Gas chromatography for in situ analysis of a cometary nucleus. IV. Study of capillary column robustness for space application.

As part of the development of the European Space Agency Rosetta space mission to investigate a cometary nucleus, the selection of columns dedicated to the gas chromatographic subsystem of the Cometary Sampling and Composition (COSAC) experiment was achieved. Once the space probe launched, these columns will be exposed to the harsh environmental constraints of space missions: vibrations, radiation (by photons or energetic particles), space vacuum, and large temperature range. In order to test the resistance of the flight columns and their stationary phases, the columns were exposed to these rough conditions reproduced in the laboratory. The comparison of the analytical performances of the columns, evaluated prior and after the environmental tests, demonstrated that all the columns withstand space constraints, and that their analytical properties were preserved. Therefore, all the selected capillary columns, even having porous layer or chiral stationary phases, were qualified for space exploration.

Gas chromatography for in situ analysis of a cometary nucleus III. Multi-capillary column system for the cometary sampling and composition experiment of the Rosetta lander probe.

The cometary sampling and composition (COSAC) experiment is one of the principal experiments of the surface lander probe of the European Space Agency Rosetta mission to be launched in January 2003. The instrument is designed for the in situ chemical analysis of a cometary nucleus as the details of the nucleus composition are of primary importance for understanding both the formation of the solar system, and the origin of life on Earth. The COSAC experiment consists of an evaporation/pyrolysis device and two analytical systems: a multi-column gas chromatograph and a high-resolution time-of-flight mass spectrometer which may either be operated alone or in a coupled mode. The gas chromatograph includes five general purpose chromatographic columns and three chiral ones, all mounted in parallel. Taking into account the chemical species potentially present in the cometary nucleus as well as the space constraints, a set of five complementary columns was selected to perform the separation and identification of the compounds present in the cometary nucleus. This set of columns includes a carbon molecular sieve porous-layer open tubular (PLOT) column used for the separation of both the noble and other permanent gases, and the C1-C2 hydrocarbons. A second PLOT column uses a divinylbenzene-ethylene glycol-dimethylacrylate porous polymer as stationary phase for the analysis of a wide range of C1-C2 organic molecules, Two complementary wall-coated open tubular (WCOT) columns with polydimethylsiloxane (PDMS) liquid stationary phases, one containing cyanopropyl-phenylsiloxane and the other diphenylsiloxane groups, are designed to target the same range of organic compounds (C3-C7) which could be representative of the widest range of cometary compounds. A third WCOT column with an apolar stationary phase made of non-substituted PDMS is used for the separation and identification of higher-molecular-mass compounds (up to C10) and aromatic species (monoaromatic and polyaromatic). This paper describes these five general-purpose capillary PLOT and WCOT columns, selected to be used in the COSAC GC system. The analytical capabilities are examined with a special emphasis on the exobiological and planetological implications.

Amino acids from ultraviolet irradiation of interstellar ice analogues.

Amino acids are the essential molecular components of living organisms on Earth, but the proposed mechanisms for their spontaneous generation have been unable to account for their presence in Earth's early history. The delivery of extraterrestrial organic compounds has been proposed as an alternative to generation on Earth, and some amino acids have been found in several meteorites. Here we report the detection of amino acids in the room-temperature residue of an interstellar ice analogue that was ultraviolet-irradiated in a high vacuum at 12 K. We identified 16 amino acids; the chiral ones showed enantiomeric separation. Some of the identified amino acids are also found in meteorites. Our results demonstrate that the spontaneous generation of amino acids in the interstellar medium is possible, supporting the suggestion that prebiotic molecules could have been delivered to the early Earth by cometary dust, meteorites or interplanetary dust particles.

Conception of the 'Chirality-Experiment' on ESA's mission ROSETTA to comet P46/Wirtanen.

