Exposure to brefeldin A induces unusual expression of hybrid- and complex-type free N-glycans in HepG2 cells.

This study determined the effect of brefeldin A (BFA) on the free N-glycomic profile of HepG2 cells to better understand the effect of blocking intracellular vesicle formation and transport of proteins from the endoplasmic reticulum to the Golgi apparatus. A series of exoglycosidase- and endoglycosidase-assisted analyses clarified the complex nature of altered glycomic profiles. A key feature of BFA-mediated alterations in Gn2-type glycans was the expression of unusual hybrid-, monoantennary- and complex-type free N-glycans (FNGs). BFA-mediated alterations in Gn1-type glycans were characterized by the expression of unusual hybrid- and monoantennary-FNGs, without significant expression of complex-type FNGs. A time course analysis revealed that sialylated hybrid- and complex-type Gn2-type FNGs were generated later than asialo-Gn2-type FNGs, and the expression profiles of Gn2-type FNGs and N-glycans were found to be similar, suggesting that the metabolic flux of FNGs is the same as that of protein-bound N-glycans. Subcellular glycomic analysis revealed that almost all FNGs were detected in the cytoplasmic extracts. Our data suggest that hybrid-, monoantennary- and complex-type Gn2-type FNGs were cleaved from glycoproteins in the cytosol by cytosolic PNGase, and subsequently digested by cytosolic endo-ß-N-acetylglucosaminidase (ENGase) to generate Gn1-type FNGs. The substrate specificity of ENGase explains the limited expression of complex Gn1 type FNGs.

Rapeseed (canola) oil aggravates metabolic syndrome-like conditions in male but not in female stroke-prone spontaneously hypertensive rats (SHRSP).

This study was conducted to investigate whether or not there are sex differences in canola oil (CAN)-induced adverse events in the rat and to understand the involvement and the role of testosterone in those events, including life-shortening. Stroke-prone spontaneously hypertensive rats (SHRSP) of both sexes were fed a diet containing 10 wt/wt% soybean oil (SOY, control) or CAN as the sole dietary fat. The survival of the males fed the CAN diet was significantly shorter than that of those fed the SOY diet. In contrast, the survival of the females was not affected by CAN. The males fed the CAN diet showed elevated blood pressure, thrombopenia and insulin-tolerance, which are major symptoms of metabolic syndrome, whereas such changes by the CAN diet were not found in the females. Plasma testosterone was significantly lower in animals of both sexes fed the CAN diet than in those fed the SOY diet, but interestingly, the lowered testosterone was accompanied by a marked increase in plasma aldosterone only in the males. These results demonstrate significant sex differences in CAN-toxicity and suggest that those sex differences may be attributable to the increased aldosterone level, which triggers aggravation of the genetic diseases specific to SHRSP, that is, metabolic syndrome-like conditions, but only in the males. The present results also suggest that testosterone may negatively regulate aldosterone production in the physiology of the males, and the inhibition of that negative regulation caused by the CAN diet is one of the possible causes of the adverse events.

Different Renal Chronotoxicity of Bromobenzene and Its Intermediate Metabolites in Mice.

Bromobenzene (BB) is known to pose a serious threat to human health. We previously demonstrated that BB showed chronotoxicity, that is, daily fluctuations in the severity of hepatotoxicity induced in mice. Although BB showed mild nephrotoxicity, a daily fluctuation was not observed in this toxicity. This might be attributed to the fact that BB-induced chronotoxicity is observed only in the liver and not in the kidneys and that the damage caused by BB is prominent in the liver, masking the daily fluctuation in nephrotoxicity. To confirm these two possibilities, we examined the daily fluctuations in nephrotoxicity due to BB intermediate metabolites that target the kidneys: 3-bromophenol, bromohydroquinone, and 4-bromocatechol. Mice were injected with 3-bromophenol, bromohydroquinone, or 4-bromocatechol intraperitoneally at six different time points in a day (zeitgeber time (ZT): ZT2, ZT6, ZT10, ZT14, ZT18, or ZT22). Mortality was monitored for 7 d post-injection. Mice were more sensitive to the acute toxicity of these metabolites around at ZT14 (dark-phase) exposure than around at ZT2 (light-phase) exposure. Furthermore, mice administered with a non-lethal dose of 4-bromocatechol showed significant increases in the levels of plasma blood urea nitrogen and renal malondialdehyde at ZT14 exposure. Moreover, glutathione peroxidase-4, a ferroptosis indicator, was attenuated at ZT14 exposure. These results indicate the toxicity of BB metabolites was higher during the dark-phase exposure, and demonstrate the reason why the diurnal variation of nephrotoxicity by BB was not observed in our previous report is that renal damage was masked due to severe hepatic damage.

