Human lymphoblastoid cell lines secreting antibodies with restricted HLA specificity

Peripheral B lymphocytes obtained from three healthy individuals who had been immunized against peripheral blood lymphocytes from appropriate HLA-incompatible donors were transformed by the use of Epstein-Barr virus. The transformed blastoid B cells were repeatedly subcultured by means of cluster picking, and the HLA antibody-producing cultures were identified by testing the culture supernatants by means of the cytotoxicity assay, using the corresponding donor cells. Thus far, four cell lines that secrete cytotoxic HLA antibodies (MP1, 3, 4, and 5) have been established. Specific immunoabsorption experiments revealed that the antibody activity is carried by lambda-type IgM for MP1, by kappa-type IgM for MP3 and MP5, and by both for MP4. Specificity analysis of a panel of HLA-pretyped cells indicated that MP1 detects DQw2, whereas MP5 recognizes B7. The specificity of MP3 was similar to a DQ specificity termed DC5 (probably equivalent to TA10) but not the same. In the case of MP4, both of the lambda-type and kappa-type antibodies appeared to be directed toward new HLA class 11 determinants.Abbreviations used in this paper HLA human major histocompatibility - EBV Epstein-Barr virus - B-LCL Blymphoblastoid cell line - NA not absorbed - PBS phosphate-buffered saline - SPA Sepharose protein A - NRS normal rabbit serum     

Regulation of murine T cell proliferation by B cell stimulatory factor-1

The proliferation of mitogen-activated primary T cells, antigen-activated memory T cells from mixed leukocyte culture, and antigen-dependent alloreactive T cell clones in response to purified murine recombinant B cell stimulatory factor-1 (also known as interleukin 4) was examined. We found that B cell stimulatory factor-1 (BSF-1) stimulated optimal proliferation of these T cells only after their recent activation by antigen or mitogen. Analysis of cell surface BSF-1 receptor expression indicated that although T cell activation is accompanied by a small increase in BSF-1 receptor expression, the cells also express BSF-1 receptors prior to activation at a time when they do not proliferate in response to BSF-1. BSF-1 was as effective a stimulus as interleukin 2 for inducing proliferation of the Lyt-2+ subpopulation of concanavalin A-activated murine spleen cells and an alloreactive cytolytic T cell clone. However, the L3T4+ subpopulation of concanavalin A-activated spleen and an alloreactive helper T cell clone were less responsive to BSF-1 than to interleukin 2. Taken together, the data indicate an important role for BSF-1 in the regulation of normal T cell proliferation.     

Granulocyte-macrophage colony-stimulating factor augments the primary antibody response by enhancing the function of antigen-presenting cells

Purified, recombinant-derived murine granulocyte-monocyte colony-stimulating factor was found to enhance the primary in vitro immune response to SRBC by murine spleen cells. In determining the mechanism of this augmentation, it was found that only splenic adherent cells and neither resting nor activated T cells nor B cells expressed specific receptors for GM-CSF. When splenic adherent cells were pulsed briefly with GM-CSF before addition to macrophage-depleted cultures, they reconstituted the PFC response to a significantly greater degree than did control macrophages. Splenic adherent cells incubated overnight with SRBC plus GM-CSF were also more efficient antigen-presenting cells than splenic adherent cells incubated with antigen alone. The mechanism of this enhanced antigen presentation was found to be due to a GM-CSF-dependent increase in the level of IL 1 secretion and Ia antigen expression. Consistent with these data was the finding that GM-CSF augmented IL 2 production by splenic T cells in response to suboptimal concentrations of Con A. Finally, the day 5 in vivo antibody response (as measured by serum titers) of mice immunized with a low dose of SRBC was enhanced by two daily inoculations of GM-CSF. Thus, the role that GM-CSF plays in augmenting immune responses may not be solely accounted for by its ability to cause the proliferation or differentiation of macrophages, but more than likely includes its ability to enhance the function of antigen-presenting macrophages.     

Proton magnetic resonance study of lysine-binding to the kringle 4 domain of human plasminogen. The structure of the binding site

The binding of L-Lys, D-Lys and epsilon-aminocaproic acid (epsilon ACA) to the kringle 4 domain of human plasminogen has been investigated via one and two-dimensional 1H-nuclear magnetic resonance spectroscopy at 300 and 600 MHz. Ligand-kringle association constants (Ka) were determined assuming single site binding. At 295 K, pH 7.2, D-Lys binds to kringle 4 much more weakly (Ka = 1.2 mM-1) than does L-Lys (Ka = 24.4 mM-1). L-Lys binding to kringle 4 causes the appearance of ring current-shifted high-field resonances within the -1 approximately less than delta approximately less than 0 parts per million range. The ligand origin of these signals has been confirmed by examining the spectra of kringle 4 titrated with deuterated L-Lys. A systematic analysis of ligand-induced shifts on the aromatic resonances of kringle 4 has been carried out on the basis of 300 MHz two-dimensional chemical shift correlated (COSY) and double quantum correlated spectroscopies. Significant differences in the effect of L-Lys and D-Lys binding to kringle 4 have been observed in the aromatic COSY spectrum. In particular, the His31 H4 and Trp72 H2 singlets and the Phe64 multiplets appear to be the most sensitive to the particular enantiomers, indicating that these residues are in proximity to the ligand C alpha center. In contrast, the rest of the indole spectrum of Trp72 and the aromatic resonances of Trp62 and Tyr74, which are affected by ligand presence, are insensitive to the optical nature of the ligand isomer. These results, together with two-dimensional proton Overhauser studies and ligand-kringle saturation transfer experiments reported previously, enabled us to generate a model of the kringle 4 ligand-binding site from the crystallographic co-ordinates of the prothrombin kringle 1. The latter, although lacking recognizable lysine-binding capability, is otherwise structurally homologous to the plasminogen kringles.     

