Astrochemistry: Complex organic matter in Titan's aerosols?

On 14 January 2005, the Huygens probe entered the atmosphere of Titan after a seven-year interplanetary flight as part of the Cassini mission to Saturn. Huygens carried, among other instruments, an aerosol collection and pyrolysis (ACP) device. Its designers, Israël et al., now claim to have detected complex organic matter in two aerosol samples collected at different altitudes (130-35 km and 25-20 km, respectively), on the basis of their detection of ammonia (NH3) and hydrogen cyanide (HCN) when the sample oven was heated to 600 degrees C. However, the authors' remarkable conclusions, which would have far-reaching consequences for our understanding of the chemical environment prevailing on Saturn's largest moon, are not supported by their limited data.

Astrochemistry: Complex organic matter in Titan's aerosols?

On 14 January 2005, the Huygens probe entered the atmosphere of Titan after a seven-year interplanetary flight as part of the Cassini mission to Saturn. Huygens carried, among other instruments, an aerosol collection and pyrolysis (ACP) device. Its designers, Israël et al., now claim to have detected complex organic matter in two aerosol samples collected at different altitudes (130-35 km and 25-20 km, respectively), on the basis of their detection of ammonia (NH3) and hydrogen cyanide (HCN) when the sample oven was heated to 600 degrees C. However, the authors' remarkable conclusions, which would have far-reaching consequences for our understanding of the chemical environment prevailing on Saturn's largest moon, are not supported by their limited data.

Characterization by tandem mass spectrometry of structural modifications in proteins.

Tandem mass spectrometry can be used to solve a number of protein structural problems that are not amenable to conventional methods for amino acid sequencing. Typical problems that use this approach involve characterization of peptides with blocked amino termini or peptides that have been otherwise posttranslationally processed, such as, by phosphorylation or sulfation. The structure and homogeneity of synthetic peptides can also be evaluated. Since peptides can be selectively characterized in the presence of other peptides or contaminants, the need for extensive purification is reduced or eliminated.

Water pollution: organic compounds in the Charles River, Boston.

The major lipophilic organic compounds present in water collected during November and December 1971 from the Charles River Basin (Boston) are as follows: normal alkanes (C(15) to C(31)), alkyl naphthalenes, alkyl anthracenes or phenanthrenes, pyrene, fluoranthene, dibutyl phthalate, and di(2-ethylhexyl) phthalate. The concentration of the naphthalenes (determined by liquid chromatography) correlates with the effective storm-water runoff content of the river. These data suggest a multiplicity of sources ranging from indigenous biological materials to automobile exhaust condensate.

Composition of the atmosphere at the surface of Mars: detection of argon-36 and preliminary analysis.

The composition of the martian atmosphere was determined by the mass spectrometer in the molecular analysis experiment. The presence of argon and nitrogen was confirmed and a value of 1 to 2750 +/- 500 for the ratio of argon-36 to argon-40 was established. A preliminary interpretation of these results suggests that Mars had a slightly more massive atmosphere in the past, but that much less total outgassing has occurred on Mars than on Earth.

Sulfur-containing nucleoside from yeast transfer ribonucleic acid: 2-thio-5(or 6)-uridine acetic acid methyl ester.

A nucleoside, isolated from yeast transfer RNA, has been assigned the structure 2-thio-5-uridine acetic acid methyl ester on the basis of high-esolution mass spectrometry, chemical properties, and ultraviolet spectra. The alternate 6-substituted isomeric structure cannot yet be completely ruled out.

Acidic components of green river shale identified by a gas chromatography-mass spectrometry-computer system.

A system consisting of a gas chromatograph coupled to a mass spectrometer and computer has been used to characterize the extractable acidic components of Green River shale. This system accumulates mass spectra at every point of the gas chromatogram in a permanent form which permits one to observe mass spectra of minor as well as major constituents. One minor component, whose identification was confirmed by synthesis, was 6,10,14-trimethyl-pentadecanoic acid.

Phenethyl alcohol and tryptophol: autoantibiotics produced by the fungus Candida albicans.

Two autoantibiotics produced by the fungus Candida albicans have been identified, with the aid of mass spectrometry, as 2-phenylethanol and 3-beta-hydroxyethylindole. The cyclodipeptides, cyclo(proline-leucine) and cyclo(proline-phenylalanine) were also isolated from cultures of Candida albicans, but did not inhibit the growth of the fungus.

Search for organic material in lunar fines by mass spectrometry.

Three kinds of experiments were performed in an effort to detect and identify organic compounds present in the lunar material (fines 10,086): (i) vaporization of the volatilizable components directly into the ion source of a high-resolution mass spectrometer, and (ii and iii) extraction of the material with organic solvents before and after dissolving most of the inorganic substrate in hydrochloric and hydrofluoric acid. The extracts were investigated by a combination of gas chromatography and mass spectrometry. Although a number of organic compounds or compound types have been detected, none appears to be indigenous to the lunar surface.

