Does aspartic acid racemization constrain the depth limit of the subsurface biosphere?

Previous studies of the subsurface biosphere have deduced average cellular doubling times of hundreds to thousands of years based upon geochemical models. We have directly constrained the in situ average cellular protein turnover or doubling times for metabolically active micro-organisms based on cellular amino acid abundances, D/L values of cellular aspartic acid, and the in vivo aspartic acid racemization rate. Application of this method to planktonic microbial communities collected from deep fractures in South Africa yielded maximum cellular amino acid turnover times of ~89 years for 1 km depth and 27 °C and 1-2 years for 3 km depth and 54 °C. The latter turnover times are much shorter than previously estimated cellular turnover times based upon geochemical arguments. The aspartic acid racemization rate at higher temperatures yields cellular protein doubling times that are consistent with the survival times of hyperthermophilic strains and predicts that at temperatures of 85 °C, cells must replace proteins every couple of days to maintain enzymatic activity. Such a high maintenance requirement may be the principal limit on the abundance of living micro-organisms in the deep, hot subsurface biosphere, as well as a potential limit on their activity. The measurement of the D/L of aspartic acid in biological samples is a potentially powerful tool for deep, fractured continental and oceanic crustal settings where geochemical models of carbon turnover times are poorly constrained. Experimental observations on the racemization rates of aspartic acid in living thermophiles and hyperthermophiles could test this hypothesis. The development of corrections for cell wall peptides and spores will be required, however, to improve the accuracy of these estimates for environmental samples.

Prevalence and clinical relevance of local allergic rhinitis.

BACKGROUND: Evidence demonstrates the existence of local allergic rhinitis (LAR) in nonatopic patients, although its prevalence in the rhinitis population remains unknown. The aim, therefore, of this study was to evaluate the prevalence, clinical characteristics, and severity of LAR in a Spanish rhinitis population, compared with patients having classical allergic rhinitis (AR) with systemic atopy or nonallergic rhinitis (NAR). METHODS: A group of 452 adult rhinitis patients were randomly selected from a total of 3860 who attended our allergy service over 1 year. A clinical questionnaire, skin prick test (SPT), spirometry, and serum total and specific IgE (sIgE) were evaluated. A nasal allergen provocation test with multiple aeroallergens (NAPT-M), including Dermatophagoides pteronyssinus, pollens, alternaria, and dog epithelia, was performed in patients with negative SPT and serum sIgE. RESULTS: A total of 428 patients completed the study; 24 were excluded because of nasal hyper-reactivity. LAR was diagnosed in 25.7%, AR in 63.1%, and NAR in 11.2%. The LAR and AR patients had a similar clinical profile: a nonsmoking woman with severe, persistent perennial rhinitis frequently associated with conjunctivitis and asthma. More than 36% of LAR patients reported rhinitis onset in childhood. NAPT-M detected aeroallergen polysensitization in 37.3% of the LAR patients. Dermatophagoides pteronyssinus was the main sensitizing aeroallergen in LAR and AR (60% vs 54%, P > 0.05). CONCLUSIONS: Local allergic rhinitis is a prevalent entity in patients evaluated with rhinitis. Persistent and severe symptoms associated with conjunctivitis and/or asthma and polysensitization were likely to be detected in LAR and AR.

Negativization rates of IgE radioimmunoassay and basophil activation test in immediate reactions to penicillins.

BACKGROUND: Skin test sensitivity in patients with immediate allergy to penicillins tends to decrease over time, but no information is available concerning in vitro tests. We analysed the negativization rates of two in vitro methods that determine specific immunoglobulin E (IgE) antibodies, the basophil activation test using flow cytometry (BAT) and the radioallergosorbent test (RAST), in immediate allergic reactions to penicillins. METHODS: Forty-one patients with immediate allergic reactions to amoxicillin were followed up over a 4-year period. BAT and RAST were performed at 6-month intervals. Patients were randomized into groups: Group I, skin tests carried out at regular intervals; Group II, skin tests made only at the beginning of the study. RESULTS: Differences were observed between RAST and BAT (P < 0.01), the latter showing earlier negativization. Considering different haptens, significant differences for the rate of negativization were only found for amoxicillin (P < 0.05). Comparisons between Groups I (n = 10) and II (n = 31) showed a tendency to become negative later in Group I with RAST. CONCLUSIONS: Levels of specific IgE antibodies tended to decrease over time in patients with immediate allergic reactions to amoxicillin. Conversion to negative took longer for the RAST assay, although the differences were only detected with the amoxicillin hapten. Skin testing influenced the rate of negativization of the RAST assay, contributing to maintenance of in vitro sensitivity. Because of the loss of sensitivity over time, the determination of specific IgE antibodies to penicillins in patients with immediate allergic reactions must be done as soon as possible after the reaction.

