Reducing the heterogeneity of chemically conjugated targeted toxins: homogeneous basic FGF-saporin.

Basic fibroblast growth factor-saporin (FGF-SAP) is an extremely potent cytotoxic agent for cells bearing the FGF receptor. For its synthesis, the two free cysteines on the surface of basic FGF are available for linking by disulfide bridge to SAP which has been derivatized with a reactive sulfhydryl. Because of the heterogeneous nature of the synthesis, the resulting conjugate is heterogeneous as judged by gel electrophoresis.

Differential control of activin, inhibin and follistatin proteins in cultured rat granulosa cells.

Follistatin, activin and inhibin proteins are produced by granulosa cells, but the mechanisms controlling their production remain unclear. Here, we examined how the protein kinase A (PKA) and protein kinase C (PKC) pathways act and interact to regulate production of these proteins. Granulosa cells from immature rats were cultured with activators of the PKA pathway (100 ng/ml FSH, 10 microM forskolin) and/or activators of the PKC pathway (100 nM GnRH agonist, 100nM 2-0-tetradecanoyl-phorbol-13-acetate, TPA).

Control of glycaemia.

Maintenance of plasma glucose concentrations within a narrow range despite wide fluctuations in the demand (e.g. vigorous exercise) and supply (e.

Increased follistatin (activin-binding protein) gene expression in rat anterior pituitary tissue after ovariectomy may be mediated by pituitary activin.

For lack of evidence to the contrary, it is now believed that the FSH-suppressing actions of follistatin are due to its ability to bind endogenous pituitary activin. Recent data have demonstrated a role for pituitary activin-B in mediating FSH hypersecretion after ovariectomy (OVX) and during the secondary FSH surge on estrus. Therefore, given that follistatin is produced within anterior pituitary tissue, and considering the potentially important function of follistatin to modulate activin bioactivity, we sought to gain insights into the regulation of follistatin gene expression in the anterior pituitary gland of adult female rats.

Differential effects of glycosylated and nonglycosylated prolactin on islet cell division and insulin secretion.

A growing body of evidence suggests that prolactin (PRL) is a potent regulator of the structure and function of the islets of Langerhans, but PRL is a polymorphic hormone that exists in several molecular forms. Therefore, it is important to know whether glycosylated PRL, a major structural variant of the hormone in several species, has an effect different from that of the nonglycosylated PRL on islet function. This in vitro study examined the differential effects of glycosylated and nonglycosylated porcine PRL on cell division and insulin secretion from neonatal rat islets, and compared these results with those produced by homologous rat PRL.

Expression of basic fibroblast growth factor and its receptor in an invasive bladder carcinoma cell line.

Basic fibroblast growth factor (bFGF) is a multifunctional growth factor that can stimulate cell proliferation, production of proteases, and angiogenesis. Loss of mechanisms that regulate bFGF activity could result in tumor development. To test this idea, cells derived from an invasive bladder carcinoma (EJ) were compared with cells derived from a noninvasive bladder carcinoma (RT4) for the expression of bFGF and high and low affinity FGF receptors.

Effects of estrous cycle stage and pregnancy on follistatin gene expression and immunoreactivity in rat reproductive tissues: progesterone is implicated in regulating uterine gene expression.

Follistatin, a monomeric protein originally isolated from ovarian follicular fluid, is now believed to be a major local regulator of the multifaceted actions of activin by virtue of its activin-binding properties. In view of the ability of follistatin to stimulate progesterone production from granulosa cells and its presence in newly formed corpora lutea, the following study was conducted to determine the effects of cycle stage and pregnancy on follistatin gene expression and immunoreactivity in the rat ovary and uterus with the intent of gaining additional insights into the regulation of follistatin in these tissues. Decidua and placentas were also examined on days 15, 18, and 21 of pregnancy.

Structural characterization of the rat insulin-like growth factor binding protein-6 gene.

Recently a family of six distinct insulin-like growth factor binding proteins (IGFBPs) have been identified and the gene structures of the first five (IGFBP-1, -2, -3, -4 and -5) characterized. We now isolated the IGFBP-6 gene from a rat genomic library and determined its organization as well as the DNA sequence at the 5' flanking region of the gene. The rat IGFBP-6 gene spans 5.

The expression of saporin, a ribosome-inactivating protein from the plant Saponaria officinalis, in Escherichia coli.

We have isolated and sequenced genomic clones from the DNA of Saponaria officinalis using a cDNA probe that encodes proteins with high homology to saporin-6, one of the most potent of the ribosome-inactivating proteins that is currently used for the construction of immunotoxins and mitotoxins. Sequence differences in the clones suggest a multigene family of proteins. These data agree with observations of several different proteins with ribosome-inactivating protein activity and similar structure.

Structural and functional characterization of the rat follistatin (activin-binding protein) gene promoter.

Follistatin was originally identified as a specific inhibitor of follicle stimulating hormone secretion and later characterized as a binding protein for activin. Since activin regulates hormone secretion and cell differentiation, the importance of understanding the mechanisms regulating the synthesis of its binding protein, follistatin, is evident. To study the regulation of follistatin gene expression, we first determined the transcription start site (cap site) of the rat follistatin gene using primer extension and ribonuclease protection assay.

