Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli

G protein-coupled receptors (GPCRs) are notoriously difficult to express, particularly in microbial systems. Using GPCR fusions with the green fluorescent protein (GFP), we conducted studies to identify bacterial host effector genes that result in a general and significant enhancement in the amount of membrane-integrated human GPCRs that can be produced in Escherichia coli. We show that coexpression of the membrane-bound AAA+ protease FtsH greatly enhances the expression yield of four different class I GPCRs, irrespective of the presence of GFP. Using this new expression system, we produced 0.5 and 2 mg/L of detergent-solubilized and purified full-length central cannabinoid receptor (CB1) and bradykinin receptor 2 (BR2) in shake flask cultures, respectively, two proteins that had previously eluded expression in microbial systems.     

Self-Assembly of Phenylalanine Oligopeptides: Insights from Experiments and Simulations

Studies of peptide-based nanostructures provide general insights into biomolecular self-assembly and can lead material engineering toward technological applications. The diphenylalanine peptide (FF) self-assembles into discrete, hollow, well ordered nanotubes, and its derivatives form nanoassemblies of various morphologies. Here we demonstrate for the first time, to our knowledge, the formation of planar nanostructures with β-sheet content by the triphenylalanine peptide (FFF). We characterize these structures using various microscopy and spectroscopy techniques. We also obtain insights into the interactions and structural properties of the FF and FFF nanostructures by 0.4-μs, implicit-solvent, replica-exchange, molecular-dynamics simulations of aqueous FF and FFF solutions. In the simulations the peptides form aggregates, which often contain open or ring-like peptide networks, as well as elementary and network-containing structures with β-sheet characteristics. The networks are stabilized by polar and nonpolar interactions, and by the surrounding aggregate. In particular, the charged termini of neighbor peptides are involved in hydrogen-bonding interactions and their aromatic side chains form “T-shaped” contacts, as in three-dimensional FF crystals. These interactions may assist the FF and FFF self-assembly at the early stage, and may also stabilize the mature nanostructures. The FFF peptides have higher network propensities and increased aggregate stabilities with respect to FF, which can be interpreted energetically.     

Analysis of xyloglucan endotransglycosylase/hydrolase (XTH) genes from allotetraploid (Gossypium hirsutum) cotton and its diploid progenitors expressed during fiber elongation

Multiple cellular pathways have been shown to be involved during fiber initiation and elongation stages in the cultivated allotetraploid cotton (Gossypium hirsutum). The cell wall enzymes xyloglucan endotransglycosylase/hydrolases (XTH) have been reported to be associated with the biosynthesis of the cell wall and the growth of cotton fibers, probably regulating the plasticity of the primary cell wall. Among various cotton fiber cDNAs found to be preferentially expressed in cotton fibers, a xyloglucan endotransglycosylase (XTH) cDNA was significantly up-regulated during the elongation stage of cotton fiber development. In the present study, we isolated and characterized genomic clones encoding cotton XTH from cultivated cotton (Gossypium hirsutum) and its diploid progenitors (Gossypium arboreum and Gossypium raimondii), designated GhXTH1-1, GhXTH1-2, GaXTH1 and GrXTH, respectively. In addition, we isolated and characterized, by in silico methods, the putative promoter of XTH1 from Gossypium hirsutum. Sequence analysis revealed more than 50% homology to XTH's at the protein level. DNA gel blot hybridization indicated that at least two copies of GhXTH1 are present in Gossypium hirsutum whereas the diploid progenitor species Gossypium arboreum and Gossypium raimondii has only a single copy. Quantitative real-time PCR and high-resolution melting experiments indicated that in Gossypium hirsutum cultivars, in cotton fibers during early stages of fiber elongation specifically expressing only the GhXTH1-1 gene and expression levels of GhXTH1-1 in fibers varies among cultivars differing in fiber percentage and fiber length.     

