Adiponectin stimulates glucose uptake in mouse blastocysts and embryonic carcinoma cells.

Preimplantation embryos are sensitive to maternal hormones affecting embryonic signal transduction and metabolic functions. We examined whether adiponectin, the most abundantly secreted adipokine, can influence glucose transport in mouse embryonic cells. In mouse blastocysts full-length adiponectin stimulated glucose uptake, while no effect of globular adiponectin was found. Full-length adiponectin stimulated translocation of GLUT8 glucose transporter to the cell membrane; we did not detect significant changes in the intracellular localization of GLUT4 glucose transporter in adiponectin-treated blastocysts. To study adiponectin signaling in detail, we used embryoid bodies formed from mouse embryonic carcinoma cell (ECC) line P19. We confirmed the expression of adiponectin receptors in these cells. Similar to mouse blastocysts, full-length adiponectin, but not globular adiponectin, stimulated glucose uptake in ECC P19 embryoid bodies. Moreover, full-length adiponectin stimulated AMPK and p38 MAPK phosphorylation. These results indicate that besides AMPK, p38 MAPK is a potential target of adiponectin in mouse embryonic cells. AMPK inhibitor did not influence the adiponectin-stimulated p38 MAPK phosphorylation, indicating independent action of these two signaling pathways. In mouse embryos adiponectin acts as a hormonal regulator of glucose uptake, which becomes especially important in phases with reduced levels of circulating insulin. Our results suggest that adiponectin maintains the glucose supply for early embryos under hypoinsulinaemic conditions, for example, in mothers suffering from type 1 diabetes mellitus.

The laboratory performance of the enFLOW(®) , buddy lite(™) and ThermoSens(®) fluid warmers.

We measured heating of isotonic saline by three fluid warmers in six experiments: saline at 5 °C or 20 °C delivered at 30, 50 or 100 ml.min(-1) . At the three flow rates, the enFLOW(®) , buddy lite(™) and ThermoSens(®) systems heated 5 °C saline to mean (SD) temperatures of: 41.1 (0.5) °C, 37.7 (0.6) °C and 39.1 (0.6) °C; to 40.3 (0.8) °C, 33.9 (1.6) °C and 39.3 (0.7) °C; and to 37.1 (0.8) °C, 24.0 (1.3) °C and 37.6 (1.0) °C, respectively, p < 0.0001 for each experiment. The mean (SD) times taken to heat 5 °C saline were: 16.6 (1.7) s, 258.4 (58.9) s and 134.2 (79.6) s; 16.9 (1.8) s, 256.2 (62.2) s and 182.5 (74.5) s; and 21.5 (1.5) s, 275.9 (49.3) s and 313.5 (18.0) s, respectively, p < 0.0003 for each experiment. The results for saline at 20 °C were similar. The enFLOW system heated saline above 36 °C faster than the ThermoSens system, whereas the buddy lite often failed to achieve 36 °C.

MicroRNA-302 induces proliferation and inhibits oxidant-induced cell death in human adipose tissue-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are a heterogeneous population of cells that proliferate in vitro as plastic-adherent cells, have a fibroblast-like morphology, form colonies in vitro and can differentiate into bone, cartilage and fat cells. The abundance, ease and repeatable access to subcutaneous adipose tissue and the simple isolation procedures provide clear advantages for the use of human adipose tissue-derived mesenchymal stem cells (hASDCs) in clinical applications. We screened microRNAs (miRNAs) that affected the proliferation and survival of hADSCs. Transfection of miR-302d mimic increased cell proliferation and protected cells from oxidant-induced cell death in hADSCs, which was supported by flow-cytometric analysis. miR-302d did not affect the expression of Bcl-2 family members or anti-oxidant molecules. The Nrf2-Keap1 system, which is one of the major mechanisms for the cellular defense against oxidative stress, was not altered by transfection of miR-302d mimic. To identify the target of the miR-302d actions on proliferation and survival of hADSCs, a microarray analysis was performed using miR-302d-overexpressing hADSCs. Real-time PCR analysis showed that transfection of miR-302d mimic inhibited the CDKN1A and CCL5 expression. Downregulation of CDKN1A with a specific siRNA mimicked the effect of miR-302d on hADSCs proliferation, but did not affect miR-302d-induced cell survival. Downregulation of CCL5 protected oxidant-induced cell death as miR-302d, inhibited oxidant-induced reactive oxygen species (ROS) generation and the addition of recombinant CCL5 inhibited the protective action of miR-302d on oxidant-induced cell death. This study indicates that miR-302 controls proliferation and cell survival of hADSCs through different targets and that this miRNA can be used to enhance the therapeutic efficacy of hADSCs transplantation in vivo.

