Regulation of endothelial nitric oxide synthase by the actin cytoskeleton

In the present study, the association ofendothelial nitric oxide synthase (eNOS) with the actin cytoskeleton inpulmonary artery endothelial cells (PAEC) was examined. We found thatthe protein contents of eNOS, actin, and caveolin-1 were significantly higher in the caveolar fraction of plasma membranes than in the noncaveolar fraction of plasma membranes in PAEC. Immunoprecipitation of eNOS from lysates of caveolar fractions of plasma membranes in PAECresulted in the coprecipitation of actin, and immunoprecipitation ofactin from lysates of caveolar fractions resulted in thecoprecipitation of eNOS. Confocal microscopy of PAEC, in which eNOS waslabeled with fluorescein, F-actin was labeled with Texasred-phalloidin, and G-actin was labeled with deoxyribonuclease Iconjugated with Texas red, also demonstrated an association betweeneNOS and F-actin or G-actin. Incubation of purified eNOS with purifiedF-actin and G-actin resulted in an increase in eNOS activity. Theincrease in eNOS activity caused by G-actin was much higher than thatcaused by F-actin. Incubation of PAEC with swinholide A, an actinfilament disruptor, resulted in an increase in eNOS activity, eNOSprotein content, and association of eNOS with G-actin and in a decrease in the association of eNOS with F-actin. The increase in eNOS activitywas higher than that in eNOS protein content in swinholide A-treatedcells. In contrast, exposure of PAEC to phalloidin, an actin filamentstabilizer, caused decreases in eNOS activity and association of eNOSwith G-actin and increases in association of eNOS with F-actin. Theseresults suggest that eNOS is associated with actin in PAEC and thatactin and its polymerization state play an important role in theregulation of eNOS activity.

     

Evidence for contractile protein translocation in macrophage spreading, phagocytosis, and phagolysosome formation

Macrophage pseudopodia that surround objects during phagocytosis contain a meshwork of actin filaments and exclude organelles. Between these pseudopodia at the base of developing phagosomes, the organelle exclusion ceases, and lysosomes enter the cell periphery to fuse with the phagosomes. Macrophages also extend hyaline pseudopodia on the surface of nylon wool fibers and secrete lysosomal enzymes into the extracellular medium instead of into phagosomes. To analyze biochemically these concurrent alterations in cytoplasmic architecture, we allowed rabbit lung macrophages to spread on nylon wool fibers and then subjected the adherent cells to shear. This procedure caused the selective release of β-glucoronidase into the extracellular medium and yielded two fractions, cell bodies and isolated pseudopod blebs resembling podosomes, which are plasma-lemma-bounded sacs of cortical cytoplasm. Cytoplasmic extracts of the cell bodies eluted from nylon fibers contained two-thirds less actin-binding protein and myosin, and approximately 20 percent less actin and two-thirds of the other two proteins were accounted for in podosomes. The alterations in protein composition correlated with assays of myosin-associated EDTA-activated adenosine triphosphatase activity, and with a diminution in the capacity of extracts of nylon wool fiber-treated cell bodies to gel, a property dependent on the interaction between actin-binding protein and F-actin. However, the capacity of the remaining actin in cell bodies to polymerize did not change. We propose that actin-binding protein and myosin are concentrated in the cell cortex and particularly in pseudopodia where prominent gelation and syneresis of actin occur. Actin in the regions from which actin-binding protein and myosin are displaced disaggregates without depolymerizing, permitting lysosomes to gain access to the plasmalemma. Translocation of contractile proteins could therefore account for the concomitant differences in organelle exclusion that characterize phagocytosis.     

Food resource allocation patterns in lactating females in a long-term selection experiment for litter size in mice

Resource allocation patterns, as quantified by residual food intake (RFI), and the consequences for offspring development were investigated during lactation in 96 females of a mouse line selected for 104 generations for high litter size at birth (S-line) and in 87 females of a non-selected control line (C-line). Litters of 45 C-line dams (Cs) and 48 S-line dams (Ss) were standardised (s) at birth; other dams (ns) supported total number of pups born (Cns and Sns, respectively). RFI during lactation was significantly lower in Sns-dams than in C-line dams and Sns-dams. After weaning Sns-dams seemed to be able to restore the negative resource situation. Sns-pups were about 25% less mature than Cns-pups at all times. Maturity was similar for Cs- and Ss-pups from 2 d in lactation on, and about 18% and 53% higher than Cns- and Sns-pups. The pre-weaning mortality rate was significantly higher in Sns-litters (35.6 ± 2.76) than in Cns-litters (4.95 ± 2.23). The results suggest that S-line dams allocated considerably more resources to maintenance of offspring than C-line dams. This was insufficient to provide the offspring with an adequate amount of resources, resulting in reduced pup development and increased pre-weaning mortality rates.     

