trans-o-Hydroxybenzylidenepyruvate hydratase-aldolase as a biocatalyst

The hydratase-aldolase-catalyzed conversion of trans-o-hydroxybenzylidenepyruvate to salicylaldehyde and pyruvate is an intermediate reaction in the conversion of naphthalene to salicylate by bacteria. Here, a variety of aromatic aldehydes and some nonaromatic aldehydes together with pyruvate have been shown to be substrates for aldol condensations catalyzed by this enzyme in extracts of the recombinant strain Escherichia coli JM109(pRE701). Some of the products of these reactions were also compared as substrates in the opposite (hydration-aldol cleavage) reaction.     

Aldosterone induces ras methylation in A6 epithelia

Aldosterone increases Na⁺ reabsorption by renalepithelial cells: the acute actions (<4 h) appear to be promoted byprotein methylation. This paper describes the relationship betweenprotein methylation and aldosterone's action and describesaldosterone-mediated targets for methylation in cultured renal cells(A6). Aldosterone increases protein methylation from 7.90 ± 0.60 to 20.1 ± 0.80 methyl ester cpm/µg protein. Aldosteronestimulates protein methylation by increasing methyltransferase activityfrom 14.0 ± 0.64 in aldosterone-depleted cells to 31.8 ± 2.60 methyl ester cpm/µg protein per hour in aldosterone-treated cells. Three known methyltransferase inhibitors reduce thealdosterone-induced increase in methyltransferase activity. One ofthese inhibitors, the isoprenyl-cysteine methyltransferase-specificinhibitor,S-trans,trans-farnesylthiosalicylic acid, completely blocks aldosterone-induced protein methylation and also aldosterone-induced short-circuit current. Aldosterone inducesprotein methylation in two molecular weight ranges: near 90 kDa andaround 20 kDa. The lower molecular weight range is the weight of smallG proteins, and aldosterone does increase both Ras protein 1.6-fold andRas methylation almost 12-fold. Also, Ras antisense oligonucleotidesreduce the activity of Na⁺ channels by about fivefold. Weconclude that 1) protein methylation is essential foraldosterone-induced increases in Na⁺ transport;2) one target for methylation is p21^ras; and3) inhibition of Ras expression or Ras methylation inhibits Na⁺ channel activity.

     

Generation and initial characterization of conditionally immortalized chromaffin cells

Adrenal chromaffin cells have been successfully used to attenuate chronic pain when transplanted near the spinal cord, but primary cells are neither homogeneous nor practical for routine use in human therapy. Conditional immortalization with the temperature-sensitive allele of the large T antigen (tsTag) and creation of stable chromaffin cell lines would advance our understanding of both the use and limits of cell lines that contain this immortalization gene for such therapies. Cultures of embryonic day 17 rat adrenal and neonatal bovine adrenal cells were immortalized with the temperature-sensitive allele of SV40 tsTag and chromaffin cell lines established. The rat chromaffin line, RAD5.2, and the bovine chromaffin cell line, BADA.20, both expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), the first enzyme in the synthetic pathway for catecholamines, dopa-beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). At permissive temperature (33 degrees C), these chromaffin cells are proliferative, have a typical rounded chromaffinlike morphology, and contain detectable TH-, DbetaH-, and PNMT-ir. At nonpermissive temperature (39 degrees C), these cells stop proliferating, decrease Tag expression, and change the expression of TH-, DbetaH-, and PNMT-ir in vitro, suggesting increased differentiation at nonpermissive temperature. The chromaffin cell lines also express immunoreactivity for the opioid met-enkephalin (ENK) at permissive and nonpermissive temperatures. The expression of TH-ir in the bovine chromaffin cells is upregulated by the addition of dexamethasone (DEX) or forskolin during differentiation; TH-ir is not affected by the addition of DEX or forskolin in the rat chromaffin cells. The addition of forskolin during differentiation upregulates the expression of DbetaH-ir in the rat chromaffin cells. PNMT-ir is not affected by differentiation or agents in either cell line. However, catecholamine synthesis was not detectable by high-performance liquid chromatography, suggesting incomplete differentiation under current conditions, or influence by continued low levels of Tag expression. Both cell lines have been carried over many passages in vitro for more than 3 years and were repeatedly frozen and thawed. These data describe an initial step in the conditional immortalization of chromaffin cells that can maintain the phenotype of primary chromaffin cells in vitro over long periods. The use of such chromaffin cell lines that are able to deliver neuroactive molecules offers a novel approach to pain management.     

