The predominant heavily glycosylated glycoproteins at the surface of rat lymphoid cells are differentiation antigens.

Rat lymphoid cells have been labeled with sodium 3H-borohydride after periodate oxidation. The labeled glycoproteins were solubilized in detergent and analyzed by fluorography after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Major bands were found at 150 000, 95 000 and 25 000 apparent mol.wt. for thymocytes; at 170 000 and 95 000 mol. wt. for T lymphocytes and at 200 000 mol.wt. for B lymphocytes. Bone marrow cells showed a diffuse band at 100 000 mol.wt. with relatively minor bands around 150 000 mol.wt. With the exception of the 95 000 mol. wt. bands, all these glycoproteins bound to lentil lectin. Using monoclonal or monospecific antibodies in immunoprecipitation and on antibody affinity columns, each of these glycoprotein bands was identified as a previously defined lymphocyte differentiation antigen. The bands at 150 000 mol.wt. on thymocytes, at 170 000 on T lymphocytes, at 200 000 on B lymphocytes, and at 130 000 to 150 000 on bone marrow cells all consist of a leukocyte-common antigen, which has previously been shown to be present on leukocytes but not on other tissues. At least a part of the 95 000 mol.wt. band on thymocytes, T lymphocytes and bone marrow cells is the W3/13 antigen previously shown to be on mature T lymphocytes, polymorphonuclear cells, and in brain. The 25 000 mol.wt. band of thymocytes is the Thy-1 antigen. Similar experiments were carried out on thymocytes labeled with ¹²⁵I by the lactoperoxidase method. An intense band at 150 000 mol. wt. was identified as the leukocyte-common antigen by immunoprecipitation. A labeled band, which did not bind to lentil lectin, was immunoprecipitated at 95 000 mol. wt. with W3/13 antibody. Rat Thy-1 antigen was not labeled with ¹²⁵I.     

Characterization of mouse thymocyte and peripheral lymphocyte xenoantigens by two-dimensional electrophoresis

Two-dimensional electrophoresis was used to analyze the surface glycoproteins of murine thymocytes and lymph node cells. Two-dimensional maps of unselected, radioiodinated lymphocyte surface proteins were complex, showing at least 20 different components, but simpler patterns were obtained by using rabbit antibodies directed against the surface proteins of a T lymphoma cell line, to precipitate xenoantigens from lysates of radioiodinated or biosynthetically labeled thymocytes and lymph node cells. These xenoantibodies precipitated 12–13 distinct components from each cell type, of which all but 3 were sialoglycoproteins. Two types of difference between the surface glycoproteins of thymocytes and peripheral lymphocytes could be detected. First, higher mol. wt. glycoproteins and Thy-1 are more acidic in peripheral lymphocytes than in thymocytes, and this difference disappears after neuraminidase treatment. One additional high mol. wt. glycoprotein is also detectable in peripheral lymphocytes, probably reflecting the greater carbohydrate complexity of these molecules, when expressed on such cells. Second, 3 glycoproteins are strongly labeled only on thymocytes, and 3 others only on peripheral lymphocytes. These 6 glycoproteins might represent genuine differentiation antigens.     

Four distinct antigen systems on human thymus and T cells defined by monoclonal antibodies: immunohistological and immunochemical studies.

Human thymus and T cell antigens were identified by using four distinct monoclonal antibodies (MoAb), designated 2D5, 5B3, 7A5 and 9D4. 2D5 antibody reacted with most human thymocytes and a few peripheral lymphocytes as well as with a subpopulation (20%) of bone marrow cells, and precipitated a 45K molecular weight (mol. wt.) component from 125I-labelled thymus cell lysate. 7A5 antibody also reacted with the majority (80%) of thymocytes but neither with peripheral lymphocytes nor with bone marrow cells. The antigen detected by 7A5 was a glycoprotein consisting of 48K and 12K mol. wt. components, which were non-covalently associated with each other. 5B3 reacted with virtually all of human thymus and T cells but not with the majority of B cells and bone marrow cells. This reagent precipitated a 72K mol. wt. glycoprotein from thymus and T cells. An additional 65K mol. wt. glycoprotein was precipitated by 5B3 together with the 72K mol. wt. component, but with poor reproducibility. 9D4 antibody, on the other hand, reacted with a 200K mol. wt. component from thymus and T cells as well as 220K and 210K components from the non-T cell fraction of tonsil lymphocytes. Whereas antigens detected by 2D5 and 7A5 appeared to be highly expressed on cortical thymocytes, the antigen defined by 5B3 occurred much more abundantly on medullary thymocytes and peripheral T cells than on cortical thymocytes. Based on the data described above, it is suggested that 7A5, 5B3 and 9D4 MoAb recognize human homologues of mouse TL, Ly-1 and Ly-5 antigens, respectively, whereas 2D5 antibody seems to resemble OKT10, as described by others.     

