Thirty years development of ¹³C metabolic flux analysis: a review / 生物工程学报

¹³C metabolic flux analysis (¹³C-MFA) enables the precise quantification of intracellular metabolic reaction rates by analyzing the distribution of mass isotopomers of proteinogenic amino acids or intracellular metabolites through ¹³C labeling experiments. ¹³C-MFA has received much attention as it can help systematically understand cellular metabolic characteristics, guide metabolic engineering design and gain mechanistic insights into pathophysiology. This article reviews the advances of ¹³C-MFA in the past 30 years and discusses its potential and future perspective, with a focus on its application in industrial biotechnology and biomedicine.

Convergent responses of fish belonging to different feeding guilds to sewage pollution

This study aimed to evaluate if the presence of pollutants promotes changes in feeding habits of fish species from different trophic guilds: the detritivorous species, Hypostomus francisci, and the piscivorous, Hoplias intermedius. Both species were sampled at 12 sites (with different degrees of pollution) in the Rio das Velhas basin, which is heavily polluted by domestic and industrial sewage from the Metropolitan Region of Belo Horizonte (MRBH). Stable isotope analyses of carbon (δ13C) and nitrogen (δ15N) of fish tissue and the main food resources were performed. Fishes from both trophic guilds altered their diets in degraded environments, but the detritivorous species showed greater trophic plasticity. The isotopic niche of both trophic guilds was broadest in unpolluted sites and more δ15N enriched in polluted regions. The detritivorous species presented high niche-breadth in unpolluted sites, probably due to the greater variety of resources consumed. In addition, the δ15N of the detritivorous was more enriched than the piscivorous species in polluted sites. In conclusion, fishes from both trophic guilds presented similar isotopic responses to environmental pollution. However, the detritivorous species was more sensitive to these alterations and therefore, is likely a better indicator of environmental condition than the piscivorous.(AU)

Este estudo teve como objetivo avaliar se a presença de poluentes promove mudanças nos hábitos alimentares de espécies de peixes de diferentes guildas tróficas: a espécie detritívora, Hypostomus francisci, e a piscívora, Hoplias intermedius. Ambas espécies foram amostradas em 12 locais (com diferentes níveis de poluição) na bacia do Rio das Velhas, que é altamente poluída por esgoto doméstico e industrial da Região Metropolitana de Belo Horizonte (RMBH). Foram realizadas análises de isótopos estáveis de carbono (δ13C) e nitrogênio (δ15N) dos tecidos dos peixes e dos principais recursos alimentares. Espécies de ambas guildas tróficas alteraram suas dietas em ambientes degradados, mas a espécie detritívora apresentou maior plasticidade trófica. O nicho isotópico de ambas as espécies foi mais amplo em locais menos perturbados e mais enriquecido em δ15N em regiões poluídas. A espécie detritívora apresentou grande amplitude em seu nicho isotópico em locais menos perturbados, provavelmente devido à maior variedade de recursos consumidos. Além disso, o δ15N da espécie detritívora foi mais enriquecido que a espécie piscívora em locais poluídos. Em conclusão, ambas as espécies apresentaram respostas isotópicas semelhantes à poluição ambiental. No entanto, a espécie detritívora foi mais sensível a essas alterações e, portanto, é provavelmente uma melhor indicadora de condição ambiental do que a espécie piscívora.(AU)

Testicular cancers in Radiation and Isotope centre, Khartoum, Sudan: a retrospective study

This is a retrospective study of 44 cases of testicular cancers treated at the Radiation and Isotopes Center ofKhartoum (RICK).The mean age was 29.9 years. Six patients (13.6%) had undescended testicles; 24 patients (54.4%) presented with stages three and four. Only 20 patients (45.4%) survived for five years and . more

Functional investigation of bone implant viability using radiotracers in a new model of osteonecrosis

