Substrate reduction therapy in mouse models of the glycosphingolipidoses

Substrate reduction therapy uses small molecules to slow the rate of glycolipid biosynthesis. One of these drugs, N-butyldeoxynojirimycin (NB-DNJ), shows efficacy in mouse models of Tay-Sachs, Sandhoff and Fabry diseases. This offers the prospect that NB-DNJ may be of therapeutic benefit, at least in the juvenile and adult onset variants of these disorders. The infantile onset variants will require an additional enzyme-augmenting modality if the pathology is to be significantly improved. A second drug, N-butyldeoxyglactonojirimycin, looks very promising for treating storage diseases with neurological involvement as high systemic dosing is achievable without any side-effects.     

Size-dependent selection on arrival times in sticklebacks: why small males arrive first

Abstract Studies on arrival time to breeding areas show that high-quality males usually arrive first and gain the highest reproductive success. This is generally assumed to be due to phenotype-dependent costs and benefits of early arrival. We show that the opposite arrival order can occur, probably due to selection on poor-quality males to increase their chances of reproduction. In a fish species, the threespine stickleback, Gasterosteus aculeatus , small males arrived before larger males at the breeding grounds. Early arrival was costly because predation risk was at its highest at the start of the season and early territory establishment was selected against, as demonstrated by selection coefficients for territory maintenance and hatching success. Large males probably postponed arrival until females were available to decrease predation risk costs and increase offspring production. An experimental study showed that a delay in arrival of large males does not decrease their probability of reproduction, because large males are able to take over nest sites from small males. Small males, on the other hand, are less likely to establish territories in competition with large males but can pay the costs of early arrival in exchange for the benefit of access to territories. Thus, whereas natural selection favors later arrival, sexual selection through competition for breeding territories favors early arrival in small, competitively inferior males. This results in the benefits of early arrival depending on the competitive ability of the male, which favors size-dependent optimal arrival times.     

DNA adducts as indicators of genotoxic exposure in indigenous and transplanted mussels, Mytilus edulis L. from Icelandic coastal sites

Indigenous mussels, Mytilus edulis, were collected at sites with supposed different amounts of pollution; Reykjavík harbour, Keflavík harbour, Grafarvogur and Hvalfj?rdur (reference), along the south-western coast of Iceland in March 2000. Mussels from Hvalfj?rdur and Reykjavík harbour were also collected in August the same year. Additionally, mussels were transplanted from the reference site to Reykjavík harbour for 6 weeks during both winter and summer for comparison. DNA adducts were analysed by 32P-post-labelling in gills and digestive gland. Highest adduct levels were found in gill tissue from indigenous mussels collected in Reykjavík harbour. Adduct levels in both tissues from mussels collected at the reference site were below or very close to the detection limit during winter, but seemed to increase a little during summer. Mussels from sites with supposed intermediate pollution had intermediate levels of DNA adducts in gills but did not differ from Reykjavík harbour in digestive gland. No increase in adduct levels was observed in mussels transplanted from the reference site to Reykjavík harbour, except for a slight increase in digestive gland during winter. This study shows that 32P-post-labelling analysis of DNA adducts is sensitive enough to be used on indigenous mussels from relatively pristine areas and that adduct levels are increased in harbours/urban sites. However, transplantation of mussels from a clean site to the harbour for 6 weeks did not result in increased adduct levels in gills, the tissue with the highest adduct levels. The results also indicate that seasonal variation in adduct levels may occur.     

Measurement of ATP production and respiratory chain enzyme activities in mitochondria isolated from small muscle biopsy samples

A set of methods suitable for assessment of respiratory chain function in mitochondria isolated from 25mg of muscle is described. This set of methods includes determination of the mitochondrial ATP production rate (MAPR) and the activities of the respiratory chain complexes I, I+III, II+III, and IV and citrate synthase. MAPR is determined with an optimized version of a luminometric method previously described. The optimized method measures 50-220% higher activities than the original method. The highest MAPRs are recorded using the substrate combinations glutamate+succinate and N,N,N(1),N(1)-tetramethyl-1,4-phenyldiamine+ascorbate. The respiratory chain complex activities are determined with standard spectrophotometric methods, adapted to an automated photometer. The sensitivity in the determination of complex I, I+III, and II+III activities was increased considerably by pretreating the samples with saponin. The set of methods was evaluated on double biopsy samples from five healthy volunteers and showed coefficients of variation between 7 and 14% when citrate synthase was used as reference base. All of the various measures of mitochondrial function showed high correlation coefficients to each other (r=0.84-0.98; p<0.01). It is concluded that the set of methods is suitable for diagnosis of mitochondrial disorders in adults and small children.     

