Insulin is a dominant suppressor of sterol 12 alpha-hydroxylase P450 (CYP8B) expression in rat liver: possible role of insulin in circadian rhythm of CYP8B

Sterol 12 alpha-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid/chenodeoxycholic acid ratio in bile acid biosynthesis. The hepatic CYP8B level was elevated in streptozotocin-induced diabetic rats, and the elevated CYP8B was suppressed by insulin administration [Ishida, H. et al. (1999) J. Biochem. 126, 19-25]. The streptozotocin-induced elevation of hepatic CYP8B mRNA concomitantly responded to the decrement of the serum insulin level. The CYP8B mRNA level in the cultivated rat hepatoma H4TG cells was strongly suppressed by insulin, although it was affected by dibutyryl cAMP or thyroxine to lesser extents. These observations demonstrate that CYP8B expression is dominantly regulated by the direct action of insulin on hepatocytes. A marked circadian rhythm (maximum at 13:00-16:00 and minimum at 1:00) was observed both on the mRNA level and the activity of CYP8B. This rhythm was shifted from that of cholesterol 7 alpha-hydroxylase, a rate-limiting enzyme of bile acid biosynthesis, showing a maximum at 22:00 and a minimum at 10:00, and this shift might oscillate the cholic acid/chenodeoxycholic acid ratio, which is increased in the late afternoon and decreased at midnight. The rhythm of CYP8B was the inverse of the circadian variation of serum insulin level and was similar to the circadian rhythm of glucose 6-phosphatase. These facts and the potent suppressive effect of insulin on CYP8B indicate that the oscillation of the serum insulin may be a factor in producing the circadian rhythm of CYP8B.     

Intracellular degradation of histidine-rich glycoprotein mutants: tokushima-1 and 2 mutants are degraded by different proteolytic systems

We reported the first case of a congenital histidine-rich glycoprotein deficiency (HRG Tokushima) in which substitution of Gly85 with Glu (G85E) in the first cystatin domain resulted in intracellular degradation and a low plasma level of HRG [Shigekiyo, T. et al. (1998) Blood 91, 128-133]. Recently, we identified the gene mutation of a second case of HRG deficiency as a Cys223 to Arg (C223R) mutation in the second cystatin domain. To investigate the molecular and cellular bases of these deficiencies, we expressed these HRG mutants in baby hamster kidney (BHK) cells. Pulse-chase experiments in the absence and presence of various proteinase inhibitors revealed that, while wild-type HRG was completely secreted during 4-h chase periods, both the G85E and C223R mutants were only partially secreted and primarily degraded within the cells. The intracellular degradation of the C223R mutant was almost completely inhibited in the presence of a proteasome inhibitor, lactacystin, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, resulting in increased secretion of the C223R mutant, and thus implicating the proteasome system in this degradation process. In contrast, the sum of the amounts of the G85E mutant inside and outside the cells decreased during the chase periods even in the presence of the proteasome inhibitor, carbobenzoxy-leucyl-leucyl-leucinal or N-acetyl-leucyl-leucyl-norleucinal, although proteasome-specific inhibitor lactacystin and one of the cysteine protease inhibitors, E-64-d, prevented the intracellular degradation. These results suggested that intracellular degradation of G85E HRG occurred to some extent through a hitherto unknown mechanism. Similar studies involving recombinant mutants in which Gly85 or Cys223 was replaced with several other amino acids revealed that proteins with mutations leading to the destruction of the predicted b-sheet structure of the cystatin domains were eliminated by the intracellular quality control system.     

MAPK upstream kinase (MUK)-binding inhibitory protein, a negative regulator of MUK/dual leucine zipper-bearing kinase/leucine zipper protein kinase

Mitogen-activated protein kinase upstream kinase/dual leucine zipper-bearing kinase/leucine-zipper protein kinase (MUK/DLK/ZPK) is a MAPKKK class protein kinase that induces JNK/SAPK activation. We report here a protein named MBIP that binds to MUK/DLK/ZPK. MUK-binding inhibitory protein (MBIP) contains two tandemly orientated leucine-zipper-like motifs with a cluster of basic amino acids located between the two motifs. MBIP interacts with one of the two leucine-zipper-like motifs of MUK/DLK/ZPK and inhibits the activity of MUK/DLK/ZPK to induce JNK/SAPK activation. Notably, no similar effect was observed with another JNK/SAPK-inducing MAPKKK, COT/Tpl-2, showing the specificity of MBIP action. Furthermore, the overexpression of MBIP partially inhibits the activation of JNK by 0.3 m sorbitol in 293T cells. Taken together, these observations indicate that MBIP can function as a regulator of MUK/DLK/ZPK, a finding that may provide a clue to understanding the molecular mechanism of JNK/SAPK activation by hyperosmotic stress.     

