentiate and is thus an early marker of differentiation. Compounds (such as antiauxins, anticytokinins, and tunicamycin) that inhibit or delay differentiation alter the expression of this isoperoxidase. 4-Coumarate:coenzyme A ligase activity increases dramatically only as cells differentiate. Together, these results suggest that the onset of lignification in differentiating Zinnia cells might be controlled by the availability of precursors synthesized by way of 4-coumarate:coenzyme A ligase. These precursors would then be polymerized into lignin in the cell wall by the induction-specific isoperoxidase.
NASA Discipline Plant Biology↗
Functional properties of slow and fast gastrocnemius muscle fibers after a 17-day spaceflight
The purpose of this investigation was to study the effects of a 17-day spaceflight on the contractile properties of individual fast- and slow-twitch fibers isolated from biopsies of the fast-twitch gastrocnemius muscle of four male astronauts. Single chemically skinned fibers were studied during maximal Ca2+-activated contractions with fiber myosin heavy chain (MHC) isoform expression subsequently determined by SDS gel electrophoresis. Spaceflight had no significant effect on the mean diameter or specific force of single fibers expressing type I, IIa, or IIa/IIx MHC, although a small reduction in average absolute force (P(o)) was observed for the type I fibers (0.68 +/- 0.02 vs. 0.64 +/- 0.02 mN, P < 0.05). Subject-by-flight interactions indicated significant intersubject variation in response to the flight, as postflight fiber diameter and P(o) where significantly reduced for the type I and IIa fibers obtained from one astronaut and for the type IIa fibers from another astronaut. Average unloaded shortening velocity [V(o), in fiber lengths (FL)/s] was greater after the flight for both type I (0.60 +/- 0.03 vs. 0.76 +/- 0.02 FL/s) and IIa fibers (2.33 +/- 0.25 vs. 3.10 +/- 0.16 FL/s). Postflight peak power of the type I and IIa fibers was significantly reduced only for the astronaut experiencing the greatest fiber atrophy and loss of P(o). These results demonstrate that 1) slow and fast gastrocnemius fibers show little atrophy and loss of P(o) but increased V(o) after a typical 17-day spaceflight, 2) there is, however, considerable intersubject variation in these responses, possibly due to intersubject differences in in-flight physical activity, and 3) in these four astronauts, fiber atrophy and reductions in P(o) were less for slow and fast fibers obtained from the phasic fast-twitch gastrocnemius muscle compared with slow and fast fibers obtained from the slow antigravity soleus [J. J. Widrick, S. K. Knuth, K. M. Norenberg, J. G. Romatowski, J. L. W. Bain, D. A. Riley, M. Karhanek, S. W. Trappe, T. A. Trappe, D. L. Costill, and R. H. Fitts. J Physiol (Lond) 516: 915-930, 1999].
NASA Experiment Number 8913020 1/2↗
Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation
Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.
NASA Discipline Radiation Health↗
Distribution of Candida albicans genotypes among family members
Thirty-three families (71 subjects) were screened for the presence of Candida albicans in mouthwash or stool specimens; 12 families (28 subjects) were culture-positive for this yeast. An enrichment procedure provided a twofold increase in the recovery of C. albicans from mouthwash specimens. Nine of the twelve culture-positive families had two positive members each, two families had three positive members each, and one family had four positive members. Genetic profiles were obtained by three methods: pulsed-field gel electrophoresis; restriction endonuclease analysis, and random amplification of polymorphic DNA analysis. DNA fingerprinting of C. albicans isolated from one body site three consecutive times revealed that each of the 12 families carried a distinct genotype. No two families shared the same strain, and two or more members of a family commonly shared the same strain. Intrafamily genotypic identity (i.e., each member within the family harbored the same strain) was demonstrated in six families. Genotypes of isolates from husband and wife differed from one another in five families. All three methods were satisfactory in determining genotypes; however, we concluded that restriction endonuclease analysis provided adequate resolving power.