In the last years extraterrestrial scenarios for the origin of homochirality in biological structures received considerable attention in the topical literature: Rubenstein and Bonner postulated a rapidly rotating neutron star emitting circularly polarised synchrotron radiation responsible for the first asymmetric synthesis; the group of Bailey published the observation of circular polarisation caused by Mie scattering from aligned dust grains in the Orion OMC-1 star-formation region that might provide an enantioselective effect on prochiral or racemic organic molecules. Rikken and Raupach observed a magnetochiral effect and considered extraterrestrial magnetic fields of sufficient strengths to introduce biomoleculars parity violation. With the aim to investigate these hypotheses among other theories describing the origin of biological asymmetry, our laboratory participates in the conception and development of ROSETTA's COSAC Experiment, that is designed to identify organic molecules in the cometary matter in situ. Within COSAC's 'Chirality Module' enantiomers will be separated gas chromatographically with the help of capillary columns coated with chirally active liquid films. This technique will allow the separation of specific chiral organic compounds out of the analysed cometary matter into their enantiomeric constituents. Both thermo conductivity and mass spectrometric detectors will be used to determine each enantiomer's amount and therefore the corresponding enantiomeric excesses. As a consequence of COSAC's 'Chirality-Experiment' far-reaching results are expected to investigate the various hypotheses about the first asymmetric synthesis.

Stereochemical investigation of cometary matter onboard the Rosetta Lander.

At present the European Space Agency is working on one of its Cornerstone Missions, named "Rosetta", to be launched in January 2003 for a visit to comet 46P/Wirtanen2 in 2011. The Rosetta spacecraft will carry a small subsatellite, the Rosetta Lander3 (RoLand), to be detached from the orbiter and land on the surface of the comet's nucleus. One of our main scientific interests is to find out whether chiral organic compounds in cometary matter brought to the Earth by cometary impacts might have had, due to corresponding enantiomeric excesses, a seed function in determining the handedness which is characteristic of homochiral compounds employed by life on Earth. For this reason we have developed an Experiment for the ROSETTA mission, named Cometary Sampling and Composition Experiment (COSAC).

Enantiomer separation of hydrocarbons in preparation for ROSETTA's "chirality-experiment".

Until now the favored method for separating racemic pairs of underivatized alcohols, diols, and phenylsubstituted amines has been gas chromatography on cyclodextrin phases. However, certain enantiomers of saturated chiral hydrocarbons could not be resolved in this way because they lack the functional groups necessary to undergo "intensive" diastereomeric interactions with the cyclodextrins. The present study describes a gas-chromatographic technique for resolution of saturated aliphatic hydrocarbons into their enantiomers and presents a brief discussion of the possible applications. The (enantiomer) separations were performed in preparation for the Cometary Sampling and Composition Experiment on board the cometary lander RoLand, part of ESA's cornerstone mission ROSETTA. This experiment has been designed to investigate the hypotheses that biomolecular asymmetry has an interstellar origin and to separate and identify a wide range of organic enantiomers in situ on the surface of a comet's nucleus.

Construction of a new multi-turn time-of-flight mass spectrometer.

A new type of multi-turn time-of-flight mass spectrometer was constructed, consisting of four cylindrical electric sectors and 28 electric quadrupole lenses, the size of the vacuum chamber being 60 x 70 x 20 cm. It was demonstrated that the mass resolution can be increased according to the number of cycles of the ions through the ion optical system.

Gas chromatography for in situ analysis of a cometary nucleus: characterization and optimization of diphenyl/dimethylpolysiloxane stationary phases.

The development of a gas chromatograph for the cometary sampling and composition (COSAC) experiment is described in the context of the preparation for the European Space Agency (ESA) Mission Rosetta for investigation of a cometary nucleus. COSAC is one out of ten experiments on the Rosetta Lander. Its scientific goal is to analyze in situ the chemical composition of the volatile constituents of the nucleus of the target comet P/Wirtanen. Constituted of several (up to eight) capillary wall-coated and porous-layer open tubular columns operating in parallel, the GC system is designed to separate and identify both organic and inorganic compounds which evolve from the comet naturally or are obtained from cometary samples through stepwise heating in a miniaturized pyrolizer. In this first part of our study, dimethylpolysiloxane (DMPS) stationary phases with increasing percentages of diphenyl substituted group (DP) have been investigated. A coupled experimental and theoretical approach has been taken in order to predict chromatographic data. By the use of a four-point experimental calibration (0 to 65% diphenyl group) in conjunction with Pro ezGC modeling software, results in prediction of multicomponent chromatograms with a mean error less than 5% for each compound retention time were obtained, irrespective of the stationary phase's diphenyl content and column physical parameters. The possibility to associate such phases is illustrated by the evolution of coelutions obtained on a non-polar (100% DMPS) and a medium polar (65% DP-DMPS) stationary phase, respectively. This study showed that with a small number of well tuned DP-DMPS columns, the separation and identification of most of the targeted compounds can be achieved with a minimum amount of coelutions and within the experiment requirements.

