Laboratory simulations of the pyrolytic release experiments: An interim report

Summary During its operation on Mars the pyrolytic release experiment (PR) detected the fixation of small amounts of CO₂ and/or CO. Laboratory simulations of the experimental conditions were made in an attempt to substantiate the previous conclusion that these reactions were chemical rather than biological. The selection of model substrata for these tests was based on the known properties of the Martian surface material. After pretreatment and incubation under various conditions, pyrolytic analysis was used to indicate the extent of surface catalyzed conversion of¹⁴CO₂ or¹⁴CO to¹⁴C-organic compounds. This abiotic synthesis was detected in experiments with three iron oxides, viz. hematite, magnetite and maghemite. When the incubation atmosphere was supplemented with water vapor, the levels of synthesis were in a range comparable to that detected in the Viking PR tests. An abiotic synthesis was also detected in experiments with a mixture of clays and minerals (Mars analog soil) or with montmorillonite artifically enriched in iron. With either substratum the reaction appeared to be the result of a photocatalytic synthesis of¹⁴C-organics from¹⁴CO and surface hydroxyl groups. This process was not dependent on the presence of water vapor in the incubation atmosphere. Although a duplication of the Viking data has not been achieved, these findings support the abiotic interpretation of the PR results.     

Degradation and metabolism of tetraethyllead in soils

Summary The objective of this study was to determine the disappearance of the leaded gasoline enhancer tetraethyllead (TEL), formation of degradation products, and mass balance in nonsterile and autoclaved Leon and Madison soils. Ethyl-1-¹⁴C-labeled TEL was used so that mineralization rates of TEL and mass balance could be determined.¹⁴C-TEL in nonsterile and autoclaved surface and subsurface samples of the two soils disappeared rapidly, and ionic ethyllead products, water soluble nonlead organic products and bound residues were rapidly formed. A small fraction (7.74%) of¹⁴C-TEL in nonsterile soil samples was mineralized to¹⁴CO₂ in 28 days. Triethyllead (TREL) was the major ionic ethyllead product detected in both nonsterile and autoclaved soils; diethyllead (DEL) was occasionally detected. Recovery of¹⁴C from mass balance studies for all nonsterile and autoclaved soil samples after 28 days of incubation was poor, less than 50% of the¹⁴C applied. It appears that unknown volatile and/or gaseous organic products were the major degradation products of TEL in soils. Based on the observations of more rapid initial disappearance of¹⁴C-TEL, more rapid formation and more rapid disappearance of¹⁴C-DEL, and occurrence of¹⁴CO₂ production in nonsterile soils, it was concluded that both biological and chemical degradation contributed to the degradation of TEL in soils, with chemical degradation being the major factor.     

Bacterial desorption from food container and food processing surfaces

The desorption ofStaphylococcus aureus, Acinetobacter calcoaceticus, and a coryneform from the surfaces of materials used for manufacturing food containers (glass, tin plate, and polypropylene) or postprocess canning factory conveyor belts (stainless steel and nylon) was investigated. The effect of time, pH, temperature, and adsorbed organic layers on desorption was studied.S. aureus did not detach from the substrata at any pH investigated (between pH 5 and 9).A. calcoaceticus and the coryneform in some cases detached, depending upon pH and substratum composition. The degree of bacterial detachment from the substrata was not related to bacterial respiration at experimental pH values. Bacterial desorption was not affected by temperature (4–30°C) nor by an adsorbed layer of peptone and yeast extract on the substrata. The results indicate that bacterial desorption, hence bacterial removal during cleaning or their transfer via liquids flowing over colonized surfaces, is likely to vary with the surface composition and the bacterial species colonizing the surfaces.     

