Purification and characterization of a tissue plasminogen activator-inhibitor complex from human umbilical vein endothelial cell conditioned medium

Tissue plasminogen activator-inhibitor complexes were purified from the conditioned medium of human umbilical vein endothelial cells by affinity chromatography followed by gel filtration. It was found that a single complex was isolated which can exist in two distinct interconvertible conformations. These may be separated by electrophoresis into a form with a 105,000 apparent molecular weight and a form with an 88,000 apparent molecular weight. The particular conformation which predominates may be altered by changing the pH at which preparations are incubated or by including dithiothreitol in incubation buffers. Plasminogen activator enzymatic activity may be partially recovered from purified complexes by incubation in the presence of fibrin. Incubation in 1.5 M NH4OH results in the dissociation of the complex into two major polypeptides of 67 and 40 kilodaltons (kDa). The 40-kDa protein was isolated by gel filtration high-pressure liquid chromatography. N-Terminal amino acid analysis of this protein revealed three distinct sequences. Two of these were nearly identical and matched the N-terminal sequence recently reported for the native plasminogen activator inhibitor from endothelial cells. The third sequence exactly matched an internal portion of the same protein. The results suggest that the internal sequence is located at the site where the inhibitor is cleaved by tissue plasminogen activator.     

Isolation and structural characterization of recombinant human neu differentiation factor expressed in Escherichia coli

Recombinant human neu differentiation factor produced in engineered E. coli was isolated and subject to structural characterization. The recombinant molecule can be prepared to apparent purity and is active in stimulating receptor tyrosine autophosphorylation in cultural cells expressing HER2 receptor. The 229 amino-acid polypeptide consists of eight cysteines, of which two cysteines near the N-terminus are disulfide-bonded to form an immunoglobulin-like domain and the remaining six cysteines at the C-terminus cross-link to form an epidermal growth factor-like structure. Detailed chemical characterization of the recombinant molecule by peptide mapping in conjunction with Edman sequencing and mass spectrometry reveals that the bacterially produced recombinant neu differentiation factor preparation is properly folded and contains the correct disulfide structure. The peptide mapping procedure is also useful in identifying abnormal peptides derived from deamidation and oxidation of Asn and Met residues, respectively.     

Purification of a multipotential colony-stimulating factor from pokeweed mitogen-stimulated mouse spleen cell conditioned medium

A factor able to stimulate the proliferation and differentiation of multipotential stem cells and progenitor cells of the granulocyte-macrophage, eosinophil, and erythroid lineages as well as being able to maintain factor-dependent cell lines in culture has been purified from pokeweed mitogen-stimulated mouse spleen cell-conditioned medium. The factor was purified over 2 million-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Sephadex G-75, ion exchange chromatography on DEAE-Sepharose, reverse-phase high performance liquid chromatography on a phenyl-silica column, and gel permeation high performance liquid chromatography. All of the biological activities ascribed to the multipotential colony-stimulating factor co-fractionated through all steps, and the other known mouse-active hemopoietic regulator in pokeweed mitogen-stimulated mouse spleen cell-conditioned medium, granulocyte-macrophage colony-stimulating factor, was separated at the ion exchange step. Two protein species having Mr = 24,000 and 19,000 were visualized by silver-staining of sodium dodecyl sulfate-polyacrylamide gels of the purified factor. Both species migrated coincidently with the biological activities. The factor was active at a half-maximal concentration of 1 X 10(-13) M when assayed on a factor-dependent cell line.     

Characterization and partial purification of a haemopoietic cell growth factor in WEHI-3 cell conditioned medium.

A myelomonocytic leukaemia cell line, WEHI-3, releases into its growth medium factors which stimulate the development of pluripotential cells, granulocyte/macrophage progenitor cells, megakaryocytic and erythroid progenitor cells. Also present is a factor which is essential for the continued proliferation in vitro of a variety of haemopoietic precursor cell lines of a granulocytic nature (FDC-P cells). Characterization of this growth factor has demonstrated that it is a glycoprotein of apparent Mr 25 800, in which the carbohydrate component appears to be important for activity. After several purification steps, there is an increase in specific activity of approx. 4000-fold over the starting material. At each stage of purification, the factor necessary for the proliferation of FDC-P cells 'co-purifies' with activity which stimulates the proliferation and development of normal multipotential haemopoietic cells as well as megakaryocytic, erythroid and granulocytic committed progenitor cells. This 'co-purification' occurs to the extent that the multilineage stimulating factor and the FDC-P growth factor can be eluted from the same region of sodium dodecyl sulphate/polyacrylamide gels. Thus, evidence so far, using different starting methods and purification regimes, suggests that one molecule may have multiple activities on diverse cell types.     

