Detection and quantitation of a type D retrovirus gag protein in ovine pulmonary carcinoma (sheep pulmonary adenomatosis) by means of a competition radioimmunoassay.

A heterologous competition radioimmunoassay (RIA) which consisted of 125I-labeled langur retrovirus major gag protein and goat anti-squirrel monkey retrovirus serum was used to detect a type D retrovirus-associated antigen in tumor cell homogenates, lung fluid, and cell culture supernatant fluids of naturally occurring and experimentally-induced ovine pulmonary carcinoma (OPC, sheep pulmonary adenomatosis). In this assay, there was no cross reactivity between structural proteins of the type D retrovirus and an ovine lentivirus, which frequently co-infects OPC-affected sheep. The sensitivity of the assay was similar to an immunoblotting assay using antiserum to Mason-Pfizer monkey virus major gag protein which had been used previously to detect the OPC retrovirus antigen in tumor homogenates and lung fluids of OPC-affected sheep. All unconcentrated samples of lung fluid collected from five sheep with naturally occurring OPC or six sheep with experimentally induced OPC competed in the competition RIA. The competition RIA titers of the type D retrovirus antigen in lung fluids of lambs with induced OPC were relatively higher than the titers of this antigen in the naturally occurring OPC cases. The competition RIA detected the retrovirus antigen associated with OPC in the culture fluids of four out of five primary lung cultures from OPC sheep tested between 1 and 56 days after culture initiation. Because this RIA is appropriate for the quantitation of OPC-associated antigen, it will provide a means for determination of the target cell type for OPC virus replication in vitro.

Lesions and retroviruses associated with naturally occurring ovine pulmonary carcinoma (sheep pulmonary adenomatosis).

Five sheep with ovine pulmonary carcinoma were markedly dyspneic and had sporadic coughing; two had copious watery nasal exudate. In four, lesions consisted of multifocal nodules of neoplastic cuboidal epithelial cells in acinar or papillary patterns. Electron microscopically, cells had microvilli, tight junctions, and cytoplasmic lamellar bodies typical of alveolar type II cells. One sheep had a single lung tumor of nonciliated bronchiolar epithelial cells. Vacuolated alveolar macrophages surrounded adenomatous foci. One sheep had a metastatic lesion in the caudal mediastinal lymph node. All sheep had histologic lesions of lymphoid interstitial pneumonia (LIP, ovine progressive pneumonia) consisting of peribronchiolar and interstitial lymphoid hyperplasia, and fibromuscular proliferation; all had serum precipitating antibodies to ovine lentivirus. Lung fluids or tumor homogenates contained a 26-kd peptide that crossreacted with a primate-derived type D retrovirus as detected by immunoblotting or interspecies competition radioimmunoassay. Ovine lentivirus was isolated from concentrated lung fluids or tumor tissues of four sheep tested and from tumor cell DNA of one animal transfected into ovine muscle cells. These studies document the presence of type D-related retrovirus antigen in ovine pulmonary carcinoma (OPC) in the United States and indicate that lentivirus-induced LIP is a lesion frequently associated with this disease.

Seroreactivity of Peruvian sheep and goats to small ruminant lentivirus-ovine progressive pneumonia virus.

Sera from 3,369 sheep and 1,394 goats in Peru were examined by agar-gel immunodiffusion for antibodies to ovine progressive pneumonia virus (OPPV). The point prevalence rates for antibodies to OPPV in sheep were 1.7% to 40.6% (mean, 19.02%) in the 7 flocks studied, whereas for goats, the point prevalence rates for antibodies that cross-reacted with OPPV in 12 herds were 0.0% to 45.1%. For sheep, a direct association between increasing age and increasing seroreactivity to OPPV was established, and there was evidence to indicate that lambs born to primiparous ewes and raised separated from all other sheep after they were weaned may have been less likely to become infected with OPPV than those lambs born to multiparous ewes and not separated from other sheep after they were weaned. For goats, antibodies to OPPV were detected in 7 of 12 herds studied, the highest infection rate being present within a herd in the Lima department (district).

Macrophages in efferent lymph of sheep and their role in lectin-induced lymphocyte blastogenesis.

Very low numbers (3.9 +/- 3.0/10(5) cells) of macrophages (cells with intense cytoplasmic staining for nonspecific esterase) were found in seven popliteal lymph samples taken at surgery from 6 sheep. 67% of these cells were phagocytic for latex beads. Dose response and limiting dilution experiments indicated that efferent lymph lymphocytes responded to concanavalin A and pokeweed mitogen when at least 10 macrophages/10(5) cells were present, but macrophages appeared not to be necessary for phytohemagglutinin responses. Further depletion of macrophages from efferent lymph lymphocytes by nylon wool columns almost abolished concanavalin A and pokeweed mitogen responses and reduced phytohemagglutinin responses by 67%. The responses to all mitogens were restored to near normal efferent lymph lymphocyte levels by addition of 1% adherent cells and were elevated to near normal cell levels by 5% adherent cells. These results indicate that macrophages are present in efferent lymph and that, despite their low numbers, they may be of functional significance in regulation of immune responses.

