肠缺血-再灌流大鼠血浆二胺氧化酶活性的变化与肠损伤的关系

观察并探讨了肠缺血 -再灌流大鼠血浆二胺氧化酶 (DAO)活性的变化规律及其与肠损伤相关指标变化的关系。结果显示 ,肠缺血 -再灌流过程中血浆DAO活性呈双峰升高 :第一峰在肠缺血期 ,时相上早于血浆内毒素 (LPS)和D -乳酸的升高 ;第二峰在再灌流后 2h ,在肠损伤相关指标变化的峰值之后。结果提示 ,肠粘膜上皮细胞和 (或 )肠屏障受到缺血和再灌流二次损伤的打击 ;血浆DAO活性作为反映肠损伤的指标特异性强、灵敏度高 ,在临床上对病程判断及并发症的防治有一定的指导意义。     

肠缺血-再灌流大鼠血浆二胺氧化酶活性的变化与肠损伤的关系

观察并探讨了肠缺血 -再灌流大鼠血浆二胺氧化酶 (DAO)活性的变化规律及其与肠损伤相关指标变化的关系。结果显示 ,肠缺血 -再灌流过程中血浆DAO活性呈双峰升高 :第一峰在肠缺血期 ,时相上早于血浆内毒素 (LPS)和D -乳酸的升高 ;第二峰在再灌流后 2h ,在肠损伤相关指标变化的峰值之后。结果提示 ,肠粘膜上皮细胞和 (或 )肠屏障受到缺血和再灌流二次损伤的打击 ;血浆DAO活性作为反映肠损伤的指标特异性强、灵敏度高 ,在临床上对病程判断及并发症的防治有一定的指导意义。     

Determination of diamine oxidase in lentil seedlings by enzymic activity and immunoreactivity

A competitive radioimmunoassay for the quantitation of diamine oxidase (EC 1.4.3.6) from Lens culinaris is reported. Specific antibodies raised in rabbits immunized with a homogeneous preparation of the enzyme were incubated with purified ¹²⁵I-enzyme and with either unlabeled diamine oxidase or plant material. Antigen-antibody complexes were isolated from the mixture by incubation with Staphylococcus protein A. The sensitivity of the test was about 5 nanograms in terms of enzyme protein. This assay was applied to the determination of the enzyme in extracts from lentil shoots grown either in the dark or in the light. Diamine oxidase activity and enzyme protein (as determined by radioimmunoassay) were measured during 7 days after germination. Both enzymic activity and enzyme protein declined slowly in the dark and rapidly in the light. These results indicate that fluctuation of the enzymic activity in this organ, both in the light and in the dark, are mediated via changes in the amount of the enzyme protein and not via the action of an inhibitor.     

RBE variation in prostate carcinoma cells in active scanning proton beams: In-vitro measurements in comparison with phenomenological models

PurposeIn-vitro radiobiological studies are essential for modelling the relative biological effectiveness (RBE) in proton therapy. The purpose of this study was to experimentally determine the RBE values in proton beams along the beam path for human prostate carcinoma cells (Du-145). RBE-dose and RBE-LET_d (dose-averaged linear energy transfer) dependencies were investigated and three phenomenological RBE models, i.e. McNamara, Rørvik and Wilkens were benchmarked for this cell line.MethodsCells were placed at multiple positions along the beam path, employing an in-house developed solid phantom. The experimental setup reflected the clinical prostate treatment scenario in terms of field size, depth, and required proton energies (127.2–180.1 MeV) and the physical doses from 0.5 to 6 Gy were delivered. The reference irradiation was performed with 200 kV X-ray beams. Respective (α/β) values were determined using the linear quadratic model and LET_d was derived from the treatment planning system at the exact location of cells.Results and ConclusionIndependent of the cell survival level, all experimental RBE values were consistently higher in the target than the generic clinical RBE value of 1.1; with the lowest RBE value of 1.28 obtained at the beginning of the SOBP. A systematic RBE decrease with increasing dose was observed for the investigated dose range. The RBE values from all three applied models were considerably smaller than the experimental values. A clear increase of experimental RBE values with LET_d parameter suggests that proton LET must be taken into consideration for this low (α/β) tissue.     