OBJECTIVES: Conventional imaging methods are excellent for the morphological characterization of the consequences of osteonecrosis; however, only specialized techniques have been considered useful for obtaining functional information. To explore the affinity of radiotracers for severely devascularized bone, a new mouse model of isolated femur implanted in a subcutaneous abdominal pocket was devised. To maintain animal mobility and longevity, the femur was harvested from syngeneic donors. Two technetium-99m-labeled tracers targeting angiogenesis and bone matrix were selected. METHODS: Medronic acid and a homodimer peptide conjugated with RGDfK were radiolabeled with technetium-99m, and biodistribution was evaluated in Swiss mice. The grafted and control femurs were evaluated after 15, 30 and 60 days, including computed tomography (CT) and histological analysis. RESULTS: Radiolabeling achieved high (>95%) radiochemical purity. The biodistribution confirmed good blood clearance 1 hour after administration. For 99mTc-hydrazinonicotinic acid (HYNIC)-E-[c(RGDfK)2, remarkable renal excretion was observed compared to 99mTc-methylene diphosphonate (MDP), but the latter, as expected, revealed higher bone uptake. The results obtained in the control femur were equal at all time points. In the implanted femur, 99mTc-HYNIC-E-[c(RGDfK)2 uptake was highest after 15 days, consistent with early angiogenesis. Regarding 99mTc-MDP in the implant, similar uptake was documented at all time points, consistent with sustained bone viability; however, the uptake was lower than that detected in the control femur, as confirmed by histology. CONCLUSIONS: 1) Graft viability was successfully diagnosed using radiotracers in severely ischemic bone at all time points. 2) Analogously, indirect information about angiogenesis could be gathered using 999mTc-HYNIC-E-[c(RGDfK)2. 3) These techniques appear promising and warrant further studies to determine their potential clinical applications.

Transfection of hypertrophic cardiac myocytes in vitro with (99)Tc(m)-labeled antisense miR208b oligonucleotide / 南方医科大学学报

<p><b>OBJECTIVE</b>To test the efficiency of transfecting (99)Tc(m)-labeled anti-miR208b oligonucleotide into early hypertrophic cardiac myocytes in vitro.</p><p><b>METHODS</b>The anti-oligonucleotide targeting miR208b (AMO) was synthesized and modified with LNA followed by conjugation with N-hydroxysuccinimidyl S-acetyl-meraptoacetyl triglycine (NHS-MAG3) and radiolabeling with (99)Tc(m). NHS-MAG3-LNA-AMO and labeled AMO were purified with Sep-Pak C18 column chromatography, and the former was examined for UV absorption at the 260 nm using Gene Quant DNA/RNA calculator. The labeling efficiency, radiochemical purity, stability and molecular hybridization activity were analyzed. An angiotensin II-induced cell model of hypertrophic cardiac myocytes was transfected with (99)Tc(m)-NHS-MAG3-LNA-AMO via liposome, and the relative expression of miRNA208b and retention ratio of the labeled AMO in early hypertrophic cells were determined.</p><p><b>RESULTS</b>The labeling efficiency and radiochemical purity of the labeled AMO after purification exceeded 84% and 86%, respectively. The radio- chemical purities of the labeled AMO incubated in serum and normal saline for 12 h were both higher than 80%, and the labeled AMO showed a capacity to hybridize with the target gene. In the hypertrophic model of cardiac myocytes, the retention ratio of labeled AMO at 6 h was higher than 20%.</p><p><b>CONCLUSION</b>The (99)Tc(m)-labeled antisense probe can be efficiently transfected into hypertrophic cardiac myocytes in vitro, which provides an experimental basis for subsequent radionuclide imaging studies.</p>

Effects of stable isotope labeled internal standard on determination of ivabradine and N-demethylivabradine in human plasma / 药学学报

This study aims to develop a liquid chromatography with tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of ivabradine and N-demethylivabradine in human plasma, and investigate effects of stable isotope labeled (SIL) internal standard (IS) on ivabradine. The analytes and IS were extracted from plasma by protein precipitation with acetonitrile, and chromatographied on a Capcell PAK C18 (100 mm x 4.6 mm, 5 μm) column using a mobile phase of methanol and 5 mmol x L(-1) ammonium acetate. Multiple reaction monitoring with electrospray ionization (ESI) was used in the positive mode for mass spectrometric detection. The effect of ivabradine isotope peak [M+H+3] + on IS and the effect of SIL IS purity on ivabradine were evaluated. An appropriate concentration of SIL IS was chosen to permit method selectivity and linearity of the assay over the required range. The standard curves were demonstrated to be linear in the range of 0.100 to 60.0 ng x mL(-1) for ivabradine, and 0.050 0 to 20.0 ng x mL(-1) for N-demethylivabradine. The intra and inter day precision and accuracy were within the acceptable limits for all concentrations. Besides, the interaction between IS and ivabradine did not impact the determination of analytes. This method was successfully applied to a pharmacokinetic study of hydrogen sulfate ivabradine sustained release tablets on Chinese healthy volunteers.

