Use of nonradioactive labeling to detect large gene rearrangements in 21-hydroxylase deficiency

PURPOSE: To establish the Southern blotting technique using hybridization with a nonradioactive probe to detect large rearrangements of CYP21A2 in a Brazilian cohort with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH-21OH). METHOD: We studied 42 patients, 2 of them related, comprising 80 non-related alleles. DNA samples were obtained from peripheral blood, digested by restriction enzyme Taq I, submitted to Southern blotting and hybridized with biotin-labeled probes. RESULTS: This method was shown to be reliable with results similar to the radioactive-labeling method. We found CYP21A2 deletion (2.5%), large gene conversion (8.8%), CYP21AP deletion (3.8%), and CYP21A1P duplication (6.3%). These frequencies were similar to those found in our previous study in which a large number of cases were studied. Good hybridization patterns were achieved with a smaller amount of DNA (5 mug), and fragment signs were observed after 5 minutes to 1 hour of exposure. CONCLUSIONS: We established a non-radioactive (biotin) Southern blot/hybridization methodology for CYP21A2 large rearrangements with good results. Despite being more arduous, this technique is faster, requires a smaller amount of DNA, and most importantly, avoids problems with the use of radioactivity.

21-Hydroxylase deficiency in Brazil

We determined the frequency of large rearrangements and point mutations in 130 Brazilian patients with 21-hydroxylase deficiency and correlated genotype with phenotype. The frequency of CYP21 deletions was lower (4.4%) than in most of the previous series described, whereas the frequency of large gene conversions was similar to the frequency reported in the literature (6.6%). The most frequent point mutations were I2 splice (41.8% in salt wasting - SW), I172N (32.6% in simple virilizing - SV) and V281L (40.2% in the late onset form - LO). The frequency of the nine most common point mutations was similar to that reported for other countries. The 93 fully genotyped patients were classified into 3 mutation groups based on the degree of enzymatic activity (A<2%, B @ 2%, C>20%). In group A, 62% of cases presented the SW form; in group B, 96% the SV form, and in group C, 88% the LO form. We diagnosed 80% of the affected alleles after screening for large rearrangements and 15 point mutations. To diagnose these remaining alleles we sequenced the CYP21 gene of one patient with the SV form and identified a heterozygous G->A transition in codon 424. This mutation leads to a substitution of glycine by serine in a conserved region and was also found in a compound heterozygous state in 4 other patients. The mutation G424S presented a linkage disequilibrium with CYP21P and C4A gene deletions and HLA DR17, suggesting a probable founder effect. Search for the G424S mutation in other populations will reveal if it is restricted to the Brazilian patients or if it has a wider ethnic distribution.

Neonatal handling and the expression of immunoreactivity to tyrosine hydroxylase in the hypothalamus of adult male rats

Neonatal handling has long-lasting effects on behavior and stress reactivity. The purpose of the present study was to investigate the effect of neonatal handling on the number of dopaminergic neurons in the hypothalamic nuclei of adult male rats as part of a series of studies that could explain the long-lasting effects of neonatal stimulation. Two groups of Wistar rats were studied: nonhandled (pups were left undisturbed, control) and handled (pups were handled for 1 min once a day during the first 10 days of life). At 75-80 days, the males were anesthetized and the brains were processed for immunohistochemistry. An anti-tyrosine hydroxylase antibody and the avidin-biotin-peroxidase method were used. Tyrosine hydroxylase-immunoreactive (TH-IR) neurons were counted bilaterally in the arcuate, paraventricular and periventricular nuclei of the hypothalamus in 30-µm sections at 120-µm intervals. Neonatal handling did not change the number of TH-IR neurons in the arcuate (1021 ± 206, N = 6; 1020 ± 150, N = 6; nonhandled and handled, respectively), paraventricular (584 ± 85, N = 8; 682 ± 62, N = 9) or periventricular (743 ± 118, N = 7; 990 ± 158, N = 7) nuclei of the hypothalamus. The absence of an effect on the number of dopaminergic cells in the hypothalamus indicates that the reduction in the amount of neurons induced by neonatal handling, as shown by other studies, is not a general phenomenon in the brain.

