Abrupt emergence and predominance in Vietnam of rotavirus A strains possessing a bovine-like G8 on a DS-1-like background.

An apparently single rotavirus A strain possessing a genotype constellation of G8-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 abruptly emerged, caused diarrhoea in children requiring hospitalisation, and increased to reach 27 % of strains detected during the first half of 2015 in Vietnam.

Molecular epidemiology of noroviruses detected in Vietnamese children with acute gastroenteritis from 2012 to 2015.

Noroviruses, an important cause of diarrhoea in humans, are genetically diverse. The recent norovirus seasons recorded the emergence of new recombinants of the capsid and polymerase genotypes, with a global dominance of GII.Pe_GII.4 Sydney 2012 and GII.P17_GII.17 in Asian countries. However, the number of papers reporting the distribution of both polymerase and capsid genotypes circulating among children is scarce, with none from Vietnam. This study described both the polymerase and capsid genotypes of noroviruses circulating in Vietnamese children using stool specimens obtained under the World Health Organization rotavirus surveillance programme from 2012 to 2015. Of 350 specimens tested, noroviruses were detected in 90 (28 %) of 319 inpatient specimens and in 9 (29 %) of 31 outpatient specimens. The polymerase and capsid genotype combinations of GII.Pe_GII.4 Sydney 2012 and GII.P21_GII.3 were co-dominant (51 and 24 %, respectively), both of which were recombinants, contributing to a high proportion (87 %) of recombinants among circulating noroviruses. GII.4 variants evolved in the same fashion in Vietnam as in other countries, with amino acid substitutions in the putative variant-specific epitopes of the protruding domain. Unlike neighbouring countries where the predominance of GII.P17_GII.17 was reported, only one GII.P17_GII.17 strain was detected from an outpatient in 2015 in Vietnam. In conclusion, a substantial burden due to norovirus gastroenteritis hospitalizations among Vietnamese children was associated with circulating co-dominant GII.Pe_GII.4 Sydney 2012 and GII.P21_GII.3 strains. Continued surveillance is necessary to monitor infection caused by GII.4 variants and that of GII.P17_GII.17 noroviruses in paediatric patients in Vietnam.

NMR-derived solution structure of a 17mer hydroxymethyluracil-containing DNA.

Incorporation of 5-(hydroxymethyl)-2'-deoxyuridine into DNA in place of thymine by SPO1, a Bacillus subtilis bacteriophage, allows the viral DNA to bind selectively to transcription factor 1. We have synthesized a TF1-binding site: d(5'-ACCHACHCHHHGHAGGT-3')-d(5'-ACCHACAAAGAGHAGGT-3') and studied this molecule using NMR spectroscopy. The chemical shifts of exchangeable and non-exchangeable protons were sequentially assigned. Absence of corresponding NOEs in the imino-imino region suggested that the end base pairs did not form Watson-Crick hydrogen bond. Restrained molecular dynamics calculation yielded a family of B-DNA structures whose r.m.s.d. was 0.66 A (all atoms) for the internal 15 bp. The helical twist was 38.5 degrees per step. The base pairs were situated directly on the helix axis (X-displacement = -0.2 A). All sugars exhibited C2'-endo puckering with P = 167.3 degrees and upsilon(max)= 38.2 degrees. The OH groups of all hmU bases resided on the 3' side of the base plane and may affect the base orientation relative to the sugar plane as the average chi value for all hmU was 4 degrees more positive than that of other nucleosides (258 degrees versus 254 degrees ). Positive roll angles (rho) and small flanking twists (omega) at hmU suggested that the two hmU-A base pair steps open toward the minor grooves.

Mechanisms for the enhanced thermal stability of a mutant of transcription factor 1 as explained by (1)H, (15)N and (13)C NMR chemical shifts and secondary structure analysis.

A variant of the bacteriophage SPO1-encoded transcription factor 1 (TF1) with two site-specific mutations (E15G and T32I) was shown to be more thermally stable and bind DNA more tightly compared to the wild-type protein. In order to understand the biochemical mechanisms underlying these properties, we are engaged in determining the solution structures of this mutant alone and in complex with DNA using nuclear magnetic resonance (NMR) spectroscopy. The first phase of this project is reported here, as we have completed most of the backbone and sidechain sequential NMR assignments of the mutant protein, TF1-G15/I32. Insights derived from the (1)H, (15)N and (13)C chemical shifts and from the secondary structure analysis provide us with an explanation for the noted increase in thermal stability of TF1-G15/I32. Compared to the structure of the wild-type protein, the beta-sheet and the C-terminal helix remain largely unaffected whereas the mutations cause great changes in the first two helices and their enclosed loop. Specifically, we have found that the second helix is extended by one residue at its N-terminus and rotated in a way that allows Ala-37 to interact with Tyr-94 of the C-terminal helix. The loop has been found to become more rigid as a result of hydrophobic interactions between the flanking second and first helices and also between the second helix and the loop itself. Furthermore, the T32I mutation allows tighter packing between the second helix and the beta-sheet. Collectively, these changes contribute to a more tightly associated dimer and hence, to a greater thermal stability.

