Screening for mutations in human alpha-globin genes by nonradioactive single-strand conformation polymorphism

Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystem™ and GenePhor™, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystem™ and 20% gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.

Interferon-alpha receptor 1 mRNA expression in peripheral blood mononuclear cells is associated with response to interferon-alpha therapy of patients with chronic hepatitis C

Interferon (IFN)-alpha receptor mRNA expression in liver of patients with chronic hepatitis C has been shown to be a response to IFN-alpha therapy. The objective of the present study was to determine whether the expression of mRNA for subunit 1 of the IFN-alpha receptor (IFNAR1) in peripheral blood mononuclear cells (PBMC) is associated with the response to IFN-alpha in patients with chronic hepatitis C. Thirty patients with positive anti-HCV and HCV-RNA, and abnormal levels of alanine aminotransferase in serum were selected and treated with IFN-alpha2b for one year. Those with HBV or HIV infection, or using alcohol were not included. Thirteen discontinued the treatment and were not evaluated. The IFN-alpha response was monitored on the basis of alanine aminotransferase level and positivity for HCV-RNA in serum. IFNAR1-mRNA expression in PBMC was measured by reverse transcription-polymerase chain reaction before and during the first three months of therapy. The results are reported as IFNAR1-mRNA/ß-actin-mRNA ratio (mean ± SD). Before treatment, responder patients had significantly higher IFNAR1-mRNA expression in PBMC (0.67 ± 0.15; N = 5; P < 0.05) compared to non-responders (0.35 ± 0.17; N = 12) and controls (0.30 ± 0.16; N = 9). Moreover, IFNAR1-mRNA levels were significantly reduced after 3 months of treatment in responders, whereas there were no differences in IFNAR1 expression in non-responders during IFN-alpha therapy. Basal IFNAR1-mRNA expression was not correlated with the serum level of alanine and aspartate aminotransferases or the presence of cirrhosis. The present results suggest that IFNAR1-mRNA expression in PBMC is associated with IFN-alpha response to hepatitis C and may be useful for monitoring therapy in patients with chronic hepatitis C.

Increased production of tumor necrosis factor-alpha in whole blood cultures from children with primary malnutrition

Because low tumor necrosis factor-alpha (TNF-alpha) production has been reported in malnourished children, in contrast with high production of TNF-alpha in experimental protein-energy malnutrition, we reevaluated the production of TNF-alpha in whole blood cultures from children with primary malnutrition free from infection, and in healthy sex- and age-matched controls. Mononuclear cells in blood diluted 1:5 in endotoxin-free medium released TNF-alpha for 24 h. Spontaneously released TNF-alpha levels (mean ± SEM), as measured by enzyme immunoassay in the supernatants of unstimulated 24-h cultures, were 10,941 ± 2,591 pg/ml in children with malnutrition (N = 11) and 533 ± 267 pg/ml in controls (N = 18) (P < 0.0001). TNF-alpha production was increased by stimulation with lipopolysaccharide (LPS), with maximal production of 67,341 ± 16,580 pg/ml TNF-alpha in malnourished children and 25,198 ± 2,493 pg/ml in controls (P = 0.002). In control subjects, LPS dose-dependently induced TNF-alpha production, with maximal responses obtained at 2000 ng/ml. In contrast, malnourished patients produced significantly more TNF-alpha with 0.02-200 ng/ml LPS, responded maximally at a 10-fold lower LPS concentration (200 ng/ml), and presented high-dose inhibition at 2000 ng/ml. TNF-alpha production a) was significantly influenced by LPS concentration in control subjects, but not in malnourished children, who responded strongly to very low LPS concentrations, and b) presented a significant, negative correlation (r = -0.703, P = 0.023) between spontaneous release and the LPS concentration that elicited maximal responses in malnourished patients. These findings indicate that malnourished children are not deficient in TNF-alpha production, and suggest that their cells are primed for increased TNF-alpha production.

