p51/p63 Inhibits ultraviolet B-induced apoptosis via Akt activation.

The epidermis must be protected against excess apoptotic cell death in response to ultraviolet-B (UV-B) irradiation. p53 is known to be critical for this protection. Although the p53 family member DeltaNp51B/DeltaNp63alpha (an N terminal-deleted form of p51/p63) is abundantly expressed in keratinocytes, its contribution to UV-B-dependent apoptosis is largely unknown. We found that, after a transient increase, DeltaNp51B is downregulated in UV-B-irradiated keratinocytes undergoing apoptosis, whereas p53 is upregulated with delayed kinetics. Furthermore, the reduction of DeltaNp51B by small interfering RNAs augmented UV-B-dependent apoptosis in keratinocytes, indicating that DeltaNp51B blocks keratinocyte apoptosis. Although the exogenous expression of DeltaNp51B in keratinocytes did not further block the UV-B-dependent apoptosis, to our surprise the expression of TAp51B (an isoform with a full NH(2)-terminal transactivation domain that is structurally and functionally similar to p53) decreased apoptosis significantly. The blockade of keratinocyte apoptosis by the p51 was dependent on the phosphorylation of Akt, resulting in the activation of a survival pathway. Thus, in addition to its indispensable roles in epithelial development, p51 acts in adult cells to protect the epidermis against UV-B irradiation by preventing excess depletion of keratinocytes.

p53 homologue, p51/p63, maintains the immaturity of keratinocyte stem cells by inhibiting Notch1 activity.

p53 homologue, p51/p63, predominantly expressed in keratinocyte stem cells, is indispensable for the formation of epidermis. Notch1, another such gene indispensable for the process, induces growth arrest and differentiation in keratinocytes. We found that exogenous expression of DeltaNp51B (DeltaNp63alpha), one of the isoforms of p51 specifically expressed in basal keratinocytes, blocked Notch 1-dependent growth arrest and differentiation in mouse keratinocytes by inhibiting p21 expression and maintaining integrins expression. Furthermore, DeltaNp51B by itself was found to have ability to induce expression of integrin alpha6beta4, which promotes attachment of basal cells to basal membrane thereby keeping the cells in immature state. Therefore, we conclude that DeltaNp51B expression warrants integrin expression even under the influence of Notch1 and that DeltaNp51B is a long-sought factor required to maintain basal cell keratinocytes immaturity by inhibiting Notch1 activity. We will postulate a plausible model explaining the maintenance of the squamous epithelium architectures as well as offering mechanistic explanations for pathological features of skin diseases, including cancers, psoriasis along with physiological wound healings.

AID in antibody perfection.

Expressed immunoglobulin (Ig) genes undergo alterations in sequence and genomic structure in order to optimize antibody function. A single B cell-specific factor, activation-induced deaminase (AID), initiates these changes by deamination of cytosine to uracil. Uracil in DNA is encountered commonly, and conserved pathways are responsible for its faithful repair. However, at the Ig loci of B cells, AID-initiated damage is processed to produce three distinct outcomes: somatic hypermutation, class switch recombination and gene conversion. This review focuses on the role of AID in Ig gene diversification, emphasizing how AID functions within the mechanism of the Ig gene diversification pathway; and highlights open questions for future research, particularly the most provocative current question: what makes a gene a target for AID-initiated mutagenesis?

Characterization of the enzymes involved in the in vitro metabolism of amrubicin hydrochloride.

The in vitro metabolism of amrubicin by rat and human liver microsomes and cytosol was examined. The main metabolic routes in both species were reductive deglycosylation and carbonyl group reduction in the side-chain. In vitro metabolism of amrubicinol by rat and human liver microsomes and cytosol was also examined and the main metabolic route of this active metabolite was reductive deglycosylation. Metabolism of amrubicin in human liver microsomes was inhibited by TlCl(3) and that in human liver cytosol was inhibited by dicumarol and quercetin. Generation of amrubicinol was inhibited only by quercetin. The results indicate that metabolism of amrubicin is mediated by NADPH-cytochrome P450 reductase, NADPH:quinone oxidoreductase and carbonyl reductase. In addition, generation of amrubicinol is mediated by carbonyl reductase. Metabolism of amrubicinol in human liver microsomes was inhibited by TlCl(3) and that in human liver cytosol was inhibited by dicumarol. The results indicate that metabolism of amrubicinol is mediated by NADPH-cytochrome P450 reductase and NADPH:quinone oxidoreductase. To investigate the influence of cisplatin on the metabolism of amrubicin and amrubicinol, human liver microsomes and cytosol were pre-incubated with cisplatin. This did not change the rates of amrubicin and amrubicinol metabolism in either human liver microsomes or cytosol.

