An optimized protocol for DNA extraction in plants with a high content of secondary metabolites, based on leaves of Mimosa tenuiflora (Willd.) Poir. (Leguminosae).

Some species are characterized by a high content of tannins, alkaloids, and phenols in their leaves. These secondary metabolites are released during DNA extraction and might hinder molecular studies based on PCR (polymerase chain reaction). To provide an efficient method to extract DNA, Mimosa tenuiflora, an important leguminous plant from Brazilian semiarid region used in popular medicine and as a source of fuelwood or forage, was used. Eight procedures previously reported for plants were tested and adapted from leaf tissues of M. tenuiflora stored at -20°C. The optimized procedure in this study encompassed the utilization of phenol during deproteinization, increased concentrations of cetyltrimethylammonium bromide and sodium chloride, and a shorter period and lower temperature of incubation concerning other methods. The extracted DNA did not present degradation, and amplification via PCR was successful using ISSR, trnL, ITS, and ETS primers. Besides M. tenuiflora, this procedure was also tested and proved to be efficient in genetic studies of other plant species.

Transferability of microsatellite markers in Syagrus coronata (Mart.) Becc. (Arecaceae), an iconic palm tree from the Brazilian semiarid region.

The licuri palm Syagrus coronata plays a key role in the ecology and economy of Brazilian semiarid region. Nonetheless, genetic data about populations of this species are absent even though the intensive and uncontrolled exploitation since colonial periods has threatened the sustainability and viability of licuri populations. Therefore, we attempted to test the efficacy of transferability of microsatellite loci isolated from three palm tree species to S. coronata to analyze the population of this species throughout their range. A set of 19 heterologous microsatellite loci was tested in three native populations of S. coronata from the State of Bahia, northeastern Brazil, which amplified using distinct annealing temperatures (50°-60°C). Based on the 10 most polymorphic loci, the selected populations exhibited a mean number of alleles per locus of 9.8, and high genetic diversity values since the expected heterozygosity ranged from 0.573 to 0.754, while the observed heterozygosity varied from 0.785 to 1.000. In conclusion, the tested loci are transferrable and highly efficient to population studies in S. coronata, thus minimizing the lack of species-specific loci to the genetic monitoring of licuri populations.

Genetic diversity and structure in natural populations of Maytenus truncata Reiss, 1861, a medicinal plant vulnerable to extractivism in Bahia State, Brazil.

Maytenus truncata (Celastraceae) is a plant species widely used in the treatment of ulcers and tumors. Despite the intensive harvest of native specimens in the State of Bahia, northeastern Brazil, there is no information about the genetic variability or structure of this species. Therefore, the goal of this study was to estimate the genetic diversity and population structure of M. truncata based on inter simple sequence repeat (ISSR) molecular markers. The samples comprised specimens from Jequié, Contendas do Sincorá, Boa Nova, and Boa Vista do Tupim in the State of Bahia. After selection of eight ISSR primers, the percentage of polymorphic loci was equal to 96.2% and genetic diversity was 0.3581. The Mantel test revealed positive correlation among genetic and geographic distances (r = 0.5462), but it was not significant (r ≥ 0, P = 0.8365). Even though AMOVA revealed that most variation was found within populations (68%), a high structuring was detected among them (ΦST = 0.31, P < 0.001). Both UPGMA and Bayesian analyses indicated that gene flow was higher between Jequié and Contendas do Sincorá, whereas samples from Boa Nova and Boa Vista do Tupim were more isolated. This result is likely because of the population decrease and restriction to gene flow associated with intensive extractivism of populations of this species.

Optimization of DNA extraction from fresh leaf tissues of Melanoxylon brauna (Fabaceae).