Comparison of surface electromyographic activity of erector spinae before and after the application of central posteroanterior mobilisation on the lumbar spine

Lumbar spine accessory movements, used by therapists in the treatment of patients with low back pain, is thought to decrease paravertebral muscular activity; however there is little research to support this suggestion. This study investigated the effects of lumbar spine accessory movements on surface electromyography (sEMG) activity of erector spinae.A condition randomised, placebo controlled, repeated measures design was used. sEMG measurements were recorded from 36 asymptomatic subjects following a control, placebo and central posteroanterior (PA) mobilisation to L3 each for 2 min. The therapist stood on a force platform while applying the PA mobilisation to quantify the force used. The PA mobilisation applied to each subject had a mean maximum force of 103.3 N, mean amplitude of force oscillation of 41.1 N, and a frequency of 1.2 Hz. Surface electromyographic data were recorded from the musculature adjacent to L3, L5 and T10.There were statistically significant reductions of 15.5% (95% CI: 8.0–22.5%) and 17.8% (95% CI: 12.9–22.4%) in mean sEMG values following mobilisation compared with the control and placebo, respectively.This study demonstrates that a central PA mobilisation to L3 results in a statistically significant decrease in the sEMG activity of erector spinae of an asymptomatic population.     

Relationship of CSF neurotransmitter metabolite levels to disease severity and disability in multiple sclerosis

Axonal degeneration and brain tissue loss occur during disease progression in multiple sclerosis (MS) and are expected to influence neurotransmitter activities, with consequences on neurologic and psychiatric symptomatology. We searched for relationships of disease duration, disability, and severity of MS patients to CSF levels of the major metabolites of noradrenaline, dopamine, and serotonin, MHPG, methoxyhydroxyphenylglycol (MHPG), homovanillic acid, and 5-hydroxyindoleacetic acid (5-HIAA), respectively, in 39 patients with relapsing–remitting (RR) MS in remission, and 26 patients with progressive (PR) MS. Disability and Disease Severity were assessed by the Expanded Disability Status Scale (EDSS) and the Multiple Sclerosis Severity Score (MSSS). Compared with the levels of 50 control subjects, MHPG levels were not different in either MS group, correlated negatively to duration of illness and number of relapses in the RRMS group, but not to EDSS score or to MSSS. Homovanillic acid levels were significantly lower only in the PRMS group, with a negative correlation to duration of illness, and a strong negative correlation to EDSS score, but not to MSSS. 5-HIAA was significantly lower in both RRMS and PRMS groups. In the RRMS group, 5-HIAA levels were negatively related to EDSS and to MSSS. Multiple regression analyses revealed a significant association of MHPG to duration of illness, and a strong negative association of 5-HIAA to MSSS rather than to EDSS. The strong negative correlation of MSSS to CSF 5-HIAA levels in RRMS group of patients indicates that deficits in central serotonergic activity are related to the rate of disability accumulation in RRMS, and could be linked to the reported reduction of disease activity by serotonergic drugs.     

Syntaxonomy and Synecology of <Emphasis Type="Italic">Quercus coccifera</Emphasis> Mediterranean Shrublands in Greece

Quercus coccifera (kermes oak) is the most common species of the Mediterranean maquis with a wide distribution across the Mediterranean Basin. This paper presents a syntaxonomic overview of the Q. coccifera plant communities in the Mediterranean zone of Greece (Quercetea ilicis) based on the classification of 221 relevés from 34 (17 continental and 17 insular) mountainous areas throughout Greece. Two associations and eight sub-associations are described and presented in a synoptic constancy table. Querco cocciferae–Pistacietum lentisci is the most widespread, is found in the entire continental Greece and most islands, and is further subdivided into five sub-associations reflecting primarily local peculiarities in the disturbance regime and the influence of local floristic elements. Rhamno lycioidis–Cocciferetum (Rivas Goday & Rivas-Martínez 1954), on the other hand, is geographically confined on the island of Crete and is further subdivided into three sub-associations, reflecting differences in the annual precipitation, and they are characterized by the presence of many phryganic and grazing-resistant species. Climate and the anthropogenic pressure have been identified to be the most important factors determining the structure and the floristic composition of Q. coccifera Mediterranean shrublands of Greece.     

How Many 3D Structures Do We Need to Train a Predictor？

It has been shown that the progress in the determination of membrane protein structure grows exponentially, with approximately the same growth rate as that of the water-soluble proteins. In order to investigate the effect of this, on the performance of prediction algorithms for both α-helical and β-barrel membrane proteins, we conducted a prospective study based on historical records. We trained separate hidden Markov models with different sized training sets and evaluated their performance on topology pred...