Effect of HSP70 induced by warm ischemia to the liver on liver function after partial hepatectomy.

BACKGROUND/AIMS: The purpose of this study was to determine if induction of HSP70 (heat shock protein 70), a stress protein which plays a cytoprotective role in response to various stimuli, protects hepatocytes from damage caused by partial hepatectomy and, if so, to elucidate the mechanism of such protection. METHODOLOGY: One hundred and eight male F344 rats weighing 190-220 g were randomly assigned to two groups with or without the presence of preconditioning. Fifteen-minute warm ischemia was applied to the liver of rats to induce HSP70, and 70% hepatectomy was performed 48 hours after the induction of HSP70 (ischemia group; n = 72). The rats in the nonischemia group did not undergo 15-min warm ischemia prior to 70% hepatectomy (nonischemia group; n = 36). Six rats, selected randomly from each group, were sacrificed at each measurement point to obtain blood and liver tissue samples. The levels of HSP70 in the liver, serum nitric oxide, levels of catalase and superoxide dismutase activity in the liver as antioxidative enzymes, and levels of Bcl-xL and Bax proteins and caspase-3-like activity in the liver as indices of apoptosis, were measured. RESULTS: The mean +/- SD level of HSP70 in the ischemia group (100 +/- 42 arbitrary unit (au)) was significantly higher than that of the nonischemia group (2 +/- 0.7 au) immediately before hepatectomy (P < 0.05). The ischemic preconditioning attenuated the liver damage caused by the subsequent partial hepatectomy. The levels of superoxide dismutase and catalase activity, serum nitric oxide level, and Bax protein level of the ischemia and nonischemia groups showed no significant differences after the partial hepatectomy. In contrast, the mean +/- SD level of Bcl-xL in the liver of the ischemia group (261 +/- 52 au) was significantly higher than that in the nonischemia group (114 +/- 33 au) 12 hours after the hepatectomy (P < 0.01). Furthermore, the mean +/- SD level of caspase-3-like activity in the liver of the ischemia group (18.1 +/- 4.6 au) was significantly lower than that of the nonischemia group (26.0 +/- 4.8 au) at 12 hours after the hepatectomy (P < 0.05). CONCLUSIONS: HSP70 induced by ischemic preconditioning prior to the partial hepatectomy was considered to protect the liver itself. In addition, the induced HSP70 may affect the Bcl-xL level after partial hepatectomy. Therefore, Bcl-xL seems to be involved in the reduction of liver damage after partial hepatectomy along with HSP.

Diesel exhaust particle-induced cell death of human leukemic promyelocytic cells HL-60 and their variant cells HL-NR6.

The cytotoxicity of diesel exhaust particles (DEPs) toward human leukemic promyelocytic cells HL-60 was examined. DEPs were toxic and cytotoxicity increased in a dose-dependent manner. All cells died with 750 microg/ml DEPs in culture media. Apoptosis occurred in HL-60 cells exposed to DEPs. The cytotoxicity of DEP extracts with organic solvents was much lower than those of DEPs and organic solvent-washed residual DEPs. HL-NR6 cells, an HL-60 variant cell line, having higher superoxide dismutase and catalase activities than HL-60 cells, were more resistant to DEP cytotoxicity. When preincubated with the fluorescent probe diacetoxymethyl 6-carboxy-2',7'-dichlorodihydrofluorescinate diacetate and then exposed to DEPs, HL-60 cells emitted green fluorescence under blue illumination, indicating that reactive oxygen species were generated within the cells. The DEP cytotoxicity correlated inversely with the cellular concentration of reduced glutathione (GSH), which had been attenuated with L-buthionine-(R,S)-sulfoximine, a gamma-glutamylcysteine synthetase inhibitor, and was lowered with ethyl reduced glutathionate, a GSH carrier across biomembranes. Further, DEPs themselves decreased the cellular concentration of GSH in a dose-dependent manner. The alpha-tocopherol model compound 2,2,5,7,8-pentamethylchroman-6-ol decreased DEP cytotoxicity, while alpha-tocopherol had no effect. In addition, quinacrine, an endocytosis inhibitor, decreased DEP cytotoxicity. These results show that DEPs are cytotoxic and suggest that the cytotoxicity results from generation of reactive oxygen species by DEPs which have been incorporated into cells.

