cis-Prenyltransferase AtCPT6 produces a family of very short-chain polyisoprenoids in planta

cis-Prenyltransferases (CPTs) comprise numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite the composition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating. The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6-overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT-Rer2 since it restores the growth but not protein glycosylation in rer2Δ yeast. This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots.     

Mechanism of stabilization of Bacillus circulans xylanase upon the introduction of disulfide bonds

The introduction of disulfide bonds has been used as a strategy to enhance the stability of Bacillus circulans xylanase. The transition temperature of the S100C/N148C (DS1), V98C/A152C (DS2), and A1GC/G187,C188 (cXl) in comparison to the wild type was increased by 5.0, 4.1 and 3.8 degrees C, respectively. Interestingly, a combination of two disulfide bonds of DS1 and cXl (cDS1, circular disulfide 1) led to a 12 degrees C increase in the transition temperature. Importantly, an increase in the melting point and DeltaDeltaG values of the cDS1 mutant was cooperative. These results suggest that the mechanism of stabilization by disulfide bonds under irreversible denaturation condition is achieved through: (1) a change in the rate-limiting step on the denaturation pathway; (2) destabilizing the unfolded state without affecting the relative rate constants on the denaturation pathway (like cXl mutant); and (3) or combination of the two (cDS1 mutant).     

Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy

Lipid rafts in the plasma membrane, domains rich in cholesterol and sphingolipids, have been implicated in a number of important membrane functions. Detergent insolubility has been used to define membrane "rafts" biochemically. However, such an approach does not directly contribute to the understanding of the size and the lifetime of rafts, dynamics of the raft-constituent molecules, and the function of rafts in the membrane in situ. To address these issues, we have developed pulse EPR spin labeling and single molecule tracking optical techniques for studies of rafts in both artificial and cell membranes. In this review, we summarize our results and perspectives obtained by using these methods. We emphasize the importance of clearly distinguishing small/unstable rafts (lifetime shorter than a millisecond) in unstimulated cells and stabilized rafts induced by liganded and oligomerized (GPI-anchored) receptor molecules (core receptor rafts, lifetime over a few minutes). We propose that these stabilized rafts further induce temporal, greater rafts (signaling rafts, lifetime on the order of a second) for signaling by coalescing other small/unstable rafts, including those in the inner leaflet of the membrane, each containing perhaps one molecule of the downstream effector molecules. At variance with the general view, we emphasize the importance of cholesterol segregation from the liquid-crystalline unsaturated bulk-phase membrane for formation of the rafts, rather than the affinity of cholesterol and saturated alkyl chains. In the binary mixture of cholesterol and an unsaturated phospholipid, cholesterol is segregated out from the bulk unsaturated liquid-crystalline phase, forming cholesterol-enriched domains or clustered cholesterol domains, probably due to the lateral nonconformability between the rigid planar transfused ring structure of cholesterol and the rigid bend of the unsaturated alkyl chain at C9-C10. However, such cholesterol-rich domains are small, perhaps consisting of only several cholesterol molecules, and are short-lived, on the order of 1-100 ns. We speculate that these cholesterol-enriched domains may be stabilized by the presence of saturated alkyl chains of sphingomyelin or glycosphingolipids, and also by clustered raft proteins. In the influenza viral membrane, one of the simplest forms of a biological membrane, the lifetime of a protein and cholesterol-rich domain was evaluated to be on the order of 100 micro, again showing the short lifetime of rafts in an unstimulated state. Finally, we propose a thermal Lego model for rafts as the basic building blocks for signaling pathways in the plasma membrane.     

Kinin B1 receptor antagonists with multi-enzymatic resistance properties

The kinin B, receptor has been implicated in a variety of pathological states; therefore, potent, selective, and specific antagonists with prolonged duration of action in vivo are needed. Using R-715 (AcLys[D-beta-Nal(7),Ile(8)] desArg9BK) as a template, new peptides containing alpha-MePhe in position 5, Oic in position 2, and AcOrn instead of AcLys at the N-terminal were prepared and tested for their antagonist potency, their selectivity, and their specificity for the kinin B1 receptor. In vitro metabolic stabilities toward aminopeptidase M (from human plasma), aminopeptidase P (from human platelets), and angiotensin-converting enzyme (purified from rabbit lung) were also investigated. The results of this study indicate that the three modifications applied separately are as well tolerated as they are when present conjointly in the template R-715. Indeed, pA2 values of R-715 (ranging from 8.40 to 8.5) do not differ significantly from the analogues R-954 and R-955 (both ranging from 8.4 to 8.6) when measured at kinin B1 receptors from rabbit aortas and human umbilical veins. Moreover, the chemical modifications utilized in the peptides R-954 and R-955 have provided resistance against aminopeptidases M and P, as well as the angiotensin-converting enzyme, unlike the early (e.g., Lys[Leu8]desArg9BK) and more recent (e.g., R-715, B-9858) generations of B, receptor antagonists. Ongoing in vivo assays will validate the assumption that the analogues R-954 and R-955 have a prolonged duration of action.     

