Probing altered enzyme activity in the biochemical characterization of cancer

Enzymes have evolved to catalyze their precise reactions at the necessary rates, locations, and time to facilitate our development, to respond to a variety of insults and challenges, and to maintain a healthy, balanced state. Enzymes achieve this extraordinary feat through their unique kinetic parameters, myriad regulatory strategies, and their sensitivity to their surroundings, including substrate concentration and pH. The Cancer Genome Atlas (TCGA) highlights the extraordinary number of ways in which the finely tuned activities of enzymes can be disrupted, contributing to cancer development and progression often due to somatic and/or inherited genetic alterations. Rather than being limited to the domain of enzymologists, kinetic constants such as k_cat, K_m, and k_cat/K_m are highly informative parameters that can impact a cancer patient in tangible ways—these parameters can be used to sort tumor driver mutations from passenger mutations, to establish the pathways that cancer cells rely on to drive patients’ tumors, to evaluate the selectivity and efficacy of anti-cancer drugs, to identify mechanisms of resistance to treatment, and more. In this review, we will discuss how changes in enzyme activity, primarily through somatic mutation, can lead to altered kinetic parameters, new activities, or changes in conformation and oligomerization. We will also address how changes in the tumor microenvironment can affect enzymatic activity, and briefly describe how enzymology, when combined with additional powerful tools, and can provide us with tremendous insight into the chemical and molecular mechanisms of cancer.     

The anti-inflammatory drug Diclofenac retains anti-listerial activity in vivo

Aims:  The interactions between nonsteroidal anti-inflammatory drugs (NSAID) and Listeria monocytogenes have not been sufficiently documented to date. The aim of this study was to investigate the possible effects of Diclofenac (Dc) in a murine listerial infection model.
Methods and Results:  Dc was administered orally at 2·5 μg g⁻¹ to female albino strain of laboratory mouse (BALB/c) thrice postinfection (1 × 10⁸ CFU ml⁻¹ oral challenge with L. monocytogenes ATCC 51774), which resulted in significantly ( P  < 0·01) reduced bacterial counts in liver and spleen, decreased (10-fold, P  < 0·05) hepatic colonization and necrosis, and caused up-regulation of the expression of inflammatory cytokines (interferon-γ, interleukin-1β, tumour necrosis factor-α), compared with drug-free control.
Conclusions:  Dc may be useful as a promising adjuvant to the existing therapies in controlling systemic listerial infection. Further, quantitative structure–activity relationship studies might contribute in manipulating it as a lead compound for the synthesis of new, more effective nonantibiotics, perhaps, devoid of side-effects that could be recommended as a compassionate therapy for listeriosis.
Significance and Impact of the study:  This is the first in vivo study designed to evaluate the antilisterial effect of the NSAID Dc with special emphasis on the immunological mechanism of action of the drug.     

Preparation of matrine ethosome,its percutaneous permeation in vitro and anti-inflammatory activity in vivo in rats

The aim of this work was to evaluate the preparation of matrine ethosome and the percutaneous permeation in vitro and the anti-inflammatory activity in vivo in the rat skin. The matrine ethosomes were prepared by the ethanol injection-sonication method. The particle size of the ethosomes was measured by a laser particle-size analyzer, and the entrapment efficiency was detected by ultracentrifugation. The anti-inflammatory activity in vivo of the matrine formulations was determined by a reflection spectrophotometer. In this study, we found that the average particle size of matrine ethosomes was in the range of 50–200?nm with a narrow distribution, and the entrapment efficiency was in the range of 40–90%. Compared with other formulations, matrine ethosomes had the largest 24-hour accumulative permeation quantity (60.5%) and with no permeation lag time. Matrine ethosomes were able to make the induced erythema disappear more rapidly than the nonethosomes formulations of matrine. This study reveals that the average particle size of matrine ethosomes decreases with the increase of ethanol concentration and increases with the increase of phospholipid concentration, while the entrapment efficiency increases with the increase of the concentration of both ethanol and phospholipid. Matrine ethosomes can increase the percutaneous permeation of matrine in the experiment in vitro and improve the anti-inflammatory activity of matrine in vivo in rat skin.     