Phylogeographic relationships of Ixodes uriae (Acari: Ixodidae) and their significance to transequatorial dispersal of Borrelia garinii.

The seabird tick Ixodes uriae (Acari: Ixodidae) has a bi- and circumpolar distribution and is commonly infected with Lyme disease Borrelia. Identical Borrelia flagellin gene sequences have been detected in I. uriae from both the Northern and Southern Hemispheres, suggesting a transequatorial transport of Borrelia. Parsimony analysis of the internal transcribed spacer 2 (ITS2) and a part of 16S rDNA of I. uriae from the Northern and Southern Hemispheres indicated that northern and southern I. uriae might be reproductively separated. We hypothesize that Borrelia is probably not dispersed from one hemisphere to the other by ticks attached to seabirds.

Local thrombus formation in the site of intravenous injection of chlorpromazine: possible colloid-osmotic lysis of the local endothelial cells.

Since amphiphilic drugs are known to interact with biomembranes, we investigated local vessel damage and thrombosis which might be brought about by intravenous dosing using chlorpromazine (CPZ) as a representative compound. CPZ-induced hemolysis was suppressed by an increase in sucrose concentration in the medium, characterizing this hemolysis to be colloid-osmotic lysis, which includes the enhancement of membrane phospholipid fluidity and consequent small pore formation in the membranes. This was supported by the observation that hemolysis by filipin, not featuring the stage of small pore formation, was not affected by sucrose. [14C]Glucose-entrapping liposomes were degraded by CPZ, and this degradation was enhanced by an increase in the intravesicle glucose concentration. These results indicated that the compound could induce colloid-osmotic lysis in erythrocytes and artificial membrane vesicles. CPZ also injured cultured porcine aortic endothelial cells (PAEC), as evidenced by lactate dehydrogenase (LDH) leakage. This injury was also suppressed by increase in sucrose concentration in the medium, suggesting that colloid-osmotic lysis again occurred. When rats were intravenously injected with CPZ, local endothelial cell (EC) injury and associated thrombus formation were observed, suggesting that CPZ's action was also evident in vivo. To our knowledge, this is the first finding which suggests that an intravenously dosed amphiphilic drug can injure local ECs based on a colloid-osmotic lysis mechanism leading to thrombosis.

Caspase activation and cytochrome c release during HL-60 cell apoptosis induced by a nitric oxide donor.

Nitric oxide (NO) from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (NOC-18) induces apoptosis in human leukemia HL-60 cells. This effect was prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), thereby implicating caspase activity in the process. NOC-18 treatment resulted in the activation of several caspases including caspase-3, -6, -8, and -9(-like) activities and the degradation of several caspase substrates such as nuclear lamins and SP120 (hnRNP-U/SAF-A). Moreover, release of cytochrome c from mitochondria was also observed during NOC-18-induced apoptosis. This change was substantially prevented by Z-VAD-FMK, thereby suggesting that the released cytochrome c might function not only as an initiator but also as an amplifier of the caspase cascade. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to NOC-18, supporting the above notion. Thus, NO-mediated apoptosis in HL-60 cells involves a caspase/cytochrome c-dependent mechanism.

Molecular phylogenetic analysis of ixodid ticks based on the ribosomal DNA spacer, internal transcribed spacer 2, sequences.

