The relationship between phospholipid methylation and calcium influx in murine lymphocytes stimulated with native and modified Con A.

Native Con A and two chemical derivatives, divalent dimeric Con A and monovalent dimeric Con A. induced a transient increase of phospholipid methylation, Ca2+ influx, and also increased DNA synthesis in murine lymphocytes. For each of the individual mitogens, the dose-response curves for these three activities were very similar. However, there were major differences between the dose-response curves for Con A and each of its two chemical derivatives. On the other hand, the time course of phospholipid methylation for each lectin reached a maximum at about 10 min after the addition of lectin, and then gradually decreased to control levels. In like manner, Ca2+ influx reached its maximum at approximately 5 min. The lectin-stimulated increase in phospholipid methylation occurred in calcium-free medium, while the inhibitor of phospholipid methylation, 3-deaza-SIBA, also suppressed the increased calcium influx. This suggests that the Ca2+ influx might be regulated by early phospholipid methylation. Further, in the absence of calcium, the methylated phospholipids do not undergo Con A-accelerated breakdown by phospholipase A2. This suggests that the increased influx of calcium is necessary for the activation of phospholipase A2, an enzyme that hydrolyses methylated phospholipids to yield arachidonic acid and lysolecithin. Blocking any of these biochemical steps also blocked subsequent DNA synthesis, suggesting that the pathway may be required for the activation of lymphocytes.

Fluorescence microscopic and flow cytometric analysis of bone marrow-derived cells in human epidermis: a search for the human analogue of the murine dendritic Thy-1+ epidermal cell.

Lymphoid cells with an affinity for the epidermis (epidermotropic lymphocytes) have been proposed to play a role in the immune functions of the epidermis. However, antigen-presenting Langerhans cells (LC) and indeterminate cells are presently the only cells in the human epidermis which have been demonstrated to originate in the bone marrow. Recent studies of murine epidermis have identified a population of bone marrow-derived cells which express Thy-1 antigen and which are present in a similar density to, but distinct from, LC. We therefore sought to identify the potential human analogue of the murine Thy-1+ epidermal cell utilizing a battery of antileukocyte reagents in immunohistochemical, flow cytometric, and cell sorting studies. A panel of antibodies failed to detect significant numbers of human Thy-1 antigen-bearing cells, T cells, B cells, monocytes/macrophages (other than LC), and natural killer cells in tissue sections, epidermal sheets, and epidermal cell (EC) suspensions. This was the case using EC suspensions either unfractionated or fractionated on Ficoll-Hypaque to enrich for leukocyte subpopulations. Since the nature of the murine Thy-1+ EC is uncertain, it is possible that antibodies directed against well-defined leukocyte subpopulations may not be of value in the detection of a potential human analogue. We therefore utilized double fluorescence staining with anti-HLe-1, an antibody which identifies all human leukocytes, and anti-HLA-Dr (Dr), which identifies epidermal LC, in order to demonstrate a potential population of HLe-1+ Dr- non-LC, bone marrow-derived cells. The vast majority of HLe-1+ cells were HLA-Dr+ LC; these were present at a density of 608 cells/mm2 in epidermal sheets. A minor population of HLe-1+ cells which did not express HLA-Dr (HLe-1+ Dr-) was observed in tissue sections, epidermal sheets, and EC suspensions. The nondendritic morphology and low density of these HLe-1+ Dr- EC in epidermal sheets (mean density of 4.2 +/- 1.6 cells/mm2) precluded their representing a strict human analogue of the murine Thy-1+ EC, since murine Thy-1+ EC are dendritic and are present in a density similar to that of LC. Purified preparations of the minor HLe-1+ Dr- EC population obtained by electronic cell sorting or panning and examined ultrastructurally were not enriched for any bone marrow-derived cell population. Thus, using currently available markers and sorting technology, we have been unable to identify a human analogue of the murine dendritic Thy-1+ epidermal cell.

Detection and enumeration of monocytes in human blood with peanut agglutinin.

Binding of peanut agglutinin (PNA) to normal human peripheral blood mononuclear cells was analyzed on a cell sorter, and compared to the binding of the monocyte specific monoclonal antibodies Mac-1 and Leu-M3. Each of the reagents labeled 9-11% of the mononuclear cells and similar binding patterns were observed. Of the PNA+ cells, 67% adhered to plastic petri dishes, whereas 76% of Mac-1+ cells were adherent. No competition for binding was observed between PNA and Mac-1 on the one hand, or PNA and Leu-M3 on the other. In double staining experiments, about 10% of the cells, comprising 80% of the monocytes, were PNA+ Leu-M3+. Our results show that PNA can serve for the identification and enumeration of monocytes in human peripheral blood.

The stimulation of immunoglobulin production in murine spleen cells by the pokeweed mitogens.

