Megakaryocyte growth and development factor. Analyses of in vitro effects on human megakaryopoiesis and endogenous serum levels during chemotherapy-induced thrombocytopenia.

The present study shows that recombinant human megakaryocyte growth and development factor (r-HuMGDF) behaves both as a megakaryocyte colony stimulating factor and as a differentiation factor in human progenitor cell cultures. Megakaryocyte colony formation induced with r-HuMGDF is synergistically affected by stem cell factor but not by interleukin 3. Megakaryocytes stimulated with r-HuMGDF demonstrate progressive cytoplasmic and nuclear maturation. Measurable levels of megakaryocyte growth and development factor in serum from patients undergoing myeloablative therapy and transplantation are shown to be elaborated in response to thrombocytopenic stress. These data support the concept that megakaryocyte growth and development factor is a physiologically regulated cytokine that is capable of supporting several aspects of megakaryopoiesis.

Identification and characterization of a soluble c-kit receptor produced by human hematopoietic cell lines.

Stem cell factor (SCF) triggers cell growth by binding to cell surface c-kit receptors. Soluble forms of several cytokine receptors have been described and may play a role in the modulation of cytokine activity in vivo. For these reasons, we investigated whether human hematopoietic cells produce soluble c-kit receptors. The human leukemia cell lines OCIM1 and MO7e display approximately 80,000 and approximately 35,000 high-affinity cell surface c-kit receptors, respectively. Soluble c-kit receptors were detected by enzyme immunoassay in OCIM1 and MO7e culture supernatants. We determined the molecular weight and binding affinity of soluble c-kit receptor produced by OCIM1 cells, soluble c-kit receptor purified from human serum, and recombinant soluble c-kit receptor expressed in CHO cells. The three soluble c-kit receptors each have a molecular weight of 98 kD. Quantitative binding experiments with 125I-SCF indicate that the soluble c-kit receptors obtained from human serum or OCIM1 cells have binding affinities for SCF of approximately 200 to 300 pmol/L, in contrast to the recombinant form, which has a binding affinity of approximately 1.5 nmol/L. All three forms of the soluble c-kit receptor were able to compete with c-kit receptors on OCIM1 cells for 125I-SCF binding. Thus human hematopoietic cells can produce a soluble form of the c-kit receptor that retains high-affinity SCF binding activity. We speculate that the soluble c-kit receptor may bind SCF and function as a receptor antagonist in vivo.

Axl receptor tyrosine kinase stimulated by the vitamin K-dependent protein encoded by growth-arrest-specific gene 6.

The Axl receptor tyrosine kinase was identified as a protein encoded by a transforming gene from primary human myeloid leukaemia cells by DNA-mediated transformation of NIH 3T3 cells. Axl is the founding member of a family of related receptors that includes Eyk, encoded by a chicken proto-oncogene originally described as a retroviral transforming gene, and c-Mer, encoded by a human proto-oncogene expressed in neoplastic B- and T-cell lines. The transforming activity of Axl demonstrates that the receptor can drive cellular proliferation. The function of Axl in non-transformed cells and tissues is unknown, but may involve the stimulation of cell proliferation in response to an appropriate signal, namely a ligand that activates the receptor. We report here the purification of an Axl stimulatory factor, and its identification as the product of growth-arrest-specific gene 6 (ref. 6). This is, to our knowledge, the first description of a ligand for the Axl family of receptors.

Soluble kit receptor in human serum.

c-kit encodes the transmembrane receptor tyrosine kinase (Kit) for the recently described ligand stem cell factor (SCF). We have developed an enzyme-linked immunosorbent assay for measuring soluble human Kit and we have used the assay to show high levels of soluble Kit in human serum. The distribution of soluble Kit levels was investigated among 112 normal human serum donors. The mean serum level (+/- SD) was found to be 324 +/- 105 ng/mL with the values falling between 163 ng/mL and 788 ng/mL. No correlation between soluble Kit levels and the sexes or ages of the donors was found. Partial purification using immunoaffinity chromatography allowed us to characterize the soluble Kit from pooled human serum. Antibodies generated to a 497-amino acid recombinant human soluble Kit corresponding to the N-terminal extracellular domain of the receptor recognized the serum-derived soluble Kit by immunoblotting. We found that the serum-derived soluble Kit is glycosylated, with mostly N-linked but also O-linked carbohydrate, and with terminal sialic acid residues. When compared with the recombinant human soluble Kit, the serum-derived material was similar both in size and glycosylation pattern. CNBr cleavage of the isolated serum-derived material followed by amino terminal sequencing confirmed the presence of five peptides expected for the extracellular portion of the Kit molecule. The immunoaffinity purified serum-derived soluble Kit inhibited binding of [125I]SCF to membrane-bound receptor in an in vitro assay. These results indicate that soluble Kit could modulate the activity and functions of SCF in vivo.

Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl.

A novel megakaryocyte growth and development factor (MGDF) has been identified in aplastic canine plasma, and its cDNAs have been cloned from canine, murine, and human sources. Purified canine MGDF isolated by procedures involving MpI receptor affinity chromatography exists in at least two forms, with apparent molecular masses of 25 kDa and 31 kDa, that share the N-terminal amino acid sequence APP-ACDPRLLNKMLRDSHVLH. Human, dog, and mouse cDNAs for MGDF are highly conserved and encode open reading frames for proteins of 353, 352, and 356 amino acids, respectively, including predicted signal peptides. Canine MGDF and recombinant human MGDF support the development of megakaryocytes from human CD34+ progenitor cell populations in liquid culture and promote the survival of a factor-dependent murine cell line (32D) engineered to express MpI. These biological activities are blocked by the soluble extracellular domain of MpI. These data demonstrate that MGDF is a novel cytokine that regulates megakaryocyte development and is a ligand for the MPI receptor.

B61 is a ligand for the ECK receptor protein-tyrosine kinase.

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Contribution of the B oligomer to the protective activity of genetically attenuated pertussis toxin.

An enzymatically deficient recombinant S1 subunit, in which Arg-9 was replaced by Lys, was combined with native B oligomer to form a mutant holotoxin molecule. This molecule exhibited decreased leukocytosis-promoting and histamine-sensitizing activities compared with those of the native toxin, supporting the view that the B oligomer is not responsible for these activities. The protective activity of this genetically attenuated pertussis toxin was compared with that of B oligomer alone. The mutant pertussis toxin and B oligomer were similarly capable of protecting mice against a respiratory infection with Bordetella pertussis, suggesting that the B oligomer makes a significant contribution to the protection afforded by the genetically attenuated holotoxin.

The molecular engineering of pertussis toxoid.

The demand for a safer pertussis vaccine has led to the development of acellular vaccine products. We have sought to manufacture a component vaccine based upon the genetic inactivation of pertussis toxin derived by recombinant DNA technology and protein engineering. Rational site-directed mutagenesis of the S1 subunit of pertussis toxin has resulted in an enzymatically-deactivated polypeptide which retains its immunogenic potential. Mutagenic analysis of the other subunits of this toxin has permitted a delineation of the structural determinants involved in its recognition of cellular receptors. The in vitro assembly of holotoxin species possessing selectively engineered subunits may facilitate the production of a molecularly-defined genetic toxoid for pertussis prophylaxis.

Isolation and structural characterization of three isoforms of recombinant consensus alpha interferon.

Recombinant DNA-derived consensus alpha interferon was expressed in Escherichia coli and purified. Isoelectric focusing of this purified protein indicated the presence of three isoelectric subforms of pI 6.1, 6.0, and 5.7. These three subforms were preparatively separated by isoelectric focusing using Immobiline polyacrylamide gel and did not exhibit apparent differences in biological activity and tertiary structure. The pI 5.7 subform could also be separated from the pI 6.1 and 6.0 subforms by reverse-phase HPLC. Automated N-terminal amino acid sequence analysis of the pI 6.1 and 6.0 subforms yielded sequences corresponding to the methionyl and des-methionyl forms of the protein, respectively. Sequence analysis of the pI 5.7 subform indicated that its N terminus is blocked. To further determine the structure of the blocking moiety in the pI 5.7 subform, a blocked N-terminal tryptic peptide was isolated from HPLC peptide mapping of the S-carboxymethylated derivative. Results obtained from mass spectroscopic and amino acid analyses of this peptide suggest that it is blocked with an acetyl group at the N-terminal cysteine residue.

Pertussis holotoxoid formed in vitro with a genetically deactivated S1 subunit.