Rapid detection of antibiotic resistance based on mass spectrometry and stable isotopes.

With the emergence and growing complexity of bacterial drug resistance, rapid and reliable susceptibility testing has become a topical issue. Therefore, new technologies that assist in predicting the effectiveness of empiric antibiotic therapy are of great interest. Although the use of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the rapid detection of antibiotic resistance is an attractive option, the current methods for MALDI-TOF MS susceptibility testing are restricted to very limited conditions. Here, we describe a technique that may allow for rapid susceptibility testing to an extent that is comparable to phenotypic methods. The test was based on a stable isotope labelling by amino acids in cell culture (SILAC)-like approach. This technique was used to visualise the growth of bacteria in the presence of an antibiotic. Pseudomonas aeruginosa was chosen as the model organism, and strains were incubated in normal medium, medium supplemented with (13)C6-(15) N2-labelled lysine and medium supplemented with labelled lysine and antibiotic. Peak shifts occurring due to the incorporation of the labelled amino acids were detected by MALDI-TOF MS. Three antibiotics with different mechanisms of action, meropenem, tobramycin and ciprofloxacin, were tested. A semi-automated algorithm was created to enable rapid and unbiased data evaluation. With the proposed test, a clear distinction between resistant and susceptible isolates was possible for all three antibiotics. The application of SILAC technology for the detection of antibiotic resistance may contribute to accelerated and reliable susceptibility testing.

Successful extracorporeal membrane oxygenation for right heart failure after heart transplantation-2 case reports and literature review.

After heart transplantation (HT), transient right heart failure (RHF) is common. If it does not improve with appropriate medical therapy, we must consider mechanical support. Recently, extracorporeal membrane oxygenation (ECMO) has shown better results than a right ventricular assist device or retransplantation. Two HT patients with hypertrophic cardiomyopathy had cold ischemic times beyond >240 minutes. After HT, their right heart function worsened and was unresponsive to medical therapy. After our application of ECMO, weaning was successful and the patients were discharged without complication. Early application of ECMO for RHF after HT is a good option.

Flow characteristics of LIMA radial composite sequential bypass grafting and single LIMA and saphenous vein sequential bypass grafting performed under OPCAB.

AIM: The present study was aimed to compare the hemodynamic flow characteristics of LIMA radial artery composite sequential bypass grafting and with single LIMA and saphenous vein sequential bypass grafting performed by off-pump coronary artery bypass grafting (OPCAB). METHODS: Between March 2007 and February 2008, 121 OPCAB patients were prospectively divided into two groups; Group I (N.=70, left internal thoracic artery [LITA]-left anterior descending [LAD] and Ao-SV sequential grafting), and Group II (N.=51, LITA-RA sequential grafting). The mean flow, pulsatility index (PI) and back flow (BF) were measured using the Transit-time flow meter (TTFM). In Group II, the proximal (p-LITA) and distal LITA (d-LITA) flow in relation to the RA side branch anastomosis were measured separately. RESULTS: The mean flow and PI of the proximal SV sequential graft and that of the RA graft were 64.4 ± 37.3 mL/min and 2.6 ± 1.6 versus 27.3 ± 18.6 mL/min and 4.1 ± 4.4, respectively (P<0.05). In Group I, the mean LITA flow, PI, and BF were 26.9 ± 16.4 mL/min, 2.6 ± 1.5, and 3.1 ± 6.1% whereas in Group II those of the p-LITA were 37.3 ± 21.6 mL/min, 2.3 ± 1.0, and 2.0 ± 3.5% and the d-LITA were 18.8 ± 12.2 mL/min, 3.9 ± 3.3 and 7.4 ± 11.8% (P<0.01). CONCLUSION: The results of the present data suggest the hemodynamic flow characteristics of composite bypass grafting to be inferior to the single LIMA and separate aorta-saphenous vein bypass grafting strategy. However, a longer follow up is warranted to assess the implications of these findings on graft durability.

Time-resolved measurements of PM2.5 carbonaceous aerosols at Gosan, Korea.

