Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: Implications for morphogenesis and organization of encapsidated RNA

The capsid protein of hepatitis B virus, consisting of an “assembly” domain (residues 1–149) and an RNA-binding “protamine” domain (residues 150–183), assembles from dimers into icosahedral capsids of two different sizes. The C terminus of the assembly domain (residues 140–149) functions as a morphogenetic switch, longer C termini favoring a higher proportion of the larger capsids, it also connects the protamine domain to the capsid shell. We now have defined the location of this peptide in capsids assembled in vitro by engineering a mutant assembly domain with a single cysteine at its C terminus (residue 150), labeling it with a gold cluster and visualizing the cluster by cryo-electron microscopy. The labeled protein is unimpaired in its ability to form capsids. Our density map reveals a single undecagold cluster under each fivefold and quasi-sixfold vertex, connected to sites at either end of the undersides of the dimers. Considering the geometry of the vertices, the C termini must be more crowded at the fivefolds. Thus, a bulky C terminus would be expected to favor formation of the larger (T = 4) capsids, which have a greater proportion of quasi-sixfolds. Capsids assembled by expressing the full-length protein in Escherichia coli package bacterial RNAs in amounts equivalent to the viral pregenome. Our density map of these capsids reveals a distinct inner shell of density—the RNA. The RNA is connected to the protein shell via the C-terminal linkers and also makes contact around the dimer axes.

Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.

Group B streptococci escape host immunity by deletion of tandem repeat elements of the alpha C protein.

Group B streptococci (GBS) are the most common cause of neonatal sepsis, pneumonia, and meningitis. The alpha C protein is a surface-associated antigen; the gene (bca) for this protein contains a series of tandem repeats (each encoding 82 aa) that are identical at the nucleotide level and express a protective epitope. We previously reported that GBS isolates from two of 14 human maternal and neonatal pairs differed in the number of repeats contained in their alpha C protein; in both pairs, the alpha C protein of the neonatal isolate was smaller in molecular size. We now demonstrate by PCR that the neonatal isolates contain fewer tandem repeats. Maternal isolates were susceptible to opsonophagocytic killing in the presence of alpha C protein-specific antiserum, whereas the discrepant neonatal isolates proliferated. An animal model was developed to further study this phenomenon. Adult mice passively immunized with antiserum to the alpha C protein were challenged with an alpha C protein-expressing strain of GBS. Splenic isolates of GBS from these mice showed a high frequency of mutation in bca--most commonly a decrease in repeat number. Isolates from non-immune mice were not altered. Spontaneous deletions in the repeat region were observed at a much lower frequency (6 x 10(-4)); thus, deletions in that region are selected for under specific antibody pressure and appear to lower the organism's susceptibility to killing by antibody specific to the alpha C protein. This mechanism of antigenic variation may provide a means whereby GBS evade host immunity.

Systematic investigation of projectile fragmentation using beams of unstable B and C isotopes

Background: Models describing nuclear fragmentation and fragmentation fission deliver important input for planning nuclear physics experiments and future radioactive ion beam facilities. These models are usually benchmarked against data from stable beam experiments. In the future, two-step fragmentation reactions with exotic nuclei as stepping stones are a promising tool for reaching the most neutron-rich nuclei, creating a need for models to describe also these reactions. Purpose: We want to extend the presently available data on fragmentation reactions towards the light exotic region on the nuclear chart. Furthermore, we want to improve the understanding of projectile fragmentation especially for unstable isotopes. Method: We have measured projectile fragments from (10,12-18C) and B10-15 isotopes colliding with a carbon target. These measurements were all performed within one experiment, which gives rise to a very consistent data set. We compare our data to model calculations. Results: One-proton removal cross sections with different final neutron numbers (1 pxn) for relativistic C-10,C-12-18 and B10-15 isotopes impinging on a carbon target. Comparing model calculations to the data, we find that the EPAX code is not able to describe the data satisfactorily. Using ABRABLA07 on the other hand, we find that the average excitation energy per abraded nucleon needs to be decreased from 27 MeV to 8.1 MeV. With that decrease ABRABLA07 describes the data surprisingly well. Conclusions: Extending the available data towards light unstable nuclei with a consistent set of new data has allowed a systematic investigation of the role of the excitation energy induced in projectile fragmentation. Most striking is the apparent mass dependence of the average excitation energy per abraded nucleon. Nevertheless, this parameter, which has been related to final-state interactions, requires further study.

A B C of gothic architecture /

Includes index.

Sumerian mythology, a study of spiritual and literary achievement in the third millennium, B.C.

Frontispiece accompanied by leaf with descriptive letterpress.

Zenon papyri; business papers of the third century B.C. dealing with Palestine and Egypt,

Vol. 2 cite as: P.Col. IV

Dedications from the Athenian Akropolis; a catalogue of the inscriptions of the sixth and fifth centuries B. C.

Includes bibliographical references.

A B C of Gothic architecture /

Includes index.

A B C of gothic architecture /

Includes index.

A B C of gothic architecture /

Mode of access: Internet.

Diagnostic motor vehicle inspection demonstration projects, program engineering support. Volume 3 - appendix B: performance analysis of Washington, D.C. inspection lane. Final report.

National Highway Traffic Safety Administration, Washington, D.C.