Effect of D-alpha-tocopherol on tubular nephron acidification by rats with induced diabetes mellitus

The objective of the present study was to determine if treatment of diabetic rats with D-alpha-tocopherol could prevent the changes in glomerular and tubular function commonly observed in this disease. Sixty male Wistar rats divided into four groups were studied: control (C), control treated with D-alpha-tocopherol (C + T), diabetic (D), and diabetic treated with D-alpha-tocopherol (D + T). Treatment with D-alpha-tocopherol (40 mg/kg every other day, ip) was started three days after diabetes induction with streptozotocin (60 mg/kg, ip). Renal function studies and microperfusion measurements were performed 30 days after diabetes induction and the kidneys were removed for morphometric analyses. Data are reported as means ± SEM. Glomerular filtration rate increased in D rats but decreased in D + T rats (C: 6.43 ± 0.21; D: 7.74 ± 0.45; D + T: 3.86 ± 0.18 ml min-1 kg-1). Alterations of tubular acidification observed in bicarbonate absorption flux (JHCO3) and in acidification half-time (t/2) in group D were reversed in group D + T (JHCO3, C: 2.30 ± 0.10; D: 3.28 ± 0.22; D + T: 1.87 ± 0.08 nmol cm-2 s-1; t/2, C: 4.75 ± 0.20; D: 3.52 ± 0.15; D + T: 5.92 ± 0.19 s). Glomerular area was significantly increased in D, while D + T rats exhibited values similar to C, suggesting that the vitamin prevented the hypertrophic effect of hyperglycemia (C: 8334.21 ± 112.05; D: 10,217.55 ± 100.66; D + T: 8478.21 ± 119.81µm²). These results suggest that D-alpha-tocopherol is able to protect rats, at least in part, from the harmful effects of diabetes on renal function.

Concentration of CCL11, CXCL8 and TNF-alpha in sputum and plasma of patients undergoing asthma or chronic obstructive pulmonary disease exacerbation

Asthma and chronic obstructive pulmonary disease (COPD) are common respiratory illnesses characterized by chronic inflammation of the airways. The characterization of induced or spontaneously produced sputum is a useful technique to assess airway inflammation. In the present study, we compared the concentrations of CCL2, CCL11, CXCL8, and tumor necrosis factor-alpha (TNF-alpha) in plasma and induced sputum of patients with severe asthma or COPD and correlated the levels of these mediators with inflammatory cells in sputum. Asthmatic patients had elevated levels of eosinophils (40.1 ± 6.24%) in sputum whereas neutrophils (63.3 ± 4.66%) predominated in COPD patients. The levels of the chemokine CCL11 were markedly increased in sputum (708.7 ± 330.7 pg/ml) and plasma (716.6 ± 162.2 pg/ml) of asthmatic patients and correlated with the percentage of eosinophils in induced sputum. The concentrations of CXCL8 (817.0 ± 105.2 pg/ml) and TNF-alpha (308.8 ± 96.1 pg/ml) were higher in sputum of COPD patients and correlated with the percentage of neutrophils in induced sputum. There was also an increase in the concentrations of CXCL8 (43.2 ± 6.8 pg/ml) in sputum of asthmatic patients. These results validate that sputum is a suitable method to assess chemokines and cytokines associated with asthma and COPD. Moreover, the mechanisms involved in the synthesis of CCL11 and CXCL8/TNF-alpha would be helpful to better understand the inflammatory profile associated with asthma and COPD, respectively.

Inducible nitric oxide synthase and tumor necrosis factor-alpha in delayed gastric emptying and gastrointestinal transit induced by lipopolysaccharide in mice

