William L. Ragland

Fluorometric scanning of fluorescamine-labeled proteins in polyacrylamide gels.

Abstract A very rapid and sensitive fluorescence technique has been described for detecting and quantitatively assaying proteins in polyacrylamide gels. Proteins were labeled with fluorescamine, subjected to SDS polyacrylamide gel electrophoresis and scanned for fluorescence with a modified Gilford 2410-S linear transport. The fluorescence signal was linear with the concentration of protein over a range of 0.5–7 μg (myoglobin), 9 μg (chymotrypsinogen A), and at least 12.5 μg (ovalbumin). The limits of sensitivity were not determined but as little as 6 ng myoglobin could be measured accurately. Several applications for fluorescence gel scanning were discussed.

Abundance of IFN-α and IFN-γ mRNA in blood of resistant and susceptible chickens infected with Marek's disease virus (MDV) or vaccinated with turkey herpesvirus; and MDV inhibition of subsequent induction of IFN gene transcription

Summary.The effects of the very virulent RB-1B strain of Marek’s disease virus (MDV) and turkey herpesvirus (HVT), a vaccinal strain, on abundance of IFN mRNA in the blood were investigated. MDV and HVT infection did not change the circulating level of IFN-γ mRNA 1 and 7 days p.i., but they increased IFN-α mRNA levels slightly in genetically susceptible (to tumour development) B13/B13 chickens. The total number of circulating leukocytes was unchanged and increase in message was accompanied by an increase in circulating CD8α+ and MHC Class II+ cells. On the contrary, both viruses slightly increased IFN-γ transcripts and decreased IFN-α transcripts in genetically resistant B21/B21 chickens. Further, oncogenic MDV was able to block the response to inactivated Newcastle disease virus, a potent inducer of IFN, in both chicken lines. The inhibiting effect on transcription was present for both IFN at days 1 and 7 p.i. in susceptible B13/B13 chickens, but only at day 7 p.i. in resistant B21/B21 chickens. By contrast, non-oncogenic HVT did not interfere with induction of either message at one day p.i. and MDV had a more suppressive effect than HVT on IFN gene transcription 7 days p.i. in B21/B21 chickens. Thus, the strong ability of MDV to block induction of IFN gene transcription detected in the blood as soon as one day after infection in susceptible chickens, as opposed to resistant chickens, not only causes immunosuppression but also may be related to the virus’s oncogenicity.

Estimation of molecular weight by polyacrylamide gel electrophoresis using heat stable fluorophors

Abstract The utilization of 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) as a fluorescent label for proteins in SDS polyacrylamide gel electrophresis is described. The procedure is very sensitive and detects disks containing as little as 1 ng of protein. The fluorescence signal is linear with concentration of protein to 500 ng. Emphasis is placed on the use of MDPF-labeled proteins as standards for the estimation of molecular weights of fluorescamine-labeled proteins. Data are provided which demonstrate the need for only three standard proteins in the molecular weight curve. Finally, the relative mobilities of proteins from nonreplicate determinations exhibited an average variation of only 3.9%.

Avian thymic hormone (ATH) is a parvalbumin

Amino acid sequence analysis of a protein from chicken thymus tissue which promotes immunological maturity in chicken bone marrow cells in culture has established sequences of a 45-residue fragment, a 24-residue fragment and a 9-residue and an 8-residue peptide. Independent comparison of the 45- and 24-residue fragments with known amino acid sequences by computer search has unequivocally identified avian thymic hormone as a parvalbumin. This is the first demonstration that a protein previously identified by a biological function is a parvalbumin.

Short Communication: Chicken Anemia Virus and Infectious Bursal Disease Virus Interfere with Transcription of Chicken IFN-α and IFN-γ mRNA

Chicken anemia virus (CAV) and infectious bursal disease virus (IBDV) are the two most important viruses that cause immunosuppression in commercial chickens. Because inapparent, subclinical infections by these viruses cause immunosuppression, there is need for assessment of the immune status of chickens. Interference with induction of transcription for chicken interferon-α (ChIFN-α) and ChIFN-γ was noted after subclinical infections with either CAV or IBDV. Because the immunosuppressive viruses of chickens may interfere with transcription for ChIFN-α and ChIFN-γ, we propose using this interference to assess the immune status of chickens.

