DNA topoisomerase targets of the fluoroquinolones: A strategy for avoiding bacterial resistance

Fluoroquinolones are antibacterial agents that attack DNA gyrase and topoisomerase IV on chromosomal DNA. The existence of two fluoroquinolone targets and stepwise accumulation of resistance suggested that new quinolones could be found that would require cells to obtain two topoisomerase mutations to display resistance. For wild-type cells to become resistant, the two mutations must be acquired concomitantly. That is expected to occur infrequently. To identify such compounds, fluoroquinolones were tested for the ability to kill a moderately resistant gyrase mutant. Compounds containing a C8-methoxyl group were particularly lethal, and incubation of wild-type cultures on agar containing C8-methoxyl fluoroquinolones produced no resistant mutant, whereas thousands arose during comparable treatment with control compounds lacking the C8 substituent. When the test strain contained a preexisting topoisomerase IV mutation, which by itself conferred no resistance, equally high numbers of resistant mutants were obtained for C8-methoxyl and control compounds. Thus C8-methoxyl fluoroquinolones required two mutations for expression of resistance. Although highly lethal, C8-methoxyl fluoroquinolones were not more effective than C8-H controls at blocking bacterial growth. Consequently, quinolone action involves two events, which we envision as formation of drug–enzyme–DNA complexes followed by release of lethal double-strand DNA breaks. Release of DNA breaks, which must occur less frequently than complex formation, is probably the process stimulated by the C8-methoxyl group. Understanding this stimulation should provide insight into intracellular quinolone action and contribute to development of fluoroquinolones that prevent selection of resistant bacteria.

p53 associates with and targets ΔNp63 into a protein degradation pathway

A human p53 homologue, p63 (p40/p51/p73L/CUSP) that maps to the chromosomal region 3q27–29 was found to produce a variety of transcripts that encode DNA-binding proteins with and without a trans-activation domain (TA- or ΔN-, respectively). The p63 gene locus was found to be amplified in squamous cell carcinoma, and overexpression of ΔNp63 (p40) led to increased growth of transformed cells in vitro and in vivo. Moreover, p63-null mice displayed abnormal epithelial development and germ-line human mutations were found to cause ectodermal dysplasia. We now demonstrate that certain p63 isotypes form complexes with p53. p53 mutations R175H or R248W abolish the association of p53 with p63, whereas V143A or R273H has no effect. Deletion studies suggest that the DNA-binding domains of both p53 and p63 mediate the association. Overexpression of wild type but not mutant (R175H) p53 results in the caspase-dependent degradation of certain ΔNp63 proteins (p40 and ΔNp63α). The association between p53 and ΔNp63 supports a previously unrecognized role for p53 in regulation of ΔNp63 stability. The ability of p53 to mediate ΔNp63 degradation may balance the capacity of ΔNp63 to accelerate tumorigenesis or to induce epithelial proliferation.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T2-biotin·dT-T2 loop. The third base was a biotinylated uracil (UB) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3′ dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3′ end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5′-FITC, or radiolabeled with [γ-33P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45°C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin–target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

p19ARF targets certain E2F species for degradation

p19ARF suppresses the growth of cells lacking p53 through an unknown mechanism. p19ARF was found to complex with transcription factors E2F1, -2, and -3. Levels of endogenous or ectopically expressed E2F1, -2, and -3, but not E2F6, were reduced after synthesis of p19ARF, through a mechanism involving increased turnover. p19ARF-induced degradation of E2F1 depended on a functional proteasome, and E2F1 was relocalized to nucleoli when coexpressed with p19ARF. Consistent with reduced levels of E2F1 and E2F3, the proliferation of cells defective for p53 function was suppressed by p19ARF, and the effect was partially reversed by ectopic overexpression of E2F1. These results suggest a broader role for p19ARF as a tumor suppressor, in which targeting of certain E2F species may cooperate with stimulation of the p53 pathway to counteract oncogenic growth signals.

Overexpression of p27Kip1 lengthens the G1 phase in a mouse model that targets inducible gene expression to central nervous system progenitor cells

We describe a mouse model in which p27Kip1 transgene expression is spatially restricted to the central nervous system neuroepithelium and temporally controlled with doxycycline. Transgene-specific transcripts are detectable within 6 h of doxycycline administration, and maximum nonlethal expression is approached within 12 h. After 18–26 h of transgene expression, the G1 phase of the cell cycle is estimated to increase from 9 to 13 h in the neocortical neuroepithelium, the maximum G1 phase length attainable in this proliferative population in normal mice. Thus our data establish a direct link between p27Kip1 and control of G1 phase length in the mammalian central nervous system and unveil intrinsic mechanisms that constrain the G1 phase length to a putative physiological maximum despite ongoing p27Kip1 transgene expression.

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

Direct modulation of calmodulin targets by the neuronal calcium sensor NCS-1.

