Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS-7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50-fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires'both 70 and 73 kDa subunits.

Glioblastoma multiforme (GBM) is an incurable brain tumor resistant to standard treatment modalities such as surgery, radiation, and chemotherapy. Since recurrent GBM tends to develop predominantly within the infiltrative rim surrounding the primary tumor focus, novel therapy strategies need in addition to focal tumor destruction to target this somewhat diffuse area. This is a phase I/II clinical study in adult patients with recurrent GBM which is aimed at evaluating biological safety, maximum tolerated dose, and antitumor efficacy of a genetically modified replication-disabled Semliki forest virus vector (SFV) carrying the human interleukin 12 (IL-12) gene and encapsulated in cationic liposomes (LSFV-IL12). The vector will be administered in doses of 1 x 10(7)-1 x 10(9) infectious particles by continuous intratumoral infusion, thus exploiting the advantages of convection-enhanced drug delivery in the brain. The present protocol is also designed to investigate systemic and local immune response and to identify factors predicting tumor response to LSFV-IL12 therapy, such as volume of extracellular space of the tumor, volume of contrast enhancing lesion, and immune status of the patients. SFV, an insect alphavirus, infects mitotic and non-mitotic cells and triggers apoptosis in tumor cells within 48-72 h. Preclinical work with the LSFV-IL12 vector in breast and prostate cancer animal models demonstrated its biosafety and some antitumor efficacy. An ongoing phase I clinical study in patients with melanoma and renal cell carcinoma seems also to confirm the biosafety of intravenously administered vectors. This protocol will be the first study of SFV-IL12 therapy of human recurrent GBM.

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Bioluminescence imaging has evolved as a powerful tool for monitoring biological processes in vivo. As transmission efficiency of light through tissue increases greatly for wavelengths above 600 nm we examined whether a redshifted codon-optimized firefly luciferase (lambdamax=615 nm) could be successfully employed as a sensitive reporter in mammalian cells. To this end, unmodified codon-optimized luciferase (lambdamax=557 nm) as well as the red-emitting S284T mutant luciferase were expressed simultaneously in human glioma cells in vitro as well as in quadriceps muscles of mice in vivo. We show here that activity of the redshifted enzyme in human glioma cell culture approached approximately one-fourth of that seen with the unmodified enzyme. In contrast, light emission by the red-emitting luciferase in vivo was generally more efficient than that produced by its unmodified counterpart, most likely due to reduced absorption of red light by tissue. The mean ratio of light emission produced by the redshifted luciferase to that of the unmodified enzyme in vivo was approximately 3. Application of this new redshifted luciferase together with other optical reporters may be of considerable importance to biological research as it allows for imaging of deeper tissues as well as simultaneous monitoring of two molecular events in vitro and in vivo if appropriate filter sets are employed.

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