The relationship of molecular weight to electrophoretic mobility of fluorescamine-labeled proteins in polyacrylamide gels

Abstract Proteins of known molecular weights were labeled with fluorescamine and then subjected to electrophoresis through polyacrylamide gels. The electrophoretic mobilities of the fluorescamine-labeled proteins were dependent upon their respective molecular weights over a range of 17,000 to 70,000 daltons. The correlation of electrophoretic mobility of fluorescamine-labeled protein to molecular weight was similar to results obtained in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The speed with which data can be obtained with the described procedure is a definite advantage over currently employed procedures. These findings encourage the use of fluorescamine for rapid, sensitive determinations of molecular weights of proteins in polyacrylamide gels.

Selective depression of hepatic cytochrome P-450 hemoprotein by interferon inducers

Abstract The interferon inducing agents, polyriboinosinic: polyribocytidylic acid and tilorone, and Freunds complete adjuvant cause a marked depression of several components of the hepatic mixed-function oxidase system. Separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitation by fluorescence gel scanning of different molecular weight species of cytochrome P-450 indicate that the depressant effect of these agents on the apoprotein moieties of cytochrome P-450 is of a specific nature.

Immune status assessment by abundance of IFN-alpha and IFN-gamma mRNA in chicken blood.

Avian diseases, including such viral infection as infectious bursal disease, infectious anemia, and Mareks disease, often cause immunosuppression, leading to more severe infection, problems with secondary infection, and inadequate responses to vaccination. Immunosuppression thus causes serious economic losses in commercial poultry production. To date, methods for assessing immune status have been too slow to be of practical help. Reasoning that immunosuppression should be reflected by reduced production of interferons (IFN) in response to a viral antigen, we have developed competitive nucleic acid hybridization microtiter plate assays for chicken IFN-alpha (ChIFN-alpha) and ChIFN-gamma mRNA. To evaluate the assay, chickens were challenged with inactivated Newcastle disease virus (iNDV). Whole blood samples were collected at various times subsequently and preserved with a cationic detergent. Later, total RNA was extracted, and mRNA for both ChIFN-alpha and ChIFN-gamma was measured. Both rose from undetectable levels to reach a peak by 4 h, remained high for about 3 days, and fell to undetectable levels by day 5. Results were similar in chickens aged between 1 and 28 days. In later experiments, blood was collected 4 h after viral challenge. When chickens were immunosuppressed by administering 4-5 mg cyclophosphamide (CY) daily for 3 days and challenged with iNDV, they transcribed less ChIFN-alpha and ChIFN-gamma mRNA, and their antibody response was impaired. Our results suggest that suspected immunosuppression in a commercial flock could be assessed within 2-3 days by challenging birds with iNDV and measuring the amounts of ChIFN-alpha and ChIFN-gamma mRNA in blood obtained 2-4 h later.

Protection against airsacculitis with sequential systemic and local immunization of chickens using killed Mycoplasma gallisepticum bacterin with iota carrageenan adjuvant

The induction of protective immunity to Mycoplasma gallisepticum (MG) by bacterins containing 0.2% iota carrageenan (iCGN) as an adjuvant has been studied. Various combinations of intracoelomic (i.c.), intratracheal (i.t.), intranasal (i.n.), intravenous (i.v.), subcutaneous (s.c.) and oral immunization routes were evaluated. Vaccinated and non-vaccinated groups were compared with a group vaccinated s.c. with a commercial bacterin. Primary i.c. immunization with the bacterin was as effective as commercial bacterin and was more effective when followed by i.n. or i.t. immunization. Oral immunization was ineffective, in contrast to observations reported with mice. The i.c./i.n. and i.c./i.t. combinations were the most effective, produced the highest levels of anti-MG IgG and IgA in serum and tracheobronchial washes, and sometimes provided 100% protection against air sac lesions. Chickens vaccinated by the i.c./i.n. or i.c./i.t. routes had the fewest virulent organisms in their respiratory tract secretions. These results demonstrated that i.c. immunization followed by local immunization with the bacterin is most efficacious in protecting chickens against airsacculitis.

Plasma membranes of bursal, thymic and circulating lymphocytes have unique proteins

Abstract The protein subunit composition of plasma membranes of chicken bursal, thymic and circulating lymphocytes was determined by polyacrylamide gel electrophoresis. Two of the proteins common to bursa and thymus were not expressed in the circulating lymphocytes. Bursal membranes had 4 unique proteins, 2 of which were still expressed in the circulating lymphocytes. Thymic membranes had 3 unique proteins, one of which was still expressed in the circulating lymphocytes. Membranes of circulating lymphocytes had one unique protein.