Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.     

Changes in Ultrastructure and Subunit Composition of Protein Body in Rice Endosperm during Germination

Changes in ultrastructure of protein bodies in subaleurone cells of rice endosperm during germination were studied by transmission electron microscopy. The subaleurone cells contained two different types of protein bodies: PB-I (spherical) and PB-II (crystalline). Both types of protein bodies were deconstructed during germination. But there was a considerable difference in digestibility between PB-I and PB-II. PB-II which did not have a dense core was easily digested from the central portion when germination began. At 6 days of germination, PB-II was almost deconstructed. On the other hand, PB-I which displayed concentric rings with a dense core was digested from the outside after 3 days of germination. At 9 days of germination, many kernels of the spherical protein bodies remained.

Changes in subunit composition of protein bodies during germination were investigated by SDS-polyacrylamide gel electrophoresis. Protein body fractions were isolated from germinating grains at various stages by enzymatic digestion and two-phase system, then subjected to SDS-polyacrylamide gel. As germination proceeded, 15 (b₁), 20 (d₁), 24 (e), 35 (f₁) and 37 (f₃) kdaltons subunits decreased. On the other hand, 16 (b₂), 21 (d₂) and 36 (f₂) k daltons subunits remained at the later stage of germination. We think that PB-I contains b₂, d₂ and f₂ subunits and is attacked only from the outside at middle and later stages of germination by de novo protease. On the contrary, PB-II contains b₁ d₁ e, f₁ and f₃ subunits is utilized at an early stage of germination. PB-II may possibly contain latent protease. The breakdown process of PB-I was by exo-type digestion, on the contrary, that of PB-II was by endo-type digestion.     

Studies on Phyto-peroxidase

Both crystalline Japanese-radish peroxidase a and c could catalyze the aerobic destruction of indole-3-acetic acid in the presence of a small amount of hydrogen peroxide as an inducer. The pH dependences of the enzymic activities of peroxidase a and c were different from each other. The optimum pH of peroxidase a was found at 3.6, while that of peroxidase c lied in a broad range over 3 to 4.8. In lower concentrations of both peroxidases at pH 3.8 and 6.0, the major product of the reaction was identical with that presented by Ray with using an Omphalia enzyme, which would probably be methyldioxindole, while, in higher concentrations of both peroxidases at pH 3.8, the major product was found to be indole-aldehyde.     

Clostridium perfringens type A enterotoxin induces release of noradrenaline from the neurosecretory PC12 cell line

Clostridium perfringens type A enterotoxin(500 ng/ml) induced extensive release of noradrenaline (1/3-2/3 of the total cell content) from PC12 cells in 2-4 min in the presence, but not the absence of extracellular Ca2+. Cells treated with toxin in the absence of Ca2+ released noradrenaline promptly on subsequent addition of Ca2+ to the medium. The amount of noradrenaline released depended on the concentrations of both Ca2+ and toxin in the medium (ED50, 0.3 mM and 420 ng/ml respectively). Ca2+ could be replaced by Ba2+ or Sr2+, and Mn2+ or Co2+, which are Ca2+ channel blockers, did not inhibit the release of the transmitter. These findings are discussed in relation to the systemic effects of enterotoxin.     

Rapid, simplified method for production and purification of tetanus toxin.

A rapid, simplified method for production and purification of tetanus toxin from bacterial extracts was described. The extracts were prepared by stirring young cells (ca. 45-h culture) of Clostridium tetani in 1 M NaCl-0.1 M sodium citrate, pH 7.5, overnight at 0 to 4 degrees C. The toxin was purified by a combination of (i) ammonium sulfate fractionation (0 to 40% saturation), (ii) ultracentrifugation for removal of particulate materials, and (iii) gel filtration by high-pressure liquid chromatography on a TSK G3000 SW-type column. This method required 6 days as follows: (i) overnight incubation of the seed culture, (ii) 2 days for growing the bacteria for toxin production, (iii) overnight extraction of the toxin from the bacteria, (iv) overnight precipitation of the toxin with ammonium sulfate, (v) 2 h for ultracentrifugation of the ammonium sulfate concentrate of the bacterial extract, and (vi) 1 h for high-pressure liquid chromatography. The minimum lethal dose of the purified toxin preparations for mice was 1.4 X 10(7) to 1.5 X 10(7) per mg of protein and they showed 360 to 390 Lf (flocculating activity) per mg protein and a 280/260 nm absorbance ratio of 2.0 to 2.1. The final recovery of the toxin from bacterial extracts was 90 to 93%. The purified preparations gave a single band of toxin protein with a molecular weight of 150,000 +/- 5,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On crossed immunoelectrophoresis, the purified toxin preparations gave a single precipitation arc against anti-crude toxin serum.     

