Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response

Trypanosoma cruzi is a protozoan parasite and the causative agent of Chagas disease. Like most living organisms, it is susceptible to oxidative stress, and must adapt to distinct environments. Hence, DNA repair is essential for its survival and the persistence of infection. Therefore, we studied whether T. cruzi has a homolog counterpart of the MutY enzyme (TcMYH), important in the DNA Base Excision Repair (BER) mechanism. Analysis of T. cruzi genome database showed that this parasite has a putative MutY DNA glycosylase sequence. We performed heterologous complementation assays using this genomic sequence. TcMYH complemented the Escherichia coli MutY- strain, reducing the mutation rate to a level similar to wild type. In in vitro assays, TcMYH was able to remove an adenine that was opposite to 8-oxoguanine. We have also constructed a T. cruzi lineage that overexpresses MYH. Although in standard conditions this lineage has similar growth to control cells, the overexpressor is more sensitive to hydrogen peroxide and glucose oxidase than the control, probably due to accumulation of AP sites in its DNA. Localization experiments with GFP-fused TcMYH showed this enzyme is present in both nucleus and mitochondrion. QPCR and MtOX results reinforce the presence and function of TcMYH in these two organelles. Our data suggest T. cruzi has a functional MYH DNA glycosylase, which participates in nuclear and mitochondrial DNA Base Excision Repair.     

马棒状杆菌（L型）的表型及分子生物学的研究

目的探讨引起败血症的Ｌ型马棒状杆菌的致病性和有效的抗菌药物及制备可疑Ｌ型特异诊断血清。方法对临床诊断为败血症的患者中共分离到１３例１７株Ｌ型马棒状杆菌，并对该菌进行了返祖诱导、生化反应、药物敏感实验及ＤＮＡＧ＋Ｃｍｏｌ％比值测定，确认分离菌株为马棒状杆菌Ｌ型。对该菌的致病毒力因子进行了动物灌胃及小鼠肠段结扎、基因探针检测。结果提示马棒状杆菌的致病毒力因子为ＳＴ和ＬＴ。不含溶血素及ＣＴ肠毒素。研究中制备的免疫血清皆与返祖菌的原代和次代发生凝集反应。结论返祖菌原代和次代具有同其亲代细菌一致的表面抗原，为制备Ｌ型特异性诊断血清建立对Ｌ型感染的快速、特异性诊断方法具有重要意义。     

Synthesis and in vitro antimalarial activity of tetraoxane-amine/amide conjugates

A series of tetraoxanes, tetraoxane-amine and tetraoxane-amide conjugates have been synthesized and screened for in vitro antimalarial activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum. Most of the conjugates showed slightly better antimalarial activity than the parent tetraoxanes. Three of the conjugate compounds were potentially active with IC₅₀ values in the range of 0.38-0.80 μM. Cytotoxicity of four selected compounds was also evaluated in a panel of four cancer (SK-MEL, KB, BT-549, SK-OV-3) and two non-cancer (Vero and LLC-PK₁₁) cell lines up to a concentration of 25 μM and none of the compounds was found toxic to any of the cells.     

In vivo and in vitro evidences that carotenoids could modulate the neutrophil respiratory burst during dietary manipulation

Summary Background The primary role of polymorphonuclear neutrophils (PMNs) is to destroy pathogenic microorganisms after phagocytosis by producing reactive oxygen species (ROS) and toxic molecules. However, PMNs produce sufficient amounts of ROS during an oxidative burst to be autotoxic and detrimental to their own functions and to possibly cause DNA damage, protein and lipid oxidation and cell membrane destructuration. Objective The aim of this study was to investigate in vivo the role of the antioxidant capacities of carotenoids in modulating ROS content in PMNs during oxidative burst. Moreover to investigate the direct or indirect effect of carotenoids, the modification of PMN ROS content was explored after in vitro supplementation with -carotene or lycopene, chosen taking account of their vitamin A and no vitamin A precursor effect, respectively. Design In vivo study: Venous blood was collected from 10 healthy male volunteers and ROS production from phorbol myristate acetate (PMA)-stimulated PMNs was determined, by flow cytometry using the fluorescent dye dihydrorhodamine 123, at baseline, after 3 weeks of carotenoid depletion (carotenoid intake limited to 25% of usual intake) and after 5 weeks of carotenoid repletion (30 mg -carotene, 15 mg lycopene and 9mg lutein per day). In vitro study: ROS content in PMA-stimulated PMNs isolated from carotenoid depleted subjects and controls was quantified after an in vitro enrichment with -carotene (1 µmol/L) or lycopene (0.3 µmol/L). Results In vivo carotenoid depletion increased PMN H₂O₂ content after PMA activation by 38% (p < 0.05 vs baseline),while supplementation for 5 weeks restored basal H₂O₂ generation (p < 0.05 vs depletion). Although H₂O₂ measurement in PMNs from non-depleted subjects was not affected by an in vitro supply with -carotene or lycopene, a significant decrease in H₂O₂ content by 78.9 % and 81.2%, respectively, was observed in PMNs from carotenoid depleted subjects (p < 0.01 vs depleted control subjects). Conclusions The carotenoid ROS quenching capacities control both in vivo and in vitro the PMNs ROS generation and probably protect these cells against DNA, membrane lipid and protein damages during oxidative burst. Moreover, these effects appear independent from the metabolic conversion of carotenoids to vitamin A.     

