Normal bone density in male pseudohermaphroditism due to 5alpha- reductase 2 deficiency

Bone is an androgen-dependent tissue, but it is not clear whether the androgen action in bone depends on testosterone or on dihydrotestosterone. Patients with 5alpha-reductase 2 deficiency present normal levels of testosterone and low levels of dihydrotestosterone, providing an in vivo human model for the analysis of the effect of testosterone on bone. OBJECTIVE: To analyze bone mineral density in 4 adult patients with male pseudohermaphroditism due to 5alpha-reductase 2 deficiency. RESULTS: Three patients presented normal bone mineral density of the lumbar column (L1-L4) and femur neck, and the other patient presented a slight osteopenia in the lumbar column. CONCLUSION: Patients with dihydrotestosterone deficiency present normal bone mineral density, suggesting that dihydrotestosterone is not the main androgen acting in bone.

Biomass accumulation, photochemical efficiency of photosystem II, nutrient contents and nitrate reductase activity in young rosewood plants (Aniba rosaeodora Ducke) submitted to different NO3-:NH4+ ratios

The rosewood (Aniba rosaeodora Ducke) is a native tree species of Amazon rainforest growing naturally in acidic forest soils with reduced redox potential. However, this species can also been found growing in forest gaps containing oxide soils. Variations in the forms of mineral nitrogen (NO3- or NH4+) may be predicted in these different edaphic conditions. Considering that possibility, an experiment was carried out to analyze the effects of different NO3-:NH4+ ratios on the growth performance, mineral composition, chloroplastid pigment contents, photochemical efficiency photosystem II (PSII), and nitrate redutase activity (RN, E.C.1.6.6.1) on A. rosaeodora seedlings. Nine-month-old seedlings were grown in pots with a washed sand capacity of 7.5 kg and submitted to different NO3-:NH4+ ratios (T1 = 0:100%, T2 = 25:75%, T3 = 50:50%, T4 = 75:25%, and T5 = 100:0%). The lowest relative growth rate was observed when the NO3-:NH4+ ratio was equal to 0:100%. In general, high concentrations of NO3- rather than NH4+ favored a greater nutrient accumulation in different parts of the plant. For the chloroplastid pigment, the highest Chl a, Chl b, Chl tot, Chl a/b and Chl tot/Cx+c contents were found in the treatment with 75:25% of NO3-:NH4+, and for Chl b and Cx+c it was observed no difference. In addition, there was a higher photochemical efficiency of PSII (Fv/Fm) when high NO3- concentrations were used. A linear and positive response for the nitrate reductase activity was recorded when the nitrate content increased on the culture substrate. Our results suggest that A. rosaeodora seedlings have a better growth performance when the NO3- concentrations in the culture substrate were higher than the NH4+ concentrations.

Comparison of the effect of two HMG CoA reductase inhibitors on LDL susceptibility to oxidation

OBJECTIVE: To study the differences between fluvastatin and pravastatin regarding LDL susceptibility to oxidation, plasma levels of total cholesterol (TC), HDL-C, LDL-C and triglycerides (TG) in hypercholesterolemic patients with established coronary heart disease (CHD). METHODS: A double-blind randomized parallel study was conducted that included 41 hypercholesterolemic outpatients with CHD treated at the Instituto de Cardiologia do Rio Grande do Sul. The inclusion criteria were LDL-C above 100 mg/dL and triglycerides below 400 mg/dL based on 2 measures. After 4 weeks on a low cholesterol diet, those patients that fullfilled the inclusion criteria were randomized into 2 groups: the fluvastatin group (fluvastatin 40 mg/day) and the pravastatin group (pravastatin 20 mg/day), for 24 weeks of treatment. LDL susceptibility to oxidation was analyzed with copper-induced production of conjugated dienes (Cu2+) and water-soluble free radical initiator azo-bis (2'-2'amidinopropanil) HCl (AAPH). Spectroscopy nuclear magnetic resonance was used for determination of lipids. RESULTS: After 24 weeks of drug therapy, fluvastatin and pravastatin significantly reduced LDL susceptibility to oxidation as demonstrated by the reduced rate of oxidation (azo and Cu) and by prolonged azo-induced lag time (azo lag). The TC, LDL-C, and TG reduced significantly and HDL-C increased significantly. No differences between the drugs were observed. CONCLUSION: In hypercholesterolemic patients with CHD, both fluvastatin and pravastatin reduced LDL susceptibility to oxidation.

