Neonatal sympathectomy reduces NADPH oxidase activity and vascular resistance in spontaneously hypertensive rat kidneys

Neonatal sympathectomy reduces arterial pressure in spontaneously hypertensive rats (SHR). In SHR transplanted with a kidney from sympathectomized SHR, arterial pressure was lower and less Na+ sensitive than in SHR transplanted with a kidney from hydralazine-treated SHR. This study was performed to identify underlying renal mechanisms. Tests for differential renal mRNA expression of nine a priori selected genes revealed robust differences for renal medullary expression of the NADPH oxidase subunit p47phox. Therefore, we investigated the effects of neonatal sympathectomy on renal mRNA expression of NADPH oxidase subunits, NADPH oxidase activity, and renal function. In 10-wk-old sympathectomized SHR fed a 0.6% NaCl diet, medullary p47phox and gp91phox expression was 40% less than in hydralazine-treated SHR. Also, after a 1.8% NaCl diet, medullary p47phox mRNA expression was lower in sympathectomized than in hydralazine-treated SHR. We found lower cortical (-30%, P<0.01) and medullary (-30%, P<0.05) NADPH oxidase activities in sympathectomized than in hydralazine-treated or untreated SHR. Glomerular filtration rate, renal blood flow, medullary blood flow, and fractional Na+ excretion in kidney grafts from sympathectomized and hydralazine-treated donors (n=8 per group) were similar at baseline and in response to a 20-mmHg rise in renal perfusion pressure. Renal vascular resistance was lower in kidneys from sympathectomized than hydralazine-treated donors (25+/-2 vs. 32+/-4 mmHg.min.ml-1, P<0.05). The results indicate that the sympathetic nervous system contributes to the level of renal NADPH oxidase activity and to perinatal programming of alterations in renal vascular function that lead to elevated renal vascular resistance in SHR.     

A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle

The regulation of striated muscle contraction involves changes in the interactions of troponin and tropomyosin with actin thin filaments. In resting muscle, myosin-binding sites on actin are thought to be blocked by the coiled-coil protein tropomyosin. During muscle activation, Ca2+ binding to troponin alters the tropomyosin position on actin, resulting in cyclic actin-myosin interactions that accompany muscle contraction. Evidence for this steric regulation by troponin-tropomyosin comes from X-ray data [Haselgrove, J.C., 1972. X-ray evidence for a conformational change in the actin-containing filaments of verterbrate striated muscle. Cold Spring Habor Symp. Quant. Biol. 37, 341-352; Huxley, H.E., 1972. Structural changes in actin and myosin-containing filaments during contraction. Cold Spring Habor Symp. Quant. Biol. 37, 361-376; Parry, D.A., Squire, J.M., 1973. Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles. J. Mol. Biol. 75, 33-55] and electron microscope (EM) data [Spudich, J.A., Huxley, H.E., Finch, J., 1972. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J. Mol. Biol. 72, 619-632; O'Brien, E.J., Gillis, J.M., Couch, J., 1975. Symmetry and molecular arrangement in paracrystals of reconstituted muscle thin filaments. J. Mol. Biol. 99, 461-475; Lehman, W., Craig, R., Vibert, P., 1994. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368, 65-67] each with its own particular strengths and limitations. Here we bring together some of the latest information from EM analysis of single thin filaments from Pirani et al. [Pirani, A., Xu, C., Hatch, V., Craig, R., Tobacman, L.S., Lehman, W. (2005). Single particle analysis of relaxed and activated muscle thin filaments. J. Mol. Biol. 346, 761-772], with synchrotron X-ray data from non-overlapped muscle fibres to refine the models of the striated muscle thin filament. This was done by incorporating current atomic-resolution structures of actin, tropomyosin, troponin and myosin subfragment-1. Fitting these atomic coordinates to EM reconstructions, we present atomic models of the thin filament that are entirely consistent with a steric regulatory mechanism. Furthermore, fitting the atomic models against diffraction data from skinned muscle fibres, stretched to non-overlap to preclude crossbridge binding, produced very similar results, including a large Ca2+-induced shift in tropomyosin azimuthal location but little change in the actin structure or apparent alteration in troponin position.     