Solution structure of a mutant of transcription factor 1: implications for enhanced DNA binding.

An NMR solution structure of a mutant of the homodimer protein transcription factor 1, TF1-G15/I32 (22 kDa), has been solved to atomic resolution, with 23 final structures that converge to an r.m. s.d. of 0.78 A. The overall shape of TF1-G15/I32 remains similar to that of the wild-type protein and other type II DNA-binding proteins. Each monomer has two N-terminal alpha-helices separated by a short loop, followed by a three-stranded beta-sheet, whose extension between the second and third beta-strands forms an antiparallel beta-ribbon arm, leading to a C-terminal third alpha-helix that is severely kinked in the middle. Close examination of the structure of TF1-G15/I32 reveals why it is more stable and binds DNA more tightly than does its wild-type counterpart. The dimeric core, consisting of the N-terminal helices and the beta-sheets, is more tightly packed, and this might be responsible for its increased thermal stability. The DNA-binding domain, composed of the top face of the beta-sheet, the beta-ribbon arms and the C-terminal helices, is little changed from wild-type TF1. Rather, the enhancement in DNA affinity must be due almost exclusively to the creation of an additional DNA-binding site at the side of the dimer by changes affecting helices 1 and 2: helix 2 of TF1-G15/I32 is one residue longer than helix 2 of the wild-type protein, bends inward, and is both translationally and rotationally displaced relative to helix 1. This rearrangement creates a longer, narrower fissure between the V-shaped N-terminal helices and exposes additional positively charged surface at each side of the dimer.

Pharmacological characterization of an unusual mGluR-evoked neuronal hyperpolarization mediated by activation of GIRK channels.

The metabotropic glutamate receptor (mGluR) agonist ACPD exerts an unusual inhibitory effect on a population of neurons of the song-control nucleus HVc of the zebra finch via activation of the GIRK channel. We report in the present study the pharmacology of this response. ACPD directly hyperpolarized the neurons by a mechanism independent of GABA(B) receptors. The group I mGluR agonist DHPG had no effect on membrane properties and the group I mGluR antagonist 4-CPG did not affect the ACPD-induced hyperpolarization. In contrast, the ACPD response was mimicked by the group II mGluR agonist LY314593 and the group II and III agonist L-CCG-I. The group II mGluR antagonist LY307452 fully antagonized the ACPD response and reduced the response induced by L-CCG-I. The group III mGluR agonist L-AP4 induced a small hyperpolarization, which was antagonized by the group III mGluR antagonist MAP-4. These data indicate that group II and group III mGluRs are present and functional in the postsynaptic membrane of these HVc neurons, and mediate the hyperpolarizing action of mGluR agonists. In contrast, group I mGluRs are absent from these neurons, nonfunctional, or coupled to different effector systems that do not influence membrane potential or input resistance.

Quaternary salts of 2-[(hydroxyimino)methyl]imidazole. 4. Effect of various side-chain substituents on therapeutic activity against anticholinesterase intoxication.

A series of quaternary salt derivatives of 2-[(hydroxyimino)methyl]-1-methylimidazole incorporating various side chains bearing ether, silyl, nitrile, ester, halogen, nitro, sulfone, amino, or aminosulfonyl substituents was prepared and evaluated in vivo for the treatment of anticholinesterase intoxication. Test results in the mouse revealed that the type and location of the side-chain substituent both have a significant influence on the toxicity and antidotal effectiveness of the compounds. Some of the more active examples represent the most potent therapeutics to date against intoxication by the powerful cholinesterase inhibitors soman and tabun. Significantly, the antidotal effectiveness of the compounds was not dependent on the inhibiting agent nor was there any correlation between in vivo efficacy and in vitro reactivation of ethyl (4-nitrophenyl)methylphosphonate inhibited human acetylcholinesterase. These observation suggested that the main mode of antidotal protection by the compounds is something other than enzyme reactivation.

Inhibition of Friend murine leukemia virus activity by guanosine/thymidine oligonucleotides.

