Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.     

Synthesis of the 3-methyl ether and 4-deoxy derivatives of 4-cyanophenyl 1,5-dithio-beta-D-xylopyranoside (Beciparcil).

Treatment of the 2,3-isopropylidene acetal of the title dithioxyloside with 2,4,5-triiodoimidazole-PPh3 caused replacement of the 4-hydroxyl group by iodine to afford 82% of the 4-axial iodide 6, converted by base into 4-cyanophenyl 2,3-O-isopropylidene-1,5-dithio-beta-D-glycero-pent-3-enopyranoside++ + (8). Acid treatment of 8 gave 87% of the deacetonated glycos-3-ulose, borohydride reduction of which afforded 63% of 4-cyanophenyl 4-deoxy-1,5-dithio-alpha-L-threo-pentopyranoside (3), together with 27% of the 3-axial epimer. The 3-methyl ether of the title dithioxyloside was satisfactorily prepared via 2,4-protection as the cyclic phenylboronate, methylation, and deprotection; alternative strategy via the 2,4-bis(triisopropylsilyl) ether was complicated because of silyl-group migration under methylation conditions.     

Spectroscopic characterization of the spinach Lhcb4 protein (CP29), a minor light-harvesting complex of photosystem II.

A spectroscopic characterization is presented of the minor photosystem II chlorophyll a/b-binding protein CP29 (or the Lhcb4 protein) from spinach, prepared by a modified form of a published protocol [Henrysson, T., Schroder, W. P., Spangfort, M. & Akerlund, H.-E. (1989) Biochim. Biophys. Acta 977, 301-308]. The isolation procedure represents a quicker, cheaper means of isolating this minor antenna protein to an equally high level of purity to that published previously. The pigment-binding protein shows similarities to other related light-harvesting complexes (LHCs), including the bulk complex LHCIIb but more particularly another minor antenna protein CP26 (Lhcb5). It is also, in the main, similar to other preparations of CP29, although some significant differences are discussed. In common with CP26, the protein binds about six chlorophyll a and two chlorophyll b molecules. Two chlorophyll b absorption bands are present at 638 and 650 nm and they are somewhat more pronounced than in a recent report [Giuffra, E., Zucchelli, G., Sandonà, D., Croce, R., Cugini, D., Garlaschi, F.M., Bassi, R. & Jennings, R.C. (1997) Biochem. 36, 12984-12993]. The bands give rise to positive and negative linear dichroism, respectively; both show negative CD bands (cf. bands with similar properties at 637 and 650 nm in CP26). Chlorophyll a absorption is dominated by a large contribution at 674 nm which also shows similarities to the major band in LHCIIb and CP26, while (as for CP26) a reduction in absorption around 670 nm is observed relative to the bulk complex. Principal differences from LHCIIb and CP26, and from other CP29 preparations, occur in the carotenoid region.     

Monoclonal antibody toward lysosomal cathepsin B cross-reacts preferentially with distinct histone classes

1. A set of monoclonal antibodies (Mab) was prepared against cathepsin B (CB) from rat preputial-gland, an organ characterized by rapidly-renewing cell populations, which is a uniquely enriched source of lysosomal enzymes, including CB. Minute amounts of CB are known to be transferred abruptly to the nuclear compartment in a variety of activated cells. 2. Since, on the basis of its stringent substrate requirements, CB was expected to function at limited protein loci in chromatin, Mab Line II-B4 was used to probe Western blots of chromatin fractions and selected proteins. 3. The Mab, which was not directed against the active site of CB, cross-reacted preferentially with histones 3 and 4 (H3 and H4) in acid-soluble fractions of chromatin from rat preputial-gland. Line II-B4 also recognized H3 and H4 selectively in calf thymus histones and among histones purified from a wide range of sources from yeast to man. HMG 1 was minimally immunoreactive among preputial gland constituents and carbonic anhydrase (CA) was also sensitive to the Mab. 4. The common determinants were not shared by any of the H1 series, nor by H2A, H2B, protein A24 or a wide range of natural and synthetic products. 5. Origin of the antigenicity was traced by chemical modifications of H3, H4 and CA to the critical contribution of arginine and hydrophobic amino acid residues in its immediate environment, indicating that Line II-B4 may be directed against an epitope comprising the specific binding-site of CB and its selective substrate(s). 6. These data suggest that certain highly conserved cellular constituents may be uniquely vulnerable to limited proteolysis in preproliferative cells responding to mitotic signals.     

The grand challenges of migration ecology that radar aeroecology can help answer

Many migratory species have experienced substantial declines that resulted from rapid and massive expansions of human structures and activities, habitat alterations and climate change. Migrants are also recognized as an integral component of biodiversity and provide a multitude of services and disservices that are relevant to human agriculture, economy and health. The plethora of recently published studies reflects the need for better fundamental knowledge on migrations and for better management of their ecological and human‐relevant effects. Yet, where are we in providing answers to fundamental questions and societal challenges? Engaging a broad network of researchers worldwide, we used a horizon‐scan approach to identify the most important challenges which need to be overcome in order to gain a fuller understanding of migration ecology, and which could be addressed using radar aeroecological and macroecological approaches. The top challenges include both long‐standing and novel topics, ranging from fundamental information on migration routes and phenology, orientation and navigation strategies, and the multitude of effects migrants may have on resident communities, to societal challenges, such as protecting or preventing migrant services and disservices, and the conservation of migrants in the face of environmental changes. We outline these challenges, identify the urgency of addressing them and the primary stakeholders – researchers, policy makers and practitioners, or funders of research.     

