The interaction of synthetic analogs of the N-terminal fusion sequence of influenza virus with a lipid monolayer. Comparison of fusion-active and fusion-defective analogs

The amino terminus of subunit-2 of influenza virus hemagglutinin (NHA2) plays a crucial role in the induction of fusion between viral and endosomal membranes leading to the infection of a cell. Three synthetic analogs with an amino acid sequence corresponding to NHA2 of variant hemagglutinins were studied in a monolayer set up. Comparison of the interaction of a fusion-active and two fusion-defective analogs with a lipid monolayer revealed a greater surface activity of the fusion-active analog. Pronounced differences were found if the pure peptides were spread at the air/water interface; the fusion-active analog showed a higher collapse pressure and a greater limiting molecular area. Circular dichroism measurements on collected lipid monolayers indicated a high content of alpha-helical structure for the fusion-active and one of the fusion-defective analogs. A simple relation between alpha-helical content and fusogenicity does not seem to exist. Instead, the extent of penetration, a defined tertiary structure or orientation of the alpha-helical peptide may be essential for its membrane perturbing activity.     

Co-localization of neuropeptide tyrosine (NPY) and its C-terminal flanking peptide (C-PON)

Neuropeptide tyrosine (NPY) is one of the most abundant and widespread peptides in the mammalian nervous system. Recent isolation and sequencing of the DNA encoding NPY has predicted the existence of a 97 amino acid precursor peptide. Proteolytic processing of this precursor could yield three separate peptide products, an N-terminal signal peptide, neuropeptide tyrosine and a 30 amino acid C-terminal flanking peptide (C-PON). Here, we present evidence that the predicted C-flanking peptide of NPY is widely distributed in both the central and peripheral nervous systems of several mammalian species including man, and has an identical distribution to NPY. It was also demonstrated, using correlative light microscopic immunostaining on serial sections and double electron microscopic immunocytochemistry, that C-PON and NPY immunoreactivities are co-localized in neuronal cell bodies of the brain cortex, sympathetic ganglion cells, norepinephrine-containing granules of the adrenal medulla and in human pheochromocytoma tumor cells.     

A novel DNA damage recognition protein in Schizosaccharomyces pombe

Toxic and mutagenic O⁶-alkylguanine adducts in DNA are repaired by O⁶-alkylguanine-DNA alkyltransferases (MGMT) by transfer of the alkyl group to a cysteine residue in the active site. Comparisons in silico of prokaryotes and lower eukaryotes reveal the presence of a group of proteins [alkyltransferase-like (ATL) proteins] showing amino acid sequence similarity to MGMT, but where the cysteine at the putative active site is replaced by tryptophan. To examine whether ATL proteins play a role in the biological effects of alkylating agents, we inactivated the gene, referred to as atl1⁺, in Schizosaccharomyces pombe, an organism that does not possess a functional MGMT homologue. The mutants are substantially more susceptible to the toxic effects of the methylating agents, N-methyl-N-nitrosourea, N-methyl-N′nitro-N-nitrosoguanidine and methyl methanesulfonate and longer chain alkylating agents including N-ethyl-N-nitrosourea, ethyl methanesulfonate, N-propyl-N-nitrosourea and N-butyl-N-nitrosourea. Purified Atl1 protein does not transfer methyl groups from O⁶-methylguanine in [³H]-methylated DNA but reversibly inhibits methyl transfer by human MGMT. Atl1 binds to short single-stranded oligonucleotides containing O⁶-methyl, -benzyl, -4-bromothenyl or -hydroxyethyl-guanine but does not remove the alkyl group or base and does not cleave the oligonucleotide in the region of the lesion. This suggests that Atl1 acts by binding to O⁶-alkylguanine lesions and signalling them for processing by other DNA repair pathways. This is the first report describing an activity that protects S.pombe against the toxic effects of O⁶-alkylguanine adducts and the biological function of a family of proteins that is widely found in prokaryotes and lower eukaryotes.     

Dual function of the Drosophila Alk1/Alk2 ortholog Saxophone shapes the Bmp activity gradient in the wing imaginal disc

Wing patterning in Drosophila requires a Bmp activity gradient created by two Bmp ligands, Gbb and Dpp, and two Bmp type I receptors, Sax and Tkv. Gbb provides long-range signaling, while Dpp signals preferentially to cells near its source along the anteroposterior (AP) boundary of the wing disc. How each receptor contributes to the signaling activity of each ligand is not well understood. Here, we show that while Tkv mediates signals from both Dpp and Gbb, Sax exhibits a novel function for a Bmp type I receptor: the ability to both promote and antagonize signaling. Given its high affinity for Gbb, this dual function of Sax impacts the function of Gbb in the Bmp activity gradient more profoundly than does Dpp. We propose that this dual function of Sax is dependent on its receptor partner. When complexed with Tkv, Sax facilitates Bmp signaling, but when alone, Sax fails to signal effectively and sequesters Gbb. Overall, our model proposes that the balance between antagonizing and promoting Bmp signaling varies across the wing pouch, modulating the level and effective range, and, thus, shaping the Bmp activity gradient. This previously unknown mechanism for modulating ligand availability and range raises important questions regarding the function of vertebrate Sax orthologs.     

Alkyl-linked bis-THTT derivatives as potent in vitro trypanocidal agents

The effect of several alkyl-linked bis tetrahydro-(2H)-1,3,5-thiadiazine-2-thione (bis-THTT) on Leishmania donovani, Trypanosoma brucei rhodesiense, and Plasmodium falciparum is reported. Most of the compounds exhibited a potent activity against the three parasitic strains but the best in vitro activity profiles were found against T. b. rhodesiense with IC(50) values ranging between 0.3 and 4 microM for the most active compounds.     