Proteomic identification and functional characterization of MYH9, Hsc70, and DNAJA1 as novel substrates of HDAC6 deacetylase activity

Histone deacetylase 6 (HDAC6), a predominantly cytoplasmic protein deacetylase, participates in a wide range of cellular processes through its deacetylase activity. However, the diverse functions of HDAC6 cannot be fully elucidated with its known substrates. In an attempt to explore the substrate diversity of HDAC6, we performed quantitative proteomic analyses to monitor changes in the abundance of protein lysine acetylation in response to HDAC6 deficiency. We identified 107 proteins with elevated acetylation in the liver of HDAC6 knockout mice. Three cytoplasmic proteins, including myosin heavy chain 9 (MYH9), heat shock cognate protein 70 (Hsc70), and dnaJ homolog subfamily A member 1 (DNAJA1), were verified to interact with HDAC6. The acetylation levels of these proteins were negatively regulated by HDAC6 both in the mouse liver and in cultured cells. Functional studies reveal that HDAC6-mediated deacetylation modulates the actin-binding ability of MYH9 and the interaction between Hsc70 and DNAJA1. These findings consolidate the notion that HDAC6 serves as a critical regulator of protein acetylation with the capability of coordinating various cellular functions.

Feeding mouse with stable isotope labelled with amino acid / 生物工程学报

The stable isotope labeling by amino acids in culture (SILAC) based quantitative proteomics serves as a gold standard because of the high accuracy and throughput for protein identifications and quantification. In this study, we discussed the application of SILAC technology in mammal model, and developed quantitative internal standard for comparative proteomics of disease model. The C57BL/6J mice fed by special diet containing the 13C6-Lysine and bred F2 generation. We identified and analyzed total proteins of 9 mice tissues of F2 generation, including brain, lung, heart, stomach, intestine, liver, spleen, kidney, and muscle. Quantitative analysis information could evaluate the mice and different tissues' labeling efficiency. Liver was the most efficient, brain the least, and the labeling efficiency were 96.34%±0.90% and 92.62%±1.98% respectively. The average of the labeling efficiency of F2 generation was 95.80%±0.64%, which met the international standard (≥ 95%) for SILAC quantitative proteomics effective study. SILAC technology was successfully extended to mammalian model system, which will provide powerful tools for the mechanism study of the pathophysiology process with mouse model.

Trace glucose fluxes in individuals with prediabetes using stable isotopes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The glucose fluxes of individuals with prediabetes in Chinese population are not clear. This study was to determine whether the endogenous glucose production (EGP), oral glucose rate of appearance (Ra) and glucose rate of disappearance (Rd) were different in Chinese individuals with prediabetes under fasting conditions and following an oral glucose challenge.</p><p><b>METHODS</b>Five subjects with type 2 diabetes, 5 subjects with prediabetes and 5 non-diabetic subjects matched for age, weight, fat free mass and body mass index underwent a 180 minute stable glucose isotope tracing ([6, 6-(2)H2] glucose, [1-(13)C] glucose, and [U-(13)C] glucose) study under fasting and after ingestion of a 75 g oral glucose load. Isotope glucose enrichment was measured by gas chromatography-mass spectrometry. Insulin sensitivity was estimated using the oral glucose tolerance test (OGTT)-derived insulin sensitivity index, β cell function was determined by the insulinogenic index (δI30/δG30).</p><p><b>RESULTS</b>The insulin sensitivity index (P = 0.043) and insulinogenic index (P = 0.021) were decreased in subjects with prediabetes compared with non-diabetes. Fasting EGP was slightly higher (P = 0.29) and postprandial EGP was comparable in subjects with prediabetes and non-diabetes during 120 minutes after glucose ingestion, but nadir EGP occurred later in prediabetic than non-diabetic subjects. Ra did not differ among the three groups. Rd was substantially lower in subjects with prediabetes than non-diabetes after glucose intake (P = 0.013).</p><p><b>CONCLUSION</b>The mild hyperglycemia observed among individuals with prediabetes may result from decreased Rd during the postprandial state.</p>

Evaluation of iron utilization in children using single stable isotopes tracer / 中华预防医学杂志

