Identification of protein complexes containing the c-rel proto-oncogene product in avian hematopoietic cells.

The c-rel proto-oncogene product has been identified as a 75 kDa protein expressed in lymphoid cells transformed by REV-T and Marek's disease virus. A 4.0 kb c-rel transcript is expressed in the bursa, spleen and thymus of chickens with highest levels of expression at 10 days post hatch. Using antiserum specific for the v-rel oncogene product, P75c-rel has been precipitated from [35S]methionine-labeled extracts of bursal, splenic and thymic lymphocytes. Additionally, proteins with the molecular mass of 40 kDa, 115 kDa, and 124 kDa co-immunoprecipitate. These proteins co-migrate with the proteins found associated with pp59v-rel in REV-T transformed lymphoid cells. Antiserum specific for pp40, the most abundant cellular protein associated with pp59v-rel, co-precipitates p75c-rel verifying the existence of p75c-rel/pp40 complexes in normal avian lymphocytes. Antiserum directed against the amino-terminal region of pp59v-rel fails to precipitate native p75c-rel complexes from normal lymphoid cells. In the presence of ionic detergents, antisera directed against the amino, middle and carboxy-regions precipitate equivalent amounts of p75v-rel. These results suggest that the amino-terminal region of p75c-rel is active in binding other proteins or is inaccessible to the antiserum due to the conformation of p75c-rel in the complex. Two p75c-rel complexes exist in the cytosol of normal lymphocytes. The most abundant complex contains 60% of the p75c-rel associated with p115 and p124. The remaining p75c-rel is associated with pp40.

Bursin localization in mammalian bone marrow and epithelial cells of intrahepatic bile ducts.

Bursin is a tripeptide (lysyl-histidyl-glycyl-amide) found in follicular and dendritic reticular epithelial cells of the avian bursa of Fabricius that selectively induces the differentiation of committed B-lymphocyte precursor cells but not of committed T-lymphocyte precursor cells. We now show, in immunoassays with tissue extracts, that bursin is also present in avian and bovine bone marrow. There was, however, a categorical difference between avian liver (bursin-negative) and bovine liver (bursin-positive). Bursin was therefore isolated from bovine liver and bone marrow and the structure of mammalian bursin was determined; it was identical to avian bursin. Immunohistochemical examination of bovine liver showed the presence of bursin within epithelial cells of the intrahepatic bile ducts. These cells have previously been suspected of having an endocrine function because of the rich periductal capillary plexus, which coalesces to form a portal system draining into the liver sinusoids. These findings suggest that bone marrow is a site of bursin production and associated B-cell differentiation in both birds and mammals. The bursin-containing cells of the intrahepatic bile ducts are not associated with developing B cells and it would appear that mammals have evolved a local hepatic function for bursin.

B-cell differentiation in the chicken: expression of immunoglobulin genes in the bursal and peripheral lymphocytes.

We have studied the expression of immunoglobulin genes in the chicken B-cell precursors, and of a B-cell surface marker (Bu-1) on the bursal and peripheral B cells during normal ontogeny. Since there is no way of distinguishing the precursor cells from the more mature bursal lymphocytes on the basis of surface markers, we chose to study the total bursal lymphocyte population at ages when the numbers of the various precursor cells (bursal, early post-bursal, and post-bursal stem cells) in the bursa are estimated to be at their highest. Thereafter, comparisons with the more mature lymphocytes in the peripheral organs were made. As a result, levels of the lambda and mu transcripts and expression of Bu-1 antigen in the chicken B-cell precursors were found to be unchanged during the post-hatching period. In the light of these experiments, the later events of B-cell differentiation, i.e. the development from the bursal to post-bursal B lymphocytes, occurs without the lambda, mu, and Bu-1 gene loci involved. On the other hand, the higher level of lambda and mu expression in the splenic B lymphocytes indicates that the post-bursal stem cells mature into highly active plasma cells after seeding to the peripheral organs.

Oncogene expression in reticuloendotheliosis virus-transformed lymphoid cell lines and avian tissues.

