Paul D. Gershon

The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell lumen formation

The combination of a candidate gene approach, column chromatography, and mass spectrometry identifies several fibroblast-derived proteins essential for endothelial cell sprouting and lumen formation. Furthermore, proteins responsible for EC lumen formation increase matrix stiffness, which correlates with EC lumenogenesis.

Sensitivity and detection limit of concentration and adsorption measurements by laser-induced surface-plasmon resonance

The shot noise limitation as well as other factors that influence the sensitivity of measurements with a surface plasmon resonance (SPR) sensor are considered. It is demonstrated that minute changes in the refractive index of a medium close to the surface of a metal film can be detected owing to a shift in the resonance angle. In particular, changes in the adsorption layer of only a fraction of a biomolecular monolayer could be measured. Data for SPR are presented with adjacent media of air, water, as well as aqueous solutions of ethanol and sodium chloride at different concentrations. The immobilization of the protein bovine serum albumin to a specially prepared surface was monitored with the SPR technique. Specific responses to changes in the concentration and thickness of the adsorption layer were determined. The angular resolution of the present apparatus is approximately 1 millidegree, corresponding to a detection limit for an adsorbed protein layer of 15 pg/mm(2), which is still 2 to 3 orders of magnitude larger than the shot-noise limit, and therefore a further improvement in sensitivity is possible.

3′ adenylation determines mRNA abundance and monitors completion of RNA editing in T. brucei mitochondria

Expression of the mitochondrial genome in protozoan parasite Trypanosoma brucei is controlled post‐transcriptionally and requires extensive U‐insertion/deletion mRNA editing. In mitochondrial extracts, 3′ adenylation reportedly influences degradation kinetics of synthetic edited and pre‐edited mRNAs. We have identified and characterized a mitochondrial poly(A) polymerase, termed KPAP1, and determined major polypeptides in the polyadenylation complex. Inhibition of KPAP1 expression abrogates short and long A‐tails typically found in mitochondrial mRNAs, and decreases the abundance of never‐edited and edited transcripts. Pre‐edited mRNAs are not destabilized by the lack of 3′ adenylation, whereas short A‐tails are required and sufficient to maintain the steady‐state levels of partially edited, fully edited, and never‐edited mRNAs. The editing directed by a single guide RNA is sufficient to impose a requirement for the short A‐tail in edited molecules. Upon completion of the editing process, the short A‐tails are extended as (A/U) heteropolymers into structures previously thought to be long poly(A) tails. These data provide the first direct evidence of functional interactions between 3′ processing and editing of mitochondrial mRNAs in trypanosomes.

Structure of Intracellular Mature Vaccinia Virus Visualized by In Situ Atomic Force Microscopy

ABSTRACT Vaccinia virus, the basis of the smallpox vaccine, is one of the largest viruses to replicate in humans. We have used in situ atomic force microscopy (AFM) to directly visualize fully hydrated, intact intracellular mature vaccinia virus (IMV) virions and chemical and enzymatic treatment products thereof. The latter included virion cores, core-enveloping coats, and core substructures. The isolated coats appeared to be composed of a highly cross-linked protein array. AFM imaging of core substructures indicated association of the linear viral DNA genome with a segmented protein sheath forming an extended ∼16-nm-diameter filament with helical surface topography; enclosure of this filament within a 30- to 40-nm-diameter tubule which also shows helical topography; and enclosure of the folded, condensed 30- to 40-nm-diameter tubule within the core by a wall covered with peg-like projections. Proteins observed attached to the 30- to 40-nm-diameter tubules may mediate folding and/or compaction of the tubules and/or represent vestiges of the core wall and/or pegs. An accessory “satellite domain” was observed protruding from the intact core. This corresponded in size to isolated 70- to 100-nm-diameter particles that were imaged independently and might represent detached accessory domains. AFM imaging of intact virions indicated that IMV underwent a reversible shrinkage upon dehydration (as much as 2.2- to 2.5-fold in the height dimension), accompanied by topological and topographical changes, including protrusion of the satellite domain. As shown here, the chemical and enzymatic dissection of large, asymmetrical virus particles in combination with in situ AFM provides an informative complement to other structure determination techniques.

Structural basis of mRNA cap recognition by proteins

Crystal structures have recently become available for two proteins (VP39 and eIF4E) complexed with their cognate ligand - the mRNA cap. Despite their total structural dissimilarity, both proteins bind N7-methylguanine between two parallel aromatic sidechains. The resulting stacked arrangement governs their high specificity for the alkylated form of the nucleobase.

Identification of rpo30, a vaccinia virus RNA polymerase gene with structural similarity to a eucaryotic transcription elongation factor

Eucaryotic transcription factors that stimulate RNA polymerase II by increasing the efficiency of elongation of specifically or randomly initiated RNA chains have been isolated and characterized. We have identified a 30-kilodalton (kDa) vaccinia virus-encoded protein with apparent homology to SII, a 34-kDa mammalian transcriptional elongation factor. In addition to amino acid sequence similarities, both proteins contain C-terminal putative zinc finger domains. Identification of the gene, rpo30, encoding the vaccinia virus protein was achieved by using antibody to the purified viral RNA polymerase for immunoprecipitation of the in vitro translation products of in vivo-synthesized early mRNA selected by hybridization to cloned DNA fragments of the viral genome. Western immunoblot analysis using antiserum made to the vaccinia rpo30 protein expressed in bacteria indicated that the 30-kDa protein remains associated with highly purified viral RNA polymerase. Thus, the vaccinia virus protein, unlike its eucaryotic homolog, is an integral RNA polymerase subunit rather than a readily separable transcription factor. Further studies showed that the expression of rpo30 is regulated by dual early and later promoters.