Influence of collagen gel substrata on certain biochemical activities of hepatocytes in primary culture

In order to study the influence of cell shape as modulated by the extracellular matrix on the cellular activity, hepatocytes isolated from liver were maintained on collagen I coated plastic substrata and collage I gel substrata and certain hepatocyte specific functions were investigated. The incorporation of³[H]-leucine into total proteins and albumin secreted by cells maintained on collagen gel was found to be significantly higher compared to those maintained on a collagen coated plastic substrata, indicating that hepatocytes on collagen gel have an enhanced albumin synthesizing capacity. Increased incorporation of³⁵[S]-sulphate into total proteoglycans (PG) and a relatively higher fraction of the³⁵[S]-PG in the extracellular space showed an increased rate of synthesis and secretion of sulphated PGs by cells maintained on collagen gels. But in contrast to the above results, the incorporation of³[H]-leucine into cytokeratins C₈, C₁₈ and actin were significantly low in cells maintained on collagen gel. The tyrosine amino transferase activity exhibited by hepatocytes preincubated with dexamethasone on collagen gel was also significantly low. The different forms of collagen substrata appeared to have no effect on the amino acid transport by hepatocytes, further suggesting that the various hepatocyte specific functions are not uniformly altered when hepatocytes are maintained on three-dimensional collagen gel substrata. These results indicate that the shape of the cell as determined by the nature of the matrix substratum influences the synthetic activity of secretory proteins and those remaining intracellularly, differently.     

Photochemical synthesis of simple organic free radicals on simulated planetary surfaces—An ESR study

Electron spin resonance spectroscopy provided evidence for formation of hydroxyl radicals during ultraviolet photolysis (254 nm) at −170°C of H₂O adsorbed on silica gel or of silica gel alone. The carboxyl radical was observed when CO or CO₂ or a mixture of CO and CO₂ adsorbed on silica gel at −170°C was irradiated. The ESR signals of these radicals slowly disappeared when the irradiated samples were warmed to room temperature. However, re-irradiation of CO or CO₂, or the mixture CO and CO₂ on silica gel at room temperature then produced a new species, the carbon dioxide anion radical, which slowly decayed and which was identical with that produced by direct photolysis of formic acid adsorbed on silica gel. The primary photochemical process may involve formation of hydrogen and hydroxyl radicals by means of (1) photodissociation of H₂O physically adsorbed on the silica gel, or (2) absorption of the excitation energy by the silica gel surface with subsequent cleavage of the silanol bonds, or (3) dissociation of H₂O molecules through photosensitization by the surfaces or a combination of (1) to (3). Subsequent reactions of these radicals with adsorbed CO or CO₂ or both yield carboxyl radicals, CO₂H, the precursors of formic acid. Our results confirm the formation of formic acid under simulated. Martian conditions and provide a mechanistic basis for gauging the potential importance of gas-solid photochemistry for chemical evolution on other extraterrestrial bodies, on the primitive earth and on dust grains in the interstellar medium.     

A glycyl free radical as the precursor in the synthesis of carbon monoxide and cyanide by the [FeFe]-hydrogenase maturase HydG

HydG uses tyrosine to synthesize the CN⁻/CO ligands of [FeFe]-hydrogenase active site. We have mutated two of the [4Fe-4S]-cluster cysteine ligands of the HydG C-terminal domain (CTD) to serine. The double mutant can still synthesize CN⁻ but not CO. In a mutant lacking the CTD both CN⁻ and CO synthesis are abolished. Like in ThiH, the initial steps of CN⁻ synthesis are carried out in the TIM-barrel domain of HydG but some component(s) of the CTD are later needed. The mutants indicate that CO synthesis is metal-based and occurs in the CTD. We postulate that CN⁻/CO synthesis is initiated by H₂N-CH-. Fragmentation of this radical into H₂N-CH₂ and CO₂ or H₂CNH and provides plausible precursors for CN⁻/CO synthesis.     

