MODES OF INACTIVATION OF NEUROHYPOPHYSIAL HORMONES: SIGNIFICANCE OF PLASMA DISAPPEARANCE RATE FOR THEIR PHYSIOLOGICAL RESPONSES

Analogues of oxytocin and deaminooxytocin with 4-glutamine replaced by 4-glutamic acid methyl ester readily lose their uterotonic activity when incubated with rat serum, presumably by hydrolysis to the much less active 4-glutamic acid derivatives. On the other hand, inactivation of the deaminooxytocin analogue in the rat uterus, as demonstrated by the 'oil-bath'technique, is only slightly more rapid than that of deaminooxytocin and distinctly slower than that of oxytocin. Its in situ/in vitro ratio of uterotonic activity is less than 0.1 whereas that for deaminooxytocin is about 3 and also the persistence of the uterotonic effect in situ is slightly less than that of deaminooxytocin. The results with these 'rapidly inactivated'analogues can be used as proof of some predictions of the three-compartment model for tissue distribution of neurohypophysial hormones and its influence upon the time course of a biological response published earlier. The potential use of analogues of neurohypophysial hormones as probes for inactivation mechanisms and the results thus far obtained are discussed.     

Evidence that a second tumor antigen coded by adenovirus early gene region E1a is required for efficient cell transformation.

The expression of the adenovirus (Ad) early coding region 1a (E1a) is required for virus-induced cell transformation and for the activation of other viral early genes and some cellular genes. Two overlapping early mRNAs of 13S and 12S that are transcribed from this region code for a 289-amino acid protein and a 243-amino acid protein, respectively. Earlier studies have shown that the 289-amino acid protein is essential for cell transformation. We have constructed an Ad type 2 (Ad2) deletion mutant (dl231) in which the intervening sequence for the 13S mRNA is precisely removed. Mutant dl231 is completely viable in human KB cells and produces normal amounts of 13S mRNA but much reduced amounts of a defective 12S mRNA. Mutant dl231 induces focal transformation of established rat embryo fibroblasts at a frequency one-fifth to one-half that of wild-type virus. However, the transformed cells are defective in their ability to form anchorage-independent colonies on semisolid medium. Therefore, our results demonstrate that the 243-amino acid protein is required for full transformation of rat embryo cells.     

Conversion of ?X174 and fd Single-Stranded DNA to Replicative Forms in Extracts of Escherichia coli

varphiX174 and M13 (fd) single-stranded circular DNAs are converted to their replicative forms by extracts of E. coli pol A1 cells. We find that the varphiX174 DNA-dependent reaction requires Mg(++), ATP, and all four deoxynucleoside triphosphates, but not CTP, UTP, or GTP. This reaction also involves the products of the dnaC, dnaD, dnaE (DNA polymerase III), and dnaG genes, but not that of dnaF (ribonucleotide reductase). The in vitro conversion of fd single-stranded DNA to the replicative form requires all four ribonucleoside triphosphates, Mg(++), and all four deoxynucleoside triphosphates. The reaction involves the product of gene dnaE but not those of genes dnaC, dnaD, dnaF, or dnaG. The reaction with fd DNA is inhibited by rifampicin or antibody to RNA polymerase, while the reaction with varphiX174 DNA is not affected by either. With the varphiX174 DNA-dependent reaction, activities have been detected that specifically complement extracts of dnaA, dnaB, dnaC, dnaD, or dnaG mutants.     

Evidence for an altered adenovirus DNA polymerase in cells infected with the mutant H5ts149

The N complementation group of adenovirus (Ad) serotype 5 mutants, which are temperature sensitive for viral DNA synthesis in vivo, has been used to study a 140,000-dalton DNA polymerase (Pol) that copurified with the 80,000-dalton terminal protein precursor (pTP). Extracts prepared from HeLa cells infected with the N group mutant H5ts149 at nonpermissive temperature were unable to synthesize viral DNA. The defect in these extracts was specifically reversed by addition of the Pol purified from wild-type Ad-infected cytosol. Addition of the pTP, free of the Pol, did not restore replicative activity to H5ts149 extracts. The reactions studied depend on the presence of the DNA template and include the initiation reaction (the covalent attachment of dCMP to the pTP) and the selective replication of Ad DNA restriction endonuclease fragments containing the origin sequences. Glycerol gradient sedimentation showed that a replicative activity representing the pTP-Pol complex was greatly reduced in H5ts149 extracts as compared with wild-type extracts, suggesting some alteration in the mutant. A pool of pTP free of Pol was detected on these gradients in extracts from both wild-type and H5ts149-infected cells. In addition, the initiation and elongation of Ad DNA catalyzed by H5ts149 extracts prepared from cells grown at permissive temperatures was more labile to urea inactivation than extracts prepared from cells infected with wild-type virus. These results, considered together with the mapping of the H5ts149 mutation within an open reading frame approximately large enough to code for the 140,000-dalton DNA polymerase [Gingeras, T. R., Sciaky, D., Gelinas, R. E., Bing-Dong, J., Yen, C. E., Kelly, M. M., Bullock, P. A., Parsons, B. L., O'Neill, K. E. & Roberts, R. J. (1982) J. Biol. Chem. 257, 13475-13491; Alestrom, P., Akusjarui, G., Pettersson, M. & Pettersson, U. (1982) J. Biol. Chem. 257, 13492-13498], suggest that the Pol is a virally encoded protein, as is the pTP.     

