Autolytic Mycobacterium leprae Hsp65 fragments may act as biological markers for autoimmune diseases

Investigating the proteolytic activity of the recombinant Mycobacterium leprae Heat Shock Protein of 65 kDa (rHsp65), chaperonin 2 (cpn2), we observed that it displays high instability. The fragmentation process starts at the C-terminus followed by progressive degradation of the N-terminus, which leads to a stable fragment comprising the middle region of the molecule. Urea was able to prevent autolysis, probably due to its denaturing action, while EDTA increased degradation levels indicating the need for metal ions. Peptides originated from autolysis were purified and analyzed by mass spectrometry, generating a continuous map. Since the bacteria and mammalian Hsp60 are known to be targets of the immune response and have been implicated in autoimmune diseases and chronic inflammation, the in vivo effect of rHsp65 peptides was evaluated in the spontaneous Systemic Lupus Erythematosus (SLE) model developed by the (NZB/NZW)F(1) mouse hybrids, and their individual anti-rHsp65 IgG2a/IgG1 antibody titer ratio was determined. The results showed orientation toward a T(H)1 responsiveness, and the treatment with the rHsp65 peptides diminished the environmental variance of the survival time of treated animals. These results outline the fact that environmental factors may also act through the modified stability expression of Heat Shock Proteins intervening during autoimmune processes. (C) 2011 Elsevier Ltd. All rights reserved.

XRCC1 haploinsufficiency in mice has little effect on aging, but adversely modifies exposure-dependent susceptibility

Oxidative DNA damage plays a role in disease development and the aging process. A prominent participant in orchestrating the repair of oxidative DNA damage, particularly single-strand breaks, is the scaffold protein XRCC1. A series of chronological and biological aging parameters in XRCC1 heterozygous (HZ) mice were examined. HZ and wild-type (WT) C57BL/6 mice exhibit a similar median lifespan of similar to 26 months and a nearly identical maximal life expectancy of similar to 37 months. However, a number of HZ animals (7 of 92) showed a propensity for abdominal organ rupture, which may stem from developmental abnormalities given the prominent role of XRCC1 in endoderm and mesoderm formation. For other end-points evaluated-weight, fat composition, blood chemistries, condition of major organs, tissues and relevant cell types, behavior, brain volume and function, and chromosome and telomere integrity-HZ mice exhibited by-and-large a normal phenotype. Treatment of animals with the alkylating agent azoxymethane resulted in both liver toxicity and an increased incidence of precancerous lesions in the colon of HZ mice. Our study indicates that XRCC1 haploinsufficiency in mammals has little effect on chronological longevity and many key biological markers of aging in the absence of environmental challenges, but may adversely affect normal animal development or increase disease susceptibility to a relevant genotoxic exposure.

Underreporting of Energy Intake in Brazilian Women Varies According to Dietary Assessment: A Cross-Sectional Study Using Doubly Labeled Water

Objective Underreporting of energy intake is prevalent in food surveys, but there is controversy about which dietary assessment method provides greater underreporting rates. Our objective is to compare validity of self-reported energy intake obtained by three dietary assessment methods with total energy expenditure (TEE) obtained by doubly labeled water (DLW) among Brazilian women. Design We used a cross-sectional study. Subjects/setting Sixty-five females aged 18 to 57 years (28 normal-weight, 10 over-weight, and 27 obese) were recruited from two universities to participate. Main outcome measures TEE determined by DLW, energy intake estimated by three 24-hour recalls, 3-day food record, and a food frequency questionnaire (FFQ). Statistical analyses performed Regression and analysis of variance with repeated measures compared TEE and energy intake values, and energy intake-to-TEE ratios and energy intake-TEE values between dietary assessment methods. Bland and Altman plots were provided for each method. chi(2) test compared proportion of underreporters between the methods. Results Mean TEE was 2,622 kcal (standard deviation [SD] =490 kcal), while mean energy intake was 2,078 kcal (SD=430 kcal) for the diet recalls; 2,044 kcal (SD=479 kcal) for the food record and 1,984 kcal (SD=832 kcal) for the FFQ (all energy intake values significantly differed from TEE; P<0.0001). Bland and Altman plots indicated great dispersion, negative mean differences between measurements, and wide limits of agreement. Obese subjects underreported more than normal-weight subjects in the diet recalls and in the food records, but not in the FFQ. Years of education, income and ethnicity were associated with reporting accuracy. Conclusions The FFQ produced greater under- and overestimation of energy intake. Underreporting of energy intake is a serious and prevalent error in dietary self-reports provided by Brazilian women, as has been described in studies conducted in developed countries.

