Radiometric studies on the oxidation of (1-14c) fatty acids by drug-susceptible and drug-resistant mycobacteria

A radiometric assay system has been used to study oxidation patterns of (1-14C) fatty acids by drug-susceptible and drug-resistant organisms of the genus Mycobacterium. Two strains of M. tuberculosis susceptible to all drugs, H37Rv and Erdman, were used. Drug-resistant organisms included in this investigation were M. tuberculosis H37Rv resistant to 5 ug/ml isoniazid, M. bovis, M. avium, M. intracellular, M. kansasii and M. chelonei. The organisms were inoculated in sterile reaction vials containing liquid 7H9 medium, 10% ADC enrichment and 1.0 uCi of one of the (1-14C) fatty acids (butyric, hexánoic, octanoic, decanoic, lauric, myristic, palmitic, stearic, oleic, linoleic, linolenic). Vials were incubated at 37°C and the 14CO2 envolved was measured daily for 3 days with a Bactec R-301 instrument. Although each individual organism displayed a different pattern of fatty oxidation, these patterns were not distinctive enough for identification of the organism. No combination of fatty acids nor preferential oxidation of long chain or of short chain fatty acids were able to separate susceptible from resistant organisms. Further investigation with a larger number of drug susceptible mycobacteria including assimilation studies and oxidation of other substrates may be required to achieve a distinction between drug-susceptible and drug-resistant mycobacteria.

Radiometric studies on the oxidation of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria

A radiometric assay system has been used to study oxidation patterns of (U-14C) L-amino acids by drug-susceptible and drug-resistant mycobacteria. Drug-susceptible M. tuberculosis (H37Rv TMC 102 and Erdman) along with the drug-resistant organism M. tuberculosis (H37 Rv TMC 303), M. bovis, M. avium, M. intracellulare, M. kansasii and M. chelonei were used. The organisms were inoculated into a sterile reaction system with liquid 7H9 medium and one of the (U-14C) L-amino acids. Each organism displayed a different pattern of amino acid oxidation, but these patterns were not distinctive enough for identification of the organism. Complex amino acids such as proline, phenylalanine and tyrosine were of no use in identification of mycobacteria, since virtually all organisms failed to oxidize them. There was no combination of substrates able to separate susceptible from resistant organisms.

VALIDATION OF 14C-UREA BREATH TEST FOR DIAGNOSIS OF Helicobacter pylori

The aim of this study was to validate the 14C-urea breath test for use in diagnosis of Helicobacter pylori infection. Thirty H. pylori positive patients, based on histologic test and thirty H. pylori negative patients by histology and anti-H. pylori IgG entered the study. Fasting patients drank 5 uCi of 14C-urea in 20 ml of water. Breath samples were collected at 0, 5, 10, 15, 20 and 30 min. The difference of cpm values between the two groups was significant at all the time intervals, besides time 0 (p<0.0001). At 20 min, the test gave 100% sensitivity and specificity with a cut-off value of 562 cpm. Females were higher expirers than males (p=0.005). 14C-urea breath test is highly accurate for Helicobacter pylori diagnosis. It is fast, simple and should be the non-invasive test used after treating Helicobacter pylori infection.

Dynamics of 14C-labeled glucose and ammonium in saline arable soils

Organic matter dynamics and nutrient availability in saline agricultural soils of the State of Guanajuato might provide information for remediation strategies. 14C labeled glucose with or without 200 mg kg-1 of NH4+-N soil was added to two clayey agricultural soils with different electrolytic conductivity (EC), i.e. 0.94 dS m-1 (low EC; LEC) and 6.72 dS m-1 (high EC; HEC), to investigate the effect of N availability and salt content on organic material decomposition. Inorganic N dynamics and production of CO2 and 14CO2 were monitored. Approximately 60 % of the glucose-14C added to LEC soil evolved as 14CO2, but only 20 % in HEC soil after the incubation period of 21 days. After one day, < 200 mg 14C was extractable from LEC soil, but > 500 mg 14C from HEC soil. No N mineralization occurred in the LEC and HEC soils and glucose addition reduced the concentrations of inorganic N in unamended soil and soil amended with NH4+-N. The NO2- and NO3- concentrations were on average higher in LEC than in HEC soil, with exception of NO2- in HEC amended with NH4+-N. It was concluded that increases in soil EC reduced mineralization of the easily decomposable C substrate and resulted in N-depleted soil.

