The isolation, partial purification and preliminary characterization of a growth factor from chick embryo brains

Chicl( brain growth factor (CBGF) is a mitogen isolated from embryonic chick brains thought to have a potential role as a trophic factor involved in nerve dependent amphibian limb regeneration. In addition, CBGF stimulates 3H-thymidine incorporation in chick embryo brain astrocytes in vitro. In this study, cultured chick embryo brain non-neuronal cells were employed in a bioassay to monitor CBGF activity throughout various stages of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal cells grew best on collagen-coated culture dishes in complete medium, were most responsive to a growth stimulus [10% fetal bovine serum (FBS)] at the second and third subcultures, and were healthiest when rendered "quiescent" in medium supplemented with 1% FBS. The most effective bioassay conditions consisted of a minimum 14.5 hour "quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary non-neuronal cells consisted of 6.63% GFAP positive cells; as a result cultures were thought to be mainly composed of astroblasts. CBGF was purified from 18-day chick embryo brains by ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion chromatography through a Biogel P6 column, and analytical reverse-phase high-performance liquid chromatography (rp-HPLC). The greatest activity resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10% FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal cells. Although other researchers report the isolation of a mitogenic fraction consisting of 5'-GMP from the embryonic chick brain, UV absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment (FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP. 2 Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine incorporation in the chick non-neuronal cells, while Sf-AMP had no effect. Upon treatment with pronase, the biological activity of fraction P6-3 increased; this increase was nearly 30% greater than what would be expected from a simple additive effect of any mitogenic activity of pronase alone together with P6-3 alone. This may suggest the presence of an inhibitor protein. The bioactive component may be a protein protected by a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone. While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal molecular weight or sequence information, the ion of highest molecular weight was observed at m/z 1610; this is consistent with previous estimations of CBGF's size. 3

Zero-sum problems in finite cyclic groups

The purpose of this thesis is to investigate some open problems in the area of combinatorial number theory referred to as zero-sum theory. A zero-sequence in a finite cyclic group G is said to have the basic property if it is equivalent under group automorphism to one which has sum precisely IGI when this sum is viewed as an integer. This thesis investigates two major problems, the first of which is referred to as the basic pair problem. This problem seeks to determine conditions for which every zero-sequence of a given length in a finite abelian group has the basic property. We resolve an open problem regarding basic pairs in cyclic groups by demonstrating that every sequence of length four in Zp has the basic property, and we conjecture on the complete solution of this problem. The second problem is a 1988 conjecture of Kleitman and Lemke, part of which claims that every sequence of length n in Zn has a subsequence with the basic property. If one considers the special case where n is an odd integer we believe this conjecture to hold true. We verify this is the case for all prime integers less than 40, and all odd integers less than 26. In addition, we resolve the Kleitman-Lemke conjecture for general n in the negative. That is, we demonstrate a sequence in any finite abelian group isomorphic to Z2p (for p ~ 11 a prime) containing no subsequence with the basic property. These results, as well as the results found along the way, contribute to many other problems in zero-sum theory.