Some physico-chemical parameters that influence proteinase K resistance and the infectivity of PrP Sc after high pressure treatment

Crude brain homogenates of terminally diseased hamsters infected with the 263 K strain of scrapie (PrP Sc) were heated and/or pressurized at 800 MPa at 60ºC for different times (a few seconds or 5, 30, 120 min) in phosphate-buffered saline (PBS) of different pH and concentration. Prion proteins were analyzed on immunoblots for their proteinase K (PK) resistance, and in hamster bioassays for their infectivity. Samples pressurized under initially neutral conditions and containing native PrP Sc were negative on immunoblots after PK treatment, and a 6-7 log reduction of infectious units per gram was found when the samples were pressurized in PBS of pH 7.4 for 2 h. A pressure-induced change in the protein conformation of native PrP Sc may lead to less PK resistant and less infectious prions. However, opposite results were obtained after pressurizing native infectious prions at slightly acidic pH and in PBS of higher concentration. In this case an extensive fraction of native PrP Sc remained PK resistant after pressure treatment, indicating a protective effect possibly due to induced aggregation of prion proteins in such buffers.

Melatonin prevents inflammation and oxidative stress caused by abdominopelvic and total body irradiation of rat small intestine

We investigated the day-night differences in intestinal oxidative-injury and the inflammatory response following total body (TB) or abdominopelvic (AP) irradiation, and the influence of melatonin administration on tissue injury induced by radiation. Rats (male Wistar, weighing 220-280 g) in the irradiated groups were exposed to a dose of 8 Gy to the TB or AP region in the morning (resting period - 1 h after light onset) or evening (activity span - 13 h after light onset). Vehicle or melatonin was administered immediately before, immediately after and 24 h after irradiation (10, 2.0 and 10 mg/kg, ip, respectively) to the irradiated rats. AP (P < 0.05) and TB (P < 0.05) irradiation applied in the morning caused a significant increase in thiobarbituric acid reactive substance (TBARS) levels. Melatonin treatment in the morning (P < 0.05) or evening (P < 0.05) decreased TBARS levels after TB irradiation. After AP irradiation, melatonin treatment only in the morning caused a significant decrease in TBARS levels (P < 0.05). Although we have confirmed the development of inflammation after radiotherapy by histological findings, neither AP nor TB irradiation caused any marked changes in myeloperoxidase activity in the morning or evening. Our results indicate that oxidative damage is more prominent in rats receiving TB and AP irradiation in the morning and melatonin appears to have beneficial effects on oxidative damage irrespective of the time of administration. Increased neutrophil accumulation indicates that melatonin administration exerts a protective effect on AP irradiation-induced tissue oxidative injury, especially in the morning.

Pharmacology and toxicology of diphenyl diselenide in several biological models

The pharmacology of synthetic organoselenium compounds indicates that they can be used as antioxidants, enzyme inhibitors, neuroprotectors, anti-tumor and anti-infectious agents, and immunomodulators. In this review, we focus on the effects of diphenyl diselenide (DPDS) in various biological model organisms. DPDS possesses antioxidant activity, confirmed in several in vitro and in vivo systems, and thus has a protective effect against hepatic, renal and gastric injuries, in addition to its neuroprotective activity. The activity of the compound on the central nervous system has been studied since DPDS has lipophilic characteristics, increasing adenylyl cyclase activity and inhibiting glutamate and MK-801 binding to rat synaptic membranes. Systemic administration facilitates the formation of long-term object recognition memory in mice and has a protective effect against brain ischemia and on reserpine-induced orofacial dyskinesia in rats. On the other hand, DPDS may be toxic, mainly because of its interaction with thiol groups. In the yeast Saccharomyces cerevisiae, the molecule acts as a pro-oxidant by depleting free glutathione. Administration to mice during cadmium intoxication has the opposite effect, reducing oxidative stress in various tissues. DPDS is a potent inhibitor of d-aminolevulinate dehydratase and chronic exposure to high doses of this compound has central effects on mouse brain, as well as liver and renal toxicity. Genotoxicity of this compound has been assessed in bacteria, haploid and diploid yeast and in a tumor cell line.