Melanoxylon brauna (Fabaceae - Caesalpinioideae) is an endemic and valuable hardwood tree species in the Brazilian Atlantic rainforest; it is comparable to African ebony wood. We tested three protocols of DNA extraction based on the citrimonium bromide (CTAB) method and evaluated the quantity, purity and integrity of the DNA. We also determined whether these procedures interfere with PCR amplification in order to develop a protocol for M. brauna. We found that the quality and integrity of DNA were improved with the use of proteinase K in the extraction buffer and by modifications in the centrifugation speed. The lowest concentration of DNA was obtained with Doyle and Doyle's protocol (5.42 ng/µL). Ferreira and Grattapaglia's protocol modified for M. brauna provided the most DNA (36.89 ng/µL) and the highest quality DNA (purity ratio of 1.80 nm). The original Ferreira and Grattapaglia protocol provided 13.42 ng/µL DNA; however, the purity ratio (1.44 nm) indicates protein contamination. PCR results showed that Ferreira and Grattapaglia's protocol modified for M. brauna gave satisfactory quantity and purity of DNA for molecular studies.

Modulatory effect of Byrsonima verbascifolia (Malpighiaceae) against damage induced by doxorubicin in somatic cells of Drosophila melanogaster.

Byrsonima verbascifolia, popularly known in Brazil as murici, is a medicinal plant widely used in the treatment of bacterial and viral infections, Chagas's disease, diarrhea, bronchitis, cough and fever, as well as for protection of the intestinal mucosa. Since chemotherapy and radiotherapy, broadly employed in the treatment of cancer, can have undesirable side effects, such as inducing DNA damage in normal cells, it would be useful to investigate compounds that inhibit or reduce these effects. A lyophilized water extract of murici, used at three different concentrations (25, 50, and 100 mg/mL), was tested to determine if it could reduce damage induced by the antineoplastic compound doxorubicin in somatic cells of Drosophila melanogaster, analyzed by SMART/wing. The frequency of mutant spots in descendants from standard and high bioactivation crosses was significantly reduced by treatment with murici extract. Further studies are needed using other experimental models, to determine if murici has the potential to be employed by cancer patients receiving chemotherapy.

Trypanocidal activity of 2-propen-1-amine derivatives on trypomastigotes culture and in animal model.

The cis and trans isomers (either E or Z isomers) of the unsubstituted and bromo-2-propen-1-amine derivatives were evaluated in vitro on Trypanosoma cruzi. The results showed that cis is the most active isomeric form against trypomastigote forms of T. cruzi, indicating that it may contribute most to the trypanocidal effect. All mice which received 5 mg kg(-1) daily for 9 consecutive days, or 200 mg kg(-1) in a single dose of the bromo derivative of 2-propen-1-amine, survived after an infection with 10(4) trypomastigotes/ml of the Y-strain of T. cruzi. They also had a significantly lower parasitemia than the controls. However, with 100 mg kg(-1) of benznidazol for 9 consecutive days, 25% of the animals died by the end of the evaluation 40 days after infection. The involvement of the biosynthesis of ergosterol in the trypanocidal effect of the unsubstituted 2-propen-1-amine derivative was investigated on proliferative epimastigote forms of the parasite. The chromatographic analyses of the lipid extracts obtained from parasites treated with 2-propen-1-amine derivatives and controls (not treated) revealed that growth inhibition is correlated with the accumulation of squalene and the decrease of ergosterol levels. These results suggest that inhibition of the biosynthesis of ergosterol is an important target for the action of the 2-propen-1-amine derivative on T. cruzi through the inhibition of the enzyme squalene epoxidase.

Effect of yeast extract on growth kinetics of Monascus purpureus.

Growth kinetics and red pigment production of Monascus purpureus CCT 3802 was studied. A reproducible inoculum with extremely dispersed hyphae for bioreactor runs was obtained through a two-step cultivation in a shaker. First, the spores were cultivated in a complex medium rendering a suspension of vegetative cells. In the second step these cells were grown in a semisynthetic medium. Two types of media were employed in the bioreactor runs: a semisynthetic (glucose, salts, and yeast extract), and a synthetic, without yeast extract. The inclusion of yeast extract, caused an increase in cell yield on glucose (Yx/s) as high as 40%. Also, yeast extract probably yielded a higher proportion of red pigment associated with the cell, relative to the synthetic medium. On the other hand, cells grown on the synthetic medium were slightly higher producers of red soluble pigments.

