Mammalian Pex14p: membrane topology and characterisation of the Pex14p-Pex14p interaction

Peroxisomal biogenesis is a complex process requiring the action of numerous peroxins. One central component of this machinery is Pex14p, an intrinsic peroxisomal membrane protein probably involved in the docking of Pex5p, the receptor for PTS1-containing proteins (peroxisomal targeting signal 1-containing proteins). In this work the membrane topology of mammalian Pex14p was studied. Using a combination of protease protection assays and CNBr cleavage, we show that the first 130 amino acid residues of Pex14p are highly protected from exogenously added proteases by the peroxisomal membrane itself. Data indicating that this domain is responsible for the strong interaction of Pex14p with the organelle membrane are presented. All the other Pex14p amino acid residues are exposed to the cytosol. The properties of recombinant human Pex14p were also characterised. Heterologous expressed Pex14p was found to be a homopolymer of variable stoichiometry. Finally, in vitro binding assays indicate that homopolymerisation of Pex14p involves a domain comprising amino acid residues 147-278 of this peroxin.     

Ring-14 and trisomy 14q in the same child

The case of a male child with three cell lines is described: one cell line with ring chromosome 14, another trisomic for 14q, due to a derived metacentric 14q;14q, and a third one with a normal male karyotype. The clinical findings are compatible with those of the r(14) syndrome.     

Metabolism of phytol-U-14C and phytanic acid-U-14C in the rat

The metabolism of uniformly-labeled (14)C-phytol, (14)C-phytenic acid, and (14)C-phytanic acid was studied in the rat. Conversion of both phytol and phytenic acid to phytanic acid was demonstrated. Tracer doses of phytol-U-(14)C given orally were well absorbed (30-66%), and approximately 30% of the absorbed dose was converted to (14)CO(2) in 18 hr. After intravenous injection, 20% appeared in (14)CO(2) in 4 hr. Phytanic acid-U-(14)C given intravenously was oxidized at a comparable rate (22-37% in 4 hr) and was as rapidly oxidized as palmitic acid-1-(14)C (21% in 4 hr). Metabolism of these substrates was also studied in rats previously maintained on a diet containing 5% phytol by weight, which causes accumulation of phytanic acid, phytenic acid, and, to a lesser extent, phytol in blood and tissues. Despite the large body pools of preformed, unlabeled substrate in these animals, the fraction of an administered dose of phytol-U-(14)C or phytanic acid-U-(14)C converted to (14)CO(2) was not significantly diminished. These studies indicate that the rat has an appreciable capacity to degrade the highly branched carbon skeleton of phytol and its derivatives. Twenty-four hours after administration of phytol-U-(14)C, the lipid radioactivity remaining in the body was widely distributed among the tissues, highest concentrations being found in liver and adipose tissue. Four hours after intravenous administration of phytanic acid-U-(14)C, all of the major lipid classes in the liver contained radioactivity, most in triglycerides and phospholipids and least in cholesterol esters and lower glycerides. There was no demonstrable incorporation of mevalonate-2-(14)C or acetate-1-(14)C into liver phytanic acid when they were given intravenously to a rat previously fed phytol. Endogenous biosynthesis, if it occurs at all, must be extremely limited.     

Development of the Assessment of Belief Conflict in Relationship-14 (ABCR-14)

Purpose

Nurses and other healthcare workers frequently experience belief conflict, one of the most important, new stress-related problems in both academic and clinical fields.

Methods

In this study, using a sample of 1,683 nursing practitioners, we developed The Assessment of Belief Conflict in Relationship-14 (ABCR-14), a new scale that assesses belief conflict in the healthcare field. Standard psychometric procedures were used to develop and test the scale, including a qualitative framework concept and item-pool development, item reduction, and scale development. We analyzed the psychometric properties of ABCR-14 according to entropy, polyserial correlation coefficient, exploratory factor analysis, confirmatory factor analysis, average variance extracted, Cronbach’s alpha, Pearson product-moment correlation coefficient, and multidimensional item response theory (MIRT).

Results

The results of the analysis supported a three-factor model consisting of 14 items. The validity and reliability of ABCR-14 was suggested by evidence from high construct validity, structural validity, hypothesis testing, internal consistency reliability, and concurrent validity. The result of the MIRT offered strong support for good item response of item slope parameters and difficulty parameters. However, the ABCR-14 Likert scale might need to be explored from the MIRT point of view. Yet, as mentioned above, there is sufficient evidence to support that ABCR-14 has high validity and reliability.

Conclusion

The ABCR-14 demonstrates good psychometric properties for nursing belief conflict. Further studies are recommended to confirm its application in clinical practice.     

Evidence for the contribution of a sterol 14-reductase to the 14 alpha-demethylation of lanosterol by yeast

Lanosterol was converted to a 14-demethylated metabolite, 4,4-dimethylzymosterol by Saccharomyces cerevisiae microsomes. This metabolism was mediated by a cytochrome P-450 (P-450/14DM). However, a reconstituted system consisting of P-450/14DM and its reductase converted lanosterol to the 14-desaturated derivative of 4,4-dimethylzymosterol, 4,4-dimethyl-5 alpha-cholesta-8, 14,24-trien-3 beta-ol (trienol). When AY-9944 was added to the reaction system with the microsomes, the trienol was formed with corresponding decrease in 4,4-dimethylzymosterol. These observations indicate that the 14 alpha-demethylation of lanosterol by yeast microsomes occurs sequentially via the trienol. Reduction of the trienol to 4,4-dimethylzymosterol is mediated by an AY-9944-sensitive reductase.     

Metabolic conversion of exogenous14C-aspartate and14C-gIutamate in the dark byFucus serratus L

The rates of uptake of exogenous L[U-¹⁴C] aspartate and glutamate into tissues of vegetative growing tips ofFucus serratus and their metabolism were studied in the dark. In these non-photosynthetic conditions, aspartate was fixed and metabolically converted more rapidly than glutamate. Radioactivity from¹⁴C-aspartate was principally transferred into glutamate. On the other hand, metabolism of absorbed¹⁴C-glutamate was very slow and its rate did not increase during incubation time, but produced more diversified soluble radioactive compounds. Thus inF. serratus, glutamate principally seems to be in the dark more a temporary¹⁴CO₂ storage product coming from β-carboxylation than a rapidly turned over intermediate.     

The Drosophila melanogaster gene vha14 encoding a 14-kDa F-subunit of the vacuolar ATPase

A Drosophila melanogaster (Dm) cDNA (vha14) encoding the 14-kDa F-subunit of the vacuolar H⁺-ATPase (V-ATPase) has been cloned via homology with the corresponding Manduca sexta (Ms) gene. Its deduced translation product is a 124-amino-acid polypeptide sharing 90% identity with the Ms polypeptide and 50% identity with an analogous polypeptide of Saccharomyces cerevisiae, and a more distant similarity to a subunit of the Na⁺-transporting ATPase of Enterococcus hirae. Homology was also found with expressed sequence tags from man, Arabidopsis thaliana, Caenorhabditis elegans and C. briggsiae, Oryza sativa and Plasmodium falciparum, indicating that the subunit is phylogenetically conserved. The Dm gene (vha14) is present as a single copy at cytological position 52B on the second chromosome, and gives rise to an mRNA species of 0.65 kb. Expression of the latter shows relatively little variation during development, or between adult head, thorax and abdomen, suggesting that the F-subunit is a relatively ubiquitous component of the V-ATPase.