Isolation and characterization of B cell differentiation factor (BCDF) secreted from a human B lymphoblastoid cell line

A subclone of a human B lymphoblastoid cell line, CESS-2, spontaneously secreted a kind of BCDF (B-BCDF) in their culture supernatant without any stimulation. B-BCDF induced IgG and IgM secretions in human B lymphoblastoid cell lines, CESS cells and CL-4 cells, respectively. BCDF-responsive CESS cells expressed IgG on their surface, whereas CESS-2, which were able to secrete B-BCDF, did not express surface IgG. B-BCDF could induce Ig-secretion in SAC-stimulated low density peripheral B cells, but did not induce Ig secretion in nonstimulated B cells. B-BCDF did not show any IL 2, BCGF, or gamma-interferon activities. B-BCDF was highly purified by gel filtration on an AcA-34 column, by chromatofocusing and ion exchange chromatography on an HPLC system, and by gel filtration on an HPLC column. Highly purified preparations showed a single protein band in SDS-PAGE analysis. The m.w. and isoelectric points of the factor were 20,000 and pH 5.1 to 5.2, respectively. The minimum protein amount required for Ig induction in B cell lines was 16 ng/ml.     

Isolation and characterization of epidermal growth factor from human milk

Epidermal growth factor (EGF) has been purified from human milk. The purification was monitored with a human placental membrane radioreceptor assay using murine salivary epidermal growth factor I (mEGF I) as a competitive ligand and was achieved exclusively by the use of reverse-phase liquid chromatography (RPLC). The sequential use of preparative, semipreparative and analytical RPLC on an octylsilica support with solvent systems of different solute selectivity such as pyridine formate, triethylammonnium phosphate or perfluorocarbonic acids in the presence of n-propanol or acetonitrile allowed purification to homogeneity with 5 consecutive runs. The molecular mass, amino acid composition and NH2-terminal sequence of human EGF were determined. Gas-phase microsequencing of residues 1-17 revealed the following sequence: Asn-Ser-Asp-Ser-Glu-X-Pro-Leu-Ser-His-Asp-Gly-Tyr-X-Leu-X-Asp which is identical with the NH2-terminof urogastrone from human urine. The purified polypeptide competes with mEGF for the placental membrane receptor with a ki of 1 ng. Furthermore, it stimulates the anchorage-dependent as well as -independent proliferation of human and rat indicator cells with half-maximal stimulation at 1 and 2.5 ng/ml, respectively. Although human epidermal growth factor has been unequivocally identified in human milk and -for the first time-shown to be identical with urogastrone from human urine, the high-resolution techniques employed have also revealed the presence of EGF-related molecules which await further characterization. It is possible that EGF and the EGF-related growth factors possess important regulatory functions in normal growth of the human breast during pregnancy and lactation as well as in abnormal growth during mammary tumor formation and progression.     

Purification and characterization of the neu/erb B2 ligand-growth factor from bovine kidney.

