Completion of the Viking labeled release experiment on Mars.

The final Labeled Release (LR) cycle on each Viking lander tested a surface sample that had been stored for several months at approximately 10 degrees C prior to the onset of the active sequence. At each lander site, activity was strongly diminished. This thermal sensitivity of the active agent on the surface of Mars is consistent with a biological explanation of the LR experiment. At the end of one of these cycles, the incubation mixture was heated to 50 degrees C to release any radioactive gas trapped in the sample matrix. The results suggest that more than one carbon substrate is involved in the LR reaction on Mars. The thermal data from the stored samples, coupled with data from previous cycles, have formed the basis for evaluation of the thermal decomposition of the Mars active agent. The slope of the resulting Arrhenius plot has been used to test the fit of other flight data and to calculate the activation energy for thermal decomposition of the Mars agent. The results and their interpretation still leave unresolved the question of whether the Mars LR data were generated by biological or chemical activity.

Laboratory simulations of the Viking labeled release experiment: kinetics following second nutrient injection and the nature of the gaseous end product.

Injection of 14C-labeled nutrient onto Mars soil produced an evolution of 14C gas in the Viking Labeled Release (LR) experiment. However, a second injection of nutrient seven days later was followed by an abrupt diminution of the amount of radioactive gas in the test cell. Simulation experiments performed in the LR Test Standards Module (TSM) have yielded a plausible explanation for this diminution. Radioactive carbon gases were injected into the TSM test cell in the presence and absence of two Mars analog soils. After equilibration, water was injected and its effect observed. The results indicate that the flight data following second nutrient injection can be explained on a physico-chemical basis involving a carbon dioxide/water/soil equilibrium in the test cell. The results also suggest that the gaseous end product of the Labeled Release reaction on Mars is more likely carbon dioxide than carbon monoxide.

Life on Mars? The Viking labeled release experiment.

Viking radiorespirometry ("Labeled Release" [LR]) experiments conducted on surface material obtained at two sites on Mars have produced results which on Earth would clearly establish the presence of microbial activity in the soil. However, two factors on Mars keep the question open. First, the intense UV flux striking Mars has given rise to several theories postulating the production of highly oxidative compounds. Such compounds might be responsible for the observed results. Second, the molecular analysis experiment has not found organic matter in the Mars surface material, and therefore, does not support the presence of roganisms. However, sensitivity limitations of the organic analysis instrument could permit as many as one million terrestrial type bacteria to go undetected. Terrestrial experiments with UV irradiation of Mars Analog Soil did not produce Mars type LR results. Gamma irradiation of silica gel did produce positive results, but not mimicking those on Mars. The life question remains open.

Evidence for incorporation of thymidine into deoxyribonucleic acid in airborne bacterial cells.

As part of an effort to discover whether bacteria might propagate within airborne particles, we studied the incorporation of thymidine into the trichloroacetic acid-insoluble fraction of airborne cells of Serratia marcescens to seek evidence of the possible formation of new DNA. Two aerosols, one of S. marcescens and another of [3H]thymidine ([3H]dT) suspended in growth medium were caused to aggregate in air just prior to directing the aerosols into rotating-drum aerosol storage chambers. The age of the S. marcescens culture and other conditions for maximizing ([3H]dT) uptake were selected on the basis of prior in vitro trials. With 10-h cultures and addition of 2-deoxyadenosine to the [3H]dT, we showed that [3H]dT is incorporated into the trichloroacetic acid-insoluble fraction of cells recovered 6 h after aerosols were stored under the conditions of high humidity and 30 degrees C. Tests conducted in the same manner with Formalin-killed S. marcescens ruled out the possibility of adsorptive carry-over of [3H]dT. As much as 20 times more activity was found in the trichloroacetic acid-insoluble fraction of live cells than of dead cells.

Viking labeled release biology experiment: interim results.

This report summarizes all results of the labeled release life detection experiment conducted on Mars prior to conjunction. Tests at both landing sites provide remarkably similar evolution of radioactive gas upon addition of a radioactive nutrient to the Mars sample. The "active" agent in the Mars sample is stable to 18 degrees C, but is substantially inactivated by heat treatment for 3 hours at 50 degrees C and completely inactivated at 160 degrees C, as would be anticipated if the active response were caused by microorganisms. Results from test and heat-sterilized control Mars samples are compared to those obtained from terrestrial soils and from a lunar sample. Possible nonbiological explanations of the Mars data are reviewed along with plans for resolution of the Mars data. Although such explanations of the labeled release data depend on ultraviolet irradiation, the labeled release response does not appear to depend on recent direct ultraviolet activation of surface material. Available facts do not yet permit a conclusion regarding the existence of life on Mars. Plans for conclusion of the experiment are discussed.

