HIV gp120- and methamphetamine-mediated oxidative stress induces astrocyte apoptosis via cytochrome P450 2E1

HIV-1 glycoprotein 120 (gp120) is known to cause neurotoxicity via several mechanisms including production of proinflammatory cytokines/chemokines and oxidative stress. Likewise, drug abuse is thought to have a direct impact on the pathology of HIV-associated neuroinflammation through the induction of proinflammatory cytokines/chemokines and oxidative stress. In the present study, we demonstrate that gp120 and methamphetamine (MA) causes apoptotic cell death by inducing oxidative stress through the cytochrome P450 (CYP) and NADPH oxidase (NOX) pathways. The results showed that both MA and gp120 induced reactive oxygen species (ROS) production in concentration- and time-dependent manners. The combination of gp120 and MA also induced CYP2E1 expression at both mRNA (1.7±0.2- and 2.8±0.3-fold in SVGA and primary astrocytes, respectively) and protein (1.3±0.1-fold in SVGA and 1.4±0.03-fold in primary astrocytes) levels, suggesting the involvement of CYP2E1 in ROS production. This was further confirmed by using a selective inhibitor of CYP2E1, diallylsulfide (DAS), and CYP2E1 knockdown using siRNA, which significantly reduced ROS production (30–60%). As the CYP pathway is known to be coupled with the NOX pathway, including Fenton–Weiss–Haber (FWH) reaction, we examined whether the NOX pathway is also involved in ROS production induced by either gp120 or MA. Our results showed that selective inhibitors of NOX, diphenyleneiodonium (DPI), and FWH reaction, deferoxamine (DFO), also significantly reduced ROS production. These findings were further confirmed using specific siRNAs against NOX2 and NOX4 (NADPH oxidase family). We then showed that gp120 and MA both induced apoptosis (caspase-3 activity and DNA lesion using TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) assay) and cell death. Furthermore, we showed that DAS, DPI, and DFO completely abolished apoptosis and cell death, suggesting the involvement of CYP and NOX pathways in ROS-mediated apoptotic cell death. In conclusion, this is the first report on the involvement of CYP and NOX pathways in gp120/MA-induced oxidative stress and apoptotic cell death in astrocytes, which has clinical implications in neurodegenerative diseases, including neuroAIDS.     

A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes

A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.     

Functional mimetic of the G-protein coupled receptor CXCR4 on a soluble antibody scaffold

G-protein coupled receptors (GPCRs) constitute major drug targets due to their involvement in critical biological functions and pathophysiological disorders. The leading challenge in their structural and functional characterization has been the need for a lipid environment to accommodate their hydrophobic cores. Here, we report an antibody scaffold mimetic (ASM) platform where we have recapitulated the extracellular functional domains of the GPCR, C-X-C chemokine receptor 4 (CXCR4) on a soluble antibody framework. The engineered ASM molecule can accommodate the N-terminal loop and all three extracellular loops of CXCR4. These extracellular features are important players in ligand recruitment and interaction for allostery and signal transduction. Our study shows that ASM^CXCR4 can be recognized by the anti-CXCR4 antibodies, MEDI3185, 2B11, and 12G5, and that ASM^CXCR4 can bind the HIV-1 glycoprotein ligand gp120, and the natural chemokine ligand SDF-1α. Further, we show that ASM^CXCR4 can competitively inhibit the SDF-1α signaling pathway, and be used as an immunogen to generate CXCR4-specific antibodies. This platform will be useful in the study of GPCR biology in a soluble receptor context for evaluating its extracellular ligand interactions.     

Hematopoietic potential of the pre-fusion allantois

We previously showed that the fetal component of the placenta has a vigorous hematopoietic activity. Whether this organ is an environmental niche where hematopoietic stem cells (HSC) proliferate and become committed to various lineages, or whether it is also a site for HSC emergence, was left open. This issue can be addressed only if the components that will give rise to the placenta are tested prior to vascularization. The fetal part of the placenta forms through the fusion of the allantois and the chorionic plate around the stage of 7 somite pairs. The allantois, a mesodermal rudiment that provides fetal blood vessels to the placenta, was retrieved before fusion. We found in this rudiment expression of CD41, a known marker of early embryonic hematopoietic progenitors. c-Kit encoding a progenitor specific receptor was also expressed. Significantly, as early as the 1-2 somite stage, the allantois yielded erythroid, myeloid and multipotent clonogenic progenitors, when pre-cultured in toto prior to seeding in a semisolid medium. These results provide evidence that the allantois has hematopoietic potential per se. Whether this potential also involves the ability to produce HSC is still to be determined.     