The genome of reticuloendotheliosis virus (REV-T) contains a unique oncogene, designated v-rel, which is inserted into the env region. Employing a cloned rel DNA probe, a single 2.9- to 3.0-kilobase v-rel mRNA was identified in poly(A)+ RNA from REV-T-transformed lymphoid cell lines. A 4.0-kilobase rel-specific transcript corresponding to the cellular homolog of the v-rel oncogene was identified in MSB-1 cells, a herpesvirus-transformed lymphoid cell line. Cytodot hybridization was used to quantitate the levels of rel, c-rel, c-myc, c-myb, c-abl, c-fms, c-Ha-ras, c-Ki-ras, c-src, c-yes, c-mos, and c-sis mRNA in REV-T-transformed cells. The levels of rel transcription in REV-T-transformed cells were elevated only two to eightfold over levels found in the transformed immature avian lymphoid cell line MSB-1. The relatively modest levels of rel transcription in REV-T-transformed cells and the significant differences between the lengths of the v-rel and c-rel mRNA suggest that REV-T transformation is the result of the production of an altered rel protein. The c-rel proto-oncogene is expressed in all avian hematopoietic tissues but is not expressed at significant levels in brain and muscle. The transcription of other proto-oncogenes is not enhanced in REV-T-transformed lymphoid cell lines.

Sex steroid and glucocorticoid receptors in the bursa of Fabricius of obese strain chickens spontaneously developing autoimmune thyroiditis.

The female sex develops autoimmune disease far more often than the male. This is claimed to be due to differences in peripheral sex steroid levels. We have examined in the bursa of Fabricius of Obese strain (OS) chickens, which spontaneously develop autoimmune thyroiditis, as well as in their healthy counterparts androgen(AR)-, estrogen(ER)-, progestin(PR)- and glucocorticoid(GR)-receptors in an attempt to elucidate possible further pathomechanisms, namely at the target site of steroid hormones. The characterization (affinity, specificity, association- and dissociation-rate, sedimentation behaviour) of all four types of receptors revealed no difference between sex or strain. Furthermore, the ontogeny study of the receptor capacity and affinity from the 7th embryonic day (i.e. before lymphocyte settlement) until bursa involution, again showed no difference between OS and healthy chickens of either sex. Thus, it can be concluded that the principal sex dependency of the susceptibility to autoimmune disease results predominantly from differences in sex steroid levels per se, although alterations in mechanisms beyond the cytosolic receptor level can presently not be excluded.

Thymic mast cell deficiency in avian muscular dystrophy.

In animals with hereditary muscular dystrophy there are thymic abnormalities which may be of etiological significance in the dystrophic process. This study investigated mast cell number and histamine levels in the thymus of normal and dystrophic chickens. For comparison, other lymphoid tissues, namely the spleen and the bursa of Fabricius, and non-lymphoid tissues including the comb and pectoralis major muscle, were similarly studied. Our results show that the thymus of dystrophic adult birds has a deficiency in both mast cell number and histamine content. In the bursa of Fabricius of dystrophic birds a significant elevation in histamine content (microgram/g) was attributed to the abnormally small size of this organ, rather than to an absolute mast cell increase. The deficiency in thymic mast cell number in dystrophic chickens may be significant in the postulated abnormal thymus-muscle interaction of the dystrophic process.

Identification of a subpopulation of chicken B lymphocytes by the lectin from Lotus tetragonolobus.

Lectin-reactive chicken lymphoid cells were detected by agglutination. The lectin from Lotus tetragonolobus agglutinated cells from bursa and spleen and did not agglutinate cells from thymus or peripheral blood of 28-day-old chickens. The percentages of Lotus tetragonolobus reactive cells were also measured by assays of lectin-induced rosette formation, binding of lectin-labelled latex beads, and binding of rhodamine-labelled lectin. The distribution of lectin reactive cells varied with the age of the chicken. The lectin appears to identify a unique subpopulation of chicken B lymphocytes.

