Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Timing of oral morphogenesis and its relation to commitment to division in Paramecium tetraurelia

The interval between commitment to division and fission in synchronous cell samples is a constant fraction of the cell cycle (0.2) in cell cycles up to 6.5 h in duration. In longer cell cycles this interval has a fixed duration of about 80 min. The point of commitment to division is associated with the six-rowed analage stage of oral primordium development (stage V). At this stage cells carrying the cc1 mutation are not blocked by transfer to restrictive conditions but rather proceed to division. Stage V is also the stabilization point for oral anlagen. When shifted to restrictive conditions prior to this stage, development is arrested and resorption of analgen is initiated. The cc1 mutation also blocks contractile vacuole duplication and migration under restrictive conditions. The cc1 gene function is required continuously prior to the transition point. The timing of morphogenetic stages in asynchronous cells is roughly similar to that in synchronous cells. There are, however, significant differences in timing as estimated by the two experimental procedures.     

Identifying the neural circuitry of alcohol craving and relapse vulnerability

With no further intervention, relapse rates in detoxified alcoholics are high and usually exceed 80% of all detoxified patients. It has been suggested that stress and exposure to priming doses of alcohol and to alcohol-associated stimuli (cues) contribute to the relapse risk after detoxification. This article focuses on neuronal correlates of cue responses in detoxified alcoholics. Current brain imaging studies indicate that dysfunction of dopaminergic, glutamatergic and opioidergic neurotransmission in the brain reward system (ventral striatum including the nucleus accumbens) can be associated with alcohol craving and functional brain activation in neuronal systems that process attentional relevant stimuli, reward expectancy and experience. Increased functional brain activation elicited by such alcohol-associated cues predicted an increased relapse risk, whereas high brain activity elicited by affectively positive stimuli may represent a protective factor and was correlated with a decreased prospective relapse risk. These findings are discussed with respect to psychotherapeutic and pharmacological treatment options.     

Two distinct classes of mitotic cyclin homologues, Cyc1 and Cyc2, are involved in cell cycle regulation in the ciliate Paramecium tetraurelia

The eukaryotic cell cycle is regulated by the sequential activation of different CDK/cyclin complexes. Two distinct classes of mitotic cyclin homologues, CYC1 and CYC2, have been identified and cloned for the first time in the ciliate Paramecium. Cyc1 is 324 amino acids long with a predicted molecular mass of 38 kDa, whereas Cyc2 is 336 amino acids long with a predicted molecular mass of 40 kDa. They display 42-51% sequence identity to other eukaryotic mitotic cyclins within the 'cyclin box' region. The conserved 'cyclin box' and 'destruction box' elements can be identified within each of the sequences. Genomic Southern blot analysis indicated that the CYC1 gene has two isoforms, with 92.3% and 85.9% identify at the amino acid level and at the nucleotide level, respectively. Both Cyc1 and Cyc2 proteins showed characteristic patterns of accumulation and destruction during the vegetative cell cycle, with Cyc1 peaking at the point of commitment to division (PCD), and Cyc2 reaching the maximal level late in the cell cycle. Immunoprecipitation experiments with antibodies specific to Cyc1 and Cyc2 indicated that Cyc1 and Cyc2 associate with distinct CDK homologues. Both immunoprecipitates exhibited histone H1 kinase activity that oscillated in the cell cycle in parallel with the respective amount of cyclins present. Histone H1 kinase activity associated with Cyc1 reached a peak at PCD while Cyc2 showed maximal activity when about 75% cells have completed cytokinesis. We propose that Cyc1 may be involved in commitment to division, in association with the CDK that binds to p13suc1, Cdk3, and that the Cyc2/Cdk2 complex may regulate cytokinesis. PCR-amplification revealed similar sequences in Tetrahymena, Sterkiella, Colpoda and Blepharisma, suggesting the conservation of the cyclin genes within ciliates. Although cell cycle regulation in ciliates differs in some respects from that of other eukaryotes, the cyclin motifs have clearly been conserved during evolution.     

Geographical variation in temporal and spatial vocalization patterns of male harbour seals in the mating season

In the aquatically mating harbour seal, Phoca vitulina, oestrous females show marked differences in spatial and temporal distribution between geographical areas. This suggests that the males' display behaviour may also vary between areas. We recorded male vocalizations in two areas, the Moray Firth and Orkney, U.K. In the Moray Firth, females haul out on a few intertidal sandbars and travel along predictable routes to forage at sea. In Orkney, female haul out sites are much less influenced by tidal availability and females are much more dispersed. In the Moray Firth, males vocalized only during a short mating season, from 1 July to 12 August. Vocalizations varied significantly with the tide, the peak at high tide clearly coinciding with the period when most females were in the water. In contrast, vocalizations in Orkney were significantly related to both tidal and diel patterns. We suggest that the timing of male vocalizations reflects differences in female availability between sites. In the inner Moray Firth, vocalizations were heard throughout the females' range, whereas vocalizations in Orkney were heard only in two discrete areas. However, at both sites the density of vocalizing males was highest in narrow channels and/or along predictable female travel routes. Therefore, males clearly adapt their temporal and spatial behaviour patterns to variations in female distribution and density. These results suggest that male mating strategies in aquatically mating pinnipeds are more variable than was previously envisaged. Copyright 1999 The Association for the Study of Animal Behaviour.     

