Article

Background: Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction.

Methods: We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg(2+)-, Mn(2+)-, or Ca(2+)-supported DNA cleavage activity of Escherichia coli Topo IV.

Results: In the absence of any drug, 20-30 mM Mg(2+) was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1mM of either Mn(2+) or Ca(2+) was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg(2+) concentrations where Topo IV alone could not efficiently cleave DNA.

Conclusions and general significance: At low Mg(2+) concentrations, fluoroquinolones may stimulate the Topo IV-catalyzed strand breakage reaction by promoting Mg(2+) binding to metal binding site B through the structural distortion in DNA. As Mg(2+) concentration increases, fluoroquinolones may inhibit the religation reaction by either stabilizing Mg(2+) at site B or inhibition the binding of Mg(2+) to site A. This study provides a molecular basis of how fluoroquinolones stimulate the Topo IV-catalyzed strand breakage reaction by modulating Mg(2+) binding.

Structural studies of topoisomerase-fluoroquinolone-DNA ternary complexes revealed a cavity between the quinolone N-1 position and the active site tyrosine. Fluoroquinolone derivatives having positively charged or aromatic moieties extended from the N-1 position were designed to probe for binding contacts with the phosphotyrosine residue in ternary complex. While alkylamine, alkylphthalimide, and alkylphenyl groups introduced at the N-1 position afforded derivatives that maintained modest inhibition of the supercoiling activity of DNA gyrase, none retained ability to poison DNA gyrase. Thus, the addition of a large and/or long moiety at the N-1 position disrupts ternary complex formation, and retained ability to inhibit supercoiling is likely through interference with the strand breakage reaction. Two derivatives were found to possess inhibitory effects on the decatenation activity of human topoisomerase II.

 Fluoroquinolone-class agents selectively target the bacterial type IIA topoisomerases DNA gyrase and topoisomerase IV, with a few exceptions that target eukaryotic type IIA topoisomerases. Fluoroquinolones bind and stabilize type IIA topoisomerase-DNA covalent complexes that contain a double-strand break. This unique mode of action is referred to as 'topoisomerase poisoning'. We discovered that two novel fluoroquinolones having aryl functionality at the N-1 position, UITT-3-217 (217) and UITT-3-227 (227), could inhibit the catalytic activity of human topoisomerase II without stabilizing topoisomerase-DNA complexes, i.e., without poisoning it. Surprisingly, these compounds are more effective in inhibiting the catalytic activities of human and bacterial topoisomerase I. The National Cancer Institute's 60 human tumor cell lines screen revealed significant anti-proliferative activities with 217 and 227 against the majority of 60 cancer cell lines. A proof of concept in vivo efficacy study using an HT-29 xenograft model of human colorectal cancer showed that 217 could inhibit the proliferation of human colorectal cancer cells to a degree comparable to fluorouracil in mice. Although 227 also exhibited anti-proliferative activity, it was not as effective as 217 in this xenograft model. These novel fluoroquinolones may serve as promising lead compounds for the development of new anticancer drugs.

A Mg2+-water bridge between the C-3, C-4 diketo moiety of fluoroquinolones and the conserved amino acid residues in the GyrA/ParC subunit is critical for the binding of a fluoroquinolone to a topoisomerase-DNA covalent complex. The fluoroquinolone UING-5-249 (249) can bind to the GyrB subunit through its C7-aminomethylpyrrolidine group. This interaction is responsible for enhanced activities of 249 against the wild type and quinolone-resistant mutant topoisomerases. To further evaluate the effects of the 249-GyrB interaction on fluoroquinolone activity, we examined the activities of decarboxy- and thio-249 against DNA gyrase and conducted docking studies using the structure of a gyrase-ciprofloxacin-DNA ternary complex. We found that the 249-GyrB interaction rescued the activity of thio-249 but not that of decarboxy-249. A C7-group that binds more strongly to the GyrB subunit may allow for modifications at the C-4 position, leading to a novel compound that is active against the wild type and quinolone-resistant pathogens.

Chiropractic was founded by D. D. Palmer, who performed his first spinal adjustments in 1895 and started the first chiropractic school, the Palmer School and Cure, in 1897, both in Iowa.1  Diplomas granted to early graduates proclaimed them competent to teach and practice chiropractic, and many of these newly minted chiropractors did just that, often tutoring individuals and small groups in offices and clinics rather than setting up school buildings.2  For many of these tutorial schools, whether they had any graduates or even any students is unknown, and some schools were known to have been diploma mills3 (teaching chiropractic by correspondence course seems like a questionable proposition).  However, many other schools operated by the standards for a quality course of study as defined at that time.4 The earliest school teaching chiropractic technique that is known to have been in operation in Minnesota was the National School of Neuropathy & Psycho-Magnetic Healing, located in Minneapolis in 1899.5  Including tutorial schools, approximately 30 schools operated in the state from 1899 to NWCC’s founding in 1941.6  The total number of schools operating is difficult to gauge with precision, as some underwent slight name changes and others adopted names nearly identical to those of other schools.  Many schools seem to have been in existence for no more than a year or two.7 Four early Minnesota chiropractic schools are known to have operated for at least a decade.