Induction of Glutathione S-transferase Activity in Triatoma infestans

Several synthetic pesticides and allelochemicals used to treat Triatoma infestans adults by topic application showed some degree of cytosolic glutathione S-transferase (GST) induction. General inducers of detoxication systems such as phenobarbital and 3-methylcholantrene topically applied on T. infestans resulted in no GST induction. Meanwhile, general insecticide synergist such as piperonyl butoxide (160 mg/insect) increased the GST-activity in the range of 120-140%. Insects injected with reduced glutathione (300 mg/insect) presented at the forth day elevated GST activity

Glutathione S-transferases of 28kDa as major vaccine candidates against schistosomiasis

For the development of vaccine strategies to generate efficient protection against chronic infections such as parasitic diseases, and more precisely schistosomiasis, controlling pathology could be more relevant than controlling the infection itself. Such strategies, motivated by the need for a cost-effective complement to existing control measures, should focus on parasite molecules involved in fecundity, because in metazoan parasite infections pathology is usually linked to the output of viable eggs. In numerous animal models, vaccination with glutathione S-transferases of 28kDa has been shown to generate an immune response strongly limiting the worm fecundity, in addition to the reduction of the parasite burden. Recent data on acquired immunity directed to 28GST in infected human populations, and new development to draw adapted vaccine formulations, are presented.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon Region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 µM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60% at 20 µg/ml (59 and 90 µM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 µg/ml (0.6-1.5 mM).

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 µg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 µM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 µ M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 µM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.

A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.

Change in mutation patterns of Plasmodium vivax dihydrofolate reductase (Pvdhfr) and dihydropteroate synthase (Pvdhps) in P. vivax isolates from malaria endemic areas of Thailand

Malaria is the most important public health problem in several countries. In Thailand, co-infections of Plasmodium vivax and Plasmodium falciparum are common. We examined the prevalence and patterns of mutations in P. vivax dihydrofolate reductase (Pvdhfr) and P. vivax dihydropteroate synthase (Pvdhps) in 103 blood samples collected from patients with P. vivax infection who had attended the malaria clinic in Mae Sot, Tak Province during 2009 and 2010. Using nested polymerase chain reaction-restriction fragment length polymorfism, we examined single nucleotide polymorphisms-haplotypes at amino acid positions 13, 33, 57, 58, 61, 117 and 173 of Pvdhfr and 383 and 553 of Pvdhps. All parasite isolates carried mutant Pvdhfr alleles, of which the most common alleles were triple mutants (99%). Eight different types of Pvdhfr and combination alleles were found, as follows: 57I/58R/117T, 57I/58R/117T, 57I/58R/117T/N, 57L/58R/117T, 57L/58R/117T, 58R/61M/117N, 58R/61M/117N and 13L/57L/58R/117T. The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). The most common Pvdhfr alleles were 57I/58R/117T (77.7%), 57I/58R/117T/N (1%), 57L/58R/117T (5.8%) and 58R/61M/117N (14.5%). Additionally, we recovered one isolate of a carrying a quadruple mutant allele, 13L/57L/58R/117T. The most prevalent Pvdhps allele was a single mutation in amino acid 383 (82.5%), followed by the wild-type A383/A553 (17.5%) allele. Results suggest that all P. vivax isolates in Thailand carry some combination of mutations in Pvdhfr and Pvdhps. Our findings demonstrate that development of new antifolate drugs effective against sulfadoxine-pyrimethamine-resistant P. vivax is required.

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.

Nitrate reductase and glutamine synthetase activity in coffee leaves during fruit development

