Developmental changes and regional distribution of phospholipase D and base exchange enzyme activities in rat brain

Phospholipase D (PL-D) activity per mg protein of whole homogenate increased 5.1 fold between Embryonic (E) day 17 and Postpartum (P) day 14 and slightly decreased by P 30 days. This was due to the decrease of PL-D activity of the P₂ fraction. The PL-D activity of P₂ and P₃ fractions increased 11.2 and 6.1 fold respectively between E 17 and P 14. The 3 base exchange enzyme (BEE) activities per mg protein of whole homogenate increased up to P 14 or P 21 and then decreased. This decrease was greater in the P₂ fraction and the P₃ fraction increased after P14. Brains from 1 day to 25 month old rats were dissected into 7 separate regions and both PL-D and BEE activities were measured. In adult rats, the hippocampus and hypothalamus had the highest PL-D activities while medulla+pons and cerebellum had the lowest PL-D activities. The developmental patterns of 5 regions except for hippocampus and hypothalamus were similar. PL-D activity in the hippocampus was maximum at P 7 followed by a steep decrease till P30 suggesting that the PL-D activity of the hypothalamus develops later and that of the hippocampus develops earlier than any other region. The distributions of BEE activities were quite different from those of PL-D activities. In adult rats, the cerebellum had the highest activity while the striatum and medulla+pons had the lowest. The BEE activities of cerebellum were lowest at P 1 and showed steep increase during the next 2 weeks.To whom to address reprint request are to be sent.     

Similarity between protein-protein and protein-carbohydrate interactions, revealed by two crystal structures of lectins from the roots of pokeweed

The roots of pokeweed (Phytolacca americana) are known to contain the lectins designated PL-A, PL-B, PL-C, PL-D1, and PL-D2. Of these lectins, the crystal structures of two PLs, the ligand-free PL-C and the complex of PL-D2 with tri-N-acetylchitotriose, have been determined at 1.8A resolution. The polypeptide chains of PL-C and PL-D2 form three and two repetitive chitin-binding domains, respectively. In the crystal structure of the PL-D2 complex, one trisaccharide molecule is shared mainly between two neighboring molecules related to each other by a crystallographic 2(1)-screw axis, and infinite helical chains of complexed molecules are generated by the sharing of ligand molecules. The crystal structure of PL-C reveals that the molecule is a dimer of two identical subunits, whose polypeptide chains are located in a head-to-tail fashion by a molecular 2-fold axis. Three putative carbohydrate-binding sites in each subunit are located in the dimer interface. The dimerization of PL-C is performed through the hydrophobic interactions between the carbohydrate-binding sites of the opposite domains in the dimer, leading to a distinct dimerization mode from that of wheat-germ agglutinin. Three aromatic residues in each carbohydrate-binding site of PL-C are involved in the dimerization. These residues correspond to the residues that interact mainly with the trisaccharide in the PL-D2 complex and appear to mimic the saccharide residues in the complex. Consequently, the present structure of the PL-C dimer has no room for accommodating carbohydrate. The quaternary structure of PL-C formed through these putative carbohydrate-binding residues may lead to the lack of hemagglutinating activity.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). II. Stimulation of T lymphocyte proliferation

A goat antibody specific for an antigenic determinant shared between guinea pig antithrombin III (AT III) and thymocytes was shown to be mitogenic for lymph node T lymphocytes in the presence of macrophages. Although the antiserum was not mitogenic for purified populations of B lymphocytes, B lymphocytes were as efficient as T lymphocytes in absorbing the mitogenic activity of the serum. The shared antigenic determinant appeared to be carbohydrate in nature in that native and guanidine-treated AT III, but not periodate oxidized AT III, were capable of inhibiting the mitogenic activity of the serum when added continuously to the cultures. The possibility that the plasma protease inhibitor AT III or an antigenically related membrane protein are involved in the regulation of T cell activation is discussed.     

Pore formation and mitogenicity in blood cells by the class 2 protein of Neisseria meningitidis.

