Thermodynamics of RNA melting,one base pair at a time

The melting of base pairs is a ubiquitous feature of RNA structural transitions, which are widely used to sense and respond to cellular stimuli. A recent study employing solution nuclear magnetic resonance (NMR) imino proton exchange spectroscopy provides a rare base-pair-specific view of duplex melting in the Salmonella FourU RNA thermosensor, which regulates gene expression in response to changes in temperature at the translational level by undergoing a melting transition. The authors observe “microscopic” enthalpy–entropy compensation—often seen “macroscopically” across a series of related molecular species—across base pairs within the same RNA. This yields variations in base-pair stabilities that are an order of magnitude smaller than corresponding variations in enthalpy and entropy. A surprising yet convincing link is established between the slopes of enthalpy–entropy correlations and RNA melting points determined by circular dichroism (CD), which argues that unfolding occurs when base-pair stabilities are equalized. A single AG-to-CG mutation, which enhances the macroscopic hairpin thermostability and folding cooperativity and renders the RNA thermometer inactive in vivo, spreads its effect microscopically throughout all base pairs in the RNA, including ones far removed from the site of mutation. The authors suggest that an extended network of hydration underlies this long-range communication. This study suggests that the deconstruction of macroscopic RNA unfolding in terms of microscopic unfolding events will require careful consideration of water interactions.     

Determination of metformin in plasma using a new ion pair solid phase extraction technique and ion pair liquid chromatography

This article describes the development of the first ion pair solid phase extraction technique (IPSPE), which has been applied to the extraction of metformin from plasma samples. In addition an ion pair chromatographic method was developed for the specific HPLC determination of metformin. Several extraction and HPLC methods have been described previously for metformin, however, most of them did not solve the problems associated with the high polarity of this drug. Drug recovery in the developed method was found to be more than 98%. The limit of detection and limit of quantification was 3 and 5 ng/ml, respectively. The intraday and interday precision (measured by coefficient of variation, CV%) was always less than 9%. The accuracy (measured by relative error, R.E.%) was always less than 6.9%. Stability analysis showed that metformin is stable for at least 3 months when stored at -70 degrees C. The method has been applied to 150 patient samples as part of a medication adherence study.     

Double replica electroblotting: a method to produce two replicas from one gel

Electroblotting is a method by which proteins or nucleic acids, separated by electrophoresis, are transferred, also by electrophoresis, from a gel to a so-called transfer medium, e.g a nitrocellulose membrane. In some experiments, it is desirable to be able to obtain more than one replica from each gel and it has now proved possible to produce two replicas, which are almost identical, from one gel. This is achieved by applying one membrane on each side of the gel and change the direction of the current several times in such a way that the efficient transfer time is short in the beginning of the electroblotting and is increased for each cycle. This procedure will be referred to as 'double replica electroblotting'. Proteins were transferred at 100 V and the duration of an experiment with 2 h efficient transfer time in each direction was 7 h. The gel was more efficiently depleted of proteins after double replica electroblotting as compared to ordinary electrotransfer in one direction. Cathodically migrating proteins are also trapped on the membranes with this technique. Double replica electroblotting was used to produce two replicas from ordinary sodium dodecyl sulfate polyacrylamide gels as well as from 2-dimensional gels.     

Using experimental information to produce a model of the transmembrane domain of the ion channel phospholamban.

Molecular models of the transmembrane domain of the phospholamban pentamer have been generated by a computational method that uses the experimentally measured effects of systematic single-site mutations as a guiding force in the modeling procedure. This method makes the assumptions that 1) the phospholamban transmembrane domain is a parallel five-helix bundle, and 2) nondisruptive mutation positions are lipid exposed, whereas 3) disruptive or partially disruptive mutations are not. Our procedure requires substantially less computer time than systematic search methods, allowing rapid assessment of the effects of different experimental results on the helix arrangement. The effectiveness of the approach is investigated in test calculations on two helix-dimer systems of known structure. Two independently derived sets of mutagenesis data were used to define the restraints for generating models of phospholamban. Both resulting models are left-handed, highly symmetrical pentamers. Although the overall bundle geometry is very similar in the two models, the orientation of individual helices differs by approximately 50 degrees, resulting in different sets of residues facing the pore. This demonstrates how differences in restraints can have an effect on the model structures generated, and how the violation of these restraints can identify inconsistent experimental data.     

The current status of the adaptive response to ionizing radiation in mammalian cells

The results of numerous studies indicate that cells can become refractory to the detrimental effect of ionizing radiation when previously exposed to a low, “adapting dose”;. This phenomenon has been termed an “adaptive response”; to ionizing radiation. It has been postulated that the induced radioresistance is due to the induction of DNA repair systems which efficiently protect the adapted cells from the effects of a subsequent, high “challenging dose”;. However, a direct proof of this hypothesis is still lacking. The analyzed endpoints include chromosomal aberrations, survival, mutations, genetic instability and DNA damage repair measured by the comet assay. Frequently contradictory results were published by different authors. For example some authors observed a reduced frequency of apoptosis in adapted cells, whereas others reported the opposite. The source of variablity of the adaptive response in human lymphocytes remains unresolved. While there is no doubt that an adapting dose can trigger some protecting mechanisms within the cell it appears that there is no single, universal mechanism of the adaptive response that is valid for all cell types and irradiation conditions.     

The current status of NMR imaging

A brief account is given of the current status of nuclear-magnetic-resonance (NMR) imaging for medical purposes. The procedures in present use for two- and three-dimensional NMR imaging are outlined and examples given. The quality of images approaches that of computerized tomography X-ray scans and demonstrates superior tissue discrimination and pathological discrimination. Guidelines for safe operation are discussed. Clinical trials of commercial NMR scanners at an advanced stage.     

The current status of REH theory

Summary The recent evaluation by Fitch (1980) of REH theory for macromolecular divergence is a severely erroneous and distorted analysis of our work over the past decade. We reply to those distortions here. At present, there is no factual basis for believing Fitch's assessment that corrections which move evolutionary estimates of total mutations fixed closer to the true distance must do so at the expense of an increased variance sufficient to compromise the value of the improvement. By direct calculation the variance in the estimates of total mutations fixed given by REH theory is comparable to that of other models now in the literature for the case in which genetic events are equiprobable. A general argument is given that suggests that, as we consider more and more carefully the selective, functional, and structural constraints on the evolution of genes and proteins, this variance may be expected to decrease toward a lower bound.     

The current status of chemical systematics

Chemical systematics sets out to interpret the phylogenetic implications of the occurrence and distribution of secondary metabolites. In this review, a number of the major contributions from the 1960's and 1970's are identified and re-assessed in the light of recent evidence gained from DNA studies. It is shown that for the most part conclusions drawn on the basis of secondary metabolite distribution have been confirmed by the new techniques and it is concluded that chemical systematics can continue to provide useful insights into plant phylogeny.