Restriction site heteroplasmy in the mitochondrial DNA of Brycon opalinus (Cuvier, 1819) (Characiformes, Characidae, Bryconiae)

Homoplasmy is a feature usually found in the mtDNA of higher animal taxa. On the other hand, the presence of two classes of mtDNA in the same cell or organism is rare and may appear in length or site variation. Data from mtDNA RFLP analysis of Brycon opalinus populations (Cuvier, 1819; Characiformes, Characidae, Bryconinae) revealed site heteroplasmy from endonuclease NheI digestion. Southern blotting hybridization was used to survey a total of 257 specimens with 24 restriction enzymes. Three different restriction fragment patterns of mtDNA were obtained from NheI digestion. Two individuals from hatchery broodstock were found to have two of them. NheI digests of heteroplasmic individuals yielded two fragments of approximately 1180 and 1260 bp. Despite the low frequency of this type of heteroplasmy in the whole B. opalinus population, the presence of site heteroplasmy in this species supports the evidence of this phenomenon in lower vertebrate groups.

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

Prevalence of deltaF508, G551D, G542X, and R553X mutations among cystic fibrosis patients in the North of Brazil

Cystic fibrosis (CF) is the most common genetic disease among Caucasians and is rare among sub-Saharan Africans. The Brazilian population is not ethnically homogeneous but it is the result of three-way ethnic admixture of Europeans, Africans and Amerindians in varying proportions, depending on the region. In the present study, we investigated 33 patients who had been diagnosed and are currently under treatment for CF at the University Hospital João de Barros Barreto, Belém, Pará State. The molecular analysis for G542X, G551D and R553X mutations was performed by PCR followed by RFLP using BstNI, HincII and MboI, respectively, in polyacrylamide gel eletrophoresis and stained with AgNO3. ThedeltaF508 mutation (a deletion of 3 bp) was only analyzed by polyacrylamide gel electrophoresis and stained with AgNO3. Each sample was analyzed for regions of interest in the CFTR gene using amplified by PCR and specific primers. The deltaF508 and G551D mutations presented frequencies of 22.7 and 3%, respectively. In 74.3% of the remaining patients, none of the mutations investigated was found. The present study characterized in a sample of patients with an established clinical diagnosis of CF (asthma, repeated bronchopneumonia, disorders of nutritional status, etc.) the most frequent mutation ( deltaF508) in the North region of Brazil and is also the first report of the G551D mutation. In spite of the wide spectrum of CF mutations and the heterogeneous ethnic origin of the Amazon population, the molecular diagnosis is a helpful additional tool for the diagnosis and treatment of CF patients.

Mutations in SRY and WT1 genes required for gonadal development are not responsible for XY partial gonadal dysgenesis

The WT1 transcription factor regulates SRY expression during the initial steps of the sex determination process in humans, activating a gene cascade leading to testis differentiation. In addition to causing Wilms' tumor, mutations in WT1 are often responsible for urogenital defects in men, while SRY mutations are mainly related to 46,XY pure gonadal dysgenesis. In order to evaluate their role in abnormal testicular organogenesis, we screened for SRY and WT1 gene mutations in 10 children with XY partial gonadal dysgenesis, 2 of whom with a history of Wilms' tumor. The open reading frame and 360 bp of the 5' flanking sequence of the SRY gene, and the ten exons and intron boundaries of the WT1 gene were amplified by PCR of genomic DNA. Single-strand conformation polymorphism was initially used for WT1 mutation screening. Since shifts in fragment migration were only observed for intron/exon 4, the ten WT1 exons from all patients were sequenced manually. No mutations were detected in the SRY 5' untranslated region or within SRY open-reading frame sequences. WT1 sequencing revealed one missense mutation (D396N) in the ninth exon of a patient who also had Wilms' tumor. In addition, two silent point mutations were found in the first exon including one described here for the first time. Some non-coding sequence variations were detected, representing one new (IVS4+85A>G) and two already described (-7ATG T>G, IVS9-49 T>C) single nucleotide polymorphisms. Therefore, mutations in two major genes required for gonadal development, SRY and WT1, are not responsible for XY partial gonadal dysgenesis.

