Iron from haemoglobin and haemin modulates nucleotide hydrolysis in Trichomonas vaginalis

Extracellular ATP may act as a danger signalling molecule, inducing inflammation and immune responses in infection sites. The ectonucleotidases NTPDase and ecto-5’-nucleotidase are enzymes that modulate extracellular nucleotide levels; these enzymes have been previously characterised in Trichomonas vaginalis. Iron plays an important role in the complex trichomonal pathogenesis. Herein, the effects of iron on growth, nucleotide hydrolysis and NTPDase gene expression in T. vaginalis isolates from female and male patients were evaluated. Iron from different sources sustained T. vaginalis growth. Importantly, iron from haemoglobin (HB) and haemin (HM) enhanced NTPDase activity in isolates from female patients and conversely reduced the enzyme activity in isolates from male patients. Iron treatments could not alter the NTPDase transcript levels in T. vaginalis. Furthermore, our results reveal a distinct ATP, ADP and AMP hydrolysis profile between isolates from female and male patients influenced by iron from HB and HM. Our data indicate the participation of NTPDase and ecto-5’-nucleotidase in the establishment of trichomonas infection through ATP degradation and adenosine production influenced by iron.

Analysis of the NTPDase and ecto-5'-nucleotidase profiles in serum-limited Trichomonas vaginalis

Trichomonas vaginalis is a parasite of the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. Ectonucleoside triphosphate diphosphohydrolase (NTPDase) family members, which hydrolyse extracellular ATP and ADP and ecto-5′-nucleotidase, which hydrolyses AMP, have been characterised in T. vaginalis. For trichomonad culture, the growth medium is supplemented with 10 percent serum, which is an important source of nutrients, such as adenosine. Here, we investigated the ATP metabolism of T. vaginalis trophozoites from long-term cultures and clinical isolates under limited bovine serum conditions (1 percent serum). The specific enzymatic activities were expressed as nmol inorganic phosphate (Pi) released/min/mg protein, the gene expression patterns were determined by reverse transcriptase-polymerase chain reaction, the extracellular adenine nucleotide hydrolysis was analysed by high performance liquid chromatography and the cell cycle analysis was assessed by flow cytometry. Serum limitation led to the profound activation of NTPDase and ecto-5'-nucleotidase activities. Furthermore, the levels of NTPDase A and B transcripts increased and extracellular ATP metabolism was activated, which led to enhanced ATP hydrolysis and the formation of ADP and AMP. Moreover, the cell cycle was arrested at the G0/G1 stage, which suggested adenosine uptake. Our data suggest that under conditions of serum limitation, NTPDase and ecto-5'-nucleotidase play a role in providing the adenosine required for T. vaginalis growth and that this process contributes to the establishment of parasitism.

Lower frequency of the low activity adenosine deaminase allelic variant (ADA1*2) in schizophrenic patients / Diminuição da frequência da variante alélica de baixa atividade da adenosina desaminase (ADA1*2) em pacientes esquizofrênicos

OBJECTIVE: Adenosine may play a role in the pathophysiology of schizophrenia, since it modulates the release of several neurotransmitters such as glutamate, dopamine, serotonin and acetylcholine, decreases neuronal activity by pos-synaptic hyperpolarization and inhibits dopaminergic activity. Adenosine deaminase participates in purine metabolism by converting adenosine into inosine. The most frequent functional polymorphism of adenosine deaminase (22G→A) (ADA1*2) exhibits 20-30 percent lower enzymatic activity in individuals with the G/A genotype than individuals with the G/G genotype. The aim of this study was to evaluate the ADA polymorphism 22G→A (ADA1*2) in schizophrenic patients and healthy controls. METHOD: The genotypes of the ADA 22G→A were identified with allele-specific PCR strategy in 152 schizophrenic patients and 111 healthy individuals. RESULTS: A significant decrease in the frequency of the G/A genotype was seen in schizophrenic patients (7/152 - 4.6 percent) relative to controls (13/111 - 11.7 percent, p = 0.032, OR = 2.6). CONCLUSION: These results suggest that the G/A genotype associated with low adenosine deaminase activity and, supposingly, with higher adenosine levels is less frequent among schizophrenic patients.

OBJETIVO: A adenosina pode ter um papel importante na fisiopatologia da esquizofrenia, uma vez que modula a liberação de vários neurotransmissores, tais como glutamato, dopamina, serotonina e acetilcolina, diminui a atividade neuronal por hiperpolarização pós-sináptica e inibe a atividade dopaminérgica. A adenosina desaminase participa do metabolismo das purinas pela conversão de adenosina em inosina. O mais frequente polimorfismo funcional da adenosina desaminase (22G →A) (ADA1*2) exibe uma diminuição de 20-30 por cento da atividade funcional em indivíduos com genótipo G/A quando comparados com indivíduos com o genótipo G/G. O objetivo deste estudo foi avaliar o polimorfismo 22G→A (ADA1*2) em pacientes esquizofrênicos e em controles saudáveis. MÉTODO: Os genótipos da ADA 22G →A foram identificados através de uma estratégia de PCR alelo-específica em 152 pacientes esquizofrênicos e 111 controles saudáveis. RESULTADOS: Foi observada uma diminuição significativa na frequência do genótipo G/A em pacientes esquizofrênicos (7 - 4,6 por cento) em relação ao grupo controle (13 - 11,7 por cento, p = 0,032, OR = 2,6). CONCLUSÃO: Estes resultados sugerem que o genótipo G/A associado com baixa atividade de adenosina desaminase, e potencialmente com níveis aumentados de adenosina, é menos frequente entre pacientes esquizofrênicos.