NASA Center JSC↗
Decorin and biglycan of normal and pathologic human corneas
PURPOSE: Corneas with scars and certain chronic pathologic conditions contain highly sulfated dermatan sulfate, but little is known of the core proteins that carry these atypical glycosaminoglycans. In this study the proteoglycan proteins attached to dermatan sulfate in normal and pathologic human corneas were examined to identify primary genes involved in the pathobiology of corneal scarring. METHODS: Proteoglycans from human corneas with chronic edema, bullous keratopathy, and keratoconus and from normal corneas were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), quantitative immunoblotting, and immunohistology with peptide antibodies to decorin and biglycan. RESULTS: Proteoglycans from pathologic corneas exhibit increased size heterogeneity and binding of the cationic dye alcian blue compared with those in normal corneas. Decorin and biglycan extracted from normal and diseased corneas exhibited similar molecular size distribution patterns. In approximately half of the pathologic corneas, the level of biglycan was elevated an average of seven times above normal, and decorin was elevated approximately three times above normal. The increases were associated with highly charged molecular forms of decorin and biglycan, indicating modification of the proteins with dermatan sulfate chains of increased sulfation. Immunostaining of corneal sections showed an abnormal stromal localization of biglycan in pathologic corneas. CONCLUSIONS: The increased dermatan sulfate associated with chronic corneal pathologic conditions results from stromal accumulation of decorin and particularly of biglycan in the affected corneas. These proteins bear dermatan sulfate chains with increased sulfation compared with normal stromal proteoglycans.
Non-NASA Center↗
Robotic multimodality stereotactic brain tissue identification: work in progress
Real-time identification of tissue would improve procedures such as stereotactic brain biopsy (SBX), functional and implantation neurosurgery, and brain tumor excision. To standard SBX equipment has been added: (1) computer-controlled stepper motors to drive the biopsy needle/probe precisely; (2) multiple microprobes to track tissue density, detect blood vessels and changes in blood flow, and distinguish the various tissues being penetrated; (3) neural net learning programs to allow real-time comparisons of current data with a normative data bank; (4) three-dimensional graphic displays to follow the probe as it traverses brain tissue. The probe can differentiate substances such as pig brain, differing consistencies of the 'brain-like' foodstuff tofu, and gels made to simulate brain, as well as detect blood vessels imbedded in these substances. Multimodality probes should improve the safety, efficacy, and diagnostic accuracy of SBX and other neurosurgical procedures.
Brain/anatomy & histology/surgery↗
Epidemiology of Staphylococcus aureus during space flight
Staphylococcus aureus was isolated over 2 years from Space Shuttle mission crewmembers to determine dissemination and retention of bacteria. Samples before and after each mission were from nasal, throat, urine, and feces and from air and surface sampling of the Space Shuttle. DNA fingerprinting of samples by digestion of DNA with SmaI restriction endonuclease followed by pulsed-field gel electrophoresis showed S. aureus from each crewmember had a unique fingerprint and usually only one strain was carried by an individual. There was only one instance of transfer between crewmembers. Strains from interior surfaces after flight matched those of crewmembers, suggesting microbial fingerprinting may have forensic application.
Flight Experiment↗
Random-breakage mapping method applied to human DNA sequences
The random-breakage mapping method [Game et al. (1990) Nucleic Acids Res., 18, 4453-4461] was applied to DNA sequences in human fibroblasts. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated calls, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks. This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment. By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a NotI fragment on the X chromosome, thus demonstrating the validity of the technique. We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding NotI fragments on chromosome 21. A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed. Since both probes D21S1 and D21S11 hybridized to a single NotI fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method.
NASA Discipline Radiation Health↗
Characterization of the proteins comprising the integral matrix of Strongylocentrotus purpuratus embryonic spicules
In the present study, we enumerate and characterize the proteins that comprise the integral spicule matrix of the Strongylocentrotus purpuratus embryo. Two-dimensional gel electrophoresis of [35S]methionine radiolabeled spicule matrix proteins reveals that there are 12 strongly radiolabeled spicule matrix proteins and approximately three dozen less strongly radiolabeled spicule matrix proteins. The majority of the proteins have acidic isoelectric points; however, there are several spicule matrix proteins that have more alkaline isoelectric points. Western blotting analysis indicates that SM50 is the spicule matrix protein with the most alkaline isoelectric point. In addition, two distinct SM30 proteins are identified in embryonic spicules, and they have apparent molecular masses of approximately 43 and 46 kDa. Comparisons between embryonic spicule matrix proteins and adult spine integral matrix proteins suggest that the embryonic 43-kDa SM30 protein is an embryonic isoform of SM30. An adult 49-kDa spine matrix protein is also identified as a possible adult isoform of SM30. Analysis of the SM30 amino acid sequences indicates that a portion of SM30 proteins is very similar to the carbohydrate recognition domain of C-type lectin proteins.