We have shown that of the five mitogens purified from the salt extracts of pokeweed, only Pa-1, the B cell mitogen, has the ability to stimulate immunoglobulin production in murine B cells in the absence of classical T cells. Furthermore, the T cell mitogens (Pa-2-Pa-5) not only are unable to stimulate DNA or immunoglobulin synthesis by B cells in the absence of T cells, but cannot stimulate immunoglobulin production by B cells even in the presence of T cells. This suggests that Pa-2-Pa-5 do not stimulate adequate helper function in T cells or that if such a helper function is stimulated, an additional stimulatory factor is required for immunoglobulin production, which is not provided for by Pa-2-Pa-5.

Isolation and identification of the major concanavalin A binding glycoproteins from murine-lymphocytes.

The major glycoproteins which bind concanavalin A have been isolated and identified from murine spleen cells, thymocytes,and purified thymus-derived (T) lymphocytes, and from the spleen cells of congenitally athymic (nude) mice. The cells were radiolabeled by lactoperoxidase catalyzed 125I iodination or by culturing the cells in media containing [3H]leucine or [3H]fucose. The cell membrane was solubilized with Nonidet P-40 and the concanavalin A binding proteins were isolated by affinity chromatography and analyzed according to their mobility on polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The major proteins from various lymphocyte preparations were identified by immunoprecipitation with specific antisera. The molecules coded by the histocompatibility-2 complex acted as concanavalin A binding proteins H-2K and H-2D were isolated from T lymphocytes, thymocytes, and bone marrow derived (B) lymphocytes. The Ia antigens were identified from B lymphocytes and tentatively identified from T lymphocytes. In addition to these H-2 complex proteins, immunoglobulin M and D on B lymphocytes also bound concanavalin A binding. All these glycoproteins have previously been identified as cell surface molecules. The presence of certain minor unidentified concanavalin A binding proteins on lymphoid cells is indicated.

Cross-linking of T cell mitogens: effects on B and T cell proliferation and immunoglobulin synthesis.

The T cell mitogens Pa-2, concanavalin A (con A) and its dimeric derivative succinyl-con A, were each cross-linked with the bifunctional reagent dimethyl suberimidate. Although the dose-response curves of these insoluble aggregated products were markedly changed from those of the soluble mitogens, each aggregate continued to stimulate DNA synthesis by murine thymus and T cells. Both aggregated Pa-2 and aggregated succinyl con A stimulated DNA synthesis by B cells from athymic (Nu/Nu) mice. Aggregated con A did not stimulate these cells and, like soluble con A, depressed the background incorporation of 3H-thymidine. Unlike soluble Pa-2, aggregated Pa-2 also greatly increased Ig production by both the B cell cultures and B + T cell cultures from normal (BALB/c) mice.

Structural composition of canine secretory component and immunoglobulin A.

Dog serum and colostral immunoglobulin A (IgA) and free secretory component from colostrum were isolated using affinity chromatography. Both serum and colostral IgA showed similar susceptibility to reduction with dithiothreitol, but only colostral IgA released the additional subunit, bound secretory component. This released secretory component was identical with free secretory component with respect to electrophoretic migration, isoelectric focusing point, and molecular weight, but lacked some antigenic determinants. The amino acid composition and the N-terminal sequence of canine free secretory component was similar to that reported for the cow.

Comparison of bovine and mouse pituitary glycoprotein hormone pre-alpha subunits synthesized in vitro.

Poly(A)-containing RNA isolated from bovine and mouse pituitaries and a mouse pituitary thyrotropic tumor was translated in a wheat germ cell-free biosynthetic system. A precursor of the glycoprotein hormone alpha subunit, "pre-alpha," was immunoprecipitated from the translation mixtures with antiserum against bovine luteinizing hormone (LH; lutropin) alpha. The specificity of the immunoprecipitation was shown by competition with authentic bovine LHalpha and lack of competition with bovine thyroid-stimulating hormone (TSH; thyrotropin) beta. Bovine and mouse pre-alpha subunits migrated identically in sodium dodecyl sulfate gradient polyacrylamide slab gels with an apparent molecular weight of about 17,000. Pre-alpha comprised 2-3% and 20-30% of the total proteins translated with pituitary and pituitary tumor mRNA, respectively. Microanalysis of amino acid sequence of the pre-alpha subunits containing various radiolabeled amino acids gave the following partial sequence for mouse tumor pre-alpha: [Formula: see text] Met was also found in positions 1, 14, and 17 in mouse pituitary pre-alpha but only in residue 1 of the bovine pituitary pre-alpha subunit. Leu was found in identical positions in bovine pituitary pre-alpha, with an additional Leu in position 17. Leu in the common positions (12, 15, 19, and 22) has also been found in human choriogonadotropin pre-alpha subunit [Birken, S., Fetherston, J., Desmond, J., Canfield, R. & Boime, I. (1978) Biochem. Biophys. Res. Commun. 85, 1247-1253]. The data demonstrate that pituitary as well as placental glycoprotein hormone alpha subunits are synthesized with an amino-terminal hydrophobic extension, in accord with the "signal hypothesis" for secreted proteins. Furthermore, the positions of the hydrophobic amino acid Leu have been strictly conserved in pre-alpha subunits from various species and in two different tissues, the pituitary and placenta.

Inter-laboratory relative fluorescence intensity measurements using FlowCal 575 calibration beads: a baseline study.