Hypoxic-ischemic injury decreases anxiety-like behavior in rats when associated with loss of tyrosine-hydroxylase immunoreactive neurons of the substantia nigra

Neonatal Sprague-Dawley rats were randomly divided into normal control, mild hypoxia-ischemia (HI), and severe HI groups (N = 10 in each group at each time) on postnatal day 7 (P7) to study the effect of mild and severe HI on anxiety-like behavior and the expression of tyrosine hydroxylase (TH) in the substantia nigra (SN). The mild and severe HI groups were exposed to hypoxia (8% O2/92% N2) for 90 and 150 min, respectively. The elevated plus-maze (EPM) test was performed to assess anxiety-like behavior by measuring time spent in the open arms (OAT) and OAT%, and immunohistochemistry was used to determine the expression of TH in the SN at P14, P21, and P28. OAT and OAT% in the EPM were significantly increased in both the mild (1.88-, 1.99-, and 2.04-fold, and 1.94-, 1.51-, and 1.46-fold) and severe HI groups (1.69-, 1.68-, and 1.87-fold, and 1.83-, 1.43-, and 1.39-fold, respectively; P < 0.05). The percent of TH-positive cells occupying the SN area was significantly and similarly decreased in both the mild (17.7, 40.2, and 47.2%) and severe HI groups (16.3, 32.2, and 43.8%, respectively; P < 0.05). The decrease in the number of TH-positive cells in the SN and the level of protein expression were closely associated (Pearson correlation analysis: r = 0.991, P = 0.000 in the mild HI group and r = 0.974, P = 0.000 in the severe HI group) with the impaired anxiety-like behaviors. We conclude that neonatal HI results in decreased anxiety-like behavior during the juvenile period of Sprague-Dawley rats, which is associated with the decreased activity of TH in the SN. The impairment of anxiety and the expression of TH are not likely to be dependent on the severity of HI.

Effects of sciatic nerve transection on ultrastructure, NADPH-diaphorase reaction and serotonin-, tyrosine hydroxylase-, c-Fos-, glucose transporter 1- and 3-like immunoreactivities in frog dorsal root ganglion

Frogs have been used as an alternative model to study pain mechanisms. Since we did not find any reports on the effects of sciatic nerve transection (SNT) on the ultrastructure and pattern of metabolic substances in frog dorsal root ganglion (DRG) cells, in the present study, 18 adult male frogs (Rana catesbeiana) were divided into three experimental groups: naive (frogs not subjected to surgical manipulation), sham (frogs in which all surgical procedures to expose the sciatic nerve were used except transection of the nerve), and SNT (frogs in which the sciatic nerve was exposed and transected). After 3 days, the bilateral DRG of the sciatic nerve was collected and used for transmission electron microscopy. Immunohistochemistry was used to detect reactivity for glucose transporter (Glut) types 1 and 3, tyrosine hydroxylase, serotonin and c-Fos, as well as nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase). SNT induced more mitochondria with vacuolation in neurons, satellite glial cells (SGCs) with more cytoplasmic extensions emerging from cell bodies, as well as more ribosomes, rough endoplasmic reticulum, intermediate filaments and mitochondria. c-Fos immunoreactivity was found in neuronal nuclei. More neurons and SGCs surrounded by tyrosine hydroxylase-like immunoreactivity were found. No change occurred in serotonin- and Glut1- and Glut3-like immunoreactivity. NADPH-diaphorase occurred in more neurons and SGCs. No sign of SGC proliferation was observed. Since the changes of frog DRG in response to nerve injury are similar to those of mammals, frogs should be a valid experimental model for the study of the effects of SNT, a condition that still has many unanswered questions.

Dengue in an elderly patient

Although elderly populations are more exposed to the risk of getting dengue, the clinical peculiarities of this disease in this age range are not well known. This report is about an 80-year-old woman with dengue complications, self-medicated with salicylate. Literature indicates a more severe clinical condition, high hospitalization rate and significant mortality. This is caused by previous infections by other serotypes of this arbovirus, presence of chronic diseases, immunosenescence and high drug consumption, especially salicylates and the like. Analyses are required in a public health perspective in order to help health professionals that care for patients with dengue in this age range.

Euglossine Bees (Hymenoptera: Apidae) of Buriticupu, Amazonia of Maranhão, Brazil1

Male euglossine bees attracted to cineole, vanillin, methyl salicylate, eugenol and benzyl benzoate, were collected from October 1995 to September 1996, twice a month, between 06.00 and 12.00 hours, at the Companhia Vale do Rio Doce Forest Reserve, Buriticupu-MA. It were sampled 1740 individuals, 37 species and 4 genera. Euglossa was the most abundant genus (23 species), followed by Eufriesea (8), Eulaema (4) and Exaerete (2). The most frequent species were Euglossa pleosticta (33% of the collected individuals), Euglossa truncata (12,7%), Euglossa avicula (6,3%), Eufriesea superba (5,2%), Euglossa fimbriata (4,8%) Euglossa violaceifrons (4,4%), Eulaema nigrita (4,1%), Euglossa cordata (4,0%), Eulaema meriana (3,4%). Cineole attracted 66% of males and 70% of species, vanillin (20%; 59%), methyl salicylate (7,4%; 54%), eugenol (5,6%; 44%) and benzyl benzoate (0,7%; 10,8%). The highest abundance of individuals (78,3%) and species (34) occurred in the rainy season (January-June). The species of the genus Eufriesea occurred only in this period. Regarding the hourly activity, the euglossine bees were more frequently found between 10.00 and 11.00 hours, accounting for 33,5% of the individuals and 86,4% of the species.

