Harutaka Tanaka

Parietal cell autoantigens involved in neonatal thymectomy-induced murine autoimmune gastritis. Studies using monoclonal autoantibodies

Autoimmune gastritis accompanied by autoantibodies to parietal cells was induced in BALB/c nu/+ mice by neonatal thymectomy 2-4 days after birth. Three monoclonal autoantibodies, designated as 2B6 (IgG1), 2G10 (IgG2b), and 1H9 (IgG1), were obtained from one of these mice. All three reacted specifically with parietal cells, 2G10 recognizing species-specific antigenic determinants and 2B6 and 1H9 recognizing interspecies-specific antigenic determinants. All three recognized antigens on the membrane of intracellular secretory canaliculi and the cytoplasmic tubulovesicular system of parietal cells. At least two different molecular groups were recognized by these antibodies; 2B6 recognizing a 65,000-79,000-mol wt group and 1H9 recognizing a 92,000-120,000-mol wt group. Sera of most mice with autoimmune gastritis reacted with either or both groups. Both groups were consistently coprecipitated by any of the three antibodies when solubilized in NP-40. Sera, from patients with pernicious anemia, containing anti-parietal cell antibodies could also precipitate these two groups of antigens. Competition assay and physicochemical studies showed that the epitopes recognized by the three monoclonal antibodies are different.

Properties of the intracytoplasmic a particles purified from mouse tumors.

A procedure for the isolation and purification of the intracytoplasmic A particles from lymphoid leukemias and mammary tumors of DBA/2 strain mice is described. The purified A particles are 71 nm in size, consist of a 10 nm thick shell and a core, have a buoyant density of 1.26 to 1.27, and contain about 9% RNA and protein but no DNA and lipid. Two antigens of mouse mammary tumor virus (MTV) are distinguished; one (MTV-a) is shared by the B particles of MTV and the intracytoplasmic A particles, but not by the intracisternal A particles of mouse myelomas or the C particles of Moloney sarcoma virus, thus clearly establishing that the intracytoplasmic A particles are immature forms of MTV. MTV-a is not soluble in its native state, and is shared by MTV of all mouse strains tested. It is single in the A particles but splits into two components of different antigenicites (a1 and a2) in the B particles. The other antigen (MTV-b) is an envelope antigen of the B particles. This is also found in mammary tumor extracts of all MTV-carrying mouse strains tested. MTV-b is easily destroyed by dithiothreitol. Comparison is made between these antigens and those reported already for MTV, and the natural distribution of the antigens in various forms of MTV in mouse tissues is discussed.

Precursor-product relationship between nonglycosylated polypeptides of a and b particles of mouse mammary tumor virus.

Abstract Polypeptides and antigens of various preparations of intracytoplasmic A particles were analyzed in detail in comparison with those of B particles of mouse mammary tumor virus (MTV). SDS-polyacrylamide-gel electrophoresis (PAGE) revealed that B particles consisted of three major nonglycosylated polypeptides (B-p25, B-P15, and B-P7; numerals indicate calculated molecular weights in 10 3 daltons) and six glycopeptides. All A-particle polypeptides were nonglycosylated. The particles purified by a conventional method contained seven major bands in the 70,000- to 37,000-molecular weight region, but their relative amounts varied from preparation to preparation. In most preparations, one other major band, A-p7 (an A-particle polypeptide with a molecular weight of 7000), was also observed. Thus, the polypeptide composition of A particles was quite different from that of B particles, having in common only one major band, p7, and three other bands of variable amounts, p43, p37, and p13. Those A particles that had been incubated at 37° for 20 hr showed a systematic change in PAGE pattern; major bands disappeared except for A-p43, A-p37, and A-p7, while a strong new band, A-p25, appeared. Consequently, incubated A particles were now similar to B particles in their PAGE pattern except for the absence of p15 in the-A particles. In spite of such a profound change in components, no ultrastructural alteration was observed in the A particles after incubation. Polypeptide conversion induced by incubation was completely inhibited by diisopropylfluorophosphonate (DFP). A particles purified in the presence of phenylmethanesulfonyl fluoride (PMSF) consisted of a single major polypeptide, A-p70. Incubation of these A particles resulted in generation of A-p15 in addition to A-p25 and A-p7 at the sacrifice of A-p70, although the rate of polypeptide conversion was much retarded. Antigenic analysis of individual polypeptides eluted from polyacrylamide gels shows that (i) B-p25, B-p15, and B-p7 carried distinct antigens; (ii) B-p25 and B-p7 were antigenically identical with A-p25 and A-p7, respectively; (iii) A-p70 carried all three of these different antigenicities of B particles; and (iv) other major polypeptides of A particles also carried these antigens in ways characteristic to each antigen. This indicates that three major internal components of B particles are generated from a common precursor, A-p70, through enzymatic cleavage and, hence, that A particles are the real pronucleocapsids of B particles. Present observations are discussed in connection with the precursor-product relationships proposed in other RNA tumor virus systems.