The cytospectrophotometrical determination of proteins by the tetrazonium coupling reaction. II. Calibration of the staining method.

The protein content of identical samples from 5 different cell types (lymphocytes of the thymus and of the bursa of FABRICIUS of the chicken, EHRLICH ascites tumor cells, YOSHIDA ascites tumor cells, and rat liver cells) have been determined both macroscopically, by the method of LOWRY (1951), and microspectrometrically, by the tetrazonium coupling method of NOHAMMER (1978). In all the different cell types, a strong correlation is shown between the protein values determined by the 2 methods. By comparing the values thus measured, a conversion factor has been obtained such that an extinction of 0.4235, determined microspectrometrically, corresponds to a protein mass of 1 pgm. To test the accuracy of the microspectrometric method of protein determination at the lowest extreme point, the protein content of rat liver mitochondria has been measured and a mean value of 0.239 pgm protein per mitochondrion obtained. This corresponds well with the value of 0.233 pgm per mitochondrion as determined macroscopically by GEAR (1972).

Viral DNA in bursal lymphomas induced by avian leukosis viruses.

Avian leukosis viruses (ALV) induce malignant lymphoma of the bursa of Fabricius. Viral DNA in tumors and normal tissues from infected birds were analyzed by using restriction endonucleases. Viral DNA fragments diagnostic of the exogenous ALV were easily detected in tumors, uninvolved bursal tissue, kidney, and erythrocyte nuclei. Exogenous viral DNA was more difficult to detect in liver. Using a restriction endonuclease (SacI) which cleaves linear unintegrated ALV DNA in a single site to define integration sites in DNA from the various tissues, we were able to detect ALV DNA only in tumor tissue. We concluded that the proviral DNA detected in the various nontumor tissue must be integrated in multiple sites. The appearance of ALV integration sites uniquely in tumors suggests that they are clonal growths. Furthermore, the data suggested the presence of a single exogenous integration site for the ALV provirus in each of six early neoplastic bursal nodules. This provirus appeared to retain the organization of EcoRI and BamHI recognition sequences present in the genome of virus used to infect the birds. The ALV integration site appeared different in each of the tumors studied. In a widespread metastatic lymphoma, multiple ALV integration sites were found as well as structural alterations in at least some copies of the ALV provirus.

[Quantitative determination of proteins in single cells with amidoblack (author's transl)]. / Quantitative Proteinbestimmung in Einzelzellen mit Amidschwarz.

In aqueous solution Amido Black B (ASB) forms stable and well-defined complexes with bovine serum albumin (RSA) at pH 5.5. The complexes can be separated by column chromatography. The formation of the complexes consist in a fast reaction during which, after 3 to 5 h approximately, 3 molecules of ASB have been bound per molecule RSA, and of a much slower reaction which, even after a laps of 24 h, is still far from approaching its final stage. With solid films of RSA, after denaturation with ethanol, fast reaction is found to approach its final stage after 10 min reaction time. With these model protein preparations, the molar extinction coefficient of the ASB-protein complexes can be determined: the soluble ASB-RSA complexes can be brought to complete dissociation at pH 12.3. After the additivity of the specific absorptions of both RSA and ASB had been proven, it was possible to determine the content of the solution of ASB and RSA, and therefrom the molar extinction coefficient of the ASB-RSA-complex at Ph 5.5: epsilon 620 = 110,000. ASB-stained ethanol-fixed RSA films show an epsilon 620 of approximately 96,000, if their thickness and specific weight are known. After incubation in watery or ethanolic/TCA solutions of ASB, also animal cells fixed with ether-ethanol show the ASB absorption band to be in the region of 600 nm after removal of the surplus of ASB by thorough washings. As already observed with the RSA films, the kinetics of the staining of the cells show the fast reaction reaching its final stage already after 15 to 20 min. When alcoholic solution of ASB is used, the extinctions are found to be twice or three times higher than those achieved by an aqueous one. After standardization of the staining procedures with both solvents the total extinctions of EATZ, YATZ, rat hepatocytes, chicken thymus and bursa cells were measured and plotted against the macroscopically determined protein content of the respective cells. Highly significant positive linear correlations resulted with staining both in watery and alcoholic solutions, respectively. From the slope of the straight lines, specific extinction coefficient of ASB stained cellular proteins could be calculated up to epsilon' 620 = 1.76 with watery ASB solution and epsilon' 620 = 3.83 with the alcoholic solvent. The soluble ASB-RSA complexes have an epsilon' 620 = 1.67 the ASB stained ethanol denaturated films of RSA an epsilon' 620 of within a range of 1.21 to 1.80.