A neu/erb B2 ligand growth factor (NEL-GF) was purified to homogeneity from bovine kidney by a procedure involving ammonium sulfate fractionation (35-70% saturation) followed by sequential column chromatography on DEAE-cellulose (DE52), Sulfadex (sulfated Sephadex G-50), heparin-Sepharose 4B, and Superdex 75 (fast protein liquid chromatography). NEL-GF was found to be a 25-kDa polypeptide according to the analysis by gel filtration on Superdex 75 and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NEL-GF stimulated the tyrosine-specific autophosphorylation of the neu/erb B2 gene product purified by immunoabsorbent and tyrosine-specific phosphorylation of the neu/erb B2 gene product in intact dihydrofolate reductase (DHFR/G-8 cells (NIH 3T3 cells transfected with rat c-neu). NEL-GF also down-regulated the cell surface neu/erb B2 gene product in DHFR/G-8 cells. NEL-GF was mitogenic toward NIH 3T3 cells, DHFR/G-8 cells, A431 cells (human epidermoid carcinoma cells), and SK-BR-3 cells (human breast carcinoma cells) but inactive toward bovine aorta endothelial cells. NEL-GF was sensitive to 0.1% trifluoroacetic acid but resistant to 5% beta-mercaptoethanol and appeared to be distinct from a neu protein-specific activating factor (Davis, J. G., Hamuro, J., Shim, C. Y., Samanta, A., Greene, M. I., and Dobashi, K. (1991) Biochem. Biophys. Res. Commun. 179, 1536-1542) and a 30-kDa glycoprotein which competed with a monoclonal antibody for binding to the neu/erb B2 gene product (Lupu, R., Colomer, R., Zugmaier, G., Sarup, J., Shepard, M., Slamon, D., and Lippman, M. E. (1990) Science 249, 1552-1555).     

Isolation and characterization of a tumor-specific T suppressor factor from a T cell hybridoma

In our laboratory we have described a monoclonal antibody, B16G, which has been shown to bind to suppressive T cell factors (TsF) in DBA/2 mice. Therefore, B16G was used as a probe to identify T cell hybridomas secreting putative TsF. Hybridomas were obtained by the fusion of DBA/2 thymocytes stimulated in vivo by P815 tumor membrane extracts with the thymoma BW5147. One such hybridoma, A10, was selected and used for additional studies. From both the supernatants and ascites fluid of this hybrid a factor could be obtained that could specifically bind to both B16G and P815 antigen immunoadsorbent columns, and that scored positively with B16G in an ELISA after elution. Such reactivity could not be obtained from A10 supernatants or ascites absorbed over irrelevant columns, nor was it obtained from supernatants or ascites from other T cell hybrids that had scored B16G nonreactive in the original screening. In vivo studies indicated that affinity-purified A10 material injected into DBA/2J mice enhanced significantly the growth of P815 tumor cells, but not the growth of other DBA/2 syngeneic tumor lines such as L1210 or M-I. Additionally, this material did not inhibit the in vitro mixed leukocyte reaction (MLR) between DBA/2 splenocytes and allogeneic B10.BR target cells (unlike B16G purified material from whole DBA/2 spleens, which has been demonstrated to be suppressive in this type of MLR). Biochemical analysis of this tumor-specific TsF from A10 was undertaken; the native m.w. was found to be in the region of 80,000 and 90,000. Under reducing conditions, affinity-purified A10 TsF was found to resolve in SDS-PAGE as what appeared to be a heterodimer of 45,000 and 43,000. In most preparations, an associated molecule resolving at about 25,000 was observed. The implications of these observations are discussed.     

Isolation and characterization of mitochondria from human B lymphoblastoid cell lines.

Mitochondria were isolated from detergent-treated Epstein-Barr virus-transformed human lymphocytes to examine their potential use in the study of the functional expression of genetic disorders of the respiratory chain. The increase of cytochrome c oxidase activity in the mitochondrial fraction indicated a 6-fold purification of intact mitochondria. Polarographic and spectrophotometric studies revealed that the isolated mitochondria were functionally well preserved. Furthermore, the isolated mitochondria supported an active in organello protein synthesis, which was dependent on the presence of a respiratory substrate generating ATP and was essentially abolished by chloramphenicol or by a specific respiratory chain inhibitor, such as antimycin. Thus, B lymphoblastoid cell lines constitute a valuable source of mitochondria to investigate mitochondrial functions in patients affected by respiratory chain disorders.     

Isolation and characterization of a low molecular weight growth-promoting factor from human plasma

A low molecular weight growth factor (LMW-GF) enriched preparation was purified from human plasma after ultrafiltration or gel filtration by means of molecular sieving chromatography low pressure reversed phase chromatography (LP-RPLC) and electrophoresis. Purification was monitored by a biological assay testing the capacity of the fractions to enhance the sulfation activity of the somatomedins/insulin-like growth factors on chick embryo cartilage. Analysis of its chemical nature show that it is hydrophilic, stable to heat, resistant to most of the proteases but that it is degraded by acid hydrolysis or carboxypeptidase Y action. UV absorption spectrum and ion-exchange chromatographic retention behavior support the hypothesis that the most purified active preparation includes a peptide structure. The presence of sugar is suggested by concanavalin A binding experiments. The fact that the purification fractions also enhance thymidine uptake by other cell lines (fibroblasts, activated lymphocytes) widens the role of such small plasma molecules in the field of growth factor activities.     