Murine mammary tumor virus deficient in the major glycoprotein: Biochemical and biological studies on virions produced by a lymphoma cell line

Abstract A cell line, designated MLA, was established from a spontaneous lymphoma of a DBA/2 mouse. These cells contained large amounts of intracytoplasmic type-A particles, the presumed precursors of mature MuMTV. Electron microscopy revealed that the MLA cells released MuMTV-like virions into the culture fluid, but these virions were devoid of the characteristic envelope “spikes” of MuMTV. Antigenic analysis of virions purified from the culture fluid of MLA cells showed that, by microimmunodiffusion, the glycoproteins of MuMTV, gp49 and gp36, were not detectable, nor was the glycoprotein gp70 of MuLV detectable. Internal structural proteins of MuMTV, p28 and p12, were however, readily detectable in these virions. By the more sensitive radioimmunoassay technique, it was found that the virions produced by MLA cells contained some gp49-related antigen, but the amount present was about 500-fold lower than in other tissue culture-derived MuMTV. MLA cells synthesized a 70,000-dalton precursor of MuMTV glycoproteins. Culture fluids from which the virions were removed by centrifugation did contain gp49, indicating processing of the precursor polypeptide, but this polypeptide was not incorporated into the virions. Molecular hybridization studies revealed that MLA cells contained a large amount of MuMTV-specific RNA; the amount of MuLV-related RNA was about 100-fold lower. Whereas 150–200 MuMTV particles were released into culture fluid per cell per day, few MuLV particles were released. Purified virions from the MLA cells were injected into BALB/c and C57BL weanling females. This resulted in mammary tumor development in BALB/c females, but not in C57BL. The tumors contained MuMTV with the normal complement of envelope spikes and gp49 was detectable in the virions.

IMMUNOHISTOCHEMICAL EXPRESSION OF MAMMARY TUMOR VIRUS ANTIGENS IN MAMMARY GLANDS OF VIRGIN MICE, IN RELATION TO Mtv GENES

MTV antigens in the resting mammary glands of GRS/A, SHN, C3H, and Balb/c virgin mice were detected by immunoperoxidase techniques using antiserum against MTV whole virion, gp 52 or p 25 to differentiate the expression between endogenous and exogenous MTV. Balb/c mice were crossed to infect MTV into each reciprocal hybrid or fosternursed inbred. Immunohisto‐chemical stainings of gp 52 in the formol‐fixed sections were almost the same as those of the whole MTV virion, and the results on various cases were as follows: In the mice with endogenous GR‐MTV, positivity was first observed at the age of 14 days, while the first expression of exogenous GR‐MTV was delayed to the age of 140 days. The mice with endogenous and/or exogenous SHN‐MTV showed the first antigen appearance at the age of 65 days, and those with exogenous C3H‐MTV did at the age of 80 days. The virgins with only endogenous C3H‐MTV came to express the antigen after the age of 200 days. Staining of p 25 in Carnoy‐fixed sections of MTV‐positive mammary glands was found in the supranuclear cytoplasm and apical surface of the glandular cells and the lumen, all of which are the site of A and B particles. By means of preembedding method for gp 52, the reaction products were ultrastructurally detected not only on the MTV‐budding apical surface, together with the intraluminal B particles, but also on the MTV‐free apical cell membrane of the glandular cells in the mammary gland of the GR virgins.

Polyclonal and monoclonal antibodies monospecific to MMTV LTR orf protein produced in E. coli.

Monoclonal and polyclonal antibodies specific to an open reading frame of the mouse mammary tumor virus long terminal repeat were generated using an open reading frame-beta-galactosidase fusion protein produced in E. coli. Both antibodies reacted with the open reading frame-beta-galactosidase fusion protein but not with beta-galactosidase alone using an immunoblotting technique. It is concluded that these antibodies were specific for the protein encoded by the open reading frame of the mouse mammary tumor virus long terminal repeat.