An evaluation of the critical parameters for abiotic peptide synthesis in submarine hydrothermal systems.

It has been proposed that oligopeptides may be formed in submarine hydrothermal systems (SHSs). Oligopeptides have been synthesized previously under simulated SHS conditions which are likely geochemically implausible. We have herein investigated the oligomerization of glycine under SHS-like conditions with respect to the limitations imposed by starting amino acid concentration, heating time, and temperature. When 10(-1) M glycine solutions were heated at 250 degrees C for < 20 min glycine oligomers up to tetramers and diketopiperazine (DKP) were detectable. At 200 degrees C, less oligomerization was noted. Peptides beyond glycylglycine (gly2) and DKP were not detected below 150 degrees C. At 10(-2) M initial glycine concentration and below, only gly2, DKP, and gly3 were detected, and then only above 200 degrees C at < 20 min reaction time. Gly3 was undetectable at longer reaction times. The major parameters limiting peptide synthesis in SHSs appear to be concentration, time, and temperature. Given the expected low concentrations of amino acids, the long residence times and range of temperatures in SHSs, it is unlikely that SHS environments were robust sources of even simple peptides. Possible unexplored solutions to the problems presented here are also discussed.

Skin testing for immediate hypersensitivity to betalactams: comparison between two commercial kits.

INTRODUCTION: Skin testing with major and minor determinants of benzylpenicillin is the recommended standard practice to evaluate subjects with immediate hypersensitivity to betalactams. The withdrawal of these products from the market has set us back to the early days, before the introduction of reagents for in vivo testing. OBJECTIVES: To compare a recently released kit of benzylpenicillin conjugated to poly-l-lysine (PPL) and minor determinants mixture (MDM) with the previously existing kit in a positive control group of subjects sensitized to major and/or minor determinants of benzylpenicillin. METHODS: Skin tests with both kits were made in a group of positive subjects previously diagnosed with immediate hypersensitivity to penicillins and with positive results to PPL and/or MDM and in a negative control group. Radioallergosorbent test (RAST) inhibition assays with a pool of sera and individual samples were carried out to compare the inhibition capacity of PPL and MDM of both kits. RESULTS: Of 22 cases selected from our historical group, 14 were positive: eight to PPL, three to MDM and three to both. These results were equivalent for both kits. RAST inhibition studies showed similar potencies in the inhibition of PPL and MDM. CONCLUSIONS: Both tests show similar results in terms of RAST inhibition assays and skin tests sensitivity and specificity in the groups selected. The new assay can be used for the same purpose and indications as the previous test.

Extraterrestrial amino acids in Orgueil and Ivuna: tracing the parent body of CI type carbonaceous chondrites.

Amino acid analyses using HPLC of pristine interior pieces of the CI carbonaceous chondrites Orgueil and Ivuna have found that beta-alanine, glycine, and gamma-amino-n-butyric acid (ABA) are the most abundant amino acids in these two meteorites, with concentrations ranging from approximately 600 to 2,000 parts per billion (ppb). Other alpha-amino acids such as alanine, alpha-ABA, alpha-aminoisobutyric acid (AIB), and isovaline are present only in trace amounts (<200 ppb). Carbon isotopic measurements of beta-alanine and glycine and the presence of racemic (D/L approximately 1) alanine and beta-ABA in Orgueil suggest that these amino acids are extraterrestrial in origin. In comparison to the CM carbonaceous chondrites Murchison and Murray, the amino acid composition of the CIs is strikingly distinct, suggesting that these meteorites came from a different type of parent body, possibly an extinct comet, than did the CM carbonaceous chondrites.

Survival of amino acids in micrometeorites during atmospheric entry.

