Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium–HCl,urea, and pH

The stability of bovine lactoperoxidase to denaturation by guanidinium–HCl, urea, or high temperature was examined by differential scanning calorimetry (DSC) and tryptophan fluorescence. The calorimetric scans were observed to be dependent on the heating scan rate, indicating that lactoperoxidase stability at temperatures near T_m is controlled by kinetics. The values for the thermal transition, T_m, at slow heating scan rate were 66.8, 61.1, and 47.2 °C in the presence of 0.5, 1, and 2 M guanidinium–HCl, respectively. The extrapolated value for T_m in the absence of guanidinium–HCl is 73.7 °C, compared with 70.2 °C obtained by experiment; a lower experimental value without a denaturant is consistent with distortion of the thermal profile due to aggregation or other irreversible phenomenon. Values for the heat capacity, C_p, at T_m and E_a for the thermal transition decrease under conditions where T_m is lowered. At a given concentration, urea is less effective than guanidinium–HCl in reducing T_m, but urea reduces C_p relatively more. Both fluorescence and DSC indicate that thermally denatured protein is not random coil. A change in fluorescence around 35 °C, which was previously reported for EPR and CD measurements (Boscolo et al. Biochim. Biophys. Acta 1774 (2007) 1164–1172), is not seen by calorimetry, suggesting that a local and not a global change in protein conformation produces this fluorescence change.     

人破伤风类毒素免疫血浆筛选试验中两种抗体检测方法的比较

应用酶联免疫法(ELISA)和间接血凝法(IHA)对1014份破伤风类毒素全程免疫后人血浆进行抗体水平检测,比较两者的收浆率、收浆符合率以及与动物实验的相关性。结果表明两者收浆率均达80%。ELISA法与IHA法的合格浆符合率达92%,与动物实验的相关性更好。ELISA法操作简便快速,结果判读客观明确,优于传统的IHA法,可作为人破伤风类毒素免疫血浆筛选的常规方法。     

原核表达人41型腺病毒(Ad41) 蛋白 V及其抗血清的制备

摘要：目的 人肠腺病毒Ad41被称为难养腺病毒,其难以培养的特性可能与次要核心蛋白V（Ad41 protein V,pV）表达不充分有关。本研究拟表达纯化Ad41 pV抗原,免疫动物制备抗血清,为研究Ad41难养性机理打下基础。方法 以野生型Ad41基因组DNA为模板,PCR扩增pV,克隆到原核表达载体pET30a(+),测序后,转化大肠杆菌BL21(DE3)菌株,异丙基-β-D-硫代半乳糖苷（IPTG）诱导目的蛋白表达,固化金属亲和层析(IMAC)方法纯化,免疫BALB/c小鼠制备抗血清,将获得的抗血清用于Western blot检测Ad41感染各细胞系后pV的表达。结果 克隆得到包括完全编码区的pV基因,表达质粒转化BL21(DE3)菌株,使用1 m mol/L IPTG 37℃诱导4 h,pV以包涵体形式表达,或使用0.5 m mol/L IPTG 25℃诱导8 h获得可溶性表达。利用皮下多点注射包涵体的方法免疫小鼠,得到抗pV抗血清;使用纯化的可溶性pV作为抗原对抗血清进行了鉴定,表明该抗血清可用于Western blot检测。等量野生型Ad41感染293或293E12细胞（一株稳定表达Ad41 E1B55K基因的293细胞）后,pV在293E12细胞的表达明显高于293细胞。结论 成功克隆了Ad41 pV基因,表达纯化了重组蛋白,获得了可用于Western blot检测的抗血清,为进一步研究Ad41难养性机理打下了基础。     

棉铃虫病毒gp41基因的克隆表达及抗体制备

根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigerasingle nucleocapsid nucleopolyhedrovirus,HaSNPV)gp41基因的序列,设计引物,引入适当的酶切位点,通过PCR的方法扩增目的片段。将扩增出的基因片段克隆至原核表达载体pET-28a,构建重组质粒并转化至大肠杆菌中,经IPTG诱导表达。纯化蛋白产物并免疫家兔产生抗血清。该抗血清可与原核表达的His-GP41融合蛋白及在感染的昆虫细胞中表达的GP41蛋白发生特异性免疫反应。该抗体的获得为深入研究GP41的功能提供了基础。     

Participation of galaninergic neurotransmission at the paraventricular hypothalamic nucleus in the suppression of baroreceptor reflex response by locus ceruleus in the rat

We evaluated the potential participation of galanin (GAL) at the paraventricular nucleus of hypothalamus (PVN) in the suppression of baroreceptor reflex (BRR) response by locus ceruleus (LC), using adult male Sprague-Dawley rats anesthetized with pentobarbital sodium. Microinjection of GAL (100 pmol) bilaterally into the PVN significantly depressed the BRR response. This suppressive effect was appreciably antagonized when GAL (100 pmol) and GAL antiserum (1:20) were coadministered into the bilateral PVN. Whereas bilateral microinjection of GAL antiserum into the PVN by itself elicited minimal effect, it nevertheless significantly attenuated the suppressive effect of either electrical or chemical activation of LC on the BRR response. Pretreatment with the same amount of normal rabbit serum (1:20), on the other hand, was ineffective. These results suggest that a galaninergic projection from the LC to PVN may participate in the suppression of BRR response by this dorsal pontine nucleus.     

