Structural and Functional Studies on the Overproduced L11 Protein from Thermus thermophilus

The L11 ribosomal protein from Thermus thermophilus (TthL11) has been overproduced and purified to homogeneity using a two-step purification protocol. The overproduced protein carries a similar methylation pattern at Lys-3 as does its homolog from Escherichia coli. Chymotrypsin digested only a small part of the TthL11 protein and did not cleave TthL11 into two peptides, as in the case of EcoL11, but produced only a single N-terminal peptide. Tryptic digestion of TthL11 also produced an N-terminal peptide, in contrast to the C-terminal peptide obtained with L11 from Bacillus stearothermophilus. The recombinant protein forms a specific complex with a 55-nt 23S rRNA fragment known to interact with members of the L11 family from several organisms. Cooperative binding of TthL11 and thiostrepton to 23S rRNA leads to an increased protection of TthL11 from tryptic digestion. The similar structural and biochemical properties as well as the significant homology between L11 from E. coli and B. stearothermophilus with the corresponding protein from Thermus thermophilus indicate an evolutionarily conserved protein important for ribosome function.     

Molecular analysis of resistance to acaricidal spirocyclic tetronic acids in Tetranychus urticae: CYP392E10 metabolizes spirodiclofen,but not its corresponding enol

Spirodiclofen is one of the most recently developed acaricides and belongs to the new family of spirocyclic tetronic acids (ketoenols). This new acaricidal family is an important chemical tool in resistance management strategies providing sustainable control of spider mites such as Tetranychus urticae. Spirodiclofen targets lipid biosynthesis mediated by direct inhibition of acetyl coenzyme A carboxylase (ACCase). In this study, we investigated two genetically distant spider mite strains with high resistance to spirodiclofen. Despite the strong resistance levels to spirodiclofen (up to 680-fold), only limited cross-resistance with other members of this group such as spiromesifen and spirotetramat could be detected. Amplification and sequencing of the ACCase gene from resistant and susceptible strains did not reveal common non-synonymous mutations, and expression levels of ACCase were similar in both resistant and susceptible strains, indicating the absence of target-site resistance. Furthermore, we collected genome-wide expression data of susceptible and resistant T. urticae strains using microarray technology. Analysis of differentially expressed genes revealed a broad response, but within the overlap of two resistant strains, several cytochrome P450s were prominent. Quantitative PCR confirmed the constitutive over-expression of CYP392E7 and CYP392E10 in resistant strains, and CYP392E10 expression was highly induced by spirodiclofen. Furthermore, stage specific expression profiling revealed that expression levels were not significantly different between developing stages, but very low in eggs, matching the age-dependent resistance pattern previously observed. Functional expression of CYP392E7 and CYP392E10 confirmed that CYP392E10 (but not CYP392E7) metabolizes spirodiclofen by hydroxylation as identified by LC–MS/MS, and revealed cooperative substrate binding and a K_m of 43 μM spirodiclofen. CYP392E10 also metabolizes spiromesifen, but not spirotetramat. Surprisingly, no metabolism of the hydrolyzed spirodiclofen-enol metabolite could be detected. These findings are discussed in the light of a likely resistance mechanism.     

Idiopathic and normal lateral lumbar curves: muscle effects interpreted by 12th rib length asymmetry with pathomechanic implications for lumbar idiopathic scoliosis

Background

The historical view of scoliosis as a primary rotation deformity led to debate about the pathomechanic role of paravertebral muscles; particularly multifidus, thought by some to be scoliogenic, counteracting, uncertain, or unimportant. Here, we address lateral lumbar curves (LLC) and suggest a pathomechanic role for quadrates lumborum, (QL) in the light of a new finding, namely of 12th rib bilateral length asymmetry associated with idiopathic and small non-scoliosis LLC.

Methods

Group 1: The postero-anterior spinal radiographs of 14 children (girls 9, boys 5) aged 9–18, median age 13 years, with right lumbar idiopathic scoliosis (IS) and right LLC less that 10°, were studied. The mean Cobb angle was 12° (range 5–22°). Group 2: In 28 children (girls 17, boys 11) with straight spines, postero-anterior spinal radiographs were evaluated similarly to the children with the LLC, aged 8–17, median age 13 years. The ratio of the right/left 12th rib lengths and it’s reliability was calculated. The difference of the ratio between the two groups was tested; and the correlation between the ratio and the Cobb angle estimated. Statistical analysis was done using the SPSS package.