Effect of aging on carotid artery stiffness and baroreflex sensitivity during head-out water immersion in man

To examine the possible age-related blood pressure (BP) deregulation in response to central hypervolemia, we measured spontaneous baroreflex sensitivity (SBRS), carotid arterial compliance (CC), and R-R interval coefficient of variation (RRICV) during basal and thermoneutral resting head-out-of-water immersion (HOWI) in 7 young (YG = 24.0 ± 0.8 years) and 6 middle-aged/older (OL = 59.3 ± 1.3 years) healthy men. Compared with basal conditions (YG = 19.6 ± 4.0 vs OL = 6.1 ± 1.5 ms/mmHg, P < 0.05), SBRS remained higher in YG than OL during rest HOWI (YG = 23.6 ± 6.6 vs OL = 9.3 ± 2.1 ms/mmHg, P < 0.05). The RRICV was significantly different between groups (YG = 6.5 ± 1.4 vs OL = 2.8 ± 0.4%, P < 0.05) under HOWI. The OL group had no increase in CC, but a significant increase in systolic BP (basal = 115.3 ± 4.4 vs water = 129.3 ± 5.3 mmHg, P < 0.05) under HOWI. In contrast, the YG group had a significant increase in CC (basal = 0.16 ± 0.01 vs water = 0.17 ± 0.02 mm²/mmHg, P < 0.05) with no changes in systolic BP. SBRS was positively related to CC (r = 0.58, P < 0.05 for basal vs r = 0.62, P < 0.05 for water). Our data suggest that age-related vagal dysfunction and reduced CC may be associated with SBRS differences between YG and OL groups, and with BP elevation during HOWI in healthy older men.

A semi-automatic computerized method to measure baroreflex-mediated heart rate responses that reduces interobserver variability

Arterial baroreflex sensitivity estimated by pharmacological impulse stimuli depends on intrinsic signal variability and usually a subjective choice of blood pressure (BP) and heart rate (HR) values. We propose a semi-automatic method to estimate cardiovascular reflex sensitivity to bolus infusions of phenylephrine and nitroprusside. Beat-to-beat BP and HR time series for male Wistar rats (N = 13) were obtained from the digitized signal (sample frequency = 2 kHz) and analyzed by the proposed method (PRM) developed in Matlab language. In the PRM, time series were low-pass filtered with zero-phase distortion (3rd order Butterworth used in the forward and reverse direction) and presented graphically, and parameters were selected interactively. Differences between basal mean values and peak BP (deltaBP) and HR (deltaHR) values after drug infusions were used to calculate baroreflex sensitivity indexes, defined as the deltaHR/deltaBP ratio. The PRM was compared to the method traditionally (TDM) employed by seven independent observers using files for reflex bradycardia (N = 43) and tachycardia (N = 61). Agreement was assessed by Bland and Altman plots. Dispersion among users, measured as the standard deviation, was higher for TDM for reflex bradycardia (0.60 ± 0.46 vs 0.21 ± 0.26 bpm/mmHg for PRM, P < 0.001) and tachycardia (0.83 ± 0.62 vs 0.28 ± 0.28 bpm/mmHg for PRM, P < 0.001). The advantage of the present method is related to its objectivity, since the routine automatically calculates the desired parameters according to previous software instructions. This is an objective, robust and easy-to-use tool for cardiovascular reflex studies.

Acetylcholine and bradykinin enhance hypotension and affect the function of remodeled conduit arteries in SHR and SHR treated with nitric oxide donors

Discrepancy was found between enhanced hypotension and attenuated relaxation of conduit arteries in response to acetylcholine (ACh) and bradykinin (BK) in nitric oxide (NO)-deficient hypertension. The question is whether a similar phenomenon occurs in spontaneously hypertensive rats (SHR) with a different pathogenesis. Wistar rats, SHR, and SHR treated with NO donors [molsidomine (50 mg/kg) or pentaerythritol tetranitrate (100 mg/kg), twice a day, by gavage] were studied. After 6 weeks of treatment systolic blood pressure (BP) was increased significantly in experimental groups. Under anesthesia, the carotid artery was cannulated for BP recording and the jugular vein for drug administration. The iliac artery was used for in vitro studies and determination of geometry. Compared to control, SHR showed a significantly enhanced (P < 0.01) hypotensive response to ACh (1 and 10 µg, 87.9 ± 6.9 and 108.1 ± 5.1 vs 35.9 ± 4.7 and 64.0 ± 3.3 mmHg), and BK (100 µg, 106.7 ± 8.3 vs 53.3 ± 5.2 mmHg). SHR receiving NO donors yielded similar results. In contrast, maximum relaxation of the iliac artery in response to ACh was attenuated in SHR (12.1 ± 3.6 vs 74.2 ± 8.6% in controls, P < 0.01). Iliac artery inner diameter also increased (680 ± 46 vs 828 ± 28 µm in controls, P < 0.01). Wall thickness, wall cross-section area, wall thickness/inner diameter ratio increased significantly (P < 0.01). No differences were found in this respect among SHR and SHR treated with NO donors. These findings demonstrated enhanced hypotension and attenuated relaxation of the conduit artery in response to NO activators in SHR and in SHR treated with NO donors, a response similar to that found in NO-deficient hypertension.

