Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector

A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.     

Molecular cloning and characterization of complementary deoxyribonucleic acids corresponding to bovine trophoblast protein-1: a comparison with ovine trophoblast protein-1 and bovine interferon-alpha II

Bovine trophoblast protein-1 (bTP-1) is a secreted glycoprotein that consists of several forms differing slightly in mol wt and isoelectric point. It is produced by bovine conceptuses after about day 15 of pregnancy and is believed to play a key role in signalling the presence of an embryo to the mother. In this study, a series of recombinant cDNA clones corresponding to the mRNA for bTP-1 have been isolated from cDNA libraries representing day 18-19 bovine conceptus poly(A)+ mRNA. Base sequencing of several cDNAs indicated that multiple mRNAs for bTP-1 exist. Northern blotting and primer extension experiments showed that the mRNAs average about 1 kilobase in length. One apparently full-length cDNA clone consisted of 1035 bases up to the beginning of the poly(A) tail. It contained an open reading frame of 195 codons which began at a position 79 bases from the 5' end. Its entire sequence was 85% identical to that of a cDNA for the immunologically related ovine trophoblast protein-1 (oTP-1) and about 79% identical to that for a bovine interferon-alpha II (IFN alpha II). The highest conservation of sequence (greater than 90%) was noted in the 3'-untranslated sequences of the bTP-1 and oTP-1 cDNAs. The deduced amino acid sequence of bTP-1 shared 80% identity with oTP-1, between 45-55% with human, rodent, porcine, and bovine IFNs of the alpha 1 subfamily and about 70% with a bovine IFN alpha II. A single potential site for N-glycosylation was noted at Asn78. These results show that bTP-1, like its ovine counterpart oTP-1, is structurally related to the IFN alpha S. We suggest that these embryonic IFNs play a role in controlling immunoreactions at the trophoblast-uterus interface as well as triggering other maternal responses to pregnancy.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

The production of distinct forms of plasminogen activator by mouse embryonic cells

We have examined cultured parietal endoderm, visceral endoderm, and extraembryonic mesoderm cells from the mouse embryo for production of the protease plasminogen activator. All of these cell types synthesize and secrete the enzyme, but the molecular characteristics of the plasminogen activators differ as defined by the apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gels, the antigenic properties as defined by two antisera to distinct plasminogen activators, and the interaction with an inhibitor present in fetal bovine serum. The parietal endoderm plasminogen activator has a predominant molecular weight of 79,000, is immunoprecipitated and inhibited by an antiserum raised against a human melanoma plasminogen activator, but not by an antiserum against mouse urokinase, and is only partially inhibited by the serum inhibitor. The visceral endoderm and extraembryonic mesoderm plasminogen activators, which are identical by all criteria, have molecular weights of 48,000, are inactivated only by the anti-urokinase antibodies, and are inhibited by an inhibitor in fetal bovine serum. These results establish the presence of at least two different forms of plasminogen activator in the early mouse embryo. The distinctive nature of the enzyme produced by parietal endoderm can be used as a diagnostic marker for this cell type at this stage of development. When F9 teratocarcinoma stem cells are induced to differentiate by retinoic acid and dibutyryl cAMP, they secrete a plasminogen activator of the parietal endoderm type.     

Osteocyte differentiation in the tibia of newborn rabbit: an ultrastructural study of the formation of cytoplasmic processes

The morphological changes undergone by the osteoblast at the ultrastructural level, during its differentiation into osteocyte, were studied in the primary parallel-fibred bone of the newborn rabbit by means of incomplete three-dimensional reconstruction from partially serial-sectioned preosteocytes. The findings obtained suggest that the formation of osteocyte cytoplasmic processes is an asynchronous and asymmetrical phenomenon that seems to precede the mineralization of the organic matrix and to give rise to an asymmetrical mature osteocyte. The functions of cytoplasmic processes as regards bone formation, cell nutrition and osteoblast modulation are discussed. The mechanism by which the osteoblast 'enters' the bone matrix is hypothesized.     

Phosphorylation of the RGS protein Sst2 by the MAP kinase Fus3 and use of Sst2 as a model to analyze determinants of substrate sequence specificity

Previously, we used mass spectrometry to demonstrate pheromone-stimulated phosphorylation of Ser-539 in Sst2, a regulator of G protein signaling in yeast Saccharomyces cerevisiae [Garrison, T. R., et al. (1999) J. Biol. Chem. 274, 36387-36391]. Here, we show that Sst2 phosphorylation is mediated by the mitogen-activated protein (MAP) kinase Fus3. Phosphorylation occurs within a canonical MAP kinase phosphorylation site (Pro-X-Ser/Thr-Pro, where "X" at the -1 position can be any amino acid), a consensus sequence deduced earlier from analysis of synthetic peptide substrates. In a direct test of the model, we compared Sst2 phosphorylation following systematic substitution of the -1 residue His-538. Each of the substitution mutants was suitable as a MAP kinase substrate, as shown by phosphorylation-dependent mobility shifts in vivo and/or by direct phosphorylation in vitro followed by peptide mapping and mass spectrometry sequencing. This analysis documents phosphorylation of Sst2 by Fus3 and demonstrates that the prevailing model for MAP kinase recognition is valid for a native substrate protein in vivo as well as for small synthetic peptides tested in vitro.     

Hormonal induction of differentiation in teratocarcinoma stem cells: generation of parietal endoderm by retinoic acid and dibutyryl cAMP

It has previously been shown that retinoic acid induces multiple phenotypic changes in cultures of F9 teratocarcinoma stem cells. In this paper we demonstrate that these retinoid-generated cells can be converted to yet another cell type by compounds that elevate cAMP concentrations. The phenotype of the new cell type is characterized by the synthesis of plasminogen activator, laminin and type IV collagen, and by very low levels of alkaline phosphatase and lactate dehydrogenase. The secretion of plasminogen activator and type IV collagen, and low levels of alkaline phosphatase and lactate dehydrogenase, have been previously shown to be properties of parietal endoderm, an extraembryonic cell which is generated early in mouse embryonesis. We show here that parietal endoderm also synthesizes laminin. The cell type generated by retinoic acid and dibutyryl cAMP treatment is therefore indistinguishable from definitive parietal endoderm. Analysis of the final phenotype indicates that it is not dependent upon the continued presence of either compound, and that cAMP agents are active only on cells that have been treated with retinoic acid.