In order to better understand the characteristics of atmospheric carbonaceous aerosol at a background site in Northeast Asia, semicontinuous organic carbon (OC) and elemental carbon (EC), and time-resolved water-soluble organic carbon (WSOC) were measured by a Sunset OC/ EC and a PILS-TOC (particle-into-liquid sampler coupled with an online total organic carbon) analyzer, respectively, at the Gosan supersite on Jeju Island, Korea, in the summer (May 28-June 17) and fall (August 24-September 30) of 2009. Hourly average OC concentration varied in the range of approximately 0.87-28.38 microgC m-3, with a mean of 4.07+/- 2.60 microgC m-3, while the hourly average EC concentration ranged approximately from 0.04 to 8.19 .microgC m-3, with a mean of 1.35 +/- 0.71 microgC m-3, from May 28 to June 17, 2009. During the fall season, OC varied in the approximate range 0.9-9.6 microgC m-3, with a mean of 2.30 +/-0.80 microgC m-3, whereas EC ranged approximately from 0.01 to 5.40 microgC m-3, with a mean of 0.66 +/- 0.38 microgC m-3. Average contributions of EC to TC and WSOC to OC were 26.0% +/- 9.7% and 20.6% +/-7.4%, and 37.6% +/- 23.5% and 57.2% +/- 22.2% during summer and fall seasons, respectively. As expected, clear diurnal variation of WSOC/OC was found in summer, varying from 0.22 during the nighttime up to 0.72 during the daytime, mainly due to the photo-oxidation process. In order to investigate the effect of air mass pathway on the characteristics of carbonaceous aerosol, 5-day back-trajectory analysis was conducted using the HYSPLIT model. The air mass pathways were classified into four types: Continental (CC), Marine (M), East Sea (ES) and Korean Peninsula (KP). The highest OC/EC ratio of 3.63 was observed when air mass originated from the Continental area (CC). The lowest OC/EC ratio of 0.79 was measured when air mass originated from the Marine area (M). A high OC concentration was occasionally observed at Gosan due to local biomass burning activities. The contribution of secondary OC to total OC varied approximately between 8.4% and 32.2% and depended on air mass type.

Neutron Laue diffraction study on the magnetic phase diagram of multiferroic MnWO4 under pulsed high magnetic fields.

We have combined time-of-flight neutron Laue diffraction and pulsed high magnetic fields at the Spallation Neutron Source to study the phase diagram of the multiferroic material MnWO(4). The control of the field-pulse timing enabled an exploration of magnetic Bragg scattering through the time dependence of both the neutron wavelength and the pulsed magnetic field. This allowed us to observe several magnetic Bragg peaks in different field-induced phases of MnWO(4) with a single instrument configuration. These phases were not previously amenable to neutron diffraction studies due to the large fields involved.

17ß-Estradiol, aging, inflammation, and the stress response in the female heart.

Heat shock proteins (HSPs) are a cardioprotective class of proteins induced by stress and regulated by the transcription factor, heat shock factor (HSF)-1. 17ß-estradiol (E(2)) indirectly regulates HSP expression through rapid activation of nuclear factor-κB (NF-κB) and HSF-1 and protects against hypoxia. As males experience a loss of protective cellular responses in aging, we hypothesized that aged menopausal (old ovariectomized) rats would have an impaired HSP response, which could be prevented by immediate in vivo E(2) replacement. After measuring cardiac function in vivo, cardiac myocytes were isolated from ovariectomized adult and old rats with and without 9 weeks of E(2) replacement. Myocytes were treated with E(2) in vitro and analyzed for activation of NF-κB, HSF-1, and HSP expression. In addition, we measured inflammatory cytokine expression and susceptibility to hypoxia/reoxygenation injury. Cardiac contractility was reduced in old ovariectomized rats and could prevented by immediate E(2) replacement in vivo. Subsequent investigations in isolated cardiac myocytes found that in vitro E(2) activated NF-κB, HSF-1, and increased HSP 72 expression in adult but not old rats. In response to hypoxia/reoxygenation, myocytes from adult, but not old, rats had increased HSP 72 expression. In addition, expression of the inflammatory cytokines TNF-α and IL-1ß, as well as oxidative stress, were increased in myocytes from old ovariectomized rats; only the change in cytokine expression could be attenuated by in vivo E(2) replacement. This study demonstrates that while aging in female rats led to a loss of the cardioprotective HSP response, E(2) retains its protective cellular properties.