Identification and characterization of cell lines with a defect in a post-adsorption stage of Sendai virus-mediated membrane fusion

In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virus-mediated membrane fusion.     

Expression of tenascin-C in stromal cells of the murine uterus during early pregnancy: induction by interleukin-1 alpha, prostaglandin E(2), and prostaglandin F(2 alpha)

Tenascin-C (TN-C), an extracellular matrix glycoprotein, is known to be expressed in uterine stroma in the peri-implantation period. Examination of the spatiotemporal pattern during early pregnancy using immunohistochemistry and in situ hybridization revealed TN-C expression in the stroma beneath the luminal epithelia of the murine endometrium on Days 0 and 1 of pregnancy, subsequent disappearance, and reappearance on Day 4. After decidualization, tissue around the deciduoma was positive. In situ hybridization demonstrated TN-C production by the stromal cells adjacent to the epithelia. To investigate the regulation of TN-C expression in vitro, murine uterine stromal and epithelial cells were isolated and cultured. Addition of interleukin-1 alpha (IL-1 alpha) and prostaglandin E(2) (PGE(2)) and F(2 alpha) (PGF(2 alpha)) induced TN-C expression in the stromal cells at both protein and mRNA levels, while the sex steroid hormones, progesterone and ss-estradiol, exerted little effect. Immunohistochemistry using anti-IL-1 alpha antibody showed epithelial cells to be positive on Days 2-4 of pregnancy, and addition of progesterone but not ss-estradiol enhanced IL-1 alpha expression in epithelial cells in vitro. In a culture insert system, TN-C expression by stromal cells cocultured with epithelial cells was induced by addition of progesterone alone that was blocked by additions of anti-IL-1 alpha antibody. Collectively, these findings indicate that TN-C expression in the preimplantation period is under the control of progesterone, but not directly, possibly by the paracrine and autocrine intervention of IL-1 alpha secreted by epithelial cells and PGE(2) and PGF(2 alpha) secreted by stromal cells.     

Fungal gliotoxin targets the onset of superoxide-generating NADPH oxidase of human neutrophils

Gliotoxin from Aspergillus, bearing a S&bond;S bond in its structure, prevented the onset of O(-)(2) generation by the human neutrophil NADPH oxidase in response to phorbol myristate acetate (PMA). Gliotoxin affected the activation process harder than the activated oxidase, as shown by its stronger inhibition when added to neutrophils prior to, than post-PMA at maximum enzyme turnover. Decreased O(-)(2) generation persisted even if cells treated with gliotoxin were subsequently washed, with half-inhibition concentrations (IC(50)) of 5.3, and 3.5 microM for treatments of 15 and 30 min, respectively. In addition, gliotoxin made neutrophils reduce cytochrome c regardless of absence of PMA, through its reaction with intracellular reductants in an oxygen-dependent process, named redox cycling. Thus, we next tested whether preincubation of neutrophils with gliotoxin under hypoxic conditions would relieve the inhibition of NADPH oxidase. Instead, this prevention of redox cycling significantly favored damage to the NADPH oxidase with an IC(50) of 0.009 microM. Moreover, conversion of gliotoxin to its dithiol derivative by addition of reduced dithiothreitol during incubation protected cells from losing oxidase activity. These findings support that the disulfide form of gliotoxin targets NADPH oxidase activation.     

Color-coding reveals tandem repeats in the Escherichia coli genome

Genomic DNA contains a wide variety of repetitive sequences. In Escherichia coli, there have been several classes of repetitive sequences reported, some of which cluster as tandem repeats. We propose a novel method for analyzing symbolic sequences by two-dimensional pattern formation with color-coding. We applied this method for searching tandem repeats in the E. coli genome and found approximately 50 repeats with periods longer than 30 bases. The longest repeat has a period of 1267 bases.     

Adenine-induced selective apoptosis toward HIV chronically infected cells in vitro

A novel strategy for anti-HIV therapy is the clearance of the residual infected cells from the body. Here, we show that 6-aminopurine, adenine, induced selective apoptosis toward HIV-1 producing chronically infected MOLT4 cells (MOLT4/HIV) without augmentation of virus production, whereas the growth of uninfected MOLT4 was stimulated. This selective apoptosis did not occur with other adenine nucleotides or with other bases. The purine ring and the amino residue of adenine were responsible for the apoptosis induction and selectivity, respectively. In addition, adenine slightly but consistently reduced viable cell numbers and the production of virus in a fraction of HIV-1 chronically infected human peripheral blood mononuclear cells (PBMCs/HIV) at day 7. On the other hand, blastogenic response of normal PBMCs to PHA, PWM and Candida albicans were potentiated in the presence of adenine. These results indicated that the effect of adenine may be attributable to activation-induced selective apoptosis toward virus-infected cells.