Apolipoprotein E polymorphism in Brazilian dyslipidemic individuals: Ouro Preto study

The influence of apolipoprotein E alleles and genotypes on plasma lipid levels was determined in 185 individuals of mixed ethnicity living in Ouro Preto, Brazil. DNA was obtained from blood samples and the genotypes were determined by an RFLP-PCR procedure. The *3 allele was the most frequent (72%), followed by *4 (20%) and *2 (8%); *4 frequency was higher and *2 frequency was lower in the dyslipidemic group than in the normal control group. The *2 carriers presented lower LDL and total cholesterol levels compared to the *3 and *4 carriers. All six expected genotypes were observed in the individuals genotyped: E2/2 (2.1%), E4/4 (2.7%), E2/4 (3.7%), E2/3 (8.0%), E3/3 (53.3%), E3/4 (29.9%); no difference in genotype frequencies was found between the normal and dyslipidemic groups. Compared with *2, the presence of *3 increases more than two times the risk for dyslipidemia (OR = 2.31; P = 0.025; 95% CI = 1.06-5.06) and the presence of *4 increases it three times (OR = 3.31; P = 0.006; 95% CI = 1.36-8.04). The only significant effect of genotype was an increased risk for dyslipidemia in the *4 genotype carriers (E3/4 + E4/4) compared with the *2 genotype carriers (E2/2 + E2/3) with OR = 3.69 (95% CI = 1.25-10.88). The present study indicates that in the Ouro Preto admixed population the presence of APOE *2 can confer a protective effect, whereas the presence of APOE *4 implies an enhanced risk for dyslipidemia.

Attenuation of phosphamidon-induced oxidative stress and immune dysfunction in rats treated with N-acetylcysteine

The effect of N-acetylcysteine, a thiolic antioxidant, on attenuation of phosphamidon-induced oxidative stress and immune dysfunction was evaluated in adult male Wistar rats weighing 200-250 g. Rats were divided into four groups, 8 animals/group, and treated with phosphamidon, N-acetylcysteine or the combination of both for 28 days. Oral administration of phosphamidon (1.74 mg/kg), an organophosphate insecticide, increased serum malondialdehyde (3.83 ± 0.18 vs 2.91 ± 0.24 nmol/mL; P < 0.05) and decreased erythrocyte superoxide dismutase (567.8 ± 24.36 vs 749.16 ± 102.61 U/gHb; P < 0.05), catalase activity (1.86 ± 0.18 vs 2.43 ± 0.08 U/gHb; P < 0.05) and whole blood glutathione levels (1.25 ± 0.21 vs 2.28 ± 0.08 mg/gHb; P < 0.05) showing phosphamidon-induced oxidative stress. Phosphamidon exposure markedly suppressed humoral immune response as assessed by antibody titer to ovalbumin (4.71 ± 0.51 vs 8.00 ± 0.12 -log2; P < 0.05), and cell-mediated immune response as assessed by leukocyte migration inhibition (25.24 ± 1.04 vs 70.8 ± 1.09%; P < 0.05) and macrophage migration inhibition (20.38 ± 0.99 vs 67.16 ± 5.30%; P < 0.05) response. Phosphamidon exposure decreased IFN-у levels (40.7 ± 3.21 vs 55.84 ± 3.02 pg/mL; P < 0.05) suggesting a profound effect of phosphamidon on cell-mediated immune response. A phosphamidon-induced increase in TNF-α level (64.19 ± 6.0 vs 23.16 ± 4.0 pg/mL; P < 0.05) suggests a contributory role of immunocytes in oxidative stress. Co-administration of N-acetylcysteine (3.5 mmol/kg, orally) with phosphamidon attenuated the adverse effects of phosphamidon. These findings suggest that oral N-acetylcysteine treatment exerts protective effect and attenuates free radical injury and immune dysfunction caused by subchronic phosphamidon exposure.

Association between apolipoprotein E genotype, serum lipids, and colorectal cancer in Brazilian individuals

We evaluated genetic variants of apolipoprotein E (APOE HhaI) and their association with serum lipids in colorectal cancer (CRC), together with eating habits and personal history. Eight-seven adults with CRC and 73 controls were studied. APOE*2 (rs7412) and APOE*4 (rs429358) were identified by polymerase chain reaction-restriction fragment length polymorphism. APOE gene polymorphisms were similar in both groups, but the ε4/ε4 genotype (6%) was present only in controls. The patients had reduced levels (mean ± SD) of total cholesterol and low-density lipoprotein cholesterol fraction (180.4 ± 49.5 and 116.1 ± 43.1 mg/dL, respectively) compared to controls (204.2 ± 55.6, P = 0.135 and 134.7 ± 50.8 mg/dL; P = 0.330, respectively) indicating that they were not statistically significant after the Bonferroni correction. The APOE*4 allele was associated with lower levels of total cholesterol, low- and high-density lipoprotein cholesterol fraction and increased levels of very low-density lipoprotein cholesterol fraction and triglycerides only among patients (P = 0.014). There was a positive correlation between the altered lipid profile and increased body mass indexes in both groups (P < 0.010). Moreover, a higher rate of hypertension and overweight was observed in controls (P < 0.002). In conclusion, the presence of the ε4/ε4 genotype only in controls may be due to a protective effect against CRC. Lower lipid profile values among patients, even those on lipid-rich diets associated with the APOE*4 allele, suggest alterations in the lipid synthesis and metabolism pathways in CRC.

