Testosterone control of endplate and non-endplate acetylcholinesterase in the rat levator ani muscle

The time course of effects of castration (5–60 days) and testosterone treatment (15–60 days) of adult male rats were examined on the endplate (+EP) and non-endplate (–EP) acetylcholinesterase (AChE) of the androgen-dependent levator ani (LA) muscle. The thiocholine method was used to determine the enzyme activity. Castration caused LA muscle atrophy within 5 days but reduced the –EP and +EP AChE after 10 and 20 days, respectively. Following 30 days castration –EP and +EP AChE reached respectively 41% and 35% of control activity. Testosterone retrieval restored the control values of both muscle weight and total AChE after 15 and 60 days, respectively. Recovery of the +EP AChE preceded that of –EP AChE by 30 days. The results showed that in the rat LA muscle, +EP and –EP AChE depend on a continuous testosterone regulation that predominates at +EP region spreading thereafter to –EP region. Those data suggest a hormone regulation of AChE exerted indirectly through the synthesis and release of neurotrophic substances.     

The role of nitric oxide in muscle fibers with oxidative phosphorylation defects

NO has been pointed as an important player in the control of mitochondrial respiration, especially because of its inhibitory effect on cytochrome c oxidase (COX). However, all the events involved in this control are still not completely elucidated. We demonstrate compartmentalized abnormalities on nitric oxide synthase (NOS) activity on muscle biopsies of patients with mitochondrial diseases. NOS activity was reduced in the sarcoplasmic compartment in COX deficient fibers, whereas increased activity was found in the sarcolemma of fibers with mitochondrial proliferation. We observed increased expression of neuronal NOS (nNOS) in patients and a correlation between nNOS expression and mitochondrial content. Treatment of skeletal muscle culture with an NO donor induced an increase in mitochondrial content. Our results indicate specific roles of NO in compensatory mechanisms of muscle fibers with mitochondrial deficiency and suggest the participation of nNOS in the signaling process of mitochondrial proliferation in human skeletal muscle.     

Hepatic Disease with Portal Hypertension and Acute Juvenile Paracoccidioidomycosis: A Report of Two Cases and Literature Review

Paracoccidioidomycosis (PCM) is a neglected systemic mycosis endemic to Latin America caused by dimorphic fungi of the genus Paracoccidioides. The acute juvenile PCM is a severe type of presentation that usually affects young vulnerable patients and rarely progresses to portal hypertension. Here, two cases of liver disease and portal hypertension as complications of acute juvenile PCM are reported. Diagnosis of PCM was performed by isolation of the fungus and molecular identification of the strains provided through partial sequencing of two protein encoding genes, arf and gp43. Genotypic analysis revealed that Paracoccidioides brasiliensis S1 was the phylogenic species involved in both cases. Patients presented a good clinical response to amphotericin B and sulfamethoxazole–trimethoprim. These results highlight the importance of the interdisciplinary approach in patients with severe forms of PCM to avoid and treat complications, and the necessity of further investigations focusing on host-pathogen interaction in order to explain the broad clinical spectrum in PCM as well as the severity and poor outcome in some clinical cases.     

Interaction with Pantoea agglomerans Modulates Growth and Melanization of Sporothrix brasiliensis and Sporothrix schenckii

Mycopathologia - Sporothrix brasiliensis and Sporothrix schenckii stand as the most virulent agents of sporotrichosis, a worldwide-distributed subcutaneous mycosis. The origin of Sporothrix...     

Racial classification regarding semen donor selection in Brazil

Brazil has not yet approved legislation on assisted reproduction. For this reason, clinics, hospitals and semen banks active in the area follow Resolution 1358/92 of the Conselho Federal de Medicina, dated 30 September 1992. In respect to semen donation, the object of this article, the Resolution sets out that gamete donation shall be anonymous, that is, that the donor and recipients (and the children who might subsequently be born) shall not be informed of each other's identity. Thus, since recipients are unaware of the donor's identity, semen banks and the medical teams involved in assisted reproduction become the intermediaries in the process. The objective of this article is to show that, in practice, this represents disrespect for the ethical principles of autonomy, privacy and equality. The article also stresses that the problem is compounded by the racial question. In a country like Brazil, where racial classification is so flexible and goes side by side with racist attitudes, the intermediary role played by semen banks and medical teams is conditioned by their own criteria of racial classification, which are not always the same as those of donors and semen recipients. The data presented in this paper were taken from two semen banks located in the city of São Paulo (Brazil). At the time of my research, they were the only semen banks in the state of São Paulo and supplied semen to the capital (São Paulo city), the state of São Paulo, and to cities in other Brazilian states where semen banks were not available.     