The class 2 outer membrane protein (MIEP) of Neisseria meningitidis has recently been shown to be mitogenic for lymphocytes (Liu, M.A., Friedman, A., Tai, J., Martinez, D., Deck, R. R., Hawe, L. A., Shieh, J. T.-C., Jenkins, T. D., Donnelly, J. J., and Oliff, A. I. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4633-4637. In this study, a possible connection between MIEP's mitogenicity and its possible action as a porin was investigated. MIEP, purified from the bacterial outer membrane protein complex under denaturing conditions, caused a modest but specific release of ions (86Rb) from both erythrocytes and lymphocytes, ultimately resulting in cell lysis. The dose-response of MIEP on erythrocyte lysis was qualitatively similar to a known porin (protein I from Neisseria gonorrhoeae) but was much less efficient. Induction or preservation of native structure in MIEP increased pore formation, resulting in levels comparable to that of the protein I porin. These observations suggest that native MIEP, free of the other outer membrane proteins of Neisseria meningitidis, can efficiently form pores in cells, but that denatured MIEP is variably and marginally effective. However, pore formation by MIEP was not related to its mitogenicity in lymphocytes, based on: (i) native MIEP was not mitogenic; (ii) denatured MIEP was highly mitogenic; and (iii) denatured MIEP was mitogenic at concentrations below the threshold level for pore formation. Therefore, mitogenicity is dependent upon MIEP being in a denatured, monomeric state and is masked by native conformation.     

Cooperativity of lectin binding to lymphocytes, and its relevance to mitogenic stimulation.

The relationship between the binding patterns of soybean agglutinin, peanut agglutinin (both in their native (unaggregated) form and in their polymerized form), and of Phaseolus vulgaris leucoagglutinin, to neuraminidase-treated lymphocytes from different sources, and the mitogenic activity of these lectins, was studied. In all cases investigated, binding of a lectin to lymphocytes which resulted in stimulation was a positive cooperative process. Our findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.     

Cooperativity of lectin binding to lymphocytes,and its relevance to mitogenic stimulation

The relationship between the binding patterns of soybean agglutinin, peanut agglutinin (both in their native (unaggregated) form and in their polymerized form), and of Phaseolus vulgaris leucoagglutinin, to neuraminidase-treated lymphocytes from different sources, and the mitogenic activity of these lectins, was studied. In all cases investigated, binding of a lectin to lymphocytes which resulted in stimulation was a positive cooperative process. Our findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.     

The lack of mitogenic response of neuraminidase-treated and untreated human blood lymphocytes to divalent, hexavalent, or insoluble Helix pomatia a hemagglutinin

The mitogenic effects on human blood lymphocytes of Helix pomatia A hemagglutinin (HP) were measured by assaying incorporation of [¹⁴C]thymidine into cellular DNA. This highly purified lectin binds to human lymphocytes, treated with neuraminidase, but not to untreated lymphocytes. HP, which is hexavalent in its native form, totally failed to induce DNA synthesis in neuraminidase-treated as well as in untreated cells. Divalent HP, prepared by partial reduction and alkylation, and HP insolubilized by coupling to nylon sheets, also lacked mitogenicity. Control experiments indicated that neuraminidase-treated lymphocytes responded well to mitogenic lectins such as PHA. The lack of mitogenicity of HP was in contrast to the effects of soy bean agglutinin (SBA), which resembled HP in regard to carbohydrate specificity and ability to bind to neuraminidase-treated lymphocytes only. SBA was strongly mitogenic for neuraminidase-treated lymphocytes. The mitogenic effects of SBA were not inhibited by including varying doses of HP in the incubation mixtures. The results indicate that binding of lectin to carbohydrate receptors on the lymphocyte surface is not by itself sufficient to trigger DNA-synthesis.     

Lens culinaris lectin is a T-cell mitogen: binding inhibition by concanavalin A and phytohemagglutinin-P.

Binding and mitogenicity of a lectin from Lens culinaris (LcH) were studied in mouse lymphocytes. Both continuous and pulse treatment of lymphocytes with LcH induced a mitogenic response selectively in T cells. LcH and Con A, which have similar binding specificities, exhibited binding inhibition both in unfixed cells and glutaraldehype-fixed cells, with native Con A and succinyl Con A and at 37 °C as well as 0 °C. On the other hand, reciprocal binding inhibition by a third T-cell mitogen, phytohemagglutinin-P (PHA-P), was found only in unfixed cells at 37 °C and with native Con A, indicating that the inhibition is a secondary effect as opposed to direct competition for receptors. The inhibition of mitogenic responses to LcH and PHA-P by pretreatment of cells with Con A was studied in relation to the two different types of binding inhibition. Only the type of binding inhibition caused by a secondary effect correlated with interference with the mitogenic response.     

Binding and functional properties of concanavalin A and its derivatives. II. A proteolytic product with saccharide-binding activity.

