Effects of IGF-I gene therapy on the injured rat pudendal nerve.

Injured nerves and their motor units may undergo enhanced recovery when exposed to recombinant human insulin-like growth factor-I (rhIGF-I). The external anal sphincter muscle in the female rat was denervated to model incontinence. The treatment-group muscle was injected with rhIGF-1 plasmid, whereas in the control group the plasmid lacked the cDNA insert and the normal group received neither surgery nor treatment. Electromyography data at 56 days post surgery indicated more reinnervation without fibrillation potentials in the treatment group (2 of 6) than in the control group (0 of 6). The histology of the regenerated axons in the pudendal nerve distal to the crush site also suggested an improved recovery in the treatment group. The number of motor neurons retrogradely labeled with horseradish peroxidase was decreased by 50% following pudendal nerve crush in both experimental groups compared to the normal group. We conclude from these preliminary results that rhIGF-I gene therapy may improve the distal recovery of structure and function.

Assay of UDP, GDP, CDP, and ADP reductase activities by column chromatography on polyethyleneimine cellulose.

Deoxyribonucleosides were separated from ribonucleosides by chromatography on polyethyleneimine cellulose columns (Pasteur pipettes. The deoxyribonucleosides were quantitatively eluted with 25 mM boric acid in less than 10 ml while the ribonucleosides were retained. The ribonucleosides were eluted with 1 M NaCl. This method was utilized to assay for GDP, UDP, ADP, and CDP reductase activities after hydrolysis of the substrate and product nucleotides to the corresponding nucleosides. All four reductase activities were assayed using identical conditions of column size, eluting solution (25 mM boric acid), and elution volume. The use of polyethyleneimine cellulose columns with boric acid can be adapted to other enzyme assays such as purine nucleoside phosphorylase and for the isolation of deoxyribonucleotides from cellular extracts.

Noncoordinate changes in the components of ribonucleotide reductase in mammalian cells.

Ribonucleotide reductase from mammalian cells consists of two nonidentical components which are both required for enzymatic activity. It was found that the addition of the effector-binding component (dye fraction) to cell-free extracts of Ehrlich tumor cells stimulated CDP reductase activity. The decrease in CDP reductase activity which accompanied the decrease in Ehrlich tumor cell proliferation in vivo could be correlated with the decrease in the dye fraction component. In regenerating liver, the reductase activity was increased maximally at 36 h following partial hepatectomy. This activity could be further stimulated by exogenous tumor cell dye fraction. The non-heme iron component (Tris fraction) was isolated and quantitated from the liver extracts of regenerating livers. The maximal increase on the Tris fraction component was observed in the 24-h regenerating liver. These data provide evidence that the components making up the active ribonucleotide reductase species are not coordinately increased at the time of the increase in reductase activity.

Mode of inhibition of tumor cell ribonucleotide reductase by 2,3-dihydro-1H-pyrazolo[2,3-a]imidazole (NSC 51143).

2,3-Dihydro-1H-pyrazolo[2,3-a]imidazole (NSC 51143; IMPY) inhibits partially purified ribonucleotide reductase from Ehrlich tumor cells. Both cytidine 5'-diphosphate and adenosine 5'-diphosphate reductase activities were inhibited by IMPY, although adenosine 5'-diphosphate reductase activity was inhibited to a greater extent than was cytidine 5'-diphosphate reductase activity at all concentrations of IMPY studied. The inhibition of the intact enzyme by IMPY could be reversed by the addition of the exogenous non-heme iron-containing subunit (tris(hydroxymethyl)aminomethane fraction) but not by the addition of the effector-binding subunit. Further, the inhibition of the intact enzyme or the tris(hydroxymethyl)aminomethane fraction by IMPY could be reversed by the addition of 6 microM Fe(NH4)2(SO4)2, and the inhibition of IMPY could be potentiated by 0.167 mM ethylenediaminetetraacetic acid. These results demonstrate that IMPY inhibits tumor cell nucleotide reductase by interaction with the iron of the non-heme iron-containing subunit.

Specific inhibitors directed at the individual components of ribonucleotide reductase as an approach to combination chemotherapy.

It had been shown previously that the ribonucleotide reductase from mouse tumor consisted of two nonidentical components (Tris and dye fractions, each prepared from the 20 to 40% (NH/)2SO4 protein fraction containing the ribonucleotide reductase activity by blue dextran-Sepharose chromatography). The individual components either separated or present in the intact enzyme can be specifically and independently inhibited by different compounds. The Tris fraction component was inhibited by 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone while the dye fraction component was inactivated by pyridoxal phosphate:BH4- and the dialdehyde derivative of inosine (Inox) and 5'-deoxyinosine prepared by the periodate oxidation of inosine and 5'-deoxyinosine. The intact enzyme could be completely inhibited by any of these compounds. Reductase activity was restored by reconstitution with the exogenous components. The individual components of the reductase in the intact Ehrlich tumor cell could also be specifically inhibited. Activity in the crude cell-free extracts prepared from 4-methyl-5-amino-1-formylisoquinoline thiosemicarbazone- or Inox-treated cells was restored by the addition of the appropriate exogenous component. These data suggest that combinations of inhibitors of ribonucleotide reductase which specifically inhibit the components may be useful in the treatment of cancer.

Reconstitution of the ribonucleotide reductase enzyme from Ehrlich tumor cells.

Ribonucleotide reductase from Ehrlich tumor cells was separated by chromatography on blue dextran/Sepharose into two protein fractions (Tris and Dye fractions). Neither fraction alone had reductase activity, but when combined, constituted an active enzyme system. Heat treatment of either fraction resulted in an inactive combination. The approximate molecular size of the active component of the Tris and Dye fractions was determined to be 5.7 S and 6.5 S, respectively, compared to 9 S for the intact enzyme. The Tris fraction was inactivated by hydroxylamine while the dye fraction was inactivated by pyridoxal phosphate/BH4-treatment. The inactivation of the Dye fraction was prevented by ATP. These data would indicate that the Tris and Dye fractions were comparable in function to the B2 and B1 proteins, respectively, of the Escherichia coli ribonucleotide reductase.