P-Glycoprotein Expression, Tumor Weight, Age, and Relapse in Patients with Stage I and II Favorable-Histology Wilms` Tumor

Fifteen percent of patients with Wilms`` tumor (WT) experience relapse. It has been suggested that weight and age may affect the chances of relapse. Few studies have investigated the role, if any, between P-glycoprotein (P-gp) and relapse. The authors assessed the prognostic value of tumor weight and age at diagnosis and asked whether some other potential biological markers, specifically P-gp protein expression, had a prognostic value in favorable-histology WT. No association between age and relapse could be found. Patients with tumor weight >= a parts per thousand yen550 g were 6 times more likely to relapse, whereas P-gp expression was positive in 18/40 (45%%) of the patients, of which 10/12 (83.3%%) relapsed and 8/28 (28.6%%) did not. Further studies are necessary to elucidate whether or not P-gp is related to relapse in patients with histologically favorable Wilms`` tumor. If confirmed, the protein may be used in the future as a target for new drugs and treatments for this group of patients.

Demographic characteristics and prevalence of serologic markers among blood donors who use confidential unit exclusion (CUE) in Sao Paulo, Brazil: implications for modification of CUE polices in Brazil

BACKGROUND: This study evaluated demographic profiles and prevalence of serologic markers among donors who used confidential unit exclusion (CUE) to assess the effectiveness of CUE and guide public policies regarding the use of CUE for enhancing safety versus jeopardizing the blood supply by dropping CUE. STUDY DESIGN AND METHODS: We conducted a cross-sectional analysis of whole blood donations at a large public blood center in Sao Paulo from July 2007 through June 2009, compared demographic data, and confirmed serologic results among donors who used and who have never used CUE (CUE never). RESULTS: There were 265,550 whole blood units collected from 181,418 donors from July 2007 through June 2009. A total of 9658 (3.6%) units were discarded, 2973 (1.1%) because CUE was used at the current donation (CUE now) and 6685 (2.5%) because CUE was used in the past (CUE past). The CUE rate was highest among donors with less than 8 years of education (odds ratio [OR], 2.78; 95% confidence interval [CI], 2.51-3.08). CUE now donations were associated with higher positive infectious disease marker rates than CUE never donations (OR, 1.41; CI, 1.13-1.77), whereas CUE past donations were not (OR, 1.04; CI, 0.75-1.45). CONCLUSION: The CUE process results in a high rate of unit discard. CUE use on an individual donation appears predictive of a high-risk marker-positive donation and, thus, appears to contribute modestly to blood safety. The policy of discarding units from donors who have previously CUE-positive donations does not improve safety and should be discontinued.

Prevalence of hepatitis B and C serological markers among first-time blood donors in Brazil: A multi-center serosurvey

Little data are available on the seroprevalence of, and risk factors for hepatitis B and C viruses (HBV and HCV) infection in Latin American countries. A multi-center serosurvey was conducted among 3,598 first-time blood donors (65% men) from Sao Paulo, Salvador and Manaus in Brazil. The gender-specific seroprevalences of antibodies against hepatitis B core antigen (anti-HBc) and of the hepatitis B surface antigen (HBsAg) in anti-HBc-positive sera were measured, and risk factors analyzed by gender. The gender-specific seroprevalences of antibodies against HCV (anti-HCV) were measured, but risk factors for HCV were not determined. Anti-HBc and HBsAg seroprevalences were not significantly different in men [101/2,341 (4.31%) and 4/2,229 (0.18%), respectively] and women [65/1,237 (5.25%) and 8/ 1,169 (0.68%), respectively], whereas the seroprevalence of anti-HCV was higher in women (12/1,238 [0.97%] vs. 9/2,353 [0.38%]; odds ratio [OR] = 2.49; 95% confidence interval [Cl]: 1.0-6.0). No significant difference for HBV infection was found across the three study sites or by ethnic group. The seroprevalence of anti-HBc increased with age, but decreased with education level in both genders. Lifetime number of sexual partners was associated with anti-HBc prevalence among men (OR = 1.95; 95% Cl: 1.2-3.1), but not women. The seroprevalence of HBV and HCV was low among Brazilian blood donors, and exposure increased with age in both genders.