Ca2+ and its ubiquitous intracellular receptor calmodulin (CaM) are required in the nervous system, among a host of cellular responses, for the modulation of several important enzymes and ion channels involved in synaptic efficacy and neuronal plasticity. Here, we report that CaM can be replaced by the neuronal calcium sensor NCS-1 both in vitro and in vivo. NCS-1 is a calcium binding protein with two Ca(2+)-binding domains that shares only 21% of homology with CaM. We observe that NCS-1 directly activates two Ca2+/CaM-dependent enzymes (3':5'-cyclic nucleotide phosphodiesterase and protein phosphatase calcineurin). Co-activation of nitric oxide synthase by NCS-1 and CaM results in a higher activity than with CaM alone. Moreover, NCS-1 is coexpressed with calcineurin and nitric oxide synthase in several neuron populations. Finally, injections of NCS-1 into calmodulin-defective cam1 Paramecium partially restore wildtype behavioral responses. With this highly purified preparation of NCS-1, we have obtained crystals suitable for crystallographic structure studies. NCS-1, despite its very different structure, distribution, and Ca(2+)-binding affinity as compared with CaM, can substitute for or potentiate CaM functions. Therefore, NCS-1 represents a novel protein capable of mediating multiple Ca(2+)-signaling pathways in the nervous system.

Mitochondria are selective targets for the protective effects of heat shock against oxidative injury.

Heat shock (HS) proteins (HSPs) induce protection against a number of stresses distinct from HS, including reactive oxygen species. In the human premonocytic line U937, we investigated in whole cells the effects of preexposure to HS and exposure to hydrogen peroxide (H2O2) on mitochondrial membrane potential, mass, and ultrastructure. HS prevented H2O2-induced alterations in mitochondrial membrane potential and cristae formation while increasing expression of HSPs and the protein product of bcl-2. Protection correlated best with the expression of the 70-kDa HSP, hsp70. We propose that mitochondria represent a selective target for HS-mediated protection against oxidative injury.

An immunoglobulin mutator that targets G.C base pairs.

Hypermutation can be defined as an enhancement of the spontaneous mutation rate which the organism uses in certain types of differentiated cells where a high mutation rate is advantageous. At the immunoglobulin loci this process increases the mutation rate > 10(5)-fold over the normal, spontaneous rate. Its proximate cause is called the immunoglobulin mutator system. The most important function of this system is to improve antibody affinity in an ongoing response; it is turned on and off during the differentiation of B lymphocytes. We have established an in vitro system to study hypermutation by transfecting a rearranged mu gene into a cell line in which an immunoglobulin mutator has been demonstrated. A construct containing the mu gene and the 3' kappa enhancer has all the cis-acting elements necessary for hypermutation of the endogenous gene segments encoding the variable region. The activity of the mutator does not seem to depend strongly on the position of the transfected gene in the genome. The mutator is not active in transformed cells of a later differentiation stage. It is also not active on a transfected lacZ gene. These results are consistent with the specificity of the mutator system being maintained and make it possible to delineate cis and trans mutator elements in vitro. Surprisingly, the mutator preferentially targets G-C base pairs. Two hypotheses are discussed: (i) the immunoglobulin mutator system in mammals consists of several mutators, of which the mutator described here is only one; or (ii) the primary specificity of the system is biased toward mutation of G-C base pairs, but this specificity is obscured by antigenic selection.

High-level production of recombinant human lysosomal acid alpha-glucosidase in Chinese hamster ovary cells which targets to heart muscle and corrects glycogen accumulation in fibroblasts from patients with Pompe disease.

Infantile Pompe disease is a fatal genetic muscle disorder caused by a deficiency of acid alpha-glucosidase, a glycogen-degrading lysosomal enzyme. We constructed a plasmid containing a 5'-shortened human acid alpha-glucosidase cDNA driven by the cytomegalovirus promoter, as well as the aminoglycoside phosphotransferase and dihydrofolate reductase genes. Following transfection in dihydrofolate reductase-deficient Chinese hamster ovary cells, selection with Geneticin, and amplification with methotrexate, a cell line producing high levels of the alpha-glucosidase was established. In 48 hr, the cells cultured in Iscove's medium with 5 mM butyrate secreted 110-kDa precursor enzyme that accumulated to 91 micrograms.ml-1 in the medium (activity, > 22.6 mumol.hr-1.ml-1). This enzyme has a pH optimum similar to that of the mature form, but a lower Vmax and Km for 4-methylumbelliferyl-alpha-D-glucoside. It is efficiently taken up by fibroblasts from Pompe patients, restoring normal levels of acid alpha-glucosidase and glycogen. The uptake is blocked by mannose 6-phosphate. Following intravenous injection, high enzyme levels are seen in heart and liver. An efficient production system now exists for recombinant human acid alpha-glucosidase targeted to heart and capable of correcting fibroblasts from patients with Pompe disease.

In vitro integration of human immunodeficiency virus type 1 cDNA into targets containing protein-induced bends.

Integration of human immunodeficiency virus type 1 cDNA into a target DNA can be strongly influenced by the conformation of the target. For example, integration in vitro is sometimes favored in target DNAs containing sequence-directed bends or DNA distortions caused by bound proteins. We have analyzed the effect of DNA bending by studying integration into two well-characterized protein-DNA complexes: Escherichia coli integration host factor (IHF) protein bound to a phage IHF site, and the DNA binding domain of human lymphoid enhancer factor (LEF) bound to a LEF site. Both of these proteins have previously been reported to bend DNA by approximately 140 degrees. Binding of IHF greatly increases the efficiency of in vitro integration at hotspots within the IHF site. We analyzed a series of mutants in which the IHF site was modified at the most prominent hotspot. We found that each variant still displayed enhanced integration upon IHF binding. Evidently the local sequence is not critical for formation of an IHF hotspot. LEF binding did not create preferred sites for integration. The different effects of IHF and LEF binding can be rationalized in terms of the different proposed conformations of the two protein-DNA complexes.