The Atmosphere of Mars near the Surface: Isotope Ratios and Upper Limits on Noble Gases.

Several new analyses of the martian atmosphere have been carried out with the mass spectrometer in the molecular analysis experiment. The ratios of abundant isotopes of carbon and oxygen are within 10 percent of terrestrial values, whereas nitrogen-15 is considerably enriched on Mars. We have detected argon-38 and set new limits on abundances of krypton and xenon. The limit on krypton is sufficiently low to suggest that the inventories of volatile substances on Mars and on Earth may be distinctly different.

The atmosphere of Mars: detection of krypton and xenon.

Krypton and xenon have been discovered in the martian atmosphere with the mass spectrometer on the second Viking lander. Krypton is more abundant than xenon. The relative abundances of the krypton isotopes appear normal, but the ratio of xenon-129 to xenon-132 is enhanced on Mars relative to the terrestrial value for this ratio. Some possible implications of these findings are discussed.

N-nitrosamines in the rubber and tire industry.

Airborne N-nitrosomorpholine (0 to 27 micrograms per cubic meter) was found in two of four rubber industry factories. N-Nitrosodimethylamine was also found in two factories, but at lower levels. These findings may be relevant to the reported increased risk of certain types of cancer in rubber workers in some of the same areas where the N-nitrosomorpholine levels were highest.

Search for organic and volatile inorganic compounds in two surface samples from the chryse planitia region of Mars.

Two surface samples collected from the Chryse Planitia region of Mars were heated to temperatures up to 500 degrees C, and the volatiles that they evolved were analyzed with a gas chromatograph-mass spectrometer. Only water and carbon dioxide were detected. This implies that organic compounds have not accumulated to the extent that individual components could be detected at levels of a few parts in 10(9) by weight in our samples. Proposed mechanisms for the accumulation and destruction of organic compounds are discussed in the light of this limit.

Selective inhibition of amino-terminal methionine processing by TNP-470 and ovalicin in endothelial cells.

BACKGROUND: The angiogenesis inhibitors TNP-470 and ovalicin potently suppress endothelial cell growth. Both drugs also specifically inhibit methionine aminopeptidase 2 (MetAP2) in vitro. Inhibition of MetAP2 and changes in initiator methionine removal in drug-treated endothelial cells have not been demonstrated, however. RESULTS: Concentrations of TNP-470 sufficient to inactivate MetAP2 in intact endothelial cells were comparable to those that inhibited cell proliferation, suggesting that MetAP2 inhibition by TNP-470 underlies the ability of the drug to inhibit cell growth. Both drug-sensitive and drug-insensitive cell lines express MetAP1 and MetAP2, indicating that drug sensitivity in mammalian cells is not simply due to the absence of compensating MetAP activity. With a single exception, detectable protein N-myristoylation is unaffected in sensitive endothelial cells treated with TNP-470, so MetAP1 activity can generally compensate when MetAP2 is inactive. Analysis of total protein extracts from cells pulse-labeled with [(35)S]-methionine following TNP-470 treatment revealed changes in the migration of several newly synthesized proteins. Two of these proteins were identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cyclophilin A. Purification and amino-terminal sequencing of GAPDH from TNP-470-treated cells revealed partial retention of its initiator methionine, indicating that methionine removal from some, but not all, proteins is affected by MetAP2 inactivation. CONCLUSIONS: Amino-terminal processing defects occur in cells treated with TNP-470, indicating that inhibition of MetAP2 by the drug occurs in intact cells. This work renders plausible a mechanism for growth inhibition by TNP-470 as a consequence of initiator methionine retention, leading to the inactivation of as yet unidentified proteins essential for endothelial cell growth.

Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM-1470 and ovalicin.

BACKGROUND: Angiogenesis, the formation of new blood vessels, is essential for tumor growth. The inhibition of angiogenesis is therefore emerging as a promising therapy for cancer. Two natural products, fumagillin and ovalicin, were discovered to be potent inhibitors of angiogenesis due to their inhibition of endothelial cell proliferation. An analog of fumagillin, AGM-1470, is currently undergoing clinical trials for the treatment of a variety of cancers. The underlying molecular mechanism of the inhibition of angiogenesis by these natural drugs has remained unknown. RESULTS: Both AGM-1470 and ovalicin bind to a common bifunctional protein, identified by mass spectrometry as the type 2 methionine aminopeptidase (MetAP2). This protein also acts as an inhibitor of eukaryotic initiation factor 2alpha (elF-2alpha) phosphorylation. Both drugs potently inhibit the methionine aminopeptidase activity of MetAP2 without affecting its ability to block elF-2alpha phosphorylation. There are two types of methionine aminopeptidase found in eukaryotes, but only the type 2 enzyme is inhibited by the drugs. A series of analogs of fumagillin and ovalicin were synthesized and their potency for inhibition of endothelial cell proliferation and inhibition of methionine aminopeptidase activity was determined. A significant correlation was found between the two activities. CONCLUSIONS: The protein MetAP2 is a common molecular target for both AGM-1470 and ovalicin. This finding suggests that MetAP2 may play a critical role in the proliferation of endothelial cells and may serve as a promising target for the development of new anti-angiogenic drugs.