The effect of chemical pretreatment on the aerobic microbial degradation of PCB congeners in aqueous systems

A series of experiments was conducted to examine the effects of chemical pretreatment on biodegradation of¹⁴C-labeled PCB congeners in aqueous systems. Fenton's reagent was used to generate hydroxyl radicals (OH) which were successful in partially oxidizing/transforming otherwise recalcitrant molecules of tetrachlorinated PCB, but had little or no impact on the biodegradation of a monochlorinated congener. Application of Fenton's reagent (1% H₂O₂, 1 mM FeSO₄) followed by inoculation with pure culturesPseudomonas sp, strain LB 400 andAlcaligenes eutrophus, strain H850 resulted in the removal of approximately 38% of 2-chlorobiphenyl and 51% of 2,2, 4,4-tetrachlorobiphenyl in the form of¹⁴CO₂. Comparison of the rate and extent of biodegradation of 2,2, 4,4-tetrachlorobiphenyl after the application of Fenton's reagent with the dynamic and final level of radioactivity in the aqueous phase of experimental system suggests two possible means of microbial utilization of tetrachlorinated PCB congener altered by chemical oxidation: (a) consumption of the partially oxidized chemical dissolved in the aqueous phase, and (b) direct microbial attack on the transformed compound, which may still be adhered to the solid surface.     

Normal and frictional interactions of purified human statherin adsorbed on molecularly-smooth solid substrata

Human salivary statherin was purified from parotid saliva and adsorbed to bare hydrophilic (HP) mica and STAI-coated hydrophobic (HB) mica in a series of Surface Force Balance experiments that measured the normal (F _n) and friction forces (F _s*) between statherin-coated mica substrata. Readings were taken both in the presence of statherin solution (HP and HB mica) and after rinsing (HP mica). F _n measurements showed, for both substrata, monotonic steric repulsion that set on at a surface separation D ～ 20 nm, indicating an adsorbed layer whose unperturbed thickness was ca 10 nm. An additional longer-ranged repulsion, probably of electrostatic double-layer origin, was observed for rinsed surfaces under pure water. Under applied pressures of ～ 1 MPa, each surface layer was compressed to a thickness of ca 2 nm on both types of substratum, comparable with earlier estimates of the size of the statherin molecule. Friction measurements, in contrast with F _n observations, were markedly different on the two different substrata: friction coefficients, μ ≡ ?F _s*/?F _n, on the HB substratum (μ ≈ 0.88) were almost an order of magnitude higher than on the HP substratum (μ ≈ 0.09 and 0.12 for unrinsed and rinsed, respectively), and on the HB mica there was a lower dependence of friction on sliding speed than on the HP mica. The observations were attributed to statherin adsorbing to the mica in multimer aggregates, with internal re-arrangement of the protein molecules within the aggregate dependent on the substratum to which the aggregate adsorbed. This internal re-arrangement permitted aggregates to be of similar size on HP and HB mica but to have different internal molecular orientations, thus exposing different moieties to the solution in each case and accounting for the very different friction behaviour.     

Embryonic nutrition,growth and energetics inZoarces viviparus L. as indication of a maternal-fetal trophic relationship

Summary The embryos ofZoarces viviparus (L.) show linear growth during their intraovarian development. In early gestation before hatching, the embryos take up very small amounts of low molecular tracer compounds such as glucose, glycine or taurine. Later in gestation (two months after hatching), the embryos accumulate substantial amounts of the tracer compounds. The uptake rates of the tracer compounds in vitro are correlated with ambient concentrations of unlabelled compounds within the natural concentration range of the ovarian fluid. The highest uptake rates are found for glucose and the lowest for taurine. Release of¹⁴CO₂ and dissolved organic carbon (DO¹⁴C) from assimilated tracers in the embryos is low. Oxygen uptake and body weight of the embryos appear to be linearly correlated, and the average oxygen uptake is 4.20 (SD 0.73) mol O₂ g^–1 h^–1 WW at 11°C. The contribution of glucose respiration to total aerobic respiration is 13.9%. A growth to respiration ratio of 0.91 indicates a relatively high efficiency for converting food to growth.Symbols and abbreviations DW dry weight - WW wet weight - DO ¹⁴ C dissolved organic carbon (¹⁴C-labelled) - t _1/2 half life time - turnover time (replacement time)     

Synthesis of biological molecules on molecular sieves

Catalytic properties of aluminosilicates may play a role in the synthesis of biological molecules from simple gaseous molecules commonly found in planetary atmospheres. Urea, amino acids and UV absorbing substances have been obtained by heating CO and NH₃ with Linde molecular sieves saturated with Ca⁺², NH₄ ⁺ or Fe⁺³. The yields of amino acids produced have been determined by an amino acid analyzer. The quantity of urea produced largely depends on the nature of the saturating cation. Experiments using¹⁴CO confirm that the amino acids are not due to contaminants adsorbed on the surface of the molecular sieves.     

