Larry E. Gentry

Differential stereospecificities and affinities of folate receptor isoforms for folate compounds and antifolates

Two membrane folate receptor (MFR) isoforms are present in human tissues i.e. MFR-1 (e.g. placenta) and MFR-2 (e.g. placenta, KB cells, CaCo-2 cells). MFR-1 was expressed in COS-1 cells and the resulting protein had the same polypeptide molecular weight as the native protein. The affinities of (6S) and (6R) diastereoisomers of N5-methyltetrahydrofolate, N5-formyltetrahydrofolate, and 5,10-dideazatetrahydrofolate as well as folic acid and methotrexate to MFR-1, MFR-2 and placental MFR (MFR-1 plus MFR-2) were determined in terms of the Ki values for their competitive inhibition of the binding of [3H]folic acid to these proteins. The results indicated a striking difference in the stereospecificity of MFR-1 and MFR-2 for reduced folate coenzymes; MFR-2 preferentially bound to the physiological (6S) diastereoisomers and MFR-1 bound preferentially to the unphysiological (6R) diastereoisomers, while dideazatetrahydrofolate did not show significant stereospecificity for MFR-1. Furthermore, MFR-2 displayed significantly (2- to 100-fold) greater affinities for all the compounds tested compared to MFR-1. Purified placental MFR, a natural source of MFR-1 which contains variable amounts of MFR-2, showed intermediate Ki values for the compounds tested compared with MFR-1 and MFR-2 and stereospecificities similar to MFR-1. These observations demonstrate striking differences in the ligand binding sites of MFR-1 and MFR-2 which could potentially be exploited in the design of MFR isoform specific antifolates.

Localization of Cells Containing Transforming Growth Factor- α Precursor Immunoreactivity in the Basal Ganglia of the Adult Rat Brain

Transforming growth factor-alpha-like immunoreactivity (TGF-alpha-ir) was visualized in the adult rat forebrain using three antisera directed against carboxyterminal sequences in the TGF-alpha precursor. Using immunoperoxidase and immunofluorescence techniques with all three antisera, TGF-alpha-ir was found to be present in a subpopulation of astrocytes in the forebrain. Striatal and pallidal regions of the basal ganglia were studied in detail. In the striatum, there was an uneven distribution of astrocytes containing TGF-alpha-ir, with the greatest number in the dorsal medial third of the caudate-putamen and the overlying corpus callosum/external capsule. In addition, the region of the caudate-putamen bordering the globus pallidus contained numerous clusters of TGF-alpha-ir astrocytes. The globus pallidus itself contained numerous and more evenly distributed TGF-alpha-ir astrocytes. Other pallidal structures--including the ventral pallidum, entopeduncular nucleus, and substantia nigra pars reticulata--contained moderate numbers of TGF-alpha-ir astrocytes. These results suggest that TGF-alpha precursor is present and, perhaps, synthesized in astrocytes. A related growth factor, epidermal growth factor (EGF), has also been reported to be present in pallidal regions of rat brain. Therefore, the TGF-alpha/EGF family of trophic factors may play a role in the function of the central nervous system.

Bone-derived and recombinant transforming growth factor β′S are potent inhibitors of tumor cell growth

Two naturally occurring chrondogenesis inducing peptides have been purified to homogeneity from demineralized bovine bone. Cartilage-inducing factors A and B are the bone-derived equivalents of transforming growth factor-beta types I and II. Both peptides exhibit identical biological activities in chondrogenesis assays and stimulate anchorage independent cell growth. In this study we show that both bone-derived factors are potent (ng/ml) inhibitors of both DNA synthesis and the anchorage independent growth of a variety of human and non-human tumor cells. Unique in this study is also a comparison of the activities of these polypeptide growth factors with recombinant transforming growth factor type I expressed in mammalian cells.

Structural requirements of diacylglycerols for binding and activating phospholipid-dependent, Ca++-sensitive protein kinase

Phospholipid-dependent, Ca++-sensitive protein kinase (protein kinase C) is activated by phorbol esters and diacylglycerols. A series of diacylglycerols was synthesized with different substituents at positions 1 and 2 in order to expand known structure-activity relationships for these compounds with respect to binding and activating purified protein kinase C. Compounds were synthesized with saturated and unsaturated long chain fatty acyl groups at position 1 and acetyl, butyryl, or hexanoyl groups at position 2. Binding to protein kinase C correlated well with in-vitro activation of the enzyme. These diacylglycerols activated protein kinase C in an intact cellular system causing the phosphorylation of pp60c-src. This indicates that the length of the fatty acyl group at C2 is critical and that the existence of unsaturation in the fatty acyl group at C1 is not essential.