Isotope Incorporation Patterns Research Articles

Effect of cocooning conditions on the structure, carbon and nitrogen isotope ratios of silks.

The stable isotope technique provides the possibility to trace ancient textiles because the technique is associated with advantages such as trace indication, fast detection, and accurate results. Since different cocooning conditions may impact cocoons even under identical habitats, it is important to investigate the effects of different cocooning temperatures and humidity on the isotope incorporation values in the cocoons. In this study, silk fibers were reeled under different conditions of temperature and humidity, followed by analysis of the secondary structure of cocoon proteins and isotope incorporation patterns. We found that the deviations in carbon isotope values of silk under different cocooning conditions could reach up to 0.76‰, while the deviation in carbon isotope values at different locations of a single silk was 2.75‰. Further, during the cocooning process, depletion of the 13C-isotope at different locations of the silk fibers was observed, reducing the δ13C values. We proposed that the changes in carbon isotopes in silk were related to the content of sericin and silk fibroin in silk. Finally, we did not observe a significant difference in isotope ratios in degummed cocoons. In summary, the 13C isotope was enriched in sericin, whereas 15N was enriched in fibroin, and these findings provide basic information for tracing the provenance of silks.

Exploring source differences on diet-tissue discrimination factors in the analysis of stable isotope mixing models

Stable isotope mixing models are regularly used to provide probabilistic estimates of source contributions to dietary mixtures. Whilst Bayesian implementations of isotope mixing models have become prominent, the use of appropriate diet-tissue discrimination factors (DTDFs) remains as the least resolved aspect. The DTDFs are critical in providing accurate inferences from these models. Using both simulated and laboratory-based experimental data, this study provides conceptual and practical applications of isotope mixing models by exploring the role of DTDFs. The experimental study used Mozambique Tilapia Oreochromis mossambicus, a freshwater fish, to explore multi-tissue variations in isotopic incorporation patterns, and to evaluate isotope mixing model outputs based on the experiment- and literature-based DTDFs. Isotope incorporation patterns were variable for both muscle and fin tissues among the consumer groups that fed diet sources with different stable isotope values. Application of literature-based DTDFs in isotope mixing models consistently underestimated the dietary proportions of all single-source consumer groups. In contrast, application of diet-specific DTDFs provided better dietary estimates for single-source consumer groups. Variations in the proportional contributions of the individual sources were, nevertheless, observed for the mixed-source consumer group, which suggests that isotope assimilation of the individual food sources may have been influenced by other underlying physiological processes. This study provides evidence that stable isotope values from different diet sources exhibit large variations as they become incorporated into consumer tissues. This suggests that the application of isotope mixing models requires consideration of several aspects such as diet type and the associated biological processes that may influence DTDFs.

The influence of diet composition and tissue type on the stable isotope incorporation patterns of a small‐bodied southern African minnow Enteromius anoplus (Cypriniformes, Cyprinidae)

In trophic ecology, the use of stable isotope data relies on the general understanding of isotope turnover rates and diet-to-tissue discrimination factors (DTDFs). Recent studies on the application of stable isotope data have shown that isotope turnover rates and DTDFs can be influenced by many factors, including diet composition and tissue type. This study investigated the influence of diet composition and tissue type on stable isotope incorporation patterns in a small-bodied African minnow, the chubbyhead barb Enteromius anoplus. The isotopic incorporation patterns of carbon (δ13 C values) and nitrogen (δ15 N values) into white muscle and caudal fin tissues of the chubbyhead barb were examined using two isotopically different diets. Controlled-diet stable isotope feeding trials using a fishmeal-based diet (diet 1) and a soya-based diet (diet 2) were conducted over a 180-day period for the chubbyhead barb. The two diets had contrasting isotopic incorporation patterns: diet 1 was associated with progressively high δ13 C and δ15 N values, whereas diet 2 was associated with progressively low δ13 C and δ15 N values over time for both muscle and fin tissues. The δ13 C turnover rates were similar for both tissues (56 and 61 days), whereas the δ15 N turnover rates differed between fin and muscle tissue in both diets (diet 1 = 4 and 130 days, and diet 2 = 72 and 300 days, respectively). The DTDFs were similar for both tissues in diet 1 (Δ13 C: -3.96 to -2.62‰, Δ15 N: 1.98 to 2.61‰) and diet 2 (Δ13 C: 4.05 to 5.24‰, Δ15 N: 8.45 to 9.69‰). These results suggest that fin tissue can potentially be used as an alternative for muscle tissue in food web studies with a reasonable level of error. The isotopic turnover rate and DTDFs estimates for E. anoplus, however, require consideration of diet composition because different diets may differ in their isotopic incorporation patterns.

