A new fluorescent PET probe for hydrogen peroxide and its use in enzymatic assays for L-lactate and D-glucose

We present a new probe for the determination of hydrogen peroxide (HP). It is based on the yellow fluorophore 4‐amino‐1,8‐napththalimide, coupled to p‐anisidine (as a redox‐active group) to form a probe that is based on photoinduced electron transfer (PET). The preparation of the probe (which we refer to as “HP Green”) was accomplished in four steps with good yield. Its fluorescence is independent of pH in the physiological range and quenched by a PET process that occurs between the p‐anisidine redox moiety and the naphthalimide luminophore. If the p‐anisidine group is oxidized by HP, PET is suppressed and fluorescence intensity is strongly increased. Addition of horseradish peroxidase (HRP) enhances the oxidation of HP Green and further improves the detection limit of HP. The use of HRP and HP Green enables the determination of HP concentration in a range of 0.1 to 5 μM , with a limit of detection (LOD) as low as 64 nM (16 pmol per well in microtiter plates). HP Green and HRP also enable sensitive enzymatic assays of oxidase substrates in a kinetic format, as shown for L ‐lactate and D ‐glucose. L ‐Lactate concentration can be rapidly determined between 0.5 and 10 μM after 6 minutes of incubation at 30 °C, with an LOD of 164 nm (41 pmol per well). This LOD is more than sixfold lower than that of the best commercial assays for lactate. The detection range for D ‐glucose is 2 to 30 μm, and the LOD is 644 nM (161 pmol per well). These are among the lowest concentrations detectable for oxidase‐based assays. The hexanoic acid moiety in HP Green may be further used to immobilize the probe in order to obtain sensor layers for continuous assays.     

固体超强酸S_2O_8~(2-)/ZrO_2-CeO_2催化合成乳酸丁酯的研究

以固体超强酸S2O82-/ZrO2-CeO2为催化剂,乳酸和正丁醇为原料合成乳酸丁酯。考察了催化剂的用量,反应物的配比、反应时间、反应温度、催化剂重复使用等因素对反应的影响。结果表明,催化合成乳酸丁酯的最佳条件为:n(丁醇)∶n(乳酸)=3.0∶1.0,w(S2O82-/ZrO2-CeO2)=12.0%(相对于乳酸),温度145℃,反应2.0 h,乳酸的酯化率达96.6%,催化剂可重复多次使用。     

头孢西丁钠结晶工艺改进

头孢西丁钠是一种具有长效、广谱抗菌特点的第二代头孢类抗生素。本文通过对成盐剂和有机溶媒的筛选,对头孢西丁钠的结晶工艺进行优化,得到晶型好、质量高、成本低的头孢西丁钠产品,收率大于95%。本方法操作简便,成本低廉,适于工业化生产。     

Cytosolic 5′-Nucleotidase II Silencing in Lung Tumor Cells Regulates Metabolism through Activation of the p53/AMPK Signaling Pathway

Cytosolic 5′-nucleotidase II (cN-II) is an allosteric catabolic enzyme that hydrolyzes IMP, GMP, and AMP. The enzyme can assume at least two different structures, being the more active conformation stabilized by ATP and the less active by inorganic phosphate. Therefore, the variation in ATP concentration can control both structure and activity of cN-II. In this paper, using a capillary electrophoresis technique, we demonstrated that a partial silencing of cN-II in a pulmonary carcinoma cell line (NCI-H292) is accompanied by a decrease in adenylate pool, without affecting the energy charge. We also found that cN-II silencing decreased proliferation and increased oxidative metabolism, as indicated by the decreased production of lactate. These effects, as demonstrated by Western blotting, appear to be mediated by both p53 and AMP-activated protein kinase, as most of them are prevented by pifithrin-α, a known p53 inhibitor. These results are in line with our previous observations of a shift towards a more oxidative and less proliferative phenotype of tumoral cells with a low expression of cN-II, thus supporting the search for specific inhibitors of this enzyme as a therapeutic tool for the treatment of tumors.     

菜籽多酚对S180小鼠肉瘤及其免疫功能的影响

研究菜籽多酚的抑瘤作用及其机理。通过接种S180细胞复制荷S180瘤小鼠模型,分别腹膜注射生理盐水、环磷酰胺、菜籽多酚。测定各组小鼠肿瘤重量与脏器指数、免疫能力、抗氧化能力、乳酸脱氢酶活力等指标。结果表明:50~200 mg/kg.d的RSPP抑瘤率在30.23%~44.19%之间;RSPP能显著提高荷瘤小鼠脾脏指数、腹腔巨噬细胞吞噬率与吞噬指数、迟发性超敏反应、小鼠血清溶血素含量与脾细胞抗体形成等指标,能显著抑制荷S180小鼠血清LDH的活性,提高红细胞CAT活性,降低血清中MDA含量。以上结果表明RSPP在小鼠体内具有抑制S180肉瘤的作用,能增进荷瘤小鼠免疫力、清除自由基与抑制LDH活性可能是其抑制肿瘤的机理。     

