Structure-Function Analysis of the Acyl Carrier Protein Synthase (AcpS) from Mycobacterium tuberculosis

We have solved the crystal structure of the acyl carrier protein synthase (AcpS) from Mycobacterium tuberculosis (Mtb) at 1.95 Å resolution. AcpS, a 4-phosphopantetheinyl transferase, activates two distinct acyl carrier proteins (ACPs) that are present in fatty acid synthase (FAS) systems FAS-I and FAS-II, the ACP-I domain and the mycobacterial ACP-II protein (ACPM), respectively. Mtb, the causal agent of tuberculosis (TB), and all other members of the Corynebacterineae family are unique in possessing both FAS systems to produce and to elongate fatty acids to mycolic acids, the hallmark of mycobacterial cell wall. Various steps in this process are prime targets for first-line anti-TB agents. A comparison of the Mtb AcpS structure determined here with those of other AcpS proteins revealed unique structural features in Mtb AcpS, namely, the presence of an elongated helix followed by a flexible loop and a moderately electronegative surface unlike the positive surface common to other AcpSs. A structure-based sequence comparison between AcpS and its ACP substrates from various species demonstrated that the proteins of the Corynebacterineae family display high sequence conservation, forming a segregated subgroup of AcpS and ACPs. Analysis of the putative interactions between AcpS and ACPM from Mtb, based on a comparison with the complex structure from Bacillus subtilis, showed that the Mtb AcpS and ACPM lack the electrostatic complementarity observed in B. subtilis. Taken together, the common characteristic of the Corynebacterineae family is likely reflected in the participation of different residues and interactions used for binding the Mtb AcpS to ACP-I and ACPM. The distinct features and essentiality of AcpS, as well as the mode of interaction with ACPM and ACP-I in Mtb, could be exploited for the design of AcpS inhibitors, which, similarly to other inhibitors of fatty acid synthesis, are expected to be effective anti-TB-specific drugs.     

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.     

Collecting and organizing systematic sets of protein data

Systems biology, particularly of mammalian cells, is data starved. However, technologies are now in place to obtain rich data, in a form suitable for model construction and validation, that describes the activities, states and locations of cell-signalling molecules. The key is to use several measurement technologies simultaneously and, recognizing each of their limits, to assemble a self-consistent compendium of systematic data.     

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.     

Bacterial swimming strategies and turbulence

Most bacteria in the ocean can be motile. Chemotaxis allows bacteria to detect nutrient gradients, and hence motility is believed to serve as a method of approaching sources of food. This picture is well established in a stagnant environment. In the ocean a shear microenvironment is associated with turbulence. This shear flow prevents clustering of bacteria around local nutrient sources if they swim in the commonly assumed "run-and-tumble" strategy. Recent observations, however, indicate a "back-and-forth" swimming behavior for marine bacteria. In a theoretical study we compare the two bacterial swimming strategies in a realistic ocean environment. The "back-and-forth" strategy is found to enable the bacteria to stay close to a nutrient source even under high shear. Furthermore, rotational diffusion driven by thermal noise can significantly enhance the efficiency of this strategy. The superiority of the "back-and-forth" strategy suggests that bacterial motility has a control function rather than an approach function under turbulent conditions.     

13-Demethyl-13-substituted-13,14-dihydroretinols as potential affinity labels of retinol-binding proteins: syntheses and stability studies

13-Demethyl-13-substituted-13,14-dihydroretinols were synthesized and their stability under various conditions was measured in order to evaluate whether they would be useful as affinity labels of retinol binding proteins and retinol metabolizing enzymes. The 13-chloro analog could not be isolated because it eliminated HCl under the Wittig reaction conditions of its preparation. The trans- and cis-13,14-epoxy analogs are stable in non-protic organic solvents, but undergo an elimination reaction under various chromatographic conditions and in mixtures of organic solvents with water or alcohol. The 13-hydroxy and 13-methoxy analogs are stable in aqueous solutions and are therefore suitable for biological studies.     

Determination of nalmefene in plasma by high-performance liquid chromatography with electrochemical detection and its application in pharmacokinetic studies

A method for measuring human plasma levels of nalmefene after oral and intravenous administration is presented. The method consists of a solid-phase extraction procedure followed by HPLC analysis. A cyanopropyl column is used for the solid-phase extraction and 60% (v/v) acetonitrile in dilute sodium pentanesulfonic acid solution for elution. The concentrated and filtered eluate is injected into the HPLC system, which is equipped with an electrochemical dual-electrode detector. A phenyl column is used in this HPLC system with a mobile phase containing 30% (v/v) acetonitrile in dilute sodium pentanesulfonic acid solution. A signal-to-noise ratio of 4.5 is obtained when a 1 ng/ml spiked plasma sample is analyzed. To determine the applicability of this method for human pharmacokinetic studies, nalmefene levels in plasma were measured at time points up to 24 h following oral and intravenous administration of 30 mg of nalmefene hydrochloride to two subjects. These studies demonstrated that the proposed method is sufficiently sensitive to study the pharmacokinetic profile of nalmefene in man.