Selective ligand-induced stabilization of active and desensitized parathyroid hormone type 1 receptor conformations

For many G protein-coupled receptors, agonist-induced activation is followed by desensitization, internalization, and resensitization. In most cases, these processes are dependent upon interaction of agonist-occupied receptor with cytoplasmic beta-arrestins. The ligand-induced intramolecular rearrangements of the receptor responsible for the desensitized versus active conformational states, which dictate both the pharmacological properties of ligands and the biological activity of G protein-coupled receptors, have not been fully elucidated. Here, we identify specific interactions between parathyroid hormone (PTH)-related protein and the human PTH type 1 receptor (PTH1Rc) and the related receptor conformational changes that lead to beta-arrestin-2-mediated desensitization. PTH-related protein analogs modified at position 1 induced selective stabilization of the active G protein-coupled state of the receptor, resulting in lack of beta-arrestin-2 recruitment to the cell membrane, sustained cAMP signaling, and absence of ligand-receptor complex internalization. Mechanistically, the ligands modified at position 1, interacting with the extracellular end of helix VI of PTH1Rc, produced a translocation of transmembrane helices V and VI that differed from that induced by the cognate agonist, resulting in significantly different conformations of the third intracellular loop. These results show how specific interactions between PTH1Rc and its ligands may stabilize distinct conformational states, representing either the active G protein-coupled or a desensitized beta-arrestin-coupled receptor state. In addition, they establish that sustained biological activity of PTH1Rc may be induced by appropriately designed agonist ligands.     

Modulation of Synapsin I Gene Expression in Rat Cortical Neurons by Extracellular Matrix

1. Neuronal differentiation depends on crosstalk between genetic program and environmental cues. In this study we tried to dissect this complex interplay by culturing neurons from fetal rat brain cortices in a chemically defined, neuron-specific, medium and on different substrata, either artificial (poly-D-lysine) or natural.2. Among the extracellular matrix compounds used in this study, two (collagen I and fibronectin) allowed only a weak attachment of cortical neurons to the substratum, while the others (collagen IV, laminin, and basal lamina from Engelbreth-Holm-Swarm sarcoma) allowed both firm attachment and moderate to extensive neurite outgrowth from neuronal cell bodies.3. By using synapsin I gene expression as a parameter of neuronal differentiation, we found that neurite outgrowth and neuronal differentiation are not linearly linked. Synapsin I gene expression, in fact, was maximal in neurons cultured on laminin, while the fastest neuritic outgrowth was recorded in cultures on poly-D-lysine.4. The data presented in this paper are consistent with the hypothesis that the extracellular matrix plays an active role in modulating the differentiative program of neurons.     

A 23-year study of mortality and development of co-morbidities in patients with obesity undergoing bariatric surgery (laparoscopic gastric banding) in comparison with medical treatment of obesity

Although obesity-related type 2 diabetes mellitus (T2DM) and sarcopenia in the elderly have been increasing worldwide, the associations among visceral fat accumulation, skeletal muscle indices (mass, strength, and quality) and cardiovascular diseases in T2DM remain poorly investigated. We enrolled 183 Japanese T2DM inpatients (126 men, 57 women; mean age 64.7 ± 12.6 years, ± SD). The estimated-visceral fat area (eVFA) and skeletal muscle mass were measured by each device using bioelectrical impedance analysis method. We also measured grip strength by dynamometer and motor nerve conduction velocity (MCV). We analyzed the difference in skeletal muscle indices between T2DM patients with and without visceral fat accumulation, and examined the impact of skeletal muscle indices on cardiovascular diseases in patients with visceral fat accumulation. The prevalence of sarcopenia defined by the Consensus of Asian Working Group for Sarcopenia and low skeletal muscle mass were both lower in the visceral fat accumulation (+) group than in (−) group. However, the prevalence of weak hand grip strength was similar in the visceral fat accumulation (−) and (+) groups, indicating that considerable patients with visceral fat accumulation had weak grip strength in spite of fair skeletal muscle mass. Muscle quality [grip strength (kg)/arm muscle mass (kg)] was significantly lower in patients with visceral fat accumulation. Multiple regression analysis identified eVFA, MCV and sex as significant and independent determinants of muscle quality. In visceral fat accumulation (+) group, the patients with low muscle quality had longer duration of diabetes, lower eGFR, higher serum adiponectin, lower MCV and higher prevalence of cardiovascular diseases, compared to the patients with high muscle quality. Finally, sex- and age-adjusted models showed significant association between low muscle quality and cardiovascular diseases in all subjects (odds ratio 2.28, p = 0.012), especially in patients with visceral fat accumulation (odds ratio 2.72, p = 0.018). T2DM patients with visceral fat accumulation had low muscle quality, and patients with low muscle quality were more affected with cardiovascular diseases.     