Autotrophic synthesis of activated acetic acid from two CO2 inMethanobacterium thermoautotrophicum

An in vitro system of autotropic synthesis of activated acetic acid from¹⁴CO₂ inMethanobacterium thermoautotrophicum was developed.

1. A recognized¹⁴CO₂-fixation product in vitro was activated [¹⁴C] acetic acid. It could be trapped enzymatically into citrate and released again as [¹⁴C] acetate by citrate synthase and citrate lyase, respectively.
2. The synthesis of both activated acetic acid and methane from CO₂ proceeded in parallel under a variety of conditions. Both of these processes were stimulated greatly and to the same extent by the addition of methyl coenzyme M to the assay.
3. Various inhibitors of methanogenesis tested also inhibited acetate synthesis, e.g. CH₂Cl₂, CHCl₃, CCl₄, N₂O, and bromoethane sulfonic acid. Cyanide specifically inhibited the synthesis of activated acetic acid, whereas methane formation was unaffected. Cyanide inhibition was relieved by adding CO, whereas the inhibition by the other compounds was not.

The data suggest: The product studied in vitro was acetyl CoA. Its synthesis involves intermediates of CO₂ reduction to methane. In addition, a cyanide-sensitive reaction is required which does not participate in CO₂ reduction to methane.     

Continuous culture with deep seawater of a benthic food diatom Nitzschia sp.

A continuous culture system for a benthic food diatom Nitzschia sp. wasestablished by using properties of high nutrient and clean of deep seawater(DSW). DSW collected from 320 m depth in Muroto City, Japan, was introducedinto a glass-pipe bioreactor (14 cm length, 3 cm diam.) containing glassbeads of 0.5 cm diam. as substrata for the alga, and it was incubated at18°C · 80Em^–2sec^–1 · L:D=14:10. The chlorophyll a yield of benthicdiatoms in a reactor as a unit of surface area of the substratum was only0.001–0.003 g cm^–2 when the flow rate of DSW was 0(batch culture conditions). However, when DSW was supplied continuously to areactor, the yield increased to 1.4 g-chl.a cm^–2 alongwith the increase in flow rate of DSW. Moreover, amounts of chl.a washed outof the system were negligible, 0.0014 to 0.0045%, even though theflow rate of DSW was as much as 25 times h^–1, suggesting thatsloughing of benthic diatoms from the substratum was minimized. Although theyield of diatoms fluctuated significantly at the time that the DSW wascollected, the variation could be minimized by increasing the flow rate ofDSW. These results indicate that the continuous culturing system with DSWsupports the stable and effective mass culture of benthic food diatom.     

Effects of adsorbed organic and primary fouling films on bryozoan settlement

Marine primary fouling films, which consist of molecular organic and microbial components, have been reported to facilitate colonization of immersed surfaces by marine fouling organisms. Larvae of the cosmopolitan fouling bryozoan Bugula neritina (Linnaeus) were offered various substrata for attachment and metamorphosis. The materials were offered (a) after detergent washing, (b) after sorption of dissolved organic molecular films, and (c) after formation of primary films consisting of both microbial and adsorbed organic material. Wettability of the substrata by sea water was determined by contact angle measurements for each substratum. On washed substrata, attachment was favored with contact angles greater than ≈45° (cos contact angle <0.7). Adsorbed surface films had no effect on the low settlement of larvae on glass and high settlement on plastics. Microbial primary films, however, made glass attractive and plastics unattractive. These settlement preference changes did not correlate with the changes in wettability observed on these substrata. Dispersion of larvae over the settlement surface was random except on wettable surfaces coated with bacterial films, where settlement was strongly clustered (contagious).     