Twenty-one laboratories participated in a baseline study of their ability to agree on the measurement of fluorescence intensities of a stable multi-peak reference material (FlowCal 575) and of stained and fixed CD4 lymphocytes. Relative fluorescence intensities were calculated as ratios to the most fluorescent bead. The good correlation between laboratories suggests that this simple approach may be useful in multi-laboratory studies. The data also provide a baseline for the evaluation of any improvement of inter-laboratory agreement gained by more rigorous and demanding approaches.

Phospholipid methylation: a biochemical signal modulating lymphocyte mitogenesis.

Phospholipid methylation in murine T lymphocytes but not B cells was stimulated by mitogenic lectins such as concanavalin A and phytohemagglutinin, and the methylation was then returned to the control level by the concomitant activation of phospholipase A2. A parallelism between dose-response curves of concanavalin A for phospholipid methylation and thymidine incorporation was found. Inhibition of either synthesis or degradation of methylated phospholipids resulted in a decrease in the thymidine incorporation. Although prostaglandins such as the E and F series were the main products of arachidonic acid released by phospholipase A2 activation, inhibition of synthesis of these compounds by indomethacin did not reduce the thymidine incorporation significantly. These results suggest that the mitogenesis of murine T lymphocytes is triggered by the activation of both phospholipid methyltransferase(s) and phospholipase A2.

Segmental homology between T-cell receptors and immunoglobulin variable regions: evidence that antisera to synthetic JH1 peptide react with murine and human T-cell products.

To determine precisely the nature of serological determinants shared between T-cell surface molecules and immunoglobulin variable regions, the capacity of antisera directed against a synthetic peptide corresponding to the entire JH 1 region of classical immunoglobulin plus five residues of the D region were tested for their capacity to bind to T-cell membranes and isolated T-cell products. The anti-JH 1 antisera reacted with normal and monoclonal in vitro grown T-cell lines as judged by microhemagglutination and binding in enzyme-linked immunosorbent assays. Immunologically cross-reactive membrane components disclosed by immunoblot transfer analysis ("Western blots") consisted of major components in the molecular weight range 30-35,000 and minor components in the range 65-70,000. The major product of the human T-cell leukemia line MOLT-3 had an approximate mass of 34,000 Da, a value consistent with the predicted size of the molecule specified by the recently described putative T-cell receptor gene YT35. The 65 to 70,000-Da components are most probably tightly associated dimers of the 30 to 35,000-Da forms. It was possible to align the JH sequences of molecules reactive with the anti-JH 1 antisera and other characterized VH sequences of molecules known to be cross-reactive with T-cell products. This facilitated a comparison disclosing clear segmental homology between the protein sequence derived from the YT35 gene and immunoglobulin VH framework regions sharing approximately 50% of sequence identity. The identification of VH-related T-cell products (termed VT-bearing molecules) with products of putative T-cell receptor genes gained further support by N-terminal sequence of the 68,000-Da product of the 70-N2 T-cell line which showed homology to the predicted N-terminal region of the YT35 product. These serological and protein chemical data, coupled with the comparison to gene sequence, show that T-cell components that bear serological determinants cross-reactive with VH show segmental homology with products of putative T-cell receptor genes and immunoglobulin VH.

The covalent and three-dimensional structural of concanavalin A. I. Amino acid sequence of cyanogen bromide fragments F1 and F2.

Concanavalin A is a lectin composed of identical subunits, each containg 237 amino acid residues. The complete amino acid sequence of the first 129 residues of the polypeptide chain has been determined by analysis of peptides obtained from digests of CNBr Fragments F1 (residues 1 to 42) and F2 (residues 40 to 129). Correlation of the chemical sequence with x-ray crystallographic results indicates that Fragment F1 contains all of the protein ligands for the binding of 2 metal ions, Mn2+ and Ca2+, and that Fragment F2 contains many of the residues involved in the interactions of the subunits to form dimers and tetramers. The site of cleavage of the polypeptide chain to yield the naturally occurring Fragments A1 and A2 has also been identified as the peptide bond between residues 118 and 119.

The covalent and three-dimensional structure of concanavalin A. II. Amino acid sequence of cyanogen bromide fragment F3.

The amino acid sequence of the COOH-terminal CNBr fragment, F3 (residues 130 to 237), of concanavalin A has been established, completing the determination of the covalent structure of this lectin. Analysis of the chemical sequence showed that the distribution of charged residues is generally more dense in the NH2-terminal half of the polypeptide chain than in the COOH-terminal portion and that in the latter region there is a linear stretch composed of many hydrophobic residues. Correlation with x-ray crystallographic results indicates that the hydrophobic region is located in the interior of the molecule, and that it forms a part of a deep cavity which is the binding site for the inhibitor, beta-(o-iodophenyl)-D-glucopyranoside. In conjunction with the three-dimensional structure, the amino acid sequence reported here provides new data for analysis of variables involved in predicting the three-dimensional folding of proteins from the primary structure. The sequence of concanavalin A is the first determined for a lectin and it serves as a reference structure for comparisons with other lectins.