The delivery of amino acids by micrometeorites to the early Earth during the period of heavy bombardment could have been a significant source of the Earth's prebiotic amino acid inventory provided that these organic compounds survived atmospheric entry heating. To investigate the sublimation of amino acids from a micrometeorite analog at elevated temperature, grains from the CM-type carbonaceous chondrite Murchison were heated to 550 degrees C inside a glass sublimation apparatus (SA) under reduced pressure. The sublimed residue that had collected on the cold finger of the SA after heating was analyzed for amino acids by HPLC. We found that when the temperature of the meteorite reached approximately 150 degrees C, a large fraction of the amino acid glycine had vaporized from the meteorite, recondensed onto the end of the SA cold finger, and survived as the rest of the grains heated to 550 degrees C. alpha-Aminoisobutryic acid and isovaline, which are two of the most abundant non-protein amino acids in Murchison, did not sublime from the meteorite and were completely destroyed during the heating experiment. Our experimental results suggest that sublimation of glycine present in micrometeorite grains may provide a way for this amino acid to survive atmospheric entry heating at temperatures > 550 degrees C; all other amino acids apparently are destroyed.

[Differential characteristics of AIDS in foreign immigrants]. / Características diferenciales del sida en inmigrantes extranjeros.

BACKGROUND: AIDS in foreign migrants can have specific characteristics compared with that of Spanish patients. METHODS: Cross-sectional study, including cases from the Barcelona AIDS register diagnosed from 1988 to 1998. Global characteristics of migrants are compared with those of Spanish patients. Adjusted odds ratio (OR) with 95% confidence interval (CI) were calculated through logistic regression. A formal analysis was also performed. RESULTS: Among the 4,663 patients included, 5% were migrants. 33% of patients were diagnosed with AIDS 10 years after having migrated to Spain. They differentiate at the multivariate level by sex (OR [male]: 1.70; CI: 1.10-2.64), district of residence (OR [district I]: 2.07, IC: 1.50-2.87) and transmission pattern (OR [other than injecting drug users]: 4.08; IC: 2.77-5.99). No significant differences in survival were showed. CONCLUSIONS: The percentage of migrants among AIDS cases in Barcelona is low. Migrants are more frequently men, more often live in the inner city and are less frequently injecting drug users.

Características diferenciales del SIDA en inmigrantes extranjeros / Differential characteristics of AIDS in foreign immigrants in Spain

Objetivo: Determinar las características diferenciales del sida en inmigrantes extranjeros residentes en Barcelona. Métodos: Estudio transversal de los casos de sida en mayores de 15 años, residentes en Barcelona, incluidos en el registro de la ciudad entre el 1-1-1988 y el 31-12-1998. Se describen características diferenciales de los inmigrantes, y se calculan Odds ratio (OR) ajustados con intervalos de confianza del 95 por ciento (IC) mediante regresión logística. Se realizó un análisis de supervivencia. Resultados: De los 4.663 pacientes, 5 por ciento eran inmigrantes extranjeros. En el 33 por ciento de ellos el diagnóstico de sida se hizo cuando llevaban más de 10 años de residencia en España. En el análisis multivariado, los casos de sida en inmigrantes extranjeros se diferenciaban de los autóctonos en las siguientes variables: sexo (OR [hombre]: 1,70; CI:1,10-2,64), lugar de residencia (OR [distrito I]: 2,07, IC: 1,50-2,87) y modo de transmisión (OR [no uso de drogas por vía parenteral]: 4,08; IC: 2,77-5,99). No se objetivaron diferencias significativas en el análisis de supervivencia. Conclusiones: El porcentaje de inmigrantes extranjeros entre los casos de sida es bajo, manteniéndose estable durante el periodo estudiado. Los casos de sida en inmigrantes extranjeros se diferenciaban de los autóctonos porque eran con más frecuencia hombres, residían en las zonas más deprimidas socioeconómicamente de la ciudad, y porque con menor frecuencia eran usuarios de drogas por vía parenteral (AU)

Prebiotic synthesis of adenine and amino acids under Europa-like conditions.