The viking biological investigation: preliminary results.

Three different types of biological experiments on samples of martian surface material ("soil") were conducted inside the Viking lander. In the carbon assimilation or pyrolytic release experiment, (14)CO(2) and (14)CO were exposed to soil in the presence of light. A small amount of gas was found to be converted into organic material. Heat treatment of a duplicate sample prevented such conversion. In the gas exchange experiment, soil was first humidified (exposed to water vapor) for 6 sols and then wet with a complex aqueous solution of metabolites. The gas above the soil was monitored by gas chromatography. A substantial amount of O(2) was detected in the first chromatogram taken 2.8 hours after humidification. Subsequent analyses revealed that significant increases in CO(2) and only small changes in N(2) had also occurred. In the labeled release experiment, soil was moistened with a solution containing several (14)C-labeled organic compounds. A substantial evolution of radioactive gas was registered but did not occur with a duplicate heat-treated sample. Alternative chemical and biological interpretations are possible for these preliminary data. The experiments are still in process, and these results so far do not allow a decision regarding the existence of life on the plonet Mars.

Purification and properties of the cathepsin D types proteinase from beef and rabbit lung and its identification in macrophages.

The acid-acting proteinase, cathepsin D (EC 3.4.4.23), was purified from extracts of homogenized rabbit lung and beef lung by autolysis at acid pH, acetone and ammonium sulfate fractionation, column chromatography, and isoelectric focusing. Four isoenzymes were obtained from each source. With acid hemoglobin as the substrate, the proteinase from rabbit lung had a pH optimum of 3.0 and that from beef lung had a pH optimum of 3.6. Their activity was not affected by thiol reagents or by Fe(2+), Mn(2+), or Mg(2+). One isoenzyme from rabbit lung was used to immunize a goat, and one from beef lung was used to immunize a rabbit. In immunoelectrophoresis, each resulting antiserum formed a single precipitin line with its homologous enzyme. They cross-reacted with the other three isoenzymes from the same species, but not with any isoenzyme from the other species. At high concentrations, each antiserum completely inhibited the proteolytic activity of its homologous enzyme. The antiserum against rabbit lung cathepsin D also inhibited the proteolytic activity of rabbit peritoneal and pulmonary macrophages. In limited quantities, this antiserum has now been made commercially available and is being used with labeled antibody techniques to identify under a microscope the presence of cathepsin D in macrophages and to study its role in the pathogenesis of tuberculosis.

Mutagenic action, loss of transforming activity, and inhibition of deoxyribonucleic acid template activity in vitro caused by chemical linkage of carcinogenic polycyclic hydrocarbons to deoxyribonucleic acid.

In a solvent system of 10(-2)m phosphate buffer (pH 6.8)-ethanol (2:1, v/v) and in an iodine-induced reaction, the polycyclic hydrocarbons [(3)H]3,4-benzpyrene and [(3)H]3,4-BP/[(3)H]9, 10-dimethyl-1,2-benzanthracene (DMBA) can be covalently linked to deoxyribonucleic acid (DNA) at room temperature. By stepwise addition of the hydrocarbon and repeating the reaction two to three times after isolating the hydrocarbon DNA adduct, it was possible to introduce as many as one covalently bound hydrocarbon molecule per 100 nucleotide bases. When 3,4-BP and DMBA were linked in this way to biologically active transforming DNA of Bacillus subtilis, they caused (i) reduction of the transforming activity of the DNA accompanied by (ii) significant increases in the frequency of forward mutations. The majority of these hydrocarbon-induced mutations were not able to revert spontaneously. These samples of DNA covalently linked with hydrocarbons showed much lower levels of survival of biological activity when assayed in recipient strains (hcr(-)) which are known to be deficient in the enzymes required for repair of ultraviolet light-induced damage to DNA. 3,4-BP covalently linked to calf thymus DNA at a level of approximately one hydrocarbon molecule per 330 bases was shown to cause up to 80% inhibition of the in vitro transcription of the DNA by highly purified ribonucleic acid polymerase prepared from Micrococcus luteus under the experimental condition of template saturation. The presence of 3,4-BP and DMBA molecules covalently bound to B. subtilis DNA samples was also found to prevent complete denaturation of the bihelical structure of certain DNA molecules and thus appears to effect a cross-link in these DNA molecules.