Measurement of erythrocyte arginase activity and the effect of lead on the activity in vitro

Measurement of arginase activity made by varying the arginine concentration and the reaction time clarified that the amount of urea formed by arginase peaked at the substrate concentration of 150 mmol/l, and increased linearly by 20 minutes. By using discs punched out from a dried blood filter paper prepared from identical human blood sample, the amount of formed urea showed linear increase up to 160 U/g.Hb. The coefficients of variation in ten measurements were 6.1% for the mean activity of 46.2 U/g.Hb and 2.1% for 152.1 U/g.Hb. No decreases in arginase activity were observed when two different dried blood filter papers were left for one week at room temperature. Intact erythrocytes were treated with various concentrations of lead from 0 to 5 mumols/l and their arginase activities were measured. The activity did not elevate linearly with the rise of lead concentration, but an almost diphasic elevation was observed. The arginase activity of erythrocytes treated with 5 mumols/l of lead was higher than that for untreated ones by 32%.     

Immunomodulating activity of arabinogalactan and fucoidan in vitro

Many polysaccharides obtained from natural sources are considered to be biological response modifiers and have been shown to enhance various immune responses. Here, we investigated the immunomodulating effects of arabinogalactan (AG) and fucoidan (FU) in vitro. Mouse spleen lymphocytes became cytotoxic to tumor cells after culture with AG and FU at concentrations of 10-100 microg/mL. Also, AG and FU were mitogenic in spleen lymphocytes and peripheral macrophages. Macrophages treated with AG and FU (10-100 microg/mL) exhibited induced tumoricidal activity and increased phagocytosis, lysosomal enzyme activity, and production of nitrite, H2O2, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-6. However, AG and FU had little effect on the level of IL-1beta. Thus, the tumoricidal effect of AG- and FU-activated macrophages appeared to be mainly mediated by production of free radicals (NO and H2O2) and cytokines (TNF-alpha and IL-6). These data suggest that AG and FU are activators of lymphocytes and macrophages. This property may contribute to their effectiveness in the immunoprevention of cancer.     

Quantification of the in vitro activity of some compounds with spermicidal activity.

The in vitro spermicidal activity of the commonly used surfactant spermicides and the antiseptic chlorhexidine, were quantified in a statistically reproducible manner, using donor semen and image capture analysis. The spermicidal activity was expressed as the ED₅₀ under defined assay conditions. Using these parameters, the order of spermicidal activity was: Menfegol > nonoxynol-9 ≈ benzalkonium chloride > sodium docusate > chlorhexidine. These differences were statistically significant.     