Lung cancer risk in mice: analysis of fractionation effects and neutron RBE with a biologically motivated model

Data from Argonne National Laboratory on lung cancer in 15,975 mice with acute and fractionated exposures to gamma rays and neutrons are analyzed with a biologically motivated model with two rate-limiting steps and clonal expansion. Fractionation effects and effects of radiation quality can be explained well by the estimated kinetic parameters. Both an initiating and a promoting action of neutrons and gamma rays are suggested. While for gamma rays the initiating event is described well with a linear dose-rate dependence, for neutrons a nonlinear term is needed, with less effectiveness at higher dose rates. For the initiating event, the neutron RBE compared to gamma rays is about 10 when the dose rate during each fraction is low. For higher dose rates this RBE decreases strongly. The estimated lifetime relative risk for radiation-induced lung cancers from 1 Gy of acute gamma-ray exposure at an age of 110 days is 1.27 for male mice and 1.53 for female mice. For doses less than 1 Gy, the effectiveness of fractionated exposure to gamma rays compared to acute exposure is between 0.4 and 0.7 in both sexes. For lifetime relative risk, the RBE from acute neutrons at low doses is estimated at about 10 relative to acute gamma-ray exposure. It decreases strongly with dose. For fractionated neutrons, it is lower, down to about 4 for male mice.     

Polyamine levels and diamine oxidase activity in hypertrophic heart of spontaneously hypertensive rats and of rats treated with isoproterenol

Polyamine levels and diamine oxidase (EC 1.4.3.6) activity were studied in hypertrophic heart of spontaneously hypertensive rats as well as in the heart of Wistar rats during the development and regression of cardiac hypertrophy induced by isoproterenol administration. In spontaneously hypertensive rats, putrescine content and diamine oxidase activity were higher than those found in normotensive Kyoto-Wistar control rats. During the development of cardiac hypertrophy induced by isoproterenol, there was an increase in polyamine content and diamine oxidase activity. The administration of cycloheximide or actinomycin D prevented the increase in diamine oxidase activity during the first 24 h after isoproterenol administration, demonstrating that the rise in diamine oxidase activity was due to synthesis of new enzyme. Following the cessation of isoproterenol treatment, cardiac hypertrophy regressed and polyamine levels and diamine oxidase activity diminished toward control values. The administration of aminoguanidine to isoproterenol-treated rats caused in the heart an inhibition of diamine oxidase activity that led to an increase in putrescine level beyond the values found in animals given isoproterenol alone. The results suggest that the enhancement of diamine oxidase activity plays a role in the regulation of putrescine level in hypertrophic heart.     

糖脂代谢性疾病模型小鼠肠道菌群特征分析

基于16S rDNA测序研究非酒精性脂肪性肝病(NAFLD)、2型糖尿病(T2D)及动脉粥样硬化(AS)小鼠的肠道菌群特征, 分析上述疾病肠道微生物的异同。

以SPF级C57BL/6J雄鼠为对象, 分别采用高脂饮食制备NAFLD模型, 高脂饮食联合小剂量链脲佐菌素腹腔注射建立T2D模型, ApoE^-/-小鼠高脂饮食诱导AS模型, 另设对照组, 每组10只。采用试剂盒测定小鼠血清中总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)的水平。收集粪便样本, 以Illumina MiSeq测序平台, 采用QIIME2软件对肠道菌群的可分类操作单元(OTUs)数量, Alpha、Beta多样性和菌群多样性指数以及差异菌门、菌属等进行综合分析与评价, 并对肠道菌群代谢功能进行预测。

与对照组小鼠比, T2D组、NAFLD组、AS组血清中TC、TG和LDL-C水平均显著升高, 菌群多样性指数显著降低(F=14.33, P < 0.01), Firmicutes/Bacteroidetes比值逐渐升高; 双歧杆菌属(Bifidobacterium)丰度在AS组、T2D组中显著增加(F=12.15, P < 0.01), 在NAFLD组中显著下降(F=12.15, P < 0.05), 乳杆菌属(Lactobacillus)丰度在NAFLD组、AS组中著降低(F=9.35, P < 0.01), 在T2D组中显著降低。关联分析表明Lactobacillus、Akkermansia等与血脂呈负相关, Faecalibaculum、Blautia等与血脂呈正相关。肠道菌群参与代谢性疾病主要涉及碳水化合物代谢、氨基酸代谢、脂质代谢以及能量代谢等通路。

本研究阐明了NAFLD、T2D、AS肠道微生物组成与变化的共性和个性特征, 为靶向调控肠道微生物治疗代谢性疾病提供科学依据。

     

Xanthine oxidase activity in mouse pancreas: effects of caerulein-induced acute pancreatitis

The development of acute pancreatitis involves a number of pathophysiological changes which result in pancreatic tissue damage. Data from several models of acute pancreatitis suggest that the in vivo conversion of the enzyme xanthine dehydrogenase to xanthine oxidase may cause tissue damage by the subsequent generation of oxygen-derived free radical products. In the present studies, acute pancreatitis was induced in mice by the administration of supramaximal secretory doses of caerulein, a cholecystokinin analogue. Pancreatic xanthine oxidase activity was observed to occur in the dehydrogenase form in both control and treated mice. Artifactual conversion to the oxidase form could be induced by exclusion of 2-mercaptoethanol and phenylmethylsulfonyl fluoride from the buffer during tissue preparation. These data indicate that no significant conversion of xanthine dehydrogenase to oxidase is associated with this model of acute pancreatitis in mice.     