The time-dependent effect of lipopolysaccharide on kainic acid-induced neuronal death in hippocampal CA3 region: possible involvement of cytokines via glucocorticoid.

It has been reported that glucocorticoid (Gc) can induce neuronal cell toxicity in the hippocampus. In addition, we examined that serum Gc increased by restraint stress aggravated kainic acid (KA)-induced neuronal death in hippocampal CA3 region. However, the effect of other stressful stimulus like lipopolysaccharide (LPS) increasing serum Gc on KA-induced neuronal death was not elucidated until now. Thus, we examined the time course effect of LPS on KA-induced neuronal death in the hippocampal CA3 region of mice, especially to address the role of Gc and inflammatory mediators. In the present study, we found that an aggravating effect of LPS on KA-induced neuronal death was correlated with an alteration of hippocampal IL-1beta mRNA level at all time points, and the serum Gc and hippocampal IL-1beta mRNA level was peak at 90 min after LPS treatment (LPS 90 min) when the aggravating effect of LPS on KA-induced neuronal death was maximum. In addition, RU38486 (glucocorticoid receptor antagonist) decreased the hippocampal IL-1beta mRNA level and abolished the aggravating effect of LPS on KA-induced neuronal death at LPS 90 min and 24 h. In the immunohistochemical study, we found activated and ramified microglia (OX-42) and astrocyte (GFAP) at 24 h after LPS treatment (LPS 24 h) in the hippocampus. These results suggest that Gc itself, cytokines triggered by Gc, or both appears to be involved in the LPS effect depending on LPS pretreatment time.

Differential expression of phosphorylated Ca2+/calmodulin-dependent protein kinase II and phosphorylated extracellular signal-regulated protein in the mouse hippocampus induced by various nociceptive stimuli.

In the present study, we characterized differential expressions of phosphorylated Ca(2+)/calmodulin-dependent protein kinase IIalpha (pCaMKIIalpha) and phosphorylated extracellular signal-regulated protein (pERK) in the mouse hippocampus induced by various nociceptive stimuli. In an immunoblot study, s.c. injection of formalin and intrathecal (i.t.) injections of glutamate, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1 beta) significantly increased pCaMKIIalpha expression in the hippocampus, but i.p. injections of acetic acid did not. pERK1/2 expression was also increased by i.t. injection of glutamate, TNF-alpha, and IL-1beta but not by s.c. injections of formalin or i.p. injections of acetic acid. In an immunohistochemical study, we found that increased pCaMKIIalpha and pERK expressions were mainly located at CA3 or the dentate gyrus of the hippocampus. In a behavioral study, we assessed the effects of PD98059 (a MEK 1/2 inhibitor) and KN-93 (a CaMKII inhibitor) following i.c.v. administration on the nociceptive behaviors induced by i.t. injections of glutamate, pro-inflammatory cytokines (TNF-alpha or IL-1beta), and i.p. injections of acetic acid. PD98059 as well as KN-93 significantly attenuated the nociceptive behavior induced by glutamate, pro-inflammatory cytokines, and acetic acid. Our results suggest that (1) pERKalpha and pCaMK-II located in the hippocampus are important regulators during the nociceptive processes induced by s.c. formalin, i.t. glutamate, i.t. pro-inflammatory cytokines, and i.p. acetic acid injection, respectively, and (2) the alteration of pERK and pCaMKIIalpha in nociceptive processing induced by formalin, glutamate, pro-inflammatory cytokines and acetic acid was modulated in a different manner.

The effect of formalin pretreatment on nicotine-induced antinociceptive effect: the role of mu-opioid receptor in the hippocampus.

Nicotine is attractive as an analgesic component despite that its antinociceptive mechanism is not well known until now. In the present study, we examined the antinociceptive effect of nicotine administered supra-spinally on acetic acid-induced visceral pain induction (writhing test), and found that the antinociceptive effect of nicotine was abolished by mu-, delta-, and kappa-opioid receptor antagonist administered i.c.v. In addition, s.c. 5% formalin pretreatment at 5 h, 20 h, 40 h, and 1 week prior to i.c.v. nicotine injection abolished the antinociceptive effect of nicotine in the writhing test, suggesting that s.c. formalin pretreatment induced tolerance to the antinociceptive effect of nicotine in the supra-spinal region. Furthermore, neuronal loss of the hippocampal cornus ammonis (CA) 3 region reduced nicotine-induced an antinociceptive effect in the writhing test. In Western blot assay, we examined s.c. formalin injection down-regulated mu-opioid receptor in the hippocampus after 40 h, and its effect was maintained for 1 week. However, various acetylcholine receptor subunits and delta-, and kappa-opioid receptors were not altered. These results suggest that s.c. formalin pretreatment can contribute to induce tolerance on nicotine-induced antinociception as down-regulating mu-opioid receptor in the hippocampus, especially 40 h after s.c. formalin injection.