头孢哌酮/舒巴坦对鲍氏不动杆菌及肺炎克雷伯菌的体外抗菌活性研究

目的探讨头孢哌酮/舒巴坦对鲍氏不动杆菌及肺炎克雷伯菌的体外抗菌活性,为临床合理用药提供参考依据。方法选取2014年12月-2016年2月医院分离的鲍氏不动杆菌92株和肺炎克雷伯菌94株,分别选用琼脂稀释法、抗菌药物浓度梯度法、纸片扩散法以及全自动微生物分析系统检测不同配比的头孢哌酮/舒巴坦对鲍氏不动杆菌和肺炎克雷伯菌体外抗菌活性。结果与琼脂稀释法相比,纸片扩散法和Vitek-2法对鲍氏不动杆菌和肺炎克雷伯菌耐药率明显提高,分别可达54.35%,42.39%,39.36%,37.23%,差异有统计学意义(P<0.05);与2∶1头孢哌酮/舒巴坦相比,琼脂稀释法和纸片扩散法中1:1头孢哌酮/舒巴坦对鲍氏不动杆菌和肺炎克雷伯菌敏感率均明显提高,可达46.74%,39.13%,44.68%,41.48;耐药率均明显下降,仅为10.87%,21.83%,11.70%,20.21%,差异均有统计学意义(P<0.05)。结论在鲍氏不动杆菌和肺炎克雷伯菌敏感性检查中,纸片扩散法和Vitek 2法不宜选择,且头孢哌酮与舒巴坦的药物配比应加以说明。     

In vitro and in vivo evidences that the malabsorption of cobalamin is related to its binding on haptocorrin (R binder) in chronic pancreatitis

The intraluminal transport of cobalamin (Cbl) remains controversial in chronic pancreatitis. We have determined the ability of intestinal juice to degrade the digestive holohaptocorrin (R binder) and the binding of endogenous Cbl in basal intestinal juice from 22 chronic pancreatitis patients and 22 controls. The intestinal juice from patients and controls degraded 34.7 +/- 32.3% and 95.2 +/- 7.2% of holohaptocorrin, respectively. This percentage was correlated with the trypsin output but not with the Schilling test. The unsaturated Cbl-binding capacity was similar in both groups. Respectively, 62.5 +/- 26.6% and 19.6 +/- 11.7% of endogenous Cbl was bound to haptocorrin in intestinal juice from patients and controls. These percentages were correlated with the Schilling test and with the ability of intestinal juice to degrade haptocorrin. We concluded that 1) the sequestration of Cbl to haptocorrin is one of the factors responsible for the malabsorption of crystalline Cbl in patients with chronic pancreatitis and 2) enterohepatic circulation of Cbl can be interrupted in some cases of chronic pancreatitis.     

临床常见致病菌对美洛西林／舒巴坦等4种抗菌药物体外抗菌活性研究

目的 对比研究美洛西林／舒巴坦（MLBT）与抗菌药物替卡西林／克拉维酸（特美汀，TMT）、美洛西林（MLXL）和哌拉西林／三唑巴坦（他唑西林，TZXL）对179株临床分离菌的体外抗菌活性。方法 采用琼脂平板稀释法测定最低抑菌浓度（MIC）。结果 MLBT对临床常见致病菌的体外抗菌活性较单用等剂量的MLXL高1－2倍，MLBT对产酶率较高的大肠埃希菌、肠杆菌属细菌、不动杆菌属细菌敏感率显著高于MLXL（P＜0．01）；对肠杆菌科属细菌、其他G^-杆菌敏感率高于TMT（P＜0．01）；MLBT和TZXL的抗菌谱和抗菌活性基本相似，敏感率基本等同，MIC50后者较前者低1－2倍，但差异无统计学意义。结论 MLBT的抗菌谱、体外抗菌活性与TZXL相似，强于TMT、MLXL。     

Management of the axilla in breast cancer: evidences and unresolved issues]