<p><b>OBJECTIVE</b>To evaluate the iron utilization in children using single stable isotopes tracer.</p><p><b>METHODS</b>57 children aged from 10 to 12 from a primary school of Beijing in 2010 were selected, 30 of them were boys and 27 were girls. All the subjects were given 5 ml artificial enriched ⁵⁷FeSO₄ twice per day within 5 days, and the total amount of ⁵⁷Fe was 30 mg. 5 ml blood were taken at 1 day before and 14 days after test, and all the feces during the test were collected. The samples were detected by AAS and MC-ICP-MS after pre-treatment to determine the content and abundances of iron in samples, then the iron utilization in whole blood were calculated.</p><p><b>RESULTS</b>The blood volume of male and female subjects 14 days after test were (3.19 ± 0.41) and (3.15 ± 0.29) ml respectively, and there was no significantly difference (t = 1.13, P > 0.05) between them; The amount of ⁵⁷Fe intake by male and female subjects were (27.46 ± 0.25) and (27.29 ± 0.15) mg (t = 1.13, P > 0.05); The amount of ⁵⁷Fe in blood were (5.92 ± 0.71) and (6.30 ± 0.65) mg respectively (t = 2.29, P < 0.05); The iron utilization in whole blood at 14 days of male and female subjects were (20.41 ± 2.03)% and (22.04 ± 0.80)% respectively, male subjects were significantly lower than females (t = 2.51, P < 0.05).</p><p><b>CONCLUSION</b>Single stable isotopes tracer can be used in iron utilization evaluation in children, and the iron utilization in whole blood of female children is higher than males.</p>

Trophic ecology of Arapaima in Guyana: giant omnivores in Neotropical floodplains

Using stable nitrogen and carbon isotope signatures, we investigated the trophic ecology and identified potential prey fish groups supporting the giant Arapaima within floodplain lakes of the Essequibo River basin in southwestern Guyana. Morphological descriptions of feeding structures and digestive tract are presented together with preliminary data on Arapaima diets. Stable isotope results suggest that algivorous/detritivorous and omnivorous fishes contributed most to Arapaima biomass, and generally, that was consistent with what is known about Arapaima diets. Stable nitrogen isotope ratios for piscivorous fishes in these lakes were higher than nitrogen isotope ratios for Arapaima, indicating that piscivorous fishes are unlikely to constitute a major source of energy for Arapaima. This population of Arapaima has an intestine averaging 1.45 times total body length, relatively small teeth, and numerous, closely-spaced gill rakers. These morphological features, together with isotope data, support our inference that Arapaima are secondary consumers and may be better characterized as omnivores and not top predators.

Utilizando firmas de isotopos estables de nitrógeno y carbón, investigamos la ecología trófica e identificamos los grupos de peces que potencialmente mantienen a la Arapaima en los lagos inundables de la cuenca del río Essequibo, al suroeste de Guyana. Presentamos descripciones morfológicas de las estructuras alimentarias y tracto digestivo de la Arapaima, conjuntamente a datos preliminares de sus dietas. Los isotopos estables sugieren que peces algívoros/detritívoros y peces omnívoros son los principales contribuyentes de la biomasa de la Arapaima, y estos resultados son compatibles con lo que se conoce actualmente de la dieta de la Arapaima. A diferencia, las proporciones del isotopo estable de nitrógeno para peces piscívoros en estos lagos resultaron más altas que los valores obtenidos para el isotopo estable de nitrógeno en la Arapaima. Esto indica que es improbable que sean peces piscívoros los que constituyan la fuente energética principal de la Arapaima. La población de Arapaima estudiada presenta un intestino que promedia 1,45 veces la longitud total del cuerpo, dientes relativamente pequeños, y agallas con branquiespinas numerosas y cercanamente espaciadas. Estas características morfológicas, conjuntamente a los datos obtenidos a través del uso de isotopos estables apoyan nuestra inferencia que la Arapaima es un consumidor secundario y que puede ser caracterizada como un pez omnívoro y no como un depredador mayor.