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.

The primary structure and properties of thioltransferase (glutaredoxin) from human red blood cells.

Thioltransferase (glutaredoxin) was purified from human red blood cells essentially as described previously (Mieyal JJ et al., 1991a, Biochemistry 30:6088-6097). The primary sequence of the HPLC-pure enzyme was determined by tandem mass spectrometry and found to represent a 105-amino acid protein of molecular weight 11,688 Da. The physicochemical and catalytic properties of this enzyme are common to the group of proteins called glutaredoxins among the family of thiol:disulfide oxidoreductases that also includes thioredoxin and protein disulfide isomerase. Although this human red blood cell glutaredoxin (hRBC Grx) is highly homologous to the 3 other mammalian Grx proteins whose sequences are known (calf thymus, rabbit bone marrow, and pig liver), there are a number of significant differences. Most notably an additional cysteine residue (Cys-7) occurs near the N-terminus of the human enzyme in place of a serine residue in the other proteins. In addition, residue 51 of hRBC Grx displayed a mixture of Asp and Asn. This result is consistent with isoelectric focusing analysis, which revealed 2 distinct bands for either the oxidized or reduced forms of the protein. Because the enzyme was prepared from blood combined from a number of individual donors, it is not clear whether this Asp/Asn ambiguity represents inter-individual variation, gene duplication, or a deamidation artifact of purification.

C-terminal specific protein degradation: activity and substrate specificity of the Tsp protease.

The activity of Tsp, a periplasmic endoprotease of Escherichia coli, has been characterized by assaying the cleavage of protein and peptide substrates, determining the cleavage sites in several substrates, and investigating the kinetics of the cleavage reaction. Tsp efficiently cleaves substrates that have apolar residues and a free alpha-carboxylate at the C-terminus. Tsp cleaves its substrates at a discrete number of sites but with rather broad primary sequence specificity. In addition to preferences for residues at the C-terminus and cleavage sites, Tsp displays a preference for substrates that are not stably folded: unstable variants of Arc repressor are better substrates than a hyperstable mutant, and a peptide with little stable structure is cleaved more efficiently than a protein substrate. These data are consistent with a model in which Tsp cleavage of a protein substrate involves binding to the C-terminal tail of the substrate, transient denaturation of the substrate, and then recognition and hydrolysis of specific peptide bonds.

Kinetic equivalence of stable-isotope-labeled and unlabeled phenytoin.

Stable isotope labeling (SIL) of a drug results in a higher molecular weight than that of the unlabeled drug. SIL tracer doses can be quantitated separately from unlabeled drug by gas chromatography-mass spectrometry (GC-MS) without exposing the patient to radiation. The higher molecular weight of SIL drug could cause a higher energy of activation for (and slowing of) metabolic reactions ("isotope effect"). To evaluate possible isotope effect, three dogs and three men were infused with a mixture containing equal amounts of SIL (2-13C-1,3-15N2) and unlabeled phenytoin (PHT). Plasma and urine were collected at regular intervals. Concentrations of SIL and unlabeled PHT and HPPH (the major metabolite of PHT) were determined by GC-MS. Within each subject there was no trend for concentrations of SIL PHT or HPPH to be higher or lower than concentrations of their unlabeled analogs (greater than 0.20 to 0.90). There was no difference in the distribution and elimination half-lifes (t 1/2s), volume of distribution, volume of central compartment, or clearance of the two forms of PHT. Thus, no isotope effect was found.

Chemical and in vivo studies of the anion oxo[N,N'-ethylenebis(2-mercaptoacetimido)]Technetate(V).

Studies of the anionic coordination complex 99Tc-oxo[N,N'-ethylene-bis(2-mercaptoacetimido)]technetate(V) ([TcO(ema)]-) are described. Syntheses performed both at carrier levels (10(-5)M) and with no carrier added (less than 10(-8)M) indicate that the complex is formed virtually quantitatively from pertechnetate ion over this range. Tissue distributions in normal rats are similar at both concentrations up to one hour after administration. It has been shown--using a combination of high-pressure liquid chromatography and field-desorption mass spectrometry--that the anion is excreted unchanged into both urine and bile. The effectiveness of this N2S2 donor set in sequestering Tc-99m, and the in vivo stability of the resulting complex, suggest that modified chelates of this structural class could provide a series of useful diagnostic agents.