Modulation by pyrophosphoric acid of DNA binding activity of the transcription factor max in murine brain

S87 711

MODULATION BY PYROPHOSPHORIC ACID OF DNA BINDING ACTIVITY OF THE TRANSCRIPTION FACTOR MAX IN MURINE BRAIN. KIYOKAZU OGITA AND YUKIO YONEDA,

Department

of Pharmacology,

Setsunan University,

Hirakata, Osaka 573-01,

Japan.

The addition of KC1 markedly potentiated binding of radiolabeled double stranded oligonucleotide probe for the transcription factor Myc in nuclear extracts of murine brain, while further inclusion of MgCl, or CaCl, diminished the potentiation by KC1 at a concentration range above 2 mM. Unlabeled probe for Myc with double stranded oligonucleotides was effective in competing for the Myc binding irrespective of the addition of MgCl,, whereas that with single stranded DNAs was ineffective. Moreover, the Myc binding was not significantly competed with double stranded probes for activator protein-l and cyclic AMP response element binding protein. Sodium pyrophosphate selectively potentiated the Myc binding only in the presence of MgCl, with concomitant inhibition of that in the absence of MgCl,. However, the Myc binding was not affected by other inhibitors of a variety of protein phosphatases. Proteolysis by S. aureus Vg protease was effective in differentiating the Myc binding in the presence of MgCl, from that in the absence of MgCl,. The addition of an antibody against Max protein induced a marked super-shift of mobility of the bound Myc probe on the gel independent of the addition of MgCl, and sodium pyrophosphate. In contrast, an antibody against c-Myc protein was inactive under any conditions. These results suggest that pyrophosphoric acid may have an ability to distinguish diverse protein dimers consisting of Max protein with affinity for the double stranded oligonucleotide probe for Myc in murine brain.

712

RESPONSIVENESS TO NMDA SIGNALS OF THE TRANSCRIPTION FAmOR APl IN MURINE HIPPOCAMPUS. YASUTAKA AZUMA, KIYOKAZU OGITA AND YUKIO YONEDA, Department

of Pharmacology,

Setsunan University,

Hirakata, Osaka 573-01,

Japan.

Nuclear extracts of murine brain contained binding of double stranded oligonucleotide probes specific for transcription factors with leucine-zipper motifs, including activator protein-l (APl), cyclic AMP response element binding protein (CREB) and Myc, as revealed by analyses using a gel retardation electrophoresis technique. An intraperitoneal injection of N-methyl-D-aspartic acid (NMDA) markedly potentiated the APl binding in a dose-dependent manner at a subconvulsive dose range of 25 to 100 mg/kg in murine hippocampus 2 h after the administration. This potentiation occurred in a fashion sensitive to prevention by in vivo administration of the gene translation inhibitor cycloheximide. Moreover, in vitro addition of antibodies against c-Jun and c-Fos proteins was effective in inducing a marked super-shift of mobility on the gel of the bound APl probe in hippocampal nuclear extracts obtained from animals injected with NMDA. However, the administration of NMDA induced less than 2-fold potentiation of the APl binding in the cerebral cortex, striatum, hypothalamus and medulla-pons, without significantly affecting that in the midbrain and cerebellum. In addition, NMDA was ineffective in markedly altering binding activities of probes for both CREB and Myc in all discrete brain regions examined. These results suggest that the nuclear transcription factor APl may be rapidly and selectively expressed in response to NMDA signals at least in part through de novo synthesis of c-Fos protein in murine hippocampus.

713

GENE TRANSFER INTO NEURONS BY VIRAL VECTORS.S_DMIO TERADA"' YUMI RANRGAE',IZUMU _-_-__ SAITOAXAO -___z.___ NAKATA' and Cell Biology,Facultyf .___.._.~~~ .AW!.._EXG!TARA _aIRQKAW&" ,'Department of Anatomy &dic&ne ,Un&~$_~s&fy_ of Tokyo,.Rongo,Bunkyo-ku ,To&yo, 113,Jsan,' IQsti>_ute for Brai_n__R_~_~e~~~h,Facu~_~~gf Medicine,DniversLty_of TokVo,Bongo,_B_u~n_~kyo-ku,Tokyo,113,Japan,f;nstitute of Medical Science,Dniversity of~T~~Shiroqaneda~,_~~~ato_ku,Tokyo.lI)S,J~pan. Viral vectors are promising methods for gene transfer because of their high efficiency, least cytotoxity and wide range of application from in vitro to in vivo. We compared two viral vector systems which are supposed to be least toxic---(A)defective herpes simplex virus type 1 vector (amplicon type) and (B)recombinant adenovirus vector---regarding their possible application for direct gene delivery into neurons. We have used prototypic vectors, each containing the E.coli b-galactosidase gene, to infect mouse embryonal hippocampal neurons in primary culture. We show in both systems, nearly all neuronal cells express b-galactosidase strongly after 5-6 hours post-infection;in system(A), there is apparent cytotoxic effect afer 3 days post-infection, while in systm(B),cultured neurons continued normal morphological development for at least 10 days without cell damage. These results demonstrate the potential usefulness of both systems for high-efficient and transient gene expression in neural cells, and that the system(A), which is rather easy to construct, is preferrable for short-term expression of multiple samples, while system(B) is appropriate for long-term (more than one week) experiments.