抑前胸腺肽在家蚕体内的活性作用

家蚕Bombyx mori抑前胸腺肽是昆虫脑神经肽的一种,体外实验表明它能抑制处于活动时期的家蚕前胸腺合成蜕皮激素,因此抑前胸腺肽可能对昆虫的变态起着重要的作用。将抑前胸腺肽以不同的浓度分单一注射和加强注射导入家蚕体内,不同的时间间隔取样,利用蜕皮激素放射免疫分析方法,观察到了抑前胸腺肽在家蚕体内的活性作用以及引起家蚕体内血淋巴中蜕皮激素浓度的动态变化,首次证明了抑前胸腺肽在体内对家蚕前胸腺合成蜕皮激素有强烈的抑制作用。     

抑前胸腺肽在家蚕体内的活性作用

家蚕Bombyx mori抑前胸腺肽是昆虫脑神经肽的一种,体外实验表明它能抑制处于活动时期的家蚕前胸腺合成蜕皮激素,因此抑前胸腺肽可能对昆虫的变态起着重要的作用。将抑前胸腺肽以不同的浓度分单一注射和加强注射导入家蚕体内,不同的时间间隔取样,利用蜕皮激素放射免疫分析方法,观察到了抑前胸腺肽在家蚕体内的活性作用以及引起家蚕体内血淋巴中蜕皮激素浓度的动态变化,首次证明了抑前胸腺肽在体内对家蚕前胸腺合成蜕皮激素有强烈的抑制作用。     

Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum

Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra‐ and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration‐based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration‐based washing. In the centrifugation‐based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin‐G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3–4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin‐G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood. Biotechnol. Bioeng. 2010;107: 105–115. © 2010 Wiley Periodicals, Inc.     

Inhibitory effects of some phenolic compounds on the activities of carbonic anhydrase: from in vivo to ex vivo

Carbonic anhydrase (CA) inhibitors have been used for more than 60 years for therapeutic purposes in many diseases table such as in medications against antiglaucoma and as diuretics. Phenolic compounds are a new class of CA inhibitor. In our study, we tested the effects of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid on esterase and the CO₂-hydratase activities of CA I and II isozymes purified from in vivo to ex vivo. The K_i values of arachidonoyl dopamine, 2,4,6-trihydroxybenzaldehyde and 3,4-dihydroxy-5-methoxybenzoic acid were 203.80, 1170.00 and 910.00?μM, respectively for hCA I and 75.25, 354.00 and 1510.00?μM, respectively for hCA II. Additionally, IC₅₀ values from in vivo studies were found to be in the range of 173.25–1360.0?μM for CA I and II, respectively, using CO₂-hydratase activity methods. These results demonstrated that phenolic compounds used in in vivo studies could be used in different biomedical applications to inhibit approximately 30% of the CO₂-hydratase activity of the total CA enzyme of rat erythrocytes.     

粘虫谷氨酰胺转胺酶(MsTGase)活力测定条件的正交优化及其在幼虫体内的分布

【目的】本研究旨在优化Grossowicz氧肟酸比色法测定粘虫Mythimna separata谷氨酰胺转胺酶(Ms TGase)活力的组合条件,以Ms TGase酶活力为依据分析其在不同龄期幼虫体内的分布规律。【方法】取4龄粘虫幼虫,通过组织匀浆和沉析纯化制备Ms TGase,采用Grossowicz比色法测定Ms TGase酶活力,并对Grossowicz比色法的多重实验因素进行正交优化,进一步结合差速离心法分析不同龄期幼虫体内和亚细胞组分(细胞核和细胞碎片,线粒体,微粒体以及胞质溶胶)中Ms TGase酶活力。【结果】结果表明,酶浓度、底物浓度、反应体系pH值、反应温度及钙离子浓度等实验因素都对Ms TGase酶活力测定结果产生显著影响,其影响大小顺序为:酶浓度>温度> p H>底物浓度>Ca2+浓度。Ms TGase酶比活力测定的最优化条件:酶浓度20 mg/m L、底物浓度0. 04 mol/L、反应体系pH值6. 5、测定温度37℃,不添加钙离子。在1-5龄幼虫中以4龄幼虫的Ms TGase酶活力最高,其比活力也显著高于其他龄期的,且在1-5龄幼虫胞质溶胶中Ms TGase酶活力分别占各亚细胞组分酶活力总和的39%,25%,48%,60%和61%。【结论】所获得的最优化条件适用于粘虫Ms TGase酶活力测定。Ms TGase在粘虫体内呈显著的龄期表达特征和亚细胞分布规律。     