BAY 41-2272防治大鼠Thy-1肾炎的实验研究

目的:观察比较BAY41-2272和己酮可可碱(PTX)这两种增强环磷酸鸟苷(cGMP)活性的物质对大鼠Thy-1诱导的进展性肾病模型的疗效,探讨肾纤维化治疗的新途径。方法:成功诱导Thy-1肾炎后,将大鼠分为四组:即肾纤维化组(RF组)、BAY 41-2272治疗组、PTX治疗组和对照组。治疗15周后,分别检测各组大鼠血清肌酐和尿蛋白排泄量,肾组织病理和免疫组化方法检测肾小球/小管间质基质堆积程度和ED1/PCNA阳性细胞数,检测肾小球和皮质转化生长因子TGF-β1、纤粘蛋白(fi- bronectin)、基质金属蛋白酶抑制物(TIMP-1)等蛋白能表达水平,ELISA方法检测肾小球/小管间质细胞的基础、刺激后cGMP水平。RT-PCR方法检测内皮细胞一氧化氮合成酶、α1/β1可溶性鸟苷酸环化酶mRNA水平。结果:观察结束时,BAY41-2272显著降低大鼠尿蛋白排泄和血肌酐水平,显著减轻肾小球/小管间质基质堆积程度,降低巨噬细胞浸润数目和TGF-β1、fibronectin表达水平,而PTX治疗仅产生轻微的变化。肾纤维化组大鼠的小管间质sGC mRNA、NO刺激的cGMP水平显著高于对照组,而肾小球的则较对照组降低;BAY41-2272治疗后肾小球/小管间质sGC mRNA、刺激后cGMP水平可显著提高,而PTX治疗的只有轻度提高而无统计学意义。结论:BAY41-2272治疗可显著改善肾组织NO-cGMP信号通路转导和cGMP的活性,可有效的延缓肾纤维化的进展,而PTX疗效则相对很弱。采用BAY41-2272提高cGMP活性的方案可能为肾纤维化提供新颖、极具前景的治疗策略。     

Dynamic domains and geometrical properties of HIV-1 gp120 during conformational changes induced by CD4 binding

The HIV-1 gp120 exterior envelope glycoprotein undergoes a series of conformational rearrangements while sequentially interacting with the receptor CD4 and coreceptor CCR5 or CXCR4 on the surface of host cells to initiate virus entry. Both the crystal structures of the HIV-1 gp120 core bound by the CD4 and antigen 17b, and the SIV gp120 core pre-bound by the CD4 are known. We have performed dynamic domain studies on the homology models of the CD4-bound and unliganded HIV-1 gp120 with modeled V3 and V4 loops to explore details of conformational changes, hinge axes, and hinge bending regions in the gp120 structures upon CD4 binding. Four dynamic domains were clustered and intricately motional modes for domain pairs were discovered. Together with the detailed comparative analyses of geometrical properties between the unliganded and liganded gp120 models, an induced fit model was proposed to explain events accompanying the CD4 engagement to the gp120, which provided new insight into the dynamics of the molecular induced binding mechanism that complements the molecular dynamics and crystallographic studies.     

CCR5 interactions with the variable 3 loop of gp120

The G-protein coupled receptor CCR5 functions pathologically as the primary co-receptor for macrophage tropic (R5) strains of HIV-1. The interactions responsible for co-receptor activity are unknown. Molecular-dynamics simulations of the extracellular and adjacent transmembrane domains of CCR5 were performed with explicit solvation utilizing a rhodopsin-based homology model. The functional unit of co-receptor binding was constructed via docking and molecular-dynamics simulation of CCR5 and the variable 3 loop of gp120, which is a dominant determinant of co-receptor utilization. The variable 3 loop was demonstrated to interact primarily with the amino terminus and the second extracellular loop of CCR5, providing novel structural information regarding the co-receptor-binding site. Alanine mutants that alter chemokine binding and co-receptor activity were examined. Molecular-dynamics simulations with and without the variable 3 loop of gp120 were able to rationalize the activities of these mutants successfully, providing support for the proposed model. Based on these results, the global complex of CCR5, gp120 including the V3 loop and CD4, was investigated. The utilization of computational analysis, in combination with molecular biological data, provides a powerful approach for understanding the use of CCR5 as a co-receptor by HIV-1.     

Sequence of the immunoregulatory early region 3 and flanking sequences of adenovirus type 35

Adenovirus type 35 (Ad35) is an important pathogen in immunosuppressed individuals such as AIDS patients and bone marrow transplant recipients. Ad35, a member of Ad subgroup B, differs with respect to pathogenic properties from the more fully characterized subgroup C Ad, such as Ad2 and Ad5. One region of human Ad which varies between subgroups and which may influence Ad pathogenesis is early region 3 (E3), a region which appears to modulate the immune response to Ad infection. In order to begin to characterize the differences between the Ad35 E3 and the E3 of other Ad, the complete DNA sequence of the Ad35 E3 promoter and coding sequence along with two flanking structural proteins, pVIII and fiber, has been determined. Ad35 contains open reading frames which are unique to the subgroup B Ad in addition to the four characterized immunoregulatory proteins encoded by the subgroup C Ad. Further evaluation of the sequence of one of these proteins, 18.5K, which is the class-I major histocompatibility complex (MHC) binding protein of 18.5 kDa, demonstrates that the amino acid sequence of this Ad2 gp19K homologue fits a proposed model of gp19K-MHC interaction. Analysis of promoter sequences demonstrates that an NF-κB site found in the subgroup C E3 promoter is absent from the Ad35 E3 promoter. In addition, the fiber genes of Ad35 and other subgroup B Ad have been shown to diverge in an unexpected way, yielding three clusters of fiber homology.