Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH 2 -THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH 2 -THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP ؉ -dependent CH 2 -THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH 2 -THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH 2 -THF to formate in adult mammalian mitochondria.
Questionnaires concerning cervical screening behaviour and attitudes were completed by a convenience sample of 70 female students aged 20-25 years. Based on the Prochaska-DiClemente definitions of 'stages of change', participants were classified into precontemplation, contemplation, action and maintenance groups according to their previous and intended screening behaviour. On average, participants rated their relative risk of personally contracting cervical cancer in the future to be below average, an apparent example of optimistic bias (p > .001). This was unrelated to their knowledge of behavioural risk factors or to their previous or intended screening behaviour. Linear trends were observed across the four groups in terms of perceived barriers to screening (precontemplators perceiving most barriers, p > .001) and on the Webster-Kruglanski measure of need for cognitive closure (precontemplators reporting lowest need, p > .01). The relationship between need for closure and stages of change appeared to be mediated by perceived barriers. It is argued that an understanding of psychological factors in screening should attend to differences in the extent to which individuals wish to reduce uncertainty about their own risk status, and their expectations concerning the screening processes.
The preferential 5-HT( 2)/5-HT(1C) receptor agonist DOI (0.1-4 mg/kg s.c.) caused an increase in locomotor activity, grooming and 'wet-dog' shakes (WDS) in the adult guinea-pig. The DOI-induced WDS behaviour was potently inhibited by several antagonists that have high affinity for the 5-HT(2) binding site. The WDS response is likely to be centrally-mediated since the effects of peripherally administered DOI were poorly antagonized by the peripherally-acting 5-HT(2) receptor antagonist BW501C67. Although these studies do not exclude an effect of DOI at 5-HT(1C) receptors, the high potency of ketanserin and spiperone in attenuating the effects of DOI would suggest an effect at the 5-HT(2) receptor. The present data suggest that antagonism of the directly-acting agonist DOI may be useful for assessing the selectivity and duration of action of centrally-acting 5-HT(2) receptor antagonists in the guinea-pig.
The use of fluorescent proteins fused to other proteins has been very useful in revealing the location and function of many proteins. However, it is very important to show that the fusion of these reporter proteins does not impact the function of the protein of interest. Plants have 2 forms of the cap-binding protein that function in initiation of translation, eIF4E and a plant specific form, eIFiso4E. In an attempt to determine the cellular localization of eIFiso4E, fusions to GFP were made, but were found to not be competent to rescue the lethal phenotype of plants lacking eIF4E and eIFiso4E. This suggested that the GFP fusions at either the N- or C-terminus of eIFiso4E were not functional. Biochemical analysis of the fusions revealed that eIFiso4E•GFP fusions were not able to bind to mGTP Sepharose indicating that they were not functional as cap-binding proteins. Analysis of eIF4E•GFP fusions, both in yeast and , showed that the N-terminal fusion may be functional, whereas the C-terminal fusion bound mGTP Sepharose very poorly and functioned poorly in yeast. These results highlight the importance of verification both biochemically and that reporter fusions of proteins maintain activity and are stable in order to prevent observations that may result in artifacts.
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