Mitochondria: a sulfhydryl oxidase and fission GTPase connect mitochondrial dynamics with pluripotency in embryonic stem cells

Mitochondria have long been recognized as cellular energy power houses that also regulate cellular redox signaling to arbitrate cell survival. Recent studies of mitochondria in stem cells (SCs) demonstrate that they have critical roles beyond this traditional view. Embryonic (E) SCs, termed pluripotent for their ability to differentiate into all cell types within an organism, maintain a limited number of morphologically undifferentiated (electron translucent and poorly formed cristae) mitochondria. As these cells differentiate, their mitochondria undergo a tightly choreographed gain of number, mass and morphological complexity. Therefore, mechanisms that regulate mitochondrial growth, localization, division and partition must play active roles in the maintenance of pluripotency and execution of differentiation. Aberrant mitochondrial dynamics are associated with a plethora of human disorders, for which SCs hold curative potential. Hence, a comprehensive understanding of the mechanisms that regulate mitochondrial dynamics and function in SCs and their overall relationship to the maintenance of pluripotency is pivotal for the progression of therapeutic regenerative medicine.     

Role of disulfides in biological activity and conformational stability of pig kidney diamine oxidase: evidence for two disulfide states

Six disulfides are found to be present in pig kidney diamine oxidase and all of these are available to reducing agents under nondenaturating conditions. Disulfide reduction with dithiothreitol followed by carbamidomethylation indicated two states of disulfides, each containing three groups, distinguishable by pH dependence. The first group of three disulfides has a functional role in catalytic activity. The another class of three disulfides showed accessibility only at higher pH values and appears to be important in maintaining the three dimensional structure of the molecule. The disulfides for these two activities appear to be independent of each other. Almost similar behaviour was noticed with copper depleted apo-enzyme.     

Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity.

1. Isoelectric focusing studies of human placental diamine oxidase showed the pI value of the active enzyme to be 6.5. This information was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH gradient elution and this, together with affinity chromatography on concanavalin A--Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration of diamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultracentrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx. 1.0 and 1.2g-atom respectively/70000mol.wt. unit. 5. The e.s.r. spectral intensity did not decrease nor did the spectral line shape change when excess of p-dimethylaminomethylbenzylamine was added to the enzyme. 6. Addition of K13CN to the enzyme eliminated the copper e.s.r. signal without affecting the manganese signal. 7. The placental enzyme therefore appears to differ from other amine oxidases in terms of its metal cofactor requirement, molecular weight and substrate specificity, and possible roles in vivo for this enzyme are discussed.     

Protective effect of probiotics on Salmonella infectivity assessed with combined in vitro gut fermentation-cellular models

Background

The alphaproteobacterium Wolbachia pipientis, the most common endosymbiont in eukaryotes, is found predominantly in insects including many Drosophila species. Although Wolbachia is primarily vertically transmitted, analysis of its genome provides evidence for frequent horizontal transfer, extensive recombination and numerous mobile genetic elements. The genome sequence of Wolbachia in Drosophila simulans Riverside (wRi) is available along with the integrated bacteriophages, enabling a detailed examination of phage genes and the role of these genes in the biology of Wolbachia and its host organisms. Wolbachia is widely known for its ability to modify the reproductive patterns of insects. One particular modification, cytoplasmic incompatibility, has previously been shown to be dependent on Wolbachia density and inversely related to the titer of lytic phage. The wRi genome has four phage regions, two WORiBs, one WORiA and one WORiC.

Results

In this study specific primers were designed to distinguish between these four prophage types in wRi, and quantitative PCR was used to measure the titer of bacteriophages in testes, ovaries, embryos and adult flies. In all tissues tested, WORiA and WORiB were not found to be present in excess of their integrated prophages; WORiC, however, was found to be present extrachromosomally. WORiC is undergoing extrachromosomal replication in wRi. The density of phage particles was found to be consistent in individual larvae in a laboratory population. The WORiC genome is organized in conserved blocks of genes and aligns most closely with other known lytic WO phages, WOVitA and WOCauB.

Conclusions

The results presented here suggest that WORiC is the lytic form of WO in D. simulans, is undergoing extrachromosomal replication in wRi, and belongs to a conserved family of phages in Wolbachia.     

Lack of D-amino-acid oxidase activity causes a specific renal aminoaciduria in the mouse

Thin-layer chromatography and amino acid analysis showed that urine of mutant ddY/DAO- mice lacking D-amino-acid oxidase activity contained more serine, proline, alanine and methionine than that of normal ddY/DAO+ mice. Among these four, an increase in alanine was conspicuous. However, the urinary levels of 11 other amino acids and glucose were not different between the ddY/DAO- and ddY/DAO+ mice. Amino acid analysis showed that the plasma levels of serine, proline and methionine were not elevated in the ddY/DAO- mice, though a slight increase in alanine was observed. Genetic crosses showed that aminoaciduria and lack of D-amino-acid oxidase activity were concomitantly transmitted as a set through generations. These results indicated that the lack of enzyme activity caused a specific renal aminoaciduria. Whether this enzyme merely diminishes the D-amino acid load presented for reabsorption, or actually participates catalytically in the reabsorption process, remains undetermined.     