Partial purification of neurofilament subunits from bovine brains and studies on neurofilament assembly.

The 200,000-dalton neurofilament subunit (P200) and the 160,000-dalton (P160) and 78,000-dalton (P78) neurofilament subunits were partially purified from bovine brain. Intact neurofilaments were prepared by high-speed and sucrose-zone centrifugation. The crude neurofilament was solubilized in 8 M urea solution containing pyridine, formic acid, and 2-mercaptoethanol. The solubilized neurofilament was purified by carboxymethyl (CM) cellulose column and hydroxylapatite column chromatography. The P200 was purified as separate from P160 and P78, but the P160 and P78 subunits were copurified on CM cellulose, hydroxylapatite, Bio-Gel A150m, and Sephadex G-150 column chromatography. Electron microscopy of these purified neurofilament subunits revealed the P200 subunit as a globular structure, and the P160 and P78 subunits as a rod-shaped structure extending up to 120 nm with a 8- to 12-nm width. In the presence of 200 mM KCl, 15 mM MgCl2, and 1 mM ATP, the purified subunits assembled into long filaments. Under the assembly condition, P160 and P78 subunits elongated up to 500 nm, but the longer filament formation required the presence of P200 subunits. The filaments formed in vitro were of two types: long straight filaments and intertwined knobby-type filaments. From these results, we have suggested that P160 and P78 form the neurofilament backbone structure and P200 facilitates the assembly of the backbone units into longer filaments.

The loss of neuron-specific proteins during the course of Wallerian degeneration of optic and sciatic nerve.

To identify axonal proteins which are unique constituents of neurons, the spectrum of detectable proteins of degenerating nerves has been compared with that of intact control nerves from the same animals. Wallerian degeneration was induced in rabbits by unilateral transection of the optic and sciatic nerves. Proteins of nerve homogenates were compared by sodium dodecyl sulfate-gel electrophoresis and by two-dimensional electrophoresis. Four non-myelin proteins disappear from degenerating nerve. These include the three neurofilament proteins (P68, P150, and P200) and a polypeptide with a molecular weight of about 65,000 daltons (P65) which is not associated with filaments.

The effect of amino acids on protein metabolism as measured in long-term experiments in immature brain explants.

In a study of a system suitable for investigating long-term effects on brain protein metabolism, we measured amino-acid incorpration into isolated immature brain explants incubated under sterile conditions up to ten days. Measurements of changes in total proteins, total DNA, cell number during the experiments, and 14C-thymidine incorporation measurements indicated no significant net growth; new cell formation was below 5% in a 5-day period; therefore, amino-acid incorporation was mainly due to protein turnover. The rate of incorporation in our immature brain preparation was similar to that of the adult brain in vivo: by ten days about one-half of the tissue protein turned over. The label incorporated was released in subsequent incubations with cold amino acids. Such release occurred in all subcellular fractions examined. Incorporation was fairly stable; at temperatures below 30 degrees C it rapidly declined, but it was not affected when phenylalanine or the branched chain amino acids (leucine, isoleucine, valine) were elevated in the incubation medium. Brief exposure to low amino-acid media had no effect; longer exposure resulted in tissue damage. Our model system indicates that overall brain protein turnover is not sensitive to such variations in the level of most amino acids, which may occur under various conditions. Protein metabolism of the nervous system occurs at a high rate. A recent long-term labeling method (Lajtha, Latzkovits, and Toth, 1976) gave a best fit to incorporation curves by assuming two compartments for adult brain proteins, one of which (about 6%) has a half-life of 15 hr and the other (94%) has a half-life of ten days. The disappearance of protein-bound label with time under conditions in which all proteins were previously labeled indicated that most, possibly all, proteins in brain are in a dynamic state (Lajtha and Toth, 1966). Incorporation of amino acids was found in all proteins and structures that have been studied to date; myelin proteins previously thought less active are also metabolized at a significant rate (Sabri, Bone, and Davison, 1974; Lajtha, Toth, Fujimoto, and Agrawal, 1977). We have fairly extensive information available in addition to turnover studies about the mechanisms of protein synthesis in brain (Roberts, 1971); protein breakdown was also studied in some detail (Marks and Lajtha, 1971). In contrast to our knowledge about protein metabolism under physiological equilibrium conditions, our information about alterations during functional demands or pathological conditions is scanty. Although a significant amount of work has been reported, largely because of technical difficulties the results are difficult to interpret unequivocally. The present report represents our effort to address some of the obstacles: to develop a system in which influences on long-term incorporation can be studied...