Cryptosporidium: Detection in water and food

Water and food are major environmental transmission routes for Cryptosporidium, but our ability to identify the spectrum of oocyst contributions in current performance-based methods is limited. Determining risks in water and foodstuffs, and the importance of zoonotic transmission, requires the use of molecular methods, which add value to performance-based morphologic methods. Multi-locus approaches increase the accuracy of identification, as many signatures detected in water originate from species/genotypes that are not infectious to humans. Method optimisation is necessary for detecting small numbers of oocysts in environmental samples consistently, and further work is required to (i) optimise IMS recovery efficiency, (ii) quality assure performance-based methods, (iii) maximise DNA extraction and purification, (iv) adopt standardised and validated loci and primers, (v) determine the species and subspecies range in samples containing mixtures, and standardising storage and transport matrices for validating genetic loci, primer sets and DNA sequences.     

Comparison of Different DNA-Based Methods for Molecular Typing of Histoplasma capsulatum

Histoplasma capsulatum is very prevalent in the environment and is one of the most common causes of mycoses in humans and diverse animals in Brazil. Multiple typing methods have been developed to study H. capsulatum epidemiology; however, there is limited information concerning comparisons of results obtained with different methods using the same set of isolates. To explore the diversity of H. capsulatum in Brazil and to determine correlations between the results of three different molecular typing techniques, we examined 51 environmental, animal, and human isolates by M13 PCR fingerprinting, PCR-restriction fragment length polymorphism (RFLP) analysis of the internal transcribed region 1 (ITS1)-5.8S-ITS2 region of the rDNA locus, and DNA sequencing and phylogenetic analysis of parts of four protein-encoding genes, the Arf (ADP ribosylation factor), H-anti (H antigen precursor), Ole (delta-9 fatty acid desaturase), and Tub1 (alpha-tubulin) genes. Each method identified three major genetic clusters, and there was a high level of concordance between the results of the typing techniques. The M13 PCR fingerprinting and PCR-RFLP analyses produced very similar results and separated the H. capsulatum isolates included in this study into three major groups. An additional approach used was comparison of our Brazilian ITS1-5.8S-ITS2 sequences with the sequences deposited previously in NCBI data banks. Our analyses suggest that H. capsulatum can be divided into different molecular types that are dispersed around the world. Our results indicate that the three methods used in this study are reliable and reproducible and that they have similar sensitivities. However, M13 PCR fingerprinting has some advantages over the other two methods as it is faster, cheaper, and more user friendly, which especially increases its utility for molecular typing of Histoplasma in situations where laboratory facilities are relatively limited.Histoplasmosis is a serious community-acquired infection in the United States (28) and in certain countries of Latin America, where it is an especially significant problem in patients with AIDS (14). This disease is one of the most common systemic mycoses in Brazil, where epidemiological surveys carried out using the histoplasmin skin test have indicated that it is endemic in all areas surveyed (15). Data suggest that the numbers of cases of histoplasmosis in Brazil may be underestimated and that the areas where it is endemic are more widespread than previously thought.Histoplasma capsulatum is a dimorphic fungus that grows as a mold and produces aerial hyphae at 25 to 30°C, but it undergoes morphogenesis to a yeast phase at 37°C. The filamentous phase of this organism is usually found in soil enriched with several compounds, such as nitrogen and phosphate compounds. When conidial or hyphal fragments are inhaled by humans or animals, H. capsulatum changes to the yeast form and continues to replicate as a yeast. Although H. capsulatum has been recognized as an important fungal pathogen in immunocompromised hosts, particularly AIDS patients (27), there are significant gaps in our knowledge of this species'' epidemiology and pathogenesis. For instance, systemic histoplasmosis has been found in patients with AIDS who do not reside in regions where it is endemic (29), leading to the suggestion that the disease can result from reactivation of a previously acquired H. capsulatum infection. The clinical manifestations of histoplasmosis range from asymptomatic infections, mild flu-like symptoms, or pneumonia to a systemic disease involving the skin, brain, intestine, adrenal glands, and/or bone marrow (6). Importantly, diverse strains of H. capsulatum have been identified worldwide, and the strains vary in virulence. In addition to classical biochemical assays, distinctions between strains may be based on colony morphology or polymorphism of the genome (19).Multiple typing methods have been developed to study the epidemiology of H. capsulatum. These methods are based on phenotypic characteristics, such as antigenic profiles (13) and multilocus enzyme electrophoresis results (2), or on DNA-based analysis. Most recently, typing has been accomplished by analysis of fatty acid profiles of H. capsulatum (34). Molecular typing methods are generally considered to have advantages over phenotypic methods in terms of the stability of genomic markers and greater levels of typeability. Several genotype-based methods, such as hybridization of target genes (probes), chromosomal DNA typing, restriction fragment length polymorphism (RFLP) analysis, random amplified polymorphic DNA (RAPD) analysis, and sequencing, have been described for H. capsulatum (4, 5, 7, 8, 11, 17, 19). Despite the abundance of previously developed molecular techniques, there is limited information concerning comparisons of the results obtained with different methods using the same set of isolates. In H. capsulatum, no single approach based on DNA assays has been the dominant method.The current study was done to explore the diversity of H. capsulatum in Brazil and to determine the correlation between the results of three different molecular typing techniques. For this analysis, we used M13 PCR fingerprinting, PCR-RFLP analysis of the internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the rDNA gene, and analysis of the nucleotide sequence polymorphism of four partial genes. M13 PCR fingerprinting (25) is based on generation of multiple PCR products with different electrophoretic mobilities. PCR fingerprinting primers are typically designed using repetitive DNA sequences (31), and the products facilitate detection of two types of genetic variations: (i) differences in the length of DNA and (ii) alterations in the sequence of the priming regions. PCR-RFLP analysis of the ITS1-5.8S-ITS2 region of the rDNA gene (9) involves use of a gene-specific PCR in combination with restriction digestion in order to generate highly stable and reproducible markers. Analysis of the nucleotide sequence polymorphism is based on the sequences of four partial protein-encoding genes (Arf, the H-anti gene, Ole, Tub1) (4). Additionally, to assess the utility of an assay to study the global epidemiology of the fungus, we performed a DNA sequencing analysis of the ITS1-5.8S-ITS2 region to compare the Brazilian H. capsulatum ITS1-5.8S-ITS2 sequences with sequences obtained for H. capsulatum strains isolated in other countries.     

