Two distinct conformational states define the interaction of human RAD51‐ATP with single‐stranded DNA

An essential mechanism for repairing DNA double‐strand breaks is homologous recombination (HR). One of its core catalysts is human RAD51 (hRAD51), which assembles as a helical nucleoprotein filament on single‐stranded DNA, promoting DNA‐strand exchange. Here, we study the interaction of hRAD51 with single‐stranded DNA using a single‐molecule approach. We show that ATP‐bound hRAD51 filaments can exist in two different states with different contour lengths and with a free‐energy difference of ~4 k_BT per hRAD51 monomer. Upon ATP hydrolysis, the filaments convert into a disassembly‐competent ADP‐bound configuration. In agreement with the single‐molecule analysis, we demonstrate the presence of two distinct protomer interfaces in the crystal structure of a hRAD51‐ATP filament, providing a structural basis for the two conformational states of the filament. Together, our findings provide evidence that hRAD51‐ATP filaments can exist in two interconvertible conformational states, which might be functionally relevant for DNA homology recognition and strand exchange.     

A cytogenetic study of papaya workers exposed to ethylene dibromide

Ethylene dibromide (EDB) has been shown to be carcinogenic in animal studies and mutagenic in vitro. One cytogenetic study of workers exposed to low levels of EDB for short durations was negative. To test whether exposure to low levels of EDB over long periods caused cytogenetic changes, we have assessed the frequencies of sister-chromatid exchanges (SCE) and chromosomal aberrations (CA) in the peripheral blood lymphocytes of 60 men occupationally exposed to EDB. These men worked in papaya-packing plants where EDB was used to fumigate the fruit after harvest to kill fruit-fly larvae. 42 other men who worked at a nearby sugar mill served as controls. The average duration of exposure of the papaya workers was 5 years. 82 full shift personal breathing-zone air samples indicated that the papaya workers were exposed to a geometric mean of 88 ppb of EDB, as an 8-h time weighted average (TWA). Peaks up to 262 ppb were measured. The proposed OSHA 8-h TWA for EDB is 100 ppb, while NIOSH recommends 45 ppb. No differences in SCE levels were found between exposed and nonexposed workers. No differences were found in the total CA frequency between exposed and nonexposed workers. SCE levels were significantly increased in men who smoked cigarettes (p = 0.0001) and in men who smoked marijuana (p = 0.01). CA levels showed a significant increasing trend with age (p = 0.03).     

DNA divergence in and around the alcohol dehydrogenase locus in five closely related species of Hawaiian Drosophila

The alcohol dehydrogenase (Adh) region from five planitibia subgroup species of Hawaiian picture-wing Drosophila has been cloned. A total of 15 kb of DNA in and around the Adh gene has been compared among the five species. Genetic distances were calculated to determine evolutionary relationships. These distances agree with previous distances determined by protein polymorphism and DNA hybridization techniques and can be interpreted in terms of specific island colonization and speciation (founder) events over the past 5 Myr. Examination of the restriction maps of the cloned Adh region from the five species shows many instances of small deletions, insertion of a transposable element in D. heteroneura, and the existence of a highly variable region on the 3' side of the Adh gene. Clustering relationships and rates of DNA change are calculated and compared with the relationship found for other species of Drosophila.      

Oxygenation of monounsaturated fatty acids by soybean lipoxygenase-1: evidence for transient hydroperoxide formation

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of polyunsaturated fatty acids to produce conjugated diene hydroperoxides. Previous work from our laboratories has demonstrated that SBLO-1 will also catalyze the oxygenation of monounsaturated acids (Clapp, C. H., Senchak, S. E., Stover, T. J., Potter, T. C., Findeis, P. M., and Novak, M. J. (2001) Soybean Lipoxygenase-Mediated Oxygenation of Monounsaturated Fatty Acids to Enones, J. Am. Chem. Soc. 123, 747-748). Interestingly, the products are alpha,beta-unsaturated ketones rather than the expected allylic hydroperoxides. In the present work, we provide evidence that the monoolefin substrates are initially converted to allylic hydroperoxides, which are subsequently converted to the enone products. The hydroperoxide intermediates can be trapped by reduction to the corresponding allylic alcohols with glutathione peroxidase plus glutathione or with SnCl2. Under some conditions, the hydroperoxide intermediates accumulate and can be detected by HPLC and peroxide assays. Kinetics measurements at low concentrations of [1-14C]-9(Z)-octadecenoic acid indicate that oxygenation of this substrate at 25 degrees C, pH 9.0 occurs with kcat/Km = 1.6 (+/-0.1) x 10(2) M-1 s-1, which is about 105 lower than kcat/Km for oxygenation of 9(Z),12(Z)-octadecadienoic acid (linoleic acid). Comparison of the activities of 9(Z)-octadecenoic acid and 12(Z)-octadecenoic acid implies that the two double bonds of linoleic acid contribute almost equally to the C-H bond-breaking step in the normal lipoxygenase reaction. The results are consistent with the notion that SBLO-1 functionalizes substrates by a radical mechanism.     