Molecular parity violation via comets?

Recent theoretical and experimental investigations referring to the origin of homochirality are reviewed and integrated into the hitherto known state of the art. Attention is directed to an extraterrestrial scenario, which describes the interaction of circularly polarized synchrotron radiation with interstellar organic matter. Following this Bonner-Rubenstein hypothesis, optically active molecules could be transferred to Earth via comets. We plan to identify any enantiomeric enhancement in organic molecules of the cometary matter in situ. The present preliminary experimental study intends to optimize gas-chromatographic conditions for the separation of racemates into their enantiomer constituents on the surface of the comet 46P/Wirtanen. Underivatized racemic pairs of alcohols, diols, and phenyl-substituted amines have been separated with the help of a stationary trifluoroacetyl-cyclodextrin phase. We are still developing a technique that will enable us to detect any enantiomeric enhancement of specific simple organic molecules both in cometary or Martian matter in situ and in meteorites found on Earth.

[Effect of various vitamin E supply sources to fattening pigs on pork and bacon quality depending on time of storage]. / Einfluss unterschiedlich hoher Vitamin-E-Gaben an Mastschweine auf die Fleisch- und Speckqualität in Abhängigkeit von der Lagerung.

99 individually kept, fattening pigs (castrated males) were divided into 3 groups. 33 animals each were supplemented with 0, 0.5 or 1.0 g alpha-tocopheryl acetate per day last 21 days before slaughtering. Samples from blood, liver, bacon, and muscle were taken to determine vitamin E content by HPLC depending on time of storing. TBARS values of muscle and bacon, induction-time of bacon ("Rancimat"), pH, drip loss, and color of muscle were determined as further criterions of quality. The Vitamin E supply increased significantly the vitamin E content of all samples (e.g., serum: 1.5, 2.4, and 2.7 mg/kg; liver: 3.8, 5.6, and 7.0 mg/kg for 0, 0.5, or 1.0 g per animal per day, respectively). Vitamin E content of pork decreased depending on time of storing (3.9, 6.2, and 7.8 mg/kg vers. 1.9, 4.1, and 5.0 mg/kg after 29 weeks of freeze storing). Storing time had no significant influence on vitamin E content of bacon. Vitamin E supply (esp. 1.0 g daily) decreased TBARS values, increased time of induction and improved meat color, but did not influence pH and drip loss of porc significantly.

TIF-IC, a factor involved in both transcription initiation and elongation of RNA polymerase I.

We have characterized a transcription factor from Ehrlich ascites cells that is required for ribosomal gene transcription by RNA polymerase I (Pol I). This factor, termed TIF-IC, has a native molecular mass of 65 kDa, associates with Pol I, and is required both for the assembly of Sarkosyl-resistant initiation complexes and for the formation of the first internucleotide bonds. In addition to its function in transcription initiation, TIF-IC also plays a role in elongation of nascent RNA chains. At suboptimal levels of TIF-IC, transcripts with heterogeneous 3' ends are formed which are chased into full-length transcripts by the addition of more TIF-IC. Moreover, on a tailed template, which allows initiation in the absence of auxiliary factors, TIF-IC was found to stimulate the overall rate of transcription elongation and suppress pausing of Pol I. Thus TIF-IC appears to serve a function similar to the Pol II-specific factor TFIIF which is required for Pol II transcription initiation and elongation.

The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation.

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.

Trans-acting factors involved in species-specificity and control of mouse ribosomal gene transcription.

Faithful and efficient transcription initiation at the mouse ribosomal gene promoter requires besides RNA polymerase I (pol I) four polypeptide trans-acting factors, termed TIF-IA, TIF-IB, TIF-IC, and mUBF. We have partially purified these proteins from cultured Ehrlich ascites cells and show that in the presence of TIF-IA and TIF-IB, pol I directs very low amounts of specific transcripts. Neither TIF-IC nor mUBF on their own significantly stimulate the efficiency of template utilization. However, both factors together strongly activate transcription. Interestingly, factor TIF-IB - the murine homologue of human SL1 - fails to program a human extract to transcribe the murine template, but requires its homologous RNA polymerase I. This finding implicates that not only some rDNA transcription factors but also pol I exhibits species-specific differences. The growth-related factor TIF-IA, on the other hand, stimulates both mouse and human rDNA transcription. This regulatory factor whose amount or activity fluctuates according to the proliferation rate of the cells, is functionally inactivated by antibodies against cdc2 protein kinase. This result together with the observation that transcription is stimulated by ATP-gamma S, an ATP analogue which is a substrate for protein kinases but not for protein phosphatases, strongly suggests that post-translational protein modification is involved in rDNA transcription regulation.