An internal transcribed spacer (ITS2) sequence between the 5.8S and 28S rRNA genes was used to estimate the phyletic relationships among Ixodes spp. tick vectors of Lyme disease-causing Borrelia spirochetes. Analysis indicates that Borrelia burgdorferi sensu lato species associated with Lyme disease are found mainly in ticks of the Ixodes ricinus species complex. Other closely related tick species are not known to transmit the Borrelia-that cause Lyme disease in humans, but they appear to have a specific association with other closely related Borrelia species. There is a high degree of concordance in the phylogenetics of Borrelia taxa and the phylogenetic relationships among Ixodes ticks.

Tissue distribution and pharmacological potential of SM-16896, a novel oestrogen-bisphosphonate hybrid compound.

Postmenopausal osteoporosis is caused mainly by a deficiency of oestrogen with rapid bone loss. To target oestrogen to the bone effectively, we have synthesized and evaluated the effects of a novel hybrid compound of oestrogen and bisphosphonate, SM-16896. The tissue distribution pattern and pharmacological potential are reported. Although the affinity for calf uterine oestrogen receptor was very low (IC50: 73.3 microM; 1/25000 of that of 17beta-oestradiol (2.84 nM)), SM-16896 showed oestrogenic activity. SM-16896 (1 microM) induced a 4.5-fold transcriptional activity in rat osteosarcoma UMR-106 cells compared with vehicle-treated control, when we used the expression vector for human oestrogen receptor and a CAT reporter plasmid containing an oestrogen-responsive element. The distribution of SM-16896 after a subcutaneous administration to 7-week-old female rats was examined by radioluminography using 3H-labelled SM-16896. At 30 min after the administration, significant radioactivity was detected in the bone. At 24 h after administration, a high level of radioactivity was detected in the bone, but in the uterus it was only at a background level. Daily subcutaneous administration of 0.5 mgkg(-1) SM-16896 for 12 weeks (five times per week) to 13-week-old ovariectomized rats suppressed the ovariectomized-induced reduction in bone mineral density. A bone mineral density ratio of 120% was maintained compared with sham-operated rats, whereas a relatively low suppression of uterine weight was observed (about 50% loss compared with sham-operated rats). In the same experiment, the implantation of a 17beta-oestradiol time-release pellet (0.25 mg/pellet/90 days) almost completely suppressed the reduction of both the bone mineral density and uterine tissue weight. It is likely that the effect of SM-16896 on bone was due to its oestrogenic activity, since 1.0 mgkg(-1) SM-18108, the bisphosphonate moiety of this compound, had no effect on bone in 7-week-old ovariectomized rats. The results suggest that SM-16896, a bisphosphonate-conjugated oestrogen, showed a preference profile in the uterus and bone due to its characteristic distribution pattern compared with the natural oestrogen analogue 17beta-oestradiol. Thus, bisphosphonate-conjugated oestrogens have the potential to improve patient compliance in oestrogen therapy by minimizing adverse effects and reducing the frequency of medication.

Selective inhibition of formyl-methionyl-leucyl-phenylalanine (fMLF)-dependent superoxide generation in neutrophils by pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.

It has been shown previously that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, such as compactin, lovastatin, and pravastatin, block cholesterol synthesis, suppress lymphocyte functions, and beneficially affect atherogenesis. Recently, it was reported that compactin and lovastatin inhibit the respiratory burst of DMSO-differentiated HL-60 cells, an effect reversed by mevalonic acid. The mode of action of these inhibitors in this role is not understood fully. Thus, we studied the mechanism of inhibition of neutrophil superoxide (O2*-) generation by pravastatin and found that pravastatin at 0.5 mM inhibited the receptor-mediated tyrosine kinase (TK)-dependent pathway of O2*- generation and also luminol chemiluminescence but not the protein kinase C (PKC)-dependent or the TK- and PKC-independent pathways of O2*- generation in neutrophils. Pravastatin also inhibited the tumor necrosis factor-alpha- and formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of a tyrosine of a 115-kDa protein. These effects were not reversed by mevalonate. From these results it is concluded that pravastatin inhibited receptor-mediated O2*-generation by decreasing tyrosine phosphorylation but not by inhibiting the formation of an intermediate in the biosynthesis of cholesterol.

