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.     

Functional analysis of norcoclaurine synthase in Coptis japonica

(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe(2+)-binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.     

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.     

Phylogenetic analysis of the internal transcribed spacer region of Japanese Lilium species

 The DNA from 16 Lilium species and one variety endemic to or naturalized in Japan were obtained and their internal transcribed spacer regions of nuclear ribosomal DNA (nrDNA) were amplified by PCR and sequenced by cycle sequencing. Phylogenetic analysis of the ITS sequences supported the validity of Comber’s classification system. It has also provided molecular evidence for the transfer of Lilium dauricum to sect. Sinomartagon. The phylogenetic relationships revealed by ITS DNA analysis were supported by previously published crossability data. The molecular phylogeny of Japanese Lilium species was discussed with reference to the putative migration routes of these species. Received: 30 June 1998 / Accepted: 19 October 1998     

Relationships among Old and New World Alliums according to ITS DNA sequence analysis

 Phylogenetic analysis of ITS DNA sequences of various Old and New World Alliums suggests the reinclusion of Nectaroscordum and affirms the reestablishment of Caloscordum as subgenera of genus Allium. The results sanction the elimination of the Old World Allium species from subg. Amerallium Traub and endorse Wendelbo’s definition of subg. Molium (excluding sect. Porphyrason). Received: 29 July 1998 / Accepted: 13 August 1998     

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.     

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.