Sulfur dynamics in mineral horizons of two northern hardwood soils. A column study with 35S

Sulfur dynamics of two Spodosols were ascertained using soil columns constructed from homogenized mineral soil from nothern hardwood ecosystems at the Huntington Forest (HF) in the Adirondack Mountains of New York and Bear Brook Watershed in Maine (BBWM). Columns were leached for 20 weeks with a simulated throughfall solution with³⁵SO₄ ^2-. Sulfur constituents were similar to those of other Spodosols, with the organic S fractions (C-bonded S and ester sulfate) constituting over 90% of total S. HF soil columns had higher total S (14.9 mol S g^-1) than that for the BBWM soil columns (7.4 mol g^-1) primarily due to higher C-bonded S in the former.Initially, adsorbed SO₄ ^- accounted for 5 and 4% of total S for the BBWM and HF soil columns, respectively. After 20 weeks, adsorbed SO₄ ^2- decreased (81%) in BBWM and increased (33%) in HF soil columns. For both HF and BBWM soil columns, C-bonded S increased and ester sulfate decreased, but only for HF columns was there a net mineralization of organic S (5.6% of total S). The greatest decrease in ester sulfate occurred at the top of the columns.Leaching of³⁵S was less than 0.5% of the³⁵S added due to its retention in various S constituents. There was an exponential decrease in³⁵S with column depth and most of the radioisotope was found in C-bonded S (70–88 and 70–91% for BBWM and HF, respectively). The rapid turnover of adsorbed SO^2- ₄ was reflected in its high specific activity (834 and 26 kBq mol^-1 S for BBWM and HF, respectively). The lower specific activity of adsorbed SO₄ ^2- in HF was attributable to greater isotopic dilution by non-radioactive SO^2- ₄ derived from greater organic S mineralization in the HF versus the BBWM columns.Both soil columns initially had high levels of NO^- ₃ which resulted in the generation of H⁺ and net retention of SO₄ ^2- in the early phase of the experiment due to pH dependent sulfate adsorption; later NO₃ ^- decreased and SO₄ ^2- was desorbed. Leaching of NIO₃ ^- and SO₄ ^2- was correlated with losses of Mg²⁺ and Ca²⁺ of which the latter was the dominant cation.Analyses using both S mass balances and radioisotopes corroborate that for BBWM soil columns, SO^2- ₄ adsorption-desorption dominated the S biogeochemistry while in HF soil columns, organic S mineralization-immobilization processes were more important. It is suggested that similar techniques can be applied to soils in the field to ascertain the relative importances of SO₄ ^2- adsorption processes and organic S dynamics.     

Time course of active Na transport and oxidative metabolism following transepithelial potential perturbation in toad urinary bladder

Summary The use of an Ussing chamber with well-defined mixing characteristics coupled to a mass spectrometer permits the concurrent evaluation of transepithelial current and oxidative metabolism with improved temporal resolution. The time-course of the amiloride-sensitive currentI ^a and the rate of suprabasal CO₂ productionJ _CO2 ^sb were observed in 10 toad urinary bladders at short-circuit and after clamping at 100 mV, serosa positive. Following perturbation of (0100mV),I ^a declined sharply within 1/2 min, remained near constant 15 min, and then increased slightly.J _CO2 ^sb declined more gradually, remained near constant at 4–7 min, and then declined further. Detailed analysis revealed an early quasi-steady state with near constancy ofJ _CO2 ^sb starting at 2.9±1.1 (sd) min and lasting 4.7 ±1.8 (sd) min, followed by relaxation to a later steady state at about 15 min. During the early quasi-steady state,I ^a was also nearly constant. Considering that in steady statesI ^a/FJ _Na ^a , the rate of transepithelial active Na transport, during the early quasi-steady state mean values ±se ofJ _Na ^a ,J _CO2 ^sb and (J _Na ^a /J _CO2 ^sb ) were, respectively, 29.9±1.7%, 59.4 ±3.2%, and 56.4±5.7% of values at short-circuit. Corresponding values during the late steady state were 41.4±6.0%, 38.2±6.1%, and 111.3±8.6%. Thus the flow ratioJ _Na ^a /J _CO2 ^sb was depressed significantly during the early quasi-steady state, but returned later to the original value. The results of measurements ofI ^a andJ _CO2 ^sb in three hemibladders were qualitatively similar. In terms of a phenomenological black-box treatment the findings are consistent with earlier studies indicating incomplete coupling between transport and metabolism. Further studies will be required to clarify the molecular basis for these observations.     

