Biomedical subjects

John J Rux

Publications and source records attributed to John J Rux.

8 recordsLinked to original sources

PubMed2006

Structure-based identification of a major neutralizing site in an adenovirus hexon.

Virus-specific neutralizing antibodies present an obstacle to the effective use of adenovirus vectors for gene therapy and vaccination. The specific sites recognized by neutralizing antibodies have not been identified for any adenovirus, but they have been proposed to reside within the hexon, in small regions of the molecule that are exposed on the capsid surface and possess sequences that vary among serotypes. We have mapped the epitopes recognized by a panel of seven hexon-specific monoclonal antibodies that neutralize the chimpanzee adenovirus 68 (AdC68). Surface plasmon resonance experiments revealed that the antibodies compete for a single hexon binding site, and experiments with synthetic peptides indicated that this site resides within just one small surface loop. Mutations within this loop (but not in other surface loops) permitted virus to escape neutralization by all seven monoclonal antibodies and to resist neutralization by polyclonal antisera obtained from animals immunized against AdC68. These results indicate that a single small surface loop defines a major neutralization site for AdC68 hexon.

Adenoviridae↗

PubMed2005

A novel four-dimensional strategy combining protein and peptide separation methods enables detection of low-abundance proteins in human plasma and serum proteomes.

A novel strategy, termed protein array pixelation, is described for comprehensive profiling of human plasma and serum proteomes. This strategy consists of three sequential high-resolution protein prefractionation methods (major protein depletion, solution isoelectrofocusing, and 1-DE) followed by nanocapillary RP tryptic peptide separation prior to MS/MS analysis. The analysis generates a 2-D protein array where each pixel in the array contains a group of proteins with known pI and molecular weight range. Analysis of the HUPO samples using this strategy resulted in 575 and 2890 protein identifications from plasma and serum, respectively, based on HUPO-approved criteria for high-confidence protein assignments. Most importantly, a substantial number of low-abundance proteins (low ng/mL - pg/mL range) were identified. Although larger volumes were used in initial prefractionation steps, the protein identifications were derived from fractions equivalent to approximately 0.6 microL (45 microg) of plasma and 2.4 microL (204 microg) of serum. The time required for analyzing the entire protein array for each sample is comparable to some published shotgun analyses of plasma and serum proteomes. Therefore, protein array pixelation is a highly sensitive method capable of detecting proteins differing in abundance by up to nine orders of magnitude. With further refinement, this method has the potential for even higher capacity and higher throughput.

Biomarkers↗

PubMed2004

Gene specific siRNA selector.

Small interfering RNA (siRNA) is used in functional genomics applications to decrease the expression of a target gene, which may yield a biological effect that suggests a function for the target gene. The siRNA design tool scans a target gene for candidate siRNA sequences that satisfy user-adjustable rules. Selected candidates are then screened to identify those siRNA sequences that are specific to the gene of interest.

Algorithms↗

PubMed2004

Adenovirus structure.

Structural studies continue to play an essential role as the focus of adenovirus research shifts in emphasis from basic biology to adenovirus-based vector technologies. A crucial step in developing novel therapeutics for gene replacement, cancer, and vaccines is often to modify the virion. Such engineered changes are designed to retarget the virus, or to reduce the immunological responses to infection. These efforts are far more effective when they are based on detailed structural knowledge. This minireview provides a brief summary of the wealth of information that has been obtained from the combined application of X-ray crystallography and electron microscopy. This knowledge now includes a good working model for the architectural organization of the virion, and atomic resolution molecular structures for all the major capsid proteins, hexon, penton, and fiber. We highlight new developments, which include the structure of the penton base and the discovery that adenovirus has several relatives. We sketch how the structural information can be used to engineer novel virions and conclude with the prospects for future progress.

Adenoviruses, Human↗

PubMed2003

Adeno-associated viruses undergo substantial evolution in primates during natural infections.

Adeno-associated viruses (AAVs) are single-stranded DNA viruses that are endemic in human populations without known clinical sequelae and are being evaluated as vectors for human gene therapy. To better understand the biology of this virus, we examined a number of nonhuman primate species for the presence of previously uncharacterized AAVs and characterized their structure and distribution. AAV genomes were widely disseminated throughout multiple tissues of a variety of nonhuman primate species. Surprising diversity of sequence, primarily localized to hypervariable regions of the capsid protein, was detected. This diversity of sequence is caused, in part, by homologous recombination of co-infecting parental viruses that modify the serologic reactivity and tropism of the virus. This is an example of rapid molecular evolution of a DNA virus in a way that was formerly thought to be restricted to RNA viruses.