TRPV1 receptors and nasal trigeminal chemesthesis

The trigeminal nerve responds to a wide variety of irritants. Trigeminal nerve fibers express several receptors that respond to chemicals, including TRPV1 (vanilloid) receptors, acid-sensing ion channels, P2X (purinergic) receptors, and nicotinic acetylcholine receptors. In order to assess whether TRPV1 plays a role in responses to a broad array of substances, TRPV1 (along with green fluorescent protein) was expressed in human embyonic kidney cells (HEK) 293t cells which were then stimulated with diverse trigeminal irritants. Calcium imaging was used to measure responses to capsaicin, amyl acetate, cyclohexanone, acetic acid, toluene, benzaldehyde, (-)-nicotine, (R)-(+)-limonene, (R)-(-)-carvone, and (S)-(+)-carvone. Three irritants (acetic acid and the 2 carvones) stimulated nontransfected controls. Two irritants (capsaicin and cyclohexanone) stimulated only transfected cells. The response could be eliminated with capsazepine, a TRPV1 blocker. The 5 remaining irritants were nonstimulatory in both nontransfected and transfected cells. Because all the compounds tested on HEK cells elicited neural responses from the ethmoid branch of the trigeminal nerve in rats, the 5 nonstimulatory compounds must do so by a non-TRPV1 receptor. These results suggest that TRPV1 serves as a receptor for both cyclohexanone and capsaicin in trigeminal nerve endings.     

C331A Mutant of Neuronal Nitric-oxide Synthase Is Labilized for Hsp70/CHIP (C Terminus of HSC70-interacting Protein)-dependent Ubiquitination

It is established that suicide inactivation of neuronal nitric-oxide synthase (nNOS) by drugs and other xenobiotics leads to ubiquitination and proteasomal degradation of the enzyme. The exact mechanism is not known, although it is widely thought that the covalent alteration of the active site during inactivation triggers the degradation. A mechanism that involves recognition of the altered nNOS by Hsp70 and its cochaperone CHIP, an E3-ubiquitin ligase, has been proposed. To further address how alterations of the active site trigger ubiquitination of nNOS, we examined a C331A nNOS mutant, which was reported to have impaired ability to bind l-arginine and tetrahydrobiopterin. We show here that C331A nNOS is highly susceptible to ubiquitination by a purified system containing ubiquitinating enzymes and chaperones, by the endogenous ubiquitinating system in reticulocyte lysate fraction II, and by intact HEK293 cells. The involvement of the altered heme cleft in regulating ubiquitination is confirmed by the finding that the slowly reversible inhibitor of nNOS, N^G-nitro-l-arginine, but not its inactive d-isomer, protects the C331A nNOS from ubiquitination in all these experimental systems. We also show that both Hsp70 and CHIP play a major role in the ubiquitination of C331A nNOS, although Hsp90 protects from ubiquitination. Thus, these studies further strengthen the link between the mobility of the substrate-binding cleft and chaperone-dependent ubiquitination of nNOS. These results support a general model of chaperone-mediated protein quality control and lead to a novel mechanism for substrate stabilization based on nNOS interaction with the chaperone machinery.     

Multifactorial approach and superior treatment efficacy in renal patients with the aid of nurse practitioners. Design of The MASTERPLAN Study [ISRCTN73187232]

Background

Patients with chronic kidney disease (CKD) are at a greatly increased risk of developing cardiovascular disease. Recently developed guidelines address multiple risk factors and life-style interventions. However, in current practice few patients reach their targets. A multifactorial approach with the aid of nurse practitioners was effective in achieving treatment goals and reducing vascular events in patients with diabetes mellitus and in patients with heart failure. We propose that this also holds for the CKD population.

Design

MASTERPLAN is a multicenter randomized controlled clinical trial designed to evaluate whether a multifactorial approach with the aid of nurse-practicioners reduces cardiovascular risk in patients with CKD. Approximately 800 patients with a creatinine clearance (estimated by Cockcroft-Gault) between 20 to 70 ml/min, will be included. To all patients the same set of guidelines will be applied and specific cardioprotective medication will be prescribed. In the intervention group the nurse practitioner will provide lifestyle advice and actively address treatment goals. Follow-up will be five years. Primary endpoint is the composite of myocardial infarction, stroke and cardiovascular mortality. Secondary endpoints are cardiovascular morbidity, overall mortality, decline of renal function, change in markers of vascular damage and change in quality of life. Enrollment has started in April 2004 and the study is on track with 700 patients included on October 15th, 2005. This article describes the design of the MASTERPLAN study.     

Guidelines for indications for coronary revascularization in The Netherlands

In 1996 the Minister of Public Health, Welfare and Sports in The Netherlands published a 'Planning Decree Special Interventions in the Heart'. She requested from the professional organizations guidelines for the indications for interventions in the heart. A working group was formed with representatives from the Dutch professional organizations for cardiology and thoracic surgery, to address this issue for patients with coronary artery disease. The working group confirmed the need to discuss all patients who are considered for either elective or emergency revascularization during a multidisciplinary consultation in (or with) one of the specialized Dutch hospitals. During this meeting of the 'heart team', at least one interventional cardiologist and one thoracic surgeon should be present. There are three possible outcomes of the heart team's consultations for each patient: drug therapy only ('conservative management'), coronary surgery or catheter intervention. For each case, the team should indicate the expected benefit, the risk of the intervention, the urgency and the estimated waiting time. The guidelines presented in this paper address these issues for three patient categories: stable angina pectoris, unstable angina pectoris and acute myocardial infarction.