Clustering of psychiatric and somatic illnesses in the general population: multimorbidity and socioeconomic correlates

The distribution of psychiatric disorders and of chronic medical illnesses was studied in a population-based sample to determine whether these conditions co-occur in the same individual. A representative sample (N = 1464) of adults living in households was assessed by the Composite International Diagnostic Interview, version 1.1, as part of the São Paulo Epidemiological Catchment Area Study. The association of sociodemographic variables and psychological symptoms regarding medical illness multimorbidity (8 lifetime somatic conditions) and psychiatric multimorbidity (15 lifetime psychiatric disorders) was determined by negative binomial regression. A total of 1785 chronic medical conditions and 1163 psychiatric conditions were detected in the population concentrated in 34.1 and 20% of respondents, respectively. Subjects reporting more psychiatric disorders had more medical illnesses. Characteristics such as age range (35-59 years, risk ratio (RR) = 1.3, and more than 60 years, RR = 1.7), being separated (RR = 1.2), being a student (protective effect, RR = 0.7), being of low educational level (RR = 1.2) and being psychologically distressed (RR = 1.1) were determinants of medical conditions. Age (35-59 years, RR = 1.2, and more than 60 years, RR = 0.5), being retired (RR = 2.5), and being psychologically distressed (females, RR = 1.5, and males, RR = 1.4) were determinants of psychiatric disorders. In conclusion, psychological distress and some sociodemographic features such as age, marital status, occupational status, educational level, and gender are associated with psychiatric and medical multimorbidity. The distribution of both types of morbidity suggests the need of integrating mental health into general clinical settings.

Leukotrienes are not essential for the efficacy of a heterologous vaccine against Mycobacterium tuberculosis infection

Leukotrienes are reported to be potent proinflammatory mediators that play a role in the development of several inflammatory diseases such as asthma, rheumatoid arthritis and periodontal disease. Leukotrienes have also been associated with protection against infectious diseases. However, the role of leukotrienes in Mycobacterium tuberculosis infection is not understood. To answer this question, we studied the role of leukotrienes in the protective immune response conferred by prime-boost heterologous immunization against tuberculosis. We immunized BALB/c mice (4-11/group) with subcutaneous BCG vaccine (1 x 10(5) M. bovis BCG) (prime) followed by intramuscular DNA-HSP65 vaccine (100 µg) (boost). During the 30 days following the challenge, the animals were treated by gavage daily with MK-886 (5 mg·kg-1·day-1) to inhibit leukotriene synthesis. We showed that MK-886-treated mice were more susceptible to M. tuberculosis infection by counting the number of M. tuberculosis colony-forming units in lungs. The histopathological analysis showed an impaired influx of leukocytes to the lungs of MK-886-treated mice after infection, confirming the involvement of leukotrienes in the protective immune response against experimental tuberculosis. However, prime-boost-immunized mice treated with MK-886 remained protected after challenge with M. tuberculosis, suggesting that leukotrienes are not required for the protective effect elicited by immunization. Protection against M. tuberculosis challenge achieved by prime-boost immunization in the absence of leukotrienes was accompanied by an increase in IL-17 production in the lungs of these animals, as measured by ELISA. Therefore, these data suggest that the production of IL-17 in MK-886-treated, immunized mice could contribute to the generation of a protective immune response after infection with M. tuberculosis.