A limited survey of aflatoxins and fumonisins in retail maizebased products in the UK using immunoassay detection

A total of 27 maize-based products destined for human consumption were collected from retail outlets within the city of Glasgow in the UK and were analysed for the presence of aflatoxins using immunoaftinity column chromatography with fluorescence detection and for fumonisins by competitive ELISA. Aflatoxins were detected at a trace level below 4 in eight (30%) of the 27 samples tested, no sample contained aflatoxins at a high level although one sample of sweetcorn did contain aflatoxins at a level of 5-10 Fumonisins were detected in eight (30%) of the samples at levels from 1 to 8mgkg^-1 and a further eight samples contained fumonisin at a level below 1 mgkg^-1 but above the detectable level. The highest concentration of fumonisins was found in a sample of fine corn meal at 8-12mgkg^-1.     

Sprint,agility, strength and endurance capacity in wheelchair basketball players

The aims of the present study were, firstly, to determine the reliability and reproducibility of an agility T-test and Yo-Yo 10 m recovery test; and secondly, to analyse the physical characteristics measured by sprint, agility, strength and endurance field tests in wheelchair basketball (WB) players. 16 WB players (33.06 ± 7.36 years, 71.89 ± 21.71 kg and sitting body height 86.07 ± 6.82 cm) belonging to the national WB league participated in this study. Wheelchair sprint (5 and 20 m without ball, and 5 and 20 m with ball) agility (T-test and pick-up test) strength (handgrip and maximal pass) and endurance (Yo-Yo 10 m recovery test) were performed. T-test and Yo-Yo 10 m recovery test showed good reproducibility values (intraclass correlation coefficient, ICC = 0.74-0.94). The WB players’ results in 5 and 20 m sprints without a ball were 1.87 ± 0.21 s and 5.70 ± 0.43 s and with a ball 2.10 ± 0.30 s and 6.59 ± 0.61 s, being better than those reported in the literature. Regarding the pick-up test results (16.05 ± 0.52 s) and maximal pass (8.39 ± 1.77 m), players showed worse values than those obtained in elite players. The main contribution of the present study is the characterization of the physical performance profile of WB players using a field test battery. Furthermore, we demonstrated that the agility T-test and the aerobic Yo-Yo 10 m recovery test are reliable; consequently they may be appropriate instruments for measuring physical fitness in WB.     

Ubiquitin fusion expression and tissue-dependent targeting of hG-CSF in transgenic tobacco

Background

The transglutaminase activated factor XIII (FXIIIa) acts to strengthen pathological fibrin clots and to slow their dissolution, in part by crosslinking active α₂-antiplasmin (α₂AP) to fibrin. We previously reported that a yeast-derived recombinant fusion protein comprising α₂AP residues 13-42 linked to human serum albumin (HSA) weakened in vitro clots but failed to become specifically incorporated into in vivo clots. In this study, our aims were to improve both the stability and clot localization of the HSA fusion protein by replacing α₂AP residues 13-42 with shorter sequences recognized more effectively by FXIIIa.

Results

Expression plasmids were prepared encoding recombinant HSA with the following N-terminal 23 residue extensions: H₆NQEQVSPLTLLAG₄Y (designated XL1); H₆DQMMLPWAVTLG₄Y (XL2); H₆WQHKIDLPYNGAG₄Y (XL3); and their 17 residue non-His-tagged equivalents (XL4, XL5, and XL6). The HSA moiety of XL4- to XL6-HSA proteins was C-terminally His-tagged. All chimerae were efficiently secreted from transformed Pichia pastoris yeast except XL3-HSA, and following nickel chelate affinity purification were found to be intact by amino acid sequencing, as was an N-terminally His-tagged version of α₂AP(13-42)-HSA. Of the proteins tested, XL5-HSA was cross-linked to biotin pentylamine (BPA) most rapidly by FXIIIa, and was the most effective competitor of α₂AP crosslinking not only to BPA but also to plasma fibrin clots. In the mouse ferric chloride vena cava thrombosis model, radiolabeled XL5-HSA was retained in the clot to a greater extent than recombinant HSA. In the rabbit jugular vein stasis thrombosis model, XL5-HSA was also retained in the clot, in a urea-insensitive manner indicative of crosslinking to fibrin, to a greater extent than recombinant HSA.

Conclusions

Fusion protein XL5-HSA (DQMMLPWAVTLG₄Y-HSAH₆) was found to be more active as a substrate for FXIIIa-mediated transamidation than seven other candidate fusion proteins in vitro. The improved stability and reactivity of this chimeric protein was further evidenced by its incorporation into in vivo clots formed in thrombosis models in both mice and rabbits.