Helix pomatia A hemagglutinin: selectivity of binding to lymphocyte surface glycoproteins on T cells and certain B cells

Human and mouse lymphocytes were surface-labeled by lactoperoxidasecatalyzed iodination, or by galactose oxidase oxidation followed by reduction with tritiated sodium borohydride. The labeled cells were lysed with Nonidet P-40. Proteins binding to Helix pomatia A hemagglutinin (HP) were isolated by affinity chromatography on HP-Sepharose and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. A major cell surface glycoprotein (apparent mol. wt. 150000, using reducing conditions) on human lymphocytes was responsible for almost all binding of HP. This protein was present on normal and malignant thymus-derived lymphocytes, e.g. thymocytes, blood T cells and T leukemia cell lines. It was also found on chronic lymphocytic leukemia cells, one null cell leukemia line, one unidentified leukemia line, one lymphoblastoid cell line of B origin and on one stem cell lymphoma line. In contrast, this protein was not found on various B cells at different steps of differentiation, e.g. four B lymphoma lines or one myeloma line. It was also absent from a histiocytic leukemia line. However, two of the four B lymphoma lines and the myeloma line had another HP-binding surface glycoprotein (mol. wt. 200000) instead of the 150000 protein. Studies of mouse lymphocytes similarly showed that thymus-derived lymphocytes (normal and malignant) but not normal adult B cells expressed a major HP-binding surface glycoprotein of apparent mol. wt. 130000 (reducing conditions).     

Identification and partial characterization of surface antigens specific for human normal and leukemic T cells

Rabbit antisera to membrane preparations of human thymocytes (anti-HTLA), T lymphocytes of the MOLT 4 cell line or B lymphocytes of the LIK cell line, have been serologically characterized and used for the identification of surface membrane antigens specifically expressed by human lymphocyte subpopulations. By immunoprecipitation of surface ¹²⁵I- or NaB (³H)₄-labeled protein followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the precipitates, the anti-HTLA antiserum was shown to detect on thymocytes three glycoproteins of apparent molecular weights 160000, 110000 and 45000. The first two only appeared to be sialylated. A similar pattern of antigens could be detected on cultured T lymphocytes, except for the 45 kDalton glycoprotein which could not be detected on the surface of these cells. None of these proteins were detected on ¹²⁵I-labeled Raji cells. The serological specificity of the anti-MOLT 4 antiserum for T lymphocytes was more restricted since it only precipitated the 160 kDalton antigen. In addition, the 45 kDalton protein was shown to be sensitive to trypsin treatment as was the sheep red blood cell (SRBC) receptor. The results shown suggest a possible activity as SRBC receptor for this particular protein. In contrast, the anti-LIK antiserum which behaves as an anti-HLA-DR reagent in cytotoxicity testing, only precipitates from cultured B lymphocyte lysates the typical Ia-like 28 and 33 kDalton polypeptides.     

Immunochemical characterization of human TL-like (T48) and Ly 1-like (T72) glycoproteins using two-dimensional polyacrylamide gel electrophoresis

Xenoantisera, designated AT48 and AT72, were developed by immunizing rabbits with human thymus cell membrane and guinea-pigs with a T-cell glycoprotein purified from leukaemic T-cell membrane. Whereas AT48, after appropriate absorption, reacted exclusively with the majority of thymocytes (mainly cortical thymocytes) among normal lymphoid populations, AT72 reacted with virtually all of the thymus and T cells but not with B cells. Thymocytes, which were strongly reactive with AT72, existed in the thymic medulla, but cortical cells were also very weakly reactive with AT72. When cultured T-cell lines, all of which were derived from patients with T-cell-type acute lymphatic leukaemias, were tested for their reactivities with AT48 and AT72 by immunofluorescence, we found that AT48 stained certain T-cell lines, whereas AT72 stained all of the T-cell lines tested so far. Immunochemical data showed that AT48 precipitated a 48K molecular weight (mol. wt) glycoprotein from 125I-labelled thymus cell surface glycoproteins, which appeared to be very weakly associated with a 12K mol. wt component. These 48K and 12K mol. wt components precipitated by AT48 showed almost identical isoelectric points (pI) to those of HLA heavy chain and beta 2-microglobulin respectively. AT72, on the other hand, precipitated a 72K mol. wt glycoprotein from thymus and T cells as well as from leukaemic T cells. A less prominent 65K mol. wt glycoprotein was also precipitated by AT72 from thymus and T cells but not from leukaemic T cells. These two components showed almost identical pI ranging approximately from 4 to 7, and this marked charge heterogeneity observed was reduced by neuraminidase treatment, suggesting that it reflects the heterogeneity in sialylation of this molecular species. We concluded from these data that AT48 and AT72 used in this work could detect human homologues of mouse TL and Ly 1 antigens respectively.     