In order to simulate prebiotic synthetic processes on Europa and other ice-covered planets and satellites, we have investigated the prebiotic synthesis of organic compounds from dilute solutions of NH4CN frozen for 25 years at -20 and -78 degrees C. In addition, the aqueous products of spark discharge reactions from a reducing atmosphere were frozen for 5 years at -20 degrees C. We find that both adenine and guanine, as well as a simple set of amino acids dominated by glycine, are produced in substantial yields under these conditions. These results indicate that some of the key components necessary for the origin of life may have been available on Europa throughout its history and suggest that the circumstellar zone where life might arise may be wider than previously thought.

Microfabricated capillary electrophoresis amino acid chirality analyzer for extraterrestrial exploration.

Chiral separations of fluorescein isothiocyanate-labeled amino acids have been performed on a microfabricated capillary electrophoresis chip to explore the feasibility of using such devices to analyze for extinct or extant life signs in extraterrestrial environments. The test system consists of a folded electrophoresis channel (19.0 cm long x 150 microns wide x 20 microns deep) that was photolithographically fabricated in a 10-cm-diameter glass wafer sandwich, coupled to a laser-excited confocal fluorescence detection apparatus providing subattomole sensitivity. Using a sodium dodecyl sulfate/gamma-cyclodextrin pH 10.0 carbonate electrophoresis buffer and a separation voltage of 550 V/cm at 10 degrees C, baseline resolution was observed for Val, Ala, Glu, and Asp enantiomers and Gly in only 4 min. Enantiomeric ratios were determined for amino acids extracted from the Murchison meteorite, and these values closely matched values determined by HPLC. These results demonstrate the feasibility of using microfabricated lab-on-a-chip systems to analyze extraterrestrial samples for amino acids.

Amino acids in the Martian meteorite Nakhla.

A suite of protein and nonprotein amino acids were detected with high-performance liquid chromatography in the water- and acid-soluble components of an interior fragment of the Martian meteorite Nakhla, which fell in Egypt in 1911. Aspartic and glutamic acids, glycine, alanine, beta-alanine, and gamma-amino-n-butyric acid (gamma-ABA) were the most abundant amino acids detected and were found primarily in the 6 M HCl-hydrolyzed, hot water extract. The concentrations ranged from 20 to 330 parts per billion of bulk meteorite. The amino acid distribution in Nakhla, including the D/L ratios (values range from <0.1 to 0.5), is similar to what is found in bacterially degraded organic matter. The amino acids in Nakhla appear to be derived from terrestrial organic matter that infiltrated the meteorite soon after its fall to Earth, although it is possible that some of the amino acids are endogenous to the meteorite. The rapid amino acid contamination of Martian meteorites after direct exposure to the terrestrial environment has important implications for Mars sample-return missions and the curation of the samples from the time of their delivery to Earth.

Preservation of key biomolecules in the fossil record: current knowledge and future challenges.

We have developed a model based on the analyses of modern and Pleistocene eggshells and mammalian bones which can be used to understand the preservation of amino acids and other important biomolecules such as DNA in fossil specimens. The model is based on the following series of diagenetic reactions and processes involving amino acids: the hydrolysis of proteins and the subsequent loss of hydrolysis products from the fossil matrix with increasing geologic age; the racemization of amino acids which produces totally racemized amino acids in 10(5)-10(6) years in most environments on the Earth; the introduction of contaminants into the fossil that lowers the enantiomeric (D:L) ratios produced via racemization; and the condensation reactions between amino acids, as well as other compounds with primary amino groups, and sugars which yield humic acid-like polymers. This model was used to evaluate whether useful amino acid and DNA sequence information is preserved in a variety of human, amber-entombed insect and dinosaur specimens. Most skeletal remains of evolutionary interest with respect to the origin of modern humans are unlikely to preserve useful biomolecular information although those from high latitude sites may be an exception. Amber-entombed insects contain well-preserved unracemized amino acids, apparently because of the anhydrous nature of the amber matrix, and thus may contain DNA fragments which have retained meaningful genetic information. Dinosaur specimens contain mainly exogenous amino acids, although traces of endogenous amino acids may be present in some cases. Future ancient biomolecule research which takes advantage of new methologies involving, for example, humic acid cleaving reagents and microchip-based DNA-protein detection and sequencing, along with investigations of very slow biomolecule diagenetic reactions such as the racemization of isoleucine at the beta-carbon, will lead to further enhancements of our understanding of biomolecule preservation in the fossil record.