Characterization of the hypothalamic proopiomelanocortin gene and beta-endorphin expression in the hypothalamic arcuate nucleus of mice elicited by inflammatory pain.

We examined proopiomelanocortin (POMC) mRNA and beta-endorphin expression in the hypothalamus of mice after various nociceptive stimuli. The time-course study (10 min, 30 min, 1 h, 2 h, and 10 h) showed that the POMC mRNA level significantly increases from 1 h after s.c. formalin injection and returns to the control level at 10 h. Intrathecal (i.t.) substance P (SP) injection also increases the hypothalamic POMC mRNA level from 1 h to 10 h. However, i.t. glutamate injection did not affect the hypothalamic POMC gene expression at all time points. We found that the POMC mRNA after s.c. formalin injection was located in the arcuate nucleus of the hypothalamus. In the same manner, beta-endorphin immunoreactivity was also increased in the hypothalamic arcuate nucleus. The expression of phosphorylated extracellular signal-regulated protein kinase 1/2 (pERK1/2), phosphorylated calcium/calmodulin-dependent protein kinase-IIalpha (pCaMK-IIalpha) protein and phosphorylated IkappaB (pIkappaB) protein was increased by s.c. formalin injection at various time points. We also found that increased pERK1/2, pCaMKIIalpha and pIkappaB protein after s.c. formalin injection was mainly located in the arcuate nucleus of hypothalamus in which cells containing beta-endorphin after s.c. formalin injection also express pERK1/2, pCaMK-IIalpha and pIkappaB immunoreactivity. In addition, formalin-induced POMC mRNA expression was significantly reduced by 10 min, pretreatment with i.c.v. PD98059 (mitogen-activated protein kinase (MAPK) pathways inhibitor; 6.6 mug) and KN93 (pCaMK-II inhibitor; 20 mug). In conclusion, POMC mRNA expression in the arcuate nucleus of the hypothalamus was increased by inflammatory pain stimuli, in which pERK1/2, pCaMK-IIalpha and NFkappaB may play an important role in the expression of the hypothalamic POMC gene and beta-endorphin expression.

Transforming growth factor-beta1 regulates the fate of cultured spinal cord-derived neural progenitor cells.

OBJECTIVES: We have evaluated the physiological roles of transforming growth factor-beta1 (TGF-beta1) on differentiation, migration, proliferation and anti-apoptosis characteristics of cultured spinal cord-derived neural progenitor cells. METHODS: We have used neural progenitor cells that had been isolated and cultured from mouse spinal cord tissue, and we also assessed the relevant reaction mechanisms using an activin-like kinase (ALK)-specific inhibitory system including an inhibitory RNA, and found that it involved potential signalling molecules such as phosphatidylinositol-3-OH kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK1/2). RESULTS AND CONCLUSIONS: Transforming growth factor-beta1-mediated cell population growth was activated after treatment and was also effectively blocked by an ALK41517-synthetic inhibitor (4-(5-benzo(1,3) dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl) benzamide (SB431542) and ALK siRNA, thereby indicating the involvement of SMAD2 in the TGF-beta1-mediated growth and migration of these neural progenitors cells (NPC). In the present study, TGF-beta1 actively induced NPC migration in vitro. Furthermore, TGF-beta1 demonstrated extreme anti-apoptotic behaviour against hydrogen peroxide-mediated apoptotic cell death. At low dosages, TGF-beta1 enhanced (by approximately 76%) cell survival against hydrogen peroxide treatment via inactivation of caspase-3 and -9. TGF-beta1-treated NPCs down-regulated Bax expression and cytochrome c release; in addition, the cells showed up-regulated Bcl-2 and thioredoxin reductase 1. They also had increased p38, Akt and ERK1/2 phosphorylation, showing the involvement of both the PI3K/Akt and MAPK/ERK1/2 pathways in the neuroprotective effects of TGF-beta1. Interestingly, these effects operate on specific subtypes of cells, including neurones, neural progenitor cells and astrocytes in cultured spinal cord tissue-derived cells. Lesion sites of spinal cord-overexpressing TGF-beta1-mediated prevention of cell death, cell growth and migration enhancement activity have been introduced as a possible new basis for therapeutic strategy in treatment of neurodegenerative disorders, including spinal cord injuries.