Gastrointestinal motility disturbances during endotoxemia are probably caused by lipopolysaccharide (LPS)-induced factors: candidates include nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), interleukin-1ß, and interleukin-6. Flow cytometry was used to determine the effects of LPS and these factors on gastric emptying (evaluated indirectly by determining percent gastric retention; %GR) and gastrointestinal transit (GIT) in male BALB/c mice (23-28 g). NO (300 µg/mouse, N = 8) and TNF-alpha (2 µg/mouse, N = 7) increased (P < 0.01) GR and delayed GIT, mimicking the effect of LPS (50 µg/mouse). During early endotoxemia (1.5 h after LPS), inhibition of inducible NO synthase (iNOS) by a selective inhibitor, 1400 W (150 µg/mouse, N = 11), but not antibody neutralization of TNF-alpha (200 µg/mouse, N = 11), reversed the increase of GR (%GR 78.8 ± 3.3 vs 47.2 ± 7.5%) and the delay of GIT (geometric center 3.7 ± 0.4 vs 5.6 ± 0.2). During late endotoxemia (8 h after LPS), both iNOS inhibition (N = 9) and TNF-alpha neutralization (N = 9) reversed the increase of GR (%GR 33.7 ± 2.0 vs 19.1 ± 2.6% (1400 W) and 20.1 ± 2.0% (anti-TNF-alpha)), but only TNF-alpha neutralization reversed the delay of GIT (geometric center 3.9 ± 0.4 vs 5.9 ± 0.2). These findings suggest that iNOS, but not TNF-alpha, is associated with delayed gastric emptying and GIT during early endotoxemia and that during late endotoxemia, both factors are associated with delayed gastric emptying, but only TNF-alpha is associated with delayed GIT.

Effect of estrogen receptor-alpha (ESR1) gene polymorphism on high density lipoprotein levels in response to hormone replacement therapy

Studies have shown that estrogen replacement therapy and estrogen plus progestin replacement therapy alter serum levels of total, LDL and HDL cholesterol levels. However, HDL cholesterol levels in women vary considerably in response to hormone replacement therapy (HRT). A significant portion of the variability of these levels has been attributed to genetic factors. Therefore, we investigated the influence of estrogen receptor-alpha (ESR1) gene polymorphisms on HDL levels in response to postmenopausal HRT. We performed a prospective cohort study on 54 postmenopausal women who had not used HRT before the study and had no significant general medical illness. HRT consisted of conjugated equine estrogen and medroxyprogesterone acetate continuously for 1 year. The lipoprotein levels were measured from blood samples taken before the start of therapy and after 1 year of HRT. ESR1 polymorphism (MspI C>T, HaeIII C>T, PvuII C>T, and XbaI A>G) frequencies were assayed by restriction fragment length polymorphism. A general linear model was used to describe the relationships between HDL levels and genotypes after adjusting for age. A significant increase in HDL levels was observed after HRT (P = 0.029). Women with the ESR1 PvuII TT genotype showed a statistically significant increase in HDL levels after HRT (P = 0.032). No association was found between other ESR1 polymorphisms and HDL levels. According to our results, the ESR1 PvuII TT genotype was associated with increased levels of HDL after 1 year of HRT.

Tumor necrosis factor alpha increases epithelial barrier permeability by disrupting tight junctions in Caco-2 cells

The objectives of this study were to determine the effect of tumor necrosis factor alpha (TNF-α) on intestinal epithelial cell permeability and the expression of tight junction proteins. Caco-2 cells were plated onto Transwell® microporous filters and treated with TNF-α (10 or 100 ng/mL) for 0, 4, 8, 16, or 24 h. The transepithelial electrical resistance and the mucosal-to-serosal flux rates of the established paracellular marker Lucifer yellow were measured in filter-grown monolayers of Caco-2 intestinal cells. The localization and expression of the tight junction protein occludin were detected by immunofluorescence and Western blot analysis, respectively. SYBR-Green-based real-time PCR was used to measure the expression of occludin mRNA. TNF-α treatment produced concentration- and time-dependent decreases in Caco-2 transepithelial resistance and increases in transepithelial permeability to the paracellular marker Lucifer yellow. Western blot results indicated that TNF-α decreased the expression of phosphorylated occludin in detergent-insoluble fractions but did not affect the expression of non-phosphorylated occludin protein. Real-time RT-PCR data showed that TNF-α did not affect the expression of occludin mRNA. Taken together, our data demonstrate that TNF-α increases Caco-2 monolayer permeability, decreases occludin protein expression and disturbs intercellular junctions.