Sulforaphane induces thioredoxin through the antioxidant-responsive element and attenuates retinal light damage in mice.

PURPOSE: Thioredoxin (Trx) is a multifunctional endogenous redox regulator that protects cells against various types of cellular or tissue stresses. This study was conducted to test whether sulforaphane (SF), a naturally occurring isothiocyanate that is highly concentrated in broccoli sprouts, induces Trx in retinal tissues and whether pretreatment with SF protects against light-induced retinal damage in mice. METHODS: Expression of Trx in mouse retina was analyzed by Western blot and immunohistochemistry. Retinal damage was induced by exposure to white light at 6000 lux for 2 hours. To estimate retinal cell damage, the number of cell nuclei and the percentage of TUNEL-positive cells were counted in the outer nuclear layer and the retinal pigment epithelial (RPE) layer and the electroretinograms recorded. To analyze further the mechanism of Trx induction by SF, cultured human K-1034 RPE cells were used. RESULTS: Both intraperitoneal and oral SF induced Trx protein in the neural retina and RPE. The maximum induction of Trx was observed with intraperitoneal SF 0.5 mg/d for 3 days. After exposure to light, mice pretreated with SF had a significantly lower percentage of TUNEL-positive RPE and photoreceptor cells, a significantly higher number of RPE and photoreceptor nuclei, and greater amplitude of ERG a- and b-waves than in the saline-treated mice. In K-1034 cells, 1 microM SF induced Trx protein, whereas 10 microM SF did not damage cells or augment cellular peroxide production, tested by a lactate dehydrogenase (LDH) release assay and 2',7'-dichlorofluorescein diacetate (DCFH-DA)/flow cytometry, respectively. In the luciferase reporter assay, the antioxidant-responsive element (ARE) played a role in SF-induced Trx expression. In the electrophoretic mobility shift assay, SF induced binding of Nrf2, small Maf, and c-Jun to the ARE of the Trx gene. CONCLUSIONS: SF induced Trx in murine retina and effectively reduced retinal light damage. Evidence suggests that the ARE is involved in the mechanism of Trx induction by SF in RPE cells.

Cloning and characterization of a transmembrane-type serine protease from rat kidney, a new sodium channel activator.

We have cloned the gene of a new transmembrane-type serine protease from rat kidney, which activates sodium channels. The amino acid sequence deduced from a full-length cDNA revealed that transmembrane serine protease-1 (TMSP-1) is a member of the clan SA/family S1 of serine proteases, comprising a 30 amino acid prepropeptide, a mature form sequence of 274 amino acids starting with the Ile-Val-Gly-Gly-Gln motif, and a common catalytic triad of serine proteases. The hydrophobic amino acid sequence in the carboxy-terminus of this enzyme suggests that it is a glycosylphosphatidylinositol-anchored protein. As revealed by quantitative reverse transcription-polymerase chain reaction analysis, it is highly expressed in kidney, small intestine, and stomach, and moderately expressed in lung, thymus, spleen and skin. The recombinant protease had an optimal pH at 9.0, selectively cleaved synthetic peptide substrates of trypsin, and was inhibited by aprotinin, leupeptin and benzamidine. Immunohistochemical studies revealed that this protease is predominantly expressed in cells from collecting ducts of the renal medulla. We also demonstrate that a C-terminally truncated variant of TMSP-1 significantly activates the epithelial sodium channel, and that its mRNA levels are upregulated by aldosterone. These observations suggest that it is a new member of the trypsin-type transmembrane proteases, which regulate sodium balance by activating the epithelial sodium channel.

Cloning, expression analysis, and tissue distribution of esp-1/testisin, a membrane-type serine protease from the rat.

Esp-1/testisin, a serine protease abundantly expressed in human and mouse testis, is presumed to play an important role in the process of spermatogenesis and fertilization. In this study, we cloned an esp-1/testisin cDNA from rats, and analyzed its expression and tissue distribution. The isolated cDNA consisted of 1099 nucleotides with a single open reading frame encoding 328 amino acids and an expected molecular mass of 36.6 kDa. The deduced amino acid sequence of rat Esp-1/Testisin had 89% and 62% identity with its murine and human counterparts, respectively, and appeared to be a trypsin-type serine protease with a hydrophobic region at the C-terminus. By quantitative real-time polymerase chain reaction analysis, rat esp-1/testisin mRNA was predominantly expressed in testis, as in human and mouse. However, its immunohistochemical distribution was predominantly in the elongated spermatids at steps 12 to 19, and not in the primary spermatocytes and round spermatids. This different distribution profile suggests that Esp-1/Testisin plays a role in species-specific proteolytic events during spermatogenesis and fertilization.