Cardiovascular risk factors in a population of Brazilian schoolchildren

Epidemiological and clinical evidence suggests that a judicious diet, regular physical activity and blood pressure (BP) monitoring must start in early childhood to minimize the impact of modifiable cardiovascular risk factors. This study was designed to evaluate BP and metabolic parameters of schoolchildren from Vitória, Espírito Santo State, Brazil, and correlate them with cardiovascular risk factors. The study was conducted on 380 students aged 10-14 years (177 boys, 203 girls) enrolled in public schools. Baseline measurements included body mass index, BP and heart rate. The students were submitted to exercise spirometry on a treadmill. VO2max was obtained from exercise testing to voluntary exhaustion. Fasting serum total cholesterol (TC), LDL-C, HDL-C, triglycerides (TG), and glucose were measured. Nine point nine percent of the boys and 11.7% of the girls were hypertensive or had pre-hypertensive levels. There was no significant correlation between VO2max and TC, LDL-C, or TG in prepubertal children, but a slight negative correlation was detected in post-pubertal boys for HDL-C and TG. In addition, children with hypertension (3.4%) or pre-hypertensive levels (6.6%) also had comorbidity for overweight and blood lipid abnormalities (14% for triglycerides, 44.7% for TC, 25.9% for LDL-C, 52% for low HDL-C). The present study shows for the first time high correlations between prehypertensive blood pressure levels and the cardiovascular risk factors high TC, high LDL-C, low HDL-C in schoolchildren. These are important for the formulation of public health policies and strategies.

PCR identification of Mycobacterium tuberculosis complex in a clinical sample from a patient with symptoms of tuberculous spondylodiscitis

A 42-year-old male complaining of thoracic spine pain was admitted to the hospital for evaluation. An X-ray and computer tomography of the thoracic spine showed spondylodiscitis of the L3 lumbar and L2-L3 intervertebral disk. The tuberculin skin test (PPD) was strongly positive. A radioscopy-guided fine needle aspirate of the affected area was cultured but did not reveal the cause of the disease. Two biopsy attempts failed to reveal the cause of the disease by culturing or by acid-fast-resistant staining (Ziehl Neelsen) of the specimens. A third biopsy also failed to detect the infectious agent by using microbiological procedures, but revealed the presence of a 245-bp amplicon characteristic of the Mycobacterium tuberculosis complex after PCR of the sample. The result demonstrates the efficacy of PCR for the identification of M. tuberculosis in situations in which conventional diagnosis by culturing techniques or direct microscopy is unable to detect the microorganism. Following this result the patient was treated with the antituberculous cocktail composed by rifampicin, pirazinamide and isoniazid during a six-month period. At the end of the treatment the dorsalgia symptoms had disappeared.

A double-blind comparison of the effect of the antipsychotics haloperidol and olanzapine on sleep in mania

The effects of haloperidol and olanzapine on polysomnographic measures made in bipolar patients during manic episodes were compared. Twelve DSM-IV mania patients were randomly assigned to receive either haloperidol (mean ± SD final dosage: 5.8 ± 3.8 mg) or olanzapine (mean ± SD final dosage: 13.6 ± 6.9 mg) in a 6-week, double-blind, randomized, controlled clinical trial. One-night polysomnographic evaluation was performed before and after the haloperidol or olanzapine treatment. Psychopathology and illness severity were rated respectively with the Young Mania Rating Scale (YMRS) and the Clinical Global Impressions - Bipolar version (CGI-BP). There was a significant improvement in the YMRS and CGI-BP scores at the end of the study for both groups. Mixed ANOVA used to compare the polysomnographic measures of both drugs demonstrated significant improvement in sleep measures with olanzapine. In the olanzapine group, statistically significant time-drug interaction effects on sleep continuity measures were observed: sleep efficiency (mean ± SEM pre-treatment value: 6.7 ± 20.3%; after-treatment: 85.7 ± 10.9%), total wake time (pre-treatment: 140.0 ± 92.5 min; after-treatment: 55.2 ± 44.2 min), and wake time after sleep onset (pre-treatment: 109.7 ± 70.8 min; after-treatment: 32.2 ± 20.7 min). Conversely, improvement of polysomnographic measures was not observed for the haloperidol group (P > 0.05). These results suggest that olanzapine is more effective than haloperidol in terms of sleep-promoting effects, although olanzapine is comparatively as effective as haloperidol in treating mania. Polysomnography records should provide useful information on how manic states can be affected by psychopharmacological agents.