Nitrate reductase is the first enzyme in the pathway of nitrate reduction by plants, followed by glutamine synthetase, which incorporates ammonia to glutamine. The purpose of this study was to evaluate the nitrate reductase and glutamine synthetase activity, total soluble protein content, N and Ni content in coffee leaves during fruit development under field conditions to establish new informations to help assess the N nutritional status and fertilizer management. The experimental design was in randomized complete blocks, arranged in a 3 x 6 factorial design, with five replications. The treatments consisted of 3 N rates (0 - control, 150 and 300 kg ha-1) and six evaluation periods (January, February, March, April, May, and June) in six-year-old coffee (Coffea arabica L.) plants of Catuaí Vermelho IAC 44 cv. The nitrate reductase and glutamine synthetase activities, leaf soluble protein, and N concentrations increased linearly with the N rates. During fruit development, the enzyme activity, leaf soluble protein and N content decreased, due to the leaf senescence process caused by nutrient mobilization to other organs, e.g, to the berries. Leaf Ni increased during fruit development. Beans and raisin-fruits of plants well-supplied with N had higher Ni contents. Enzyme activities, total leaf N and leaf soluble protein, evaluated during the green fruit stage in March, were significantly correlated with coffee yield. These variables can therefore be useful for an early assessment of the coffee N nutritional status as well as coffee yield and N fertilization management.

Nitrate reductase activity and spad readings in leaf tissues of guinea grass submitted to nitrogen and potassium rates

Nitrogen and K deficiency are among the most yield limiting factors in Brazilian pastures. The lack of these nutrients can hamper the chlorophyll biosynthesis and N content in plant tissues. A greenhouse experiment was carried out to evaluate the relationship among N and K concentrations, the indirect determination of chlorophyll content (SPAD readings), nitrate reductase activity (RNO3-) in newly expanded leaf lamina (NL) and the dry matter yield for plant tops of Mombaça grass (Panicum maximum Jacq.). A fractionated 5² factorial design was used, with 13 combinations of N and K rates in the nutrient solution. The experimental units were arranged in a randomized block design, with four replications. Plants were harvested twice. The first harvest occurred 36 days after seedling transplanting and the second 29 days after the first. Significance occurred for the interaction between the N and K rates to SPAD readings and to RNO3- assessment taken on the NL during the first growth. Besides, RNO3- and SPAD readings increased only with the NL N concentration, reaching the highest values of both variables up to about 25 g kg-1, but were ratively constant at higher leaf N. Significant relationships either between SPAD readings or RNO3- activity and shoot dry mass weight were also observed. The critical levels of N concentration in the NL were, respectively, 22 and 17g kg-1 in the first and second harvest. Thus, SPAD instrument and RNO3- assessment can be used as complementary tools to evaluate the N status in forage grass.

N-partitioning, nitrate reductase and glutamine synthetase activities in two contrasting varieties of maize

In order to identify useful parameters for maize genetic breeding programs aiming at a more efficient use of N, two maize varieties of contrasting N efficiency, Sol da Manhã NF (efficient) and Catetão (inefficient) were compared. Experiments were carried out under field and greenhouse conditions, at low and high N levels. The parameters analysed included total and relative plant and grain N content, biomass and the activities of nitrate reductase and glutamine synthetase in different parts of the plant. It was found that the translocation efficiency of N and photoassimilates to the developing seeds and the source-sink relations were significantly different for the two varieties. N content of the whole plant and grain, cob weight and the relative ear dry weight were useful parameters for characterizing the variety Sol da Manhã NF as to its efficient use of N. Enzymes activity of glutamine synthetase (transferase reaction) and nitrate reductase did not differ among the varieties.

Expression of P-glycoprotein, multidrug resistance-associated protein, glutathione-S-transferase pi and p53 in canine transmissible venereal tumor

The overexpression of proteins P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP1), mutant p53, and the enzyme glutathione-S-transferase (GSTpi) are related to resistance to chemotherapy in neoplasms. This study evaluated the expression of these markers by immunohistochemistry in two groups of canine TVT, without history of prior chemotherapy (TVT1, n=9) and in TVTs presented unsatisfactory clinical response to vincristine sulfate (TVT2, n=5). The percentage of specimens positively stained for P-gp, MRP1, GSTpi and p53 were, respectively 88.8%, 0%, 44.5% and 22.2% in TVT1 and 80%, 0%, 80% and 0% in TVT2. In TVT1, one specimen presented positive expression for three markers and four specimens for two markers. In TVT2, three specimens expressed P-gp and GSTpi. In conclusion, the canine TVTs studied expressed the four markers evaluated, but just P-gp and GSTpi were significantly expressed, mainly at cytoplasm and cytoplasm and nuclei, respectively, either before chemotherapy as after vincristine sulfate exposure. Future studies are needed to demonstrate the function of these two markers in conferring multidrug resistance (MDR) or predict the response to chemotherapy in canine TVT.