Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism.

G-quadruplexes play important roles in cellular regulatory functions, but despite significant experimental and theoretical efforts, their folding mechanisms remain poorly understood. In this context, we developed a T-jump experiment to access the thermal denaturation and renaturation dynamics of short intramolecular G-quadruplexes in vitro, on the time scale of a few hundred milliseconds. With this new setup, we compared the thermal denaturation and renaturation kinetics of three antiparallel topologies made of the human telomeric sequences d[(5'-GGG(TTAGGG)3-3']/Na+ and d[5'-AGGG(TTAGGG)3-3']/Na+ and the thrombin-binding aptamer sequence d[5'-GGTTGGTGTGGTTGG-3']/K+, with those of the parallel topology made of the human CEB25 minisatellite d[5'-AAGGGTGGGTGTAAGTGTGGGTGGGT-3']/Na+. In all cases, exponential kinetics of the order of several hundred milliseconds were observed. Measurements performed for different initial temperatures revealed distinct denaturation and renaturation dynamics, ruling out a simple two-state mechanism. The parallel topology, in which all guanines adopt an anti conformation, displays much slower dynamics than antiparallel topologies associated with very low activation barriers. This behavior can be explained by the constrained conformational space due to the presence of the single-base propeller loops that likely hinders the movement of the coiled DNA strand and reduces the contribution of the entropy during the renaturation process at high temperatures.

Calcium fluoride micro cells for synchrotron radiation circular dichroism spectroscopy.

De-mountable calcium fluoride micro cells have been developed for use in synchrotron radiation circular dichroism (SRCD) spectroscopy. They have the advantages that they require only very small sample volumes (as low as 1 microliter) and have short path lengths (as small as 2 micrometers). Highly reproducible spectra can be obtained in consecutive loadings with these spacer-free, fixed path length cells, which enable low wavelength data collection.

Structure to 1.9 A resolution of a complex with herpes simplex virus type-1 thymidine kinase of a novel, non-substrate inhibitor: X-ray crystallographic comparison with binding of aciclovir.

Treatment of herpes infections with nucleoside analogues requires as an initial step the activation of the compounds by thymidine kinase. As an aid to developing more effective chemotherapy, both for treatment of recurrent herpes infection and in gene therapy systems where thymidine kinase is expressed, two high-resolution X-ray structures of thymidine kinase have been compared: one with the relatively poor substrate aciclovir (Zovirax), the other with a synthetic inhibitor having an N2-substituted guanine. Both compounds have similar binding modes in spite of their size difference and apparently distinct ligand properties.

Abnormal alterations in the metabolic patterns of patients on valproate therapy.

Four cases of abnormal metabolic patterns which were obtained from three infantile patients and one adult on valproate (valproic acid; 2-n-propyl-pentanoic acid) therapy are reported. Serum levels of valproate and 15 metabolites were measured by gas chromatography/mass spectrometry. A mentally retarded, 11-month-old boy developed an extremely altered metabolic profile after having been treated with valproate polytherapy for 3 months. The altered pattern included strongly elevated serum levels of the 4-ene as well as of the omega-/omega1-metabolites, with the beta-metabolites (2-ene; 2,3'-diene) being diminished. Two samples obtained previously had shown a common pattern. The infant died 3 weeks after the last sample had been taken. Two boys of the same age showed similar but less intense deviations in their metabolic profiles at the onset of valproate therapy. Within a few weeks they approached, in a step-wise fashion, the average pattern common for children under 3 years of age. The striking alterations were paralleled by the metabolic profiles of an adult patient who suffered from intrahepatic metastasis and renal insufficiency. From the close resemblance of the abnormal metabolic patterns it was concluded that liver dysfunction results in alteration of the whole metabolic system. Regular inspection of the entire profile of an individual might help to recognize conspicuous alterations in time to avoid severe side effects.

Establishing individual metabolite patterns for patients on valproate therapy.

