Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisomedeficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N-and Cterminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40 -42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

The 41-kDa membrane-anchored peroxin Pex14p functions as the peroxisome targeting signal (PTS) receptor-mediated, initial import site for matrix proteins. We here identify the functional domains of Pex14p involved in the assembly of import site subcomplexes. The minimal region of Pex14p required for restoring impaired protein import in pex14 Chinese hamster ovary cell mutant lies at residues 21-260 in the primary sequence. A highly conserved N-terminal region, encompassing residues 21-70, interacts with the PTS1 receptor Pex5p, Pex13p, and Pex19p that is essential for membrane biogenesis. N-terminal residues 21-140, including a hydrophobic segment at 110 -138, function as a topogenic sequence. Site-directed mutagenesis, size fractionation, and chemical cross-linking analyses demonstrate that the coiled-coil domain at residues 156 -197 regulates homodimerization of Pex14p. Moreover, AXXXA and GXXXG motifs in the transmembrane segment mediate homomeric oligomerization of Pex14p, giving rise to assembly of high molecular mass complexes and thereby assuring Pex13p-dependent localization of Pex14p to peroxisomes. Pex5p, Pex13p, and Pex19p bind to Pex14p homo-oligomers with different molecular masses, whereas cargo-unloaded Pex5p apparently disassembles Pex14p homo-oligomers. Thus, Pex14p most likely forms several distinct peroxin complexes involved in peroxisomal matrix protein import.Peroxisome is a ubiquitous, spherical organelle present in virtually all of eukaryotes, from yeast to mammals. Peroxisome functions in a wide variety of metabolic pathways, including ␤-oxidation of very long chain fatty acids and biosynthesis of plasmalogens (1). More than 30 peroxins have been identified (2-4). The import processes of matrix proteins include: 1) recognition of peroxisomal targeting signals 1 and 2 (PTS1 and PTS2) 2 of proteins by cytosolic receptors, Pex5p and Pex7p; 2) translocation and docking of the cargo protein-PTS receptor complexes to a potential import machinery on peroxisome membranes; and 3) unloading of cargoes into the matrix. In mammals, the longer isoform of Pex5p, termed Pex5pL, also functions as Pex7p-PTS2 transporter (5, 6). Potential import machinery complexes on peroxisome membranes contain several peroxins, including Pex14p, Pex13p, and RING peroxins such as Pex2p, Pex10p, and Pex12p (6, 7). Pex14p plays a central role in matrix protein import (5, 6, 8 -11). Biochemical functions of other members of the import machinery, such as Pex13p and RING peroxins remain to be better defined.We used genetic phenotype complementation screening of the peroxisome-deficient Chinese hamster ovary (CHO) cell mutants, ZP110 and ZP161, to isolate mammalian PEX14 that encoded a 41-kDa integral membrane peroxin (11). Pex14p comprises three distinct parts, a highly conserved N-terminal region containing a hydrophobic sequence, a typical coiled-coil domain in the middle region, and a C-terminal part. In mammals, interactions of Pex14p with Pex5p (6, 12) and Pex13p (6, 13) as well as Pex19p (14 -16) have been reported. How...

Six influenza A(H1N1)pdm09 viruses were detected in Sapporo, Japan, between November and December 2013. All six viruses possessed an H275Y substitution in the neuraminidase protein, which confers cross-resistance to oseltamivir and peramivir. No epidemiological link among the six cases could be identified; none of them had received neuraminidase inhibitors before specimen collection. The haemagglutinin and neuraminidase genes of the six viruses were closely related to one another, suggesting clonal spread of a single resistant virus.

To evaluate whether habitual excess sodium intake is a significant risk factor for calcium loss, we studied the relation between calcium excretion and sodium excretion in 410 male and 476 female Japanese aged 20-79 y. They were apparently healthy, free-living, and consuming diets of their own choosing. We divided the subjects into two groups: 20-49 y olds and 50-79 y olds. In each group, we observed significant positive correlation between daily calcium excretion and daily sodium excretion in both sexes. Multivariate analyses revealed that in each age group the relation was still significant after sex, age, body weight, and protein, calcium, and phosphorus intakes were adjusted for. The increases in urinary calcium excretion were estimated to be approximately 0.6 and 1.0 mmol for a 100-mmol increment in urinary sodium excretion for the 20-49 y olds and 50-79-y olds, respectively. We also observed significant positive correlations between daily hydroxyproline excretion and daily sodium excretion in both sexes for both age groups. The relation was still significant after sex, age, body weight, and protein intake from meat and fish were adjusted for. The results suggest that individuals with high sodium intake may lose more calcium in their urine than those with low sodium intake.

A 5'-nucleotidase (EC 3.1.3.5) was highly purified from the soluble fraction of rat heart. The preparation appeared homogeneous by the criterion of polyacrylamide-gel electrophoresis. The enzyme was activated by ATP and ADP, and inhibited by Pi. When AMP was used as substrate, the velocity/substrate-concentration plot was sigmoidal. ATP or ADP changed the plot to hyperbolic and decreased S0.5. Pi increased both the sigmoidicity of the plot and S0.5. When IMP was used as substrate, the velocity/substrate plot was hyperbolic. ATP or ADP decreased Km and increased V. Pi changed the plot to sigmoidal and increased S0.5. Within the range of adenylate energy charge observed in surviving mammalian cells (0.7-0.9), the rate of AMP-hydrolysing activity catalysed by the 5'-nucleotidase increased sharply with decreasing energy charge. The highest activity was observed at an energy-charge value of about 0.6. The response was also observed in the presence of Pi. No change in IMP-hydrolysing activity was observed in the physiological range of adenylate energy charge, but in the presence of Pi the activity gradually increased with increasing energy charge. These results suggest the possibility that this enzyme participates in production of adenosine, a vasodilator, during hypoxia and in removal of IMP, which accumulates during the hypoxia, in the heart.