Determination of the geometrical diarylpropenamine isomers in feces by high-performance liquid chromatography.

Diarylpropenamine derivatives are a class of compounds which have been evaluated as potential drug candidates. Here a specific and reproducible HPLC method for the determination of cis- and trans-isomers of the unsubstituted derivative, 3-(4'-bromo-[1,1'-biphenyl]-4-yl)-3-(4-X-phenyl-N,N-dimethyl-2-propen -1-amine (I, where X=H) in feces is described. The analyte I and internal standard, nitro derivative (II, where X=NO2), were isolated from the basified biological matrix using a liquid-liquid extraction with ethyl acetate followed by a solid-phase procedure performed on a silica cartridge. The organic phase was evaporated to dryness, the residue was reconstituted in mobile phase and injected into the HPLC system. The analytes were eluted with ethyl acetate-hexane-triethylamine (59:40:1) in HPLC column (silica) and detected by UV spectrophotometry at 272 nm. Linearity, precision and accuracy data for feces standards after extraction were acceptable. The method has been applied to analyses of feces samples from rats dosed with I, in which it could be anticipated that fecal excretion is quantitatively the major route for I elimination.

Activity of N,N-dimethy-1-2-propen-1-amine derivatives in mice experimentally infected with Trypanosoma cruzi.

Propenamine derivatives 3-(4'-bromo-[1,1'-biphenyl]-4-yl)-3-(4-X-phenyl)-N,N-dimethyl-2-prope n-1-amine (where X = H or Br) were tested in experimentally infected mice with Trypanosoma cruzi (Y strain). When a daily dose of 20 mg kg-1 of the bromo (X = Br) derivative for 9 consecutive days was used, no parasite by optical microscopy was found. Significant parasitemic decrease was also observed with a single dose (100 mg kg-1) of this compound. Moreover, both treatment schemes displayed a strong protective effect characterized by decreased of mortality. On the other hand, similar treatment schedules using the unsubstituted (X = H) derivative led to parasitemic and mortality curves similar to the control group. These results indicate that the bromo derivative has a remarkable activity against T. cruzi infection. Due to the potentiality of this derivative, further investigation of this class of compounds as chemotherapeutic agents for Chagas' disease is of prime importance.

Massive rectal bleeding due to jejunal and colonic tuberculosis.

A case of massive rectal bleeding due to colonic tuberculosis in advanced pregnancy with intrauterine foetal death is reported. Patient was treated with resection of the left colon and left transverse end colostomy with closure of the rectal stump. Hysterotomy for the removal of the dead foetus was performed. The patient improved in health with antitubercular treatment. The colorectal anastomosis was performed after 4 months. Massive rectal bleeding in intestinal tuberculosis, though rare should be kept in mind.

Effects of naltrexone and cross-tolerance to morphine in a learned helplessness paradigm.

Opiates have been implicated in learned helplessness (LH), a phenomenon known to be related to opiate stress-induced analgesia (SIA). In the present study, we investigated the role of opiates in the induction of LH and SIA under different conditions. Adult female Wistar rats were trained either by receiving 60 inescapable 1-mA footshocks (IS group, N = 114) or by confinement in the shock box (control or NS group, N = 92). The pain threshold of some of the animals was immediately evaluated in a tail-flick test while the rest were used 24 h later in a shuttle box experiment to examine their escape performance. The opiate antagonist naltrexone (0 or 8 mg/kg, ip) and the previous induction of cross-tolerance to morphine by the chronic administration of morphine (0 or 10 mg/kg, sc, for 13 days) were used to identify opiate involvement. Analysis of variance revealed that only animals in the IS group demonstrated antinociception and an escape deficit, both of which were resistant to the procedures applied before the training session. However, the escape deficit could be reversed if the treatments were given before the test session. We conclude that, under our conditions, induction of the LH deficit in escape performance is not opiate-mediated although its expression is opiate-modulated.