Genotyping hepatitis C virus from hemodialysis patients in Central Brazil by line probe assay and sequence analysis

The present study examined the distribution of hepatitis C virus (HCV) genotypes and subtypes in a hemodialysis population in Goiás State, Central Brazil, and evaluated the efficiency of two genotyping methods: line probe assay (LiPA) based on the 5' noncoding region and nucleotide sequencing of the nonstructural 5B (NS5B) region of the genome. A total of 1095 sera were tested for HCV RNA by RT-nested PCR of the 5' noncoding region. The LiPA assay was able to genotype all 131 HCV RNA-positive samples. Genotypes 1 (92.4%) and 3 (7.6%) were found. Subtype 1a (65.7%) was the most prevalent, followed by subtypes 1b (26.7%) and 3a (7.6%). Direct nucleotide sequencing of 340 bp from the NS5B region was performed in 106 samples. The phylogenetic tree showed that 98 sequences (92.4%) were classified as genotype 1, subtypes 1a (72.6%) and 1b (19.8%), and 8 sequences (7.6%) as subtype 3a. The two genotyping methods gave concordant results within HCV genotypes and subtypes in 100 and 96.2% of cases, respectively. Only four samples presented discrepant results, with LiPA not distinguishing subtypes 1a and 1b. Therefore, HCV genotype 1 (subtype 1a) is predominant in hemodialysis patients in Central Brazil. By using sequence analysis of the NS5B region as a reference standard method for HCV genotyping, we found that LiPA was efficient at the genotype level, although some discrepant results were observed at the subtype level (sensitivity of 96.1% for subtype 1a and 95.2% for subtype 1b). Thus, analysis of the NS5B region permitted better discrimination between HCV subtypes, as required in epidemiological investigations.

Reduced plasma levels of angiotensin-(1-7) and renin activity in preeclamptic patients are associated with the angiotensin I- converting enzyme deletion/deletion genotype

The relationship between preeclampsia and the renin-angiotensin system (RAS) is poorly understood. Angiotensin I-converting enzyme (ACE) is a key RAS component and plays an important role in blood pressure homeostasis by generating angiotensin II (Ang II) and inactivating the vasodilator angiotensin-(1-7) (Ang-(1-7)). ACE (I/D) polymorphism is characterized by the insertion (I) or deletion (D) of a 287-bp fragment, leading to changes in ACE activity. In the present study, ACE (I/D) polymorphism was correlated with plasma Ang-(1-7) levels and several RAS components in both preeclamptic (N = 20) and normotensive pregnant women (N = 20). The percentage of the ACE DD genotype (60%) in the preeclamptic group was higher than that for the control group (35%); however, this percentage was not statistically significant (Fisher exact test = 2.86, d.f. = 2, P = 0.260). The highest plasma ACE activity was observed in the ACE DD preeclamptic women (58.1 ± 5.06 vs 27.6 ± 3.25 nmol Hip-His Leu-1 min-1 mL-1 in DD control patients; P = 0.0005). Plasma renin activity was markedly reduced in preeclampsia (0.81 ± 0.2 vs 3.43 ± 0.8 ng Ang I mL plasma-1 h-1 in DD normotensive patients; P = 0.0012). A reduced plasma level of Ang-(1-7) was also observed in preeclamptic women (15.6 ± 1.3 vs 22.7 ± 2.5 pg/mL in the DD control group; P = 0.0146). In contrast, plasma Ang II levels were unchanged in preeclamptic patients. The selective changes in the RAS described in the present study suggest that the ACE DD genotype may be used as a marker for susceptibility to preeclampsia.