A regulatory role for proline metabolism in stimulating Arabidopsis thaliana seed germination

Analysis of the rate-limiting dehydrogenases of the oxidative pentosephosphate pathway (OPPP), as well as changes in the contributions of¹⁴C₁- and ¹⁴C₆-labelled glucose torespired CO₂ during germination, indicated activation of the OPPPduring Arabidopsis seed germination. An approximatelyfour-fold increase in free proline (Pro) was also observed prior to radicleemergence in most seeds. Delayed radicle emergence in transgenicArabidopsis seeds which expressed an antisense copy of thegene encoding the Pro biosynthetic enzyme¹-pyrroline-5-carboxylate synthetase correlated with anapproximately 35% reduction in the maximal concentration of Pro accumulated bythe antisense lines during germination. A dose-dependent inhibition ofArabidopsis seed germination by millimolar concentrationsof Pro capable of feedback inhibition of Pro synthesis reinforced the role ofPro synthesis in promoting germination. The ability of the artificial oxidantsmethylene blue and phenazine ethosulphate to overcome the inhibitory effects ofPro suggests a functional link between elevated Pro synthesis and increasedOPPPactivity and the importance of coupling of both pathways in stimulatinggermination.     

Induction of cell spreading by substratum-adsorbed ligands directed against the cell surface

Studies were carried out to compare the spreading of baby hamster kidney (BHK) cells, which occurs by an interaction between the cells and a specific serum glycoprotein (ASF) adsorbed onto the substratum surface, with the spreading of BHK cells that occurs by an interaction between the cells and substrata coated with ligands directed at various cell surface determinants. The ligands tested were polycationic ferritin, concanavalin A (ConA) and antibody directed against BHK plasma membranes. Cell spreading onto ASF and ligand-coated substrata were similar even though different cell surface components were apparently involved. The similarities were:

1. 1. The shape of the spread cells.

2. 2. The inhibition of cell spreading by conditions that interfere with metabolic activity, block free sulfhydryl groups, or interfere with microtubules and microfilaments.

3. 3. The similar reorganization of certain cell surface antigenic determinants during cell spreading onto any of the substrata.

The results indicate that cell spreading is a general cellular response to specific cell-substratum interactions but does not depend upon binding between a unique cell surface receptor and the substratum.     

Thermodynamic aspects of cell spreading on solid substrata

To verify the validity of thermodynamic approaches to the prediction of cellular behavior, cell spreading of three different cell types on solid substrata was determined in vitro. Solid substrata as well as cell types were selected on the basis of their surface free energies, calculated from contact angle measurements. The surface free energies of the solid substrata ranged from 18–116 erg cm⁻². To measure contact angles on cells, a technique was developed in which a multilayer of cells was deposited on a filter and air dried. Cell surface free energies ranged from 60 erg cm⁻² for fibroblasts, and 57 for smooth muscle cells, to 91 for HeLa epithelial cells. After adsorption of serum proteins, cell surface free energies of all three cell types converged to approx 74 erg cm⁻². The spreading of these cell types from RPMI 1640 medium on the various solid substrata showed that both in the presence and in the absence of serum proteins in the medium, cells spread poorly on low energy substrata (Y _s <50 erg cm⁻²), whereas good cell spreading was observed on the higher energy substrata. Calculations of the interfacial free energy of adhesion (ΔF _adh) show that ΔF _adh decreases with increasingY _s , and equals zero around 45 erg cm⁻² for all three cell types in the presence of serum proteins and for HeLa epithelium cells in the absence of serum proteins. This explains the spreading of these cells on the various substrata upon a thermodynamic basis. The results clearly show that substratum surface free energy has a predictive value with respect to cell spreading in vitro, both in the presence and absence of serum proteins. It is noted, however, that interfacial thermodynamics fail to explain the behavior of fibroblasts and smooth muscle cells in the absence of serum proteins, most likely because of the relatively high surface charges of these two cell types.