Robbery in progress: Historical museum collections bring to light a mitochondrial capture within a bird species widespread across southern Australia,the Copperback Quail‐thrush Cinclosoma clarum

We surveyed mitochondrial, autosomal, and Z chromosome diversity within and between the Copperback Quail‐thrush Cinclosoma clarum and Chestnut Quail‐thrush C. castanotum, which together span the arid and semi‐arid zones of southern Australia, and primarily from specimens held in museum collections. We affirm the recent taxonomic separation of the two species and then focus on diversity within the more widespread of the two species, C. clarum. To guide further study of the system and what it offers to understanding the genomics of the differentiation and speciation processes, we develop and present a hypothesis to explain mitonuclear discordance that emerged in ourdata. Following a period of historical allopatry, secondary contact has resulted in an eastern mitochondrial genome replacing the western mitochondrial genome in western populations. This is predicted under a population‐level invasion in the opposite direction, that of the western population invading the range of the eastern one. Mitochondrial captures can be driven by neutral, demographic processes, or adaptive mechanisms, and we favor the hypothesized capture being driven by neutral means. We cannot fully reject the adaptive process but suggest how these alternatives may be further tested. We acknowledge an alternative hypothesis, which finds some support in phenotypic data published elsewhere, namely that outcomes of secondary contact have been more complex than our current genomic data suggest. Discriminating and reconciling these two alternative hypotheses, which may not be mutually exclusive, could be tested with closer sampling at levels of population, individual, and nucleotide than has so far been possible. This would be further aided by knowledge of the genetic basis to phenotypic variation described elsewhere.     

PRECONVULSIVE CHANGES IN BRAIN GLUCOSE METABOLISM FOLLOWING DRUGS INHIBITING GLUTAMATE DECARBOXYLASE

—Brain glucose and glycogen concentrations have been studied in mice treated with allylglycine, 4-deoxypyridoxine and isoniazid, and the effects compared with the preconvulsive increase in brain glucose and glycogen concentration that follows d , l -methionine sulphoximine treatment. Allylglycine (180 mg/kg), 4-deoxypyridoxine (250 mg/kg), isoniazid (150 mg/kg) and d ,l -methionine sulphoximine (300 mg/kg) when given to mice at room temperature, cause a fall in rectal temperature which can be prevented by maintaining the mice in an incubator at 33-34°C. An increase in brain glucose concentration is seen after allylglycine (+ 133%), d ,l -methionine sulphoximine (+ 113%) and 4-deoxypyridoxine (+ 70%) treatment when mice are kept at room temperature and killed before convulsions occur. This is associated with a rise in blood glucose concentration after allylglycine, but not after the other drugs. Preventing the fall in rectal temperature reduces, but does not abolish, the rise in brain glucose concentration seen after allylglycine, d ,l -methionine sulphoximine and 4-deoxypyridoxine. Brain glycogen concentration increases at room temperature after D,L-methionine sulphoximine and 4-deoxypyridoxine, but in mice with maintained body temperature only 4-deoxypyridoxine produces an increase in brain glycogen. Isoniazid does not increase brain glucose or glycogen at room temperature, but reduces their concentration in mice kept in the incubator. All four drugs are known to act on amino acid metabolism; d ,l -methionine sulphoximine potently inhibits glutamine synthetase whereas 4-deoxypyridoxine, allylglycine and isoniazid inhibit glutamate decarboxylase. The connection, if any, between a block in the further metabolism of glutamate and an increase in brain glucose and glycogen is unknown.     

Brain Adrenoceptor Binding Sites in Mice Susceptible (DBA/2J) and Resistant (C57 Bl/6) to Audiogenic Seizures

We have examined if the age-related susceptibility of DBA/2J mice to audiogenic seizures is the result of an abnormality in the number or sensitivity of brain adrenoceptors. The binding of alpha 1-, alpha 2-, and beta-adrenoceptor ligands to membranes prepared from whole brain or regions of brain of DBA/2J mice was measured at various ages, corresponding to the periods before, during, and after the maximal sensitivity to audiogenic seizures. For comparison, we have studied concurrently age-matched C57 Bl/6 mice, a strain resistant to audiogenic seizures at all ages. There was no difference in the binding of alpha 2- or beta-adrenoceptor ligands to whole brain membranes between the two strains of mice at any age. The maximal number of alpha 1-adrenoceptor binding sites was lower in whole brains of DBA/2J mice than of C57 Bl/6 mice at all ages studied except 13-15 days of age. The differences were small (maximally 17%) but were statistically significant at 21-23 days of age, the time of maximal sensitivity of DBA/2J mice to audiogenic seizures. No difference between the two strains was found in the number or affinity of alpha 1- or alpha 2-adrenoceptor binding sites at any age in any of the brain regions studied. The age-related susceptibility of DBA/2J mice to audiogenic seizures is not the result of an abnormality in number or sensitivity of alpha 2- or beta-adrenoceptor binding sites, but a reduced number of alpha 1-adrenoceptor binding sites may be involved.