Generation of Rhesus Macaque-Tropic HIV-1 Clones That Are Resistant to Major Anti-HIV-1 Restriction Factors

Human immunodeficiency virus type 1 (HIV-1) replication in macaque cells is restricted mainly by antiviral cellular APOBEC3, TRIM5α/TRIM5CypA, and tetherin proteins. For basic and clinical HIV-1/AIDS studies, efforts to construct macaque-tropic HIV-1 (HIV-1mt) have been made by us and others. Although rhesus macaques are commonly and successfully used as infection models, no HIV-1 derivatives suitable for in vivo rhesus research are available to date. In this study, to obtain novel HIV-1mt clones that are resistant to major restriction factors, we altered Gag and Vpu of our best HIV-1mt clone described previously. First, by sequence- and structure-guided mutagenesis, three amino acid residues in Gag-capsid (CA) (M94L/R98S/G114Q) were found to be responsible for viral growth enhancement in a macaque cell line. Results of in vitro TRIM5α susceptibility testing of HIV-1mt carrying these substitutions correlated well with the increased viral replication potential in macaque peripheral blood mononuclear cells (PBMCs) with different TRIM5 alleles, suggesting that the three amino acids in HIV-1mt CA are involved in the interaction with TRIM5α. Second, we replaced the transmembrane domain of Vpu of this clone with the corresponding region of simian immunodeficiency virus SIVgsn166 Vpu. The resultant clone, MN4/LSDQgtu, was able to antagonize macaque but not human tetherin, and its Vpu effectively functioned during viral replication in a macaque cell line. Notably, MN4/LSDQgtu grew comparably to SIVmac239 and much better than any of our other HIV-1mt clones in rhesus macaque PBMCs. In sum, MN4/LSDQgtu is the first HIV-1 derivative that exhibits resistance to the major restriction factors in rhesus macaque cells.     

miR‐197 induces epithelial–mesenchymal transition in pancreatic cancer cells by targeting p120 catenin

Invasive ductal adenocarcinoma (IDA) of the pancreas manifests poor prognosis due to the early invasion and distant metastasis. In contrast, intraductal papillary mucinous adenoma or carcinoma (IPMA or IPMC) reveals better clinical outcomes. Various molecular mechanisms contribute to these differences but entire picture is still unclear. Recent researches emphasized the important role of miRNA in biological processes including cancer invasion and metastasis. We previously described that miR‐126 is down‐regulated in IDA compared with IPMA or IPMC, and miR‐126 regulates the expression of invasion related molecule disintegrin and metalloproteinase domain‐containing protein 9 (ADAM9). Assessing the difference of miRNA expression profiles of IDA, IPMA, and IPMC, we newly identified miR‐197 as an up‐regulated miRNA specifically in IDA. Expression of miR‐197 in pancreatic cancer cells resulted in the induction of epithelial–mesenchymal transition (EMT) along with the down‐regulation of p120 catenin which is a putative target of miR‐197. Direct interaction between miR‐197 and p120 catenin mRNA sequence was confirmed by 3′UTR assay, and knockdown of p120 catenin recapitulated EMT induction in pancreatic cancer cells. In situ hybridization of miR‐197 and immunohistochemistry of p120 catenin showed mutually exclusive patterns suggesting pivotal role of miR‐197 in the regulation of p120 catenin. This miR‐197/p120 catenin axis could be a novel therapeutic target. J. Cell. Physiol. 228: 1255–1263, 2013. © 2012 Wiley Periodicals, Inc.     

Sensitivity to RNA interference-mediated gene silencing in intact and ligated larvae of the armyworm, Mythimna separata (Lepidoptera: Noctuidae)

RNA interference (RNAi) is a common tool for analysis of gene function in both model and non-model insects, but it is becoming evident that RNAi efficiency varies considerably from species to species. We examined RNAi efficiency in larvae of the armyworm Mythimna separata (Walker) using multiple genes and tissues. First, we showed that five different target genes exhibited distinct tissue distribution patterns by quantitative determination of mRNA in total hemocytes, foregut, midgut, hindgut, Malpighian tubules and fat body: neuroglian mRNA was most abundant in fat body; inhibitor of apoptosis proteins mRNA was found to be ubiquitous; aquaporin 4 mRNA was most enriched in hindgut; cueball and prophenoloxidase 2 were mainly expressed in hemocytes. Second, we assessed sensitivity to gene silencing by double-strand RNA injection of these five genes in the six different tissues. We found that these genes generally showed refractoriness to double-strand RNA-mediated gene knockdown irrespective of the tissue tested. Finally, we demonstrated that appreciable gene knockdown was achieved at least in the adhering hemocyte fraction when larval isolated abdomen was prepared by ligation and subjected to dsRNA injection. Our study thus added detailed information on the refractoriness of larval tissues of a lepidopteran insect to gene silencing through RNAi and provided a new potential approach to improve RNAi efficiency.     

