2' Derivatives of guanosine and inosine cyclic 3',5'-phosphates. Synthesis, enzymic activity, and the effect of 8-substituents.

A series of representative derivatives of guanosine cyclic 3',5'-phosphate (cGMP) and inosine cyclic 3',5'-phosphate (cIMP) which contained modifications in either the 2' position or the 8 and 2' positions were synthesized. Three types of derivatives were investigated: (1) derivatives in which the 2' position has been altered to produce a 2'-deoxynucleoside cyclic 3',5'-phosphate or a 9-beta-D-arabinofuranosylpurine cyclic 3',5'-phosphate; (2) 2'-omicron-acyl derivatives; and (3) doubly modified derivatives containing a 2' modification [as in (1) and (2)] and an 8-substitution. 2'-Deoxyinosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosylhypoxanthine cyclic 3',5'-phosphate were obtained by HNO2 deamination of 2'-deoxyadenosine cyclic 3',5'-phosphate and 9-beta-D-arabinofuranosyladenine cyclic 3',5'-phosphate (ara-cAMP), respectively. Treatment of 8-bromo-2'-omicron-(p-toluenesulfonyl) adenosine cyclic 3',5'-phosphate with NaSH yielded the intermediate 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoadenine cyclic 3',5-phosphate, which was converted directly to 2'-deoxyadenosine cyclic 3',5'-phosphate (dcAMP) by treatment with Raney nickel. 8-Bromo-2'-omicron-(p-toluenesulfonyl) guanosine cyclic 3',5'-phosphate was converted to 8,2'-anhydro-9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate, and the latter was desulfurized with Raney nickel to give 2-deoxyguanosine cyclic 3',5'-phosphate. Ara-cAMP, 9-beta-D-arabinofuranosylguanine cyclic 3',5'-phosphate, and 9-beta-D-arabinofuranosyl-8-mercaptoguanine cyclic 3',5'-phosphate have been previously reported (Mian et al. (1974), J. Med. Chem. 17, 259). 8-Bromo-2'-omicron-acetylinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]-2'-omicron-acetylinosine cyclic 3',5'-phosphate were produced by acylation of 8-bromoinosine cyclic 3',5'-phosphate and 8-[(p-chlorophenyl)thio]inosine cyclic 3',5'-phosphate, respectively; while 8-bromo-2'-omicron-butyrylguanosine cyclic 3',5'-phosphate was synthesized by bromination of 2'-omicron-butyrylguanosine cyclic 3',5'-phosphate.     

The nature of the stimuli causing digestive juice secretion in Dionaea muscipula Ellis (venus's flytrap)

Summary An investigation into the stimuli of the secretory system in the carnivorous plant Dionaea muscipula is presented. Secretion of fluid and protein are both stimulated by various nitrogenous small molecules. These secretions are studied as a function of time. A new method is described for the collection of secreted juice. Significant differences are found between the quantities of fluid and protein produced in response to different stimuli. The results are discussed in comparison to the mammalian gastro-intestinal secretory systems.     

Suicide inactivation of the E. coli O6-methylguanine-DNA methyltransferase

The O6-methylguanine-DNA methyltransferase of Escherichia coli acts rapidly and stoichiometrically to convert a mutagenic O6-methylguanine residue in DNA to unsubstituted guanine. Even at low protein concentrations and in the absence of any cofactors, the transfer of a methyl group to one of the protein's own cysteine residues occurs in less than 2 s at 37 degrees C. The entire kinetic process can be followed experimentally at 5 degrees C. Formation of S-methylcysteine in the protein is accompanied by loss of activity and accounts for the exceptional suicide kinetics of this enzyme as well as for the sharp saturation of O6-methylguanine repair observed in vivo. The enzyme can remove greater than 98% of the methyl groups from O6-methylguanine present in alkylated DNA, but leaves N-alkylated purines untouched. Single-stranded DNA containing O6-methylguanine is a poor substrate, with the methyl transfer occurring at approximately 0.1% of the rate for duplex DNA. This latter observation may explain the high frequency of mutations induced by alkylating agents at DNA replication forks.     