Antibacterial Activity of the Lactoperoxidase System Combined with Edible Laminaria Hot-Water Extract as a Source of Halide Ions

Hot-water extracts prepared from nine out of 12 samples of dried edible Laminaria reduced the viable numbers of Aggregatibacter actinomycetemcomitans, Staphylococcus aureus, and Esherichia coli below the detection limit after incubation for 5 min when combined with lactoperoxidase, glucose oxidase, and glucose. Some extracts showed higher bactericidal activity and a higher OI^? concentration in the assay mixture after ultrafiltration.     

Effect of oxidative iodination of epidermal growth factor on its binding and secretion by hepatocytes

Experiments were undertaken to determine whether the method of iodination of epidermal growth factor (EGF) affects its binding to rat liver plasma membranes and its uptake, processing, and secretion into bile by intact rat hepatocytes. EGF was iodinated using one of three oxidative reagents: chloramine T (CT), lactoperoxidase (LP), or monochloride (MC). Quantitative receptor binding studies on plasma membranes isolated from male rat livers with either CT-, LP-or MC-125I-EGF indicated no significant difference in the apparent binding constants of the three preparations. To determine whether these three preparations were capable of forming a covalent-like complex with the EGF receptor, they were individually incubated with isolated plasma membranes and subjected to polyacrylamide gel electrophoresis under reducing conditions, followed by autoradiography. Each preparation formed a major radioactive protein band of approximately 180 kD, identified as the EGF receptor by immunoprecipitation with monoclonal anti-EGF receptor antibodies. Furthermore, even unlabeled EGF incubated with plasma membranes formed this same 180 kD band, as revealed on Western blots using anti-EGF antibody. The biliary secretion of CT-, LP-, and MC-125I-EGF was compared by injecting each one into rat portal veins and measuring the total and immunoprecipitable radioactivity in bile. The amount of immunologically intact CT-125I-EGF in bile was significantly greater than the others, whereas MC-125I-EGF transport was significantly reduced. We conclude that the method of iodination does not affect the covalent-like binding properties of EGF. Furthermore, since unlabeled EGF displayed these same binding properties, oxidative iodination procedures per se do not account for the covalent-like association between EGF and its receptor. However, the method of iodination used did affect the intracellular transport and processing of EGF by hepatocytes. The structural modification responsible for this alteration in transport properties has yet to be determined.     

Immunological identification of four different polypeptides in 'subunit VII' of mammalian cytochrome c oxidase

Rat liver cytochrome c oxidase was separated by SDS-gel electrophoresis into 13 polypeptide bands. Monospecific antisera against the isolated polypeptides VIIa, VIIb and VIIc were raised in rabbits. Cytochrome c oxidase was blotted on nitrocellulose and incubated with the antisera. The antisera reacted only with their corresponding polypeptides, indicating no immunological relationship between polypeptides VIIa, VIIb and VIIc. The data also exclude that these polypeptides are proteolytic breakdown products of larger subunits.     

DNA-binding proteins specified by bacteriophage T1

Abstract A type A Clostridium perfringens enterotoxin was chially purified by ammonium sulfate precipitation (0 to 15%) and was submitted to polyacrylamide gel electrophoresis (7%). A specific enterotoxin antiserum was obtained by inoculating a rabbit with the polyacrylamide gel strip containing the enterotoxin. This serum gave only one precipitin line with purified enterotoxin and cellular extract in immunodiffusion and immunoelectrophoresis. The titer (1:8) in counter-immunoelectrophoresis was sufficient to detect 0.39 μg/ml enterotoxin by this technique. This serum neutralized the mouse lethality, cytotoxicity and plating efficiency of Vero cells.     

In vitro effects of standard antioxidants on lactoperoxidase enzyme–A molecular docking approach

Lactoperoxidase (LPO), an antioxidant enzyme, is a natural antimicrobial system that eliminates the harmful effects of microorganisms in milk. It has a wide range of applications and is also preferred in cosmetic and clinical applications, as well as used in foods. The use of antioxidants is well recognized in the food and feed industries to improve the shelf life of products. This study aimed to determine the in vitro inhibition effects of Trolox, α‐tocopherol, butylated hydroxyanisole, butylated hydroxytoluene, and propyl gallate, which are commonly used as antioxidants in food and pharmaceutical products. For this purpose, LPO was first purified in a single step using sepharose‐4B‐l ‐tyrosine‐sulfanilamide affinity gel chromatography. Also, some inhibition parameters, including half‐maximal inhibitory concentration (IC₅₀), K_i values, and inhibition types, were calculated for each antioxidant molecule. The IC₅₀ values of these molecules, which exhibited competitive inhibition, varied between 377.7 and 3397.8 nM. Molecular docking studies were also performed for all compounds. According to the binding scores, α‐tocopherol was shown to exhibit the most effective inhibitor property (IC₅₀: 377.7 nM and K_i: 635.8 ± 16.8 nM) among the standard antioxidants used in this study. Inhibiting the LPO activity by standard antioxidants results in the weakening of the immune system during lactation, which is important for metabolism.