Analyzed the molecular interaction network of tumor suppressor gene 14-3-3 sigma in lung cancer cell based on stable isotope labeling by amino acids in cell culture technology / 中华预防医学杂志

<p><b>OBJECTIVE</b>To analysis the molecular interaction network of 14-3-3 sigma in non small cell lung cancer (NSCLC) cells.</p><p><b>METHODS</b>Established stable over-expressed 14-3-3 sigma protein PG cells, MTT assay was used to assess the growth rate of PG cells. Though stable isotope labeling by amino acids in cell culture (SILAC) and Mass spectrometry (MS) technology, to identify difference expressed proteins caused by over expressed 14-3-3 sigma. The protein expressed >2 or <0.5 times was termed as the differential protein. By searching Human protein reference database (HPRD) and Kyoto encyclopedia of genes and genomes (KEGG), established the molecular interaction network of tumor suppressor gene 14-3-3 sigma.</p><p><b>RESULTS</b>The growth rate of over-expressed 14-3-3 sigma PG cell was obviously slower down compared to vector PG cells. A database including 147 differential protein was established. And a molecular interaction network of 14-3-3 sigma containing 26 protein was constructed.In this network, the expression of CSNK2A1 (casein kinase II subunit alpha), involved in numerous cellular processes, such as cell cycle progression, apoptosis and transcription, was the most significantly increased. A DNA repair protein, MEN1 (Menin) which functions as a transcriptional regulator was the most significantly decreased.</p><p><b>CONCLUSION</b>After stable transfected with 14-3-3 sigma gene, growth rate of PG cells was inhibited, the proteins associated with cell cycle, DNA damage repair mechanisms were significantly changed, and constructed the molecular interaction network.</p>

Total energy expenditure of 16 Chinese young men measured by the doubly labeled water method / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Doubly labeled water (DLW) method is the gold standard for measuring total energy expenditure (TEE). We used this method to measure TEE in Chinese young men.</p><p><b>METHODS</b>Sixteen healthy young men age 23±1 years with body mass index 22.0±1.4 kg/m2 were recruited. TEE was measured by the DLW method, and basal energy expenditure (BEE) was determined by indirect calorimetry. We also conducted 24-h activity, energy balance and factorial approach to estimate energy requirements of the subjects.</p><p><b>RESULTS</b>TEE of subjects by DLW method was 9.45±0.57 MJ/day (2258±180 kcal/day). The 24-h activity was 10.80±0.33 MJ/day (2582±136 kcal/day). The energy requirement, derived from energy balance observations, was 9.93±1.32 MJ/day (2373±315 kcal/day). The BEE of 6.65±0.28 MJ/day (1589±67 kcal/day), calculated by the adjusted Schofield equation, was significantly higher (P<0.001) than that measured by indirect calorimetry, 5.99±0.66 MJ/day (1433±158 kcal/day). The TEE derived from the factorial approach was 10.31±0.43 MJ/day (2463±104 kcal/day).</p><p><b>CONCLUSION</b>The TEE of Chinese young men measured by the DLW method was about 10% lower than the current recommended nutrient intake (RNI), suggesting that the RNI for Chinese men maybe overestimated. Further studies are warranted to determine the value of the estimated energy requirement.</p>

Dietary habits of juveniles of the Mayan cichlid, Cichlasoma urophthalmus, in mangrove ponds of an offshore islet in Belize, Central America

Foraging habitats of juveniles of the Mayan cichlid, Cichlasoma urophthalmus (Günther, 1862), were investigated in two mangrove ponds located in Twin Cays offshore islet in Belize: Sink Hole pond (SH) and Hidden Lake pond (HL). Sink Hole pond is a semiclosed body of water, whereas Hidden Lake pond is connected by a channel to adjacent seagrass beds that surround the islet. Gut contents of 21 juvenile C. urophthalmus (9.8-13.2 cm total length) were analyzed, and five prey taxa were identified. In both mangrove ponds, C. urophthalmus were opportunistic carnivores and consumed primarily crustaceans. Plant material and detritus present in gut contents were most likely ingested incidentally when the fish foraged on small invertebrates. Carbon isotopic values of fish specimens from the two ponds were similar (mean ± SD of -19.2 ± 0.4‰ in SH and -19.4 ± 0.4‰ in HL), and were close to those of mangrove prey (mean ± SD = -20.2 ± 1.5‰), suggesting that this fish species forages in this habitat. Mixing models showed a higher contribution of mangrove food sources to the fish diet than seagrass food sources. This study reveals that young Mayan cichlids, inhabiting two Belize mangrove ponds, are generalists and opportunistic carnivores that forage on mangrove food sources and do not appear to move to adjacent seagrass beds to complement their diets. Understanding trophic linkages between aquatic consumers and food resources may contribute to better management of threatened coastal ecosystems.