Comparative study of the glycosphingolipids of chicken bursa of Fabricius and of chicken, rat and human thymus.

1. The glycosphingolipid compositions of the thymus and bursa of Fabricius of young male chickens were compared. The two tissues were found to contain complex mixtures of both neutral glycosphingolipids and gangliosides. Both tissues contained mono-, di-, tri-, tetra- and penta-glycosylceramides; the pentaglycosylceramide displayed a reaction of identity with authentic Forssman antigen when tested against a specific anti-(Forssman antigen) serum. The ganglioside G(m3) containing N-acetylneuraminic acid was the principle ganglioside of both tissues. 2. The thymus contained appreciable amounts of the simple ganglioside N-acetylneuraminylgalactosylceramide, a compound not found in the bursa. The ganglioside G(d3) (disialohaematoside) was detected in both tissues. 3. Rat and human thymus, like sheep thymus (Narasimhan, Hay, Greaves & Murray (1976) Biochim, Biophys. Acta 431, 578-591), both contained a tetraglycosylceramide species as their most complex neutral glycosphingolipid and possessed little or no Forssman antigen. They also contained a complex mixture of gangliosides. 4. The possible significance of these results is briefly discussed.

A combined autoradiography--immunofluorescence technique for the study of lymphocyte traffic in relation to antigen localisation.

A combined autoradiography-immunofluorescence method is described for the simultaneous detection of antigens and lymphoid cells on cryostat tissue sections. The method is discussed here with reference to studies on cell traffic and antigen localisation in the chicken spleen.

[Effect of Mecadox therapy on various organs of the reticulohistiocytary system in chickens]. / Einfluss der Behandlung mit Mecadox auf einige Organe des retikulohistiozytären Systems bei Küken

Chickens received Mecadox treatment for 28 days, beginning at the age of five days, with the daily rations having been 1 g in 1 kg feedstuff. Changes were caused in two periods. The first therapeutic period, first to 14th days, was characterised by rise in total protein, DNA, RNA, amino nitrogen, and ascorbic acid in the adrenal gland as well as by rise in body mass, however, with no change of thymus and bursa mass. The second therapeutic period, between the 14th and 28 th days, led to decline in the above parameters and to involution of the lymphatic organs. The conclusion is suggested that biological stimulation can be achieved only in response to short-time Mecadox treatment.

Surface carbohydrate composition of different types of chicken lymphocytes.

A simple and quick procedure was used to analyse the carbohydrate composition of surface glycoproteins from chicken lymphocytes. The procedure included papain digestion of the cells, a two-step purification of the supernatant material and a sugar analysis by gaschromatography. The method made it possible to analyse samples of about 10(9) lymphocytes. The surface glycoproteins from different chicken lymphocyte preparations were found to differ significantly in their sugar composition. Lymphocytes from thymus, bursa, spleen or blood were characterized by typical relative amounts of glucose, galactosamine, fucose and sialic acid. The values for mannose, glucosamine and galactose, however, were approximately 1:1:1 for all four lymphocyte preparations. Bursectomy or thymectomy in combination with whole body irradiation altered the carbohydrate composition significantly. The results suggest the possibility that the carbohydrate composition can be used as a marker for different lymphocyte populations. The results are also discussed in respect to the hypothesis that carbohydrate determinants on the cell surface determine the migration of lymphocytes.