Limited digestion of the intact subunit of concanavalin A (Mr = 26,000) with trypsin followed by affinity chromatography on Sephadex G-100 has yielded a highly purified product designated here as Tn-Con A. Chemical studies have shown that Tn-Con A is composed of several components: a large fragment (Tn I, Mr = 19,000) spanning residues 1 to 172, and lower molecular weight polypeptides that are noncovalently associated with Tn I to form the active molecule. The molecular weight of Tn-Con A at pH 7 was 90,000, suggesting that, like native concanavalin A, it was a tetramer at physiological pH. Equilibrium dialysis experiments showed that Tn-Con A bound 1 molecule of alpha-methyl-D-glucoside/22,000 g atoms of protein and therefore that four saccharides are bound by the tetrameric molecule. Tn-Con A and native concanavalin A competed for the same receptors on the lymphocyte surface. Moreover, Tn-Con A was mitogenic for both mouse and human lymphocytes with dose-response curves similar to those of the native lectin. All of these results indicate that tryptic hydrolysis of concanavalin A produces a fragmented molecule retaining the saccharide-binding, subunit association, and mitogenic capacity of the native protein.     

Effect of overexpressing fibroblast growth factor 2 protein isoforms in osteoblastic ROS 17/2.8 cells

Fibroblast growth factor-2 (FGF-2) is made by osteoblasts and modulates their function. There are high molecular weight (HMW) protein isoforms of FGF-2 that have nuclear localization sequences and a low molecular weight (LMW) 18 kDa FGF-2 protein that is exported from cells. Since FGF-2 is a trophic factor and potent mitogen for osteoblasts, the goal of this study was to utilize targeted overexpression of FGF-2 as a novel means of assessing different FGF-2 isoforms on osteoblastic cell viability and proliferation. Either LMW or HMW human Fgf2 cDNAs were cloned downstream of 3.6 kb alpha1(I)-collagen 5' regulatory elements (Col 3.6). A set of expression vectors, called Col3.6-Fgf2 isoforms-IRES-GFPsaph, capable of concurrently overexpressing either LMW or HMW FGF-2 isoforms concomitant with GFPsaph from a single bicistronic mRNA were built. Viable cell number in ROS 17/2.8 cells stably transfected with Vector (Col3.6-IRES-GFPsaph) versus each of the Col3.6-Fgf2-IRES-GFPsaph constructs were compared. In the presence of 1 or 10% serum, DNA synthesis was increased in cells expressing any isoform of FGF-2 compared with vector. However, cells transfected with HMW isoform had augmented DNA synthesis in 1 or 10% serum compared with cells expressing either ALL or LMW FGF-2 isoforms. A neutralizing FGF-2 antibody significantly reduced the mitogenic response in cells harboring ALL or the LMW FGF-2 isoforms but did not block the mitogenic effect of cells harboring the HMW isoforms. In summary, overexpression of any isoform of FGF-2 protein increased viable cell number and OB proliferation in the presence of low or high concentrations of serum. However, the HMW/nuclear isoforms preferentially mediate augmented OB proliferation. We conclude that differential expression of FGF-2 proteins isoforms is important in modulating OB function.     

Limits to the early increase in free cytoplasmic calcium concentration during the mitogenic stimulation of lymphocytes.

Three aspects of the calcium hypothesis we have proposed previously [Metcalfe, Pozzan, Smith & Hesketh (1980) Biochem. Soc. Symp. 45, 1-26] for the control of mitogenic stimulation of lymphocytes are examined in studies on the mitogenic action of the Ca2+ ionophore A23187 and its effect on cap formation. (1) Pig lymphocytes that were mitogenically stimulated by continuous incubation with 3H-labelled A23187 for 48 h contained between 3 and 15 amol of ionophore per cell. Lymphocytes exposed to 3H-labelled A23187 for 2h before washing the cells and resuspending them in ionophore-free medium were only stimulated mitogenically at 48h if the residual ionophore associated with the cells after washing was in the concentration range 3-15 amol per cell. When the cells were washed repeatedly after 2h incubation with ionophore to reduce the cell-associated ionophore below the critical concentration range, no mitogenic stimulation occurred as a result of short-term exposure to any ionophore concentration. Re-addition of ionophore to within the indicated range of cell-associated concentrations restored mitogenic stimulation at 48h. We conclude that large, short-term Ca2+ fluxes into the cells induced by the ionophore cannot generate a mitogenic signal that commits the cells to enter the cell cycle. (2) Further experiments with the ionophore showed that detectable mitogenic stimulation at 48h required a minimum of 3h exposure to optimal ionophore concentrations, and that maximal stimulation required at least 20h exposure. This is consistent with the view that a prolonged increase in the free cytoplasmic calcium concentration is required to stimulate the maximum proportion of the cells into the cell cycle. (3) Mouse splenic lymphocytes treated for short periods with very high ionophore concentrations (30 microM) in the presence of various external Ca2+ concentrations showed significant inhibition of cap formation of surface immunoglobulin receptors in the range 1-10 microM-Ca2+ in normal or depolarizing medium. We conclude that mitogens at optimal concentrations for the stimulation of lymphocytes do not cause any early increase in the free cytoplasmic Ca2+ concentration above 10 microM.     