Isolation and characterization of a butyrylesterase from human erythrocytes.

Human erythrocytes contain a butyrylesterase which, judging from the ease with which it can be solubilized, is present in the cytoplasm of these cells. This enzyme has been isolated and a number of its properties characterized. The purified enzyme hydrolyzed butyryl esters with both a lower Km and higher V than is seen with esters containing longer or shorter acyl groups. It has a molecular weight of 320 000 and an isoelectric point of 4.1. This low isoelectric point is apparently a result of the relatively high content of glutamic and aspartic acids. The stability of the isolated butyrylesterase has been examined under a number of different conditions. The enzyme is inhibited by low concentrations of Hg2+, Cd2+, Zn2+ and the organophosphorus compound Mipafox, but is insensitive to eserine. The properties of this butyrylesterase, including its ability to hydrolyze thiocholine esters at a relatively rapid rate (albeit with a high Km), are a mixture of those expected for an arylesterase and a cholinesterase.     

Glycoproteins from human colonic adenocarcinoma. Isolation and characterization of cell surface carcinoembryonic antigen from a cultured tumor cell line.

Alterations in cell surface glycoproteins have been implicated in malignancy. We examined surface membrane proteins of a cultured cell line, SKCO-1, which had been derived from a human colonic adenocarcinoma. Cell surface labeling of SKCO-1 cells with galactose oxidase, followed by reduction with sodium borotritide, revealed five major labeled glycoproteins upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. At least three additional labeled glycoproteins could be detected if galactose oxidase treatment was preceded by neuraminidase treatment. Some, but not all, of the glycoproteins could be iodinated by lactoperoxidase. The predominantly labeled glycoprotein (GPI) had a molecular weight of 200,000 and co-migrated in SDS gel with carcinoembryonic antigen (CEA). GPI was not removed from the cell surface by EDTA, hypertonic saline, or sonication but was released from the membrane by detergents. This glycoprotein was subsequently purified using lectin-agarose columns and gel filtration. GPI was judged homogenous by protein- and carbohydrate-stained SDS-polyacrylamide gels and had an amino acid composition similar to that of CEA. The carbohydrate composition of GPI was qualitatively similar to CEA but quantitatively distinct. GPI had a greater proportion of sialic acid and galactosamine and less fucose and glucosamine than CEA. Immunological studies, however, demonstrated identity between GPI and CEA. A study of the turnover rate of GPI showed it to have a half-life of 5 days.     

Further evidence for a human B cell activating factor distinct from IL-4

Supernatants from activated human T cell clones were previously shown to contain B cell-activating factor (BCAF), an activity which results in polyclonal resting B cell stimulation. In the present study, we investigate the relationship between this activity and human interleukin-4 which was also shown to act on resting B cells. The supernatant of the T cell clone TT9 contains IL-4 but anti-IL-4 antiserum does not affect the response of B cells as measured by thymidine uptake or cell volume increase. Furthermore, IL-4 induces Fc epsilon-receptor (CD23) expression on 30% of unstimulated human B cells, whereas BCAF-containing supernatants from clone P2, that do not contain detectable amounts of IL-4, promote B cell proliferation without inducing CD23 expression. Our results therefore establish that IL-4 and BCAF are distinct activities and suggest that they trigger different activation pathways in human B cells. In addition, culture of B cells with T cell supernatants for 72 hr induces a three- to fourfold increase in the expression of HLA-DR, -DP, and -DQ antigens in 50% of B cells. The addition of inhibiting concentrations of anti-IFN-gamma, LT, or IL-4 antisera to the cultures does not change these results. Finally, 30% of B cells cultured with T cell supernatants leave the G1 phase of the cell cycle and 20% reach mitosis. Taken together, our findings further support the existence of a B cell-activating factor responsible for the activation of resting human B cells.     

Isolation and characterization of mouse homolog of the neutrophil activating peptide-2.