Effects of 17β-estradiol replacement on the apoptotic effects caused by ovariectomy in the rat hippocampus

AimsThe aim of the present study was to investigate the effects of different periods of ovariectomy and 17β-estradiol replacement on apoptotic cell death and expression of members of the Bcl-2 family in the rat hippocampus.Main methodsHippocampi were obtained from rats in proestrus, ovariectomized (15 days, 21 days and 36 days), ovariectomized for 15 days and then treated with 17β-estradiol for 7 or 21 days, and rats ovariectomized and immediately treated with 17β-estradiol for 21 days.The expression of Bcl-2 and Bax and the number of apoptotic cells were determined.Key findingsOvariectomy decreased Bcl-2 expression and increased Bax expression and the number of apoptotic cells. Replacement with 17β-estradiol (21 days) throughout the post-ovariectomy period reduced the number of apoptotic cells to the control levels, and prevented the effects of ovariectomy on Bax expression, but only partially restored the Bcl-2 expression. After 15 days of ovariectomy, the replacement with 17β-estradiol for 21 days, but not for 7 days, restored the Bcl-2 and Bax expression and the percentage of apoptotic cells to the levels found in the proestrus control.SignificanceThe present results show that a physiological concentration of 17β-estradiol may help maintain long-term neuronal viability by regulating the expression of members of the Bcl-2 family. Even after a period of hormonal deprivation, treatment with 17β-estradiol is able to restore the expression of Bax and Bcl-2 to control levels, but the duration of the treatment is a key factor to obtain the desired effect. These data provide new understanding into the mechanisms contributing to the neuroprotective action of estrogen.     

The major salivary gland antigens of Culex quinquefasciatus are D7-related proteins

The sera of persons with strong allergic responses to the bites of the mosquito, Culex quinquefasciatus, contained IgE antibodies reactive with two major salivary gland proteins with molecular weights of 35 and 28 kDa. These antigens were purified, their amino termini sequenced, and the sequences were used to search for similar sequences in public databases. Two cDNAs, CuQu-D7Clu1 and CuQu-D7Clu12, which encode D7-related proteins, were identified as containing predicted amino acid sequences identical to the 35 and 28 kDa antigens, respectively. These proteins are expressed specifically in adult female salivary glands and, their predicted tertiary structures are consistent with a role as carriers of hydrophobic molecules in mosquito saliva.