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

Background

All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.

Methods

Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.

Results

Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (π_s) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (π_a) was 7.0 × 10^-4 in the M segment and 17.3 × 10^-4 in the S segment sequences. The low π_a relative to π_s suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10^-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10^-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.

Conclusion

Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.     

Altered Receptor Specificity and Cell Tropism of D222G Hemagglutinin Mutants Isolated from Fatal Cases of Pandemic A(H1N1) 2009 Influenza Virus

Mutations in the receptor-binding site of the hemagglutinin of pandemic influenza A(H1N1) 2009 viruses have been detected sporadically. An Asp222Gly (D222G) substitution has been associated with severe or fatal disease. Here we show that 222G variants infected a higher proportion of ciliated cells in cultures of human airway epithelium than did viruses with 222D or 222E, which targeted mainly nonciliated cells. Carbohydrate microarray analyses showed that 222G variants bind a broader range of α2-3-linked sialyl receptor sequences of a type expressed on ciliated bronchial epithelial cells and on epithelia within the lung. These features of 222G mutants may contribute to exacerbation of disease.Although the majority of disease cases have been mild, the pandemic influenza A(H1N1) 2009 (H1N1pdm) virus has caused a substantial number of severe and fatal infections (2). Mutants with a D222G or D222E substitution (D225G or D225E in the H3 numbering system) in the receptor-binding site of the virus hemagglutinin (HA) have been detected sporadically (1), and the D222G substitution has been observed to correlate with cases of severe or fatal disease (1, 3, 9, 14). Cell surface receptors for influenza viruses are sialyl glycans (α2-3 Sia or α2-6 Sia) with terminal sialic acid linked α2-3 or α2-6, respectively, to a penultimate galactose. These differ in distribution in the tissues and cells of different species. The sialyl glycans are differentially recognized by the HAs of human and animal influenza viruses and are critical determinants of host range and tissue tropism (16). Using an experimental system of differentiated cultures of human tracheobronchial epithelial cells (HTBE) for studying influenza virus cell tropism, we and others have established that in the initial stages of infection, seasonal human influenza viruses which recognize α2-6 Sia receptors infect mainly nonciliated cells, whereas avian viruses which recognize α2-3 Sia receptors predominantly infect ciliated cells (8, 17, 22).Previous analyses of human and swine influenza H1N1 viruses (5, 15, 21) and preliminary studies of H1N1pdm viruses (24) have indicated that amino acid substitutions in the HA at position 222 may affect the specificity of receptor binding. This, in turn, would be predicted to determine the range of cell types in human respiratory tissues infected by the viruses (17, 20, 22, 23). We have therefore examined the influence of the D222G and D222E substitutions on the cell tropism of H1N1pdm viruses in HTBE cultures (Table ​(Table1).1). Five viruses were isolated from clinical material in MDCK cells and passaged solely in these cells. Two of these, A/Hamburg/5/2009 (Ham) (4) isolated from a case of mild infection and A/Moldova/G186/2009 (Mol) from a serious but nonfatal infection, had 222D. A/Dakar/37/2009 (Dak) isolated from a mild case of the disease had 222E. Two isolates from fatal cases, A/Lviv/N6/2009 (Lvi) and A/Norway/3206-3/2009 (Nor), had 222G. A sixth virus tested, A/Hamburg/5/2009-e (Ham-e), was derived from Ham by egg passage and plaque purification in MDCK cells and differed by a single substitution, D222G.

TABLE 1.

Differences in amino acid sequence of the HAs of the H1N1pdm viruses and cell tropism in HTBE cultures

LPS-induced cytokine levels are repressed by elevated expression of HSP70 in rats: possible role of NF-κB

Heat shock protein (HSP)70 provides a spectrum of protection against any of a variety of stresses, preventing damage measured at the level of molecules, cells, as well as whole organism. We have previously reported that lipopolysaccharide (LPS)-induced lethality in rats is prevented by a previous exposure to a mild thermal stress and that a thermal stress sufficient to induce HSP70 expression in the liver is accompanied by an inhibition of endotoxin-mediated cytokines and modulation of febrile response. However, the effect of HSP70 upregulation on cytokine expression in animals is unknown. The aim of the present study was to demonstrate the effect of HSP70 overexpression with adenovirus administration on LPS-induced increase in cytokines levels in animals. In the present study, Sprague–Dawley rats were infected with either the control AdTrack or Ad70 virus that directs the expression of human HSP70. After a 5-day incubation, animals were injected with either saline alone or LPS (50 μg/kg). Four hours later, blood samples were drawn and plasma levels of interleukin (IL)-6 or tumor necrosis factor (TNF)-α were measured by enzyme-linked immunosorbent assay. Our data demonstrate for the first time that HSP70 overexpression with adenovirus injection prevented the LPS-induced increase in TNF-α and IL-6 levels in rats. Repression of LPS-induced cytokines expressions by HSP70 upregulation was associated with inhibited IκBα degradation and nuclear factor kappa-B (NF-κB) p65 nuclear translocation in liver, suggesting that HSP70 overexpression may regulate LPS-induced cytokines expression through NF-κB pathway. We conclude that the effects of heat stress-induced increase in HSP70 protein expression on LPS-induced cytokine elaboration in whole animals can be reproduced by the actions of a single gene product.