Effect of aerobic training on EEG alpha asymmetry and depressive symptoms in the elderly: a 1-year follow-up study

The effect of physical exercise on the treatment of depressive elderly adults has not been investigated thus far in terms of changes in cortical hemispheric activity. The objective of the present study was to identify changes in depressive symptoms, quality of life, and cortical asymmetry produced by aerobic activity. Elderly subjects with a diagnosis of major depressive disorder (DSM-IV) were included. Twenty patients (70% females, 71 ± 3 years) were divided into an exercise group (pharmacological treatment plus aerobic training) and a control group (undergoing pharmacological treatment) in a quasi-experimental design. Pharmacological treatment was maintained stable throughout the study (antidepressants and anxiolytics). Subjects were evaluated by depression scales (Beck Depression Inventory, Hamilton Depression Rating Scale, Montgomery-Asberg Depression Rating Scale) and the Short Form Health Survey-36, and electroencephalographic measurements (frontal and parietal alpha asymmetry) before and after 1 year of treatment. After 1 year, the control group showed a decrease in cortical activity on the right hemisphere (increase of alpha power), which was not observed in the exercise group. The exercise group showed a significant decrease of depressive symptoms, which was not observed in the control group. This result was also accompanied by improved treatment response and remission rate after 1 year of aerobic exercise associated with treatment. This study provides support for the effect of aerobic training on alpha activity and on depressive symptoms in elderly patients. Exercise facilitates the treatment of depressive elderly adults, leading to clinical and physical improvement and protecting against a decrease in cortical activity.

Characterization of alpha thalassemic genotypes by multiplex ligation-dependent probe amplification in the Brazilian population

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.

Effect of thymosin alpha-1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro

Thymosin alpha 1 (Tα1) has been shown to have beneficial effects on numerous immune system parameters, but little is known about the effects of Tα1 on patients with gastric carcinoma. The objective of this study was to determine the effect of Tα1 on subpopulations of Th1, Th2, Th17, and regulatory T cells (Tregs) in vitro, and to evaluate its efficacy as an immunoregulatory factor in patients with gastric carcinoma. We compared the effect of Tα1 on the frequency of CD4+ and CD8+ T cells, especially the CD4+CD25+Foxp3+ Tregs in peripheral blood mononuclear cells (PBMCs) from gastric carcinoma patients (N = 35) and healthy donors (N = 22). We also analyzed the changes in the proliferation of PBMCs in response to treatment with Tα1, and examined the production of Th1, Th2, and Th17 cytokines by PBMCs and tumor-infiltrating lymphocytes. The treatment of PBMCs from gastric cancer patients, with Tα1 (50 µg/mL) alone increased the percentage of CD4+CD25+Foxp3+ (suppressive antitumor-specific Tregs) from 1.68 ± 0.697 to 2.19 ± 0.795% (P < 0.05). Our results indicate that Tα1 increases the percentage of Tregs and IL-1β, TNF-α, and IL-6 in vitro.

Proteomic data show an increase in autoantibodies and alpha-fetoprotein and a decrease in apolipoprotein A-II with time in sera from senescence-accelerated mice

We evaluated changes in levels by comparing serum proteins in senescence-accelerated mouse-prone 8 (SAMP8) mice at 2, 6, 12, and 15 months of age (SAMP8-2 m, -6 m, -12 m, -15 m) to age-matched SAM-resistant 1 (SAMR1) mice. Mice were sacrificed, and blood was analyzed by 2-dimensional electrophoresis combined with mass spectrometry. Five protein spots were present in all SAMP8 serum samples, but only appeared in SAMR1 samples at 15 months of age except for spot 3, which also showed a slight expression in SAMR1-12 m sera. Two proteins decreased in the sera from SAMP8-2 m, -6 m, and -12 m mice, and divided into 2 spots each in SAMP8-15 m sera. Thus, the total number of altered spots in SAMP8 sera was 7; of these, 4 were identified as Ig kappa chain V region (M-T413), chain A of an activity suppressing Fab fragment to cytochrome P450 aromatase (32C2_A), alpha-fetoprotein, and apolipoprotein A-II. M-T413 is a monoclonal CD4 antibody, which inhibits T cell proliferation. We found that M-T413 RNA level was significantly enhanced in splenocytes from SAMP8-2 m mice. This agreed with serum M-T413 protein alterations and a strikingly lower blood CD4+ T cell count in SAMP8 mice when compared to the age-matched SAMR1 mice, with the latter negatively correlating with serum M-T413 protein volume. Age-related changes in serum proteins favored an increase in autoantibodies and alpha-fetoprotein and a decrease of apolipoprotein A-II, which occurred in SAMP8 mice at 2 months of age and onwards. These proteins may serve as candidate biomarkers for early aging.