As massage therapy experiences a resurgence of use for hospitalized patients, it is appropriate to consider the competencies needed by practitioners to practice safely and effectively in the inpatient setting. Hospitals differ vastly from other massage practice locations such as private offices, spas, and sports clubs. The variety of conditions encountered in an acute care setting require the knowledge and ability to adapt massage protocols appropriately. The Academic Collaborative for Integrative Health (ACIH) created the Hospital Based Massage Therapy (HBMT) Task Force to determine if there is a need for HBMT specific competencies and then, if needed, to develop peer reviewed competencies that hospital staff, massage therapy educators, and massage therapists all may find useful.

The members of the task force identified massage therapists who worked in hospitals generally, as well as in hospitals known to have HBMT programs. A spreadsheet was created listing the individuals and a survey was distributed to those on the spreadsheet. These individuals were also asked to identify others who might be interested in participating in the project. The purpose of the survey was to assess various elements of HBMT programs such as educational/experience requirements, employment model, orientation, and supervision. 32 out of 37 hospitals (87%) completed the survey. The Task Force considered the high response rate and the extent to which respondents provided in-depth answers to the open-ended questions as evidence of the need for specific competencies for safe and effective massage therapy for hospitalized patients.

In addition to the survey, the task force used a Delphi technique to engage survey participants and other experts in the field to shape the initial draft of the competencies. As these competencies are shared with hospitals, massage therapists, and massage educators, the Task Force members expect that additional development of the competencies will take place as various groups implement them.

The “Mystery Lab” is a laboratory experience that allows students to apply critical thinking, writing, and organic chemical laboratory skills. Students are challenged to solve a medical mystery presented as a short story that includes both situational information as well as clues to needed procedures. The students must identify which of a number of samples is tainted with a poison, separate the poison from the rest of the sample, then identify the poison using provided analytical data. Students complete the lab report by writing the end of the story presented in the lab handout. Creativity is encouraged!

Background—Fluoroquinolones target bacterial type IIA topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Fluoroquinolones trap a topoisomerase-DNA covalent complex as a topoisomerase-fluoroquinolone-DNA ternary complex and ternary complex formation is critical for their cytotoxicity. A divalent metal ion is required for type IIA topoisomerase-catalyzed strand breakage and religation reactions. Recent studies have suggested that type IIA topoisomerases use two metal ions, one structural and one catalytic, to carry out the strand breakage reaction. 

Methods—We conducted a series of DNA cleavage assays to examine the effects of fluoroquinolones and quinazolinediones on Mg2+-, Mn2+-, or Ca2+-supported DNA cleavage activity of Esherichia coli Topo IV. 

Results—In the absence of any drug, 20–30 mM Mg2+ was required for the maximum levels of the DNA cleavage activity of Topo IV, whereas approximately 1 mM of either Mn2+ or Ca2+ was sufficient to support the maximum levels of the DNA cleavage activity of Topo IV. Fluoroquinolones promoted the Topo IV-catalyzed strand breakage reaction at low Mg2+ concentrations where Topo IV alone could not efficiently cleave DNA.

 DNA gyrase and topoisomerase IV control bacterial DNA topology by breaking DNA, passing duplex DNA through the break, and then resealing the break. This process is subject to reversible corruption by fluoroquinolones, antibacterials that form drug-enzyme-DNA complexes in which the DNA is broken. The complexes, called cleaved complexes because of the presence of DNA breaks, have been crystallized and found to have thefluoroquinolone C-7ring systemfacing the GyrB/ParE subunits. As expected from x-ray crystallography, a thiol-reactive, C-7-modifiedchloroacetylderivativeofciprofloxacin(CipAcCl) formed cross-linked cleaved complexes with mutant GyrB-Cys466 gyrase as evidenced by resistance to reversal by both EDTAandthermaltreatments.Surprisingly,cross-linking was also readily seen with complexes formed by mutant GyrAG81Cgyrase, thereby revealing a novel drug-gyrase interaction not observed in crystal structures. The cross-link between fluoroquinolone and GyrA-G81C gyrase correlated with exceptional bacteriostatic activity for Cip-AcCl with a quinoloneresistant GyrA-G81C variant of Escherichia coli and its Mycobacteriumsmegmatisequivalent(GyrA-G89C).Cip-AcClmediated, irreversible inhibition of DNA replication provided further evidence for a GyrA-drug cross-link. Collectively these data establish the existence of interactions between the fluoroquinoloneC-7ringandbothGyrAandGyrB.BecausetheGyrAGly81andGyrB-Glu466residuesarefarapart(17Å)inthecrystal structureofcleavedcomplexes,twomodesofquinolonebinding mustexist. The presence of two binding modesraises the possibility that multiple quinolone-enzyme-DNA complexes can form, a discovery that opens new avenues for exploring and exploiting relationships between drug structure and activity with type II DNAtopoisomerases.

Numerous therapeutic applications have been proposed for molecules that bind heparin-binding proteins. Development of such compounds has primarily focused on optimizing the degree and orientation of anionic groups on a scaffold, but utility of these polyanions has been diminished by their typically large size and nonspecific interactions with many proteins. In this study, N-arylacyl O-sulfonated aminoglycosides were synthesized and evaluated for their ability to selectively inhibit structurally similar bacterial and human topoisomerases. It is demonstrated that the structure of the aminoglycoside and of the N-arylacyl moiety imparts selective inhibition of different topoisomerases and alters the mechanism. The results here outline a strategy that will be applicable to identifying small, structurally defined oligosaccharides that bind heparin-binding proteins with a high degree of selectivity.