Sodium taste threshold in children and its relationship to blood pressure

Popular science has emphasized the risks of high sodium intake and many studies have confirmed that salt intake is closely related to hypertension. The present mini-review summarizes experiments about salt taste sensitivity and its relationship with blood pressure (BP) and other variables of clinical and familial relevance. Children and adolescents from control parents (N = 72) or with at least one essential hypertensive (EHT) parent (N = 51) were investigated. Maternal questionnaires on eating habits and vomiting episodes were collected. Offspring, anthropometric, BP, and salt taste sensitivity values were recorded and blood samples analyzed. Most mothers declared that they added "little salt" when cooking. Salt taste sensitivity was inversely correlated with systolic BP (SBP) in control youngsters (r = -0.33; P = 0.015). In the EHT group, SBP values were similar to control and a lower salt taste sensitivity threshold. Obese offspring of EHT parents showed higher SBP and C-reactive protein values but no differences in renin-angiotensin-aldosterone system activity. Salt taste sensitivity was correlated with SBP only in the non-obese EHT group (N = 41; r = 0.37; P = 0.02). Salt taste sensitivity was correlated with SBP in healthy, normotensive children and adolescents whose mothers reported significant vomiting during the first trimester (N = 18; r = -0.66; P < 0.005), but not in "non-vomiter offspring" (N = 54; r = -0.18; nonsignificant). There is evidence for a linkage between high blood pressure, salt intake and sensitivity, perinatal environment and obesity, with potential physiopathological implications in humans. This relationship has not been studied comprehensively using homogeneous methods and therefore more research is needed in this field.

Genetic variations of EBV-LMP1 from nasopharyngeal carcinoma biopsies: potential loss of T cell epitopes

To find Epstein-Barr virus (EBV) strains with genetic variations of EBV latent membrane protein 1 (EBV-LMP1) from nasopharyngeal carcinoma (NPC), the full-length DNA of LMP1 genes from 21 NPC biopsies obtained in Hunan province in southern China was amplified and sequenced. Our sequences were compared to those previously reported by the Clustal V method. Results showed that all 21 sequences displayed two amino acid changes most frequently in LMP1 of CD4+ T cell epitopes at codons 144 (F®I, 21/21) and 212 (G®S, 19/21) or (G®N, 2/21). We also show that type A EBV strain is prevalent in the cases of NPC from Hunan province with a 30-bp 18/21 deletion, and we highlight that this deletion resulted in loss of one of the CD4+ T cell-restricted epitopes. The other 3 sequences without this deletion all had a change at codon 344 (G®D). Furthermore, in the major epitope sequence of CD8+ T cells restricted by HLA-A2, all 21 sequences showed changes at codons 126 (L®F) and 129 (M®I). Our study discovered that one of the 21 sequence variations harbored a new change at codon 131 (W®C), and 5/21 specimens showed another novel change at codon 115 (G®A) in the major epitope sequence of CD8+ T cells restricted by HLA-A2. Our study suggests that these sequence variations of NPC-derived LMP1 may lead to a potential escape from host cell immune recognition, protecting latent EBV infection and causing an increase in tumorigenicity.

Cloning, sequence analysis, and expression of cDNA coding for the major house dust mite allergen, Der f 1, in Escherichia coli

Our objective was to clone, express and characterize adult Dermatophagoides farinae group 1 (Der f 1) allergens to further produce recombinant allergens for future clinical applications in order to eliminate side reactions from crude extracts of mites. Based on GenBank data, we designed primers and amplified the cDNA fragment coding for Der f 1 by nested-PCR. After purification and recovery, the cDNA fragment was cloned into the pMD19-T vector. The fragment was then sequenced, subcloned into the plasmid pET28a(+), expressed in Escherichia coli BL21 and identified by Western blotting. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Sequence analysis showed the presence of an open reading frame containing 966 bp that encodes a protein of 321 amino acids. Interestingly, homology analysis showed that the Der p 1 shared more than 87% identity in amino acid sequence with Eur m 1 but only 80% with Der f 1. Furthermore, phylogenetic analyses suggested that D. pteronyssinus was evolutionarily closer to Euroglyphus maynei than to D. farinae, even though D. pteronyssinus and D. farinae belong to the same Dermatophagoides genus. A total of three cysteine peptidase active sites were found in the predicted amino acid sequence, including 127-138 (QGGCGSCWAFSG), 267-277 (NYHAVNIVGYG) and 284-303 (YWIVRNSWDTTWGDSGYGYF). Moreover, secondary structure analysis revealed that Der f 1 contained an a helix (33.96%), an extended strand (17.13%), a ß turn (5.61%), and a random coil (43.30%). A simple three-dimensional model of this protein was constructed using a Swiss-model server. The cDNA coding for Der f 1 was cloned, sequenced and expressed successfully. Alignment and phylogenetic analysis suggests that D. pteronyssinus is evolutionarily more similar to E. maynei than to D. farinae.