Characterization of Mg2+ binding to the DNA repair protein apurinic/apyrimidic endonuclease 1 via solid-state 25Mg NMR spectroscopy.

Apurinic/apyrimidinic endonuclease 1 (APE1), a member of the divalent cation-dependent phosphoesterase superfamily of proteins that retain the conserved four-layered alpha/beta-sandwich structural core, is an essential protein that functions as part of base excision repair to remove mutagenic and cytotoxic abasic sites from DNA. Using low-temperature solid-state (25)Mg NMR spectroscopy and various mutants of APE1, we demonstrate that Mg(2+) binds to APE1 and a functional APE1-substrate DNA complex with an overall stoichiometry of one Mg(2+) per mole of APE1 as predicted by the X-ray work of Tainer and co-workers (Mol, C. D.; Kuo, C. F.; Thayer, M. M.; Cunningham, R. P.; Tainer, J. A. Nature 1995, 374 , 381-386). However, the NMR spectra show that the single Mg(2+) site is disordered. We discuss the probable reasons for the disorder at the Mg(2+) binding site. The most likely source of this disorder is arrangement of the protein-ligands about the Mg(2+) (cis and trans isomers). The existence of these isomers reinforces the notion of the plasticity of the metal binding site within APE1.

Base excision repair and the central nervous system.

Reactive oxygen species generated during normal cellular metabolism react with lipids, proteins, and nucleic acid. Evidence indicates that the accumulation of oxidative damage results in cellular dysfunction or deterioration. In particular, oxidative DNA damage can induce mutagenic replicative outcomes, leading to altered cellular function and/or cellular transformation. Additionally, oxidative DNA modifications can block essential biological processes, namely replication and transcription, triggering cell death responses. The major pathway responsible for removing oxidative DNA damage and restoring the integrity of the genome is base excision repair (BER). We highlight herein what is known about BER protein function(s) in the CNS, which in cooperation with the peripheral nervous system operates to control physical responses, motor coordination, and brain operation. Moreover, we describe evidence indicating that defective BER processing can promote post-mitotic (i.e. non-dividing) neuronal cell death and neurodegenerative disease. The focus of the review is on the core mammalian BER participants, i.e. the DNA glycosylases, AP endonuclease 1, DNA polymerase beta, X-ray cross-complementing 1, and the DNA ligases.

Passive immunization of cyclic mares against androgen: gonadotropin and progesterone concentrations and estrous characteristics.

Antiserum generated in a horse against testosterone conjugated to bovine serum albumin (BSA) was administered to six lighthorse mares (androgen-immunized mares) 1 to 3 d before a prostaglandin-induced estrus and twice again at 2-d intervals. Six control mares were administered antiserum generated against BSA on the same schedule. Relative to testosterone, cross-reactivities of other steroids with the testosterone antiserum were (%): dihydrotestosterone, 52; 5 alpha-androstane-3 alpha,17 beta-diol, 8.6; androst-4-ene-3,17-dione, 1.2; and all others tested less than .1. Tritiated testosterone binding in plasma increased (P less than .01) in androgen-immunized mares within 1 h and remained elevated (P less than .01) relative to controls for greater than 21 d. There was no effect (P greater than .10) of passive immunization against androgen on interval to estrus after prostaglandin injection, duration of estrus, ovarian volume, number of palpable follicles or follicular volume during estrus. In contrast, concentrations of luteinizing hormone (LH) were higher (P less than .05) in androgen-immunized mares than in control mares during estrus and early diestrus. Concentrations of follicle stimulating hormone (FSH) and progesterone at those times were not affected (P greater than .10). From these data, we conclude that androgens in the mare during estrus may be involved with the regulation of LH secretion. In contrast, no involvement with FSH secretion was apparent under these short-term conditions.

Secretion of luteinizing hormone and follicle stimulating hormone in intact and ovariectomized mares in summer and winter.

Sequential samples of blood were drawn via jugular catheters every 15 min for 24 h from four mares in each of five reproductive states: intact anestrous mares in winter, intact diestrous mares in summer, intact estrous mares in summer, ovariectomized mares in winter and ovariectomized mares in summer. Estrous mares were sampled on d 4 or 5 of estrus and diestrous mares on d 10 or 11 of diestrus. Each sample of plasma was assessed for concentrations of luteinizing hormone (LH) and follicle stimulating hormone (FSH) in two independent radioimmunoassays. A computer program was developed that determined peak hormone concentrations based on assay sensitivity, assay variability and repeatability of peaks in both independent assays. Peaks in LH and FSH were observed for mares in all five reproductive states, except for FSH concentrations in estrous mares. High frequency peaks of short duration were observed only in ovariectomized mares. Low frequency peaks of relatively long duration were observed in both intact and ovariectomized mares in both seasons. With the exception of estrous mares, there was variation among mares in the patterns of LH and(or) FSH within any one group; all estrous mares exhibited high, variable LH concentrations and low, constant FSH concentrations. In general, peaks in both gonadotropins occurred simultaneously. Ovariectomized mares exhibited more (P less than .05) peaks/24 h than intact mares for both gonadotropins. Ovariectomized mares also exhibited more (P less than .05) FSH peaks/24 h in summer than in winter.

Active immunization of intact mares against gonadotropin-releasing hormone: differential effects on secretion of luteinizing hormone and follicle-stimulating hormone.

Five lighthorse mares were actively immunized against gonadotropin releasing hormone (GnRH) to determine the relative importance of this hypothalamic hormone in the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Five mares immunized against the conjugation protein served as controls. Mares were initially immunized in November and received secondary immunizations 4 wk later, and then at 6-wk intervals until ovariectomy in June. All mares immunized against GnRH exhibited an increase (p less than 0.01) in the binding of tritiated GnRH by plasma, an indication that antibodies against this hormone had been elicited. Concentrations of LH, FSH and progesterone in weekly blood samples were lower (p less than 0.05) in GnRH-immunized mares than in controls after approximately 4 mo of immunization. However, the LH concentrations were affected to a greater degree than were FSH concentrations. All five control mares exhibited normal cycles of estrus and diestrus in spring, whereas no GnRH-immunized mare exhibited cyclic displays of estrus up to ovariectomy. All mares were injected intravenously with a GnRH analog (which cross-reacted less than 0.1% with the anti-GnRH antibodies) in May, after all control mares had displayed normal estrous cycles, to characterize the response of LH and FSH in these mares; two days later, the mares were injected with GnRH. The LH response to the analog, which was assessed by net area under the curve, was lower (p less than 0.01) by approximately 99% in mares immunized against GnRH than in control mares. In contrast, the FSH response to the analog was similar for both groups.(ABSTRACT TRUNCATED AT 250 WORDS)