Guava flavonoid glycosides prevent influenza A virus infection via rescue of P53 activity.

Influenza is a highly infectious disease caused by three types of viruses, including influenza A virus (IAV), influenza B virus, and, rarely, influenza C virus. IAV is a major, global public health threat, causing approximately 500 000 deaths per year worldwide. The new strains of IAV have emerged due to a mutation called antigenic shift, which results in a new subtype of the virus that shows resistance to common antiviral drugs. Here, guava and lemon extracts, including green leaves and flowers, were investigated for their activity against IAV replication in human A549 cells. Concomitantly, the cytotoxicity of a potent extract on host-cell multiplication was assessed. Our results reveal that guava extracts inhibit IAV replication, indicated by viral nucleoprotein expression profile and traditional plaque assay. Interestingly, treatment with guava extract inactivates Akt protein kinase and stimulates the pro-apoptotic protein P53, at early stages of infection. Furthermore, purified guava flavonoid glycosides (GFGs) show competitive inhibition of IAV-virus replication via early regulation of IL-1ß and IL-8 in association with P53 gene expression. The docking analysis of GFGs and the protein structure of upstream targets for the Akt signaling pathway indicates a sufficient interaction and stabilization with Gbr2 protein. These data indicate that treatment with GFGs disturbs IAV replication via activation of P53 and its apoptotic related factors after infection. Collectively, these data show that targeting of essential host kinases that are involved in the replication cycle of IAV and rescue of P53 activity by GFGs could represent a new strategy to eradicate IAV.

Immunomodulatory properties of quercetin-3-O-α-L-rhamnopyranoside from Rapanea melanophloeos against influenza a virus.

BACKGROUND: Influenza infection is a major public health threat. The role of influenza A virus-induced inflammatory response in severe cases of this disease is widely recognized. Drug resistance and side effects of chemical treatments have been observed, resulting in increased interest in alternative use of herbal medications for prophylaxis against this infection. The South African medicinal plant, Rapanea melanophloeos (RM) (L.) Mez of the family Myrsinaceae was selected owing to its traditional use for the treatment of several diseases such as respiratory ailments and also previous preliminary studies of anti-influenza activity of its methanolic extract. The aim of this study was to investigate the immunomodulatory properties of a glycoside flavone isolated from RM against influenza A virus. METHODS: The non-cytotoxic concentration of the quercetin-3-O-α-L-rhamnopyranoside (Q3R) was determined by MTT assay and tested for activity against influenza A virus (IAV) in simultaneous, pre-penetration and post-penetration combination treatments over 1 h incubation on MDCK cells. The virus titer and viral load targeting NP and M2 viral genes were determined using HA and qPCR, respectively. TNF-α and IL-27 as pro- and anti-inflammatory cytokines were measured at RNA and protein levels by qPCR and ELISA, respectively. RESULTS: Quercetin-3-O-α-L-rhamnopyranoside at 150 µg/ml decreased the viral titer by 6 logs (p < 0.01) in the simultaneous procedure. The NP and M2 genes copy numbers as viral target genes, calculated based on the Ct values and standard formula, significantly decreased in simultaneous treatment (p < 0.01). The expression of cytokines was also considerably affected by the compound treatment. CONCLUSIONS: This is the first report of quercetin-3-O-α-L-rhamnopyranoside from RM and its immunomodulatory properties against influenza A virus. Further research will focus on detecting the specific mechanism of virus-host interactions.

Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-µM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants at 21 days after fertilization. Moreover, the body cavities of MO-treated and nontreated fish were examined by histology and in situ hybridization, showing gonads which had no germ cells in morphants at various stages (60, 150, and 210 days after fertilization). Taken together, these results report the first known and functional method of sturgeon sterilization.

Identifying small molecule inhibitors of eukaryotic translation initiation.

In eukaryotes, translation initiation is rate-limiting with much regulation exerted at the ribosome recruitment and ternary complex (eIF2.GTP.Met-tRNA(i)(Met)) formation steps. Although small molecule inhibitors have been extremely useful for chemically dissecting translation, there is a dearth of compounds available to study the initiation phase in vitro and in vivo. In this chapter, we describe reverse and forward chemical genetic screens developed to identify new inhibitors of translation. The ability to manipulate cell extracts biochemically, and to compare the activity of small molecules on translation of mRNA templates that differ in their factor requirements for ribosome recruitment, facilitates identification of the relevant target.