青藏高原高寒草甸不同季节土壤理化性质及酶活性对施肥处理的响应

分析了青藏高原东缘高寒草甸不同施肥处理对土壤全量养分、速效养分、pH、含水量、有机碳和土壤脲酶活性的影响,以揭示高寒草甸土壤养分和酶活性对施肥的响应。结果表明:(1)随施肥量的增加,土壤pH明显趋于降低,施肥引起高寒草甸土壤酸化;全磷、速效磷均显著增大;(2)土壤全氮、有机碳和脲酶活性随施肥量增加呈单峰曲线变化,在施肥量为30或60g·m-2时最高,施肥量增加到90g·m-2时土壤资源逐渐降低;(3)季节变化对土壤养分也有一定的影响,全氮和全磷含量均于9月份较高,而速效氮含量一般于9月份较低,而速效磷含量5月份较低;(4)施肥对土壤养分的影响并不是简单的线性正相关关系,30~60g·m-2施肥量可作为高寒草甸最佳施肥水平。施肥处理下土壤有机碳和脲酶活性可作为衡量土壤肥力和土壤质量变化的重要指标。高施肥量(≥90g·m-2)可作为影响高寒草甸土壤养分及土壤酶活性的阈值。     

DNA-repair by photolyase reveals dynamic properties of nucleosome positioning in vivo

Within the Alpha class of the mammalian glutathione transferases two variants of subunit interfaces exist. One is conserved among the A4 subunits, whereas the second one is found in all other members of the Alpha class. The ability of the two Alpha class subunit interfaces to adopt a functional heterodimeric structure has been investigated here.The heterodimer GST A1-4 was obtained by co-expression of the two human Alpha class subunits A1 and A4 in Escherichia coli. A histidine tail was added to the N terminus of the A1 subunit to facilitate the purification of the heterodimer. The heterodimer was formed in a small proportion implying that the efficiency of the hybridization between subunit A1 and A4 is less than the propensity for homodimer formation. The hybrid enzyme was stable at low temperatures, but the two subunits dissociated and reassociated into homodimers at 40 degrees C.Three different substrates were used for subunit-selective kinetic characterization of the GST A1-4 heterodimer: 1-chloro-2,4-dinitrobenzene, nonenal and Delta(5)-androstene-3,17-dione. Both subunit A1 and subunit A4 were active in GST A1-4, but the specific activities and k(cat) values were lower than the average values of the two parental isoenzymes. However, at high temperatures the subunits of the hybrid enzyme dissociated and formed homodimers, and the activities increased to expected values. Hence, the low activities of the individual subunits in the heterodimer were reversible. The non-additive kinetic properties of the subunits in the heterodimer therefore highlight the importance of fine-tuned subunit interactions for optimal catalytic efficiency of GST A1-1 and GST A4-4.     

Effects of time-varying uniform magnetic fields on Natural Killer cell activity and antibody response in mice

Natural Killer cell activity and antibody response were studied in Balb/c mice which were exposed in vivo to uniform pulsed magnetic fields (square-wave, 0.8 Hz, 120 mT maximum field strength, 0.1 s rise-time) for 5 days, 10 h/day. No effects were found in antibody response to sheep red blood cell (SRBC) immunization as assayed by counting the plaque-forming cells (PFC) in the spleens of animals on the sixth day. Following 5-day exposures, the activity of Natural Killer (NK) cells was measured in vitro by challenge with YAC-1 cells, in experiments in which mice were not immunized. An increase of NK cytotoxic activity due to exposure was found which depended on the age of the mice (effect observed above 12 weeks) and on the strength of the applied field (effect observed above 30 mT). © 1993 Wiley-Liss. Inc.     

Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.     

