Acute phase proteins and biomarkers of oxidative status in feline spontaneous malignant mammary tumours

Acute phase proteins (APP) and biomarkers of oxidative status change in human and canine mammary tumours, however, they have not been studied in feline mammary tumours. The aims of this study were to investigate the APP and antioxidant responses in feline malignant mammary tumours, to evaluate their relation with tumour features, and to assess their prognostic value. Serum amyloid A (SAA), haptoglobin (Hp), albumin, butyrylcholinesterase (BChE), insulin‐like growth factor1 (IGF1), paraoxonase1 (PON1), total serum thiols (Thiol), glutathione peroxidase (GPox) and total antioxidant capacity determined by different assays, including trolox equivalent antioxidant capacity assessed by two different methodologies (TEAC1/2), ferric reducing ability of plasma (FRAP), and cupric reducing antioxidant capacity (CUPRAC), were determined in serum of 50 queens with spontaneous mammary carcinomas and of 12 healthy female cats. At diagnosis, diseased queens presented significantly higher SAA and Hp, and lower albumin, BChE, GPox, TEAC1, TEAC2 and CUPRAC than controls. Different tumour features influenced concentrations of APP and antioxidants. Increases in serum Hp, and decreases in albumin, Thiol and FRAP were significantly associated with neoplastic vascular emboli, metastasis in regional lymph nodes and/or in distant organs. Distant metastasis development during the course of the disease was associated with increases in SAA and TEAC1. At diagnosis, decreased albumin was associated with a longer survival, and BChE <1.15 μmoL/mL.minute was associated with a shorter survival time on multivariate analysis. Feline malignant mammary tumours are associated with an APP response and oxidative stress, and different tumour features influence the inflammatory response and the oxidative damage. Furthermore, some of these analytes proved to have prognostic value.     

Linarin, a selective acetylcholinesterase inhibitor from Mentha arvensis

Linarin (acacetin-7-O-beta-d-rutinoside) from the flower extract of Mentha arvensis showed selective dose dependent inhibitory effect on acetylcholinesterase.     

毕赤酵母产人源丁酰胆碱酯酶的发酵条件优化

测定不同条件下重组毕赤酵母的蛋白表达量及酶活力,优选毕赤酵母分泌表达人源丁酰胆碱酯酶的发酵条件。在单因素试验的基础上,采用响应面法优化表达条件,确定了毕赤酵母分泌表达人源丁酰胆碱酯酶的最佳发酵条件:发酵时间4.8d,甲醇浓度13.90%,甘油浓度2.1%。使用最佳发酵条件对毕赤酵母进行培养,最终得到的人源丁酰胆碱酯酶的表达量为368.2mg/L,表达量提高了27%。     

In vitro and in vivo effects of chlorpyrifos and cypermethrin on blood cholinesterases in sheep

The combination of the organophosphate (OP) chlorpyrifos (CPF) and the pyrethroid cypermethrin (CPM) is commonly marketed as pour‐on formulations for the control of sheep lice, ked, and blowflies. CPF irreversibly inhibits acetylcholinesterases (AChE), while pyrethroids are not AChE inhibitors. However, combinations of pyrethroids with OPs showed a highly synergistic effect on AChE inhibition. Thus, the aim of the current work was to evaluate in vitro and in vivo the inhibitory potency of both pesticides, alone and in combination with AChE and butyrylcholinesterase (BChE) activities in sheep blood. In vitro, IC50 values were similar after CPF or CPF plus CPM incubations. The pour‐on coadministration of recommended doses of CPF and CPM did not cause a significant inhibition of AChE and BChE in sheep blood. Only slight percentages of inhibition of their catalytic activities were observed when both drugs were given at 4‐fold higher dose rates. The lower systemic availability of topical administration of OPs in sheep may help to explain the lower degree of inhibition of blood AChE and BChE in vivo. The results emerged from this research are a further contribution to the knowledge of the risks of implementing higher dosage regimens of OPs‐containing antiparasitic formulations.     

大蒜头提取物对丁酰胆碱酯酶抑制作用的测定分析

试验采用蒸馏水提取得到的大蒜头中干扰酶抑制剂检测有机磷与氨基甲酸酯农药残留的提取物,以2,6-二氯靛酚作为显色剂,分析了提取物对丁酰胆碱酯酶活性的抑制类型.结果表明,大蒜头提取物对丁酰胆碱酯酶的抑制为可逆型抑制.     

Expression of recombinant human butyrylcholinesterase in the milk of transgenic mice

Butyrylcholinesterase (BCHE) is a natural bioscavenger that protects humans against organophosphate toxicity. Due to the limited yield of human BCHE (hBCHE) when purifying from human plasma, it is necessary to find an alternative method to produce this protein. One potential method is to produce transgenic livestock that make modified milk containing high concentration of hBCHE. In this study, we cloned the hBCHEgene into a human lactoferrin (hLF) bacterial artificial chromosome (BAC) construct to make a hLF-hBCHE BAC construct. Subsequently, we injected the BAC construct into pronuclei of mouse fertilized embryos and generated transgenic mice. Expression analysis showed that recombinant hBCHE (rhBCHE) was expressed efficiently in the mammary gland of the transgenic mice and the concentration of rhBCHE in the milk of individual mice ranged from 76±12 to 159±28 mg·L^-1. Protein function tests showed that rhBCHE has the same enzymatic activity as the native hBCHE. Our results pave the way for making transgenic livestock to produce large quantities of rhBCHE.     