A growth-dependent transcription initiation factor (TIF-IA) interacting with RNA polymerase I regulates mouse ribosomal RNA synthesis.

Control of mouse ribosomal RNA synthesis in response to extracellular signals is mediated by TIF-IA, a regulatory factor whose amount or activity correlates with cell proliferation. Factor TIF-IA interacts with RNA polymerase I (pol I), thus converting it into a transcriptionally active holoenzyme, which is able to initiate specifically at the rDNA promoter in the presence of the other auxiliary transcription initiation factors, designated TIF-IB, TIF-IC and UBF. With regard to several criteria, the growth-dependent factor TIF-IA behaves like a bacterial sigma factor: (i) it associates physically with pol I, (ii) it is required for initiation of transcription, (iii) it is present in limiting amounts and (iv) under certain salt conditions, it is chromatographically separable from the polymerase. In addition, evidence is presented that dephosphorylation of pol I abolishes in vitro transcription initiation from the ribosomal gene promoter without significantly affecting the polymerizing activity of the enzyme at nonspecific templates. The involvement of both a regulatory factor and post-translational modification of the transcribing enzyme provides an efficient and versatile mechanism of rDNA transcription regulation which enables the cell to adapt ribosome synthesis rapidly to a variety of extracellular signals.

Evidence for methane and ammonia in the coma of comet P/Halley.

Methane and ammonia abundances in the coma of Halley are derived from Giotto IMS data using an Eulerian model of chemical and physical processes inside the contact surface to simulate Giotto HIS ion mass spectral data for mass-to-charge ratios (m/q) from 15 to 19. The ratio m/q = 19/18 as a function of distance from the nucleus is not reproduced by a model for a pure water coma. It is necessary to include the presence of NH3, and uniquely NH3, in coma gases in order to explain the data. A ratio of production rates Q(NH3)/Q(H2O) = 0.01-0.02 results in model values approximating the Giotto data. Methane is identified as the most probable source of the distinct peak at m/q = 15. The observations are fit best with Q(CH4)/Q(H2O) = 0.02. The chemical composition of the comet nucleus implied by these production rate ratios is unlike that of the outer planets. On the other hand, there are also significant differences from observations of gas phase interstellar material.

A transcription terminator located upstream of the mouse rDNA initiation site affects rRNA synthesis.

Mouse ribosomal genes have a short sequence upstream of the transcription initiation site that is related in structure and function to the terminator boxes previously identified at the 3' end of the transcription unit. This upstream terminator recognizes the same protein factor as the 3'-terminal sites and is able to terminate RNA polymerase I transcription in vitro. S1 mapping and nucleolar run-on experiments reveal the presence of 5'-terminal spacer transcripts that are terminated at this site. These transcripts probably derive from spacer promoters, one of which has been identified approximately 2 kb upstream of the pre-rRNA start site. The interaction of a specific nuclear factor with the upstream terminator increases the efficiency of initiation, suggesting that transcription termination and initiation at the adjacent promoter work in an interrelated fashion.

A repeated 18 bp sequence motif in the mouse rDNA spacer mediates binding of a nuclear factor and transcription termination.

DNA sequences and protein factors directing termination of mouse rDNA transcription in a nuclear extract system were examined. Termination is specific and requires a sequence element AGGTCGACCAGATTANTCCG (the Sall box) that is present eight times in the spacer region downstream of the 3' end of pre-rRNA. Exonuclease III protection experiments reveal the binding of a nuclear protein to the Sall box. Deletions, insertions, and point mutations in the Sall box reduce or abolish the interaction with the nuclear factor and disrupt transcription termination. A synthetic oligonucleotide corresponding to the Sall box consensus sequence governs transcription termination in vitro, although with reduced activity. Therefore, other sequences normally surrounding the Sall box appear to contribute to the accuracy and efficiency of termination.