Effects of CCA (copper-chrome-arsenic) preservative treatment of wood on the settlement and recruitment of barnacles and tube building polychaete worms

The effect of the anti-marine-borer treatment of wood using CCA (a pressure impregnated solution of copper, chromium and arsenic compounds) on non-target fouling animals was investigated. Panels treated to target retentions of 12, 24 and 48 kg CCA m(-3) of wood, together with untreated controls were exposed for 6, 12 and 18 months at coastal sites in Greece, Portugal, France and Sweden. General linear model (GLM) analysis revealed significant increases in numbers of certain fouling organisms (the serpulids Ficopomatus enig-maticus, Hydroides spp., Pomatoceros lamarkii and an unidentified species, three species of spirorbid, and the balanids Balanus perforatus and Elminius modestus) with increase in retention of CCA. The effect of CCA on the numbers of recruits may be due to effects on their settlement and survival, but may also be due to suppression of competitors. Significant differences in settlement density of barnacle spat occurred on newly exposed wood and on wood that had been exposed for 6 and 18 months. The relationships between settlement density and retention could be described by logarithmic curves of the form settlement density = a 1n(l + retention)+b. The effects of CCA on settlement are ascribed either to modification of wood surface chemistry leading to changes in surface charge, the availability of Cu, Cr or As at the wood surface, or to modifications to the microbial film. Barnacle settlement was between 6.5 and 14 times more intense on latewood than on earlywood, an effect that was evident in both untreated and preservative-treated wood.     

The first UK survey and population estimate of breeding Snow Bunting Plectrophenax nivalis

Capsule: The first national survey for Snow Bunting Plectrophenax nivalis in the UK was carried out in 2011 and estimated the breeding population at 60 territories (95% confidence intervals?=?48–83).

Aims: To estimate breeding population size for Snow Buntings in the UK by surveying all sites with a history of breeding season occupation.

Methods: Surveys were carried out in June 2011 to detect males on territory at sites where Snow Bunting had been recorded during the breeding season since 1970. Each site was visited at least once during the survey period; suitable habitat was searched and vantage point watches were conducted in order to detect singing males. Repeated visits to a sample of sites allowed a correction factor to be calculated in order to account for birds undetected during surveys.

Results: In 2011, the number of Snow Bunting breeding in the UK (including adjusting for imperfect detection) is a minimum of 60 territories (95% CI?=?48–83) using confirmed and probable breeding records of males. A less conservative estimate of 99 territories (95% CI?=?88–114) results from including all records of males in suitable habitat. The vast majority of the population was found in the Cairngorm region, with isolated records in the north and west Highlands.

Conclusion: The results of the 2011 survey are consistent with well-informed estimates of the Snow Bunting population made previously. This work provides a baseline and repeatable fieldwork and analytical methods enabling future change in the population to be quantified more rigorously.     

Factors constraining the distribution of Golden Eagles Aquila chrysaetos in Scotland

Capsule Between 1992 and 2003 persecution appeared to be the main influential factor.

Aims To utilize temporal changes in the distribution and occupation of Golden Eagle territories in Scotland between the 1992 and 2003 national censuses to assess potential causes of regional and national population trends, by examining spatial associations with a number of potential constraints on the population.

Methods The distribution of occupied and vacant territories in the 1992 and 2003 censuses were entered as layers in a Geographical Information System (GIS), along with boundaries of biogeographical regions (Natural Heritage Zones) for regional analyses. Additional GIS layers were created for potential factors that may constrain the eagle population: the distribution and abundance of persecution incidents, new commercial conifer forests, popular mountains for hillwalkers (as surrogates for recreational activity), and the density of sheep and Red Deer (as surrogates for carrion abundance), drawn from comparable time periods to the national eagle censuses. Analyses then looked for spatial associations between eagle territory status and those constraint factors that may have influenced change in territory status.