Multicenter study of intravenous busulfan, cyclophosphamide, and etoposide (i.v. Bu/Cy/E) as conditioning regimen for autologous stem cell transplantation in patients with non-Hodgkin's lymphoma.

The current study aimed to evaluate the efficacy and toxicity of a combination of intravenous busulfan, cyclophosphamide and etoposide (i.v. Bu/Cy/E) as a conditioning regimen prior to autologous hematopoietic stem cell transplantation in patients with non-Hodgkin's lymphoma (NHL). Sixty-four patients with relapsed/refractory (n=36) or high-risk (n=28) lymphoma were enrolled. The high-dose chemotherapy consisted of i.v. Bu (0.8 mg kg(-1) i.v. q 6 h from day -7 to day -5), Cy (50 mg kg(-1) i.v. on day -3 and day -2) and E (400 mg m(-2) i.v. on day -5 and day -4). The median age was 43 (range 18-65) years, and 39 patients were male. Diffuse large B-cell lymphoma (40.6%) was the most common histological subtype. All evaluable patients achieved an engraftment of neutrophils (median, day 12) and platelets (median, day 13). Hepatic veno-occlusive disease was observed in four patients (three mild, one moderate grade), and two patients (3.1%) died from treatment-related complications. At a median follow-up of 16.4 months, 15 patients (23.4%) exhibited a relapse or progression, while 13 patients (20.3%) had died of disease. The estimated 3-year overall and progression-free survival for all patients was 72.1 and 70.1%, respectively. In conclusion, the conditioning regimen of i.v. Bu/Cy/E was well tolerated and seemed to be effective in patients with aggressive NHL.

Relationship between retroviral replication and RNA interference machineries.

Small interfering RNAs (siRNAs) associated with gene silencing are cellular defense mechanisms against invading viruses. The viruses fight back by suppressors or escape mechanisms. The retroviruses developed a unique escape mechanism by disguising as DNA proviruses. An evolutionary relationship between the siRNA machinery and the replication machinery of retroviruses is likely. The RNA cleavage enzymes PIWI and RNase H proteins are structurally related. This relationship can be extended from structure to function, since the retroviral reverse transcriptase (RT)/RNase H can also cause silencing of viral RNA by siRNA. Thus, both enzymes can cleave RNA-DNA hybrids and double-stranded RNA (dsRNA) with various efficiencies shown previously and here, demonstrating that their specificities are not absolute. Other similarities may exist, for example between PAZ and the RT and between RNA-binding proteins and the viral nucleocapsid protein. Dicer has some similarities with the viral integrase, since both specifically generate dinucleotide 3'-overhanging ends. We described previously the destruction of the human immunodeficiency virus (HIV) RNA by a DNA oligonucleotide ODN (oligodeoxynucleotide). Variants of the ODN indicated high length and sequence specificities, which is reminiscent of siRNA and designated here as "siDNA." Cleavage of the viral RNA in the presence of the ODN is caused by the retroviral RT/RNase H and cellular RNase H activities. Several siRNA-mediated antiviral defense mechanisms resemble the interferon system.

Roles of NF-kappaB and bcl-2 in two differential modes of cell death of mouse cortical collecting duct cells.