Glutathione S transferase M1 andT1 genotypes and susceptibility to smoking related larynx cancer

Susceptibility to smoking related larynx cancer has been suggested to be associated with genetically determined differences in the ability to detoxify carcinogens present in tobacco smoke. The genetic polymorphisms of glutathione S-transferases, involved in the metabolic inactivation of, for example, tobacco derived carcinogens, have been recognized as potential risk modifiers in various environmentally induced malignancies, including larynx cancer. We employed PCR-based methods to determine the distribution of the GSTM 1 and G STT1 null genotypes in 171 larynx cancer patients and 180 controls to examine further their potential role in individual susceptibility to this neoplasm. The GSTM 1 null genotype was found in 49 1 % of the cases and 57 7 % of the controls and the GSTT1 null genotype in 17 5 % of the cases and 21 7 % of the controls, respectively. Larynx cancer risk associated with the lack of GST M 1 (OR = 0 7; 95 % CI: 0 5-1 1) or GSTT1 (OR = 0 8; 95 % CI: 0 5-1 3) was not significantly affected by age, smoking status, or cancer progression. Although this study thus suggests no role for the G STM 1 and GSTT1 gene polymorphisms in individual susceptibility to smoking-related larynx cancer, due to its relatively small sample size more data are required before any definite conclusions can be drawn.     

The GW/WG repeats of Drosophila GW182 function as effector motifs for miRNA-mediated repression

The control of messenger RNA (mRNA) function by micro RNAs (miRNAs) in animal cells requires the GW182 protein. GW182 is recruited to the miRNA repression complex via interaction with Argonaute protein, and functions downstream to repress protein synthesis. Interaction with Argonaute is mediated by GW/WG repeats, which are conserved in many Argonaute-binding proteins involved in RNA interference and miRNA silencing, from fission yeast to mammals. GW182 contains at least three effector domains that function to repress target mRNA. Here, we analyze the functions of the N-terminal GW182 domain in repression and Argonaute1 binding, using tethering and immunoprecipitation assays in Drosophila cultured cells. We demonstrate that its function in repression requires intact GW/WG repeats, but does not involve interaction with the Argonaute1 protein, and is independent of the mRNA polyadenylation status. These results demonstrate a novel role for the GW/WG repeats as effector motifs in miRNA-mediated repression.     

Functions of Cholesterol and the Cholesterol Bilayer Domain Specific to the Fiber-Cell Plasma Membrane of the Eye Lens

The most unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content. Cholesterol saturates the bulk phospholipid bilayer and induces formation of immiscible cholesterol bilayer domains (CBDs) within the membrane. Our results (based on EPR spin-labeling experiments with lens-lipid membranes), along with a literature search, have allowed us to identify the significant functions of cholesterol specific to the fiber-cell plasma membrane, which are manifest through cholesterol–membrane interactions. The crucial role is played by the CBD. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly. The CBD raises the barrier for oxygen transport across the fiber-cell membrane, which should help to maintain a low oxygen concentration in the lens interior. It is hypothesized that the appearance of the CBD in the fiber-cell membrane is controlled by the phospholipid composition of the membrane. Saturation with cholesterol smoothes the phospholipid-bilayer surface, which should decrease light scattering and help to maintain lens transparency. Other functions of cholesterol include formation of hydrophobic and rigidity barriers across the bulk phospholipid-cholesterol domain and formation of hydrophobic channels in the central region of the membrane for transport of small, nonpolar molecules parallel to the membrane surface. In this review, we provide data supporting these hypotheses.     

The influence of the type of contraction on the masseter muscle EMG power spectrum

Different behaviours of the EMG power spectrum across increasing force levels have been reported for the masseter muscle. A factor that could explain these different behaviours may be the type of contraction used, as was recently shown for certain upper limb muscles⁵. The purpose of this study was to compare, between two types of isometric contractions, the behaviour of EMG power spectrum statistics (median frequency (MF) and mean power frequency (MPF)) obtained across increasing force levels. Ten women exerted, while biting in the intercuspal position, three 5 s ramp contractions that increased linearly from 0 to 100% of the maximal voluntary contraction (MVC). They also completed three step contractions (constant EMG amplitude) at each of the following levels: 20, 40, 60 and 80% MVC. EMG signals from the masseter muscle were recorded with miniature surface electrodes. The RMS, as well as the MPF and MF of the power spectrum were calculated at 20, 40, 60 and 80% MVC for each type of contraction. As expected, the RMS values showed similar increases with increasing levels of effort for both types of contractions. Different behaviours for both MPF (contraction^*force interaction, ANOVA, P<0.05) and MF (contraction^*force interaction, ANOVA, P>0.05) across increasing levels of effort were found between the two types of contraction. The use of step contractions gave rise to a decrease of both MPF and MF with increasing force, while the use of ramp contractions gave rise to an increase in both statistics up to at least 40% MVC followed by a decrease at higher force levels. These findings suggest that the type of contraction used does influence the behaviour of the spectral statistics across increasing force levels and that this could explain the differences obtained in previous studies for the masseter muscle.     

Repression of protein synthesis by miRNAs: how many mechanisms?

MicroRNAs are approximately 21-nucleotide-long regulators of gene expression that gain access to their target mRNAs by complementary base pairing. Recent studies have revealed that animal microRNAs might take diverse routes to repress gene expression, affecting both target mRNA levels and translation. Mechanistic details of microRNA-mediated repression are starting to emerge but a comprehensive picture of the inhibition, and particularly the effects on mRNA translation, is still lacking. Recent data support different microRNA mechanisms and a role for cytoplasmic processing bodies in the degradation and storage of mRNAs targeted by microRNA regulators.