Species specificity and developmental patterns of expression of the beta amyloid precursor protein (APP) gene in brain, liver and choroid plexus in birds.

1. Human APP cDNA hybridized to a 3.5 kb mRNA in liver and brain RNA from chickens, pigeons, quail and ducks as well as in RNA from choroid plexus of chicken and quail. In contrast to all other species hitherto examined a 1.6 kb mRNA hybridizing to APP cDNA was found in abundant amounts in RNA from chicken and quail livers. 2. In the chicken, before hatching, the levels of APP mRNA in total RNA from liver and choroid plexus were higher than those in RNA from liver and choroid plexus of adults. However, RNA from the rest of the brain of chicken embryos contained less APP mRNA than RNA from brain of adults. 3. In the chicken, between 10 and 40 days after hatching, APP mRNA levels in RNA from liver were higher than adult levels, APP mRNA levels in RNA from choroid plexus were similar to adult levels and APP mRNA levels in RNA from the rest of brain were below the adult levels.     

Transfection of Chicken Embryo Cells with DNA Extracted from Avian Virus-Producing Neoplastic Cells

DNA isolated from avian virus-producing leukemic myeloblasts induced the production of viruses, but not morphological transformation, in cultivated chicken fibroblasts. The recovered virus had the same biological characteristics as the original avian myeloblastosis virus (AMV) and produced myeloblastosis and nephroblastomas when injected into chickens. Neutralization experiments with chicken anti-AMV-BAI strain A sera showed an antigenic community between the DNA-transfected virus and the original virus. Virus induced in fibroblasts after treatment with DNA from a viral nephroblastic nephroblastoma line only gave nephroblastoma when injected into chicken. Treatment of chicken embryo cells with DNA extracted from normal chicken embryos did not induce viral production.     

Study of the reproduction conditions of the avian myeloblastosis virus for separation of the "reverse transcriptase" enzyme]

Sensitivity of eight chick lines to the avian myeloblastosis virus as the main source for RNA-dependent DNA-polymerase recovery was studied in the course of the "revertase" project. The virus (0.1 ml) was inoculated intracardially or intraperitoneally to one-day chicks, and then the virus titer was determined according to the ATP-activity. C and D-lines of the Enya-cross were shown to be most sensitive (sensitivity 75%) among the lines studied.     

Target cells for avian myeloblastosis virus in embryonic yolk sac and relationship of cell differentiation to cell transformation

The yolk sac of the 12-day chicken embryo retains the blast stage progenitors to cells of the myeloid lineages with a very low level of contamination by more mature myeloid cells which have begun to express the characteristic myeloid cell markers. Both in vivo and in vitro experiments have supported the hypothesis that target cells for the BAI-A strain of avian myeloblastosis virus are contained within the myeloid lineages. An assay system for avian myeloblastosis virus was developed which utilizes this yolk sac cell system and which appears to be more sensitive than previous published assays. In addition, the kinetics of a liquid culture transformation system is presented in which at least 4% of the yolk sac cell population was transformed in a relatively synchronous fashion at 2 days after infection. The morphological transformation preceded an increased rate of cell proliferation. Cell separation procedures provided a 10- to 20-fold enrichment of target cells and demonstrated that the target cell population copurifies with macrophage colony-forming cells which are the committed progenitors to the macrophage lineage. In combination with earlier work, this work demonstrated that cells committed to the macrophage lineage at all stages of differentiation may serve as target cells for infection by avian myeloblastosis virus.     

env-encoded residues are not required for transformation by p48v-myb.

The v-myb oncogene of avian myeloblastosis virus induces acute myeloblastic leukemia in chickens and transforms avian myeloid cells in vitro. The protein product of this oncogene, p48v-myb, is partially encoded by the retroviral gag and env genes. We demonstrated that the env-encoded carboxyl terminus of p48v-myb is not required for transformation. Our results showed, in addition, that a coding region of c-myb which is not essential for transformation was transduced by avian myeloblastosis virus.     

Identification of DNA in the core component of avian myeloblastosis virus.

Avian myeloblastosis virus (AMV) was found to contain DNA associated with the virion. The viral envelope was removed by treating the virus with a nonionic detergent and the DNA was found in the core fraction. These experiments indicate that the DNA associated with tumor virus is not contaminant associated with the viral envelope and suggest that the DNA is part of the internal core component. The DNA from avian myeloblastosis virus has a density of 1.70 g/cm3.     

Quantitation of RNA tumor viruses by spectroscopy of density gradient gels.