Molecular and antigenic heterogeneity of the rat leukocyte-common antigen from thymocytes and T and B lymphocytes

The molecular forms and antigenic heterogeneity of the leukocyte-common antigen (L-CA) of rat lymphocytes have been analyzed. Thymocytes show one main band at 180 kDa, T cells four bands at 180, 190, 200 and 220 kDa and B cells one broad band at about 240 kDa. T helper and T cytotoxic cell subsets show the same four bands with some differences in the proportion of each. Four mouse monoclonal antibodies (MRC OX-1, 28, 29 and 30) reacted with all molecular forms of L-CA and fell into two sets that were noncompetitive in binding to L-CA (MRC OX-1, 28, 29 vs. OX-30). The antigenic determinants seen by all these antibodies were lost when L-CA was reduced and alkylated. Three antibodies (MRC OX-22, 31 and 32) reacted selectively with B cells, T cytotoxic cells and about 2/3 of T helper cells. OX-22 and OX-31 competed for binding but were noncompetitive with OX-32. All these antibodies bound to a subfraction of the 190, 200 and 220-kDa forms of T cell L-CA but not at all to the 180-kDa form of T cells or thymocytes. One antibody bound to B cells only (MRC OX-33) and precipitated a subfraction of B cell L-CA. With all the antibodies that did not label thymocytes the antigenic determinants survived reduction and alkylation. Subsequent proteolysis with trypsin then destroyed all determinants except the one reacting with MRC OX-22 antibody. In this case tryptic peptides retained full antigenic activity which was, however, destroyed by further proteolysis with pronase.     

Characterization of T cell surface glycoproteins T1 and T3 present on all human peripheral T lymphocytes and functionally mature thymocytes

The monoclonal antibodies, anti-T1 and anti-T3, both react with all human peripheral thymus-derived lymphocytes and with 10% of thymocytes; each, however, recognizes different cell surface structures. It was determined that the target antigen of anti-T1 is a 69000 molecular weight cell surface glycoprotein and that the T3 antigen is a 19000 mol. wt. glycoprotein.     

Analysis of structural differences between Ly-5 molecules of T- and B-cells

A group of closely related high mol. wt (Mr) membrane glycoproteins is expressed with varying Mr on different subpopulations of lymphocytes, but the different Mr forms share the Ly-5 and T200 antigenic determinants. The Ly-5 molecule expressed by thymocytes has an Mr of 175,000 (175ly5). The antigenically related molecule on B-cells has an Mr of 210,000 (210ly5). It is not known whether the variations in size are due to differences in the polypeptide chain, post-translational modifications such as glycosylation, or both. In this report we examine the glycosylation of 175ly5 and 210ly5 to determine whether differences in carbohydrate moieties may account for the different Mr of these two Ly-5 species. Pronase digestion and alkaline borohydride treatment of these molecules labeled in the terminal galactose residues revealed that 210ly5 molecules have a more complex oligosaccharide pattern than 175ly5 molecules. While Ly-5 oligosaccharides from a T-cell tumor line were very similar to those of normal thymocytes, the pattern of Ly-5 carbohydrates from a B-cell tumor were somewhat different than those from normal B-cells. This report also presents evidence for O-linked sugars on Ly-5 molecules.     