The aim of this study was to establish individual metabolic profiles of patients receiving valproate (VPA) mono- or polytherapy in order to estimate inter- and intraindividual variability under normal conditions. Serum levels of VPA and 15 metabolites were measured by gas chromotography/mass spectrometry (GC/MS) with selected ion monitoring (SIM). Because of a huge inter-subject variability, calculating means for large epileptic populations resulted in broad and vague ranges for serum levels of VPA and its metabolites. It therefore remained difficult to recognize any significant alteration in the individual metabolic profile. Over long term periods, within-patient changes appeared to be much less intense than inherent interindividual differences. In epileptics consecutively receiving various forms of polytherapy, alterations in the metabolic profiles occurred. Therefore, integrating different kinds of co-medication into a single polytherapy group seemed to be inadequate. An adult patient on VPA monotherapy, suffering form intrahepatic metastasis and renal insufficiency, showed an extremely altered metabolic pattern, with the 4-ene and the omega-/omega1-metabolites being strongly elevated and the major beta-metabolites (E)-2-ene and (E,E)-2,3'-diene being significantly diminished. We suggest determining the individual metabolic profile, consisting of accessible major and minor metabolites, for every patient when VPA therapy has been started or been modified. The moment any clinical complications arise, the previously obtained specific pattern of the individual can be taken as reference in order to assess the possible presance of significant alterations which might indicate or even cause any severe side effects. There seems to be no need of monitoring metabolite levels of the average patient continuously except for the high risk group (e.g. infants under 3 years age receiving polytherapy) which exhibited the highest between-subject as well as within-patient variability.

Gastric hemorrhage and Kaposi's sarcoma treated with radiotherapy.

A 66-year-old man, with advanced alcoholic liver disease with Kaposi's sarcoma involving the stomach and the skin of the lower extremities, developed upper gastrointestinal hemorrhage. He was treated with radiation therapy to the stomach and the skin with rapid resolution of the gastric lesions, as demonstrated by serial endoscopy.

Amphipols from A to Z.

Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep integral membrane proteins (MPs) water soluble. In this review, we discuss their structure and solution behavior; the way they associate with MPs; and the structure, dynamics, and solution properties of the resulting complexes. All MPs tested to date form water-soluble complexes with APols, and their biochemical stability is in general greatly improved compared with MPs in detergent solutions. The functionality and ligand-binding properties of APol-trapped MPs are reviewed, and the mechanisms by which APols stabilize MPs are discussed. Applications of APols include MP folding and cell-free synthesis, structural studies by NMR, electron microscopy and X-ray diffraction, APol-mediated immobilization of MPs onto solid supports, proteomics, delivery of MPs to preexisting membranes, and vaccine formulation.

VUV irradiation effects on proteins in high-flux synchrotron radiation circular dichroism spectroscopy.

Synchrotron radiation circular dichroism (SRCD) spectroscopy is emerging as an important new tool in structural molecular biology. Previously we had shown that in lower-flux SRCD instruments, such as UV1 at ISA and beamline 3.1 at the SRS, vacuum ultraviolet (VUV) radiation damage to proteins was not evident after exposure over a period of hours. No effects were detected in either the protein primary or the secondary structures. However, with the development of high-flux beamlines, such as CD12 at the SRS, this issue has been revisited because of changes observed in the SRCD spectra of consecutive scans of protein samples obtained on this high-flux beamline. Experiments have been designed to distinguish between two different possible mechanisms: (i) photoionization causing free radicals or secondary electrons producing degradation of the protein, and (ii) local heating of the sample resulting in protein denaturation. The latter appears to be the principal source of the signal deterioration.

A DNA-porphyrin minor-groove complex at atomic resolution: the structural consequences of porphyrin ruffling.

The crystal structure of a B-type DNA hexanucleotide duplex complexed with the porphyrin molecule nickel-[tetra-N-methyl-pyridyl] porphyrin has been solved by multiwavelength anomalous diffraction phasing and refined to an R factor of 11.5% at a resolution of 0.9 A. The structure has been solved and refined as two crystallographically independent duplexes, stacked end to end. Contrary to expectation, the porphyrin molecule is not intercalated into the duplex but is stacked onto the ends of the two-duplex stack. The porphyrin molecule is highly buckled as a consequence of the nickel coordination, which produces large changes in local DNA structure. A second mode of porphyrin binding is apparent as a consequence of crystal packing, which places the ligand in the minor groove of an adjacent duplex. This structure thus provides, to our knowledge, the first atomic visualization of minor-groove binding for a porphyrin molecule. The geometry of groove binding provides a ready explanation for porphyrin-induced DNA strand cleavage at deoxyribose residues.

CDtool-an integrated software package for circular dichroism spectroscopic data processing, analysis, and archiving.

