Detection of point mutations by non-isotopic single strand conformation polymorphism

We have developed a procedure for nonradioactive single strand conformation polymorphism analysis and applied it to the detection of point mutations in the human tumor suppressor gene p53. The protocol does not require any particular facilities or equipment, such as radioactive handling, large gel units for sequencing, or a semiautomated electrophoresis system. This technique consists of amplification of DNA fragments by PCR with specific oligonucleotide primers, denaturation, and electrophoresis on small neutral polyacrylamide gels, followed by silver staining. The sensitivity of this procedure is comparable to other described techniques and the method is easy to perform and applicable to a variety of tissue specimens.

Carbon isotope analysis in apple nectar beverages

The aims of this study were to use the isotope analysis method to quantify the carbon of C3 photosynthetic cycle in commercial apple nectars and to determine the legal limit to identify the beverages that do not conform to the safety standards established by the Brazilian Ministry of Agriculture, Livestock and Food Supply. These beverages (apple nectars) were produced in the laboratory according to the Brazilian legislation. Adulterated nectars were also produced with an amount of pulp juice below the permitted threshold limit value. The δ13C values of the apple nectars and their fractions (pulp and purified sugar) were measured to quantify the C3 source percentage. In order to demonstrate the existence of adulteration, the values found were compared to the limit values established by the Brazilian Law. All commercial apple nectars analyzed were within the legal limits, which enabled to identify the nectars that were in conformity with the Brazilian Law. The isotopic methodology developed proved efficient to quantify the carbon of C3 origin in commercial apple nectars.

The satellite band patterns in the near ultraviolet absorption spectra of isotopic compounds of acetaldehyde

The 3700 A - 3000 A absorption spectra of CH3CHO and its isotopic compounds such as CH3CDO, CD3CHO and CD3CDO were studied in the gas phase at room temperature and low temperatures. The low resolution spectra of the compounds were recorded by a 1.5 m Baush and Lomb grating spectrograph. The high resolution spectra were recorded by a Ebert spectrograph with the Echelle grating and the holographic grating separately. The multiple reflection cells were used to achieve the long path length. The pressure-path length used for the absorption spectrum of CH 3CHO was up to 100 mm Hg )( 91 . 43mo The emission spectrum and the excitation spectrum of CH3CHO were also recorded in this research. The calculated satellite band patterns \vhich were ob-tailied by the method of Lewis were used to compare with the observed near UV absorption spectrum of acetaldehyde. These calculated satellite band patterns belonged to two cases: namely, the barriers-in-phase case and the barriers- out-of-phase case. Each of the calculated patterns corresponded to a stable conformation of acetaldehyde in the excited state . The comparisons showed that the patterns in the observed absorption spectra corresponded to the H-H eclipsed conformations of acetaldehyde in the excited state . The least squares fitting analysis showed that the barrier heights in the excited state were higher than in the ground state. Finally, the isotopic shifts for the isotopic compounds of acetaldehyde were compared to the compounds with the similar deuterium substitution.

A compositional approach to stable isotope data analysis

Isotopic data are currently becoming an important source of information regarding sources, evolution and mixing processes of water in hydrogeologic systems. However, it is not clear how to treat with statistics the geochemical data and the isotopic data together. We propose to introduce the isotopic information as new parts, and apply compositional data analysis with the resulting increased composition. Results are equivalent to downscale the classical isotopic delta variables, because they are already relative (as needed in the compositional framework) and isotopic variations are almost always very small. This methodology is illustrated and tested with the study of the Llobregat River Basin (Barcelona, NE Spain), where it is shown that, though very small, isotopic variations comp lement geochemical principal components, and help in the better identification of pollution sources

Diet and diversity at later medieval Fishergate: The isotopic evidence

We present the results of stable carbon and nitrogen isotope analysis of bone collagen for 155 individuals buried at the Later Medieval (13th to early 16th century AD) Gilbertine priory of St. Andrew, Fishergate in the city of York (UK). The data show significant variation in the consumption of marine foods between males and females as well as between individuals buried in different areas of the priory. Specifically, individuals from the crossing of the church and the cloister garth had consumed significantly less marine protein than those from other locations. Isotope data for four individuals diagnosed with diffuse idiopathic skeletal hyperostosis (DISH) are consistent with a diet rich in animal protein. We also observe that isotopic signals of individuals with perimortem sharp force trauma are unusual in the context of the Fishergate dataset. We discuss possible explanations for these patterns and suggest that there may have been a specialist hospital or a local tradition of burying victims of violent conflict at the priory. The results demonstrate how the integration of archaeological, osteological, and isotopic data can provide novel information about Medieval burial and society.