Habitats de alimentação de juvenis do ciclídeo-maia, Cichlasoma urophthalmus (Günther, 1862), foram investigados em duas lagoas de mangue localizadas nas ilhas Twin Cays em alto mar em Belize: Sink Hole Lake (SH) e Hidden Lake (HL). Sink Hole é um corpo d'água parcialmente isolado, enquanto Hidden Lake é ligada por um canal com bancos de sargaços que cercam a ilhota. O conteúdo estomacal de 21 juvenil de C. urophthalmus (9,8-13,2 cm de comprimento total) foram analisados e cinco táxons de presas foram identificados. Em ambas as lagoas de mangue, 'C.' urophthalmus foram carnívoros oportunistas e consumiram principalmente crustáceos. Material vegetal e detritos presentes no conteúdo digestivo foram provavelmente ingeridos acidentalmente quando o peixe se alimentava de pequenos invertebrados. Os valores de isótopos estáveis do carbono em espécimes de peixes das duas lagoas foram semelhantes (média ± SD -19,2 ± 0,4 ‰ em SH e -19,4 ± 0,4 ‰ em HL), e foram próximos aos de presas de mangue (mean ± SD = -20.2 ± 1.5 ‰), sugerindo que esta espécie de peixe vai à procura de alimentos neste habitat. Modelos mistos mostraram uma maior contribuição de fontes alimentares de mangue para a dieta dos peixes do que de fontes alimentares de algas marinhas. Este estudo revela que juvenis do ciclídeo-maia que habitam duas lagoas de mangue em Belize, são carnívoros generalistas e oportunistas que se ingerem alimentos dos manguezais e não parecem se mover para leitos de algas marinhas adjacentes para complementar suas dietas. Compreender as ligações tróficas entre consumidores aquáticos e recursos alimentares pode contribuir para uma melhor gestão dos ecossistemas costeiros ameaçados.

Radiolabeling of methanol extracts of yarrow (Achillea millefolium l) in rats / Radiomarcação do extrato metanólico de yarrow (Achillea millefolium l) em ratos

PURPOSE: Current study is focused on extraction with methanol, purification, labeling with 131I using iodogen method of the yarrow plant and investigating in vivo biological activity using biodistribution and imaging studies on healthy animal models. The aim of the study is to contribute plant extracts to discover new drugs in the diagnosis and treatment of several diseases. METHODS: Nine female and nine male healthy Wistar albino rats, which were approximately 100-150 g in weight, were used for biodistribution studies. For imaging studies four healthy male Balb-C mice were used. Quality control studies were done utilizing thin layer radio chromatography (TLRC) and high performance liquid chromatography (HPLC) methods. For biodistribution studies, 131I radiolabeled Peak 7 (131I-Peak 7) was sterilized and injected into the tail veil of rats and imaging studies were obtained using Kodak FX PRO in vivo Imaging System. RESULTS: The radiolabeling yield of each purified the bioactive extracts of the yarrow plant, seven peaks was between 79 and 92%. The highest radiolabeling yield was calculated for 131I radiolabeled seventh peak (131I-Peak 7) (92.78±5.04, n=5). For this reason the biodistribution and imaging studies were done for 131I-Peak 7. That's why; these studies with Peak 7 were carried out. CONCLUSION: Peak 7 was radiolabeled with 131I in high yield for using imaging and therapeutic studies in nuclear medical applications.

OBJETIVO: O atual estudo tem por objetivo a extração com metanol, purificação, marcação com I131 usando o método direto de marcação da planta Achillea, para investigar in vivo a atividade biológica usando biodistribuição e estudos de imagem em modelos animais saudáveis. O objetivo do estudo é contribuir com extratos de plantas para descobrir novas drogas para o diagnóstico e tratamento de várias doenças. MÉTODOS: Nove fêmeas e nove machos ratos Wistar albino saudáveis, com aproximadamente 100 a 150g de peso foram usados para estudos de biodistribuição. Para estudos de imagem, quatro camundongos Balb-C machos e saudáveis foram usados. Estudos de controle de qualidade foram realizados usando métodos de cromatografia de camada fina e cromatografia líquida de alta performance. Para estudos de biodistribuição, pico 5 radiografado com I131 (I131-Peak 7) foi esterilizado e injetado na veia da cauda dos ratos e estudos de imagem foram obtidos usando Sistema de Imagem Kodak FX PRO in vivo. RESULTADOS: O retorno radiomarcado de cada extrato bioativo purificado da planta Achillea sete picos estavam entre 79 e 92%. O retorno com maior marcação foi calculado para I131 sétimo pico (I131-Peak 7) (92,78±5,04, n=5). Por esta razão os estudos de biodistribuição e de imagem foram feitos para I131-Peak 7. CONCLUSÃO: Peak 7 foi radiomarcado com I131 em alto retorno para uso em estudos terapêuticos e de imagens nas aplicações médicas nucleares.