Association of DNA-dependent and -independent Ribonucleoside Triphosphatase Activities with dnaB Gene Product of Escherichia coli

Preparations of E. coli dnaB gene product contain ribonucleoside triphosphatase activity that is stimulated 10-fold by DNA. The products of the triphosphatase activity are nucleoside diphosphates and P(i). The dnaB complementing activity in the varphiX174 DNA-dependent system and these triphosphatase activities copurify over the last 20-fold of an extensive (about 40,000-fold) purification procedure. Acrylamide gel electrophoresis of the purified material shows a single band of protein coincident with eluted dnaB complementing and DNA-dependent and -independent nucleoside triphosphatase activities.     

Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase.

The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G1 extract (obtained from HeLa cells in G1 phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation of cyclin A-activated G1 extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G1 extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p21cip1, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G1 extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G1 extract.     

Response to 2'-deoxycoformycin after failure of interferon-alpha in nonsplenectomized patients with hairy cell leukemia

Two patients with hairy cell leukemia with massive splenomegaly and severe pancytopenia were treated with recombinant alpha-A interferon (IFN-alpha-2a). There was no significant response to a trial of IFN- alpha-2a (11 and 20 weeks) with respect to blood counts or spleen size. Subsequent treatment with 2'-deoxycoformycin (dCF) for 8 consecutive weeks (4 mg/m2/wk) resulted in normalization of spleen size and a normalization of peripheral blood counts and bone marrow in one patient. The second patient demonstrated a reduction in spleen size and improved blood counts following 9 weeks of dCF therapy but eventually became refractory. This demonstrates that dCF is non-cross-resistant with interferon and confirms the efficacy of dCF in nonsplenectomized patients.     

Evaluation of drugs for elimination of leukemic cells from the bone marrow of patients with acute leukemia

Relatively nonmyelotoxic drugs and drug combinations were investigated for their ability to eliminate malignant cells from human bone marrow. In vitro 90% inhibitory concentration (IC90) doses were established on granulocyte macrophage colony-forming units (GM-CFU) in culture of bone marrow by using the GM-CFU assay for the following drugs: 4- hydroperoxycyclophosphamide (4-HC), Adriamycin, L-asparaginase, bleomycin, hydrocortisone, VP-16, spirogermanium, Taxol, and vincristine. The leukemic cell kill efficiency of these drugs at IC90 doses was compared with that of 4-HC on acute lymphoid leukemia (ALL) cell lines by using the limiting-dilution assay. Under these conditions, no single drug was superior to 4-HC. To increase the in vitro effect in leukemic cell kill, combinations of vincristine with hydrocortisone, Adriamycin, VP-16, and 4-HC were investigated. Vincristine at 1 to 5 micrograms/mL increased the marrow cytotoxicity of hydrocortisone, Adriamycin, and VP-16, but it was protective (subadditive) with 4-HC. Vincristine and 4-HC in combination was additive to supraadditive on ALL cell lines, increased the leukemic cell kill by one to two logs above 4-HC alone at IC90 doses (P less than .05), and was not affected by the addition of excess marrow cells. The recommended doses for chemopurging in clinical studies are vincristine, 1 to 5 micrograms/mL, plus 4-HC, 5 micrograms/mL.     

Studies on In Vitro DNA Synthesis Purification of dna C Gene Product Containing dna D Activity from Escherichia coli

The conversion of varphiX174 single-stranded DNA to duplex DNA by extracts of E. coli requires products of the E. coli DNA replication genes. By use of this complementation system, the dna C gene product has been purified from wild-type E. coli as well as from a dna C temperature-sensitive mutant. The latter preparations are temperature sensitive when compared to the wild-type gene product. The dna C and dna D gene products copurify, have similar characteristics, are both temperature sensitive in preparations from dna C temperature-sensitive cells, and are both undetectable in preparations from dna D temperature-sensitive cells.