Comparison of membrane-associated proteins of murine cytolytic and helper cloned T-cell lines: identification of a protein, p24, prominent in membrane fractions from cytolytic but not helper T-cells

It has recently been reported that liposomes containing membrane components from cytolytic T-cell (TC) clones could transfer lytic activity to noncytolytic T- and B-cell lines, strongly suggesting that TC possess membrane-associated molecules which noncytolytic lymphocytes lack and which play a critical role in the lytic mechanism. It was thus of interest to compare the membrane-associated proteins from TC-lines to those of noncytolytic helper T-cell (TH) lines to determine whether any membrane-associated proteins unique to TC could be identified. Cells from three TC-lines and four TH-lines were internally labelled with [35S]methionine and then disrupted by hypotonic lysis. Low-density (plasma membrane enriched) and high-density (endoplasmic reticulum enriched) membrane fractions were isolated from each cloned cell line and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two proteins were identified which were prominent in the membrane fractions from each of the three TC-lines but not in the membrane fractions from any of the four TH-lines. One of these, p215, migrated as a broad band with an apparent mol. wt of 215,000. The other, p24, migrated as a sharp band, or tightly spaced doublet, with an apparent mol. wt of 24,000. Immunoprecipitation studies using monoclonal antibodies to T200, LFA-1, Thy 1 and Lyt 2 suggested that p215 was a variant of T200 found on TC-lines but not on TH-lines. Treatment of solubilized membrane proteins from TH-lines with anti-T200 precipitated a 185-kD protein seen on each of the TH-lines but on none of the TC-lines. In contrast, p24 was not precipitated by any of these monoclonal antibodies. It therefore appears that p24 represents a previously unidentified protein which is strongly expressed by TC but not by TH and is thus deserving of further study as to its functional significance.     

Identification of Ia glycoproteins in rat thymus and purification from rat spleen.

A fraction of la-like glycoproteins was prepared from rat thymocytes by lentil lectin affinity chromatography and gel filtration in deoxycholate. Spleen cells from mice immunized with this preparation were fused with myeloma cells to produce antibody-secreting hybrid cell lines. Antibody from four lines called MRC OX, 3, 4, 5, 6 reacted with the la-like glycoproteins, and MRC OX 3 antibody recognized an antigenie determinant polymorphic in the rat. All four antibodies also bound to mouse spleen cells and all detected polymorphisms. Studies on recombinant mouse strains suggest that the determinants are coded by the I-A subregion of the H-2 complex. MRC OX 3 correlates with Ia specificity 9, while MRC OX 4, 5, 6 correlate with specificity 17 or 18. MRC OX 4 monoclonal antibody was used for affinity chromatography to purify Ia glycoproteins from rat spleen. The rat Ia glycoprotein complex was composed of two noncovalently linked polypeptide chains of apparent mol. wt. (unreduced) 30 000 and 24 000 as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The purified Ia glycoprotein partially inhibited the binding to thoracic duct lymphocytes of an alloantiserum which detects Ia antigens linked to the major histocompatibility complex. The monoclonal anti-la antibodies bound to the majority of peripheral B lymphocytes and 18% of thymocytes, but did not significantly bind to peripheral T lymphocytes. There were on average 150000 molecules of Ia glycoprotein per la-positive B lymphocyte, and 45 000 molecules per la-positive thymocyte. From the same fusion, another cell line was prepared called MRC OX 2 which secretes monoclonal antibody to a previously undefined thymus glycoprotein of apparent mol. wt. 60000. Preliminary studies showed that the antigen was expressed on all thymocytes and on peripheral B lymphocytes in smaller amounts. It was also present in brain, but not liver or kidney homogenate.     

Isolation of lymphocyte surface antigens. II. Identification of non-beta 2 -microglobulin-associated human T cell-specific antigen.

Iodinated cell surface components from human thymus lymphocytes labeled by the lactoperoxidase method, were solubilized by papain digestion and then 3 M KCl extraction of the residual cell pellet. Antiserum to human thymus bound three components from this material, mol. wt. approximately equal to 40 000, 20 000 and 12 000 daltons. This antiserum was absorbed with cultured human lymphoblasts (CHL) until it no longer bound CHL antigens or the HLA-beta 2-microglobulin complex. It continued to bind labeled antigens from thymus, peripheral blood lymphocytes and "T" cell-enriched fraction of tonsil lymphocytes. The absorbed antiserum bound a component from papain-solubilized thymus antigens which had an estimated molecular weight of approximately 40 000 daltons and which was not associated with beta 2-microglobulin. This component seemed to be a human T cell-specific antigen.     

Lymphocyte cell surface glycoproteins which bind to soybean and peanut lectins.