Degradação de 14C-atrazina em solo sob condições semicontroladas

Objetivou-se neste trabalho, avaliar a mineralização e a formação de resíduos extraível e não-extraível de 14C-atrazina em um solo intensivamente utilizado para fins agrícolas no Estado de São Paulo. Atrazina radiomarcada foi aplicada (5 L ha-1 ou 2 mg kg-1 de i.a.) em um Latossolo Vermelho-Escuro, álico, A moderado, textura média. Frascos erlenmeyer contendo 200 g (peso seco) do solo assim preparado e com umidade ajustada para 2/3 da capacidade de campo foram enterrados na Estação Experimental de Lisímetros do CENA-USP, onde se iniciou, ao mesmo tempo, uma plantação de milho. O 14CO2 desprendido foi avaliado a cada 15 dias, durante 150 dias, e atingiu, no final desse período de incubação, 36% da atividade total aplicada, e meia-vida de 168 dias. Os resíduos formados no solo foram determinados, em termos de dessorção, com o uso de cloreto de cálcio, e, em termos de extração, com o sistema-solvente acetonitrila/água (80:20); os resíduos não-extraíveis foram avaliados por combustão. Durante o período de incubação, os resíduos extraíveis diminuíram para 1/3, enquanto os resíduos ligados (não-extraíveis) permaneceram ao redor de 34%. Os metabólitos foram identificados por cromatografia em camada delgada, e foram detectadas, nas frações dessorvidas, hidroxiatrazina (44%), desisopropilatrazina (3,28%) e atrazina (52,72%) e nas frações extraídas, hidroxiatrazina (16,22%), desisopropilatrazina (2,25%), desetilatrazina (2,24%) e atrazina (79,29%). Conclui-se que a mineralização ocorreu somente na fração de resíduos extraíveis e foi favorecida pelas condições de incubação, em que o principal fator foi a variação de temperatura.

Datação da fração humina da matéria orgânica do solo e sua comparação com idades 14C de carvões fósseis

The radiocarbon dating of the soil organic matter (SOM) is a polemic subject, due mainly to the complexity of the formation of the soils and to the variable contamination from several sources. Soil samples from 4 different Brazilian localities were submitted to physical and chemical pre-treatment for the extraction of humin fraction, which is the most stable organic compound and theoretically the oldest and representative of the age of the SOM. The radiocarbon dating obtained from the total SOM and their humin fractions are compared to the 14C ages from buried charcoals at similar depths. The radiocarbon ages obtained from such charcoals are, in most of the cases, concordant within the experimental errors of those obtained on humin fractions, or are in average 10% higher, with one exception. Thus, the ages on humin fractions could be assumed as the minimum ages for the associated soils, while the results obtained on total SOM, even at depths until 200 cm, exhibit pronounced contamination effect by modern carbon, rejuvenating their ages.

Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants

(Comparative uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid in callus cultures of monocot (Dioscorea spp.) and dicot (Nicotiana tabacum L.) plants). The uptake and metabolism of 2-[14C]-2,4-dichlorophenoxyacetic acid (2,4-D) were investigated in leaf calluses of Nicotiana tabacum, tuber calluses of Dioscorea opposita and calluses derived from zygotic embryos, leaves and petioles of Dioscorea composita. Striking similarities were evident in the patterns of 2,4-D metabolites and their chemical characteristics in the three callus types of D. composita compared, but significant differences were detected among the patterns of rnetabolites in the three species studied. Preliminary investigations on the stability of various metabolites (separated using TLC) by hydrolysis showed that sugar esters appeared to be the major metabolites in tobacco whilst in yams (D. opposita) glycosides were shown to be the main ones, which indicated a similarity between plants of Gramineae and Dioscoreaceae in terms of 2,4-D metabolism. Release of 2,4-D from tobacco callus cells upon their transfer to 2,4-D-free medium was detected and the implications of this are discussed in relation to the cultural conditions necessary to induce morphogenesis in vitro.