We have developed a system for virus particle quantitation based on the measurement of the optical absorbance of stained viruses which first have been banded at their buoyant density in an equilibrum 24 to 53% (wt/wt) sucrose density gradient, then fixed in position in the gradient by photopolymerizing an acrylamide-riboflavin mixture in the sucrose, and finally stained and destained. Using plasma from mice infected with leukemia virus (Rauscher) or chickens infected with avian myeloblastosis virus (BAI strain) or suitable controls, we have shown that this technique specifically detects RNA tumor viruses. By using virus stock solutions for which the absolute concentrations were determined by laser beat frequency spectroscopy, we have calibrated the absorbance of the viral bands in terms of virus particle concentration. Using 0.8-ml gradients gels (4 by 45 mm) we can detect as low as 2 x 10(7) viral particles with Coomassie blue staining and 6 x 10(6) viral particles with a more sensitive staining procedure using amido black.     

Isolation of two subgroup-specific leukemogenic viruses from standard avian myeloblastosis virus.

Two populations of virus having subgroup-specific homogeneity (A and B) were isolated from standard avian myeloblastosis virus stocks by passage in vivo through genetically defined chickens. Each possesses leukemogenic activity in vivo. Other properties and potential usefulness of these agents are discussed.     

Expression of endogenous avian myeloblastosis virus information in different chicken cells.

Uninfected chicken cells were found to contain endogenous avian myeloblastosis virus (AMV)-specific information. Different tissues from chicken embryos and chickens expressed different amounts of the AMV-specific information. The endogenous AMV-related RNA was most abundant in bone marrow cells, which contained about 20 copies per cell. About 5 to 10 copies of AMV endogenous RNA per cell were found in embryonic yolk sac cells and bursa cells. The spleen, muscle, liver, and kidney cells of chickens and the fibroblasts of chicken embryos contained about two copies per cell. The amounts of AMV endogenous RNA in bone marrow, yolk sac, and bursa varied with age. From 19-day-old embryos to 2-week-old chickens, the bone marrow contained 20 copies of AMV RNA per cell. Bone marrow cells from 2-year-old chickens contained five copies per cell. Yolk sac cells of 10-day-old embryos and 1-day-old chickens were found to contain two copies per cell, whereas in 15- to 17-day-old embryos, these cells contained 5 to 10 copies. These results indicate that the level of endogenous AMV expression correlates with the development of granulopoiesis of the chicken hemopoietic system. The results of experiments on the thermostability of RNA-DNA hybrids indicated that the endogenous AMV RNA is closely related to viral AMV RNA. The expression of endogenous AMV information is independent of the activity of the chick helper factor. This endogenous AMV information is expressed as 20 to 21S RNA in both bone marrow and yolk sac cells.     

The expression of transthyretin mRNA in the developing rat brain

Specific cDNA and oligonucleotide probes were used to study the appearance of transthyretin mRNA in developing rat brain using Northern gel analysis, cytoplasmic dot hybridization, and in situ hybridization. Transthyretin mRNA in embryonic rat brain was found to be confined to the epithelial layer of the choroid plexus primordia appearing first in the fourth ventricle, followed by appearance in the lateral ventricles, and subsequently in the third ventricle. Transthyretin mRNA was localized in these cells from early stages of neuroepithelium differentiation, showing that it is a sensitive marker for the differentiation of the choroid plexus within the fetal brain.     

Evaluation of the ESPLINE INFLUENZA A&B-N Kit for the diagnosis of avian and swine influenza

The ESPLINE INFLUENZA A&B-N kit was evaluated for its applicability to the rapid diagnosis of influenza in chickens and pigs. The kit specifically detected viral antigens in tracheal swabs and tissue homogenates of the trachea, liver, spleen, and colon of chickens inoculated with a highly pathogenic avian influenza virus strain, A/chicken/Yamaguchi/7/04 (H5N1), at 48 hr post-inoculation (p.i.) as well as in the tracheal and cloacal swabs and tissue homogenates of dead chickens. For those infected with a low pathogenic strain, A/chicken/aq-Y-55/01 (H9N2), antigens were detected only in the samples from tracheal swabs and organs 1-4 days p.i. The kit also detected viral antigens in the nasal swabs of miniature pigs infected with swine and avian influenza viruses. The kit was found to be sensitive and specific enough for the rapid diagnosis of infections of influenza A virus in chickens and pigs.     