Functional role of c-Src in gap junctions of the cardiomyopathic heart.

Given the essential role played by gap junctions in the coordination of cardiac muscle contraction, it is plausible that down-regulation of gap junctional conduction is in part responsible for the contractile dysfunction observed in hypertrophied and failing hearts. In the present study, we analyzed the expression and function of the gap junction protein, connexin43, in the ventricular myocardium of hereditary cardiomyopathic, Syrian BIO 14.6 hamsters. Immunoprecipitation and immunoblot analyses revealed that levels of tyrosine phosphorylated connexin43 were increased in BIO 14.6 hamsters at the late stage of congestive heart failure. Furthermore, the increased tyrosine phosphorylation was correlated with increased c-Src activity. The functional consequences of tyrosine phosphorylation of connexin43 in gap junction were assessed using transfected cells expressing constitutively active c-Src. It was found that constitutively active c-Src diminished propagation of Ca(2+) waves in HEK293 cells and reduced gap junctional conductance between pairs of cardiac myocytes. We, therefore, conclude that during the progression of cardiac dysfunction in the cardiomyopathic heart, gap junctional communication is reduced via c-Src-mediated tyrosine phosphorylation of connexin43.

Geranylgeranylacetone induces apoptosis in HL-60 cells.

Geranylgeranylacetone (GGA) induces apoptosis in human leukemia HL-60 cells in a dose- and time-dependent manner. This effect was completely prevented by the pan-caspase inhibitor z-Val-Ala-Asp(OMe) fluoromethylketone, thereby implicating the caspase cascade in the process. Prior to DNA fragmentation, GGA treatment markedly activated caspase-3(-like) proteases, which might be responsible for the observed apoptosis. In addition, GGA treatment interfered with the processing and membrane localization of Rap1 and Ras, and these changes may be a result of apoptosis. Moreover, nitric oxide donors significantly accentuated the GGA-induced apoptosis, suggesting that the apoptotic pathway induced by GGA might be regulated by a redox-sensitive mechanism. Taken together, these data suggest that the isoprenoid, GGA, is an effective inducer of apoptotic cell death in HL-60 cells.

Thyroxine enhancement and the role of reactive oxygen species in tadpole tail apoptosis.

Our objective is to clarify the role of reactive oxygen species (ROS) in the atrophying tail of anuran tadpoles (tail apoptosis). Changes in catalase, superoxide dismutase (SOD) and caspase activity, genomic DNA, and nitric oxide (NO) generation were investigated biochemically using Rana japonica tadpole tails undergoing regression during thyroid hormone enhancement. DNA fragmentation and ladder formation with concomitant shortening of tadpole tail were induced by DL-thyroxine (T4) in culture medium. Catalase activity was also decreased by T4 treatment. T4 was also found to increase NO synthase (NOS) activity in cultured tadpole tail with concomitant increase in the concentration of NO2- plus NO3- (NOx) in the culture medium. Additional treatment with N-monomethyl-L-arginine (NMMA), a potent inhibitor of NOS, suppressed the enhancing effects of T4 on tail shortening and catalase activity reduction. It was also found that treatment with isosorbide dinitrate (ISDN), a NO generating drug, alone also had an enhancing effect on tail shortening and catalase activity reduction similar to that seen with T4. Both NO and an NO donor (ISDN) strongly suppressed catalase activity. Kinetic analysis revealed that catalase activity decreased and caspase-3-like activity increased during normal tadpole tail atrophy (apoptosis). These results suggested that T4 enhances NO generation, thereby strongly inhibiting catalase activity, resulting in an increase in hydrogen peroxide, and that the oxidative stress elicited by excess hydrogen peroxide might activate cysteine-dependent aspartate-directed protease-3 (caspase-3-like protease), which is thought to cause DNA fragmentation, leading to apoptosis.

X-irradiation enhances the expression of Bcl-2 in HL-60 cells: the resulting effects on apoptosis and radiosensitivity.