Ovariectomy does not affect the cardiac sympathovagal balance of female SHR but estradiol does

The low incidence of cardiovascular diseases, including hypertension, in premenopausal women has led to the conclusion that ovarian hormones may have a protective effect on the cardiovascular system. We evaluated the effects of ovariectomy and/or estradiol on sympathovagal balance and heart rate variability (HRV) in female spontaneously hypertensive rats (SHR) with tachycardia and compared them to Wistar rats (12 weeks old; N = 8-12). Ovariectomy (OVX) and/or estradiol (10 µg/kg) did not affect basal arterial pressure in either rat strain, but estradiol increased basal heart rate (HR) in OVX SHR (454 ± 18 vs 377 ± 9 bpm). HR changes elicited by methylatropine and propranolol were used to evaluate the sympathovagal balance. Ovariectomy did not affect the cardiac sympathovagal balance of any group, while estradiol increased sympathetic tone in OVX SHR (120 ± 8 vs 56 ± 10 bpm) and sham-operated Wistar rats (57 ± 7 vs 28 ± 4 bpm), and decreased the parasympathetic tone only in OVX SHR (26 ± 7 vs 37 ± 5 bpm). HRV was studied in the frequency domain (Fast Fourier Transformation). Spectra of HR series were examined at low frequency (LF: 0.2-0.75 Hz) and high frequency (HF: 0.75-3 Hz) bands. The power of LF, as well as the LF/HF ratio, was not affected by ovariectomy, but estradiol increased both LF (29 ± 4 vs 18 ± 3 nu in Wistar sham-operated, 26 ± 5 vs 15 ± 3 nu in Wistar OVX, 50 ± 3 vs 38 ± 4 nu in SHR sham-operated, and 51 ± 3 vs 42 ± 3 nu in SHR OVX) and LF/HF (0.48 ± 0.08 vs 0.23 ± 0.03 nu in Wistar sham-operated, 0.41 ± 0.14 vs 0.19 ± 0.05 nu in Wistar OVX, 0.98 ± 0.11 vs 0.63 ± 0.11 nu in SHR sham-operated, and 1.10 ± 0.11 vs 0.78 ± 0.1 nu in SHR OVX). Thus, we suggest that ovariectomy did not affect the cardiac sympathovagal balance of SHR or Wistar rats, while estradiol increased the sympathetic modulation of HR.

CHRNA5 polymorphism and susceptibility to lung cancer in a Chinese population

Polymorphisms in the nicotinic acetylcholine receptor subunit CHRNA5 gene have been associated with lung cancer positive susceptibility in European and American populations. In the present hospital-based, case-control study, we determined whether polymorphism in rs503464 of CHRNA5 is associated with lung cancer risk in Chinese individuals. A single nucleotide polymorphism in CHRNA5 rs503464, c.-166T>A (hereafter T>A), was identified using TaqMan-MGB probes with sequencing via PCR in 600 lung cancer cases and 600 healthy individuals. Genotype frequencies for rs503464 (T>A) were in Hardy-Weinberg equilibrium for the control population. However, genotype frequencies were significantly different between cases and controls (P < 0.05), while allele frequencies were not significantly different between groups. Compared to homozygous genotypes (TT or AA), the risk of lung cancer in those with the heterozygous genotype (TA) was significantly lower (OR = 0.611, 95%CI = 0.486-0.768, P = 0.001). Using genotype AA as a reference, the risk of lung cancer for those with genotype TA was increased 1.5 times (OR = 1.496, 95%CI = 1.120-1.997, P = 0.006). However, no difference in risk was observed between T allele carriers and A allele carriers (OR = 0.914, 95%CI = 0.779-1.073, P = 0.270). Stratification analysis showed that the protective effect of TA was more pronounced in those younger than 60 years, nonsmokers, or those without a family history of cancer, as well as in patients with adenocarcinoma or squamous cell carcinoma in clinical stages III or IV (P < 0.05). Therefore, the heterozygous genotype c.-166T>A at rs503464 of CHRNA5 may be associated with reduced risk of lung cancer, thus representing a susceptibility allele in Chinese individuals.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and ofXpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.

The hydrogen sulfide donor, Lawesson's reagent, prevents alendronate-induced gastric damage in rats

