Induction of DNA bending by bifunctional intercalating agents of the 7H-pyridocarbazole family

The structures and binding energetics of selected complexes formed between the deoxynucleotides d(CpGpGpCpG).d(CpGpCpCpG), d(CpGpApTpCpG)2, d(GpCpGpCpCpG).d(CpGpGpCpGpC), and d(CpGpCpCpCpG)2 with the DNA bifunctional intercalating agent ditercalinium and three of its rigid linking chain derivatives have been investigated theoretically by means of a molecular mechanics approach that takes into account nucleic acid flexibility, ligand flexibility and solvent dielectric effects (R. Lavery, in: Unusual DNA structures, eds S. Harvey and R. Wells (Pergamon, New York, 1988) p. 189; R. Lavery, in: DNA bending and curvature, eds W.K. Olson et al. (Adenine Press, New York, 1988) p. 191). The piperidinium chains of the bis-intercalating ligands are always located in the major groove of DNA. For the energy-minimized complexes the ligand proceeds to bind following preferentially the 5'-pyrimidine-purine-3' alternating sequence, thus dictating the number of internal exclusion sites. The complexes with three exclusion sites will present (i) a bending of the structure towards the major groove, and (ii) a non-ideal distribution of unwinding angles; complexes with less than three exclusion sites will remain essentially linear. The absence of a bend does not preclude other types of local deformations of the base-pairs such as inclination, buckle and tip. The proposed structures of the d(CpGpApTpCpG)2 complexes are in agreement with NMR structural results. The possible relevance of these findings to a previously proposed mode of interaction for ditercalinium-like DNA ligands is discussed.     

In Situ Analysis of Na,K-ATPase α1- and α3-Isoform mRNAs in Aging Rat Hippocampus

Abstract: Age-related changes in the expression of Na,K-ATPase α1- and α3-isoform mRNAs were analyzed by in situ hybridization in the Fischer-344 rat hippocampus. Quantification of signal density with cRNA probes in rat hippocampus at 3 months of age showed (a) α1 content is 1.5 times higher in granule than in pyramidal cell layers, whereas α3 content shows the opposite ratio and (b) α3 label is found in large clusters related to mossy cells and basket cells and in medium clusters corresponding to interneurons within the dendritic fields of CA1–3. In the 24-month-old rats as compared with the young animals, the α1 signal is increased more than sevenfold in the dendritic fields and is not significantly changed in perikaryal layers. The α3 signal is reduced about threefold ( p < 0.0001, ANOVA, n = 6) in perikaryal layers, is almost completely absent over the interneurons, basket cells, and mossy cells, and is not significantly changed in dendritic fields. These data indicate age-related, cell- and isoform-specific alterations in pretranslational regulation of Na,K-ATPase α isoforms. The striking changes in the dendritic fields, mossy cells, and GABAergic basket cells and interneurons may constitute early and sensitive markers for age-related alterations in hippocampal function, before cell loss.     

Expression of human prostate-specific antigen (PSA) in a mouse tumor cell line reduces tumorigenicity and elicits PSA-specific cytotoxic T lymphocytes

 Human prostate-specific antigen (PSA) has a highly restricted tissue distribution. Its expression is essentially limited to the epithelial cells of the prostate gland. Moreover, it continues to be synthesized by prostate carcinoma cells. This makes PSA an attractive candidate for use as a target antigen in the immunotherapy of prostate cancer. As a first step in characterizing the specific immune response to PSA and its potential use as a tumor-rejection antigen, we have incorporated PSA into a well-established mouse tumor model. Line 1, a mouse lung carcinoma, and P815, a mouse mastocytoma, have been transfected with the cDNA for human PSA. Immunization with a PSA-expressing tumor cell line demonstrated a memory response to PSA which protected against subsequent challenge with PSA-expressing, but not wild-type, tumors. Tumor-infiltrating lymphocytes could be isolated from PSA-expressing tumors grown in naive hosts and were specifically cytotoxic against a syngeneic cell line that expressed PSA. Immunization with tumor cells resulted in the generation of primary and memory cytotoxic T lymphocytes (CTL) specific for PSA. The isolation of PSA-specific CTL clones from immunized animals further demonstrated that PSA can serve as a target antigen for antitumor CTL. The immunogenicity studies carried out in this mouse tumor model provide a rationale for the design of methods to elicit PSA-specific cell-mediated immunity in humans. Received: 4 April 1996 / Accepted: 31 May 1996