Recent data have implicated nuclear factor-kappaB (NF-kappaB) and Bcl-2 in the regulation of apoptotic and necrotic cell death in various cells. However, mechanisms of their effects on cell death of renal epithelial cells are not clear. First, we investigated the effect of specific inhibition of NF-kappaB and overexpression of Bcl-2 on necrotic cell death induced by hydrogen peroxide or cisplatin in renal collecting duct cells. M-1 cells, which were derived from outer cortical collecting duct, were stably transfected with the non-phosphorylatable mutant of inhibitory-kappaBalpha (I-kappaBalpha) and Bcl-2. Overexpression of I-kappaBalpha and Bcl-2 did not affect cisplatin-induced necrotic cell death, but overexpression of I-kappaBalpha significantly decreased H2O2-induced cell death. Regarding apoptotic cell death induced by cisplatin, serum deprivation and contact inhibition was increased by overexpression of I-kappaBalpha, whereas overexpression of bcl-2 inhibited the apoptotic cell death. I-kappaBalpha overexpression increased Bax expression and decreased cIAP-1 and -2 expression compared to vector-transfected cells, but did not alter SAPK/JNK activity in the presence or absence of cisplatin. NF-kappaB activity was significantly higher in bcl-2-overexpressing cells than in control cells. These data show that activation of NF-kappaB mediates H2O2-induced necrotic injury, but inhibits apoptotic cell death in renal collecting duct cells, and that Bcl-2 selectively protects apoptotic cell death in M-1 cells.

Unique structural features of a BCL-2 family protein CED-9 and biophysical characterization of CED-9/EGL-1 interactions.

The interactions between B-cell lymphoma 2 (BCL-2) family members are known to be mediated through the binding of the BH3 domain of a proapoptotic member to the BH3-binding groove of an antiapoptotic member. We determined the crystal structure of antiapoptotic CED-9, which reveals a unique C-terminal helix altering the common BH3-binding region. A coexpression system to produce CED-9 in complex with proapoptotic EGL-1 enabled us to show that the binding of EGL-1 to CED-9 is extremely stable, raising the melting temperature (T(M)) of CED-9 by 25 degrees C, and that the binding surface of CED-9 extends beyond the BH3-binding region and reaches the BH4 domain. Consistently, the T(M) and a 1H-15N correlation NMR spectrum of CED-9 in complex with EGL-1 are drastically different from those of CED-9 in complex with the EGL-1 BH3 peptide. The data suggest that the recognition between other BCL-2 family members may also involve much wider protein surfaces than is previously thought.

The role of inducible nitric oxide synthase in aseptic loosening after total hip arthroplasty.

We collected 16 samples of the membrane which surrounds loose hip prostheses from patients undergoing revision operations for aseptic loosening. To serve as the control group, samples of the synovial tissue and the fibrous capsular tissue were collected from 11 patients undergoing primary hip arthroplasties. Analyses of the expression levels of inducible nitric oxide synthase (iNOS), tumour necrosis factor-alpha (TNF-alpha), and cytosolic phospholipase A2 (cPLA2) mRNAs were performed by a reverse transcription polymerase chain reaction, and the content of nitrite was measured by the Griess reaction using sodium nitrite as the standard. The expression levels of iNOS, TNF-alpha, and cPLA2 mRNAs in the membranes were significantly higher than those in the control samples (p < 0.05). The expression levels of iNOS mRNA and the nitrite content in the membranes significantly correlated with those of TNF-alpha and cPLA2 mRNAs, respectively. In addition, the expression levels of iNOS, TNF-alpha, and cPLA2 mRNAs were significantly higher in membranes from cementless than in those from cemented implants (p < 0.05). Our results suggest that the expression levels of iNOS, TNF-alpha, and cPLA2 mRNAs in the membranes are regulated by closely-related mechanisms and that these have a significant role in aseptic loosening.

Differential expression of a WD protein during squamous differentiation of tracheal epithelial cells.

The lining of the trachea consists of a pseudostratified, mucociliary epithelium that under a variety of conditions, such as vitamin A deficiency, toxic and mechanical injury, becomes a stratified squamous epithelium. Several in vitro cell culture models have been established to study the process of differentiation of airway epithelium. Such studies have indicated that mucosecretory differentiation of tracheal epithelial cells can be modulated by substratum. This study was undertaken to understand molecular mechanisms of squamous differentiation in tracheal epithelia. Primary cultured tracheal cells grown on uncoated filters were differentiated to single layer of squamous cells, whereas cells were grown as stratified columnar cells on collagen-I coated filters. The responses to secretagogues were altered according to culture conditions. DD-PCR revealed that FAK and a WD protein expression was increased in squamous tracheal epithelia. Expression of a WD protein was changed by the treatment of retinoic acid in various epithelial cells. These results indicated that squamous differentiation of tracheal cells changes the expression of a variety of genes, and that the experimental model for this study can be employed to study molecular mechanisms of squamous differentiation in airway epithelial cells.