The origin of organic matter in the Martian meteorite ALH84001.

Stable carbon isotope measurements of the organic matter associated with the carbonate globules and the bulk matrix material in the ALH84001 Martian meteorite indicate that two distinct sources are present in the sample. The delta 13C values for the organic matter associated with the carbonate globules averaged -26% and is attributed to terrestrial contamination. In contrast, the delta 13C values for the organic matter associated with the bulk matrix material yielded a value of -15%. The only common sources of carbon on the Earth that yield similar delta 13C values, other then some diagenetically altered marine carbonates, are C4 plants. A delta 13C value of -15%, on the other hand, is consistent with a kerogen-like component, the most ubiquitous form of organic matter found in carbonaceous chondrites such as the Murchison meteorite. Examination of the carbonate globules and bulk matrix material using laser desorption mass spectrometry (LDMS) indicates the presence of a high molecular weight organic component which appears to be extraterrestrial in origin, possibly derived from the exogenous delivery, of meteoritic or cometary debris to the surface of Mars.

The origin of organic matter in the Martian meteorite ALH84001.

Stable carbon isotope measurements of the organic matter associated with the carbonate globules and the bulk matrix material in the ALH84001 Martian meteorite indicate that two distinct sources are present in the sample. The delta 13C values for the organic matter associated with the carbonate globules averaged -26% and is attributed to terrestrial contamination. In contrast, the delta 13C values for the organic matter associated with the bulk matrix material yielded a value of -15%. The only common carbon sources on the Earth that yield similar delta 13C values, other then some diagenetically altered marine carbonates, are C4 plants. A delta 13C value of -15%, on the other hand, is consistent with a kerogen-like component, the most ubiquitous form of organic matter found in carbonaceous chondrites such as the Murchison meteorite. Examination of the carbonate globules and bulk matrix material using laser desorption mass spectrometry (LDMS) indicates the presence of a high molecular weight organic component which appears to be extraterrestrial in origin, possibly derived from the exogenous delivery of meteoritic or cometary debris to the surface of Mars.

A search for extraterrestrial amino acids in carbonaceous Antarctic micrometeorites.

Antarctic micrometeorites (AMMs) in the 100-400 microns size range are the dominant mass fraction of extraterrestrial material accreted by the Earth today. A high performance liquid chromatography (HPLC) based technique exploited at the limits of sensitivity has been used to search for the extraterrestrial amino acids alpha-aminoisobutyric acid (AIB) and isovaline in AMMs. Five samples, each containing about 30 to 35 grains, were analyzed. All the samples possess a terrestrial amino acid component, indicated by the excess of the L-enantiomers of common protein amino acids. In only one sample (A91) was AIB found to be present at a level significantly above the background blanks. The concentration of AIB (approximately 280 ppm), and the AIB/isovaline ratio (> or = 10), in this sample are both much higher than in CM chondrites. The apparently large variation in the AIB concentrations of the samples suggests that AIB may be concentrated in rare subset of micrometeorites. Because the AIB/isovaline ratio in sample A91 is much larger than in CM chondrites, the synthesis of amino acids in the micrometeorite parent bodies might have involved a different process requiring an HCN-rich environment, such as that found in comets. If the present day characteristics of the meteorite and micrometeorite fluxes can be extrapolated back in time, then the flux of large carbonaceous micrometeorites could have contributed to the inventory of prebiotic molecules on the early Earth.

A search for endogenous amino acids in martian meteorite ALH84001.

Trace amounts of glycine, serine, and alanine were detected in the carbonate component of the martian meteorite ALH84001 by high-performance liquid chromatography. The detected amino acids were not uniformly distributed in the carbonate component and ranged in concentration from 0.1 to 7 parts per million. Although the detected alanine consists primarily of the L enantiomer, low concentrations (<0.1 parts per million) of endogenous D-alanine may be present in the ALH84001 carbonates. The amino acids present in this sample of ALH84001 appear to be terrestrial in origin and similar to those in Allan Hills ice, although the possibility cannot be ruled out that minute amounts of some amino acids such as D-alanine are preserved in the meteorite.