A chemoinformatics analysis of hit lists obtained from high-throughput affinity-selection screening

The high-throughput affinity-selection screening platform SpeedScreen was recently reported by the Novartis Institutes for BioMedical Research as a homogeneous, label-free screening technology with mass-spectrometry readout. SpeedScreen relies on the screening of compound mixtures with various target proteins and uses fast size-exclusion chromatography to separate target-bound from unbound substances. After disintegration of the target-binder complex, the binder molecules are identified by their molecular masses using liquid chromatography/mass spectrometry. The authors report an analysis of the molecular properties of hits obtained with SpeedScreen on 26 targets screened within the past few years at Novartis using this technology. Affinity-based SpeedScreen is a robust high-throughput screening technology that does not accumulate frequent hitters or potential covalent binders. The hits are representative of the most commonly identified scaffold classes observed for known drugs. Validated SpeedScreen hits tend to be enriched on more lipophilic and larger-molecular-weight compounds compared to the whole library. The potential for a reduced SpeedScreen screening set to be used in case only limited protein quantities are available is evaluated. Such a reduced compound set should also maximize the coverage of the high-performing regions of the chemical property and class spaces; chemoinformatics methods including genetic algorithms and divisive K-means clustering are used for this aim.     

p28, A Novel ERGIC/cis Golgi Protein, Required for Golgi Ribbon Formation

The mammalian Golgi apparatus consists of individual cisternae that are stacked in a polarized manner to form the compact zones of the Golgi. Several stacks are linked to form a ribbon via dynamic lateral bridges. The determinants required for maintaining the characteristic Golgi structure are incompletely understood. Here, we have characterized p28, a new γ-subfamily member of p24 membrane proteins. p28 localized to endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and cis Golgi and accumulated in the ERGIC upon Brefeldin A treatment, typical for a protein cycling in the early secretory pathway. p28 interacted with a subset of p24 proteins. Its depletion by small interfering RNA (siRNA) led to fragmentation of the Golgi without affecting the overall organization of microtubules but considerably reducing the amount of acetylated tubulin. The distribution of COPI and tethers, including GM130, was not affected. At the ultrastructural level, the Golgi fragments appeared as mini-stacks with apparently unchanged cis - trans topology. Golgi fragmentation did not impair anterograde or retrograde traffic. Fluorescence recovery after photobleaching (FRAP) experiments revealed that silencing p28 prevents protein exchange between Golgi stacks during reassembly after Brefeldin A-induced Golgi breakdown. These results show that the formation of a Golgi ribbon requires the structural membrane protein p28 in addition to previously identified SNAREs, coat proteins and tethers.     

Vulnerable taxa of European Plecoptera (Insecta) in the context of climate change

We evaluated 516 species and/or subspecies of European stoneflies for vulnerability to climate change according to autoecological data. The variables considered were stream zonation preference, altitude preference, current preference, temperature range preference, endemism and rare species. Presence in ecoregions was used to analyse the vulnerability of taxa in relation to their distribution. We selected the variables that provided information on vulnerability to change in climate. Thus, we chose strictly crenal taxa, high-altitude taxa, rheobionts, cold stenotherm taxa, micro-endemic taxa and rare taxa. Our analysis showed that at least 324 taxa (62.79%) can be included in one or more categories of vulnerability to climate change. Of these, 43 taxa would be included in three or more vulnerability categories, representing the most threatened taxa. The most threatened species and the main factors affecting their distribution are discussed. Endangered potamal species, with populations that have decreased mainly as a consequence of habitat alteration, also could suffer from the effects of climate change. Thus, the total number of taxa at risk is particularly high. Not only are a great diversity of European stoneflies concentrated in the Alps, Pyrenees and Iberian Peninsula, but so are the most vulnerable taxa. These places are likely to be greatly affected by climate change according to climate models. In general, an impoverishment of European Plecoptera taxa will probably occur as a consequence of climate change, and only taxa with wide tolerance ranges will increase in abundance, resulting in lower overall faunal diversity.     

Tautomerization of 2-nitroso-N-arylanilines by coordination as N,N′-chelate ligands to rhenium(i) complexes and the anticancer activity of newly synthesized oximine rhenium(i) complexes against human melanoma and leukemia cells in vitro