Evaluation of isotopic labeling of lysine residues of peptides for quantitative proteomics / 生物工程学报

To evaluate the reagent 2-methoxy-4,5-dihydro-1H-imidazole used for isotopic labeling in quantitative proteomics, we synthesized 2-methoxy-4,5-dihydro-1H-imidazole and its tetradeuterated analog in three steps. Prior to tryptic cleavage, bovine serum albumin (BSA) was reduced and alkylated. Tryptic peptides were derivatized with an equal volume of either DO or D4 and D4-derivatized peptides were mixed with at variable ratio (from 10:1 to 1:5) prior to MS and MS/MS analysis. We used matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and Electro spray ionization-mass spectrometry (ESI-MS) to evaluate the quantitative capability of labeling. The specificity of the reagent is excellent: only lysine side chains were modified among tryptic peptides. MALDI and ESI ionization modes not only could achieve the quantification of differentially expressed proteins but also facilitate the de novo sequencing. This side-chain modification can be used for quantitative analysis with proteomic strategies involving liquid chromatography. Reverse phase liquid chromatography (RPLC) kept a good resolution, and the introduction of D atoms did not introduce a variation of retention time between heavy and light peptides in RPLC.

Pharmacokinetics of nicotine in blood and brain using microdialysis and stable labelled isotope / 中国中药杂志

<p><b>OBJECTIVE</b>Using the stable isotopes as the internal standard of microdialysis technology to establish a new method to study the whole and local brain dynamics of nicotine percutaneous preparations.</p><p><b>METHOD</b>Using th healthy rats as experimental animals, administrating nicotine in abdominal transdermal way, then sample in the blood and brain simultaneously by microdialysis which use deuterium nicotine (DL-nicotine) as internal standard. Detecting the samples by LC-MS/MS method.</p><p><b>RESULT</b>The configuration process in blood and brain both conforms to 2 compartments model, t1/2 is 29.38 min, t1/2beta is 208.51 min, AUC(0-infinity) is 152 127.10 microg x min x L(-1) in the blood t1/2 is 86.64 min, t1/2beta is 386.00 min, AUC(0-infinity) is 152 820.90 microg x min x L(-1) in the brain.</p><p><b>CONCLUSION</b>Dl-nicotine can be used as internal standard of nicotine to correcte the recovery; Stable isotopes internal standard microdialysis technology can be used for studing the whole and the local pharmacokinetic of nicotine and also provide new ideas and methods to studing the process of new drug delivery system.</p>

Differential proteins in esophageal squamous cell line EC9706/CDDP identified by SILAC quantitative proteomic approach / 药学学报

Multidrug resistance (MDR) is one of the main causes leading to the failure in cancer treatment. Differential proteins between esophageal squamous cell carcinoma (ESCC) cell line EC9706 and its cisdiamminedichloroplatinum (CDDP)-resistant subline EC9706/CDDP revealed by quantitative analysis may provide deeper insights into the molecular mechanisms of MDR implicated in ESCC. EC9706/CDDP was generated by exposure of its parental sensitive EC9706 to a step-wise increase of CDDP concentration during EC9706 cultivation. The stable isotope labeling with amino acids in cell culture (SILAC) was used to label EC9706 and EC9706/CDDP with heavy and light medium, separately. Mixed peptides derived from EC9706 and EC9706/CDDP were analyzed by high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS/MS) and subsequently subjected to bioinformatics analysis to identify differential proteins between EC9706 and EC9706/CDDP. Compared to parental EC9706, EC9706/CDDP manifested phenotypes of slow proliferation, cell pleomorphology, atypia and increased resistant-index 3.23. Seventy-four differential proteins identified in the present study belongs to various families with multiple functions, such as cytoskeleton (20%), energy metabolism (11%), transcription regulation and DNA repair (11%), redox homeostasis (9.5%), protein biosynthesis and mRNA processing (12%), ribosome constituent (8.1%), molecular chaperone (8.1%), immunity/inflammation (5.4%), intracellular transport (5.4%) and nucleosome assembly (2.7%), which indicated that development of MDR is a complicated process involving dysregulation of multiple molecules and pathways. The data is of great value for in-depth elucidation of molecular mechanisms of the MDR implicated in ESCC and may represent potential molecular targets for future therapeutic development.

