Regulation of peroxisomal lipid metabolism by catalytic activity of tumor suppressor H-rev107

H-rev107 is a mammalian protein belonging to the HRAS-like suppressor family. Although the protein was originally found as a tumor suppressor, currently it is receiving considerable attention as a regulator of adipocyte lipolysis. We recently revealed that purified recombinant H-rev107 has phospholipase A(1/2) activity, releasing free fatty acids from glycerophospholipids with a preference for esterolysis at the sn-1 position. In the present study, we constitutively expressed H-rev107 in cloned HEK293 cells to examine its biological function in living cells. Initially, the cells accumulated free fatty acids. We also found a remarkable decrease in the levels of ether-type lipids, including plasmalogen and ether-type triglyceride, with a concomitant increase in fatty alcohols, substrates for the biosynthesis of ether-type lipids. Considering that peroxisomes are involved in the ether-type lipid biosynthesis, we next focused on peroxisomes and found that the peroxisomal markers 70-kDa peroxisomal membrane protein and catalase were abnormally distributed in the transfected cells. These biochemical and morphological abnormalities were not seen in HEK293 cells stably expressing a catalytically inactive mutant of H-rev107. When H-rev107 or its fusion protein with enhanced green fluorescence protein was transiently expressed in mammalian cells, both proteins were associated with peroxisomes in some of the observed cells. These results suggest that H-rev107 interferes with the biosynthesis of ether-type lipids and is responsible for the dysfunction of peroxisomes in H-rev107-expressing cells.     

Identification of Novel α1,3-Galactosyltransferase and Elimination of α-Galactose-containing Glycans by Disruption of Multiple α-Galactosyltransferase Genes in Schizosaccharomyces pombe

The oligosaccharides from fission yeast Schizosaccharomyces pombe contain large amounts of d-galactose (Gal) in addition to d-mannose (Man), in contrast to the budding yeast Saccharomyces cerevisiae. Detailed structural analysis has revealed that the Gal residues are attached to the N- and O-linked oligosaccharides via α1,2- or α1,3-linkages. Previously we constructed and characterized a septuple α-galactosyltransferase disruptant (7GalTΔ) anticipating a complete lack of α-Gal residues. However, the 7GalTΔ strain still contained oligosaccharides consisting of α1,3-linked Gal residues, indicating the presence of at least one more additional unidentified α1,3-galactosyltransferase. In this study we searched for unidentified putative glycosyltransferases in the S. pombe genome sequence and identified three novel genes, named otg1⁺–otg3⁺ (α one, three-galactosyltransferase), that belong to glycosyltransferase gene family 8 in the Carbohydrate Active EnZymes (CAZY) database. Gal-recognizing lectin blotting and HPLC analyses of pyridylaminated oligosaccharides after deletion of these three additional genes from 7GalTΔ strain demonstrated that the resultant disruptant missing 10 α-galactosyltransferase genes, 10GalTΔ, exhibited a complete loss of galactosylation. In an in vitro galactosylation assay, the otg2⁺ gene product had Gal transfer activity toward a pyridylaminated Man₉GlcNAc₂ oligosaccharide and pyridylaminated Manα1,2-Manα1,2-Man oligosaccharide. In addition, the otg3⁺ gene product exhibited Gal transfer activity toward the pyridylaminated Man₉GlcNAc₂ oligosaccharide. Generation of an α1,3-linkage was confirmed by HPLC analysis, α-galactosidase digestion analysis, ¹H NMR spectroscopy, and LC-MS/MS analysis. These results indicate that Otg2p and Otg3p are involved in α1,3-galactosylation of S. pombe oligosaccharides.     

Expression of (pro)renin receptor in human erythroid cell lines and its increased protein accumulation by interferon-γ

The renin-angiotensin system is known to enhance erythropoiesis. (Pro)renin receptor ((P)RR), a specific receptor for renin and prorenin, has recently been identified. However, expression of (P)RR in erythroid cells has not been studied. The aim of the present study is to clarify expression of (P)RR in erythroid cells, and the effects of erythropoietin, angiotensin II, transforming growth factor-β1 (TGF-β1), interferon-γ (IFN-γ) and interleukin-1β (IL-1β) on its expression. Western blot analysis showed that (P)RR protein was expressed in human cultured erythroid cell lines, YN-1 and YN-1-0-A (a clonal variant cell line of YN-1). Erythropoietin (1IU/ml) increased (P)RR mRNA expression levels in YN-1-0-A cells (1.7-fold increase compared with control), but angiotensin II did not. Treatment of YN-1-0-A cells with IFN-γ (10ng/ml) for 48h increased the expression levels of (P)RR protein significantly (1.4-fold increase compared with control), whereas it had no significant effects on expression levels of (P)RR mRNA. Treatment of YN-1-0-A cells with TGF-β1 or IL-1β for 24 or 48h had no significant effects on expression levels of (P)RR. The present study has shown for the first time expression of (P)RR in erythroid cells, raising the possibility that (P)RR may have a role in erythropoiesis and the pathophysiology of certain types of anemia.     