Distribution of diamine oxidase activity and polyamine pattern in bean and soybean seedlings at different stages of germination

Diamine oxidase (DAO, EC 1.4.3.6.) activity and polyamine content were measured in the shoot apex, leaves, epicotyl, cotyledons, hypocotyl and roots of light-grown bean ( Phaseolus vulgaris L. cv. Lingot) and soybean ( Glycine max L. cv. Sakai) seedlings at 3 different stages of germination (5, 8 and 14 days) as well as in embryos and cotyledons from soaked seeds. No DAO activity was detected in embryos and cotyledons of either plants. In bean seedlings DAO activity was only detectable in the shoot apex, primary leaves and cotyledons, while in soybean the activity was only detectable in the hypocotyl and roots. During seedling growth, in both plants, a different pattern of DAO activity was observed. In both species spermidine and spermine were the most abundant polyamines in embryos and cotyledons. Cadaverine, absent in bean, was only detected in soybean embryos. In the seedlings of both plants, increasing gradients of putrescine, spermidine and spermine from base to shoot apex were found. A high concentration of cadaverine was present in soybean hypocotyls and roots. A possible correlation between DAO activity and the endogenous content of the preferential substrate is discussed in relation to the possible involvement of the enzyme in regulating the cellular level of polyamines.     

Association of diamine oxidase and ornithine decarboxylase with maturing cells in rapidly proliferating epithelium

Diamine oxidase and ornithine decarboxylase activities are shown to have a parallel distribution across rat small intestine mucosa; levels of both enzyme activities are sharply higher in mature cells in the villus tip region than in proliferating cells in the crypt areas. Histidine decarboxylase levels were not measurable in the same cell preparations and aromatic-L-amino-acid decarboxylase activity was distributed in an opposite pattern to diamine oxidase and ornithine decarboxylase. The results suggest that intestinal diamine oxidase could be involved with polyamine metabolism. The new findings for ornithine decarboxylase suggest an in vivo role for polyamines in non-proliferative cells; rat small intestinal mucosa may be an excellent model for investigating the function of polyamines in regenerating cells.     

Polyamines and diamine oxidase activity in maternal, embryonal, and fetal tissues of rat after chronic ethanol consumption

The effects of maternal ethanol consumption for 4 weeks before and throughout gestation on polyamine content and diamine oxidase activity of maternal, embryonal and fetal tissues are reported. At the 12th day of pregnancy, a decrease of putrescine in the liver of the mother and marked increases in putrescine, cadaverine and spermidine in embryos were observed. At day 18, putrescine and cadaverine diminished in maternal liver and placenta, and no changes in amine content in fetal liver and brain were found. At day 12, diamine oxidase activity increased in maternal liver and placenta, whereas it greatly diminished in embryos. At day 18, enzyme activity decreased in maternal liver, placenta, fetal liver and brain. These results indicate that chronic ethanol ingestion induces alterations in polyamine concentrations and metabolism in growing and developing tissues during pregnancy that might contribute to the adverse effect of ethanol on conceptual development.     

Development and evaluation of multiple tendon injury models in the mouse

The mouse has proven to be an advantageous animal model system in basic science research focused on aiding in development and evaluation of potential treatments; however, the small size of mouse tendons makes consistent and reproducible injury models and subsequent biomechanical evaluation challenging for studying tendon healing. In this study, we investigated the feasibility and reproducibility of multiple mouse tendon injury models. Our hypothesis was that incisional (using a blade) and excisional (using a biopsy punch) injuries would result in consistent differences in tendon material properties. At 16 weeks of age, 17 C57BL/6 mice underwent surgery to create defects in the flexor digitorum longus, Achilles, or patellar tendon. Each animal received 1-2 full-thickness, central-width incisional or excisional injuries per limb; at least one tendon per limb remained uninjured. The injuries were distributed such that each tendon type had comparable numbers of uninjured, incisionally injured, and excisionally injured specimens. Three weeks after injury, all animals were euthanized and tendons were harvested for mechanical testing. As hypothesized, differences were detected for all three different tendon types at three weeks post-injury. While all models created injuries that produced predictable outcomes, the patellar tendon model was the most consistent in terms of number and size of significant differences in injured tendons compared to native properties, as well as in the overall variance in the data. This finding provides support for its use in fundamental tendon healing studies; however, future work may use any of these models, based on their appropriateness for the specific question under study.