Comparison of the efficacy of biodegradable and non-biodegradable scintillation liquids on the counting of tritium- and [14C]-labeled compounds

The widespread use of ³H and 14C in research has generated a large volume of waste mixed with scintillation liquid, requiring an effective control and appropriate storage of liquid radioactive waste. In the present study, we compared the efficacy of three commercially available scintillation liquids, Optiphase HiSafe 3, Ultima-Gold™ AB (biodegradable) and Insta-Gel-XF (non-biodegradable), in terms of [14C]-glucose and [³H]-thymidine counting efficiency. We also analyzed the effect of the relative amount of water (1.6 to 50%), radioisotope concentration (0.1 to 100 nCi/ml), pH (2 to 10) and color of the solutions (samples containing 0.1 to 1.0 mg/ml of Trypan blue) on the counting efficiency in the presence of these scintillation liquids. There were few significant differences in the efficiency of 14C and ³H counting obtained with biodegradable or non-biodegradable scintillation liquids. However, there was an 83 and 94% reduction in the efficiency of 14C and ³H counting, respectively, in samples colored with 1 mg/ml Trypan blue, but not with 0.1 mg/ml, independent of the scintillation liquid used. Considering the low cost of biodegradable scintillation cocktails and their efficacy, these results show that traditional hazardous scintillation fluids may be replaced with the new safe biodegradable fluids without impairment of ³H and 14C counting efficiency. The use of biodegradable scintillation cocktails minimizes both human and environmental exposure to hazardous solvents. In addition, some biodegradable scintillation liquids can be 40% less expensive than the traditional hazardous cocktails.

Radiometric studies of mycobacterium tuberculosis

An in vitro assay system that included automated radiometric quantification of 14CO2 released as a result of oxidation of 14C- substrates was applied for studying the metabolic activity of M. tuberculosis under various experimental conditions. These experiments included the study of a) mtabolic pathways, b) detection times for various inoculum sizes, c) effect of filtration on reproducibility of results, d) influence of stress environment e) minimal inhibitory concentrations for isoniazid, streptomycin, ethambutol and rifampin, and f) generation times of M. tuberculosis and M. bovis. These organisms were found to metabolize 14C-for-mate, (U-14C) acetate, (U-14C) glycerol, (1-14C) palmitic acid, 1-14C) lauric acid, (U-14C) L-malic acid, (U-14C) D-glucose, and (U-14C) D-glucose, but not (1-14C) L-glucose, (U-14C) glycine, or (U-14C) pyruvate to 14CO2. By using either 14C-for-mate, (1-14C) palmitic acid, or (1-14C) lauric acid, 10(7) organisms/vial could be detected within 24 48 hours and as few as 10 organisms/vial within 16-20 days. Reproducible results could be obtained without filtering the bacterial suspension, provided that the organisms were grown in liquid 7H9 medium with 0.05% polysorbate 80 and homogenized prior to the study. Drugs that block protein synthesis were found to have lower minimal inhibitory concentrations with the radiometric method when compared to the conventional agar dilution method. The mean generation time obtained for M. bovis and different strains of M. tuberculosis with various substrates was 9 ± 1 hours.

Radiometric detection of metabolic activity of Paracoccidioides brasiliensis and its susceptibility to amphotericin B and diethylstilbestrol

Paracoccidioidomycosis (South American blastomycosis) is a systemic disease, strikingly more frequent in males, caused by the dimorphic fungus Paracoccidioides brasiliensis. A radiometric assay system has been applied to study the metabolic activity and the effect of drugs on this fungus "in vitro". The Y form of the yeast, grown in liquid Sabouraud medium was inoculated into sterile reaction vials containing the 6B aerobic medium along with 2.0 μCi of 14C-substrates. Control vials, prepared in the same way, contained autoclaved fungi. To study the effects of amphotericin B (AB) (0.1 and 10 μg/ml) and diethylstilbestrol (DSB) (1.0, 5.0 and 10 μg/ml) extra controls with live fungi and no drug were used. All vials were incubated at 35°C and metabolism measured daily with a Bactec instrument. 14CO2 production by P. brasiliensis was slow and could be followed for as long as 50 days. AB at 10mg/ml and DSB at 5 μg/ml inhibited the metabolism and had a cidal effect on this fungus. The results with DSB might explain the low incidence of the disease in females. This technique shows promise for studying metabolic pathways, investi gating more convenient 14C-substrates to expedite radiometric detection and for monitoring the effects of other drugs and factors on the metabolism of P. brasiliensis "in vitro".