Melatonin and IP(3)-induced Ca(2+) Release from Intracellular Stores in the Malaria Parasite Plasmodium falciparum within Infected Red Blood Cells

IP(3)-dependent Ca(2+) signaling controls a myriad of cellular processes in higher eukaryotes and similar signaling pathways are evolutionarily conserved in Plasmodium, the intracellular parasite that causes malaria. We have reported that isolated, permeabilized Plasmodium chabaudi, releases Ca(2+) upon addition of exogenous IP(3). In the present study, we investigated whether the IP(3) signaling pathway operates in intact Plasmodium falciparum, the major disease-causing human malaria parasite. P. falciparum-infected red blood cells (RBCs) in the trophozoite stage were simultaneously loaded with the Ca(2+) indicator Fluo-4/AM and caged-IP(3). Photolytic release of IP(3) elicited a transient Ca(2+) increase in the cytosol of the intact parasite within the RBC. The intracellular Ca(2+) pools of the parasite were selectively discharged, using thapsigargin to deplete endoplasmic reticulum (ER) Ca(2+) and the antimalarial chloroquine to deplete Ca(2+) from acidocalcisomes. These data show that the ER is the major IP(3)-sensitive Ca(2+) store. Previous work has shown that the human host hormone melatonin regulates P. falciparum cell cycle via a Ca(2+)-dependent pathway. In the present study, we demonstrate that melatonin increases inositol-polyphosphate production in intact intraerythrocytic parasite. Moreover, the Ca(2+) responses to melatonin and uncaging of IP(3) were mutually exclusive in infected RBCs. Taken together these data provide evidence that melatonin activates PLC to generate IP(3) and open ER-localized IP(3)-sensitive Ca(2+) channels in P. falciparum. This receptor signaling pathway is likely to be involved in the regulation and synchronization of parasite cell cycle progression.

Investigation of whole blood of SJL/J mice using neutron activation analysis

The Br (0.0022 +/- A 0.0006 gL(-1)), Ca (0.113 +/- A 0.012 gL(-1)), Cl (3.07 +/- A 0.36 gL(-1)), K (2.63 +/- A 0.14 gL(-1)), Mg (0.045 +/- A 0.002 gL(-1)) and Na (2.09 +/- A 0.10 gL(-1)) concentrations were determined in whole blood of SJL/J mice using the Neutron Activation Analysis (NAA) technique. Eleven whole blood samples were analyzed in the IEA-R1 nuclear reactor at IPEN (So Paulo, Brazil). These data contribute for applications in veterinary medicine related to biochemistry analyses using whole blood. Moreover, the correlation with human blood estimation allows to checking the similarities for studying muscular dystrophy using this model animal.

Thyroid hormones alter the adenine nucleotide hydrolysis in adult rat blood serum

The effects of ATP, ADP, and adenosine in the processes of platelet aggregation, vasodilatation, and coronary flow have been known for many years. The sequential hydrolysis of ATP to adenosine by soluble nucleotidases constitutes the main system for rapid inactivation of circulating adenine nucleotides. Thyroid disorders affect a number of biological factors including adenosine levels in different fractions. Then, we intend to investigate if the soluble nucleotidases responsible for the ATP, ADP, and AMP hydrolysis are affected by variations in the thyroid hormone levels in blood serum from adult rats. Hyperthyroidism was induced by daily intraperitoneal injections of L-thyroxine (T4) (2.5 and 10.0 mu g/100 g body weight, respectively) for 7 or 14 days. Hypothyroidism was induced by thyroidectomy and methimazole (0.05%) added to their drinking water during 7 or 14 days. The treatments efficacy was confirmed by determination of hemodynamic parameters and cardiac hypertrophy evaluation. T4 treatment predominantly inhibited, and hypothyroidism (14 days after thyroidectomy) predominantly increased the ATP, ADP, and AMP hydrolysis in rat blood serum. These results suggest that both excess and deficiency of thyroid hormones can modulate the ATP diphosphohydrolase and 5`-nucleotidase activities in rat blood serum and consequently modulate the effects mediated by these enzymes and their products in vascular system. (C) 2010 International Union of Biochemistry and Molecular Biology, Inc.

