HIV-1 infection is associated with a progressive loss of T cell functional capacity and reduced responsiveness to antigenic stimuli. The mechanisms underlying T cell dysfunction in HIV-1/AIDS are not completely understood. Multiple studies have shown that binding of program death ligand 1 (PD-L1) on the surface of monocytes and dendritic cells to PD-1 on T cells negatively regulates T cell function. Here we show that neutrophils in the blood of HIV-1-infected individuals express high levels of PD-L1. PD-L1 is induced by HIV-1 virions, TLR-7/8 ligand, bacterial lipopolysaccharide (LPS), and IFNα. Neutrophil PD-L1 levels correlate with the expression of PD-1 and CD57 on CD4+ and CD8+ T cells, elevated levels of neutrophil degranulation markers in plasma, and increased frequency of low density neutrophils (LDNs) expressing the phenotype of granulocytic myeloid-derived suppressor cells (G-MDSCs). Neutrophils purified from the blood of HIV-1-infected patients suppress T cell function via several mechanisms including PD-L1/PD-1 interaction and production of reactive oxygen species (ROS). Collectively, the accumulated data suggest that chronic HIV-1 infection results in an induction of immunosuppressive activity of neutrophils characterized by high expression of PD-L1 and an inhibitory effect on T cell function.

Recent observational studies indicate an association between the use of hormonal contraceptives and acquisition and transmission of HIV-1. The biological and immunological mechanisms underlying the observed association are unknown. Depot medroxyprogesterone acetate (DMPA) is a progestin-only injectable contraceptive that is commonly used in regions with high HIV-1 prevalence. Here we show that medroxyprogesterone acetate (MPA) suppresses the production of key regulators of cellular and humoral immunity involved in orchestrating the immune response to invading pathogens. MPA inhibited the production of interferon (IFN)-γ, IL-2, IL-4, IL-6, IL-12, TNFα, macrophage inflammatory protein-1α (MIP-1α), and other cytokines and chemokines by peripheral blood cells and activated T cells and reduced the production of IFNα and TNFα by plasmacytoid dendritic cells in response to Toll-like receptor-7, -8, and -9 ligands. Women using DMPA displayed lower levels of IFNα in plasma and genital secretions compared with controls with no hormonal contraception. In addition, MPA prevented the down-regulation of HIV-1 coreceptors CXCR4 and CCR5 on the surface of T cells after activation and increased HIV-1 replication in activated peripheral blood mononuclear cell cultures. The presented results suggest that MPA suppresses both innate and adaptive arms of the immune system resulting in a reduction of host resistance to invading pathogens.

Recombinant membrane proteins in Escherichia coli are either expressed at relatively low level in the cytoplasmic membrane or they accumulate as inclusion bodies. Here, we report that the abundant over-production of subunit b of E. coli

The six-subunit origin recognition complex (ORC) is a DNA replication initiator protein in eukaryotes that defines the localization of the origins of replication. We report here that the smallest Drosophila ORC subunit, Orc6, is a DNA binding protein that is necessary for the DNA binding and DNA replication functions of ORC. Orc6 binds DNA fragments containing Drosophila origins of DNA replication and prefers poly(dA) sequences. We have defined the core replication domain of the Orc6 protein which does not include the C-terminal domain. Further analysis of the core replication domain identified amino acids that are important for DNA binding by Orc6. Alterations of these amino acids render reconstituted Drosophila ORC inactive in DNA binding and DNA replication. We show that mutant Orc6 proteins do not associate with chromosomes in vivo and have dominant negative effects in Drosophila tissue culture cells. Our studies provide a molecular analysis for the functional requirement of Orc6 in replicative functions of ORC in Drosophila and suggest that Orc6 may contribute to the sequence preferences of ORC in targeting to the origins.Eukaryotic cells duplicate their genomes with remarkable precision during the course of growth and division. This process depends on stringent regulatory molecular mechanisms that couple DNA replication and cell cycle progression. To efficiently duplicate large genomes, eukaryotes have evolved a mechanism for the initiation of DNA replication that involves multiple origins of replication (ori) along the chromosomal DNA. The utilization of such sites in multicellular organisms changes during development, and this process affects both gene expression and chromosome folding. The program of such spatial and temporal activation is not understood. Although not necessarily random, the origin site selection during early Drosophila and Xenopus development appears to be less dependent on specific DNA sequences (5, 25). In agreement with this idea, a number of studies suggest that specific replicator sequences might be dispensable (22,38,52,53). Later in development origin usage becomes more specific (26, 49) and depends on many mechanisms for selection of the initiation events. Overall, with an exception of the budding yeast Saccharomyces cerevisiae, DNA sequences that define eukaryotic and especially metazoan replication origins are poorly characterized, mainly because of a lack of definitive biochemical or genetic assays (13,17,18).The hexameric origin recognition complex (ORC) is an important component for eukaryotic DNA replication. It was originally discovered in the budding yeast S. cerevisiae, and subsequent studies both in yeast and higher eukaryotes laid the foundation for understanding the functions of this important key initiation factor. ORC binds to origin sites in an ATPdependent manner and serves as a scaffold for the assembly of other initiation factors (3). ORC also directly participates in the loading of initiation factors (6, 45). Sequence rules for ORC DNA binding appear to vary ...

Correlations of exocytosis with increased turnover of inositol phospholipids represent long-standing observations (1,2). The first clear functional indication that lipids are not passive participants in membrane trafficking came from demonstrations in yeast that the SEC14-encoded phosphatidylinositol (PtdIns)/phosphatidylcholine (PtdCho) transfer protein (Sec14p) is essential for trafficking of proteins from the yeast Golgi complex (3), and that this essential requirement can be efficiently bypassed by individual perturbation of a specific pathway for phosphatidylcholine biosynthesis (4) or of a pathway that is involved in regulating inositol phospholipid metabolism (5,6). The general proposal was put forth that Sec14p functions to regulate the lipid composition of yeast Golgi membranes, that this regulation minimally involves coordinate regulation of both PtdIns and PtdCho metabolism, and that a proper membrane lipid composition is an essential factor in vesicle budding from the yeast Golgi complex (3,4,7). This hypothesis was superficial in the sense that it did not precisely describe how lipid metabolism interfaces with protein factors controlling membrane trafficking processes; other than proposing that lipid microdomains might provide distinguishing platforms for effecting spatially regulated assembly of protein factors at the sites of vesicle budding (3,7). Today, because of the important contributions of many laboratories, the nature of the multifaceted functional interfaces that exist between lipid metabolism and membrane trafficking are becoming ever more clear. In retrospect, it is satisfying to note that aspects of PtdIns and PtdCho metabolism do indeed interface with specific components of the eukaryotic protein transport machinery. Other facets of lipid metabolism are also intimately involved in regulating secretory pathway function. 4-Phosphorylated Inositol Lipids and Membrane TraffickingElegant studies in both mammalian and yeast systems provide clear evidence that PtdIns-4-P, PtdIns-4,5-P 2 , PtdIns-3-P, and PtdIns-3,5-P 2 all participate in regulating key functions of both the secretory and endosomal pathways. For the purpose of this review, we will discuss the 4-and 3-phosphorylated inositol lipids separately.