In the presence of thrombopoietin (TPO), megakaryocytes mature by polyploidization and cytoplasmic maturation, and the matured megakaryocytes induce drastic morphological change and proplatelet formation and release a number of platelets. However, the regulatory mechanism of this unique differentiation process is still obscure. We therefore attempted to identify the factors, expression of which is induced by TPO stimulation in mouse bone marrow megakaryocytes. We isolated the mouse homolog of the neutrophil activating peptide-2 (NAP-2). Mouse NAP-2 cDNA encodes a predicted sequence of 113 amino acids and contains the Cys motif (CXC) found in other members of the alpha-chemokine family. At the amino acid level, the predicted mouse NAP-2 has 50.4%, 51.8%, and 72.6% identity with the predicted human, pig, and rat NAP-2, respectively. Northern blot analysis demonstrates that mouse NAP-2 is expressed only in spleen. Furthermore, the RT-PCR technique shows that the mouse NAP-2 gene is clearly upregulated by TPO stimulation in mouse megakaryocytes.     

Separation and partial characterization of neuropeptide-inducing factors in heart cell conditioned medium

Various conditioned media contain multiple factors that regulate the expression of the neurotransmitters acetylcholine, serotonin, and catecholamines and the neuropeptides substance P, somatostatin, vasoactive intestinal polypeptide-related peptides, cholecystokinin, and enkephalins in cultured sympathetic neurons. Using biochemical and immunological methods, we identify at least three distinct factors in heart cell conditioned medium: one induces acetylcholine, substance P, somatostatin, and vasoactive intestinal polypeptide-related peptides while suppressing catecholamine expression, a second factor induces only vasoactive intestinal polypeptide-related peptides, and a third factor induces only somatostatin expression. These observations demonstrate the existence of a group of biochemically and immunologically distinct factors involved in phenotypic specification with unique, but partially overlapping activities. The analogy with the family of differentiation factors in the hematopoietic system is discussed.     

Isolation and characterization of stem-like cells from a human ovarian cancer cell line

Increasing evidence supports the existence of a subpopulation of cancer cells capable of self-renewal and differentiation into diverse cell lineages. These cancer stem-like or cancer-initiating cells (CICs) also demonstrate resistance to chemo- and radiotherapy and may function as a primary source of cancer recurrence. We report here on the isolation and in vitro propagation of multicellular ovarian cancer spheroids from a well-established ovarian cancer cell line (OVCAR-3). The spheroid-derived cells (SDCs) display self-renewal potential, the ability to produce differentiated progeny, and increased expression of genes previously associated with CICs. SDCs also demonstrate higher invasiveness, migration potential, and enhanced resistance to standard anticancer agents relative to parental OVCAR-3 cells. Furthermore, SDCs display up-regulation of genes associated with epithelial-to-mesenchymal transition (EMT), anticancer drug resistance and/or decreased susceptibility to apoptosis, as well as, down-regulation of genes typically associated with the epithelial cell phenotype and pro-apoptotic genes. Pathway and biological process enrichment analyses indicate significant differences between the SDCs and precursor OVCAR-3 cells in TGF-beta-dependent induction of EMT, regulation of lipid metabolism, NOTCH and Hedgehog signaling. Collectively, our results indicate that these SDCs will be a useful model for the study of ovarian CICs and for the development of novel CIC-targeted therapies.     

Generation of human class I major histocompatibility complex activating factor in serum free medium and its partial characterization

Human peripheral blood mononuclear cells (PBMCs) activated with Con-A release a soluble factor which augments the expression of class I major histocompatibility complex (MHC) antigens by a variety of tumour cells. Previous attempts to purify this factor called MHC-activating factor (AF) (MHC-AF) made us realize that the presence of large numbers and quantities of irrelevant fetal calf serum proteins in the culture supernatants of the activated human PBMCs, interfered with the purification procedure. It was therefore necessary to standardize the use of a serum free culture medium to generate human MHC-AF. In the present communication we have tried several types of culture media and have identified DCCM-2 as the most suitable culture medium to generate human MHC-AF. MHC-AF generated in DCCM-2 medium appears to be a protein molecule resistant to pH 2 treatment but sensitive to heat treatment (56°C × 45 min) and treatment with proteolytic enzymes trypsin and chymotrypsin.