Lipophilic amines as potent inhibitors of N-acylethanolamine-hydrolyzing acid amidase

N-Acylethanolamines (NAEs) including N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine are endogenous lipid mediators. These molecules are degraded to the corresponding fatty acids and ethanolamine by fatty acid amide hydrolase (FAAH) or NAE-hydrolyzing acid amidase (NAAA). Lipophilic amines, especially pentadecylamine (2c) and tridecyl 2-aminoacetate (11b), were found to exhibit potent NAAA inhibitory activities (IC(50)=5.7 and 11.8μM), with much weaker effects on FAAH. These simple structures would provide a scaffold for further improvement in NAAA inhibitory activity.     

Anti-parallel membrane topology of two components of EbrAB, a multidrug transporter

EbrAB is a multidrug-resistance transporter in Bacillus subtilis that belongs to the small multidrug resistance, and requires two polypeptides of both EbrA and EbrB, implying that it functions in the hetero-dimeric state. In this study, we investigated the transmembrane topologies of EbrA and EbrB. Various single-cysteine mutants were expressed in Escherichia coli cells, and the efflux activity was measured. Only mutants having a high activity were used for the topology experiments. The reactivity of a membrane impermeable NEM-fluorescein against the single cysteine of these fully functional mutants was examined when this reactive fluorophore was applied either from the outside or both sides of the cell membrane or in the denatured state. The results clearly showed that EbrA and EbrB have the opposite orientation within the membrane or an anti-parallel configuration.     

Effects of Disruption of Homocitrate Synthase Genes on Nostoc sp. Strain PCC 7120 Photobiological Hydrogen Production and Nitrogenase

In the case of nitrogenase-based photobiological hydrogen production systems of cyanobacteria, the inactivation of uptake hydrogenase (Hup) leads to significant increases in hydrogen production activity. However, the high-level-activity stage of the Hup mutants lasts only a few tens of hours under air, a circumstance which seems to be caused by sufficient amounts of combined nitrogen supplied by active nitrogenase. The catalytic FeMo cofactor of nitrogenase binds homocitrate, which is required for efficient nitrogen fixation. It was reported previously that the nitrogenase from the homocitrate synthase gene (nifV) disruption mutant of Klebsiella pneumoniae shows decreased nitrogen fixation activity and increased hydrogen production activity under N₂. The cyanobacterium Nostoc sp. strain PCC 7120 has two homocitrate synthase genes, nifV1 and nifV2, and with the ΔhupL variant of Nostoc sp. strain PCC 7120 as the parental strain, we have constructed two single mutants, the ΔhupL ΔnifV1 strain (with the hupL and nifV1 genes disrupted) and the ΔhupL ΔnifV2 strain, and a double mutant, the ΔhupL ΔnifV1 ΔnifV2 strain. Diazotrophic growth rates of the two nifV single mutants and the double mutant were decreased moderately and severely, respectively, compared with the rates of the parent ΔhupL strain. The hydrogen production activity of the ΔhupL ΔnifV1 mutant was sustained at higher levels than the activity of the parent ΔhupL strain after about 2 days of combined-nitrogen step down, and the activity in the culture of the former became higher than that in the culture of the latter. The presence of N₂ gas inhibited hydrogen production in the ΔhupL ΔnifV1 ΔnifV2 mutant less strongly than in the parent ΔhupL strain and the ΔhupL ΔnifV1 and ΔhupL ΔnifV2 mutants. The alteration of homocitrate synthase activity can be a useful strategy for improving sustained photobiological hydrogen production in cyanobacteria.     