Non-NASA Center↗
Production of RNA by a polymerase protein encapsulated within phospholipid vesicles
Catalyzed polymerization reactions represent a primary anabolic activity of all cells. It can be assumed that early cells carried out such reactions, in which macromolecular catalysts were encapsulated within some type of boundary membrane. In the experiments described here, we show that a template-independent RNA polymerase (polynucleotide phosphorylase) can be encapsulated in dimyristoyl phosphatidylcholine vesicles without substrate. When the substrate adenosine diphosphate (ADP) was provided externally, long-chain RNA polymers were synthesized within the vesicles. Substrate flux was maximized by maintaining the vesicles at the phase transition temperature of the component lipid. A protease was introduced externally as an additional control. Free enzyme was inactivated under identical conditions. RNA products were visualized in situ by ethidium bromide fluorescence. The products were harvested from the liposomes, radiolabeled, and analyzed by polyacrylamide gel electrophoresis. Encapsulated catalysts represent a model for primitive cellular systems in which an RNA polymerase was entrapped within a protected microenvironment.
Non-NASA Center↗
Phosphate cycling on the basic protein of Plodia interpunctella granulosis virus
The presence of infected cell-specific phosphoproteins was investigated in Plodia interpunctella granulosis virus (PiGV)-infected fat body using [32P]orthophosphoric acid labeling. One infected cell-specific phosphoprotein had a mobility similar to that of the basic protein (VP12) of PiGV. Further analysis, using immunoblotting and acid-urea gel analysis of infected fat body, confirmed that this phosphoprotein was VP12. However we did not detect phosphorylated VP12 in 32P-labeled nucleocapsids. Phosphoamino acid analysis of 32P-labeled VP12 revealed that phosphoserine was present in the basic protein. Since VP12 is phosphorylated in the infected cell, but not in the nucleocapsid, it appears that dephosphorylation of VP12 is a critical event in the life cycle of the virus. We therefore assayed virus nucleocapsids and infected fat body for the presence of phosphatase activity. Phosphatase activity was not detected in the virus, but the infected fat body had more activity than uninfected fat body. A model for nucleocapsid assembly and uncoating is presented which takes into account the phosphorylation state of VP12, the role of Zn2+ in the nucleocapsid, and the role of the capsid-associated kinase.
NASA Discipline Cell Biology↗
Platelet factor XIII increases the fibrinolytic resistance of platelet-rich clots by accelerating the crosslinking of alpha 2-antiplasmin to fibrin
Platelet clots resist fibrinolysis by plasminogen activators. We hypothesized that platelet factor XIII may enhance the fibrinolytic resistance of platelet-rich clots by catalyzing the crosslinking of alpha 2-antiplasmin (alpha 2AP) to fibrin. Analysis of plasma clot structure by polyacrylamide gel electrophoresis and immunoblotting revealed accelerated alpha 2AP-fibrin crosslinking in platelet-rich compared with platelet-depleted plasma clots. A similar study of clots formed with purified fibrinogen (depleted of factor XIII activity), isolated platelets, and specific factor XIII inhibitors indicated that this accelerated crosslinking was due to the catalytic activity of platelet factor XIII. Moreover, when washed platelets were aggregated by thrombin, there was evidence of platelet factor XIII-mediated crosslinking between platelet alpha 2AP and platelet fibrin(ogen). Specific inhibition (by a monoclonal antibody) of the alpha 2AP associated with washed platelet aggregates accelerated the fibrinolysis of the platelet aggregate. Thus in platelet-rich plasma clots, and in thrombin-induced platelet aggregates, platelet factor XIII actively formed alpha 2AP-fibrin crosslinks, which appeared to enhance the resistance of platelet-rich clots to fibrinolysis.
Non-NASA Center↗
Phosphorylation of the budgerigar fledgling disease virus major capsid protein VP1
The structural proteins of the budgerigar fledgling disease virus, the first known nonmammalian polyomavirus, were analyzed by isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major capsid protein VP1 was found to be composed of at least five distinct species having isoelectric points ranging from pH 6.45 to 5.85. By analogy with the murine polyomavirus, these species apparently result from different modifications of an initial translation product. Primary chicken embryo cells were infected in the presence of 32Pi to determine whether the virus structural proteins were modified by phosphorylation. SDS-PAGE of the purified virus structural proteins demonstrated that VP1 (along with both minor capsid proteins) was phosphorylated. Two-dimensional analysis of the radiolabeled virus showed phosphorylation of only the two most acidic isoelectric species of VP1, indicating that this posttranslational modification contributes to VP1 species heterogeneity. Phosphoamino acid analysis of 32P-labeled VP1 revealed that phosphoserine is the only phosphoamino acid present in the VP1 protein.
Non-NASA Center↗