GC/multiple collector-ICPMS method for chlorine stable isotope analysis of chlorinated aliphatic hydrocarbons

Stable isotopic characterization of chlorine in chlorinated aliphatic pollution is potentially very valuable for risk assessment and monitoring remediation or natural attenuation. The approach has been underused because of the complexity of analysis and the time it takes. We have developed a new method that eliminates sample preparation. Gas chromatography produces individually eluted sample peaks for analysis. The He carrier gas is mixed with Ar and introduced directly into the torch of a multicollector ICPMS. The MC-ICPMS is run at a high mass resolution of >= 10 000 to eliminate interference of mass 37 ArH with Cl. The standardization approach is similar to that for continuous flow stable isotope analysis in which sample and reference materials are measured successively. We have measured PCE relative to a laboratory TCE standard mixed with the sample. Solvent samples of 200 nmol to 1.3 mu mol ( 24- 165 mu g of Cl) were measured. The PCE gave the same value relative to the TCE as measured by the conventional method with a precision of 0.12% ( 2 x standard error) but poorer precision for the smaller samples.

Carbon and nitrogen stable isotope ratio analysis of freshwater, brackish and marine fish from Belgian archaeological sites (1st and 2nd millennium AD)

Carbon and nitrogen stable isotope ratios were measured in 157 fish bone collagen samples from 15 different archaeological sites in Belgium which ranged in ages from the 3rd to the 18th c. AD. Due to diagenetic contamination of the burial environment, only 63 specimens produced results with suitable C:N ratios (2.9–3.6). The selected bones encompass a wide spectrum of freshwater, brackish, and marine taxa (N = 18), and this is reflected in the δ13C results (−28.2‰ to −12.9%). The freshwater fish have δ13C values that range from −28.2‰ to −20.2‰, while the marine fish cluster between −15.4‰ and −13.0‰. Eel, a catadromous species (mostly living in freshwater but migrating into the sea to spawn), plots between −24.1‰ and −17.7‰, and the anadromous fish (living in marine environments but migrating into freshwater to spawn) show a mix of freshwater and marine isotopic signatures. The δ15N results also have a large range (7.2‰ to 16.7‰) indicating that these fish were feeding at many different trophic levels in these diverse aquatic environments. The aim of this research is the isotopic characterization of archaeological fish species (ecology, trophic level, migration patterns) and to determine intra-species variation within and between fish populations differing in time and location. Due to the previous lack of archaeological fish isotope data from Northern Europe and Belgium in particular, these results serve as an important ecological backdrop for the future isotopic reconstruction of the diet of human populations dating from the historical period (1st and 2nd millennium AD), where there is zooarchaeological and historical evidence for an increased consumption of marine fish.

Inferring animal husbandry strategies in coastal zones through stable isotope analysis: new evidence from the Flemish coastal plain (Belgium, 1st-15th century AD)

In a proof-of-concept study, Britton et al. (2008) demonstrated that the isotopic composition of halophytic plants can be traced in the skeletal tissues of their animal consumers. Here we apply the method to domestic herbivore remains (n = 303) from nine archaeological sites in or near the Flemish coastal plain (Belgium), where, prior to embankments, salt-marshes offered extensive pasture grounds for domestic herbivores. The sites span a period of ∼1500 years (Roman to late medieval period), during which the coastal landscape was progressively transformed from little managed wetlands to a fully embanked polder area. The bulk collagen data show variations between sites and over time, which are consistent with this historical framework and are interpreted as reflecting environmental change and differences in animal management in the coastal plain throughout the late Holocene. The study demonstrates the immense value of faunal stable isotope analysis for characterising coastal husbandry strategies beyond the means of traditional zooarchaeological techniques.