Simultaneous localization of transcription and early processing markers allows dissection of functional domains in the plant cell nucleolus.

Nucleolar transcription in isolated onion cell nuclei was visualized, after Br-UTP incorporation, under the conventional fluorescence microscope, the confocal microscope, and the transmission electron microscope. The confocal microscopy study of transcription was combined with immunodetection of fibrillarin, a component of the RNP complex involved in the early processing of pre-rRNA. Superposition of transcription and fibrillarin images from the same optical section showed some small "black holes" in the nucleolus, around which a lateral and radial differentiation of labeling was observed: laterally, zones corresponding to transcription labeling alternated with zones of fibrillarin labeling; radially, areas of transcription gradually became areas of colocalization of transcription and fibrillarin, and, further outward, of fibrillarin alone, which occupied the major part of the labeled nucleolar area. Three-dimensional reconstruction of the nucleolar transcription labeling, from confocal optical sections, showed clusters of foci arranged around an area of low or no labeling. Thin labeled extensions, connecting single foci, were observed. Visualization of transcription at the ultrastructural level identified the black holes as fibrillar centers, in view of their size and the absence of labeling in them. In fact, most of the labeling was observed in discrete areas of the dense fibrillar component, near fibrillar centers, including the transition area between these two components. This observation was supported by a quantitative study. Otherwise, the outline of fibrillar centers did not appear entirely surrounded by particles, and a minor proportion of particles was detected dispersed throughout the dense fibrillar component. As a complementary study, the transcription factor upstream binding factor (UBF) and the protein NopA64, a plant nucleolin homologue, were immunolocalized. Small foci of UBF localization alone and other foci in which the two protein markers overlapped were observed. The outer areas of the nucleolus showed the exclusive presence of NopA64. Under the electron microscope, UBF labeling, quantitatively assessed, appeared as clusters of particles, most of them surrounding fibrillar centers. A graphic model is presented to give a molecular interpretation of these data.

Structure and subcellular localization of a small RNA-binding protein from tobacco.

In this study, a cDNA encoding a small RNA-binding protein was isolated from a Nicotiana sylvestris cDNA library. The predicted protein (RGP-3) is 144 amino acid residues long, and contains a consensus sequence-type RNA binding domain (CS-RBD) of 83 amino acids and a short glycine-rich region of 15 amino acids. RGP-3 synthesized in Escherichia coli has high affinity for poly(U). Immunocytochemical analysis indicated that RGP-3 is localized in the nucleoplasm, and that RGP-1b, a related protein reported previously, is localized in the nucleolus. Possible roles of these proteins in pre-mRNA or pre-rRNA processing are discussed.

Identification, nuclear localization, and binding activities of OZF, a human protein solely composed of zinc-finger motifs.

The OZF cDNA was identified in a human mammary cell line and encodes a polypeptide solely composed of ten zinc-finger motifs which belongs to the Kruppel family of zinc-finger proteins. The OZF protein produced in Escherichia coli binds zinc ions, DNA and heparin. These binding activities are characteristic of zinc-finger proteins. Immunochemical analysis using antibodies produced against the recombinant protein detected its expression in human mammary epithelial cells but not in stroma cells, which is consistent with the pattern of expression observed at the RNA level in cell cultures. Western blot analysis demonstrated the expression of a 33-kDa nuclear protein similar in size to the predicted protein and therefore excluded the presence of an additional trans-acting domain. These data establish the unique structure of the OZF protein which is distinct from previously identified zinc-finger proteins. In addition, OZF protein overexpression was found in a tumor cell line, which suggests a possible involvement in carcinogenesis.

Osmium ammine-B and electron spectroscopic imaging of ribonucleoproteins: correlation of stain and phosphorus.

Ultra-thin sections of Chironomus salivary glands were stained in a non-Feulgen procedure with osmium ammine-B and imaged at several electron energy-loss windows. For two types of RNP-containing structures (ie Balbiani ring granules and endoplasmic reticulum), a significant spatial correlation was observed between stain distribution and net phosphorus distribution. Non-Feulgen osmium ammine-B staining does not require the use of ultra-thin sections and can approximate the distribution of nucleic acid phosphorus.