Immunisation against a serine protease inhibitor reduces intensity of Plasmodium berghei infection in mosquitoes

The mosquito innate immune response is able to clear the majority of Plasmodium parasites. This immune clearance is controlled by a number of regulatory molecules including serine protease inhibitors (serpins). To determine whether such molecules could represent a novel target for a malaria transmission-blocking vaccine, we vaccinated mice with Anopheles gambiae serpin-2. Antibodies against Anopheles gambiae serpin-2 significantly reduced the infection of a heterologous Anopheles species (Anopheles stephensi) by Plasmodium berghei, however this effect was not observed with Plasmodium falciparum. Therefore, this approach of targeting regulatory molecules of the mosquito immune system may represent a novel approach to transmission-blocking malaria vaccines.     

A CD4 mimic as an HIV entry inhibitor: Pharmacokinetics

To date, several small molecules of CD4 mimics, which can suppress competitively the interaction between an HIV-1 envelope glycoprotein gp120 and a cellular surface protein CD4, have been reported as viral entry inhibitors. A lead compound 2 (YYA-021) with relatively high potency and low cytotoxicity has been identified previously by SAR studies. In the present study, the pharmacokinetics of the intravenous administration of compound 2 in rats and rhesus macaques is reported. The half-lives of compound 2 in blood in rats and rhesus macaques suggest that compound 2 shows wide tissue distribution and relatively high distribution volumes. A few hours after the injection, both plasma concentrations of compound 2 maintained micromolar levels, indicating it might have promise for intravenous administration when used combinatorially with anti-gp120 monoclonal antibodies.     

Effects of Lipids on the Rheological Properties of Aqueous Soybean Protein Dispersions

The flow properties of soybean protein–lipid–water suspension systems and coagulated gels were related to a protein–lipid interaction. For powdered soybean lecithin-added soybean protein suspension systems and their heat-induced gels, the yield stress (σ_y) and the consistency index (K) increased with increasing amounts of added lipid, but the flow behavior index (n) and the thixotropy index (TI) decreased. On the other hand, there were only small changes in the magnitudes of the thixotropic parameters and the viscometric parameters (σ_y, K and n) after adding soybean oil at various concentrations to soybean protein dispersions. These facts suggested that the formation of a protein–phospholipid complex increased the effective particle size, and that the intermolecular entanglements and linkages among the protein molecules or among the protein-phospholipid complexes were weakened by the addition of polar lipids. The thixotropy index defined in this study is available for characterizing the stress decay that occurs within soybean protein dispersions and heat-induced gels as they are sheared.     

SIZ1 deficiency causes reduced stomatal aperture and enhanced drought tolerance via controlling salicylic acid‐induced accumulation of reactive oxygen species in Arabidopsis

Transpiration and gas exchange occur through stomata. Thus, the control of stomatal aperture is important for the efficiency and regulation of water use, and for the response to drought. Here, we demonstrate that SIZ1‐mediated endogenous salicylic acid (SA) accumulation plays an important role in stomatal closure and drought tolerance. siz1 reduced stomatal apertures. The reduced stomatal apertures of siz1 were inhibited by the application of peroxidase inhibitors, salicylhydroxamic acid and azide, which inhibits SA‐dependent reactive oxygen species (ROS) production, but not by an NADPH oxidase inhibitor, diphenyl iodonium chloride, which inhibits ABA‐dependent ROS production. Furthermore, the introduction of nahG into siz1, which reduces SA accumulation, restored stomatal opening. Stomatal closure is generally induced by water deficit. The siz1 mutation caused drought tolerance, whereas nahG siz1 suppressed the tolerant phenotype. Drought stresses also induced expression of SA‐responsive genes, such as PR1 and PR2. Furthermore, other SA‐accumulating mutants, cpr5 and acd6, exhibited stomatal closure and drought tolerance, and nahG suppressed the phenotype of cpr5 and acd6, as did siz1 and nahG siz1. Together, these results suggest that SIZ1 negatively affects stomatal closure and drought tolerance through the accumulation of SA.