Effect of short-term creatine supplementation on markers of skeletal muscle damage after strenuous contractile activity

The protective effect of short-term creatine supplementation (CrS) upon markers of strenuous contractile activity-induced damage in human and rat skeletal muscles was investigated. Eight Ironman triathletes were randomized into the placebo (Pl; n = 4) and creatine-supplemented (CrS; n = 4) groups. Five days prior to the Ironman competition, the CrS group received creatine monohydrate (20 g day(-1)) plus maltodextrin (50 g) divided in two equal doses. The Pl group received maltodextrin (50 g day(-1)) only. The effect of CrS (5 g day(-1)/kg body weight for 5 days) was also evaluated in a protocol of strenuous contractile activity induced by electrical stimulation in rats. Blood samples were collected before and 36 and 60 h after the competition and were used to determine plasma activities of creatine kinase (CK), lactate dehydrogenase (LDH), aldolase (ALD), glutamic oxaloacetic acid transaminase (GOT), glutamic pyruvic acid transaminase (GPT), and C-reactive protein (CRP) level. In rats, plasma activities of CK and LDH, muscle vascular permeability (MVP) using Evans blue dye, muscle force and fatigue were evaluated. Activities of CK, ALD, LDH, GOT, GTP, and levels of CRP were increased in the Pl group after the competition as compared to basal values. CrS decreased plasma activities of CK, LDH, and ALD, and prevented the rise of GOT and GPT plasma activities. In rats, CrS delayed the fatigue, preserved the force, and prevented the rise of LDH and CK plasma activities and MVP in the gastrocnemius muscle. CrS presented a protective effect on muscle injury induced by strenuous contractile activities.

Effects of Low-Level Laser Therapy (LLLT) in the Development of Exercise-Induced Skeletal Muscle Fatigue and Changes in Biochemical Markers Related to Postexercise Recovery

STUDY DESIGN: Randomized crossover double-blinded placebo-controlled trial. OBJECTIVE: To investigate if low-level laser therapy (LLLT) can affect biceps muscle performance, fatigue development, and biochemical markers of postexercise recovery. BACKGROUND: Cell and animal studies have suggested that LLLT can reduce oxidative stress and inflammatory responses in muscle tissue. But it remains uncertain whether these findings can translate into humans in sport and exercise situations. METHODS: Nine healthy male volleyball players participated in the study. They received either active LLLT (cluster probe with 5 laser diodes; A = 810 nm; 200 mW power output; 30 seconds of irradiation, applied in 2 locations over the biceps of the nondominant arm; 60 J of total energy) or placebo LLLT using an identical cluster probe. The intervention or placebo were applied 3 minutes before the performance of exercise. All subjects performed voluntary elbow flexion repetitions with a workload of 75% of their maximal voluntary contraction force until exhaustion. RESULTS: Active LLLT increased the number of repetitions by 14.5% (mean +/- SD, 39.6 +/- 4.3 versus 34.6 +/- 5.6; P = .037) and the elapsed time before exhaustion by 8.0% (P = .034), when compared to the placebo treatment. The biochemical markers also indicated that recovery may be positively affected by LLLT, as indicated by postexercise blood lactate levels (P<.01), creatine kinase activity (P = .017), and C-reactive protein levels (P = .047), showing a faster recovery with LLLT application prior to the exercise. CONCLUSION: We conclude that pre-exercise irradiation of the biceps with an LLLT dose of 6 J per application location, applied in 2 locations, increased endurance for repeated elbow flexion against resistance and decreased postexercise levels of blood lactate, creatine kinase, and C-reactive protein. LEVEL OF EVIDENCE: Performance enhancement, level 1b. J Orthop Sports Phys Ther 2010;40(8):524-532. doi:10.2519/jospt.2010.3294

Effect of low-level laser therapy (GaAs 904 nm) in skeletal muscle fatigue and biochemical markers of muscle damage in rats

We wanted to test if pre-exercise muscle irradiation with 904 nm laser affects the development of fatigue, blood lactate levels and creatine kinase (CK) activity in a rat model with tetanic contractions. Thirty male Wistar rats were divided into five groups receiving either one of four different laser doses (0.1, 0.3, 1.0 and 3.0 J) or a no-treatment control group. Laser irradiation was performed immediately before the first contraction for treated groups. Electrical stimulation was used to induce six tetanic tibial anterior muscle contractions with 10 min intervals between them. Contractions were stopped when the muscle force fell to 50% of the peak value for each contraction; blood samples were taken before the first and immediately after the sixth contraction. The relative peak forces for the sixth contraction were significantly better (P < 0.05) in the two laser groups irradiated with highest doses [151.27% (SD +/- A 18.82) for 1.0 J, 144.84% (SD +/- A 34.47) for 3.0 J and 82.25% (SD +/- A 11.69) for the control group]. Similar significant (P < 0.05) increases in mean performed work during the sixth contraction for the 1.0 and 3.0 J groups were also observed. Blood lactate levels were significantly lower (P < 0.05) than the control group in all irradiated groups. All irradiated groups except the 3.0 J group had significantly lower post-exercise CK activity than the control group. We conclude that pre-exercise irradiation with a laser dose of 1.0 J and 904 nm wavelength significantly delays muscle fatigue and decreases post-exercise blood lactate and CK in this rat model.