Chemical profiling and biological screening of Thymus lotocephalus extracts obtained by supercritical fluid extraction and hydrodistillation

Essential oil and extracts from the aerial parts of Thymus lotocephalus were obtained by hydrodistillation (HD) and supercritical fluid extraction (SFE) in two different collectors, respectively. SFE was conducted at 40 °C and a working pressure of 12 or 18 MPa. The chemical profiles were determined using GC-FID and GC-IT-MS. Oxygen-containing monoterpenes were the primary constituents in the essential oil and SFE extracts collected in the second separator, while the extracts obtained in the first separator were predominantly oxygen-containing sesquiterpenes. A large number of compounds were identified by hydrodistillation and, in contrast, the highest extraction yields were obtained using SFE. Linalool (10.43 ± 1.63%) was the main component in essential oil, whereas camphor (7.91 ± 0.84%) and cis-linalool oxide (7.25 ± 1.45%) were the major compounds in the extracts-2nd separator obtained at pressures of 12 and 18 MPa, respectively. Caryophyllene oxide was the primary constituent identified in the extracts-1st separator (4.34 ± 0.51 and 4.41 ± 1.25% obtained at 12 and 18 MPa, respectively). The antioxidant activity was assessed by ORAC and DPPH assays, and the anti-cholinesterase activity was evaluated in vitro using Ellman's method. The essential oil and SFE extracts (first separator) of T. lotocephalus possessed antioxidant activity and strongly inhibited cholinesterases. We also demonstrated that the acetylcholinesterase and butyrylcholinesterase inhibitory activities of the essential oil could be attributed to 1,8-cineole and caryophyllene oxide, respectively.     

超声波辅助提取木薯皮活性物质工艺

木薯皮乙醇提取物同时具有抗乙酰胆碱酯酶（AChE）和抗丁酰胆碱酯酶（BuChE）的活性,为了能充分提取和利用其抗胆碱酯酶活性物质,研究了超声波提取木薯皮活性物质的工艺。通过单因素试验和Box-Behnken Design法设计的响应面优化试验确定了超声波辅助乙醇提取木薯皮中抗胆碱酯酶活性物质的最优工艺条件,并比较了超声波提取与传统溶剂浸提的提取效果。试验结果表明：超声波辅助乙醇提取木薯皮中抗胆碱酯酶活性物质的最优工艺条件为：乙醇体积分数为95%,超声波功率550W,提取时间90min,提取温度60℃,液料比12mL/g,在此工艺条件下,木薯皮活性物质的平均得率为8.97%,质量浓度为0.5mg/mL时的AChE抑制率为54.62%,BuChE抑制率为45.7%;影响木薯皮活性物质得率的4个主要因素的显著程度排列顺序为：超声波提取功率＞提取温度＞提取时间＞液料比;与传统溶剂浸提法相比,超声波提取法具有提取速度快,得率高,提取物活性好,溶剂用量少等明显优势。     

Selenium and vitamin E, natural antioxidants, protect rat cerebral cortex against dimethoate-induced neurotoxicity

Pesticides have been used in agriculture to enhance food production by eradicating unwanted insects and controlling disease vectors, nevertheless occupational exposure to high levels of these compounds can lead to neurodegenerative disorders, characterized by serious oxidative and neurotoxic effects. However, there is a lack of consensus as to which determinations are best used to quantify future risks arising from xenobiotic exposure and natural antioxidant interventions. Our study aims to determine the potential ability of selenium and/or vitamin E, used as nutritional supplements, to alleviate oxidative stress in cerebral cortex tissue induced by dimethoate, an organophosphorus pesticide. Adult Wistar rats were exposed either to dimethoate (0.2 g/L of drinking water), dimethoate + selenium (0.5 mg/kg of diet), dimethoate + vitamin E (100 mg/kg of diet), or dimethoate + selenium + vitamin E, for 30 days. Exposure to dimethoate increased malondialdehyde levels, protein carbonyl groups and advanced oxidation protein products, while Na⁺K⁺-ATPase, acetylcholinesterase and butyrylcholinesterase activities decreased in the cerebral cortex. An increase in glutathione peroxidase, superoxide dismutase and catalase activities and a decrease in glutathione, non-protein thiols and vitamin C levels were observed. Administration of selenium and/or vitamin E through the diet in dimethoate treated rats ameliorated the biochemical parameters cited above. The histological findings confirmed the biochemical results. The model of this study that we employed characterized the relationships between dimethoate-induced neurotoxicity and its alleviation by natural antioxidants like selenium and vitamin E. These elements may be considered beneficial for the protection of cerebral cortex against injury induced by dimethoate.     

Oxidative damage in bone and erythrocytes of suckling rats exposed to 2,4-dichlorophenoxyacetic acid

Nowadays, people’s exposure to pesticides such as 2,4-dichlorophenoxyacetic acid (2,4-D) is increasing continuously. This compound is suspected to produce in excess free radicals which have adverse effects on human health causing several cell alterations in the organism. The present study investigated oxidative stress in the bone and erythrocytes of suckling rats whose mothers were treated with 2,4-D. Experiments were carried out on adult Wistar rats given 600 mg/L of 2,4-D in their drinking water from the 14th day of pregnancy until day 14 after delivery. Exposing dams to 2,4-D caused disorders in the bone of their progeny. Indeed, it induced changes in bone mineralization, especially calcium and phosphorus levels. Moreover, total tartrate-resistant acid phosphatase, which reflected bone resorption, was enhanced while total alkaline phosphatase, which reflected bone formation, was reduced suggesting that this herbicide accelerated bone resorption. The impairment of bone function corresponded histologically.Rats exposed to 2,4-D showed in both bone and erythrocytes an increase in malondialdehyde, advanced oxidation protein products and protein carbonyl levels and a decrease in non-enzymatic (glutathione, non-protein thiol, and vitamin C) and enzymatic (superoxide dismutase, catalase, and glutathione peroxidase) antioxidant system.