Inactivation of a Cdk2 inhibitor during interleukin 2-induced proliferation of human T lymphocytes.

Peripheral blood T lymphocytes require two sequential mitogenic signals to reenter the cell cycle from their natural, quiescent state. One signal is provided by stimulation of the T-cell antigen receptor, and this induces the synthesis of both cyclins and cyclin-dependent kinases (CDKs) that are necessary for progression through G1. Antigen receptor stimulation alone, however, is insufficient to promote activation of G1 cyclin-Cdk2 complexes. This is because quiescent lymphocytes contain an inhibitor of Cdk2 that binds directly to this kinase and prevents its activation by cyclins. The second mitogenic signal, which can be provided by the cytokine interleukin 2, leads to inactivation of this inhibitor, thereby allowing Cdk2 activation and progression into S phase. Enrichment of the Cdk2 inhibitor from G1 lymphocytes by cyclin-CDK affinity chromatography indicates that it may be p27Kip1. These observations show how sequentially acting mitogenic signals can combine to promote activation of cell cycle proteins and thereby cause cell proliferation to start. CDK inhibitors have been shown previously to be induced by signals that negatively regulate cell proliferation. Our new observations show that similar proteins are down-regulated by positively acting signals, such as interleukin 2. This finding suggests that both positive and negative growth signals converge on common targets which are regulators of G1 cyclin-CDK complexes. Inactivation of G1 cyclin-CDK inhibitors by mitogenic growth factors may be one biochemical pathway underlying cell cycle commitment at the restriction point in G1.     

Direct role of viral hemagglutinin in B-cell mitogenesis by influenza viruses

The mitogenic activity of influenza virus is a function of the hemagglutinin (HA) molecule. Purified HA is mitogenic for murine B lymphocytes but not T lymphocytes. Furthermore, like the intact virus, HA of the H2 (but not H3) subtype is mitogenic only for B cells expressing the class II major histocompatibility complex glycoprotein I-E. Since virus bearing uncleaved HA is as mitogenic as virus bearing cleaved HA, the membrane fusion activity of the HA molecule is not involved.     

Mitogenic effect on human lymphocytes of insolubilized anti-immunoglobulins. I. Specificity of the stimulating agent

The mitogenic response of peripheral blood lymphocytes to various anti-immunoglobulin reagents has been studied by measuring incorporation of a radioactive thymidine into macromolecules. Coupling of anti-F(ab')2 or anti-light chain antibodies to Sepharose beads leads to a 5-fold increase in their mitogenic capacity with 50-fold less antibodies per culture. Pepsin-digested F(ab')2 fragments had a mitogenic capacity similar to intact antibody molecules. Anti-F(ab')2 antibodies purified by immunoabsorbent columns were found to be more effective as mitogen than unpurified antibody fractions. Antibodies to kappa- or lambda-light chains were found to be mitogenic, whereas antibodies specific to various heavy chain classes failed to induce a significant response. Isolated light chains were much more effective in inhibiting the reaction than isolated mu-chains. It is concluded that insolubilized anti-light chain antibodies are mitogenic to human peripheral blood lymphocytes.     

Urokinase plasminogen activator induces human smooth muscle cell migration and proliferation via distinct receptor-dependent and proteolysis-dependent mechanisms