Comparison of two peptide radiotracers for prostate carcinoma targeting

OBJECTIVES: Scintigraphy is generally not the first choice treatment for prostate cancer, although successful studies using bombesin analog radiopeptides have been performed. Recently, a novel peptide obtained using a phage display library demonstrated an affinity for prostate tumor cells. The aim of this study was to compare the use of a bombesin analog to that of a phage display library peptide (DUP-1) radiolabeled with technetium-99m for the treatment of prostate carcinoma. The peptides were first conjugated to S-acetyl-MAG3 with a 6-carbon spacer, namely aminohexanoic acid. METHODS: The technetium-99m labeling required a sodium tartrate buffer. Radiochemical evaluation was performed using ITLC and was confirmed by high-performance liquid chromatography. The coefficient partition was determined, and in vitro studies were performed using human prostate tumor cells. Biodistribution was evaluated in healthy animals at various time points and also in mice bearing tumors. RESULTS: The radiochemical purity of both radiotracers was greater than 95 percent. The DUP-1 tracer was more hydrophilic (log P = -2.41) than the bombesin tracer (log P = -0.39). The biodistribution evaluation confirmed this hydrophilicity by revealing the greater kidney uptake of DUP-1. The bombesin concentration in the pancreas was greater than that of DUP-1 due to specific gastrin-releasing peptide receptors. Bombesin internalization occurred for 78.32 percent of the total binding in tumor cells. The DUP-1 tracer showed very low binding to tumor cells during the in vitro evaluation, although tumor uptake for both tracers was similar. The tumors were primarily blocked by DUP1 and the bombesin radiotracer primarily targeted the pancreas. CONCLUSION: Further studies with the radiolabeled DUP-1 peptide are recommended. With further structural changes, this molecule could become an efficient alternative tracer for prostate tumor diagnosis.

Marcación de un derivado de glucosa con 99mTc mediante la formación de un complejo Tc(V)-nitruro: estudios preliminares / Labelling of a glucose derivative with 99mTc through the formation of a Tc(V)-nitride symmetrical complex: preliminary studies

La preparación de derivados de glucosa marcados con 99mTc reviste gran interés para la evaluación del consumo de glucosa in vivo en oncología y cardiología nuclear. Este trabajo presenta la marcación de un análogo de glucosa (GLU-DTC) mediante la formación de un complejo Tc(V)-nitruro simétrico. Para ello se incorporó a la biomolécula un grupo ditiocarbamato capaz de coordinar al metal. La marcación fue realizada mediante sustitución de ligandos, obteniéndose una única especie con pureza radioquímica superior al 90 por cientp, la que se mantuvo durante al menos 4 hs. La caracterización fisicoquímica y biológica muestra que el complejo 99mTc(V)-nitruro(GLU-DTC)2 es un compuesto estable y altamente hidrofílico, aunque su unión a proteínas plasmáticas es mayor a la esperada, hecho que justificaría la alta actividad retenida en sangre y en hígado durante la evaluación biológica en ratones CD1 normales. Estos resultados indican que la marcación con 99mTc de este derivado de glucosa produce una alteración significativa de su comportamiento biológico.

The preparation of 99mTc-labeled glucose derivatives is of great interest to evaluate the in vivo glucose uptake in nuclear oncology and cardiology. This paper presents the labelling of a glucose analogue (GLU-DTC) through the formation of a Tc(V)-nitride symmetrical complex. For this purpose, a dithiocarbamate group was incorporated to the biomolecule, in order to coordinate the metal. The labelling reaction was carried out by substitution yielding a single complex with radiochemical purity above 90 percent. This complex was stable for at least 4 hours. The physicochemical and biological characterization shows that the 99mTc(V)-nitride(GLU-DTC)2 complex is a stable and highly hydrophilic compound, although its plasma protein binding is greater than expected, a fact which justifies the high activity retained in blood and liver during the biological evaluation in normal CD1 mice. These results indicate that 99mTc labelling of this glucose derivate alters significantly its biological behaviour.