Members of the actin-depolymerizing factor (ADF)/cofilin family of proteins are expressed in all eukaryotic cells. In higher vertebrates, cells often express as many as three different ADF/cofilin genes and each of these proteins may be phosphorylated on serine 3, giving rise to up to six different species. Also, many avian, amphibian, and invertebrate systems have been useful in studying different aspects of ADF/cofilin function. Antibodies have been prepared against different members of the ADF/cofilin family, but no systematic examination of their cross-reactivity has been reported. Although ADF and cofilins within a single vertebrate species have about a 70% sequence homology, antibodies often differentiate between these proteins. Here, Western blotting was used with chemiluminescence substrates of different sensitivities to determine the relative immunoreactivities of different polyclonal rabbit antibodies and a mouse monoclonal antibody to purified ADF/cofilins from plants, protists, nematodes, insects, echinoderms, birds, and mammals. From immunocross-reactivities and sequence alignments, the principal epitope in mammalian ADF and cofilin-1 recognized by an antibody raised against avian ADF was identified. The specificity of an antibody to the phosphopeptide epitope of metazoan ADF/cofilins was confirmed by two-dimensional (2-D) immunoblot analysis. Futhermore, this bank of antibodies was used to identify by Western blotting a putative member of the ADF/cofilin family in the sea slug, Aplysia californica.
RNA polymerase II-associating protein 74 (RAP74) is the large subunit of transcription factor IIF (TFIIF), which is essential for accurate initiation and stimulates elongation by RNA polymerase II. Mutations within or adjacent to the ␣1 helix of the RAP74 subunit have been shown to decrease both initiation and elongation stimulation activities without strongly affecting the interactions of RAP74 with the RAP30 subunit or the interaction between TFIIF and RNA polymerase II. In this manuscript, mutations within the ␣1 helix are compared with mutations made throughout the neighboring conserved N-terminal domain of RAP74. Changes within the N-terminal domain include disruptions of specific contacts with the ␣1 helix, which were revealed in the recently published x-ray crystal structure (Gaiser, F., Tan TFIIF1 appears to be an ␣ heterodimer of RAP74 and RAP30 subunits, and previous reports that TFIIF may be an ␣ 2  2 heterotetramer are not supported by the x-ray crystal structure (1). Although not evident from primary sequence, RAP74 and RAP30 subunits are structurally similar, with an intricate series of N-terminal-sheets that form a RAP74-RAP30 dimer interface. RAP74 and RAP30 also have similar C-terminal regions with winged helix-turn-helix structures (2, 3). The larger size of the human RAP74 subunit can be attributed to an extensive loop rich in Gly, Pro, Ser, Thr, and charged residues separating more structured N-and C-terminal domains (4, 5).TFIIF is an RNA polymerase II-specific transcription factor restricted to the eukaryotic kingdom. Fig. 1, therefore, shows an amino acid sequence alignment of the N-terminal regions of several RAP74 homologues spaced throughout eukaryotic evolution. Beneath the alignment, the primary sequence is correlated with regions of secondary structure. Regions of ␣-helix and -sheet are derived from the crystal structure of human TFIIF (1) or else from secondary structure predictions (6 -9) in the regions where structural information is not available. The x-ray crystal structure indicates a unique dimer interface made up of RAP74 -sheets 1, 2, 3, 6, 7, 8, and the corresponding -sheets in RAP30. The 4 and 5 sheets of RAP74 interact to form the structured base of a loop that diverges from the dimer core. In the crystal structure, the RAP74 ␣1 helix makes intimate contacts with the 4 and 5 sheets.The ␣1 helix has been shown previously to be highly sensitive to mutation (10, 11). Several single amino acid changes, particularly in hydrophobic residues, cause significant defects in both accurate initiation and elongation. Because of symmetrical effects on initiation and elongation, ␣1 should function by contacting a molecular target common to both processes, which would implicate RNA polymerase II, TFIIF, and DNA as the most likely targets. Mutations in ␣1 do not appear to affect interaction with the RAP30 subunit, and no RAP30 contacts are identified for ␣1 in the crystal structure (1). As far as can be discerned, ␣1 mutations do not have an effect on assembly of gel-shif...
Early detection of highly pathogenic avian influenza (HPAI) infection in commercial poultry flocks is a critical component of outbreak control. Reducing the time to detect HPAI infection can reduce the risk of disease transmission to other flocks. The timeliness of different types of detection triggers could be dependent on clinical signs that are first observed in a flock, signs that might vary due to HPAI virus strain characteristics. We developed a stochastic disease transmission model to evaluate how transmission characteristics of various HPAI strains might effect the relative importance of increased mortality, drop in egg production, or daily real-time reverse transcriptase (RRT)-PCR testing, toward detecting HPAI infection in a commercial table-egg layer flock. On average, daily RRT-PCR testing resulted in the shortest time to detection (from 3.5 to 6.1 days) depending on the HPAI virus strain and was less variable over a range of transmission parameters compared with other triggers evaluated. Our results indicate that a trigger to detect a drop in egg production would be useful for HPAI virus strains with long infectious periods (6-8 days) and including an egg-drop detection trigger in emergency response plans would lead to earlier and consistent reporting in some cases. We discuss implications for outbreak control and risk of HPAI spread attributed to different HPAI strain characteristics where an increase in mortality or a drop in egg production or both would be among the first clinical signs observed in an infected flock.
Emergency response during a highly pathogenic avian influenza (HPAI) outbreak may involve quarantine and movement controls for poultry products such as eggs. However, such disease control measures may disrupt business continuity and impact food security, since egg production facilities often do not have sufficient capacity to store eggs for prolonged periods. We propose the incorporation of a holding time before egg movement in conjunction with targeted active surveillance as a novel approach to move eggs from flocks within a control area with a low likelihood of them being contaminated with HPAI virus. Holding time reduces the likelihood of HPAI-contaminated eggs being moved from a farm before HPAI infection is detected in the flock. We used a stochastic disease transmission model to estimate the HPAI disease prevalence, disease mortality, and fraction of internally contaminated eggs at various time points postinfection of a commercial table-egg layer flock. The transmission model results were then used in a simulation model of a targeted matrix gene real-time reverse transcriptase (RRT)-PCR testing based surveillance protocol to estimate the time to detection and the number of contaminated eggs moved under different holding times. Our simulation results indicate a significant reduction in the number of internally contaminated eggs moved from an HPAI-infected undetected flock with each additional day of holding time. Incorporation of a holding time and the use of targeted surveillance have been adopted by the U.S. Department of Agriculture in their Draft Secure Egg Supply Plan for movement of egg industry products during an HPAI outbreak.
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