Effects of naltrexone and cross-tolerance to morphine in a learned helplessness paradigm

Opiates have been implicated in learned helplessness (LH), a phenomenon known to be related to opiate stress-induced analgesia (SIA). In the present study, we investigated the role of opiates in the induction of LH and SIA under different conditions. Adult female Wistar rats were trained either by receiving 60 inescapable 1-mA footshocks (IS group, N = 114) or by confinement in the shock box (control or NS group, N = 92). The pain threshold of some of the animals was immediately evaluated in a tail-flick test while the rest were used 24 h later in a shutttle box experiment to examine their escape performance. The opiate antagonist naltrexone (0 or 8 mg/kg, ip) and the previous induction of cross-tolerance to morphine by the chronic administration of morphine (0 or 10 mg/kg, sc, for 13 days) were used to identify opiate involvement. Analysis of variance revealed that only animals in the IS group demonstrated antinociception and an escape deficit, both of which were resistant to the procedures applied before the training session. However, the escape deficit could be reversed if the treatments were given before the test session. We conclude that, under our conditions, induction of the LH deficit in escape performance is not opiate-mediated although its expression is opiate-modulated.

Chronic but not acute Li+ treatment prevents behavioral depression in rats.

The effect of lithium administration on the learned helplessness model of depression was investigated. Female Wistar rats (190-210 g) received either tap water alone (N = 156) or 20 mM LiCL provided chronically (30 days; N = 127) or acutely (5 days; N = 22) in the drinking water. Three days before the end of treatment, each group was divided into two subgroups which received either inescapable shock (IS) or no shock (NS) treatment in shuttle boxes. All groups were subsequently submitted to an escape test on the following day and then sacrificed one day after the escape test, when blood samples were taken to measure serum Li+, Na+ and K+ concentrations by flame photometry. There were no significant differences in serum Na+ amongst the 4 groups. Chronically treated NS and IS rats both presented an increase in serum K+ compared to the control rats. The IS and not the NS chronically treated rats presented increased serum Li+ levels which cannot be accounted for in terms of differences in Li+ intake. The IS group treated chronically with lithium had a better escape performance than the IS group receiving either tap water or acute Li+ administration. We conclude that chronic but not acute Li+ treatment at a serum level within the prophylactic range (0.5 mEq/l) is able to prevent learned helplessness in the rat. These results agree with the data obtained in clinical practice indicating that Li+ is only effective after chronic administration and that Li(+)-induced hyperkalemia is a side effect.

Chronic but not acute Li+ treatment prevents behavioral depression in rats

The effect of lithium administration on the learned hellessness model of depression was investigated. Female Wistar rats (190-210g) received either tap water alone (N=156) or 20 mM LiCL, provided chronically (30 days; N = 127) or acutely (5 days; N = 22), in the drinking water. Three days before the end od treatment, each group was divided into two subgroups which received either inescapable shock (IS) or no shock (NS) treatment in shuttle boxes. All groups were subsequenty submitted to an escape test on the following day and then sacrificed one day after the escape test, when blood samples were taken to measure serum Li+, Na+ and K+ concentrations by flame photometry. There were no significant differences in serum Na+ amongst the 4 groups. chronically treated NS and IS rats both presented an increase in serum K+ compared to the control rats. The IS and not the NS chronically treated rats presented increased serum Li+ levels which cannot be accounted for in terms of differences in Li+ intake. The IS group treated chronically with lithium had a better escape performance than the IS group receiving either tap water or acute Li+ administration. We conclude that chronic but not acute Li+ treatment at a serum level within the prophylactic range (0,5 mEq) is able to prevent learned helplessness in the rat. These results agree with the data obtained in clinical practice indicating that li+ is only effective after chronic administration and that Li+ - induced hyperkalemia is a side effect

Lithium treatment prolongs shock-induced hypoalgesia.

