Determination of <Superscript>13</Superscript>C CSA Tensors: Extension of the Model-independent Approach to an RNA Kissing Complex Undergoing Anisotropic Rotational Diffusion in Solution

Chemical shift anisotropy (CSA) tensor parameters have been determined for the protonated carbons of the purine bases in an RNA kissing complex in solution by extending the model-independent approach [Fushman, D., Cowburn, D. (1998) J. Am. Chem. Soc. 120, 7109–7110]. A strategy for determining CSA tensor parameters of heteronuclei in isolated X–H two-spin systems (X = ¹³C or ¹⁵N) in molecules undergoing anisotropic rotational diffusion is presented. The original method relies on the fact that the ratio κ₂=R₂^auto/R₂^cross of the transverse auto- and cross-correlated relaxation rates involving the X CSA and the X–H dipolar interaction is independent of parameters related to molecular motion, provided rotational diffusion is isotropic. However, if the overall motion is anisotropic κ₂ depends on the anisotropy D_||/D_⊥ of rotational diffusion. In this paper, the field dependence of both κ₂ and its longitudinal counterpart κ₁=R₁^auto/R₁^cross are determined. For anisotropic rotational diffusion, our calculations show that the average κ_av = 1/2 (κ₁+κ₂), of the ratios is largely independent of the anisotropy parameter D_||/D_⊥. The field dependence of the average ratio κ_av may thus be utilized to determine CSA tensor parameters by a generalized model-independent approach in the case of molecules with an overall motion described by an axially symmetric rotational diffusion tensor.     

High genetic diversity despite drastic bottleneck in a critically endangered,long‐lived seabird,the Mascarene Petrel Pseudobulweria aterrima

The Mascarene Petrel Pseudobulweria aterrima is a critically endangered seabird endemic to Reunion Island, with an extremely small population suffering several threats. Fifteen polymorphic microsatellite loci were isolated from this species to analyse genetic diversity, estimate contemporary effective population size, search for evidence of a population bottleneck and see whether results support the hypothesis that life history traits could preserve allelic diversity in small populations. Results from 22 individuals found grounded as a consequence of light pollution highlight a surprisingly high genetic diversity, an absence of inbreeding, a contemporary effective population size estimated at approximately 1211 individuals and a probable bottleneck around 10 000 generations ago. Additional studies on genetic diversity and structure from a larger number of samples are thus required to evaluate the evolutionary potential of this critically endangered species.     

Neuropeptide Y and α-MSH Circadian Levels in Two Populations with Low Body Weight: Anorexia Nervosa and Constitutional Thinness

Context

Anorexia nervosa (AN) presents an adaptive appetite regulating profile including high levels of ghrelin and 26RFa (orexigenic) and low levels of leptin and PYY (anorexigenic). However, this adaptive mechanism is not effective in promoting food intake. The NPY/proopiomelanocortin (POMC) system plays a crucial role in the regulation of feeding behavior as NPY is the most potent orexigenic neuropeptide identified so far and as the POMC-derived peptide α-MSH drastically reduces food intake, and this peptidergic system has not been thoroughly studied in AN.

Objective

The aim of the present study was thus to investigate whether a dysfunction of the NPY/POMC occurs in two populations with low body weight, AN and constitutional thinness (CT).

Design and Settings

This was a cross-sectional study performed in an endocrinological unit and in an academic laboratory.

Investigated Subjects

Three groups of age-matched young women were studied: 23 with AN (AN), 22 CT and 14 normal weight controls.

Main Outcome Measures

Twelve-point circadian profiles of plasma NPY and α-MSH levels were measured in the three groups of investigated subjects.

Results

No significant circadian variation of NPY was detected between the three groups. Plasma α-MSH levels were significantly lower in AN (vs controls) all over the day. The CT group, compared to controls, presented lower levels of α-MSH in the morning and the evening, and an important rise during lunchtime.

Conclusion

In AN patients, the NPY system is not up-regulated under chronic undernutrition suggesting that this may play a role in the inability of anorectic women to adapt food intake to their energy demand. In contrast, low circadian α-MSH levels integrate the adaptive profile of appetite regulation of this disease. Finally, in CT women, the important α-MSH peak detected during lunchtime could explain why these patients are rapidly food satisfied.     