Ação dos sucos digestivos sobre a absorção e destino do bcg Oral

Estudou-se a ação do suco gástrico artificial e suco duodenal humano sobre a vacina BCG, bem como a absorção e destino desta após administração intragástrica em camundongos. O contato de 2 horas do bacilo com o suco gástrico provocou uma diminuição significante do consumo de oxigênio e uma moderada perda da viabilidade. O suco duodenal induziu marcante decréscimo da respiração bacilar egrande redução da viabilidade. O BCG foi marcado com carbono-14 usando-se 14C-glicerol como precursor dos lipidios micobacterianos. Níveis similares de radioatividade foram obtidos nos órgãos dos animais, 24 horas após administração intragástrica de 14C- BCG, 14C-BCG rompido por ultra-som e 14C-glicerol. Os níveis de 14C-BCG permaneceram estáveis do 6º ao 24º dia, enquanto o sonicado de 14C-BCG e 14C-glicerol definiram um processo de decaimento biológico. As curvas de biodecaimento no intestino delgado e no fígado indicaram que o processo de absorção foi desencadeado rapidamente e alcançou seu nível máximo às 24 horas, decaindo em seguida de acordo com a complexidade química do material dado aos camundongos. Não foram isolados bacilos viáveis dos órgãos dos animais que receberam BCG não marcado. Pode-se concluir, portanto, que a maioria dos bacilos foram absorvidos intactos mas não viáveis.

Dinâmica do ecótono floresta-campo no sul do estado do Amazonas no Holoceno, através de estudos isotópicos e fitossociológicos

Com o emprego dos isótopos do carbono (12C, 13C, 14C) da matéria orgânica do solo (MOS) e das plantas, é apresentado um estudo comparativo entre perfis orgânicos de solos formados em depressões de áreas cobertas por ecossistemas de campos e florestas ao sul do estado do Amazonas, visando o entendimento da dinâmica da paleovegetação. A dinâmica da vegetação atual na região foi avaliada utilizando-se estudos fitossociológicos e caracterizações botânica e isotópica (delta13C) das espécies de plantas presentes em duas bordas floresta-campo. Teores de carbono orgânico total foram superiores nas camadas superficiais no campo, quando comparados com a floresta. Dados de delta13C associados à cronologia do 14C indicaram predomínio de plantas C3 no início do Holoceno em ambos os ecótonos. Entre aproximadamente 7.000-3.000 anos AP verificou-se a influência crescente de plantas C4, indicando regressão da floresta com possível presença de um clima mais seco. A partir de aproximadamente 3.000 anos AP os dados sugeriram expansão da floresta provavelmente relacionada ao retorno a um clima mais úmido. A presença de algumas espécies características da borda, como a Sclerolobium paniculatum e Himatanthus sucuuba, nos campos, sugere o atual avanço da floresta sobre os mesmos. Estas espécies estariam sendo as bioindicadoras desse avanço.

Palinologia holocênica do testemunho Bom Jesus, margem leste da ilha do Marajó, Pará, Amazônia

A porção superior (1,25m) do testemunho de sondagem Bom Jesus (TBJ), coletado no limite campo-mangue da Fazenda Bom Jesus, município de Soure, ilha do Marajó, Pará, Brasil, foi estudada através de análise palinológica de alta resolução objetivando a determinação da composição, abundância e diversidade de tipos polínicos bioindicadores de modificações na paleovegetação durante o Holoceno. 16 amostras sedimentares de 2cm³ foram tratadas de acordo com metodologia padrão em palinologia. Os programas Tilia e Tilia Graph foram utilizados para a construção dos diagramas palinológicos de abundância e concentração. A base do testemunho foi datada por 14C em 2730 ± 40 anos A.P. Foram definidas três zonas palinológicas. A presença de pólen de Rhizophora com abundância máxima de 88% apontou dominância de mangue ao longo de todo o testemunho sedimentar. Variações recorrentes na hidrodinâmica da baía do Marajó, caracterizadas por pulsos erosivos de curto período, parecem ter provocado redução na dominância de mangue. O incremento na abundância de tipos polínicos bioindicadores de campo inundável associados a elementos de restinga e floresta, evidenciam a migração do campo inundável sobre o manguezal. Os dados indicam correlação com outros registros polínicos holocênicos de transgressão marinha para a costa norte amazônica no Holoceno Superior.