Efficacy of the biocontrol yeast Pichia anomala during long-term storage of moist feed grain under different oxygen and carbon dioxide regimens

The yeast Pichia anomala inhibits the spoilage mold Penicillium roqueforti in laboratory experiments with high-moisture wheat in malfunctioning airtight storage. The ability of P. anomala to prevent mold growth during 14 months of grain storage was evaluated in outdoor silos with different air permeabilities. Freshly harvested wheat in 160-kg portions was inoculated with 10(2) colony-forming units (cfu) g(-1) P. roqueforti, alone or together with 10(4) cfu g(-1) P. anomala. During the first month P. anomala increased to about 10(6) cfu g(-1) in the treated silos to reach 10(7) cfu g (-1) after 9 months. Naturally occurring P. anomala in the untreated silos increased from 10(2) to about 10(3) cfu g(-1) during the first month and reached the same level as the treated silos after 9 months. Oxygen levels were reduced below the detection limit within 1 day, while carbon dioxide levels increased to 80-90% during the first month. P. roqueforti did not grow in wheat treated with P. anomala, regardless of silo permeability, but had increased to 10(5) cfu g(-1) in the untreated silos after 14 months of storage.     

Systematic affinities of the lyrebirds (Passeriformes: Menura), with a novel classification of the major groups of passerine birds

Phylogenetic relationships of the lyrebirds are investigated using DNA sequence data. The aligned data matrix consists of 4027 bp obtained from three nuclear genes (c-myc, RAG-1 and myoglobin intron II) and two mitochondrial genes (cytochrome b and ND2). Both maximum-likelihood and parsimony analyses show that the lyrebirds unambiguously belong to the oscine radiation, and that they are the sister taxon to all other oscines. The results do not support the suggestion based on DNA-DNA hybridization data (Sibley and Ahlquist, 1990) that the treecreepers and bowerbirds are part of the lyrebird clade. Nevertheless, treecreepers and bowerbirds are sister taxa to all other oscines (except the lyrebirds) and may constitute a monophyletic group, although bootstrap support values for this clade are low. A major disagreement between the present analysis and that based on DNA-DNA hybridization data is that the Corvida (sensu Sibley and Ahlquist, 1990) and Passerida are not reciprocally monophyletic, as we find the latter group be nested within the Corvida. Also, the superfamilies Meliphagoidea and Corvoidea sensu, are not recovered as monophyletic in the present study. Within the oscine radiation, all taxa belonging to the earliest splits are confined to the Australo-Papuan region. This suggests strongly that the origins and early radiation of the oscines occurred in the southern supercontinent Gondwana. A new classification of the major groups of passerines is presented following from the results presented in the present study, as well as those published recently on analyses of sequence data from the nuclear c-myc and RAG-1 genes (Ericson et al., 2002; Irestedt et al., 2001).     

Determination of ximelagatran,an oral direct thrombin inhibitor,its active metabolite melagatran,and the intermediate metabolites,in biological samples by liquid chromatography-mass spectrometry

Analytical methods for the determination of ximelagatran, an oral direct thrombin inhibitor, its active metabolite melagatran, and intermediate metabolites, melagatran hydroxyamidine and melagatran ethyl ester, in biological samples by liquid chromatography (LC) positive electrospray ionization mass spectrometry (MS) using selected reaction monitoring are described. Isolation from human plasma was achieved by solid-phase extraction on octylsilica. Analytes and isotope-labelled internal standards were separated by LC utilising a C(18) analytical column and a mobile phase comprising acetonitrile-4 mmol/l ammonium acetate (35:65, v/v) containing 0.1% formic acid, at a flow-rate of 0.75 ml/min. Absolute recovery was approximately 80% for ximelagatran, approximately 60% for melagatran ethyl ester and >90% for melagatran and melagatran hydroxyamidine. Limit of quantification was 10 nmol/l, with a relative standard deviation <20% for each analyte and <5% above 100 nmol/l. Procedures for determination of these analytes in human urine and breast milk, plus whole blood from rat and mouse are also described.     

Recombinant expression of Munc18c in a baculovirus system and interaction with syntaxin4

Two protein families that are critical for vesicle transport are the Syntaxin and Munc18/Sec1 families of proteins. These two molecules form a high affinity complex and play an essential role in vesicle docking and fusion. Munc18c was expressed as an N-terminally His-tagged fusion protein from recombinant baculovirus in Sf9 insect cells. His-tagged Munc18c was purified to homogeneity using both cobalt-chelating affinity chromatography and gel filtration chromatography. With this simple two-step protocol, 3.5 mg of purified Munc18c was obtained from a 1L culture. Further, the N-terminal His-tag could be removed by thrombin cleavage while the tagged protein was bound to metal affinity resin. Recombinant Munc18c produced in this way is functional, in that it forms a stable complex with the SNARE interacting partner, syntaxin4. Thus we have developed a method for producing and purifying large amounts of functional Munc18c--both tagged and detagged--from a baculovirus expression system. We have also developed a method to purify the Munc18c:syntaxin4 complex. These methods will be employed for future functional and structural studies.