Results We found little evidence to suggest that recreational disturbance was influential on the occupation of Golden Eagle territories, although some local effects may have occurred and further analyses are warranted. We could find evidence of only a limited number of territories having being abandoned recently due to the planting of commercial conifer forests. We also rejected the hypothesis that changes in territory occupation between national Golden Eagle censuses were influenced by change in carrion abundance. By contrast, results were consistent with the hypothesis that persecution was influential in the observed change in territory occupation between censuses, so that occupied eagle territories tended to decline where persecution was probably still influential and tended to increase where persecution had probably declined.

Conclusion In accordance with earlier predictions based on models of the demographic influence of persecution, in the central and eastern Highlands where grouse moor management predominates, the eagle population continued to decline to levels where increasingly large areas of suitable habitat are unoccupied by breeding pairs.     

Using the first European Breeding Bird Atlas for science and perspectives for the new Atlas

Capsule: The first European Bird Census Council (EBCC) Atlas of European Breeding Birds has been widely used in scientific publications.

Aims: To quantify how scientific publications have used data from the first European Bird Census Council (EBCC) Atlas of European Breeding Birds, what the topics of these studies have been, and to identify key aspects in which a second European Breeding Bird Atlas will provide new opportunities for basic and applied science.

Methods: We searched Google Scholar to find papers published in scientific journals that cited the first atlas. We analysed the contents of a random selection of 100 papers citing this atlas and described the way these papers used information from it.

Results: The first atlas has been cited in 3150 scientific publications, and can be regarded as a fundamental reference for studies about birds in Europe. It was extensively used as a key reference for the studied bird species. A substantial number of papers re-analysed atlas data to derive new information on species distribution, ecological traits and population sizes. Distribution and ecology were the most frequent topics of studies referring to the atlas, but this source of information was used in a diverse range of studies. In this context, climate change, impact of agriculture and habitat loss were, by order, the most frequently studied environmental pressures. Constraints in the atlas, such as the poor coverage in the east of Europe, the lack of information on distribution change and the coarse resolution were identified as issues limiting the use of the atlas for some purposes.

Conclusions: This study demonstrates the scientific value of European-wide breeding bird atlases. A second atlas, with its almost complete coverage across Europe, the incorporation of changes in distribution between the two atlases and the inclusion of modelled maps at a resolution of 10?×?10?km will certainly become a key data source and reference for researchers in the near future.     

Modulation of methuselah expression targeted to Drosophila insulin‐producing cells extends life and enhances oxidative stress resistance

Ubiquitously reduced signaling via Methuselah (MTH), a G‐protein‐coupled receptor (GPCR) required for neurosecretion, has previously been reported to extend life and enhance stress resistance in flies. Whether these effects are due to reduced MTH signalling in specific tissues remains unknown. We determined that reduced expression of mth targeted to the insulin‐producing cells (IPCs) of the fly brain was sufficient to extend life and enhance oxidative stress resistance. Paradoxically, we discovered that overexpression of mth targeted to the same cells has similar phenotypic effects to reduced expression due to MTH's interaction with β‐arrestin, which uncouples GPCRs from their G‐proteins. We confirmed the functional relationship between MTH and β‐arrestin by finding that IPC‐targeted overexpression of β‐arrestin alone mimics the longevity phenotype of reduced MTH signaling. As reduced MTH signaling also inhibits insulin secretion from the IPCs, the most parsimonious mechanistic explanation of its longevity and stress‐resistance enhancement might be through reduced insulin/IGF signaling (IIS). However, examination of phenotypic features of long‐lived IPC‐mth modulated flies as well as several downstream IIS targets implicates enhanced activity of the JNK stress‐resistance pathway more directly than insulin signaling in the longevity and stress‐resistance phenotypes.