A refined sampling strategy for intra-tooth stable isotope analysis of mammalian enamel

Serial sampling and stable isotope analysis performed along the growth axis of vertebrate tooth enamel records differences attributed to seasonal variation in diet, climate or animal movement. Because several months are required to obtain mature enamel in large mammals, modifications in the isotopic composition of environmental parameters are not instantaneously recorded, and stable isotope analysis of tooth enamel returns a time-averaged signal attenuated in its amplitude relative to the input signal. For convenience, stable isotope profiles are usually determined on the side of the tooth where enamel is thickest. Here we investigate the possibility of improving the time resolution by targeting the side of the tooth where enamel is thinnest. Observation of developing third molars (M3) in sheep shows that the tooth growth rate is not constant but decreases exponentially, while the angle between the first layer of enamel deposited and the enamel–dentine junction increases as a tooth approaches its maximal length. We also noted differences in thickness and geometry of enamel growth between the mesial side (i.e., the side facing the M2) and the buccal side (i.e., the side facing the cheek) of the M3. Carbon and oxygen isotope variations were measured along the M3 teeth from eight sheep raised under controlled conditions. Intra-tooth variability was systematically larger along the mesial side and the difference in amplitude between the two sides was proportional to the time of exposure to the input signal. Although attenuated, the mesial side records variations in the environmental signal more faithfully than the buccal side. This approach can be adapted to other mammals whose teeth show lateral variation in enamel thickness and could potentially be used as an internal check for diagenesis.

New-on-line method for water isotope analysis of speleothem fluid inclusions using laser absorption spectroscopy (WS-CRDS)

A new online method to analyse water isotopes of speleothem fluid inclusions using a wavelength scanned cavity ring down spectroscopy (WS-CRDS) instrument is presented. This novel technique allows us simultaneously to measure hydrogen and oxygen isotopes for a released aliquot of water. To do so, we designed a new simple line that allows the online water extraction and isotope analysis of speleothem samples. The specificity of the method lies in the fact that fluid inclusions release is made on a standard water background, which mainly improves the δ D robustness. To saturate the line, a peristaltic pump continuously injects standard water into the line that is permanently heated to 140 °C and flushed with dry nitrogen gas. This permits instantaneous and complete vaporisation of the standard water, resulting in an artificial water background with well-known δ D and δ18O values. The speleothem sample is placed in a copper tube, attached to the line, and after system stabilisation it is crushed using a simple hydraulic device to liberate speleothem fluid inclusions water. The released water is carried by the nitrogen/standard water gas stream directly to a Picarro L1102-i for isotope determination. To test the accuracy and reproducibility of the line and to measure standard water during speleothem measurements, a syringe injection unit was added to the line. Peak evaluation is done similarly as in gas chromatography to obtain &delta D; and δ18O isotopic compositions of measured water aliquots. Precision is better than 1.5 ‰ for δ D and 0.4 ‰ for δ18O for water measurements for an extended range (−210 to 0 ‰ for δ D and −27 to 0 ‰ for δ18O) primarily dependent on the amount of water released from speleothem fluid inclusions and secondarily on the isotopic composition of the sample. The results show that WS-CRDS technology is suitable for speleothem fluid inclusion measurements and gives results that are comparable to the isotope ratio mass spectrometry (IRMS) technique.

Diet and herding strategies in a changing environment: stable isotope analysis of Bronze Age and Late Antique skeletal remains from Ya'amūn, Jordan

Carbon and nitrogen stable isotope ratios of 45 human and 23 faunal bone collagen samples were measured to study human diet and the management of domestic herbivores in past Jordan, contrasting skeletal remains from the Middle and Late Bronze Age and the Late Roman and Byzantine periods from the site of Ya'amūn near Irbid. The isotope data demonstrate that the management of the sheep and goats changed over time, with the earlier animals consuming more plants from semi-arid habitats, possibly because of transhumant herding strategies. The isotope data for fish presented here are the first from archaeological contexts from the Southern Levant. Although fish of diverse provenance was available at the site, human diet was predominately based on terrestrial resources and there was little dietary variability within each time-period. Isotopic variation between humans from different time-periods can mostly be explained by ‘baseline shifts’ in the available food sources; however, it is suggested that legumes may have played a more significant role in Middle and Late Bronze Age diet than later on.

Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative peptidomics

P>Neuropeptides are produced from larger precursors by limited proteolysis, first by endopeptidases and then by carboxypeptidases. Major endopeptidases required for these cleavages include prohormone convertase (PC) 1/3 and PC2. In this study, quantitative peptidomics analysis was used to characterize the specific role PC1/3 plays in this process. Peptides isolated from hypothalamus, amygdala, and striatum of PC1/3 null mice were compared with those from heterozygous and wild-type mice. Extracts were labeled with stable isotopic tags and fractionated by HPLC, after which relative peptide levels were determined using tandem mass spectrometry. In total, 92 peptides were found, of which 35 were known neuropeptides or related peptides derived from 15 distinct secretory pathway proteins: 7B2, chromogranin A and B, cocaine- and amphetamine-regulated transcript, procholecystokinin, proenkephalin, promelanin concentrating hormone, proneurotensin, propituitary adenylate cyclase-activating peptide, proSAAS, prosomatosatin, provasoactive intestinal peptide, provasopressin, secretogranin III, and VGF. Among the peptides derived from these proteins, similar to 1/3 were decreased in the PC1/3 null mice relative to wild-type mice, similar to 1/3 showed no change, and similar to 1/3 increased in PC1/3 null. Cleavage sites were analyzed in peptides that showed no change or that decreased in PC1/3 mice, and these results were compared with peptides that showed no change or decreased in previous peptidomic studies with PC2 null mice. Analysis of these sites showed that while PC1/3 and PC2 have overlapping substrate preferences, there are particular cleavage site residues that distinguish peptides preferred by each PC.

Traceability of bovine meat and bone meal in poultry by stable isotope analysis

Bovine meat and bone meal (MBM) was widely used in animal diets until outbreaks of Bovine Spongiform Encefalopathy (BSE) occurred in some countries. It has not been confirmed yet whether or not BSE may be transmitted to man through chicken meat originated from poultry that had been fed diets containing MBM. Therefore, consumers nowadays express preference for meat originated from birds fed exclusively vegetable diets. This study analyzed samples of major breast muscle (Pectoralis major) using mass spectrometry of stable isotopes (carbon and nitrogen) as a means to assess the presence of MBM in broiler diets, a technique that might be used in the certification of poultry quality. A total of 150 day-old chicks were reared in five randomized treatments with increasing MBM dietary inclusion levels (0, 1, 2, 4 and 8%). on day 42, breast muscle samples were collected from three birds per treatment and used in the determination of 13C/12C and 15N/14N isotope ratios. The breast muscle isotope values were expressed as delta in parts per thousand (delta ). The following carbon isotope values (13C) were found: 18.74 ±0.11, 18.51 ±0.19, 18.24 ±0.10, -17.79 ±0.12, and -17.15 ±0.15 for 0, 1, 2, 4 and 8% MBM dietary levels, respectively. Nitrogen isotope values (15N) were 1.65 ±0.14, 1.65 ±0.28, 1.72 ±0.08, 1.95 ±0.16, and 2.52 ± 0.09 for 0, 1, 2, 4 and 8% MBM dietary levels, respectively. This study showed important differences in delta13C and delta15N values in breast meat, evidencing a simultaneous enrichment of this isotopic pair, which allowed tracing MBM in bird diets. Analysis of carbon and nitrogen stable isotopes may be used to ensure feeding with exclusively vegetable diets, and might also be used as a reliable evaluation tool in broiler meat certification. The diet with 1% inclusion level of MBM and the exclusively vegetable diet showed similar results.

Carbon isotope analysis in apple nectar beverages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Carbon Stocks and Isotopic Composition of the Organic Matter in Soils Covered by Native Vegetation and Pasture in Sorocaba, SP, Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)