In cellular immunology, peanut (Arachis hypogaea) lectin has been used to selectively agglutinate immature lymphoid cells and soybean (Glycine max-lectin to agglutinate B lymphocytes. We have used affinity chromatography to study the surface glycoproteins of rat and mouse lymphoid cells which bind to these lectins. Thymocyte and T and B lymphocyte glycoproteins were analysed either without modification (native) or after the removal of sialic acid with neuraminidase (asialo). The only native glycoprotein which was seen to bind to peanut lectin was the 95,000 mol. wt sialoglycoprotein from thymocytes. The equivalent molecules from T lymphocytes bound to peanut lectin only after neuraminidase digestion. Thus the selective agglutination of thymocytes by peanut lectin would seem to be due to a partial lack of sialic acid residues on the O-glycosidically-linked oligosaccharides of the thymocyte sialoglycoprotein. The B lymphocyte form of the leucocyte-common antigen was the only prominent native glycoprotein which was seen to bind to soybean lectin and this probably accounts for the specific binding of this lectin to B cells. The leucocyte-common antigens, in their asialo forms, from thymocytes and B and T lymphocytes differed in their binding to the lectins and this establishes that these glycoproteins which share antigenic determinants differ in their carbohydrate structures.     

Surface molecules of cultured human lymphoid cells.

Cell surface molecules of cultured human lymphoid cells were selectively labeled by lactoperoxidase-catalyzed iodination and examined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Two major iodinated species with apparent mol. wts. of 27 000 and 35 000 daltons were detected on autoradiographs of the labeled proteins of human lymphoid cell lines believed to be of thymus-independent (B) cell origin. Neither molecule was detected on putative thymus-dependent (T) lymphoid cell lines. Metabolic labeling studies showed that both molecules are glycoproteins. Rabbit antisera against cultured B lymphoid cells made specific by absorption for B cells reacted with several labeled species from iodinated B cells including the 27 000 and 35 000 mol. wt. glycoproteins. These molecules were also detected on tonsillar lymphocytes but not on peripheral blood lymphocytes. Reciprocal absorption with B cells of rabbit antisera against cultured T cells gave antisera specifically cytotoxic for T cells. However, these sera did not precipitate iodinated proteins from Nonidet P-40 lysates of T cells.     

Purification with monoclonal antibody of a predominant leukocyte-common antigen and glycoprotein from rat thymocytes.

A leukocyte-common (L-C) antigen which can be dominant as an immunogen in rabbit anti-rat thoracic duct lymphocyte serum has been purified from rat thymocytes. Initially, an antigenic fragment of 100,000 apparent mol. wt. was prepared at 400 to 900-fold purification by lentil lectin affinity chromatography and gel filtration in deoxycholate. Mice were then immunized with this fraction, and a hybrid myeloma cell line secreting antibody to the L-C antigen was prepared by cell fusion. This antibody was used for affinity chromatography and gave pure L-C antigen at 1400-fold purification compared with thymocytes.The L-C antigen is a major membrane glyco-protein of rat thymocytes and has an apparent mol. wt. of 150,000 as determined by electrophoresis on polyacrylamide gels in sodium dodecyl sulfate. The antigen constitutes one of the three thymocyte glycoproteins which stain intensely for carbohydrate with periodic acid Schiff stain. It is present on greater than 95% of thymocytes, bone marrow cells and thoracic duct lymphocytes.     

Human B cell line deficient in the expression of B cell-specific glycoproteins (GP 27,35).

A human B lymphoid cell line, P3HR-1, expresses only low levels of the 27 000 and 35 000 mol.wt. B cell-specific glycoproteins (GP 27,35). Indirect antibody-binding and quantitative absorption tests with a xenoantiserum against the antigens showed that P3HR-1 cells have on their surface about 1% of the amount found on other human B lymphoblastoid cell lines. The deficit of the glycoproteins on the surface of P3HR-1 cells could be accounted for by a reduced rate of synthesis in these cells. A simple relationship between the reduced expression of GP 27,35 on P3HR-1 cells and their inability to bind Epstein-Barr virus (EBV) or express complement receptors was excluded because other B lymphoid cells which expressed neither virus-binding sites nor complement receptor had normal amounts of GP 27,35 on their surface. However, antibodies against GP 27,35 could block the absorption of EBV by EBV receptor-positive B cells.     

Relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA)

The relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) has been investigated by immunofluorescence (cocapping) and radiolabeling. In neuraminidase-treated and untreated thymocytes there are two groups of glycoproteins which bind roughly equivalent amounts of PNA. One group also carries all the detectable receptors for HPA, the other binds only PNA. Binding inhibition experiments suggest that PNA and HPA receptors are in close proximity on the shared glycoproteins. The same two groups of receptors are present on 35–10% of neuraminidase-treated spleen lymphoid cells, mainly immunoglobulin (Ig)-negative lymphocytes. Almost all B cells have only PNA-specific receptors. Five–12% of the untreated spleen cells appreciably bind PNA and only a few bind HPA. Solubilized glycoproteins specific for PNA or HPA were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major PNA-specific radioiodinated glycoproteins of neuraminidase-treated thymocytes, as isolated by affinity chromatography, consist of the 185-kDa and 195-kDa components of the T200 antigen and of two (diffuse) components of about 140 and 120–125 kDa. All these molecules also bind to HPA-Sepharose, with the exception of the 185 kDa component, which is probably the main constituent of the “pure” PNA receptors on the intact thymocytes. In gels directly labeled with radioactive lectins, the only band strongly labeled by PNA and HPA is the diffuse 140-kDa band. The band at 120 kDa is well labeled by PNA, but all the other components are weakly labeled. The mobility of the 140- and 120-kDa bands depends strongly on neuraminidase-treatment. These bands cannot be detected in gels of untreated thymocytes, but a major HPA-and PNA-specific band of lower molecular weight can be labeled after treating the gels with neuraminidase. The factors determining the differences in labeling pattern obtained by different methods as well as the nature of PNA and HPA binding sites are discussed. The same major PNA- and HPA-binding glycoproteins (apart from minor differences) are present on neuraminidase-treated Ig-negative spleen lymphocytes. The major PNA-binding protein of B lymphocytes appears to correspond to the 225-kDa (“B220”) antigen specific for these cells.     

Characterization of a coronavirus: II. Glycoproteins of the viral envelope: Tryptic peptide analysis

Two species of membrane-associated glycoproteins have been identified in the coronavirus virion. They are readily distinguished on the basis of size, radiolabeling characteristics, and location in relation to the lipid bilayer. The larger glycoprotein is highly labeled by both radiolabeled fucose and glucosamine. This species is found in two forms, GP180 and GP90, with apparent molecular weights of 180,000 and 90,000. GP180 can be converted to GP90 in vitro by treatment of virions with trypsin. Analysis of tryptic digests of GP90 and GP180 give identical peptide patterns. Based on pronase and bromelain sensitivities, GP180/90 is the only protein which is located entirely external to the viral envelope. It appears to comprise the characteristic long, petal-shaped peplomers of the virion. The smaller glycoprotein, GP23, has an apparent molecular weight of 23,000 and is labeled by radiolabeled glucosamine but not by fucose. The level of glucosamine-labeling of GP23 is about 1/10 that of GP180/90. GP23 appears to possess two distinct domains: a smaller, carbohydrate containing region which is found outside the viral envelope, and a larger portion, highly labeled by methionine, which is integrally associated with the viral membrane. A new nomenclature is proposed for the three major coronavirus structural proteins. The two envelope glycoproteins, GP23 and GP180/90 are designated E1 and E2, respectively; the inner core protein, VP50, is designated N.     

In-vitro synthesis and release of immunoglobulins by thymocytes of inbred rats.

Thymic and splenic lymphocytes were isolated from inbred female rats, 9–16 weeks of age, and cultured in Basal Medium Eagle's supplemented with 0.4% bovine serum albumin. Cells were cultured for 20 hr at a density of 5 × 10⁷ per ml of medium containing ¹⁴C-labeled amino acids. The culture supernatants were analyzed for labeled immunoglobulins released into the medium by double antibody precipitation. Thymocytes which contained only 0.2% immunoglobulin-bearing lymphocytes (B-cells). released an IgG-like immunoglobulin which constituted 3.7–7.3% of the total labeled protein. Splenic lymphocytes, which contained about 42% B-cells, secreted IgG which amounted to about 20–25% of the total secreted protein. IgM synthesis and secretion were not detected in thymic cultures, but accounted for 4.5–6.2% of the total labeled protein in splenic cultures. The antigenic reactivity and mol. wt of the thymocyte immunoglobulin and of serum IgG chains were similar though the behavior of thymocyte Ig in sodium dodecyl sulfate-polyacrylamide gels indicated that the heavy and light chains may be non-covalently linked. Primary immunization in vivo with low doses of keyhole limpet hemocyanin resulted in increased immunoglobulin and total protein synthesis by thymocytes in vitro. The synthesis and release of this IgG-like immunoglobulin by normal thymic lymphocytes correlates with our earlier finding of a high content of this immunoglobulin in isolated thymocyte plasma membranes and indicates that it is synthesized by thymocytes.