Generation of a highly pathogenic avian influenza A virus from an avirulent field isolate by passaging in chickens

Highly virulent avian influenza viruses can arise from avirulent strains maintained in poultry, but evidence to support their generation from viruses in wild birds is lacking. The most likely mechanism for the acquisition of virulence by benign avian viruses is the introduction of mutations by error-prone RNA polymerase, followed by the selection of virulent viruses. To investigate whether this mechanism could apply to wild waterfowl, we studied an avirulent wild-swan virus that replicates poorly in chickens. After 24 consecutive passages by air sac inoculation, followed by five passages in chicken brain, the avirulent virus became highly pathogenic in chickens, producing a 100% mortality rate. Sequence analysis at the hemmaglutinin cleavage site of the original isolate revealed a typical avirulence type of sequence, R-E-T-R, which progressed incrementally to a typical virulence type of sequence, R-R-K-K-R, during repeated passages in chickens. These results demonstrate that avirulent viruses maintained in wild waterfowl in nature and bearing the consensus avirulence type sequence R-E-T-R have the potential to become highly pathogenic while circulating in chickens.     

Localization of immunoreactive transthyretin (prealbumin) and of transthyretin mRNA in fetal and adult rat brain

We used a combination of immunohistochemical and molecular-biological techniques to investigate the localization of transthyretin (TTR) in the brains of adult and fetal rats. The immunohistochemical studies employed antibodies purified by immunosorbent affinity chromatography, permitting the specific staining and localization of TTR using the unlabeled peroxidase-antiperoxidase method. TTR mRNA levels were measured by Northern-blot analysis of poly (A+) RNA, followed by hybridization to 32P-labeled TTR cDNA; TTR mRNA was localized in brain tissue sections by in situ hybridization. Immunoreactive TTR was found to be specifically localized in the choroid plexus epithelial cells of adult rat brain. High levels of TTR mRNA were found in poly (A+) RNA samples obtained from the choroid plexus. In addition, the specific localization of TTR mRNA in the epithelial cells of the choroid plexus was demonstrated by in situ hybridization. Neither immunoreactive TTR nor TTR mRNA were found in other regions of adult rat brains. The levels of TTR mRNA in the choroid plexus were at least 30 times higher than those observed in the adult liver. Immunoreactive TTR was observed in the brains of fetal rats on as early as the 11th day of gestation. This immunoreactive TTR was localized in the tela choroidea, the developmental forerunner of the choroid plexus. Immunoreactive TTR was also observed in the fetal choroid plexus as it began to form (14th day of gestation) as well as in the more completely developed choroid plexus (18th day of gestation).(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino-terminal amino acid sequence of p10, the fifth major gag polypeptide of avian sarcoma and leukemia viruses.

We have identified p10 as a fifth gag protein of avian sarcoma and leukemia viruses. Amino-terminal protein sequencing of this polypeptide purified from the Prague C strain of Rous sarcoma virus and from avian myeloblastosis virus implies that it is encoded within a stretch of 64 amino acid residues between p19 and p27 on the gag precursor polypeptide. For p10 from the Prague C strain of Rous sarcoma virus the first 30 residues were found to be identical with the predicted amino acid sequence from the Prague C strain of Rous sarcoma virus DNA sequence, whereas for p10 from avian myeloblastosis virus the protein sequence for the same region showed two amino acid substitutions. Amino acid composition data indicate that there are no gross composition changes beyond the region sequenced. The amino terminus of p10 is located two amino acid residues past the carboxy terminus of p19, whereas its carboxy terminus probably is located immediately adjacent to the first amino acid residue of p27.     

In situ localization of transthyretin-mRNA in the adult human liver, choroid plexus and pancreatic islets and in endocrine tumours of the pancreas and gut

In situ hybridization with 35S-labeled single stranded RNA probes was used on sections from formaldehyde-fixed and paraffin-embedded tissue specimens to provide semiquantitative data on the occurrence of transthyretin(TTR)-mRNA in human liver, choroid plexus and pancreatic islets as well as in 15 endocrine tumours of the pancreas and gut. A monoclonal antibody to TTR was used for immunocytochemical identification of the protein in consecutive sections. The amount of TTR-mRNA in hepatocytes was found to be much less than that in epithelial cells of the choroid plexus. Glucagon cells of the pancreatic islets were also specifically labeled and the level of TTR-mRNA in these cells was intermediate between that of hepatocytes and choroid plexus epithelial cells. Four glucagonomas, one malignant insulinoma and two midgut carcinoids were shown to contain TTR-mRNA. The 'in situ' labeled cells were also found to be TTR immunoreactive. These findings present the first conclusive evidence for TTR synthesis in pancreatic islets and in endocrine tumours. They also establish that the high serum concentration of TTR found in some patients with endocrine tumours (notably glucagonomas) is most likely due to tumour production of TTR.