Evaluation of total carotenoids, alpha- and beta-carotene in carrots (Daucus carota L.) during home processing

This study aims to analyze the influence of dehydration and different preparation methods during home processing related toalpha-carotene, beta-carotene and total carotenoids stability in carrots. Vitamin A values were evaluated after different treatments. Thus, carrots were submitted to steam cooking, water cooking with and without pressure, moist/dry cooking and conventional dehydration. Determination of alpha- and beta-carotenes was made by High-Performance Liquid Chromatography (HPLC) (conditions were developed by us) using spectrophotometric detection visible-UV at 470 nm; a RP-18 column and methanol: acetonitrile: ethyl acetate (80: 10: 10) as mobile phase. Total carotenoids quantification was made by 449 nm spectrophotometer. The retention of the analyzed carotenoids ranged from 60.13 to 85.64%. Water cooking without pressure promoted higher retention levels of alpha- and beta-carotene and vitamin A values, while water cooking with pressure promoted higher retention levels of total carotenoids. Dehydration promoted the highest carotenoid losses. The results showed that, among the routinely utilized methods under domestic condition, cooking without pressure, if performed under controlled time and temperature, is the best method as it reduces losses in the amount of alpha- and beta-carotene, the main carotenoids present in the carrots. Despite the significant carotenoid losses, carrots prepared through domestic methods, remain a rich source of provitamin A.

Incorporation of conjugated linoleic and alpha linolenic fatty acids into Pimedolus maculatus fillets

The objective of this study was to investigate the fatty acid composition of Pimedolus maculatus fillets of fish fed with different diets. The fish were submitted to an adaptation period (over 30 days) and fed only a control diet (diet I). After this period, the fish were divided into two groups: one group received a diet enriched with flaxseed oil (diet II), and the other received a diet enriched with conjugated linoleic acid (CLA) (diet III). Subsequent collections were undertaken after 25 and 50 days. Compared to the control samples, fish fed diet II showed an increase in concentrations of total fatty acid omega-3 (n-3) in the fillets, with an improvement ratio of n-6/n-3. The incorporation of CLA into fish fed diet III was successful. Supplementing the diet of Pimedolus maculatus with CLA and flaxseed oil led to improvements in nutritional quality and fatty acid profile of the fish fillets.

In-house method validation and occurrence of alpha-, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone and trifluralin residues in strawberry

A method for determination of organohalogen pesticides in strawberry by gas chromatography with electron capture detection was validated and applied in a monitoring program. Linearity, matrix effects, and day effect were evaluated for the analytes alpha-endosulfan, beta-endosulfan, endosulfan sulphate, lambda-cyhalothrin, procymidone, and trifluralin. The linear range varied according to the chromatographic response of the analyte. Significant matrix effects were observed. The mean recoveries ranged from 74.6 to 115.4%, with repeatability standard deviations between 1.6 and 21.0% and intermediate precision between 5.9 and 21.0%. Detection, quantification and decision limit, and detection capacity ranged from 0.003 to 0.007 mg/kg, 0.005 to 0.013 mg/kg; 0.003 to 3.128 mg/kg; and 0.005 to 3.266 mg/kg, respectively. The method was fit for the purpose of monitoring organohalogen residues in strawberries. Residues of these pesticides were detected in 124 of the 186 samples analyzed between 2009 and 2011 in the state of Minas Gerais. Nine of them did not comply with the current legislation requirements; among them, seven (3.8%) had residues of unauthorized pesticide for the culture of strawberry, one (0.5%) had residues above the maximum residue limit, and another one (0.5%) exhibited both non-conformities.