The cytotoxicity of pertussis toxin, a multisubunit exotoxin produced by Bordetella pertussis, is believed to be due to the ADP-ribosyltransferase activity of the S1 subunit. We have previously described the recombinant expression of each of the five individual pertussis toxin subunits in Escherichia coli and the production of an enzymatically deficient form of the S1 subunit by site-directed mutagenesis. We now report the in vitro assembly of holotoxin from native pertussis toxin B oligomer and recombinant S1 subunits, the latter purified and refolded from insoluble inclusion bodies. Holotoxin assembled with recombinant S1 of authentic amino acid sequence was indistinguishable from native pertussis toxin in its electrophoretic migration and ability to elicit a cytopathic response in cultured Chinese hamster ovary cells; in contrast, holotoxin assembled with the genetically deactivated analog of recombinant S1 displayed greatly diminished cytopathicity. These results verify that the in vitro cytopathic effects of pertussis toxin are the result of the enzymatic activity of the S1 subunit and illustrate the potential for constructing complex quaternary protein structures in vitro from insoluble, unfolded polypeptides derived from expression in recombinant systems.

Structural characterization of recombinant consensus interferon-alpha.

Recombinant consensus interferon-alpha is derived from genetically modified Escherichia coli containing a synthetic gene constructed from a consensus of interferon sequences. The purified and biologically active protein has been subjected to detailed structural characterization including sequence determination and peptide isolation and identification. The homogeneous consensus interferon-alpha preparation contains two chromatographically indistinguishable homologous polypeptides with one containing an extra methionyl residue at the amino terminus. The delineated amino acid sequence of the protein is identical to that expected from the coding sequence of the gene. Correct oxidation of the molecule has been confirmed with two intramolecular disulfide linkages observed at Cys(1)-Cys(99) and Cys(29)-Cys(139).

A method for the detection and differentiation of cellulase components in polyacrylamide gels.

Endoglucanase and exoglucanase components of cellulase can be detected and differentiated after polyacrylamide gel electrophoresis by performing activity stains. Endoglucanase activity was visualized in carboxymethyl cellulose agar replicas of gels by staining with Congo red. General beta-1,4-glucanase activity was located by soaking the gel in a solution of NaBH4-reduced cellulooligosaccharides, and detecting the formation of reducing sugars by reaction with triphenyl tetrazolium chloride. Endoglucanases are active in both assays, while exoglucanases can be distinguished by their activity in the cellulo-oligosaccharide assay only. This methodology has facilitated the purification and characterization of cellulase components from Trichoderma reesei and Microbispora bispora.

Membrane filter technique for the isolation of Yersinia enterocolitica.

A membrane filter procedure was developed for the isolation of Yersinia enterocolitica from aquatic environments. Primary differentiation was based on the fermentation of sorbitol, the absence of lysine decarboxylase and arginine decarboxylase-dihydrolase activities, and the production of urease. Sodium deoxycholate was incorporated as an inhibitor of background organisms. The presumptive identification of Y. enterocolitica was accomplished in 50 h, and the rate of identity confirmation of typical colonies was 88%. The mean recovery rate of 15 strains from phosphate buffer suspensions was 91%, and quantitative recovery was demonstrated for low populations of the organism in both laboratory-prepared and naturally occurring mixed cultures. The technique was used to isolate 33 strains of Y. enterocolitica from 15 of 27 river water samples and from prechlorinated sewage effluent. Nine (27%) of the isolates were rhamnose positive, and only five (15%) were serotypable. Two isolates were identified as serotype O:4 (or O:4,32), two were O:17, and the fifth was O:40.

Long-term follow-up of sequential aortocoronary venous grafts.

In 1972 we reported myocardial revascularization of 130 patients using multiple sequential aortocoronary anastomoses to a single saphenous vein ]1]. Of the 122 survivors described in that report, 121 (99%) have been followed an additional 3 years. Twelve deaths occurred during the interval. The 110 currently followed patients represent 290 anastomoses; 54 have been studied angiographically since operation. Angiographic patency at 3 years in the studied group (18) was 70%. These figures exceed our follow-up data for 135 patients revascularized during the same period using individual vein grafts. Comparison of life table survival curves demonstrates this result. We believe the improved patency and decreased operating time that have resulted from employing this technique have outweighed the likelihood of a proximal stenosis causing closure of the whole graft system. We continue to use this technique in combination with internal mammary artery grafts in the management of multiple-vessel coronary disease. Good early results using this technique have been reported by other authors [2, 3, 5].