Development of specific antibodies to rat insulin-like growth factor-binding proteins (IGFBP-2 to -6): analysis of IGFBP production by rat granulosa cells.

Six insulin-like growth factor-binding proteins (IGFBPs) have been isolated and their cDNAs cloned in the rat and human species. The next step is to develop antibodies to each IGFBP. Toward this goal, we generated rabbit polyclonal antibodies to rat IGFBP-2, -4, -5, and -6, using synthetic peptide fragments of the IGFBPs.

Inhibition of the tyrosine kinase activity of the fibroblast growth factor receptor by the methyltransferase inhibitor 5'-methylthioadenosine.

Stimulation of fibroblasts with basic fibroblast growth factor (bFGF) led to the rapid tyrosine phosphorylation of a number of cellular proteins, including a major substrate of 90 kDa. The methyltransferase inhibitor 5'-methylthioadenosine (MTA) was found to be a specific inhibitor of bFGF-stimulated protein tyrosine phosphorylation in fibroblasts, blocking both receptor autophosphorylation and substrate phosphorylation. MTA had no effect on either epidermal growth factor- or platelet-derived growth factor-stimulated protein tyrosine phosphorylation in fibroblasts.

Structure of the rat insulin-like growth factor binding protein-4 gene.

A family of six distinct insulin-like growth factor binding proteins (IGFBPs) have been isolated and their cDNA sequences characterized from rat and human species. Recently, the gene structures of the first three IGFBPs (IGFBP-1, -2 and -3) have also been determined. We now report the isolation of the rat IGFBP-4 gene and its genomic organization, as well as the DNA sequence of the promoter region.

Cloning of the rat insulin- like growth factor binding protein-5 gene and DNA sequence analysis of its promoter region.

To understand the regulation of the insulin-like growth factor binding protein-5 (IGFBP-5) gene expression, we have cloned the IGFBP-5 gene from rat genomic libraries and determined its genomic organization as well as the DNA sequence at the 5' flanking region of the gene. The rat IGFBP-5 gene spans at least 17 kilobases (kb) of the genome and contains 4 exons interrupted by 3 introns of approximately 10, 0.6 and 0.

Modulation of the epidermal growth factor receptor by basic fibroblast growth factor.

Treatment of Swiss 3T3 fibroblasts with basic fibroblast growth factor (bFGF) lead to a rapid reduction in epidermal growth factor (EGF) binding and a slower inhibition of EGF receptor autophosphorylation. The reduction in binding was due to a complete loss of the highest affinity EGF binding sites and a reduction in the lower affinity binding sites. Neither the inhibition of EGF binding nor the inhibition of EGF receptor autophosphorylation required protein kinase C.

Different members of the fibroblast growth factor receptor family are specific to distinct cell types in the developing chicken embryo.

Single-stranded RNA probes for the three chicken fibroblast growth factor (FGF) receptors, cek-1, cek-2, and cek-3, in conjunction with in situ hybridization were used to characterize the distribution of the corresponding mRNAs in the developing chicken embryo. Cek-1 was expressed diffusely in most tissues examined, whereas the expression of cek-2 and cek-3 was more restricted. The highest levels of FGF receptor expression were seen in the developing bones; in skeletal, cardiac, and smooth muscle; and in some areas of the brain.

Characterization of a saporin mitotoxin specifically cytotoxic to cells bearing the granulocyte-macrophage colony-stimulating factor receptor.

When granulocyte-macrophage colony-stimulating factor (GM-CSF) is chemically conjugated to the ribosome-inactivating protein saporin, the resulting protein conjugate is highly toxic for cells expressing the GM-CSF receptor. Structural and Western blot analyses of the purified conjugate establish that it contains equimolar amounts of the starting materials and is free of any contamination by the non-conjugated components. The resulting bifunctional reagent is specifically cytotoxic to cells expressing the GM-CSF receptor, but is ineffective to cells that do not express the receptor.

Phosphotyrosine-containing proteins are concentrated in differentiating cells during chicken embryonic development.

Protein tyrosine phosphorylation may be an important indicator of both the proliferative status and differentiation status of cells during embryonic development. To determine how each of these factors contributes to the level of phosphotyrosine-containing proteins detectable in embryonic tissues we have used immunohistochemistry with anti-phosphotyrosine antibodies on sections of developing chicken embryos. In contrast to an earlier study (Takata and Singer, 1988) we found proteins phosphorylated on tyrosine residues to be present in many different cells of the developing chicken embryo.

Localization of the heparin binding site of follistatin.

To define the heparin-binding site of follistatin, the reduced and S-carboxymethylated recombinant human follistatin containing 288 amino acids was digested by Staphylococcus aureus V8. The digested product was subjected to sulfate cellufine column chromatography and the adsorbed peptide fragments eluted with a stepwise gradient of sodium chloride. The recovered column fractions were further purified by reversed-phase high-performance liquid chromatography (HPLC) and the HPLC peaks subjected to amino-terminal sequence analysis.