The nucleotide sequence around the capripoxvirus thymidine kinase gene reveals a gene shared specifically with leporipoxvirus

We have extended previous comparisons of genetic organization between poxvirus genera by sequencing a 2.5K genomic fragment from isolate KS-1 (Kenya sheep-1) of the genus capripoxvirus. The fragment is located in the central region of the capripoxvirus genome and contains three complete and two incomplete open reading frames (ORFs). One of the complete ORFs is a gene for thymidine kinase (TK). This gene, with one of the other two complete ORFs and both the incomplete ORFs, are homologous to four contiguous ORFs from the central region of vaccinia virus (VV) DNA. They also match four ORFs of fowlpox virus (FPV) DNA, three of which are contiguous and the fourth, the FPV TK gene, is located elsewhere on the FPV genome. The third complete ORF of the capripoxvirus DNA fragment is located between the TK gene and the capripoxvirus homologue of the ORF immediately downstream of the VV TK gene. We show that a homologue to this third ORF is absent from VV and FPV DNAs, but is present downstream of the TK gene on Shope fibroma virus DNA. The sequence immediately upstream of the capripoxvirus homologue of a VV late gene contains a motif which is required for VV late gene expression. The motif required for VV early gene transcription termination is present in eight positions in the capripoxvirus sequence, and five of these positions are consistent with the motif having an equivalent function in capripoxvirus to that in VV.

An RNA virus hijacks an incognito function of a DNA repair enzyme

A previously described mammalian cell activity, called VPg unlinkase, specifically cleaves a unique protein–RNA covalent linkage generated during the viral genomic RNA replication steps of a picornavirus infection. For over three decades, the identity of this cellular activity and its normal role in the uninfected cell had remained elusive. Here we report the purification and identification of VPg unlinkase as the DNA repair enzyme, 5′-tyrosyl–DNA phosphodiesterase-2 (TDP2). Our data show that VPg unlinkase activity in different mammalian cell lines correlates with their differential expression of TDP2. Furthermore, we show that recombinant TDP2 can cleave the protein–RNA linkage generated by different picornaviruses without impairing the integrity of viral RNA. Our results reveal a unique RNA repair-like function for TDP2 and suggest an unusual role in host–pathogen interactions for this cellular enzyme. On the basis of the identification of TDP2 as a potential antiviral target, our findings may lead to the development of universal therapeutics to treat the millions of individuals afflicted annually with diseases caused by picornaviruses, including myocarditis, aseptic meningitis, encephalitis, hepatitis, and the common cold.

Surface-plasmon resonance spectrometry and characterization of absorbing liquids

The effect of absorption of the sample medium on the surface-plasmon resonance (SPR) characteristics is analyzed by approximate analytical and exact numerical models. We show that absorption leads to specific changes in the value of reflectivity near the SPR angle and that these can be used for absorbance detection. The strongest absorption-induced change in reflectivity occurs at two values of metal film thickness (28 and 55 nm for a gold film and lambda = 632.8 nm). Using a sample solution of Rhodamine 700 in ethanol, we measured the characteristic changes in the SPR angle and in reflectivity over the wavelength interval encompassing the strong absorption band at 610-680 nm. The possibility of the simultaneous determination of the refractive index and absorption from SPR measurements is demonstrated and has the potential for substance-specific detection.

A comparison of the genomes of capripoxvirus isolates of sheep, goats, and cattle.

HindIII, PstI, AvaI, and SalI sites were mapped on the genomes of six isolates of capripoxvirus from sheep, goats, and cattle. Genome pairs were aligned by the alignment of cross-hybridizing HindIII fragments and the introduction of padding fragments (pads) at specific locations. The majority of these pads represent the approximate positions of relative deletions or insertions. Three possible phylogenetic networks were generated for seven capripoxvirus isolates by Wagner parsimony analysis of the nonconserved HindIII sites on their genomes, and confidence limits were calculated for the network nodes. Nucleotide sequence divergence values, calculated from the numbers of nonconserved HindIII, PstI, AvaI, and SalI sites on the genomes of typical sheep, goat, and cattle isolates, indicated that the typical sheep and cattle isolates are more closely related to one another than to the typical goat isolate. Nonconserved HindIII, PstI, AvaI, and SalI sites were shown to be distributed throughout the genomes. Evidence that isolates YG-1 and OS-1 are descended from an isolate whose genome arose by recombination is discussed. Terminally repeated regions were identified on each of the capripoxvirus genomes mapped here. By mapping BamHI, ClaI, EcoRI, HindII, HindIII, and SalI sites present within the terminal 10 kb of the genome of isolate InS-1, the terminal repeats of this genome were shown to be between 2.25 and 3.40 kb in length and inverted with respect to one another.