MAIMS: a software tool for sensitive metabolic tracer analysis through the deconvolution of 13C mass isotopologue profiles of large composite metabolites

Metabolic tracer analysis (MTA) is a collection of principles, rules and tools for the interpretation of stable isotope incorporation patterns. One example is the GAIMS algorithm for the deconvolution of the UDP-GlcNAc 13C mass isotopologue profile. GAIMS has been presented as a powerful, yet currently unavailable, proof-of-concept-only technique. We aimed to build a tool inspired by the original GAIMS algorithm, providing identical functionality and straightforward extensibility towards alternative composite metabolites. We implemented MAIMS by applying Multistart metaheuristics combined with an efficient hybrid stopping rule to solve the non-convex optimization underlying the deconvolution problem. By testing our tool on several theoretical datasets, we were able to confirm its robust and reproducible performance. MAIMS is capable of finding the individual contributions of specifically labeled molecular subunits to large composite metabolites (such as UDP-GlcNAc and ATP) upon U-13C-glucose administration and thereby hinting on the activity of several metabolic pathway activities. Applied to proliferating endothelial cells (ECs), MAIMS led to several interesting metabolic insights and generally proved to be a sensitive way for relatively measuring specific pathway activities and for detecting compartmentalized pools of precursor metabolites. MAIMS is a powerful and extendible tool for isotopologue profile deconvolution tasks and is freely available on github as an open-source Python (Python 2.7 and 3.5 + compliant) script for command line usage. ( http://github.com/savantas/MAIMS ).

Stable Isotope Peptide Mass Spectrometry To Decipher Amino Acid Metabolism in Dehalococcoides Strain CBDB1

Dehalococcoides species are key players in the anaerobic transformation of halogenated solvents at contaminated sites. Here, we analyze isotopologue distributions in amino acid pools from peptides of Dehalococcoides strain CBDB1 after incubation with (13)C-labeled acetate or bicarbonate as a carbon source. The resulting data were interpreted with regard to genome annotations to identify amino acid biosynthesis pathways. In addition to using gas chromatography-mass spectrometry (GC-MS) for analyzing derivatized amino acids after protein hydrolysis, we introduce a second, much milder method, in which we directly analyze peptide masses after tryptic digest and peptide fragments by nano-liquid chromatography-electrospray ionization-tandem mass spectrometry (nano-LC-ESI-MS/MS). With this method, we identify isotope incorporation patterns for 17 proteinaceous amino acids, including proline, cysteine, lysine, and arginine, which escaped previous analyses in Dehalococcoides. Our results confirmed lysine biosynthesis via the α-aminoadipate pathway, precluding lysine formation from aspartate. Similarly, the isotopologue pattern obtained for arginine provided biochemical evidence of its synthesis from glutamate. Direct peptide MS/MS analysis of the labeling patterns of glutamine and asparagine, which were converted to glutamate and aspartate during protein hydrolysis, gave biochemical evidence of their precursors and confirmed glutamate biosynthesis via a Re-specific citrate synthase. By addition of unlabeled free amino acids to labeled cells, we show that in strain CBDB1 none of the 17 tested amino acids was incorporated into cell mass, indicating that they are all synthesized de novo. Our approach is widely applicable and provides a means to analyze amino acid metabolism by studying specific proteins even in mixed consortia.

Carbon dynamics of logarithmetic and stationary phase phytoplankton as determined by track autoradiography.

Track autoradiographic analysis of photosynthetic radiocarbon incorporation at the cellular level indicated that the carbon uptake rate and carbon pool size of exponentially growing (log phase) Scenedesmus cells was threefold that of stationary phase cells, while carbon turnover rates were similar. Carbon fixation was uncoupled from growth and cell division in the stationary phase cells, which were larger and contained less chlorophyll per unit volume than log phase cells. Changes in the temporal pattern of isotope incorporation were evident at the cell level prior to the cessation of division and transition to stationary phase, while bulk carbon fixation responded only the second day after cell division ceased. The carbon uptake patterns of a marine nanoplankter from a nutrient-enriched natural sample resembled that of log phase cells while the control population pattern resembled that of stationary cells. The physical, biochemical, and metabolic differences between log and stationary phase cells are potentially measurable by flow cytometry procedures currently in use and under development. The use of flow cytometry to sort cell types for analysis by track autoradiography and subsequent correlation of metabolic characteristics with flow cytometry signatures is a feasible means of investigating the heterogeneity of phytoplankton metabolic state in the marine environment.

