心脏特异表达LMNA^E82K转基因小鼠的建立

目的建立心脏特异表达LMNAE82K转基因小鼠,为研究LMNAE82K与心肌病发病机制的关系提供工具动物。方法把LMNAE82K基因插入α-MHC启动子下游,构建转基因表达载体,显微注射法建立C57BL/6JLMNAE82K转基因小鼠,PCR鉴定转基因小鼠的基因型,采用Western Blot鉴定LMNAE82K在心脏组织中的表达,H＆E染色和超声检测转基因小鼠心脏的病理改变。结果建立了2个心脏组织特异表达LMNAE82K的转基因小鼠品系。超声检查显示转基因小鼠心室壁变薄,收缩期容积和舒张期容积增加,射血分数及短轴缩短率降低。结论LMNAE82K转基因小鼠具有LMNAE82K引起的家族性扩心病有类似的病理变化,为研究LMNAE82K与心肌病发病机制的关系的研究提供了有价值的疾病动物模型。     

Influence of Pistachios on Performance and Exercise-Induced Inflammation,Oxidative Stress,Immune Dysfunction,and Metabolite Shifts in Cyclists: A Randomized,Crossover Trial

Objectives

Pistachio nut ingestion (3 oz./d, two weeks) was tested for effects on exercise performance and 21-h post-exercise recovery from inflammation, oxidative stress, immune dysfunction, and metabolite shifts.

Methods

Using a randomized, crossover approach, cyclists (N = 19) engaged in two 75-km time trials after 2-weeks pistachio or no pistachio supplementation, with a 2-week washout period. Subjects came to the lab in an overnight fasted state, and ingested water only or 3 oz. pistachios with water before and during exercise. Blood samples were collected 45 min pre-exercise, and immediately post-, 1.5-h post-, and 21-h post-exercise, and analyzed for plasma cytokines, C-reactive protein (CRP), F₂-isoprostanes (F₂-IsoP), granulocyte phagocytosis (GPHAG) and oxidative burst activity (GOBA), and shifts in metabolites.

Results

Performance time for the 75-km time trial was 4.8% slower under pistachio conditions (2.84±0.11 and 2.71±0.07 h, respectively, P = 0.034). Significant time effects were shown for plasma cytokines, CRP, F₂-IsoP, GPHAG, and GOBA, with few group differences. Metabolomics analysis revealed 423 detectable compounds of known identity, with significant interaction effects for 19 metabolites, especially raffinose, (12Z)-9,10-Dihydroxyoctadec-12-enoate (9,10-DiHOME), and sucrose. Dietary intake of raffinose was 2.19±0.15 and 0.35±0.08 mg/d during the pistachio and no pistachio periods, and metabolomics revealed that colon raffinose and sucrose translocated to the circulation during exercise due to increased gut permeability. The post-exercise increase in plasma raffinose correlated significantly with 9,10-DiHOME and other oxidative stress metabolites.

Conclusions

In summary, 2-weeks pistachio nut ingestion was associated with reduced 75-km cycling time trial performance and increased post-exercise plasma levels of raffinose, sucrose, and metabolites related to leukotoxic effects and oxidative stress.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01821820","term_id":"NCT01821820"}}NCT01821820     

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.     

Cessation of neutrophil influx in C5a-induced acute experimental arthritis is associated with loss of chemoattractant activity from the joint space

Neutrophil emigration is a critical component of the inflammatory process and is generally thought to play a role in host defense as well as in the tissue injury that often accompanies inflammation. Most inflammatory reactions exhibit a sequence of emigrating cell types, thus clearly demonstrating that the neutrophil influx eventually ceases and that the neutrophils are then removed from the lesion. It has been our premise that in order to understand the processes that lead to the progressive inflammatory reactions that underly so many disease processes, it is important to determine the mechanism by which the "normal" inflammatory response resolves. The purpose of this study was to identify the time of cessation of neutrophil influx in experimental arthritis induced by the injection of C5 fragments (C5f) and to investigate mechanisms underlying the cessation process. The migration of i.v. delivery pulses to inflamed joints was assessed by lavage of the joint space and by external scintigraphy. We found no evidence for the development of inhibitory systems against chemotactic factors or "desensitization" of the inflamed site, because a second injection of C5f into joints which had been injected previously with C5f resulted in enhancement rather than inhibition of migration. Neither was evidence found for altered tissue barriers to migration or for desensitization of neutrophils as possible explanations for cessation of influx. The major mechanism appeared to be a loss of chemoattractant activity in the joint space between 2 h and 6 h after C5f injection which was detected by transfer into a fresh joint. Radiolabeled C5a des-Arg had a t1/2 of disappearance from the joint of less than 1 h, which suggested that the transferred chemoattractant must, in part, have been due to the generation of new chemotaxins by C5f injection. These observations suggest that continued generation of chemoattractants or failure of their subsequent removal may be mechanisms leading to persistent neutrophil influx in chronic inflammation.     

Validation of signature polarlipid fatty acid biomarkers for alkane-utilizing bacteria in soils and subsurface aquifer materials

Abstract Extractable cell membrane-derived polarlipid ester-linked fatty acids (PLFA) obtained from aerated soils gassed with methane or propane and from methane- and propane-oxidizing bacteria isolated from the soils were analyzed by capillary gas chromatography/mass spectrometry. Exposure of aerated soils to methane resulted in the formation of a high proportion of an unusual 18-carbon mono-unsaturated PLFA, 18:lw8c. High proportions of this fatty acid biomarker are found in monocultures from this soil grown in minimal media with methane. This PLFA has been previously established as associated with authentic type II methane-oxidizing bacteria. The microbiota in aerated soils exposed to hydrocarbons containing propane, formed a suite of PLFA characterized by high proportions of a 16-carbon mono-unsaturated acid, 16:lw6c, and an 18-carbon saturated fatty acid with an additional methyl branch at the 10 position, 10 Me 18:0. This PLFA pattern has been detected in several monocultures enriched from the soil with propane-amended minimal media. The correspondence of high proportions of these unusual mono-unsaturated PLFA in the isolated monocultures and in situ in the soils after stimulation with the appropriate hydrocarbon is a strong validation of the utility of these biomarkers in defining the community structure of the surface soil microbial community.     

Sensitivity of genetically engineered organisms to selective media.

Eighteen strains of Escherichia coli used in genetic studies were tested for their ability to grow on several selective media. Highest recoveries were obtained with m-T7 agar. The SOS system, particularly the recA gene, may play some role in the sensitivity of E. coli to selective agents. These results may be important in the selection of media used to detect genetically engineered organisms released into the environment.     

Molecular differentiation of the heterocystous cyanobacteria,Nostoc and Anabaena,based on complete NifD sequences

The segregation of Nostoc and Anabaena into separate genera has been debated for some time. The nitrogen fixation gene nifD was completely sequenced from representatives of these genera and analyzed phylogenetically, by using the representatives of other genera of the heterocystous cyanobacteria as outgroups. We were clearly able to differentiate between Nostoc and Anabaena in all analyses used. Our data suggest that Nostoc and Anabaena should remain as separate genera. Received: 16 November 2001 / Accepted: 14 December 2001