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in many experimental models using phagocytic and non-phagocytic cells. Currently, there is some controversy about the efficacy of apocynin in non-phagocytic cells, but in phagocytes the reported results are consistent, which could be due to the presence of myeloperoxidase in these cells. This enzyme has been proposed as responsible for activating apocynin by generating its dimer, diapocynin, which is supposed to be the active compound that prevents NADPH oxidase complex assembly and activation. Here, we synthesized diapocynin and studied its effect on inhibition of gp91(phox) RNA expression. We found that diapocynin strongly inhibited the expression of gp91(phox)mRNA in peripheral blood mononuclear cells (PBMC). Only at a higher concentration, apocynin was able to exert the same effect. We also compared the apocynin and diapocynin efficacy as inhibitors of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production in response to lipopolysaccharide (LPS)-activated PBMC. Although apocynin did inhibit TNF-alpha production, diapocynin had a much more pronounced effect, on both TNF-alpha and IL-10 production. In conclusion, these findings suggest that the bioconversion of apocynin to diapocynin is an important issue not limited to enzymatic activity inhibition, but also for other biological effects as gp91(phox) mRNA expression and cytokine production. Hence, as diapocynin can be easily prepared from apocynin, a one-step synthesis, we recommend its use in studies where the biological effects of apocynin are searched. (C) 2010 Elsevier Inc. All rights reserved.

Shape and size of red blood cells from the Pygoscelid penguins of Antarctica using atomic force microscopy

Antarctic biodiversity is evolutionarily complex, reflecting the extreme ambient conditions. Therefore, Antarctic organisms exhibit sophisticated adaptations in all organization levels, including organs, tissues, and cells. Since red blood cells (RBCs) travel through the vertebrates blood delivering O(2) to all tissues and organs and purging the unwanted CO(2), they represent an interesting model to investigate biological adaptations. We have used atomic force microscopy (AFM) to compare the shape and size of RBCs of the Pygoscelid penguins. A total of 18 landmarks were measured in AFM images. When analyzed individually, the parameters were not capable of discriminating the RBCs of each species. However, the simultaneous use of multiple parameters discriminated (74%) among the RBCs. In addition, the use of RBC measurements was sufficient to hierarchically cluster the species in accordance to other common and reliable phylogenetic strategies. In light of these results, the use of RBC characters could effectively benefit taxonomic inferences.

Early Increase in Autoantibodies Against Human Oxidized Low-Density Lipoprotein in Hypertensive Patients After Blood Pressure Control

BACKGROUND Oxidized lipoproteins and antioxidized low-density lipoprotein (anti-oxLDL) antibodies (Abs) have been detected in plasma in response to blood pressure (BP) elevation, suggesting the participation of the adaptive immune system. Therefore, treatment of hypertension may act on the immune response by decreasing oxidation stimuli. However, this issue has not been addressed. Thus, we have here analyzed anti-oxLDL Abs in untreated (naive) hypertensive patients shortly after initiation of anti hypertensive therapeutic regimens. METHODS Titers of anti-oxLDL Abs were measured in subjects with recently diagnosed hypertension on stage 1 (n = 94), in primary prevention of coronary disease, with no other risk factors, and naive of anti hypertensive medication at entry. Subjects were randomly assigned to receive perindopril, hydrochlorothiazide (HCTZ), or indapamide (INDA) for 12 weeks, with additional perindopril if necessary to achieve BP control. Abs against copper-oxidized LDL were measured by enzyme-linked immunosorbent assay. RESULTS Twelve-week antihypertensive treatment reduced both office-based and 24-h ambulatory BP measurements (P < 0.0005). The decrease in BP was accompanied by reduction in thiobarbituric acid-reactive substances (TBARS) (P < 0.05), increase in anti-oxLDL Ab titers (P < 0.005), and improvement in flow-mediated dilation (FMD) (P < 0.0005), independently of treatment. Although BP was reduced, we observed favorable changes in anti-oxLDL titers and FMD. CONCLUSIONS We observed that anti-oxLDL Ab titers increase after antihypertensive therapy in primary prevention when achieving BP targets. Our results are in agreement with the concept that propensity to oxidation is increased by essential hypertension and anti-oxLDL Abs may be protective and potential biomarkers for the follow-up of hypertension treatment.