The human topoisomerase 1B Arg634Ala mutation results in camptothecin resistance and loss of inter-domain motion correlation

Human topoisomerase 1B, the unique target of the natural anticancer compound camptothecin, catalyzes the unwinding of supercoiled DNA by introducing transient single strand nicks and providing covalent protein–DNA adducts. The functional properties and the drug reactivity of the single Arg634Ala mutant have been investigated in comparison to the wild type enzyme. The mutant is characterized by an identical relaxation and cleavage rate but it displays resistance to camptothecin as indicated by a viability assay of the yeast cells transformed with the mutated protein. The mutant also displays a very fast religation rate that is only partially reduced by the presence of the drug, suggesting that this is the main reason for its resistance. A comparative analysis of the structural–dynamical properties of the native and mutant proteins by molecular dynamics simulation indicates that mutation of Arg634 brings to a loss of motion correlation between the different domains and in particular between the linker and the C-terminal domain, containing the catalytic tyrosine residue. These results indicate that the loss of motion correlation and the drug resistance are two strongly correlated events.     

Usefulness Of Ivabradine To Treat "unexpected" Heart Failure Caused By "acute" Right Ventricular Pacing

We present the case of a patient with a heart failure episode induced by acute right ventricular pacing. After reversal of beta-blockers because of chronic obstructive pulmonary disease (COPD) exacerbation, the following sinus tachycardia caused a 2:1 atrioventricular block and consequent continuous right ventricular pacing. He was treated with the selective I(f) inhibitor ivabradine, that reduced both ventricular pacing percentage and heart rate without affecting atrioventricular conduction. Ivabradine may be a valuable option in treatment of patients with atrioventricular conduction disturbances.     

Critical role of Ca2+ ions in the reaction mechanism of Euphorbia characias peroxidase

A cationic peroxidase was isolated and characterized from the latex of the perennial Mediterranean plant Euphorbia characias. The purified enzyme contained one heme prosthetic group identified as ferric iron-protoporphyrin IX. In addition, the purified peroxidase contained 1 mol of endogenous calcium per mol of enzyme; removal of this calcium ion resulted in almost complete loss of the enzyme activity. However, when excess Ca(2+) was added to the native enzyme the catalytic efficiency was enhanced by 3 orders of magnitude. The mechanism of activation was studied using a wide range of spectroscopic and analytic techniques. Analysis of the steady state by stopped-flow measurements suggests that the main effect of calcium ions is to favor the oxidation of the ferric enzyme by hydrogen peroxide to form compound I, whereas the other steps of the catalytic cycle seem to be affected to a lesser extent. UV/vis absorption spectra and CD measurements show that the heme iron is pentacoordinated high-spin in native enzyme and remains so after the binding of Ca(2+). Only minor changes in the secondary or tertiary structure of the protein could be detected by fluorescence or CD measurements in the presence of Ca(2+) ions, except for a significant perturbation of the Fe(3+) inner sphere geometry, as detected by EPR measurements. We propose that Ca(2+) binding to a low affinity site induces a reorientation of the distal histidine changing the almost inactive form of Euphorbia peroxidase to a high activity form. This is the first example of a peroxidase that responds as an on/off switch to variations in the external Ca(2+) level.     

Vegetation of tuscan ultramafic soils in relation to edaphic and physical factors

Vegetation and soil sampling were carried out in 80 plots located in five different ultramafic (serpentine) sites of Tuscany, central Italy. The physical and chemical features of each plot were determined and the species composition and cover recorded. The exchangeable fraction of soil metals was analysed because it gives a measure of their concentrations available to plants. The plots were classified by cluster analysis and ANOVA was used to compare the environmental variables of the groups of plots. Canonical correspondence analysis was used to detect the principal factors for gradients of species composition within the plant communities. A higher content of exchangeable metals was found under the more evolved and structured plant communities, suggesting that serpentine vegetation of Tuscany is not strongly limited by soil metals, such as chromium, cobalt, nickel and magnesium, typically associated with ultramafic soils. The low nutrient content of the soils and drought stress mainly due to topographical features, appear to have a more significant role in determining the typical scattered vegetation of the Tuscan ultramafics.     

