In previous work, the direct injection of 50 micrograms of a plasmid DNA vector encoding firefly luciferase (VR1205) into murine quadriceps muscle produced an average of 6.5 ng of luciferase per muscle at 7 days postinjection. In this report, various elements of the VR1205 vector were modified to increase gene expression levels or to eliminate undesired viral sequences. Expression of the modified vectors was then compared to VR1205 using the intramuscular injection assay. In general, modifications to promoter, enhancer, and intronic sequences either decreased luciferase expression levels or had no effect. However, modifications to the polyadenylation and transcriptional termination sequences, plasmid backbone elements, and the luciferase gene itself each increased luciferase expression levels. The best-expressing vector, designated VR1255, contained a combination of these incrementally beneficial changes. A single intramuscular injection of 50 micrograms of VR1255 produced 300 ng of luciferase at 7 days postinjection, an expression level 46-fold higher than the VR1205 vector (or 22-fold higher, excluding modifications to the luciferase gene) and 154-fold higher than a commercially available luciferase expression vector. Thus, VR1255 represents an improved plasmid DNA vector that may be useful for gene therapy applications.

Inorg. Chem. 1987, I 0 -0.4 0.8 V vs. Ag/AgCl Figure 12. Cyclic voltammogram (200 mV/s) for a GClpoly-CobNMez)TPP film (r = 1.4 X mol/cm2) in a CH$N + 0.1 M Et4NCI04 solution (solid curve) and for the same solution after addition of 2 volumes of HzO (dashed curve).potentials for O2 reduction and larger slope. The slope for poly-Co(o-NH2)TPP in 1 M N a O H is in fact very close to the four-electron value; we have confirmed by the rotating ring-disk techniqueI4 that this situation produces virtually no H202 product. 26, 1017-1025 1017Assuming (as we have14) that the two waves for poly-Co(o-NH2)TPP reflect monomeric and dimeric species in the film, then the lower activity of the other cobalt porphyrins may be connected to their lower dimerization constants, as found for Co(p-0H)TPP and Cob-pyr)TPP solutions from absorbance spectroscopy (Table 11) * showed that Co(1) porphyrins placed in solutions and adsorbed or bound to G C electrodes34 react rapidly with water. Figure 12 shows a test for water reduction using an electropolymerized poly-Cob-NMe2)TPP film (GC electrode). Co(II/I) voltammetry of the film in dry 0.1 M Et4NCI04/CH3CN (solid curve) occurs at Eo' = -0.92 V, whereas upon addition of two volumes of water (dashed curve), an irreversible, split (ca. -0.68 and -1.01 V) wave, with greatly enhanced cathodic current and no reverse anodic wave, is observed. While it is reasonable to suppose that these waves represent H2 evolution, they are not very stable (as S p i r~~~ also experienced) and gradually decayed. N o further studies were done. This instability is not characteristic of the polymeric porphyrin films in dry solvent, which in the potential window 0 to -1.5 V are very stable to repeated potential cycling. Acknowledgment.A series of 19 mononuclear oxomolybdenum(V) compounds of the general formula LMoOXY (L = hydrotris(3,5-dimethyl-lpyrazoly1)borate; (X,Y) = C1-, NCS-, NC, OR-, SR-, and the dianions of ethylene glycol, mercaptoethanol, dimercaptoethane, catechol, o-mercaptophenol, o-aminophenol, o-aminobenzenethiol, and toluenedithiol) have been prepared and characterized by electron paramagnetic resonance spectroscopy (EPR), cyclic voltammetry, UV-visible spectroscopy, and mass spectrometry. The LMoO(SR)~ complexes possess the cis sulfur ligands proposed for the molybdenum cofactor. The structure of LMoO(SPh), was determined by X-ray crystallography: space group P2,/c, Z = 4, u = 13.079 (6) A, 6 = 14.438 (7) A, c = 15.836 (9) A, p = 104.73 (4)O. The molecule adopts the expectedfuc six-coordinate stereochemistry with cis thiolate groups. The average Mo-S distance is 2.382 (2) A, and the average Mo-0 distance is 1.676 (4) A. The Mo-N distances range from 2.164 (5) to 2.357 (5) A. The isotropic ( g ) and (A)(95*97Mo) values show an inverse correlation and cluster according to the nature of the donor atoms.Sulfur donor atoms lead to large ( g ) and small ( A ) values. The anisotropies of the g values for LMoO(OR), and LMoO(SR)~ complexes show marked differences between monodentate and chelated ligands. All ...

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The molecular mechanisms for the development of multiple distinct endocrine cell types in the anterior pituitary have been an area of intensive investigation. Though the homeodomain protein Pit-1/GHF-1 is known to be involved in differentiation of the somatotrope and lactotrope lineages, which produce growth hormone and prolactin, respectively, little is known of the transcriptional regulators important for the gonadotrope cell lineage, which produces the glycoprotein hormones luteinizing hormone and follicle-stimulating hormone. Using transgenic mice and transfection into a novel gonadotrope lineage cell line, we have identified a regulatory element that confers gonadotrope-specific expression to the glycoprotein hormone a-subunit gene.

A cancer treatment is described in which i.m. injection of plasmid DNA (pDNA) encoding murine interferon ␣ (mIFN-␣) leads to potent antitumor effects on primary and metastatic tumors in mice. Mice bearing s.c. B16F10 melanoma, Cloudman melanoma, or glioma 261 tumors were injected i.m. with mIFN-␣ pDNA. In all three tumor models, a significant reduction in tumor volume and enhancement of survival was found after IFN pDNA therapy. The mIFN-␣ pDNA could be injected as infrequently as once every other week and still produce a significant antitumor effect, and, in a metastatic tumor model, the therapy markedly reduced the number of lung tumor metastases. Depletion of immune cell subsets indicated that CD8 ؉ T cells were required for the antitumor response. These studies demonstrate that primary and metastatic tumors can be treated systemically by i.m. injection of a plasmid encoding a cytokine gene.