Determination of interproton distances from NOESY spectra in the active site of paramagnetic metalloenzymes: cyanide-inhibited horseradish peroxidase

Two dimensional (2D) nuclear Overhauser effect (NOE) or NOESY experiments are performed on cyanide inhibited horseradish peroxidase in order to assess the prospects for obtaining quantitative interproton distances for the hyperfine shifted and paramagnetically relaxed active site signals in an intermediate sized (-44 kDa) paramagnetic metalloenzyme. This protein represents an ideal test case for such experiments because a series of structurally defined proton pairs on the heme and axial His 170 have been previously assigned. The relaxation properties of hyperfine shifted signals relevant to the experimental setup of 2D experiments and interpretation of both 1D and 2D NOE data are also investigated. NOESY spectra as a function of mixing time show that quantitative rise curves can be obtained that clearly differentiate between primary and secondary NOES even among the most strongly relaxed protons, but this requires very short mixing times in the range 0.5-3.0 ms. The sensitivity of the weak cross peaks at these short mixing times is improved by the relatively rapid pulse repetition rate and concommitant increase in the number of scans allowed by the rapid relaxation of active site protons. The paramagnetic relaxation influence, as well as the size of the protein, results in rise curves that are linear to only 1.5 ms for geminal protons and to only 3-5 ms for more weakly dipolar coupled proton pairs. However, the cross peak intensities in the linear region are shown to yield cross relaxation rates and internuclear distances for a series of assigned and orientationally invariant proton pairs that are in good agreement with their known distances. The patterns of NOESY rise curves are used both to determine the orientations of one propionate and both vinyl groups relative to the heme and to show that the axial His exhibits an orientation relative to its helix that is similar but not identical to that in crystallographically characterized cytochrome c peroxidase. Selective and nonselective 1D as well as 2D selective relaxation rate measurements for hyperfine shifted signals show that only selective or intrinsic relaxation rates can be used to optimize the setup of NOESY experiments and interpret 1D NOE data. The results of the study indicate that NOESY spectra can be expected to yield valuable quantitative structural information on the hyperfine shifted active site residues in a variety of cyanide-inhibited heme peroxidases

Studies in the Physiology of Leaf Growth: I. The Effect of Various Accessory Growth Factors on the Growth of the first Leaf of isolated Stem Tips of Rye

RESP-254

Studies in the Physiology of Leaf Growth: II. Growth and Structure of the First Leaf of Rye when cultivated in Isolation or attached to the Intact Plant

RESP-256

Brazilian Vaccinia Viruses and Their Origins

Genetic diversity enables this virus to persist in Brazil and other parts of the world

Desalination as a Negative Emissions Technology

To limit global warming, governments and industries are engaged in reducing emissions of CO2. There is increasing evidence, however, that it may be necessary to go a step further by removing CO2 already emitted. For the purpose of Carbon Dioxide Removal (CDR), a number of Negative Emissions Technologies (NET) have been proposed. These generally make extensive usage of land, energy and water – if they are to be implemented at the large scales needed. It is therefore important to seek, investigate and compare alternative approaches to NET. Desalination plants, though normally seen as sources rather than sinks of CO2, could be modified to provide a new type of NET. In this study, we propose treating desalination reject brine by electrolysis to form Mg(OH)2 and thus absorb CO2 via the oceans. The energy and water penalties associated with the electrolysis are calculated as 1.8 GJ/tCO2 and 13.7 m3/tCO2 respectively, making it an interesting option in comparison with some other types of NET. However, NET-modification more than doubles the specific energy consumption of a reverse-osmosis desalination plant. It is concluded that NET-desalination has potential to contribute to CDR in arid countries (especially if solar energy is used) thus helping to meet Intended Nationally Determined Contributions (INDCs) following the COP21 summit

Vaccinia Virus Infection in Monkeys, Brazilian Amazon

To detect orthopoxvirus in the Brazilian Amazon, we conducted a serosurvey of 344 wild animals. Neutralizing antibodies against orthopoxvirus were detected by plaque-reduction neutralizing tests in 84 serum samples. Amplicons from 6 monkey samples were sequenced. These amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in Brazil

Passatempo Virus, a Vaccinia Virus Strain, Brazil

Passatempo virus was isolated during a zoonotic outbreak. Biologic features and molecular characterization of hemagglutinin, thymidine kinase, and vaccinia growth factor genes suggested a vaccinia virus infection, which strengthens the idea of the reemergence and circulation of vaccinia virus in Brazil. Molecular polymorphisms indicated that Passatempo virus is a different isolate

Genetic dissection of complete genomes of Type 2 PRRS viruses isolated in Denmark over a period of 15 years

AbstractType 2 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) was first detected in Europe in 1996 co-incident with the introduction of a live attenuated vaccine. Since then, only limited ORF5 and ORF7 sequences of Type 2 PRRS viruses have been reported throughout Europe. In the present study, the genetic and antigenic diversity of 11 complete genomes and 49 ORF5 and 55 ORF7 nucleotide sequences obtained from 57 viruses in Denmark from 2003 to 2012 were examined. The genetic identity of the 11 complete genomes to the vaccine strain (Ingelvac PRRS MLV) ranged between 93.6 and 99.6% while the 49 ORF5 sequences examined were 94.0–99.8% identical to the vaccine strain. Among the Danish sequences, the pairwise nucleotide identity was 90.9–100% and 93.0–100.0% for ORF5 and ORF7, respectively. Analysis of the genetic region encoding NSP2 revealed high diversity among the Danish viruses with an 86.6–98.9% range in similarity. Furthermore, several of the sequenced viruses harbored deletions in the NSP2 coding region. Phylogenetic analysis in a global Type 2 PRRSV framework classified all Danish isolates to a single cluster (sub-lineage 5.1) which comprised strains closely-related to the Type 2 prototype isolate VR2332