A single-run, real-time PCR for detection and identification of Borrelia burgdorferi sensu lato species, based on the hbb gene sequence

Lyme borreliosis is the most important vector-borne disease caused by spirochetes within the Borrelia burgdorferi sensu lato (B. burgdorferi sl) complex. There is strong evidence that different species of this group of genetically diverse spirochetes are involved in distinct clinical manifestations of the disease. In order to differentiate species within this bacterial complex, we developed a real-time-PCR protocol, which targets the hbb gene. We designed a fluorescein-labeled probe specific of a region of this gene harboring a polymorphism linked to species. An internally Red640 labeled primer allowed a fluorescence resonance energy transfer to occur. The sensitivity of this method was in the range of 10 bacteria per assay. After amplification, a melting curve was generated for genotyping. Analysis of these melting curves clearly allowed the distinction between the main European species of B. burgdorferi sl. One hundred seventy tick extracts were analysed by this hbb-based method and in parallel by amplification of the 5S-23S intergenic spacer and RFLP analyses. There was a good correlation between these two methods. We conclude that this hbb-based real-time-PCR is suitable for epidemiological studies on field-collected ticks, although rare mutations in the genomic sequence spanned by the probe could lead to misidentification.     

Structure of a human pre-40S particle points to a role for RACK1 in the final steps of 18S rRNA processing

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3′-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.     

Active immunization to tumor necrosis factor-α is effective in treating chronic established inflammatory disease: a long-term study in a transgenic model of arthritis

Introduction  

Passive blockade of tumor necrosis factor-alpha (TNF-α) has demonstrated high therapeutic efficiency in chronic inflammatory diseases, such as rheumatoid arthritis, although some concerns remain such as occurrence of resistance and high cost. These limitations prompted investigations of an alternative strategy to target TNF-α. This study sought to demonstrate a long-lasting therapeutic effect on established arthritis of an active immunotherapy to human (h) TNF-α and to evaluate the long-term consequences of an endogenous anti-TNF-α response.     

Flow-conditioned HUVECs support clustered leukocyte adhesion by coexpressing ICAM-1 and E-selectin

Endothelial sequestration of circulating monocytes is a key event in early atherosclerosis. Hemodynamics is proposed to regulate monocyte-endothelial cell interactions by direct cell activation and establishment of flow environments that are conducive or prohibitive to cell-cell interaction. We investigated fluid shear regulation of monocyte-endothelial cell adhesion in vitro using a disturbed laminar shear system that models in vivo hemodynamics characteristic of lesion-prone vascular regions. Human endothelial cell monolayers were flow conditioned for 6 h before evaluation of monocyte adhesion under static and dynamic flow conditions. Results revealed a distinctive clustered cell pattern of monocyte adhesion that strongly resembles in vivo leukocyte adhesion in early- and late-stage atherosclerosis. Clustered monocyte cell adhesion correlated with endothelial cells coexpressing intercellular adhesion molecule-1 (ICAM-1) and E-selectin as result of a flow-induced, selective upregulation of E-selectin expression in a subset of ICAM-1-expressing cells. Clustered monocyte cell adhesion assayed under static conditions exhibited a spatial variation in size and frequency of occurrence, which demonstrates differential regulation of endothelial cell adhesiveness by the local flow environment. Dynamic adhesion studies conducted with circulating monocytes resulted in clustered cell adhesion only within the disturbed flow region, where the monocyte rate of motion is sufficiently low for cell-cell interaction. These studies provide evidence and reveal mechanisms of local hemodynamic regulation of endothelial adhesiveness and endothelial monocyte interaction that lead to localized monocyte adhesion and potentially contribute to the focal origin of arterial diseases such as atherosclerosis.     

3-Methylcrotonylglycine Disrupts Mitochondrial Energy Homeostasis and Inhibits Synaptic Na+,K+-ATPase Activity in Brain of Young Rats

Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an inherited metabolic disease biochemically characterized by accumulation and high urinary excretion of 3-methylcrotonylglycine (3MCG), and also of 3-hydroisovalerate in lesser amounts. Affected patients usually have neurologic dysfunction, brain abnormalities and cardiomyopathy, whose pathogenesis is still unknown. The present study investigated the in vitro effects of 3MCG on important parameters of energy metabolism, including CO₂ production from labeled acetate, enzyme activities of the citric acid cycle, as well as of the respiratory chain complexes I–IV (oxidative phosphorylation), creatine kinase (intracellular ATP transfer), and synaptic Na⁺,K⁺-ATPase (neurotransmission) in brain cortex of young rats. 3MCG significantly reduced CO₂ production, implying that this compound compromises citric acid cycle activity. Furthermore, 3MCG diminished the activities of complex II-III of the respiratory chain, mitochondrial creatine kinase and synaptic membrane Na⁺,K⁺-ATPase. Furthermore, antioxidants were able to attenuate or fully prevent the inhibitory effect of 3MCG on creatine kinase and synaptic membrane Na⁺,K⁺-ATPase activities. We also observed that lipid peroxidation was elicited by 3MCG, suggesting the involvement of free radicals on 3MCG-induced effects. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production, creatine kinase for intracellular energy transfer, and Na⁺,K⁺-ATPase for the maintenance of the cell membrane potential, the present data indicate that 3MCG potentially impairs mitochondrial brain energy homeostasis and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by 3-methylcrotonyl-CoA carboxylase deficiency.