Inhibitory effect of p53 on mitochondrial content and function during adipogenesis

The p53 protein is known as a guardian of the genome and is involved in energy metabolism. Since the metabolic system is uniquely regulated in each tissue, we have anticipated that p53 also would play differential roles in each tissue. In this study, we focused on the functions of p53 in white adipose tissue (adipocytes) and skeletal muscle (myotubes), which are important peripheral tissues involved in energy metabolism. We found that in 3T3-L1 preadipocytes, but not in C2C12 myoblasts, p53 stabilization or overexpression downregulates the expression of Ppargc1a, a master regulator of mitochondrial biogenesis. Next, by using p53-knockdown C2C12 myotubes or 3T3-L1 preadipocytes, we further examined the relationship between p53 and mitochondrial regulation. In C2C12 myoblasts, p53 knockdown did not significantly affect Ppargc1a expression and mtDNA, but did suppress differentiation to myotubes, as previously reported. However, in 3T3-L1 preadipocytes and mouse embryonic fibroblasts, p53 downregulation enhanced both differentiation into adipocytes and mitochondrial DNA content. Furthermore, p53-depleted 3T3-L1 cells showed increase in mitochondrial proteins and enhancement of both Citrate Synthase and Complex IV activities during adipogenesis. These results show that p53 differentially regulates cell differentiation and mitochondrial biogenesis between adipocytes and myotubes, and provide evidence that p53 is an inhibitory factor of mitochondrial regulation in adipocyte lineage.     

A novel copper(II) coordination at His186 in full-length murine prion protein

To explore Cu(II) ion coordination by His¹⁸⁶ in the C-terminal domain of full-length prion protein (moPrP), we utilized the magnetic dipolar interaction between a paramagnetic metal, Cu(II) ion, and a spin probe introduced in the neighborhood of the postulated binding site by the spin labeling technique (SDSL technique). Six moPrP mutants, moPrP(D143C), moPrP(Y148C), moPrP(E151C), moPrP(Y156C), moPrP(T189C), and moPrP(Y156C,H186A), were reacted with a methane thiosulfonate spin probe and a nitroxide residue (R1) was created in the binding site of each one. Line broadening of the ESR spectra was induced in the presence of Cu(II) ions in moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) but not moPrP(D143R1). This line broadening indicated the presence of electron-electron dipolar interaction between Cu(II) and the nitroxide spin probe, suggesting that each interspin distance was within 20 Å. The interspin distance ranges between Cu(II) and the spin probes of moPrP(Y148R1), moPrP(Y151R1), moPrP(Y156R1), and moPrP(T189R1) were estimated to be 12.1 Å, 18.1 Å, 10.7 Å, and 8.4 Å, respectively. In moPrP(Y156R1,H186A), line broadening between Cu(II) and the spin probe was not observed. These results suggest that a novel Cu(II) binding site is involved in His186 in the Helix2 region of the C-terminal domain of moPrP^C.     

A novel immunosuppressant FTY720 ameliorates proteinuria and alterations of intrarenal adrenomedullin in rats with autoimmune glomerulonephritis

FTY720 has been originally developed as a new immunosuppressive agent, which prolongs graft survival in organ transplantation. Adrenomedullin (AM) participates in the regulation of sodium homeostasis and has renoprotective effects. The possible involvement of renal AM in the pathophysiology of glomerulonephritis (GN) and the effect of FTY720 has been evaluated in rats. HgCl2 (1 mg/kg body weight) was inoculated subcutaneously 3 times/week for a total of 2 weeks. FTY720 (3 or 10 mg/kg) was inoculated subcutaneously daily. The proteinuria, urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion and serum total cholesterol levels were increased and serum albumin level was reduced in rats with HgCl2-induced GN compared with controls. FTY720 reduced proteinuria (3 mg/kg: -25%; 10 mg/kg: -41%), urinary NAG excretion (-11%; -52%) and total cholesterol level (-21%; -55%) in a dose-dependent manner. Renal AM level and its mRNA expression were increased in rats with GN compared with controls (Peptide Cortex: +69%; Medulla: +82%; mRNA Cortex: +25%). Interestingly, FTY720 additionally increased these levels (Peptide Cortex: +38%; Medulla: +39%; mRNA Cortex: +20%). Renal AM levels correlated with urinary NAG excretion and creatinine clearance. These results suggest that FTY720 suppresses the renal damage in rats with GN and renal AM may participate in the pathophysiology of GN and the renoprotective effects of FTY720.     

Insertion/deletion polymorphism in clusterin gene influences serum lipid levels and carotid intima-media thickness in hypertensive Japanese females

Clusterin has been implicated in lipid metabolism and atherogenesis, however, the influence of genetic variation has not been examined in Japanese. In this study, we identified 11 single nucleotide polymorphisms (SNPs) of clusterin gene by direct sequencing. Among them, one promoter SNP (-4453T>G), one missense SNP (4183G>A), and 2 common SNPs (5608T>C and 6316delT) were genotyped in 525 asymptomatic hypertensives not treated with lipid lowering agents. -4453T>G, 4183G>A, and 5608T>C showed no correlation with the clinical characteristics, however, in the 6316delT, an insertion (I)/deletion (D) polymorphism, D/D subjects had significantly higher levels of total cholesterol and low-density lipoprotein (LDL)-cholesterol than I/I subjects in females but not in males. Female subjects with the D allele (D/D+I/D) had greater intima-media thickness of the carotid artery than I/I subjects. In a multiple logistic regression analysis, the D allele of 6316delT was detected as an independent predictor for the plaque prevalence. In conclusion, the clusterin gene polymorphism may contribute to the serum lipid levels and the progression of carotid atherosclerosis in hypertensive Japanese females.