Intestinal lipoprotein synthesis in control and hypercholesterolemic rats

Previous studies in our laboratory have shown that very-low-density lipoproteins (VLDL) synthesized by the intestine of the diet-induced hypercholesterolemic rat are enriched in cholesteryl esters and unesterified cholesterol compared with intestinal VLDL from control rats. In these studies, we isolated and characterized nascent intestinal Golgi intermediate-density lipoproteins (IDL, d 1.006–1.040 g/ ml) and studied isotope incorporation into apolipoproteins of Golgi VLDL from control and hypercholesterolemic rats. IDL were triacylglycerol-rich lipoproteins but contained more cholesteryl ester and protein than the corresponding Golgi VLDL fractions. IDL from hypercholesterolemic rats were enriched in cholesteryl esters to a greater extent than IDL from control rats. The apolipoprotein patterns of IDL fractions were the same as those of intestinal Golgi VLDL, consisting of apolipoproteins (apo) B-48, A-I and A-IV. Time-course isotope incorporation curves for apo A-I and A-IV in Golgi VLDL were similar, but they differed from curves for apo B-48. None of these curves was markedly altered in the hypercholesterolemic rat. We conclude that the major effect of increased dietary cholesterol on intestinal lipoprotein biosynthesis is to increase the percentage of cholesteryl esters in Golgi lipoproteins. Dietary cholesterol does not alter the apolipoprotein composition of Golgi lipoproteins, nor does it have a significant effect on the pattern of isotope incorporation into apolipoproteins of Golgi VLDL. The effect of cholesteryl ester enrichment on the subsequent metabolism of these particles in the circulation and the effect of these particles on hepatic lipoprotein production remain to be determined.

Effects of octanoic acidemia on palmitic acid metabolism in rat liver.

AbstractThis study examined the effects of octanoic acidemia on the metabolism of radioactive palmitic acid in rat liver. Octanoic acidemia, produced either by an in-traperitoneal bolus injection or sustained intravenous infusion of sodium octanoate, markedly influenced biochemical pathways which are concerned with the hepatic disposition of palmitic acid into the different classes of rat liver lipids. During acute octanoic acidemia, the incorporation of [16-14C]palmitic acid into hepatic triacylglycerols was greatly increased, while uptake of this fatty acid into phospholipids, especially phosphatidylcholine, was sharply reduced. When the octanoic acidemia resulted from intravenous infusion of sodium octanoate for 4 hr prior to the injection of [16-14C]palmitic acid, the pattern of isotope incorporation was one which would support hepatic steatosis.

Amino acid incorporation into proteins of degenerating and regenerating goldfish optic tracts

The goldfish optic system was used to examine the patterns of incorporation of 3H-amino acid into proteins of the degenerating and regenerating optic tract. On various days after unilateral enucleation, groups of fish were injected intraperitoneally with [ 3H]-leucine and the contralateral optic tracts were examined for protein specific radioactivity 5.5 h later. Labeling of proteins in experimental tracts showed a biphasic increase over unoperated controls with the greatest increments occurring at 1 to 5 days and again at 10 to 14 days. Labeling increases of approximately 2.5-fold and 4- to 5-fold were observed in the first and second phase, respectively. Enhanced labeling was shown not to be due to increases in precursor pools and sham-operated animals showed only minimal increases in tract labeling. Parallel experiments were conducted on fish receiving optic nerve crush in which degeneration is followed by tract reinnervation. Results showed a biphasic pattern of isotope incorporation that was very similar in time course and magnitude to that seen in the totally degenerating tracts. The second phase was found to correspond in time of the entry of regenerating axons into the tract. These results have implications with reference to the probable origin of labeled cells, the possible function of the first and second phase of labeling, and the similarity in metabolic pattern between the degenerating and regenerating tracts.