The synthesis, structural characterization and biological activity of eight ortho-quinone(N-aryl)-oximine rhenium(i) complexes are described. The reaction of the halogenido complexes (CO)₅ReX (X=Cl (4), Br (5)) with 2-nitroso-N-arylanilines {(C₆H₃ClNO)NH(C₆H₄R)} (R = p-Cl, p-Me, o-Cl, H) (3a–d) in tetrahydrofurane (THF) yields the complexes fac-(CO)₃XRe{(C₆H₃ClNO)NH(C₆H₄R)} (6a–d, 7a–d) with the tautomerized ligand acting as a N,N′-chelate. The substitution of two carbonyl ligands leads to the formation of a nearly planar 5-membered metallacycle. During coordination the amino-proton is shifted to the oxygen of the nitroso group which can be observed in solution for 6 and 7 by ¹H NMR spectroscopy and in solid state by crystal structure analysis. After purification, all compounds have been fully characterized by their ¹H and ¹³C NMR, IR, UV/visible (UV/Vis) and mass spectra. The X-ray structure analyses revealed a distorted octahedral coordination of the CO, X and N,N′-chelating ligands for all Re(i) complexes. Biological activity of four oximine rhenium(i) complexes was assessed in vitro in two highly aggressive cancer cell lines: human metastatic melanoma A375 and human chronic myelogenous leukemia K562. Chlorido complexes (6a and 6c) were more efficient than bromido compounds (7d and 7b) in inducing apoptotic cell death of both types of cancer cells. Melanoma cells were more susceptible to tested rhenium(i) complexes than leukemia cells. None of the ligands (3a–d) showed any significant anticancer activity.     

Socio-Spatial Relationships in Dairy Cows

Farm animals may serve as models for evaluating social networks in a controlled environment. We used an automated system to track, at fine temporal and spatial resolution (once per minute, ±50 cm) every individual in six herds of dairy cows ( Bos taurus ). We then analysed the data using social network analyses. Relationships were based on non-random attachment and avoidance relationships in respect to synchronous use and distances observed in three different functional areas (activity, feeding and lying). We found that neither synchrony nor distance between cows was strongly predictable among the three functional areas. The emerging social networks were tightly knit for attachment relationships and less dense for avoidance relationships. These networks loosened up from the feeding and lying area to the activity area, and were less dense for relationships based on synchronicity than on median distance with respect to node degree, relative size of the largest cluster, density and diameter of the network. In addition, synchronicity was higher in dyads of dairy cows that had grown up together and shared their last dry period. This last effect disappeared with increasing herd size. Dairy herds can be characterized by one strongly clustered network including most of the herd members with many non-random attachment and avoidance relationships. Closely synchronous dyads were composed of cows with more intense previous contact. The automatic tracking of a large number of individuals proved promising in acquiring the data necessary for tackling social network analyses.     

Synthesis, X-ray structures, electrochemical properties and cytotoxic effects of Co(II) complexes

1-Benzothiazol-2-yl-3,5-dimethyl-1H-pyrazole (1a) and 1-benzothiazol-2-yl-5-(2-hydroxyphenyl)-3-methyl-1H-pyrazole-4-carboxylic acid methyl ester (1b) were reacted with the hexahydrates of cobalt(II) chloride, cobalt(II) nitrate and cobalt(II) perchlorate to give the corresponding complexes 2a-4a and 2b-5b, respectively. Obtained compounds differ in coordination spheres of central atoms. The complex 2a includes a fivefold coordinated cobalt(II) ion, whereas 3a shows a distorted octahedral configuration around the cobalt(II) ion. All complexes were characterised by FTIR spectroscopy, MS and elemental analysis. The X-ray structures of 2a, 3a and 5b complexes were also solved. The cytotoxic properties of the ligand 1a and both series of Co(II) complexes were examined on human leukemia NALM-6 and HL-60 cells and melanoma WM-115 cells. The ligands, were found to have very low cytotoxicity. Complex 3b exhibited the highest cytotoxic activity with IC₅₀ values in the range of 6.9-17.1 μM for three examined cell lines.     

Development of a novel Gateway-based vector system for efficient, multiparallel protein expression in Escherichia coli

We describe a cloning and expression system which is based on the Escherichia coli T7 expression system and Gateway recombination technology. We have produced numerous destination vectors with selected fusion tags and an additional set of entry vectors containing the gene of interest and optional labeling tags. This powerful system enables us to transfer a cDNA to several expression vectors in parallel and combine them with various labeling tags. To remove the attached amino terminal tags along with the unwanted attB1 site, we inserted PreScission protease cleavage sites. In contrast to the commercially available destination vectors, our plasmids provide kanamycin resistance, which can be an advantage when expressing toxic proteins in E. coli. Some small-scale protein expression experiments are shown to demonstrate the usefulness of these novel Gateway vectors. In summary, this system has some benefits over the widely used and commercially available Gateway standard system, and it enables many different combinations for expression constructs from a single gene of interest.