Transferências lipídicas para HDL em diabéticos tipo 2: associações com microalbuminúria, estatina e insulina

FUNDAMENTO: O diabete melito tipo 2 (DM2) é um fator de risco isolado para coronariopatia, principalmente quando associado à microalbuminúria (MA). Alterações estruturais e funcionais das lipoproteínas não são totalmente esclarecidas nesse contexto. OBJETIVO: Avaliar a transferência de lípides para HDL (T) em pacientes DM2 e a associação com a presença da MA e com o tratamento com estatina ou insulina. MÉTODOS: Estudamos 33 pacientes com DM2 e 34 controles pareados para idade. Uma nanoemulsão lipídica artificial radiomarcada com ³H-Triglicéride (TG) e 14C-colesterol livre (CL) ou ³H-colesterol éster (CE) e 14C-fosfolípide (FL) foi incubada com plasma. A nanoemulsão e as lipoproteínas foram precipitadas, exceto a HDL, que teve sua radioatividade contada. RESULTADOS: A TFL (%) foi maior no grupo com DM2 que no grupo-controle (25,2±3,2 e 19,7±3,2 respectivamente; p < 0,001), assim como a TCL (%): 9,1±2,7 e 6,3±1,5 respectivamente; p < 0,001. O diagnóstico de MA não se associou a mudanças da propriedade de transferência. O uso da insulina associou-se à menor TFL (%): 23,5±2,1 contra 26,1±3,3; p = 0,018. Já o uso da estatina associou-se à queda de todas - TCE (%): 3,5±0,9; TFL (%):23,8±2,0; TTG (%): 3,9±0,8; TCL (%):7,4±1,3 - quando comparado ao grupo que não usava estatina (TCE (%):5,9±2,4; TFL (%):26,9±3,6; TTG (%):6,4±2,2; TCL (%):11,1±2,6). CONCLUSÃO: O DM2 aumentou a transferência de lípides de superfície para HDL, enquanto o uso de estatina diminuiu todas as transferências de lípides. A presença de MA não se associou às alterações das transferências de lípides.

Estudo sôbre as temperaturas médias em Campinas

This paper deals with the study by orthogonal polynomials of trends in the mean annual and mean monthly temperatures (in degrees Centigrade) in Campinas (State of São Paulo, Brasil), from 1890 up to 1956. Only 4 months were studied (January, April, July and October) taken as typical of their respective season. For the annual averages both linear and quadratic components were significant, the regression equation being y = 19.95 - 0.0219 x + 0.00057 x², where y is the temperature (in degrees Centigrade) and x is the number of years after 1889. Thus 1890 corresponds to x = 1, 1891, to x = 2, etc. The equation shows a minimum for the year 1908, with a calculated mean y = 19.74. The expected means by the regression equation are given below. Anual temperature means for Campinas (SP, Brasil) calculated by the regression equation Year Annual mean (Degrees Centigrade) 1890 19.93 1900 10.78 1908 19.74 (minimum) 1010 19.75 1920 19.82 1930 20.01 1940 20.32 1950 20.74 1956 21.05 The mean for 67 years was 20.08°C with standard error of the mean 0.08°G. For January the regression equation was y = 23.08 - 0.0661 x + 0.00122 x², with a minimum of 22.19°C for 1916. The average for 67 years was 22.70°C, with standard error 0.12°C. For April no component of regression was significant. The average was 20.42°C, with standard error 0.13°C. For July the regression equation was of first degree, y = 16.01 + 0.0140X. The average for 67 years was 16.49°C, with standard error of the mean 0.14°C. Finally, for October the regression equation was y = 20.55 - 0.0362x + 0.00078x², with a minimum of 20.13°C for 1912. The average was 20.52°C, with standard error of the mean equal to 0.14°C.