Effect of human papillomavirus type 16 oncogenes on MAP kinase activity.

The mitogen-activated protein (MAP) kinase signal transduction pathway is an intracellular signaling cascade which mediates cellular responses to growth and differentiation factors. The MAP kinase pathway can be activated by a wide range of stimuli dependent on the cell types, and this is normally a transient response. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells in part by prolonging the activated stage of components within this signaling pathway. The human papillomavirus (HPV) oncogenes E6 and E7 play an essential role in the in vitro transformation of primary human keratinocytes and rodent cells. The HPV type 16 E5 gene has also been shown to have weak transforming activity and may enhance the epidermal growth factor (EGF)-mediated signal transduction to the nucleus. In the present study, we have investigated the effects of the oncogenic HPV type 16 E5, E6, and E7 genes on the induction of the MAP kinase signaling pathway. The E5 gene induced an increase in the MAP kinase activity both in the absence and in the presence of EGF. In comparison, the E6 and E7 oncoproteins do not alter the MAP kinase activity or prolong the MAP kinase activity induced with EGF. These findings suggest that E5 may function, at least in part, to enhance the cell response through the MAP kinase pathway. However, the transforming activity of E6 and E7 is not associated with alterations in the MAP kinase pathway. These findings are consistent with E5 enhancing the response to growth factor stimulation.     

Biochemistry and pharmacology of 7α-substituted androstenediones as aromatase inhibitors

The inhibition of aromatase, the enzyme responsible for converting androgens to estrogens, is therapeutically useful for the endocrine treatment of hormone-dependent breast cancer. Research by our laboratory has focused on developing competitive and irreversible steroidal aromatase inhibitors, with an emphasis on synthesis and biochemistry of 7α-substituted androstenediones. Numerous 7α-thiosubstituted androst-4-ene-3,17-diones are potent competitive inhibitors, and several 1,4-diene analogs, such as 7α-(4′-aminophenylthio)-androsta-1,4-diene-3,17-dione (7α-APTADD), have demonstrated effective enzyme-activated irreversible inhibition of aromatase in microsomal enzyme assays. One focus of current research is to examine the effectiveness and biochemical pharmacology of 7α-APTADD in vivo. In the hormone-dependent 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary carcinoma model system, 7α-APTADD at a 50 mg/kg/day dose caused an initial decrease in mean tumor volume during the first week, and tumor volume remained unchanged throughout the remaining 5-week treatment period. This agent lowers serum estradiol levels and inhibits ovarian aromatase activity. A second research area has focused on the synthesis of more metabolically stable inhibitors by replacing the thioether linkage at the 7α position with a carbon-carbon linkage. Several 7α-arylaliphatic androst-4-ene-3,17-diones were synthesized by 1,6-conjugate additions of appropriate organocuprates to a protected androst-4,6-diene or by 1,4-conjugate additions to a seco-A-ring steroid intermediate. These compounds were all potent inhibitors of aromatase with apparent K_is ranging between 13 and 19 nM. Extension of the research on these 7α-arylaliphatic androgens includes the introduction of a C₁---C₂ double bond in the A-ring to provide enzyme-activated irreversible inhibitors. The desired 7α-arylaliphatic androsta-1,4-diene-3,17-diones were obtained from their corresponding 7α-arylaliphatic androst-4-ene-3,17-diones by oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). These inhibitors demonstrated enzyme-mediated inactivation of aromatase with apparent k_inacts ranging from 4.4 × 10⁻⁴ to 1.90 x 10⁻³ s⁻¹. The best inactivator of the series was 7α-phenpropylandrosta-1,4-diene-3,17-dione, which exhibited a T_1/2 of 6.08 min. Aromatase inhibition was also observed in MCF-7 human mammary carcinoma cell cultures and in JAr human choriocarcinoma cell cultures, exhibiting IC₅₀ values of 64-328 nM. The 7α-arylaliphatic androgens thus demonstrate potent inhibition of aromatase in both microsomal incubations and in choriocarcinoma cell lines expressing aromatase enzymatic activity. Additionally, the results from these studies provide further evidence for the presence of a hydrophobic binding pocket existing near the 7α-position of the steroid in the active site of aromatase. The size of the 7α-substituent influences optimal binding of steroidal inhibitors to the active site and affects the extent of enzyme-mediated inactivation observed with androsta-1,4-diene-3,17-dione analogs.     

Glycine and asparagme tRNA sequences from the archaebacteriuin,Methanobacterium thermoautotrophicum

Two tRNA sequences from Methanobacterium thermoautotrophium are reported. Both tRNA^Gly_GCC and tRNA_NUU^Asn, the first tRNA sequences from methanogens, were determined by partial hydrolyses (both chemical and enzymatic) and analyzed by gel electrophoresis. The two tRNAs contain the unusual T-loop modifications, Cm and m¹I, which are present in other archaebacterial tRNAs. Finally the presence of an unknown modification in the D-loop has been inferred by a large jump in the sequence ladder. These tRNAs are approximately equidistant from eubacterial or eukaryotic tRNAs.     

Recovery of the in vitro reactivity of splenic cells of CBA/N mice toward type 2 thymus-independent antigens

CBA/N mice bearing a chromosome X linked immunological deficiency (Xid) cannot respond to type 2 thymus independent antigens (TI-2). However, when their spleen cells are in vitro simultaneously stimulated by both a TI-2 (Fluorescein conjugated polyacrylamide, Flu-PAA) antigen and a type 1 thymus independent (Trinitrophenyl conjugated Brucella abortus, TNP-BA) antigen, their capacity to respond to the TI-2 antigen is recovered. On the contrary, thymus dependent (TD) Sheep red blood cells (SRBC) antigen did not produce any significant increase of the anti-TI-2 response.     

Assembly of the mammalian muscle acetylcholine receptor in transfected COS cells

We have investigated the mechanisms of assembly and transport to the cell surface of the mouse muscle nicotinic acetylcholine receptor (AChR) in transiently transfected COS cells. In cells transfected with all four subunit cDNAs, AChR was expressed on the surface with properties resembling those seen in mouse muscle cells (Gu, Y., A. F. Franco, Jr., P.D. Gardner, J. B. Lansman, J. R. Forsayeth, and Z. W. Hall. 1990. Neuron. 5:147-157). When incomplete combinations of AChR subunits were expressed, surface binding of 125I-alpha-bungarotoxin was not detected except in the case of alpha beta gamma which expressed less than 15% of that seen with all four subunits. Immunoprecipitation and sucrose gradient sedimentation experiments showed that in cells expressing pairs of subunits, alpha delta and alpha gamma heterodimers were formed, but alpha beta was not. When three subunits were expressed, alpha delta beta and alpha gamma beta complexes were formed. Variation of the ratios of the four subunit cDNAs used in the transfection mixture showed that surface AChR expression was decreased by high concentrations of delta or gamma cDNAs in a mutually competitive manner. High expression of delta or gamma subunits also each inhibited formation of a heterodimer with alpha and the other subunit. These results are consistent with a defined pathway for AChR assembly in which alpha delta and alpha gamma heterodimers are formed first, followed by association with the beta subunit and with each other to form the complete AChR.     

Transformation and allelic replacement in Francisella spp.

We describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella. Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-Tn10-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.