Role of fibrosis-related genes and pancreatic duct obstruction in rat pancreatitis models: implications for chronic pancreatitis

Human chronic pancreatitis is characterized by irreversible fibrosis, whereas pancreatic fibrosis in animal models is reversible. In this study, we compare the development of pancreatic fibrosis in the dibutyltin dichloride (DBTC) model, WBN/Kob rats and bile duct-ligated (BDL) rats. DBTC (8 mg/kg) was administered to LEW rats, and the pancreas was histopathologically investigated sequentially. Male and female WBN/Kob rats aged 4, 6 and 8 months were also examined. BDL rats were prepared by ligation of the bile duct at the duodenal portion and sacrificed at 3 or 7 days after ligation. Fibrosis in the DBTC model peaked after 1 week and was limited to the areas around the pancreatic ducts after 2 weeks, and was composed of both type I and type III collagen. In contrast, fibrosis in male WBN/Kob rats peaked at age 4 months, expanded into intralobular area, and was composed of type III collagen. It exhibited almost no type I collagen and a marked tendency to regress. Pancreatic fibrosis in BDL rats was somewhat difficult to induce and required increased stimulation. This suggests that fibrosis in human biliary pancreatitis may gradually form based on weak, continuous stimulation. We conclude that type I collagen may be involved in the progression of irreversible fibrosis. The imbalance between synthesis and degradation of extracellular matrix molecules or degree of stimulation over a certain period may lead to pancreatic fibrosis. Gene expressions of prolyl hydroxylase and tissue inhibitors of matrix metalloproteinase-2 were elevated.     

Truncations of amphiphysin I by calpain inhibit vesicle endocytosis during neural hyperexcitation

Under normal physiological conditions, synaptic vesicle endocytosis is regulated by phosphorylation and Ca(2+)-dependent dephosphorylation of endocytic proteins such as amphiphysin and dynamin. To investigate the regulatory mechanisms that may occur under the conditions of excessive presynaptic Ca(2+) influx observed preceding neural hyperexcitation, we examined hippocampal slices following high-potassium or high-frequency electrical stimulation (HFS). In both cases, three truncated forms of amphiphysin I resulted from cleavage by the protease calpain. In vitro, the binding of truncated amphiphysin I to dynamin I and copolymerization into rings with dynamin I were inhibited, but its interaction with liposomes was not affected. Moreover, overexpression of the truncated form of amphiphysin I inhibited endocytosis of transferrin and synaptic vesicles. Inhibiting calpain prevented HFS-induced depression of presynaptic transmission. Finally, calpain-dependent amphiphysin I cleavage attenuated kainate-induced seizures. These results suggest that calpain-dependent cleavage of amphiphysin I inhibits synaptic vesicle endocytosis during neural hyperexcitation and demonstrate a novel post-translational regulation of endocytosis.     

PCR method of detecting pork in foods for verifying allergen labeling and for identifying hidden pork ingredients in processed foods

A PCR method to detect porcine DNA was developed for verifying the allergen labeling of foods and for identifying hidden pork ingredients in processed foods. The primer pair, F2/R1, was designed to detect the gene encoding porcine cytochrome b for the specific detection of pork with high sensitivity. The amplified DNA fragment (130 bp) was specifically detected from porcine DNA, while no amplification occurred with other species such as cattle, chicken, sheep, and horse. When the developed PCR method was used for investigating commercial food products, porcine DNA was clearly detected in those containing pork in the list of ingredients. In addition, 100 ppb of pork in heated gyoza (pork and vegetable dumpling) could be detected by this method. This method is rapid, specific and sensitive, making it applicable for detecting trace amounts of pork in processed foods.     

Evidence for new beta1-3 galactosyltransferase activity involved in biosynthesis of unusual N-glycan harboring T-antigen in Apis mellifera

In a previous study (Y. Kimura et al., Biosci. Biotechnol. Biochem., 70, 2583-2587, 2006), we found that new complex type N-glycans harboring Thomsen-Friedenreich antigen (Galbeta1-3GalNAc) unit occur on royal jelly glycoproteins, suggesting the involvement of a new beta1-3galactosyltransferase in the synthesis of the unusual complex type N-glycans. So far, such beta1-3galactosyltransferase activity, which can transfer galactosyl residues with the beta1-3 linkage to beta1-4 GalNAc residues in N-glycan, has not been found among any eucaryotic cells. But using GalNAc(2)GlcNAc(2)Man(3)GlcNAc(2)-PA as acceptor N-glycan, we detected the beta1-3 galactosyltransferase activity in membrane fraction prepared from honeybee cephalic portions. This result indicates that honeybee expresses a unique beta1-3 galactosyltransferase involved in biosynthesis of the unusual N-glycan containing a tumor related antigen in the hypopharyngeal gland.