Oligonucleotides consisting of only deoxyguanosine and deoxythymidine were stable in culture and were able to significantly inhibit Friend Murine Leukemia Virus (FMLV) production in acute cell culture assay systems. The oligonucleotides did not share homology with, or possess any complementary (antisense) sequence motifs to the FMLV genome. The guanosine/thymidine-containing oligonucleotides (GTOs) which demonstrated anti-FMLV activity in acute infection assays were synthesized with natural phosphodiester (PD) linkages (backbones). The observed antiviral activities of these oligonucleotides increased significantly when the PD backbone was replaced with a phosphorothioate (PT) backbone. Experiments designed to investigate a potential antiviral mechanism of action demonstrated that oligonucleotides tested were capable of blocking virus adsorption. In addition, GTOs with PD backbones were competitive inhibitors of FMLV reverse transcriptase (RT). When the same experiments were performed using oligonucleotides with PT backbones, all compounds tested demonstrated significant competitive inhibition of FMLV RT. The measured inhibitory activity of all compounds tested in culture assays was enhanced by at least a factor of 10 when the PD linkages were replaced with PT. The enhanced antiviral activity exhibited by the sulfur group on the oligonucleotide backbone, and the lack of any designed, sequence-specific interactions, suggest that a large percentage of the reported antiviral activity of oligonucleotides containing a phosphorothioate backbone is due to factors other than rationally designed, sequence-specific interactions. The ability of GTOs to inhibit FMLV in culture, potentially via a number of different mechanisms, makes this a class of compounds which warrants investigation as therapeutic agents to be used against retroviral infections.

Alpha-(Ac)AKRHRKV, a model of the histone H4 amino terminus, uses an unprotonated histidine in phosphate binding.

The 1H NMR spectrum of the title peptide at pH 3.3 in 90% H2O was assigned by HOHAHA and NOESY 2D methods. Titration studies in D2O at 300 MHz indicated a histidine side-chain pKa of 6.3. Peptide backbone NH resonances were studied in 90% H2O at 500 MHz as a function of pH and added phosphate. In acidic solution the peptide was free from conventional secondary structural elements, but near neutrality the valine amide proton resonance remained a sharp doublet, which suggests that it may form a hydrogen bond with some backbone carbonyl group. The other amide resonances broadened and showed significant saturation transfer from the water signal indicating that they exchange with solvent although not all to the same extent. Marked changes in the chemical shift of the histidine aromatic protons in the presence of phosphate and a 70-fold increase in the 31P line width of inorganic phosphate in the presence of peptide only at pH values above the pKa (6.3) of the histidine imidazole side-chain implied that the unprotonated imidazole group is specifically involved in phosphate binding. The peptide binds inorganic phosphate with a dissociation constant of 1.6 x 10(-5) M(-1) at pH 7.4.

Multiple cell types distinguished by physiological, pharmacological, and anatomic properties in nucleus HVc of the adult zebra finch.

Nucleus HVc of the songbird is a distinct forebrain region that is essential for song production and shows selective responses to complex auditory stimuli. Two neuronal populations within HVc give rise to its efferent projections. One projection, to the robust nucleus of the archistriatum (RA), serves as the primary motor pathway for song production, and can also carry auditory information to RA. The other projection of HVc begins a pathway through the anterior forebrain, (area X --> medial portion of the dorsolateral nucleus of the thalamus (DLM) --> lateral portion of the magnocellular nucleus of the anterior neostriatum (L-MAN) --> RA) that is crucial for song learning but, although active during singing, is not essential for adult song production. To test whether these different projection neuron classes have different functional properties, we recorded intracellularly from neurons in nucleus HVc in brain slices. We observed at least three classes of neuron based on intrinsic physiological and pharmacological properties as well as on synaptic inputs. We also examined the morphological properties of the cells by filling recorded neurons with neurobiotin. The different physiological cell types correspond to separate populations based on their soma size, dendritic extent, and axonal projection. Thus HVc neurons projecting to area X have large somata, show little spike-frequency adaptation, a hyperpolarizing response to the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-trans-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), and exhibit a slow inhibitory postsynaptic potential (IPSP) following tetanic stimulation. Those HVc neurons projecting to motor nucleus RA have smaller somata, show strong accommodation, are not consistently hyperpolarized by ACPD, and exhibit no slow IPSP. A third, rarely recorded class of neurons fire in a sustained fashion at very high-frequency and may be interneurons. Thus the neuronal classes within HVc have different functional properties, which may be important for carrying specific information to their postsynaptic targets.

Specificity of hydroxylmethyluracil-containing DNA for transcription factor 1: structural insights.

The genomic materials from some Bacillus subtilis bacteriophages are found to contain 5-(hydroxymethyl)-2'-deoxyuridine in place of thymine. Phage-encoded proteins such as transcription factor 1 specifically and preferentially bind to the minor grooves of these hmU-containing DNA but not to thymine-containing DNA. Data from electrophoretic mobility shift assays suggest that the inherent, localized flexibility of hmU-DNA, which is sequence-specific, is responsible for its discriminative binding. We discuss here, from the NMR-derived structural point of view, how differential DNA flexibility can contribute to specific binding of TF1 to hmU-DNA.