In order to define the relative contribution of the proteolytic domain and the receptor-binding domain of urokinase plasminogen activator (uPA) toward its mitogenic properties we studied the effects of different uPA isoforms on migration and proliferation of human aortic smooth muscle cells (hSMC). The isoforms tested included native human glycosylated uPA, and two recombinant uPA forms, namely a recombinant uPA with wild type structure (r-uPA), and a uPA-mutant in which the first 24 N-terminal amino acid residues of the receptor binding domain were replaced by 13 foreign amino acid residues (r-uPAmut). Cell migration was evaluated using a micro-Boyden chamber assay, and cell proliferation assessed by measurement of [3H]-thymidine incorporation into DNA. Competition binding studies on hSMC using 125I-r-uPA as ligand demonstrated that r-uPA and r-uPAmut exhibited equivalent displacement profiles. However, migration of hSMC was promoted by r-uPA and not by r-uPAmut. r-uPA-induced migration occurred at concentrations (half-maximally effective concentration of 2 nM) approximating the Kd for uPA-uPAR binding (1 nM). r-uPA-induced migration was not affected by the plasmin inhibitor aprotinin. In contrast to their differential chemotactic properties, uPA, r-uPA and r-uPAmut, which possess similar proteolytic activities, all stimulated [3H]-thymidine incorporation in hSMC. Since the [3H]-thymidine incorporation response to each isoform occurred at concentrations (> 50 nM) much higher than necessary for uPAR saturation by ligand (1 nM), this mitogenic response may be independent of binding to uPAR. [3H]-thymidine incorporation responses to r-uPA and -uPAmut were sensitive to the plasmin inhibitor aprotinin, and uPA stimulated DNA synthesis was inhibited by plasminogen activator inhibitor. We conclude that hSMC migration in response to uPA depends upon on its binding to uPAR, whereas uPA-stimulated DNA synthesis in these cells requires proteolysis and plasmin generation.     

Dipeptidyl peptidase IV in the immune system. Cytofluorometric evidence for induction of the enzyme on activated T lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase with essential functional significance in thymus derived lymphocytes. This conclusion is drawn from 1) the induction of this enzyme after stimulation of T lymphocytes in vitro and 2) the impairment of T cell functions in presence of active site-specific inhibitors of the enzyme. The first item will be addressed in this paper, whereas the second one will be treated in a forthcoming article. Using flow cytofluorometry we investigated the expression of dipeptidyl peptidase IV on activated lymphocytes and the phenotype of lymphocytes expressing this enzyme. After stimulation by mitogenic lectins the number of epitopes on the cell surface binding polyclonal antibodies against DP IV increases 4 to 6 times. By means of double fluorescence staining the enzyme has been shown to be restricted nearly exclusively to T lymphocytes even after mitogenic stimulation. The highest density of DP IV epitopes has been found in cells coexpressing activation markers like receptors for interleukin 2 or transferrin in a high density.     

Interleukin 1 and protein kinase C activator are dissimilar in their effects on Il-2 receptor expression and Il-2 secretion by T lymphocytes

T lymphocytes respond to mitogenic stimulation by expressing the receptor for interleukin 2 (Il-2) and secreting Il-2; once the receptor is expressed, Il-2 induces these cells to proliferation. In the present report using mouse T lymphocytes, thymocytes, and the lymphoma cell line EL4, we studied receptor expression and Il-2 secretion as early parameters for T-lymphocyte activation in response to ionomycin, concanavalin A (Con A), 12-O-tetradecanoyl-phorbol 13-acetate (TPA), and interleukin 1 (Il-1). Il-1 is required for mitogenic response of lymphocyte preparations that are rigorously depleted of macrophages. On its own, Il-1 had very little effect on Il-2 secretion and Il-2 receptor expression by T lymphocytes. TPA strongly synergized with ionomycin both for Il-2 secretion and for Il-2 receptor expression whereas Il-1 did not. Il-1 required the simultaneous presence of ionomycin and TPA to have any demonstrable effect on T lymphocytes from spleen and on thymocytes. However, on EL4 cells which were also partially responsive to TPA alone, Il-1 showed strong synergy with TPA to induce Il-2 secretion and Il-2 receptor expression. The effect of Il-1 on EL4 cells was dose dependent where increasingly higher concentrations of Il-1 in the presence of a fixed concentration of TPA caused higher percentage of EL4 cells to become Il-2 receptor positive. The present results suggest that Il-1 does not cause its effect on T lymphocytes via the same mechanism of protein kinase C activation that has been proposed for TPA.     

Ascaris suum: human lymphocyte mitogenic factor content

Ascaris suum extract was found to contain a mitogenic factor which stimulates human lymphocytes. The extract (100 micrograms protein/ml) induced an increase in [3H]thymidine incorporation into human lymphocytes at a level similar to that obtained with pokeweed mitogen (11 micrograms/ml). Stimulation of mitosis appeared to be more effective with T lymphocytes than non-T lymphocytes. The mitogenic activity of the extract was reduced by only 27% when treated at 56 C for 30 min or when immersed into boiling water for 1 min. The extract was fractionated into four protein peaks by Sephacryl S-200 column chromatography. Lymphocyte mitogenic activity was observed in the first half of the first protein peak. Allergenic activity, assessed by the passive cutaneous anaphylaxis test in rats, was observed in the latter half of the same peak. These results suggest that A. suum contains both an allergen and a mitogenic substance.