The role of niche breadth, resource availability and range position on the life history of butterflies

We analysed the relationship between three life history characteristics (mobility, length of flight period and body size) and niche breadth (larval host plant specificity and adult habitat breadth), resource availability (distribution and abundance of host plants) and range position (distance between the northernmost distribution record and southernmost point of Finland) of the butterfly fauna of Finland. The data is based on literature and questionnaires. Often in across species studies phylogeny may create spurious relationships between life-history and ecological variables. We took the phylogenetic relatedness of butterfly species into account by analysing the data with phylogenetically independent contrasts (CAIC method). Butterfly mobility was positively related to the niche breadth, resource availability and range position. The length of the flight period was negatively related to the range position, indicating that the species at the northern edge of their distribution range have shorter flight period than species which are further way from the range edge. After controlling for the phylogenetic relatedness we found no significant correlations between body size and niche breadth, resource availability or range position. We suggest that the relationship between the length of the flight period and range position may arise as a consequence of lower hatching asynchrony in edge species as a result of lower environmental variance in larval growth conditions. Our results on the mobility suggest that there is selection pressure towards lower migration rate in species that have restricted niche breadth, low resource availability and in species that are on the northern edge of their geographical distribution range. In such species, selection against mobile individuals is likely to result from the decreased probability of finding another habitat patch suitable for egg laying.     

Iron-nickel mixed metal nitrido clusters: Synthesis and solid state structure of the anions [HFe5NiN(CO)14] and [HFe4Ni2N(CO)13]

The two clusters [HFe₅NiN(CO)₁₄]²⁻ (1) and [HFe₄Ni₂N(CO)₁₃]²⁻ (2) were obtained by reaction of [Fe₄N(CO)₁₂]⁻ and [Ni₆(CO)₁₂]²⁻ in refluxing MeCN and EtCN, respectively, along with other Fe-Ni mixed metal clusters. Their solid state structures were determined on the [PPh₄]⁺ salts, and both have an octahedral metal cage, containing an interstitial nitrogen atom. The two Ni atoms in 2 are cis, with a Ni-Ni separation of 2.724(1) Å. The two anions have different stereochemistry of the carbonyl ligands: in 1, five CO’s are semi-bridging, and the remaining nine are terminal; in 2 there are three asymmetric bridging and ten terminal ligands (two for each iron and one for each nickel). The hydride ligands were located in the final difference maps, both bridging a Ni-Fe edge of the clusters but, thanks to the better quality of the diffraction data, the metal-hydrogen distances were refined only in 2. In this cluster, the Fe-H and Ni-H bond lengths are 1.77(2) and 1.79(2) Å, respectively.     

High efficiency transient and stable transformation by optimized DNA microinjection into Nicotiana tabacum protoplasts

An efficient system has been established that allows well controlledDNA microinjection into tobacco (Nicotiana tabacum) mesophyllprotoplasts with partially regenerated cell walls and subsequentanalysis of transient as well as stable expression of injectedreporter genes in particular targeted cells or derived clones.The system represents an effective tool to study parametersimportant for the successful transformation of plant cells bymicroinjection and other techniques. Protoplasts were immobilizedin a very thin layer of medium solidified with agarose or alginate.DNA microinjection was routinely monitored by coinjecting FITC-dextranand aimed at the cytoplasm of target cells. The injection procedurewas optimized for efficient delivery of injection solution intothis compartment. Cells were found to be at the optimal stagefor microinjection about 24 h after immobilization in solidmedium. Embedded cells could be kept at this stage for up to4 d by incubating them at 4 C in the dark. Within 1 h successfuldelivery of injection, solution was routinely possible into20–40 cells. Following cytoplasmic coinjection of FITC-dextran and pSHI913,a plasmid containing the neo (neomycin phosphotransferase II)gene, stably transformed, paromomycin-resistant clones couldbe recovered through selection. Transgenic tobacco lines havebeen established from such clones. Injection solutions containingpSHI913 at a concentration of either 50 µg ml^–1or 1 mg ml^–1 have been tested. With 1 mg ml^–1 plasmidDNA the percentage of resistant clones per successfully injectedcell was determined to be about 3.5 times higher. Incubationof embedded protoplasts at 4C before microinjection was foundto reduce the percentage of resistant clones obtained per injectedcell Protoplasts were immobilized above a grid pattern and the locationof injected cells was recorded by Polaroid photography. Thefate of particular targeted cells could be observed. Isolationand individual culture of clones derived from injected cellswas possible. Following cytoplasmic coinjection of FITC-dextranand 1 mg ml^–1 plasmid DNA on average about 20% of thetargeted cells developed into microcalli and roughly 50% ofthese calli were stably transformed. Transient expression ofthe firefly luciferase gene (Luc) was nondestructively analysed24 h after injection of pAMLuc. Approximately 50% of the injectedcells that were alive at this time point expressed the Luc genetransiently. Apparently, stable integration of the injectedgenes occurred in essentially all transiently expressing cellsthat developed into clones. Key words: DNA microinjection, firefly luciferase, FITCdextran, Nicotiana tabacum, protoplast transformation