Genotyping of hepatitis B virus in a cohort of patients evaluated in a hospital of Porto Alegre, south of Brazil

CONTEXT: In recent years the hepatitis B virus (HBV) genotyping has been considered a relevant factor in the natural history of the disease. OBJECTIVE: To determine hepatitis B virus genotypes and its epidemiological and clinical implications, in a cohort of patients in a hospital in Porto Alegre, South of Brazil. Methods - Sixty seven patients with HBV chronic infection markers who were being treated at ''Complexo Hospitalar Santa Casa'', in Porto Alegre, RS, Brazil, were evaluated. Demographic and epidemiological data were collected from these group of patients by following a standard protocol and ALT and HBeAg were determined. The genotypes and subtypes were determined by in-house PCR and, finally, the samples were sequenced. The level of significance used was 5 percent. RESULTS: The qualitative analysis for HBV-DNA by PCR was positive in 79.1 percent of the samples (53/67). The genotype was determined in all positive VHB-DNA samples and the genotypes A (34 percent), D (60.4 percent) and F (5.4 percent) as well as the subtypes adw, ayw and adw4 were found. No significant correlation was found between the hepatitis B virus genotypes and demographic variables considered as risk factors for hepatitis B virus infection. There was also no correlation between the genotypes and the serological and laboratory variables related to liver disease. CONCLUSION: We concluded that the most prevalent genotype found was D. However, further studies are needed to allow us to evaluate the implications of genetic variability in the clinical evolution of HBV carriers.

CONTEXTO: Nos últimos anos a genotipagem do vírus da hepatite B (VHB) tem sido considerado fator relevante para a história natural da doença. OBJETIVOS: Determinar os genótipos do VHB e suas implicações clínicas e epidemiológicas, em uma coorte de pacientes em um hospital de Porto Alegre, RS, sul do Brasil. MÉTODOS: Foram avaliados 67 pacientes com marcadores de infecção crônica pelo VHB que estavam sendo tratados no Complexo Hospitalar Santa Casa de Porto Alegre, RS. Foi aplicado um protocolo com dados demográficos e epidemiológicos dos pacientes, e AgHBe e ALT foram determinadas. Os genótipos e subtipos foram determinados por PCR in-house e, finalmente, as amostras foram sequenciadas. O nível de significância utilizado foi de 5 por cento. RESULTADOS: A análise qualitativa de VHB-DNA por PCR foi positiva em 79,1 por cento das amostras (53/67). O genótipo foi determinado em todas as amostras de VHB-DNA positivo. A análise demonstrou a presença dos genótipos A (34 por cento), D (60,4 por cento) e F (5,4 por cento). Foram encontrados os seguintes subtipos: adW, ayw e adw4. Nenhuma correlação significativa foi encontrada entre os genótipos do VHB e as variáveis demográficas estudadas como fator de risco para infecção pelo VHB, e com os exames sorológicos e laboratoriais de doença hepática. CONCLUSÃO: O genótipo mais prevalente encontrado foi o D. No entanto, mais estudos são necessários para que se possa avaliar as implicações da variabilidade genética na evolução clínica de portadores do VHB.

Association between 894G>T endothelial nitric oxide synthase gene polymorphisms and metabolic syndrome / Associação entre o polimorfismo 894g>T do gene óxido nítrico sintetase endotelial e síndrome metabólica

Metabolic syndrome (MS) is a cluster of cardiovascular risk factors such as hypertension, dyslipidemia, obesity and type II diabetes. Here, we performed a case-control study analyzing the association between 894G>T endothelial nitric oxide synthase gene polymorphism (NOS3) and MS in 616 subjects. Genotype frequencies were TT= 9.3 percent, GG= 37.2 and TG= 53.6 percent and the allelic frequencies were T=0.36 and G= 0.64. We observed a higher TT genotype frequency in the male MS group than control subjects (p=0.02), independent of other variables. We found an association between hypertension and TT genotype in females. Our data suggests that 894G>T plays a significant role in the mechanistic interaction between metabolic risk such as hypertension and MS, although sex-related differences may exist.

A síndrome metabólica (SM) é um agrupamento de fatores de riscos cardiovasculares, tais como hipertensão, dislipidemia, obesidade e diabetes tipo 2. Realizou-se um estudo caso-controle para analisar a associação entre o polimorfismo (894G>T do gene da enzima endotelial oxido nítrico sintetase (NOS3) e a SM em 616 voluntários. As freqüências genotípicas foram: TT = 9,3 por cento, GG = 37,2 por cento e TG = 53,6 por cento, e as freqüências alélicas T = 0,36 e G = 0,64. Observou-se freqüência mais alta do genótipo TT em homens com SM do que nos controles, independentemente de outros fatores (p = 0,02). Também observou-se associação entre hipertensão e o genótipo TT nas mulheres. Os dados do estudo sugerem que o polimorfismo 894G>T pode ter papel significativo na interação entre riscos metabólicos, tais como a hipertensão e a SM, ainda que existam diferenças relacionadas ao sexo.