硫氧还蛋白抗体柱的制备

目的:探索硫氧还蛋白(Trx)抗体柱对Trx融合蛋白纯化的可行性。方法与结果:对含有Trx基因的质粒表达载体pTrxFus进行改造,在Trx读框之后加入6×His序列,并在大肠杆菌中表达C端带有6×His标签的Trx,经Ni2+柱亲和纯化后制备多克隆抗体;把经蛋白A纯化后的抗体偶联在溴化氰活化的琼脂糖凝胶上,制成Trx抗体柱;用此抗体柱纯化与Trx融合表达的豇豆胰蛋白酶抑制剂(CpTI),SDS-PAGE结果显示获得了纯度较高的Trx-CpTI。结论:用Trx抗体制成的免疫亲和层析柱可以有效纯化Trx融合蛋白。     

Disrupting the CH1 domain structure in the acetyltransferases CBP and p300 results in lean mice with increased metabolic control

Opposing activities of acetyltransferases and deacetylases help regulate energy balance. Mice heterozygous for the acetyltransferase CREB binding protein (CBP) are lean and insulin sensitized, but how CBP regulates energy homeostasis is unclear. In one model, the main CBP interaction with the glucagon-responsive factor CREB is not limiting for liver gluconeogenesis, whereas a second model posits that Ser436 in the CH1 (TAZ1) domain of CBP is required for insulin and the antidiabetic drug metformin to inhibit CREB-mediated liver gluconeogenesis. Here we show that conditional knockout of CBP in liver does not decrease fasting blood glucose or gluconeogenic gene expression, consistent with the first model. However, mice in which the CBP CH1 domain structure is disrupted by deleting residues 342-393?(ΔCH1) are lean and insulin sensitized, as are p300ΔCH1 mutants. CBP(ΔCH1/ΔCH1) mice remain metformin responsive. An intact CH1 domain is thus necessary for normal energy storage, but not for the blood glucose-lowering actions of insulin and metformin.     

Ultrasound-Triggered Phase Transition Sensitive Magnetic Fluorescent Nanodroplets as a Multimodal Imaging Contrast Agent in Rat and Mouse Model

Ultrasound-triggered phase transition sensitive nanodroplets with multimodal imaging functionality were prepared via premix Shirasu porous glass (SPG) membrane emulsification method. The nanodroplets with fluorescence dye DiR and SPIO nanoparticles (DiR-SPIO-NDs) had a polymer shell and a liquid perfluoropentane (PFP) core. The as-formed DiR-SPIO-NDs have a uniform size of 385±5.0 nm with PDI of 0.169±0.011. The TEM and microscopy imaging showed that the DiR-SPIO-NDs existed as core-shell spheres, and DiR and SPIO nanoparticles dispersed in the shell or core. The MTT and hemolysis studies demonstrated that the nanodroplets were biocompatible and safe. Moreover, the proposed nanodroplets exhibited significant ultrasound-triggered phase transition property under clinical diagnostic ultrasound irradiation due to the vaporization of PFP inside. Meanwhile, the high stability and R₂ relaxivity of the DiR-SPIO-NDs suggested its applicability in MRI. The in vivo T₂-weighted images of MRI and fluorescence images both showed that the image contrast in liver and spleen of rats and mice model were enhanced after the intravenous injection of DiR-SPIO-NDs. Furthermore, the ultrasound imaging (US) in mice tumor as well as MRI and fluorescence imaging in liver of rats and mice showed that the DiR-SPIO-NDs had long-lasting contrast ability in vivo. These in vitro and in vivo findings suggested that DiR-SPIO-NDs could potentially be a great MRI/US/fluorescence multimodal imaging contrast agent in the diagnosis of liver tissue diseases.