Antibody fragments selected by phage display against the nuclear localization signal of the HIV-1 Vpr protein inhibit nuclear import in permeabilized and intact cultured cells

The HIV-1 Vpr protein harbors a nuclear localization signal in its N-terminal domain. A peptide bearing this domain and which is designated VprN has been used as a target to screen a phage display single chain Fv (scFv) library. Here we report the isolation of anti-VprN scFv fragments from this library. The purified scFv fragments were able to bind the VprN peptide in an ELISA-based system and to inhibit VprN-mediated nuclear import in permeabilized as well as in intact microinjected cells. Furthermore, the anti-VprN scFv fragments recognized the full-length recombinant Vpr protein and inhibited its nuclear import. The same scFv fragments did not inhibit nuclear import mediated by the nuclear localization signal of the SV40 large T-antigen demonstrating a specific effect. The use of the described inhibitory anti-VprN scFv fragments to study nuclear import of viral karyophilic proteins and their therapeutic potential is discussed.     

Ethiopia: An Emerging Family Planning Success Story

From 1990 to 2011, contraceptive use in Ethiopia increased ninefold and the total fertility rate fell from 7.0 to 4.8. These are two dramatic illustrations of a family planning success story that has emerged over the last two decades and is still emerging. What are the main elements of this success? We posit that the four most significant factors are: political will, generous donor support, nongovernmental and public–private partnerships, and the government's establishment of a network of health extension workers. In this study, we look at these factors and how their interaction increased the proportion of women having both the desire to use and ability to access contraceptives. Also highlighted are some of the key lessons learned in Ethiopia that are relevant to other African countries interested in emulating the country's success.     

Glutamate Signaling via the AMPAR Subunit GluR4 Regulates Oligodendrocyte Progenitor Cell Migration in the Developing Spinal Cord

Oligodendrocyte progenitor cells (OPCs) are specified from discrete precursor populations during gliogenesis and migrate extensively from their origins, ultimately distributing throughout the brain and spinal cord during early development. Subsequently, a subset of OPCs differentiates into mature oligodendrocytes, which myelinate axons. This process is necessary for efficient neuronal signaling and organism survival. Previous studies have identified several factors that influence OPC development, including excitatory glutamatergic synapses that form between neurons and OPCs during myelination. However, little is known about how glutamate signaling affects OPC migration before myelination. In this study, we use in vivo, time-lapse imaging in zebrafish in conjunction with genetic and pharmacological perturbation to investigate OPC migration and myelination when the GluR4A ionotropic glutamate receptor subunit is disrupted. In our studies, we observed that gria4a mutant embryos and larvae displayed abnormal OPC migration and altered dorsoventral distribution in the spinal cord. Genetic mosaic analysis confirmed that these effects were cell-autonomous, and we identified that voltage-gated calcium channels were downstream of glutamate receptor signaling in OPCs and could rescue the migration and myelination defects we observed when glutamate signaling was perturbed. These results offer new insights into the complex system of neuron-OPC interactions and reveal a cell-autonomous role for glutamatergic signaling in OPCs during neural development.SIGNIFICANCE STATEMENT The migration of oligodendrocyte progenitor cells (OPCs) is an essential process during development that leads to uniform oligodendrocyte distribution and sufficient myelination for central nervous system function. Here, we demonstrate that the AMPA receptor (AMPAR) subunit GluR4A is an important driver of OPC migration and myelination in vivo and that activated voltage-gated calcium channels are downstream of glutamate receptor signaling in mediating this migration.     

Evidence for host-driven selection of the HIV type 1 vpr gene in vivo during HIV disease progression in a transfusion-acquired cohort

An epidemiologically linked HIV-1-infected cohort, in which a nonprogressor donor infected two recipients who progressed to AIDS, was examined. Sequence analysis, over time, of HIV-1 vpr gene quasispecies from uncultured peripheral blood cells revealed an insertion of arginine at position 90 altering a highly conserved C-terminal motif, believed to play a role in Vpr nuclear targeting. Full genome analysis from each patient showed no gene defects in other gene regions, implying that the mutational selection was unique to the vpr gene. A detailed analysis of the vpr quasispecies showed very little amino acid diversity in the nonprogressing donor, whereas, following viral transmission, the amino acid diversity increased dramatically over time in tandem with disease progression in the two recipients. Although the R insertion at position 90 was present in all three individuals, the variable degree of additional amino acid changes over time may have influenced HIV disease in the nonprogressor donor and the two progressing recipients. These data provide the first evidence in favor of vpr gene evolution over time, which was host-driven. The status of the nonprogressing donor was consistent with a highly protective B-57 HLA type, which was absent in the two progressing recipients, implying a role for host HLA type and other immunologic selective pressures in vpr gene selection in vivo.     

缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性的变化

目的:制备大鼠在体缺血再灌注模型,观察缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性的变化。方法:实验于2005-03/2006-10在解放军沈阳军区总医院医学实验动物中心和全军心血管研究所实验室完成。实验分组:选用健康雌性SD大鼠36只,根据预适应程序分为第1,2,3次缺血,第1,2,3次再灌注,每一时间点6只大鼠。实验过程:用手术套管法造成左冠状动脉主干缺血及再灌注。所有实验动物在实验程序结束后,取出心脏迅速置液氮保存备用。实验评估:用放射免疫法测环磷酸腺苷水平,生化法测环磷酸腺苷依赖蛋白激酶活性变化。结果:36只大鼠均进入结果分析。①环磷酸腺苷含量:第1次再灌注组低于第1次缺血组[(0.325±0.015),(0.395±0.024)pmol/g,t=6.06,P<0.001],第2次再灌注组低于第2次缺血组[(0.523±0.017),(0.708±0.067)pmol/g,t=6.56,P<0.001],第3次再灌注组低于第3次缺血组[(0.567±0.031),(0.712±0.038)pmol/g,t=7.24,P<0.001]。②环磷酸腺苷依赖蛋白激酶活性:第1次再灌注组低于第1次缺血组[(10.115±1.000),(16.351±0.849)pkat/g,t=11.12,P<0.001],第2次再灌注组低于第2次缺血组[(11.877±2.213),(14.869±0.619)pkat/g,t=3.31,P<0.01],第3次再灌注组低于第3次缺血组[(11.745±0.987),(14.766±0.329)pkat/g,t=7.09,P<0.001]。③缺血预处理程序中心肌环磷酸腺苷含量及环磷酸腺苷依赖蛋白激酶活性随缺血及再灌注呈周期性波动。在5min缺血预处理时表现为明显增高,而在间隔的再灌注程序中恰呈相反改变,有明显下降的趋势。结论:环磷酸腺苷及环磷酸腺苷依赖蛋白激酶的周期性波动变化可能是激发心肌缺血预处理的机制之一,环磷酸腺苷可能在预处理保护作用中起一些作用。     

Effect of opioid active therapeutics on the ascending reflex pathway in the rat ileum

For a long time therapeutic agents that interact with opioid receptors have been used in antidiarrheal therapy. The action of the opioid active substances on motility and transit have already been characterized; however, their effects on myenteric reflexes and their possible luminal action have not yet been investigated. Loperamide, fedotozine and beta-casomorphin-4, as well as the casomorphin-analogue beta-CM-4027, are, or have been, suggested as therapeutic agents and were studied in the isolated rat ileum for their effect on the ascending reflex pathway. beta-CM-4027 > fedotozine > loperamide > beta-casomorphin-4 caused a concentration-dependent inhibition of the ascending contractile reflex response with an IC(50)of 1.4x10(-7)M, 1.5x10(-6)M, 4.1x10(-6)M and 4.5x10(-6)M respectively. At the same time as the oral contractile reflex response was inhibited, all four opioid agonists (CM-4027 > beta-casomorphin-4 > fedotozine > loperamide) increased the latency of the reflex response. Both effects were blocked by naloxone, indicating the involvement of opioid receptors. These results demonstrate that opioid-active drugs and substances modify the peristaltic reflex by reducing the efficacy of the reflex response and modulating the timing of the reflex pathway. In a second series of experiments, luminal application of opioid-active drugs was compared with serosal application. beta-casomorphine-4 caused a concentration-dependent inhibition of the oral reflex response with an IC(50)of 3x10(-3)M which was 750 times higher than after serosal application. In contrast, a stable and highly selective kappa opioid agonist (U-50,488), which caused potent inhibition upon serosal application (IC(50): 2.3x10(-7)M), showed no inhibitory effect after luminal application up to a concentration of 10(-2)M. Thus casomorphins could have a local effect on the gut wall with no need for systemic absorption. This might be used for a possible therapeutic application.