Acetate incorporation into the lipids of the anemone Anthopleura elegantissima and its associated zooxanthellae

Anthopleura elegantissima containing zooxanthellae, as well as isolated zooxanthellae, incubated with acetate-1-14C under both light and dark conditions readily incorporate radioactivity into their total lipid pools. In both cases, the specific activity was greatly increased in the light. Dark-incubated anemones and isolated zooxanthellae incorporate activity predominantly into polar lipid; the remainder being present principally in the triglyceride moiety. Light-incubated organisms, however, show a dramatic redistribution of isotope towards greatly increased triglyceride and was ester incorporation, with a concomitant drop in polar lipid. onder light conditions, 70 to 75% of the radioactivity found in the fatty acids of the total zooxanthellae lipid was present in hexadecanoic (16:0) and octadecenoic (18:1) fatty acids. These are also the two major fatty acids by mass. Octadecanoic acid (18:0) is less than 5% by mass. Isotope incorporation patterns suggest that octadecenoic acids arise by elongation of hexadecenoic acids and that this conversion is blocked in the dark. Isotope incorporation patterns for anemones suggest that fatty acids, primarily in the form of saturated or monoenoic fatty acids, are translocated from algal to animal cells. No activity was found in either octadecadienoic (18:2) or octadecatrienoic (18:3) acids. The significance of these data is discussed.

Cytoplasmic inclusions in Xenopus spermatogenic cells. ultrastructural and cytochemical analysis of the action of antimitotic agents on subcellular elements

ABSTRACT The effects of vinblastine sulphate, alone and in combination with other antimitotic agents, were tested in vitro on testicular tissue from the anuran, Xenopus laevis. Treatment with vinblastine alone resulted in dissolution of spindle and intercellular bridge microtubules, but centrioles and sperm tail axonemes remained intact. In addition, no paracrystalloid inclusions typical of those reported in other cell types treated with vinblastine alone were observed in Xenopus spermatogenic cells. Such inclusions were formed, however, when vinblastine was administered in combination with colchicine, colcemid, or puromycin. Under optimal conditions, paracrystalloids were formed in all cells from primary spermatogonia to late spermatids. The maximum size paracrystalloid formed differed in different spermatogenic cell stages, with late primary spermatocytes containing the largest such inclusions, often averaging over 34 μ m3 each. Autoradiographic analysis of spermatogenic cells labelled with [3H]leucine prior to drug treatment indicated that some radioactive material was incorporated into paracrystalloids in all cell stages. Treatment of Xenopus spermatogenic cells with vinblastine and puromycin was also employed to investigate the possible relationship between paracrystalloid material and a naturally occurring spermatogenic cell inclusion, the chromatoid body, since previous experiments indicated that microtubule protein might contribute to the mass of this structure. It was found that under certain conditions, both paracrystalloids and giant chromatoid bodies can occur together in the same cell, each inclusion reaching a characteristic size independent of the other. This suggests that the two inclusions are aggregates of materials drawn from different subcellular precursor pools. In addition, autoradiographic analysis of isotope incorporation indicated that giant chromatoid bodies could be clearly labelled with tritiated amino acids, but not with [3H]uridine. The pattern of isotope incorporation observed in giant chromatoid bodies, coupled with their histochemical staining characteristics, rules out the possibility that these structures function as cytoplasmic nucleoli.

Collagen-like fragments: excretion in urine of patients with Paget's disease of bone.

Patients with Paget's disease of bone excrete, in the urine, polypeptides that have amino acid composition and other properties resembling those of fragments of collagen. The pattern of isotope incorporation in vivo suggests that these fragments are derived from collagen that has been synthesized and rapidly degraded, or that they are rapidly synthesized but not incorporated into tropocollagen molecules.

The Biosynthesis of the 7-Deazaadenine Ribonucleoside, Tubercidin, by Streptomyces tubercidicus

Studies on the biosynthesis of the 7-deazaadenine base of the antibiotic, tubercidin, in growing cultures of Streptomyces tubercidicus are described. Isotope incorporation patterns suggest that the pyrimido moiety of a purine serves as the precursor of the pyrimido portion of 7-deazaadenine and that carbon atoms 1 and 2 of ribose may be incorporated into the pyrrole moiety of 7-deazaadenine. These conclusions are based on the following experimental observations. Carbon atom 8 of adenine-8-14C is metabolized to 14CO2 during the biosynthesis of tubercidin. 14C from adenine-2-14C, but not adenine-8-14C, is incorporated into 7-deazaadenine. The extents of incorporation of glycine and formate into cellular adenine and 7-deazaadenine are similar. The addition of adenine to cultures of S. tubercidicus inhibits the incorporation of 14C from glycine-1-14C into both cellular adenine and 7-deazaadenine. Ribose-1-14C is incorporated into the 7-deazaadenine ring. The amide nitrogen of the glutamine and the amino nitrogen of aspartic acid are incorporated into 7-deazaadenine. The implications of these findings with respect to the biological transformation of purines to 7-deazaadenine are discussed.