我国主要蜜环菌生物种胞外酶活性对温度的种特异性响应

蜜环菌生物种与天麻的共生关系受到温度的调节。为了揭示我国蜜环菌生物种的温度适应性,本研究以我国8个蜜环菌生物种为实验材料,采用2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐（ABTS）法和3,5-二硝基水杨酸（DNS）法研究了胞外漆酶、果胶酶和纤维素酶的活性对温度的响应特征。结果表明,各生物种的胞外酶活性存在显著性差异,各胞外酶活性具有随时间先升后降的变化趋势;各胞外酶的分泌时间和活性大小对温度的响应存在着很大的差异;且在培养过程中,首先分泌的是果胶酶和纤维素酶,然后才是漆酶。温度对蜜环菌胞外漆酶和果胶酶活性大小有影响,对产酶时间没有影响;对纤维素酶活性达到峰值的时间和活性大小都有很大影响。除此之外,蜜环菌胞外酶活性对温度的响应和我国蜜环菌的地域分布特点具有相关性,分布于秦岭-淮河线以南和以北的蜜环菌生物种胞外酶活性对温度的响应具有差异。本研究对开展蜜环菌生物学和我国天麻的野外种植区划的研究具有指导意义。     

芽胞杆菌抗菌活性和纤维素酶活性的测定

目的筛选鸡用益生菌。方法通过牛津杯法、混菌法和DNS分光光度法对从鸡肠道分离的10株芽胞杆菌进行抑菌效果及纤维素酶活性测定。结果编号为Y1、Y5、Y7的3株芽胞杆菌对测试菌具有较好抑菌效果,编号为Y2、Y4、Y5的3株芽胞杆菌具有较高的产酶活性。结论从分离的10株芽胞杆菌中筛选出1株（编号为Y5）兼具较好抑菌效果和较高产酶活性的地衣芽胞杆菌,具有作为饲用益生菌开发的潜能。     

Fibroblast growth factor 2 internal ribosome entry site (IRES) activity ex vivo and in transgenic mice reveals a stringent tissue-specific regulation

Fibroblast growth factor 2 (FGF-2) is a powerful mitogen involved in proliferation, differentiation, and survival of various cells including neurons. FGF-2 expression is translationally regulated; in particular, the FGF-2 mRNA contains an internal ribosome entry site (IRES) allowing cap-independent translation. Here, we have analyzed FGF-2 IRES tissue specificity ex vivo and in vivo by using a dual luciferase bicistronic vector. This IRES was active in most transiently transfected human and nonhuman cell types, with a higher activity in p53 -/- osteosarcoma and neuroblastoma cell lines. Transgenic mice were generated using bicistronic transgenes with FGF-2 IRES or encephalomyocarditis virus (EMCV) IRES. Measurements of luciferase activity revealed high FGF-2 IRES activity in 11-d-old embryos (E11) but not in the placenta; activity was high in the heart and brain of E16. FGF-2 IRES activity was low in most organs of the adult, but exceptionally high in the brain. Such spatiotemporal variations were not observed with the EMCV IRES. These data, demonstrating the strong tissue specificity of a mammalian IRES in vivo, suggest a pivotal role of translational IRES- dependent activation of FGF-2 expression during embryogenesis and in adult brain. FGF-2 IRES could constitute, thus, a powerful tool for gene transfer in the central nervous system.     

青藏高原典型冰川小流域土壤微生物生物量及胞外酶活性分布特征

土壤微生物释放的胞外酶是决定碳(C)、氮(N)、磷(P)生物地球化学循环的关键因素,为了阐明青藏高原典型小流域土壤微生物生物量和胞外酶活性沿海拔和土层的分布特征并揭示影响该分布格局的主要养分限制状况,于2021年8月采集了青藏高原廓琼岗日冰川小流域5个海拔梯度(4900 m; 5000 m; 5100 m; 5200 m; 5300 m)中4个土壤发生层(A层：腐殖质层、E层：淋溶层、B层：淀积层和C层：母质层)的土壤样品,定量分析了土壤基本理化性质、微生物生物量、胞外酶活性等指标。结果表明：1)微生物生物量碳氮磷的海拔差异变化规律不同,随着土层加深微生物生物量碳氮磷随海拔变化越小。同时,各海拔之间微生物生物量均有随土层加深而显著降低的趋势(P<0.05);2)四种酶活性的海拔间变化规律各异,但整体呈现随海拔升高而升高的趋势且在表层(A和E层)增长趋势更明显,而且随土层加深显著降低(P<0.05);3)该区域土壤微生物受到碳和磷共同限制,土层越深限制越高,而且海拔越高C限制越强,但P限制降低;4)青藏高原典型冰川小流域土壤微生物生物量和胞外酶活性在海拔和土层之间存在较明显的...