Structure of the precursor to an enzyme mediating COOH-terminal amidation in peptide biosynthesis

Many bioactive peptides terminate with an amino acid alpha-amide at their COOH terminus. The enzyme responsible for this essential posttranslational modification is known as peptidyl-glycine alpha-amidating monooxygenase or PAM. We identified cDNAs encoding the enzyme by using antibodies to screen a bovine intermediate pituitary lambda gt11 expression library. Antibodies to a beta-galactosidase/PAM fusion protein removed PAM activity from bovine pituitary homogenates. The 108,207 dalton protein predicted by the complete cDNA is approximately twice the size of purified PAM. An NH2-terminal signal sequence and short propeptide precede the NH2 terminus of purified PAM. The sequences of several PAM cyanogen bromide peptides were localized in the NH2-terminal half of the predicted protein. The cDNA encodes an additional 430 amino acid intragranular domain followed by a putative membrane spanning domain and a hydrophilic cytoplasmic domain. The forms of PAM purified from bovine neurointermediate pituitary may be generated by endoproteolytic cleavage at a subset of the 10 pairs of basic amino acids in the precursor. High levels of PAM mRNA were found in bovine pituitary and cerebral cortex. In corticotropic tumor cells, levels of PAM mRNA and pro-ACTH/endorphin mRNA were regulated in parallel by glucocorticoids and CRF.     

Protein kinase C in the regulation of smooth muscle contraction

The cellular and molecular mechanisms underlying smooth muscle contraction are reviewed in the light of recent studies of smooth muscle ultrastructure and of the role of polyphosphoinositide turnover and protein kinase C function in smooth muscle contraction. A new model of smooth muscle contraction is proposed that differs radically from accepted views, particularly the latch bridge hypothesis, in terms of both Ca2+ messenger function and the molecular events underlying this process. A coordinate fibrillar domain model of contraction is proposed in which the initial and sustained phases of contraction are mediated by different cellular and molecular events. The initial phase of response is mediated by a rise in [Ca2+]c and the resulting calmodulin-dependent activation of both myosin light chain kinase and the dissociation of caldesmon from the actin-caldesmon-tropomyosin-myosin fibrillar domain. These events lead to an interaction between actin and the phosphorylated light chains of myosin just as in previous models. However, this initial phase is followed by a sustained phase in which a rise in [Ca2+]sm stimulates the plasma membrane-associated, Ca2+-sensitive form of protein kinase C that results in the phosphorylation of both structural and regulatory components of the filamin-actin-desmin fibrillar domain. These events underlie the tonic phase of contraction.     

Ovomucoid third domains from 100 avian species: isolation, sequences, and hypervariability of enzyme-inhibitor contact residues

Ovomucoids were isolated from egg whites of 100 avian species and subjected to limited proteolysis. From each an intact, connecting peptide extended third domain was isolated and purified. These were entirely sequenced by single, continuous runs in a sequencer. Of the 106 sequences we report (five polymorphisms and chicken from the preceding paper [Kato, I., Schrode, J., Kohr, W. J., & Laskowski, M., Jr. (1986) Biochemistry (preceding paper in this issue)]), 65 are unique. In all cases except ostrich (which has Ser45), the third domains are either partially or fully glycosylated at Asn45. The majority of the third domain preparations we isolated are carbohydrate-free. Alignment of the sequences shows that their structurally important residues are strongly conserved. On the other hand, those residues that are in contact with the enzyme in turkey ovomucoid third domain complex with Streptomyces griseus proteinase B [Read, R., Fujinaga, M., Sielecki, A. R., & James, M. N. G. (1983) Biochemistry 22, 4420-4433] are not conserved but instead are by far the most variable residues in the molecule. These findings suggest that ovomucoid third domains may be an exception to the widely accepted generalization that in protein evolution the functionally important residues are strongly conserved. Complete proof will require better understanding of the physiological function of ovomucoid third domains. This large set of variants differing from each other in the enzyme-inhibitor contact area and augmented by several high-resolution structure determinations is useful for the study of our sequence to reactivity (inhibitory activity) algorithm. It is also useful for the study of several other protein properties. In the connecting peptide fragment most phasianoid birds have the dipeptide Val4-Ser5, which is absent in most other orders. This dipeptide is often present in only 70-95% of the molecules and appears to arise from ambiguous excision at the 5' end of the F intron of ovomucoid. Connecting peptides from the ovomucoids of cracid birds contain the analogous Val4-Asn5 peptide. In laughing kookaburra ovomucoid third domain we found (in 91% of the molecules) Gln5A, which we interpret as arising from ambiguous intron excision at the 3' end of the F intron.