Results

The ratio’s reliability study showed intra-observer +/−0,036 and the inter-observer error +/−0,042 respectively in terms of 95 % confidence limit of the error of measurements. The 12th rib was longer on the side of the curve convexity in 12 children with LLC and equal in two patients with lumbar scoliosis. The 12th rib ratios of the children with lumbar curve were statistically significantly greater than in those with straight spines. The correlation of the 12th rib ratio with Cobb angle was statistically significant. The 12th thoracic vertebrae show no axial rotation (or minimal) in the LLC and no rotation in the straight spine group.

Conclusions

It is not possible, at present, to determine whether the 12th convex rib lengthening is congenitally lengthened, induced mechanically, or both. Several small muscles are attached to the 12th ribs. We focus attention here on the largest of these muscles namely, QL. It has attachments to the pelvis, 12th ribs and transverse processes of lumbar vertebrae as origins and as insertions. Given increased muscle activity on the lumbar curve convexity and similar to the interpretations of earlier workers outlined above, we suggest two hypotheses, relatively increased activity of the right QL muscle causes the LLCs (first hypothesis); or counteracts the lumbar curvature as part of the body’s attempt to compensate for the curvature (second hypothesis). These hypotheses may be tested by electrical stimulation studies of QL muscles in subjects with lumbar IS by revealing respectively curve worsening or correction. We suggest that one mechanism leading to relatively increased length of the right 12 ribs is mechanotransduction in accordance with Wolff’s and Pauwels Laws.

     

Ca2+ Binding/Permeation via Calcium Channel,CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein,CD36, and Fatty Acid Metabolism

Ca²⁺ permeation and/or binding to the skeletal muscle L-type Ca²⁺ channel (Ca_V1.1) facilitates activation of Ca²⁺/calmodulin kinase type II (CaMKII) and Ca²⁺ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α₁ subunit of Ca_V1.1) gene that abolishes Ca²⁺ binding within the Ca_V1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that Ca_V1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Ca_v1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized Ca_V1.1-mediated pathway that regulates energy utilization in skeletal muscle.     

Zinc(II) complexes derived from pyridine-2-carbaldehyde thiosemicarbazone and (1E)-1-pyridin-2-ylethan-1-one thiosemicarbazone. Synthesis, crystal structures and antiproliferative activity of zinc(II) complexes

The complexes [ZnCl(2)(HFoTsc)xH(2)O], [Zn(FoTsc)(2)], [ZnCl(2)(HAcTsc)xH(2)O] and [Zn(AcTsc)(2)], where HFoTsc and HAcTsc is pyridine-2-carbaldehyde thiosemicarbazone and (1E)-1-pyridin-2-ylethan-1-one thiosemicarbazone respectively, have been prepared and structurally characterized by means vibrational, and NMR ((1)H and (13)C) spectroscopy. The crystal structures of the complexes [ZnCl(2)(HFoTsc)xH(2)O], [Zn(AcTsc)(2)] and [ZnCl(2)(HAcTsc)xH(2)O] have been determined by X-ray crystallography. The metal co-ordination geometry of [ZnCl(2)(HFoTsc)xH(2)O] and [ZnCl(2)(HAcTsc)xH(2)O] is described as distorted square pyramidal and the two complexes are self-assembled via pi-->pi stacking interactions and intermolecular hydrogen bonds. In these two cases molecular recognition of the hydrogen bonds leads to aggregation and a supramolecular assembly of infinite two-dimensional network. The metal co-ordination geometry of [Zn(AcTsc)(2)] is described as distorted octahedral configuration in a trans-N(2)-cis-N(1)-cis-S configuration. HFoTsc and HAcTsc and the zinc complexes have been evaluated for antiproliferative activity in vitro against the cells of two human cancer cell lines: MCF-7 (human breast cancer cell line), T24 (bladder cancer cell line) and a mouse fibroblast L-929 cell line. The cytotoxic activity shown by these compounds indicates that coupling of HFoTsc and HAcTsc to Zn(II) metal center result in metallic complexes with important biological properties since they display IC(50) values in a microM range similar to or better than that of the antitumor drug cis-platin and are considered as agents with potential antitumor activity candidates for further stages of screening in vitro and/or in vivo.