Our objective was to investigate the protective effect of Lawesson's reagent, an H2S donor, against alendronate (ALD)-induced gastric damage in rats. Rats were pretreated with saline or Lawesson's reagent (3, 9, or 27 µmol/kg, po) once daily for 4 days. After 30 min, gastric damage was induced by ALD (30 mg/kg) administration by gavage. On the last day of treatment, the animals were killed 4 h after ALD administration. Gastric lesions were measured using a computer planimetry program, and gastric corpus pieces were assayed for malondialdehyde (MDA), glutathione (GSH), proinflammatory cytokines [tumor necrosis factor (TNF)-α and interleukin (IL)-1β], and myeloperoxidase (MPO). Other groups were pretreated with glibenclamide (5 mg/kg, ip) or with glibenclamide (5 mg/kg, ip)+diazoxide (3 mg/kg,ip). After 1 h, 27 µmol/kg Lawesson's reagent was administered. After 30 min, 30 mg/kg ALD was administered. ALD caused gastric damage (63.35±9.8 mm2); increased levels of TNF-α, IL-1β, and MDA (2311±302.3 pg/mL, 901.9±106.2 pg/mL, 121.1±4.3 nmol/g, respectively); increased MPO activity (26.1±3.8 U/mg); and reduced GSH levels (180.3±21.9 µg/g). ALD also increased cystathionine-γ-lyase immunoreactivity in the gastric mucosa. Pretreatment with Lawesson's reagent (27 µmol/kg) attenuated ALD-mediated gastric damage (15.77±5.3 mm2); reduced TNF-α, IL-1β, and MDA formation (1502±150.2 pg/mL, 632.3±43.4 pg/mL, 78.4±7.6 nmol/g, respectively); lowered MPO activity (11.7±2.8 U/mg); and increased the level of GSH in the gastric tissue (397.9±40.2 µg/g). Glibenclamide alone reversed the gastric protective effect of Lawesson's reagent. However, glibenclamide plus diazoxide did not alter the effects of Lawesson's reagent. Our results suggest that Lawesson's reagent plays a protective role against ALD-induced gastric damage through mechanisms that depend at least in part on activation of ATP-sensitive potassium (KATP) channels.

Therapeutic concentration of morphine reduces oxidative stress in glioma cell line

Morphine is a potent analgesic opioid used extensively for pain treatment. During the last decade, global consumption grew more than 4-fold. However, molecular mechanisms elicited by morphine are not totally understood. Thus, a growing literature indicates that there are additional actions to the analgesic effect. Previous studies about morphine and oxidative stress are controversial and used concentrations outside the range of clinical practice. Therefore, in this study, we hypothesized that a therapeutic concentration of morphine (1 μM) would show a protective effect in a traditional model of oxidative stress. We exposed the C6 glioma cell line to hydrogen peroxide (H2O2) and/or morphine for 24 h and evaluated cell viability, lipid peroxidation, and levels of sulfhydryl groups (an indicator of the redox state of the cell). Morphine did not prevent the decrease in cell viability provoked by H2O2 but partially prevented lipid peroxidation caused by 0.0025% H2O2 (a concentration allowing more than 90% cell viability). Interestingly, this opioid did not alter the increased levels of sulfhydryl groups produced by exposure to 0.0025% H2O2, opening the possibility that alternative molecular mechanisms (a direct scavenging activity or the inhibition of NAPDH oxidase) may explain the protective effect registered in the lipid peroxidation assay. Our results demonstrate, for the first time, that morphine in usual analgesic doses may contribute to minimizing oxidative stress in cells of glial origin. This study supports the importance of employing concentrations similar to those used in clinical practice for a better approximation between experimental models and the clinical setting.

Relationship between deep venous thrombosis and inflammatory cytokines in postoperative patients with malignant abdominal tumors

Deep venous thrombosis (DVT) is a common surgical complication in cancer patients and evidence that inflammation plays a role in the occurrence of DVT is increasing. We studied a population of cancer patients with abdominal malignancies with the aim of investigating whether the levels of circulating inflammatory cytokines were associated with postoperative DVT, and to determine the levels in DVT diagnoses. The serum levels of C-reactive protein (CRP), interleukins (IL)-6 and IL-10, nuclear transcription factor-κB (NF-κB) and E-selectin (E-Sel) were determined in 120 individuals, who were divided into 3 groups: healthy controls, patients with and patients without DVT after surgery for an abdominal malignancy. Data were analyzed by ANOVA, Dunnet's T3 test, chi-square test, and univariate and multivariate logistic regression as needed. The CRP, IL-6, NF-κB, and E-Sel levels in patients with DVT were significantly higher than those in the other groups (P<0.05). The IL-10 level was higher in patients with DVT than in controls but lower than in patients without DVT. Univariate analysis revealed that CRP, IL-6, NF-κB, and E-Sel were statistically associated with the risk of DVT (OR=1.98, P=0.002; OR=1.17, P=0.000; OR=1.03, P=0.042; and OR=1.38, P=0.003; respectively), whereas IL-10 had a protective effect (OR=0.94, P=0.011). Multivariate analysis showed that E-Sel was an independent risk factor (OR=1.41, P=0.000). Thus, this study indicated that an increased serum level of E-Sel was associated with increased DVT risk in postoperative patients with abdominal malignancy, indicating that E-Sel may be a useful predictor of diagnosis of DVT.

Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C2H2 zinc finger protein

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C2H2 motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.