Animals↗

PubMed2003

Structural and phylogenetic analysis of adenovirus hexons by use of high-resolution x-ray crystallographic, molecular modeling, and sequence-based methods.

A major impediment to the use of adenovirus as a gene therapy vector and for vaccine applications is the host immune response to adenovirus hexon-the major protein component of the icosahedral capsid. A solution may lie in novel vectors with modified or chimeric hexons designed to evade the immune response. To facilitate this approach, we have distinguished the portion of hexon that all serotypes have in common from the hypervariable regions that are responsible for capsid diversity and type-specific immunogenicity. The common hexon core-conserved because it forms the viral capsid-sets boundaries to the regions where modifications can be made to produce nonnative hexons. The core has been defined from the large and diverse set of known hexon sequences by an accurate alignment based on the newly refined crystal structures of human adenovirus types 2 (Ad2) and Ad5 hexon. Comparison of the two hexon models, which are the most accurate so far, reveals that over 90% of the residues in each have three-dimensional positions that closely match. Structures for more distant hexons were predicted by building molecular models of human Ad4, chimpanzee adenovirus (AdC68), and fowl adenovirus 1 (FAV1 or CELO). The five structures were then used to guide the alignment of the 40 full-length (>900 residues) hexon sequences in public databases. Distance- and parsimony-based phylogenetic trees are consistent and reveal evolutionary relationships between adenovirus types that parallel those of their animal hosts. The combination of crystallography, molecular modeling, and phylogenetic analysis defines a conserved molecular core that can serve as the armature for the directed design of novel hexons.

Adenoviruses, Human↗

PubMed2002

Mutations in Rab3a alter circadian period and homeostatic response to sleep loss in the mouse.

Rab3a is the most abundant Rab (ras-associated binding) protein in the brain and has a regulatory role in synaptic vesicle trafficking. Mice with a targeted loss-of-function mutation in Rab3a have defects in Ca(2+)-dependent synaptic transmission: the number of vesicles released in response to an action potential is greater than in wildtype mice, resulting in greater synaptic depression and the abolishment of CA3 mossy-fiber long term potentiation. The effect of these changes on behavior is unknown. In a screen for mouse mutants with abnormal rest-activity and sleep patterns, we identified a semidominant mutation, called earlybird, that shortens the circadian period of locomotor activity. Sequence analysis of Rab3a identified a point mutation in the conserved amino acid (Asp77Gly) within the GTP-binding domain of this protein in earlybird mutants, resulting in significantly reduced levels of Rab3a protein. Phenotypic assessment of earlybird mice and a null allele of Rab3a revealed anomalies in circadian period and sleep homeostasis, providing evidence that Rab3a-mediated synaptic transmission is involved in these behaviors.

Alleles↗

PubMed2002

A common DNA-binding site for SZF1 and the BRCA1-associated zinc finger protein, ZBRK1.

More than 220 Kruppel-associated box-zinc finger protein (KRAB-ZFP) genes are encoded in the human genome. KRAB-ZFPs function as transcriptionalrepressors by binding DNA through their tandem zinc finger motifs.Gene silencing is mediated by the highly conserved KRAB domain, which recruits histone deacetylase complexes, histone methylases, and heterochromatin proteins. However, little is known of the biological programs regulated by KRAB-ZFPs, in large part because of the difficulty in identifying DNA-binding sites recognized by long arrays of zinc fingers. In an attempt to identify the natural target genes for a KRAB-ZFP, we chose SZF1, a hematopoietic progenitor-restricted, KRAB-ZFP that contains only four C(2)H(2) zinc finger motifs. Using recombinant SZF1 protein and a PCR-based binding site selection strategy, we identified a 15-bp consensus DNA sequence recognized by SZF1. Remarkably, this sequence is similar to the core DNA-binding site described recently for ZBRK1, a KRAB-ZFP that binds to BRCA1 and is involved in coordinating the cellular DNA damage response. The SZF1 and ZBRK1 proteins bind to both the experimentally derived SZF1 site and the canonical ZBRK1 site. The KRAB domain from SZF1 bound directly to the KAP-1 corepressor and displayed intrinsic silencing activity. Moreover, full-length SZF1 repressed a promoter containing ZBRK1 recognition sequences. Thus, SZF1 and ZBRK1 may regulate a common set of target genes in vivo.

3T3 Cells↗