Conformational changes in the Arp2/3 complex leading to actin nucleation

The two actin-related subunits of the Arp2/3 complex, Arp2 and Arp3, are proposed to form a pseudo actin dimer that nucleates actin polymerization. However, in the crystal structure of the inactive complex, they are too far apart to form such a nucleus. Here, we show using EM that yeast and bovine Arp2/3 complexes exist in a distribution among open, intermediate and closed conformations. The crystal structure docks well into the open conformation. The activator WASp binds at the cleft between Arp2 and Arp3, and all WASp-bound complexes are closed. The inhibitor coronin binds near the p35 subunit, and all coronin-bound complexes are open. Activating and loss-of-function mutations in the p35 subunit skew conformational distribution in opposite directions, closed and open, respectively. We conclude that WASp stabilizes p35-dependent closure of the complex, holding Arp2 and Arp3 closer together to nucleate an actin filament.     

Dendritic cells are dysfunctional in patients with operable breast cancer

Background: Dendritic cells (DCs) play a crucial role in presenting antigens to T lymphocytes and inducing cytotoxic T cells. DCs have been studied in patients with breast cancer to define the factors leading to failure of an effective systemic and locoregional anticancer host response. Methods: Purified DCs were obtained from peripheral blood (PB) and lymph nodes (LNs) of women with operable breast cancer, using immunomagnetic bead selection. The stimulatory capacity of DCs in the allogeneic mixed leukocyte reaction (MLR) and autologous T cell proliferation test (purified protein derivative (PPD) as stimulator), the expression of surface markers on DCs and the production of cytokines in vitro by DCs from patients with operable breast cancer and from healthy donors (controls) were studied. Results: 70–75% purified DCs were isolated from PB and LNs. PBDCs and LNDCs from patients with operable breast cancer demonstrated a reduced capacity to stimulate in an MLR, compared with PBDCs from normal donors (p<0.01). Autologous T cell proliferation in patients had a decreased ability to respond to PPD, when compared with controls (p<0.01). However, T cells from patients responded as well as control T lymphocytes in the presence of control DCs. PBDCs and LNDCs from patients expressed low levels of HLA-DR and CD86, and induced decreased interleukin-12 (IL-12) secretion in vitro, compared with DCs from normal donors (p<0.01). Conclusion: These data suggest a defective DC function in patients with operable breast cancer. Switched-off DCs in patients with early breast cancer and decreased IL-12 production may be important factors for progressive tumour growth.     

Molecular ecology and biological control: the mating system of a marsupial pest

Many studies in molecular ecology have focused on the use of repeat DNA markers to determine the nature of mating systems in a wide variety of animal species. Whilst these studies typically have focused on important issues such as the evolutionary consequences of fitness variation among males, genetic studies of mating systems are potentially also important because they can generate information of significance to wider issues in wildlife management. For example, genetically modified, sexually transmitted viral diseases have been suggested as potential agents for the control of vertebrate pest species. An understanding of the epidemiology of such agents requires an intimate knowledge of the sexual contact rates between individuals of the target species. Here, we report the use of minisatellite DNA profiling to reveal the mating system in two New Zealand populations of the introduced Australian brushtail possum. The brushtail possum is New Zealand's most important mammalian pest and a species for which control by a sexually transmitted immunocontraceptive has been proposed. Encouragingly, we report considerable variation in the reproductive success of males at both study sites, with one male siring offspring from four females in one year (mean no. of offspring/reproductively successful male/year at the two sites is 1.95-2.15), while many sired none. This bias in the pattern of reproductive success among males will probably facilitate the spread of an immunocontraceptive agent and thereby increase the power of this approach to biological control.     

Synthesis of novel DNA cross-linking antitumour agents based on polyazamacrocycles

We are seeking to develop more effective alkylating agents as antitumour agents. In previous work conformationally restricted nitrogen mustards were synthesised containing piperidine or pyrrolidine rings. The free bases were designed to be bifunctional alkylating agents via aziridinium ion formation and the effects of varying the distances between the two alkylating sites were studied. Some efficient cross-linkers of naked DNA were prepared but few of these compounds exhibited significant cytotoxicity in human tumour cells in vitro. We have extended this work by making tri- and tetra-azamacrocyclic compounds containing two to four potential alkylating sites. Most of these compounds were powerful DNA alkylating agents and showed cytotoxicity (IC(50) values 6-100microM) comparable with chlorambucil (45microM) and melphalan (8.5microM). In particular the cyclen derivative 2a was more than 10(4) times more effective at cross-linking DNA (2a XL(50)<10nM) than chlorambucil (XL(50) 100microM), and showed significant cytotoxicity in human tumour cells in vitro.