Aquaporin-11 containing a divergent NPA motif has normal water channel activity

Recently, two novel mammalian aquaporins (AQPs), AQPs 11 and 12, have been identified and classified as members of a new AQP subfamily, the “subcellular AQPs”. In members of this subfamily one of the two asparagine-proline-alanine (NPA) motifs, which play a crucial role in selective water conduction, are not completely conserved. Mouse AQP11 (mAQP11) was expressed in Sf9 cells and purified using the detergent Fos-choline 10. The protein was reconstituted into liposomes, which were used for water conduction studies with a stopped-flow device. Single water permeability (pf) of AQP11 was measured to be 1.72 ± 0.03 × 10^− 13 cm³/s, suggesting that other members of the subfamily with incompletely conserved NPA motifs may also function as water channels.     

Changes in the components of biliary and plasma lipids in selenium-deficient rats

We constructed a chronic oxidative stress model in which Se-deficient diet was fed to male Wister rats for 8 weeks. As expected, effects of oxidative damage, including Fe accumulation and increase in peroxidized lipids, were identified in the liver owing to the lack of glutathione peroxidase. Although the oxidative stress caused Fe accumulation in the liver, the Fe concentration in bile of the SeD rat was almost the same as that in the control rats. The constant excretion of Fe into bile supported the Fe accumulation in the liver. No differences were observed in the principal components of biliary lipids, i.e., bile acids, phospholipids, and cholesterol, between the two groups; moreover, these trends were also reflected in the plasma. Due to the trapping of reactive oxygen species, only bilirubin concentrations in the bile and plasma were decreased in the SeD group, when compared with those in the control group. Measurement of bilirubin concentration may be used as a supplemental oxidative stress marker.     

Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, Marsupenaeus japonicus

In penaeid shrimps, vitellogenin (VTG), the precursor of vitellin, is synthesized in the ovary and hepatopancreas and accumulated in oocytes during ovarian development. In the present study, VTG gene expression levels and hemolymph VTG levels were determined throughout ovarian development in female kuruma prawn, Marsupenaeus japonicus. Hemolymph VTG levels and VTG mRNA levels in the ovary and hepatopancreas were high during vitellogenesis, remained high until final maturation, and then decreased after oviposition. This profile suggests that VTG synthesis activity increases during vitellogenesis and decreases after oviposition. Absence of a significant increase in ovary size in final maturation suggests cessation of yolk accumulation and low activity of VTG synthesis in spite of high VTG mRNA levels. VTG mRNA levels in ovary and hepatopancreas were both highly correlated during vitellogenesis. Thus, their contribution to yolk accumulation seems to be similar. In contrast, VTG mRNA levels in the hepatopancreas increased more slowly at the start of vitellogenesis and declined more sharply after oviposition than in the ovary. This suggests a difference in the regulation of VTG synthesis between the ovary and the hepatopancreas.     

Porcine germinal angiotensin I-converting enzyme: isolation, characterization and molecular cloning

Germinal angiotensin I-converting enzyme (gACE) was purified to homogeneity from porcine seminal plasma. The molecular weight of the purified enzyme was calculated to be 182,000 on non-denaturing PAGE and 94,000 and 93,000 on SDS-PAGE in the absence and presence of beta-ME, respectively. These findings suggest that the enzyme is composed of two identical subunits in seminal plasma. The K(m), V(max), K(cat) and K(cat)/K(m) values of gACE at optimal pH (pH 7.2) were 680 microM, 1.0 micromol/mg/min, 33.1 s(-1) and 4.87 x 10(4) s(-1) M(-1) for Z-Val-Lys-Met-MCA, respectively. gACE was potently inhibited by EDTA, 1,10-phenanthroline, captopril and lisinopril, and it promptly released the dipeptides His-Leu and Phe-Arg from angiotensin I and bradykinin. Met- and Leu-enkephalins, neuromedine B and beta-neo-endorphin were also good natural substrates for gACE. We determined the structure of gACE cDNA from the porcine testis, and deduced the amino acid sequence of gACE. The cDNA is composed of 2508 bp of nucleotides in length and encodes 745 amino acids in the coding region. The overall homology of amino acid sequences between porcine, human, sheep and rat gACEs is 72.6 to 84.7%. Zinc-binding motif, chloride-binding site and positions of cysteine residues were well conserved.     

RFPL4A Increases the G1 Population and Decreases Sensitivity to Chemotherapy in Human Colorectal Cancer Cells

Cell cycle-arrested cancer cells are resistant to conventional chemotherapy that acts on the mitotic phases of the cell cycle, although the molecular mechanisms involved in halting cell cycle progression remain unclear. Here, we demonstrated that RFPL4A, an uncharacterized ubiquitin ligase, induced G₁ retention and thus conferred decreased sensitivity to chemotherapy in the human colorectal cancer cell line, HCT116. Long term time lapse observations in HCT116 cells bearing a “fluorescence ubiquitin-based cell cycle indicator” identified a characteristic population that is viable but remains in the G₁ phase for an extended period of time (up to 56 h). Microarray analyses showed that expression of RFPL4A was significantly up-regulated in these G₁-arrested cells, not only in HCT116 cells but also in other cancer cell lines, and overexpression of RFPL4A increased the G₁ population and decreased sensitivity to chemotherapy. However, knockdown of RFPL4A expression caused the cells to resume mitosis and induced their susceptibility to anti-cancer drugs in vitro and in vivo. These results indicate that RFPL4A is a novel factor that increases the G₁ population and decreases sensitivity to chemotherapy and thus may be a promising therapeutic target for refractory tumor conditions.     

An Archaeal Glutamate Decarboxylase Homolog Functions as an Aspartate Decarboxylase and Is Involved in β-Alanine and Coenzyme A Biosynthesis

β-Alanine is a precursor for coenzyme A (CoA) biosynthesis and is a substrate for the bacterial/eukaryotic pantothenate synthetase and archaeal phosphopantothenate synthetase. β-Alanine is synthesized through various enzymes/pathways in bacteria and eukaryotes, including the direct decarboxylation of Asp by aspartate 1-decarboxylase (ADC), the degradation of pyrimidine, or the oxidation of polyamines. However, in most archaea, homologs of these enzymes are not present; thus, the mechanisms of β-alanine biosynthesis remain unclear. Here, we performed a biochemical and genetic study on a glutamate decarboxylase (GAD) homolog encoded by TK1814 from the hyperthermophilic archaeon Thermococcus kodakarensis. GADs are distributed in all three domains of life, generally catalyzing the decarboxylation of Glu to γ-aminobutyrate (GABA). The recombinant TK1814 protein displayed not only GAD activity but also ADC activity using pyridoxal 5′-phosphate as a cofactor. Kinetic studies revealed that the TK1814 protein prefers Asp as its substrate rather than Glu, with nearly a 20-fold difference in catalytic efficiency. Gene disruption of TK1814 resulted in a strain that could not grow in standard medium. Addition of β-alanine, 4′-phosphopantothenate, or CoA complemented the growth defect, whereas GABA could not. Our results provide genetic evidence that TK1814 functions as an ADC in T. kodakarensis, providing the β-alanine necessary for CoA biosynthesis. The results also suggest that the GAD activity of TK1814 is not necessary for growth, at least under the conditions applied in this study. TK1814 homologs are distributed in a wide range of archaea and may be responsible for β-alanine biosynthesis in these organisms.