Euglossine bees (Hymenoptera, Apidae) in a remnant of Atlantic Forest in Paraná State, Brazil

Species composition, relative abundance, seasonal changes in the species abundance and scent association of male Euglossini collected in a semi-deciduous forest fragment in the north of the State of Paraná, southern Brazil, were recorded. Euglossine males were collected twice a month, for twelve months, from 8:00 am to 3:00 pm. The scents eucalyptol, eugenol, vanillin, methyl salicylate and benzyl acetate were used as baits. A total of 434 males distributed among 3 genera and 9 species were attracted to the chemical baits. Eufriesea violacea (Blanchard, 1840) (49.8%), Eulaema nigrita Lepeletier, 1841 (23.0%) and Euglossa pleosticta Dressler, 1982 (13.8%) were dominant in number of individuals. Among the non-dominant species, Euglossa fimbriata Rebêlo & Moure, 1995 was more common (9.0%), followed by E. cordata (L., 1758) (1.8%), E. truncata Rebêlo & Moure, 1995 (1.4%), E. melanotricha Moure, 1967 (0.7%), E. townsendi Cockerell, 1904 (0.23%) and Eufriesea auriceps Friese, 1899 (0.23%). In general, bees were more abundant in warm-wet season (September-March). Eufriesea violacea was the most seasonal species, showing activity through the warm-wet season, from October to February. Eucalyptol was the most attractive fragrance, which was responsible for 92.6% of all visits by euglossine bees.

Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis

In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs) and praziquantel (PZQ) kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW) mice of both genders were infected (100 cercariae) on postnatal day 10 and killed on post-infection days (PIDs) 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD)], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD)], 2E1 [p-nitrophenol-hydroxylase (PNPH)] and 3A11 [erythromycin N-demethylase (END)] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction). On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally) levels were measured (high-performance liquid chromatography) at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice.

Comunidade de Euglossini (Hymenoptera, Apidae) das dunas litorâneas do Abaeté, Salvador, Bahia, Brasil

Community of Euglossini (Hymenoptera, Apidae) from the coastal sand dunes of Abaeté, Salvador, Bahia, Brazil. The Euglossini community structure was analyzed by attracting males with the scents eucalyptol, eugenol, vanillin, benzyl benzoate and methyl salicylate, and by netting bees on flowers. The samplings took place three times a month along one year from 6:00 a.m. to 6:00 p.m. The scent baits attracted 670 individuals belonging to seven species of three genus. The predominant species were Euglossa cordata (Linnaeus, 1758) (76.6%) and Eulaema nigrita Lepeletier, 1841 (21.8%). Euglossini males visited the scents along the whole year, being more abundant in May and in August. The most efficient fragrance was eucalyptol, attracting 624 individuals of five species. The males abundance fluctuated along the day, being the highest frequency observed between 8:00 a.m. to 10:00 a.m. Forty eigth Euglossini females of four species were netted visiting flowers of 14 plant species belonging to 13 families. Solanaceae and Caesalpiniaceae were the most visited. The species catched on flowers were Euglossa cordata, Eulaema nigrita, Euplusia mussitans (Fabricius, 1787) and Eulaema meriana flavescens Friese 1899. Euglossa cordata was the predominant species on flowers (64.6%), being collected during almost the whole year. Euplusia mussitans was the only species netted on flowers which males were not sampled on the scents.

Euglossine bee communities in small forest fragments of the Atlantic Forest, Rio de Janeiro state, southeastern Brazil (Hymenoptera, Apidae)

Euglossine bee communities in small forest fragments of the Atlantic Forest, Rio de Janeiro state, southeastern Brazil (Hymenoptera, Apidae). Euglossine bees are important pollinators in forests and agricultural areas. Although the structure of their communities is critically affected by anthropogenic disturbances, little is known about these bees in small forest fragments. The objectives of this study were to analyze the composition, abundance, and diversity of euglossine bee species in nine small fragments of different phytophysiognomies of the Atlantic Forest in southeastern Brazil, and to identify the environmental variables that may be related to the species composition of these communities. Males were sampled quarterly from May 2007 to May 2009 with aromatic traps containing methyl cinnamate, vanillin, eucalyptol, benzyl acetate, and methyl salicylate. A total of 1558 males, belonging to 10 species and three genera of Euglossina were collected. The richness ranged from five to seven species per fragment. Euglossa cordata, E. securigera, Eulaema nigrita e E. cingulata were common to all fragments studied. The diversity differed significantly among areas, ranging from H' = 1.04 to H' = 1.65. The precipitation, phytophysiognomy, and altitude had the highest relative importance over the species composition variation. The results presented in this study demonstrate that small forest fragments are able to support populations of euglossine bee species, most of which are widely distributed and reportedly tolerant to open and/or disturbed areas and suggest that the conservation of such areas is important, particularly in areas that are regenerating and in regions with agricultural matrices where these bees can act as important pollinators