L-phenylalanyl-L-glutamate-stimulated, chloride-dependent glutamate binding represents glutamate sequestration mediated by an exchange system.

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.

Slow synaptic inhibition mediated by metabotropic glutamate receptor activation of GIRK channels.

Glutamate is the predominant excitatory neurotransmitter in the vertebrate CNS. Ionotropic glutamate receptors mediate fast excitatory actions whereas metabotropic glutamate receptors (mGluRs) mediate a variety of slower effects. For example, mGluRs can mediate presynaptic inhibition, postsynaptic excitation, or, more rarely, postsynaptic inhibition. We previously described an unusually slow form of postsynaptic inhibition in one class of projection neuron in the song-control nucleus HVc of the songbird forebrain. These neurons, which participate in a circuit that is essential for vocal learning, exhibit an inhibitory postsynaptic potential (IPSP) that lasts several seconds. Only a portion of this slow IPSP is mediated by GABA(B) receptors. Since these cells are strongly hyperpolarized by agonists of mGluRs, we used intracellular recording from brain slices to investigate the mechanism of this hyperpolarization and to determine whether mGluRs contribute to the slow synaptic inhibition. We report that mGluRs hyperpolarize these HVc neurons by activating G protein-coupled, inwardly-rectifying potassium (GIRK) channels. MGluR antagonists blocked this response and the slow synaptic inhibition. Thus, glutamate can combine with GABA to mediate slow synaptic inhibition by activating GIRK channels in the CNS.

Conformational preferences of a chimeric peptide HIV-1 immunogen from the C4-V3 domains of gp120 envelope protein of HIV-1 CAN0A based on solution NMR: comparison to a related immunogenic peptide from HIV-1 RF.

A critical problem to overcome on HIV vaccine design is the variability among HIV strains. One strategy to solve this problem is the construction of multicomponent immunogens reflective of common HIV motifs. Currently, it is not known if these motifs should be based primarily on amino acid sequence or higher-order structure of the viral proteins of a combination of the two. In this paper, we report NMR-derived solution conformations for a sympathetic peptide taken from the C4 and V3 domains of HIV-1 CAN0A gp120 envelope protein. This peptide, designated T1-SP10CAN0(A), is compared to a recently reported C4-V3 peptide. T1-SP10RF(A) from the HIV-1 RF strain [de Lorimier et al. (1994) Biochemistry 33, 2055-2062], in terms of conformational features and immune responses in mice [Haynes et al. (1995) AIDS Res. Hum. Retroviruses 11, 211-221]. The T1 segment of 16 amino acids from the gp120 C4 domain is identical in both peptides and exhibits nascent helical character. The SP10 region, taken from the gp120 V3 loop, differs from that of T1-SP10RF(A) in both sequence and conformations. A reverse turn is observed at the conserved GPGX sequence. The rest of the Sp10 domain is extended with the exception of the last three residues which show evidence for a helical arrangement. Modeling of the turn region of the T1-SP10CAN0(A) peptide shows exposure of a continuous apolar stretch of side chains similar to that reported in the crystal structure of a V3 peptide from HIV-1 MN complexed with a monoclonal antibody [Rini et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 6325-6329]. this hydrophobic patch is interrupted by a charged Lys residue in the T1-SP10RF(A) peptide. This observation suggests that the HIV-1 CAN0A and HIV-1 RF C4-V3 peptides can induce widely different anti-HIV antibodies. consistent with immunogenic results.

Triple helix formation at distant sites: hybrid oligonucleotides containing a polymeric linker.

An oligonucleotide hybrid is described which possesses two triple helix forming oligonucleotides which have been connected by a flexible polymeric linker chain. As a prototype, binding of this class of oligonucleotide to duplex DNA has been studied using a segment of the HSV-1 D-glycoprotein promoter, which possesses a pair of 12bp target sites for stable triple helix formation, separated by a duplex spacer region which is one helical turn long. Band shift and footprinting analysis show that such hybrids can bind to both 12bp elements simultaneously, if flexible linkers are included which are longer than 20-25 rotatable bonds. Molecular modeling confirms that a flexible polymeric linker as short as 22 rotatable bonds is enough to link the two distant segments of triple helix, providing that the linker element travels a path which is external to the helix grooves and parallel to the long helix axis.

Nuclear magnetic resonance analysis of solution conformations in C4-V3 hybrid peptides derived from human immunodeficiency virus (HIV) type 1 gp120: relation to specificity of peptide-induced anti-HIV neutralizing antibodies.

Immunogenic peptides containing epitopes of the gp120 C4 and V3 regions from human immunodeficiency virus strains MN and EV91 have been studied by nuclear magnetic resonance and molecular modeling and used as immunogens in rhesus monkeys. The results, combined with those for other peptides, suggest a correlation between solution conformation and immunologic cross-reactivity.