Analysis of hemin effect on lactate reduction in Lactococcus lactis

Lactococcus lactis is a facultative anaerobic microorganism that produces lactate as the major product, and acetate and acetoin as by-products; some strains of this species produce an antimicrobial compound, nisin. Lactate has a strong inhibitory effect on L. lactis growth. On the other hand, hemin has a suppressive effect on lactate production during L. lactis growth under aerobic condition. To achieve the optimum effect of hemin on lactate amount reduction in L. lactis ATCC11454, cultures entailing various conditions were performed with and without hemin. In the culture with hemin, L. lactis growth and lactate reduction improved compared with those in the culture without hemin; that is, lactate production was suppressed by 1.8- and 1.3-fold under batch and fed-batch cultures, respectively. In microaerobic fed-batch culture with hemin, lactate production was sufficiently suppressed. This result suggests that microaerobic fed-batch culture could be applied to the maintenance of the low lactate amount. Under this condition, metabolic shift was observed from lactate to acetoin and acetate. However, no increase in nisin production was observed even though lactate production could significantly decrease in L. lactis ATCC11454.     

Lactate Dehydrogenase Activity as an Endpoint Heating Indicator in Cooked Beef

Lactate dehydrogenase (LDH, E.C. 1.1.1.27) activity, specifically LDH5 isoenzyme, declined significantly when bovine Semimembranosus (SM) slurries were heated from 57° to 66°C. When SM whole muscle roasts were brine pumped and oven-cooked, total LDH and specific activities declined proportionately from 57° to 63°C resulting in almost no activity at 63°± 0.5°C. LDH activities were consistently higher in younger cattle and lower in older cattle for SM, Infraspinatus (IN) and Longissimus dorsi (LD) muscles and were lowest overall in the IN. LDH colorimetric and fluorescent assays were more accurate indicators for predicting beef roast heating temperature endpoints between 60° and 63°C than the USDA-FSIS method.     

Microbiological and chemical quality of ground beef treated with sodium lactate and sodium chloride during refrigerated storage

The effects of sodium lactate (NaL) and sodium chloride (NaCl), either alone (30 g/kg) or in combination (20+20 g/kg), on the microbiological and chemical quality of raw ground beef during vacuum-packaged storage at 2°C were investigated. The results showed that addition of NaL alone or in combination with NaCl significantly delayed the proliferation of aerobic plate counts, psychrotrophic counts, lactic acid bacteria and Enterobacteriaceae and extended the shelf life of the product up to 15 and 21 days, respectively, versus 8 days only for control. Over the storage time (21 days), NaL maintained the ground beef at almost constant pH, while the pH of control or NaCl-treated samples significantly decreased. Lipid oxidation (TBA value) was not affected by addition of NaL. At storage day 21 however, TBA values of both NaL-treated (0.309) and control (0.318) samples were significantly lower than those of samples treated with NaCl (0.463). The combination of NaCl with NaL significantly reduced the oxidative changes caused by NaCl (0.384 versus 0.463). Therefore, NaL alone or in combination with NaCl could be utilized successfully to reduce the microbial growth, maintain the chemical quality, and extend the shelf life of ground beef during refrigerated storage.     

2-(4-苯氧基苯基)乳酸乙酯的合成研究

以二苯醚与丙酮酸乙酯为原料,经傅克酰基化一步合成恶唑菌酮关键中间体2-(4-苯氧基苯基)乳酸乙酯,考察了溶剂、投料比、AlCl3用量、缚酸剂、温度对收率的影响。-25℃,CH2Cl2为溶剂,NaHCO3为缚酸剂,n(二苯醚)∶n(丙酮酸乙酯)∶n(AlCl3)=1.4∶1.0∶1.2,2-(4-苯氧基苯基)乳酸乙酯收率86.2%,目标产物结构经IR、1 H NMR确证。     

生物催化不对称还原制备(R)-2-羟基-4-苯基丁酸

以克隆表达的乳酸脱氢酶(D-LDH)为催化剂来还原前手性化合物2-羰基-4-苯基丁酸(OPBA),制备重要的手性中间体(R)-2-羟基-4-苯基丁酸(R-HPBA)。通过考察影响反应的各因素,确定了反应pH、初始底物浓度,酶加量及NAD+加量等参数。最后,以分批补料的方式进行产物制备,4 h内即可完全转化100 mmol/L底物。经验证,所得产物的构型完全正确,且e.e值>99.9%。