CDtool is a software package written to facilitate circular dichroism (CD) spectroscopic studies on both conventional lab-based instruments and synchrotron beamlines. It takes format-independent input data from any type of CD instrument, enables a wide range of standard and advanced processing methods, and, in a single user-friendly graphics-based package, takes raw data through the entire processing procedure and, importantly, uses data-mining techniques to retain in the final output all the information associated with the processing. It permits the facile comparison of data obtained from different instruments without the need for reformatting and displays it in graphical formats suitable for publication. It also includes the ability to automatically archive the processed data. This latter feature may be especially useful in light of recent funding institution directives with regard to data sharing and archiving and requirements for "good practice" and "traceability" within the pharmaceutical industry. In addition, CDtool includes a means of interfacing with protein data bank coordinate files and calculating secondary structures from them using alternate definitions and algorithms. This feature, along with a function that permits the facile production of new reference databases, enables the creation of specialized databases for secondary structural analyses of specific types of proteins. Thus the CDtool software not only enables rapid data processing and analyses but also includes many enhanced features not available in other CD data processing/analysis packages.

[The effect of valproic acid monotherapy on behavior and cognitive performance of children with idiopathic generalized epilepsy]. / Der Einfluss einer Valproinsäure-Monotherapie auf das Verhalten und die kognitive Leistungsfähigkeit von Kindern mit idiopathischen generalisierten Epilepsien.

Valproic acid (VPA) currently plays an important role in the treatment of several different types of epilepsy. Especially in children and adolescents, VPA is used because of a minimum of adverse effects and generally little impact on cognitive and psychomotor functions. However, reports in the literature regarding the influence of VPA on behavior and cognitive performance and on EEG parameters vary widely. We investigated the effect of VPA monotherapy on behavioral components (attention, concentration, inhibitory control), cognitive efficiency (motor reaction time, learning, retention) and evoked potentials in 19 children aged 6 to 14 years with idiopathic generalized epilepsy and compared the results with those of healthy controls matched for age. In addition, we analyzed the serum levels of VPA and some of the VPA metabolites in all of the children with epilepsy immediately before the psychophysiological assessments. Our results show marked differences between the children with epilepsy and the healthy controls in all types of behavior and cognitive performance assessed. Abnormal behavior (disturbances of attention and concentration, impulsive behavior patterns) and significant changes in evoked potentials appear to be correlated with serum levels of VPA and certain VPA metabolites.

mtDNA depletion and impairment of mitochondrial function in a case of a multisystem disorder including severe myopathy.

The ratio of mtDNA and a nuclear reference gene was estimated by Southern blotting in the skeletal muscle DNA of a 3-year-old girl who suffered from congenital brain damage, focal epilepsy, hepatomegaly, malabsorption syndrome and severe myopathy. The signal ratio of mtDNA versus 18S rDNA was 22% of the mean value obtained from controls. No major deletions or insertions were found and the MERRF, MELAS and NARP mutations were ruled out. Mitochondrial DNA-encoded enzyme activities and mitochondrial respiration were reduced. The analysis of the NAD(P)H and flavoprotein redox states of intact fibres revealed the presence of mitochondrial dysfunction. In tissue sections a moderate elevation of type I and type II fibre diameter variation was detected, aberrant NADH- and succinate dehydrogenase staining and some ragged red fibres. This suggested that a mitochondrial disorder caused by a decrease in the amount of intact wild-type mtDNA was responsible for the severe myopathy.

Exploring the active site of herpes simplex virus type-1 thymidine kinase by X-ray crystallography of complexes with aciclovir and other ligands.

Antiherpes therapies are principally targeted at viral thymidine kinases and utilize nucleoside analogs, the triphosphates of which are inhibitors of viral DNA polymerase or result in toxic effects when incorporated into DNA. The most frequently used drug, aciclovir (Zovirax), is a relatively poor substrate for thymidine kinase and high-resolution structural information on drugs and other molecules binding to the target is therefore important for the design of novel and more potent chemotherapy, both in antiherpes treatment and in gene therapy systems where thymidine kinase is expressed. Here, we report for the first time the binary complexes of HSV-1 thymidine kinase (TK) with the drug molecules aciclovir and penciclovir, determined by X-ray crystallography at 2.37 A resolution. Moreover, from new data at 2.14 A resolution, the refined structure of the complex of TK with its substrate deoxythymidine (R = 0.209 for 96% of all data) now reveals much detail concerning substrate and solvent interactions with the enzyme. Structures of the complexes of TK with four halogen-containing substrate analogs have also been solved, to resolutions better than 2.4 A. The various TK inhibitors broadly fall into three groups which together probe the space of the enzyme active site in a manner that no one molecule does alone, so giving a composite picture of active site interactions that can be exploited in the design of novel compounds.