Although p53 has been shown to directly activate transcriptional bax gene and to inhibit expression of bcl-2 gene during radiation-induced apoptosis, it is poorly understood how the Bcl-2 family changes in p53-deficient cells during radiation-induced apoptosis. The present work describes the effect of X-irradiation on the apoptosis of p53-deficient HL-60 cells as assessed by means of several methods. Apoptosis of HL-60 cells was induced by X-irradiation in a dose- and time-dependent manner. 18 h after 5 Gy irradiation, G2 cells underwent apoptosis, while 15 Gy X-irradiation induced the death of G1/S cells by 6 h. After X-irradiation, expression of Bcl-2 was elevated, while Bax expression was unchanged. We have isolated a clonal HL-60 variant following twice 5 Gy irradiation of HL-XR3 cells. These cells highly expressed Bcl-2 (about 2-fold), showed a reduced activation of caspase-3, and were not only more resistant to X-irradiation-induced apoptosis but also more radioresistant. These results suggest that HL-60 cells may resist apoptosis and radiation by increasing Bcl-2 expression, and that this elevated Bcl-2 expression might be one of the causes of the phenomenon, often seen clinically, that tumor cells gradually acquire radioresistance during fractionated radiation therapy.

Resistance to nitric oxide-mediated apoptosis in HL-60 variant cells is associated with increased activities of Cu,Zn-superoxide dismutase and catalase.

Nitric oxide (NO) released from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO or NOC-18) induces apoptosis in human leukemia HL-60 cells. In this study, we isolated a HL-60 variant cell line, HL-NR6, that is resistant to DETA/NO toxicity as assessed by DNA fragmentation, morphology, and colony forming ability. The variant cells also showed resistance to reactive oxygen species (ROS) such as superoxide and hydrogen peroxide as well as NO donors, but not to anti-tumor drugs. We found that HL-NR6 cells when compared with HL-60 cells possessed twice the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase, but no change in Mn-SOD nor in glutathione peroxidase. Immunoblotting confirmed the high levels of both enzymes in the variant cell. We also observed that ROS generation following DETA/NO exposure was substantially higher in HL-60 cells than in HL-NR6 cells, using the 2',7'-dichlorofluorescein fluorometric method. Moreover, the SOD mimetic Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin and exogenous catalase effectively attenuated DETA/NO-elicited DNA fragmentation in HL-60 cells. Taken together, these data suggested that the NO resistance in HL-NR6 cells is associated with the increased Cu,Zn-SOD/catalase and that NO-mediated apoptosis in HL-60 cells is correlated with the generation of ROS and derived molecules like peroxynitrite.

Role of nuclear lamins in nuclear segmentation of human neutrophils.

Nuclear breakdown leading to the formation of apoptotic bodies has been postulated to involve degradation of nuclear structural proteins, such as lamins A/C and B. Although nuclear segmentation occurs during the maturation of polymorphonuclear leukocytes (neutrophils), its mechanism is not known. We found that human neutrophils have lamin B but lack lamins A/C while mononuclear cells possess all three types of lamin as assessed by immunoblotting. Differentiation of human promyelocytic HL-60 cells into neutrophil-like cells was also accompanied by the down-regulation of lamins A/C but not of lamin B. Moreover, when compared with normal cells, neutrophils with the Pelger-Huët anomaly of nuclear hyposegmentation exhibited significantly lower activity of caspase-6, a lamin A/C-cleaving enzyme. Differentiated HL-60 cells showed higher activity of caspase-6 than that of untreated cells. These observations allow us to speculate that remodeling of nuclear lamins might underlie the mechanism for nuclear segmentation of neutrophils.

Sequence analysis of 18S-28S rRNA spacer regions from Saccharomyces kunashirensis, S. martiniae, S. rosinii, and S. transvaalensis.

Sequences of two internal transcribed spacer regions between 18S and 28S rRNA for recently described yeasts species, Saccharomyces kunashirensis, S. martiniae, S. rosinii, and S. transvaalensis, were determined to assess their phylogenetic relationship to the other Saccharomyces species. In the two phylogenetic trees constructed by the neighbor-joining method, independent branches reflected that delimitation of the four new species was valid.