Orchid bee baits attracting bees of the genus Megalopta (Hymenoptera, Halictidae) in Bauru region, São Paulo, Brazil: abundance, seasonality, and the importance of odors for dim-light bees

Nocturnal bees in the genus Megalopta Smith, 1853 are generally collected using artificial light sources. However, between 1993 and 2000, a total of 946 females (no males were captured) were captured using aromatic baits commonly used for orchid bees (Euglossini) in five localities in Bauru region, São Paulo, Brazil. Aromatic compounds used in bait traps were: benzyl acetate, eucalyptol, eugenol, skatole, methyl salicylate, and vanillin. The Megalopta species collected were: M. guimaraesi (71.2% of total number of specimens), M. amoena (28.1%), and M. aegis (0.6%). Using the data from these traps, we showed that there was a positive and significant correlation between the abundance of individuals and meteorological factors, rainfall and temperature. Bees were more commonly collected in the spring (September to December) and summer (December to March) than in the autumn and winter, the latter characterized for being a drier and colder period. Variations in the abundance were also detected among localities and years. The most attractive compounds were eugenol (54%), methyl salicylate (22%), and eucalyptol (16%). The ability to detect smells may have an important role in searching for flowers during dim-light conditions. We suggest the use of aromatic compounds in future studies on the biology of Megalopta in the Neotropical region.

Sistema em fluxo para determinação espectrofotométrica de uréia em plasma de sangue animal empregando leguminosa como fonte natural da enzima urease

A spectrophotometric flow injection analysis (FIA) procedure employing natural urease enzyme source for the determination of urea in animal blood plasma was developed. Among leguminous plants used in the Brazilian agriculture, the Cajanus cajan specie was selected as urease source considering its efficiency and availability. A minicolumn was filled with leguminous fragments and coupled to the FIA manifold, where urea was on-line converted to ammonium ions and subsequently it was quantified by spectrophotometry. The system was employed to determine urea in animal plasma samples without any prior treatment. Accuracy was assessed by comparison results with those obtained employing the official procedure and no significant difference at 90 % confidence level was observed. Other profitable features such as an analytical throughput of 30 determinations per hour, a reagent consumption of 19.2 mg sodium salicylate, 0.5 mg sodium hipochloride and a relative standard deviation of 1.4 % (n= 12) were also obtained.

Determinação espectrofotométrica por injeção em fluxo de paracetamol (acetaminofeno) em formulações farmacêuticas

A flow injection spectrophotometric procedure is proposed for the determination of paracetamol (acetaminophen) in pharmaceutical formulations. Powdered and liquid samples were previously dissolved/diluted in 0.05 mol L-1 hydrochloric acid solution and a volume of 250 µL was injected directly into a carrier stream of this same acid solution, flowing at 2.5 mL min-1. Paracetamol reacts with sodium hypochlorite forming N-acetyl-p-benzoquinoneimine which then reacts with sodium salicylate in sodium hydroxide solution yielding a blue indophenol dye which was measured at 640 nm in the pH range of 9.5-10.0. Paracetamol was determined in pharmaceutical products in the 1.0 to 100.0 mg L-1 (3.3x10-6 a 6.6x10-4 mol L-1) concentration range, with a detection limit of 0.5 mg L-1 (1.6x10-6 mol L-1). The recovery of this analyte in five samples ranged from 98.0 to 103.6 %. The analytical frequency was 80 determinations per hour and the RSDs were less than 1% for paracetamol concentrations of 25.0, 50.0 and 75.0 mg L-1 (n=10). A paired t-test showed that all results obtained for paracetamol in commercial formulations using the proposed flow injection procedure and a spectrophotometric batch procedure agree at the 95% confidence level.

PARAFAC: uma ferramenta quimiométrica para tratamento de dados multidimensionais. Aplicações na determinação direta de fármacos em plasma humano por espectrofluorimetria

Since the last decade, the combined use of chemometrics and molecular spectroscopic techniques has become a new alternative for direct drug determination, without the need of physical separation. Among the new methodologies developed, the application of PARAFAC in the decomposition of spectrofluorimetric data should be highlighted. The first objective of this article is to describe the theoretical basis of PARAFAC. For this purpose, a discussion about the order of chemometric methods used in multivariate calibration and the development of multi-dimensional methods is presented first. The other objective of this article is to divulge for the Brazilian chemical community the potential of the combination PARAFAC/spectrofluorimetry for the determination of drugs in complex biological matrices. For this purpose, two applications aiming at determining, respectively, doxorrubicine and salicylate in human plasma are presented.