In this study the evidences governing the management of the axilla were examined and on the base of these evidences, the optimal clinical practice was outlined. Computerized searches for publications, debating specific treatment of axilla, were done of MEDLINE data. Level of evidence was determined using standard criteria: 1. metaanalysis of randomized trials, 2. randomized trial, 3. prospective and retrospective studies, 4. reports and opinion of expert committees and working teams. The probability of lymph node involvement is related directly to the size of the primary tumour, and even with small tumour (up to 10 mm), the risk of nodal metastases is in the order of 10-20%. To date, the best strategy for determining complete lymph node status (qualitative and quantitative information) is through axillary dissection. For an accurate staging, at least ten nodes have to be obtained. Formal axillary sampling does not provide total quantitative data in patients with involved axilla. Sentinel node biopsy is a promising alternative to axillary dissection for staging but it is still under way. Axillary dissection should be omitted in patients with ductal carcinoma in situ since the probability of nodal involvement is less than 1%. In invasive breast cancer, the risk of axillary recurrence in the untreated axilla varies from about 10% to 40%. For women with stage I-II breast cancer at least level I and II axillary node dissection should be offered as the standard procedure to reduce the risk of regional recurrence. Women at high risk of axillary recurrence (> or = 4 involved nodes, < 6 nodes were obtained from a positive axilla) will require axillary irradiation after axillary dissection. However, there is a lack of higher level evidence to support the benefit of post-dissection axillary irradiation. Evidences suggest that axillary irradiation is as effective as axillary dissection in preventing regional recurrence. The following factors have to be considered for decisions regarding dissection or irradiation: patient wishes, general condition, age, the necessity of pathological nodal status for systemic therapy and the risk of post-treatment morbidity. At this time, there is no well defined subgroup of patients in whom axillary intervention can be safely omitted. In selected patients with clinically negative axilla, the decision to observe the axilla rather than use surgery or irradiation should be made jointly between the women and her specialists (surgeon, radiation and medical oncologist). The benefits of axillary treatment in prolonging survival are unclear. Studies have reported different effects on survival. Until evidences remain insufficient, the risk of axillary recurrence has to be minimized, and more and more patients have to be provide to get treatments in randomized clinical trials. Patient should be fully informed about the benefits and the potential side effects of treatments. A combination of radiotherapy and axillary dissection results an increased morbidity rate compared with either alone.     

Aqueous extracts of Lentinula edodes and Pleurotus sajor-caju exhibit high antioxidant capability and promising in vitro antitumor activity

Mushroom extracts are increasingly sold as dietary supplements because of several of their properties, including the enhancement of immune function and antitumor activity. We hypothesized that soluble polar substances present in mushroom extracts may show antioxidant and anticancer properties. This report shows that Brazilian aqueous extracts of Lentinula edodes and Pleurotus sajor-caju exert inhibitory activity against the proliferation of the human tumor cell lines laryngeal carcinoma (Hep-2) and cervical adenocarcinoma (HeLa). Cell viability was determined after using 3 different temperatures (4°C, 22°C, and 50°C) for mushroom extraction. Biochemical assays carried out in parallel indicated higher amounts of polyphenols in the L edodes extracts at all extraction temperatures investigated. The scavenging ability of the 2,2-diphenyl-1-picrylhydrazyl radical showed higher activity for L edodes extracts. Superoxide dismutase–like activity showed no statistically significant difference among the groups for the 2 tested extracts, and catalase-like activity was increased with the L edodes extracts at 4°C. The results for the cytotoxic activity from P sajor-caju extracts at 22°C revealed the half maximal inhibitory concentration values of 0.64% ± 0.02% for Hep-2 and 0.25% ± 0.02% for HeLa. A higher cytotoxic activity was found for the L edodes extract at 22°C, with half maximal inhibitory concentration values of 0.78% ± 0.02% for Hep-2 and 0.57% ± 0.01% for HeLa. Substantial morphological modifications in cells were confirmed by Giemsa staining after treatment with either extract, suggesting inhibition of proliferation and induction of apoptosis with increasing extract concentrations. These results indicate that the aqueous extracts of Brazilian L edodes and P sajor-caju mushrooms are potential sources of antioxidant and anticancer compounds. However, further investigations are needed to exploit their valuable therapeutic uses and to elucidate their modes of action.     

Comparative analysis of the in vitro antioxidant activity of white and black pepper

As a result of normal metabolic processes, the human body produces reactive oxygen species capable of oxidizing biomolecules that can damage DNA, cells, and contribute to chronic disease. This process can be attenuated or perhaps reversed by diets containing spices that have the ability to scavenge reactive oxygen species. The present study measured the concentration of polyphenols in peppercorn (black and white) using the Folin-Ciocalteu method and investigated the radical scavenging activities of hydrolyzed and nonhydrolyzed pepper extracts using 1,1-diphenyl-2-picrylhydrazyl, the superoxide radical, and the hydroxyl radical as substrates. The hydrolyzed and nonhydrolyzed extracts of black pepper contained significantly (P < .001) more polyphenols compared with those of white pepper. For either of these peppercorns, the hydrolyzed extract contained significantly (P < .001) more polyphenols compared with the nonhydrolyzed extract. A dose-dependent effect was observed in the free radical and reactive oxygen species scavenging activities of all the extracts, with the black pepper extracts being the most effective. Peppercorns especially black pepper, which constitutes an important component in the diet of many sub-Saharan and oriental countries, can therefore be promoted for their nutritional importance as antioxidants and radical scavengers.