We have investigated the effect of chronic lithium (Li+) treatment on stress-induced hypoalgesia, a phenomenon known to be dependent on the activation and sensitization of the central opioid system. Adult female Wistar rats received either 20 mM LiCl in the drinking water (serum level of 0.5 mEq/l, N = 110) or tap water (controls, N = 113) for 28 days. The rats were divided into three subgroups and were trained either by receiving 60 inescapable 1-mA footshocks (IS) while yoked to an escapable (ES) group, or by confinement (NS) to the shock box. As a control for the activation of the opioid system, we included rats injected with 0.9% saline (N = 24) or morphine (4 mg/kg, sc, N = 20) before confinement. Twenty-four hours later, the rats (N = 187) were either submitted to five inescapable (1 s, 0.6 mA) footshocks (shock reexposure) or received no shocks over the same period (N = 80). The pain threshold was estimated using a tail-flick apparatus after the training session and immediately after the shock reexposure. ANOVA followed by Duncan's test indicated that hypoalgesia was produced soon after the training session in the morphine and shocked groups and persisted in the Li(+)-IS group for up to three days. Hypoalgesia was reinstated in the control IS and morphine groups by reexposure to the shocks, but was not modified in the Li(+)-IS groups. We conclude that Li+ treatment prolongs the hypoalgesia induced by inescapable shocks.

Lithium treatment prolongs shock-induced hypoalgesia

We have investigated the effect of chronic lithium (Li+) treatment on stress-induced hypoalgesia, a phenomenon known to be dependent on the activation and sensitization of the central opioid system. Adult female Wistar rats received either 20 mM LiCl in the drinking water (serum level of 0.5 mEq/l,N = 110) or tap water (controls N = 113) for 28 days. The rats were divided into three subgroups and were trained either by receiving 60 inescapable 1-mA footshocks (IS) while yoked to a escapable (ES) group, or by confinement (NS) to the shock box. As a control for the activation of the opioid system, we included rats injected with 0.9 percent saline (N = 24) or morphine (4 mg/kg, sc, N =20) before confinement. Twenty-four hours later, the rats (N = 187) were either submitted to five inescapable (1 s,0.6 mA) footshocks (shock reexposure) or received no shocks over the same period (N = 80). The pain threshold was estimated using a tail-flick apparatus after the training session and immediately after the shock reesposure. ANOVA followed by duncan's test indicated that hypoalgesia was produced soon after the training session in the morphine and shocked groups and persisted in the Li+-IS group for up to three days. Hypoalgesia was reinstated in the control IS and morphine groups by reexposure to the shochs, but was not modified in the Li+-IS groups. We conclude that Li+ treatment prolongs the hypoalgesia induced by inescapable shocks

Avaliaçäo clínica do antagonista benzodiazepínico: estudo duplo-cego do Ro 15-1788 revertendo hipnose pelo midazolam em anestesia condutiva / Clinical evaluating of bezodiazepine antagonist: a double-blind study Ro 15-1788 antagonizing midazolam hypnosis in block

Foi realizado um estudo duplo-cego com 40 pacientes, ASA I-II, de ambos os sexos anestesiados com anestesia peridural, espinhal ou bloqueio do plexo braquial. Os pacientes receberam diazepam 10 mg via oral na noite anterior. Na sala de cirurgia, eram injetados 0,05 mg.Kg-1 de midazolam antes do bloqueio. Doses iguais eram repetidas após o bloqueio com o objetivo de induzir e manter o sono durante todo o procedimento. No final da cirurgia, o antagonista ou palcebo era injetado em doses fracionadas até o paciente despertar ou o conteúdo da ampola terminar. No grupo que recebeu o antagonista houve despertar mais precoce e sensaçäo agradável em uma parcela significativamente maior de pacientes. O Ro 15-1788 mostrou-se eficaz e seguro