Preferential Fas-mediated apoptotic execution at G1 phase: the resistance of mitotic cells to the cell death.

Apoptosis is induced by various stresses generated from the extracellular and intracellular environments. The fidelity of the cell cycle is monitored by surveillance mechanisms that arrest its further progression if any crucial process has not been completed or damages are sustained, and then the cells with problems undergo apoptosis. Although the molecular mechanisms involved in the regulation of the cell cycle and that of apoptosis have been elucidated, the links between them are not clear, especially that between cell cycle and death receptor-mediated apoptosis. By using the HeLa.S-Fucci (fluorescent ubiquitination-based cell cycle indicator) cells, we investigated the relationship between the cell cycle progression and apoptotic execution. To monitor apoptotic execution during cell cycle progression, we observed the cells after induction of apoptosis with time-lapse fluorescent microscopy. About 70% of Fas-mediated apoptotic cells were present at G(1) phase and about 20% of cells died immediately after cytokinesis, whereas more than 60% of etoposide-induced apoptotic cells were at S/G(2) phases in random culture of the cells. These results were confirmed by using synchronized culture of the cells. Furthermore, mitotic cells showed the resistance to Fas-mediated apoptosis. In conclusion, these findings suggest that apoptotic execution is dependent on cell cycle phase and Fas-mediated apoptosis preferentially occurs at G(1) phase.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS): novel compound heterozygous mutations in the SACS gene.

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a neurodegenerative disorder first described among French Canadians in Quebec. To date, 24 mutations have been reported in the SACS gene of ARSACS patients. The authors report a clinical and genetic analysis of a Japanese family with ARSACS with novel compound heterozygous mutations in the SACS gene (N161fsX175, L802P). The phenotype is similar to that of previously reported ARSACS patients.

A fast response and gamma-insensitive neutron detector based on parallel-plate avalanche counter.

The use of the parallel-plate avalanche counter for slow-neutron counting is described. The choice of a suitable neutron converter is discussed on the basis of Monte Carlo simulation, and some experimental results are shown. Excellent gamma-insensitivity, high rate capability, possibility of construction in large sensitive area and low production cost are among the promising features of this neutron detector.

Installation and application of an intense 7Li(p,n) neutron source for 20-90 MeV region.

An intense (7)Li(p,n) neutron source was installed with the K = 110-MeV azimuthally varying field (AVF) cyclotron at the Cyclotron and Radioisotope Center (CYRIC), Tohoku University, by employing a special arrangement to allow a short target-sample distance down to 1 m. The source can deliver a neutron flux around 1.5 x 10(6) n cm(-2) s(-1) with a lithium target of thickness equivalent to 2-MeV energy loss and 3-microA proton beam, which is the highest intensity in the world. The source is successfully used for (1) measurement of neutron cross sections relevant to radiation safety and radiation effect and (2) semiconductor irradiation test for single-event effect and (3) characterisation of neutron detectors and dosemeters.

Differential cross sections of neutron-induced fragment-emission reactions for a microdosimetry study.

Experimental differential cross sections of fragment emission (p, d, t, alpha, Li, Be and B), which were obtained for tens of mega electron volt neutrons on carbon and aluminum, using a counter telescope array and a Bragg-curve counter specially developed for neutron-induced reactions, are presented and compared with theoretical calculations using various reaction models. A calculation with the ISOBAR and GEM models was found to reproduce the experimental data except for an underestimation in non-vaporation processes. Calculations of the energy deposition by neutrons in a thin silicon layer show significant differences among the model employed.

Beam-beam limit in e+e- circular colliders.

Beam-beam effects limit the luminosity of circular colliders. Once the bunch population exceeds a threshold, the luminosity increases at a slower rate. This phenomenon is called the beam-beam limit. Onset of the beam-beam limit has been analyzed with various simulation methods based on the weak-strong and strong-strong models. We have observed that an incoherent phenomenon is mainly concerned in the beam-beam limit. The simulation have shown that equilibrium distributions of the two colliding beams are distorted from Gaussians when the luminosity is limited. The beam-beam limit is estimated to be xi approximately 0.1 for a B factory with damping time of several thousand turns.

Extremely low vertical-emittance beam in the accelerator test facility at KEK.

Electron beams with the lowest, normalized transverse emittance recorded so far were produced and confirmed in single-bunch-mode operation of the Accelerator Test Facility at KEK. We established a tuning method of the damping ring which achieves a small vertical dispersion and small x-y orbit coupling. The vertical emittance was less than 1% of the horizontal emittance. At the zero-intensity limit, the vertical normalized emittance was less than 2.8 x 10(-8) rad m at beam energy 1.3 GeV. At high intensity, strong effects of intrabeam scattering were observed, which had been expected in view of the extremely high particle density due to the small transverse emittance.

Decreasing process variation in the care of carotid endarterectomy patients.

Avoidable complications after successful carotid endarterectomy surgery typically occur in the immediate postoperative period; most of these complications are related to hemodynamic instability. At Saint Agnes Medical Center, process variation resulted from 22 anesthesiologists and 11 surgeons doing the same process 242 ways. We introduced a Post-Anesthesia Care Unit Carotid Order Set to standardize the process, drug sequence, and drug choices for postoperative carotid endarterectomy patients. Ongoing monitoring demonstrated that this reduction in process variability resulted in a lower complication rate for stroke and wound hematoma.

Human gelsolin prevents apoptosis by inhibiting apoptotic mitochondrial changes via closing VDAC.

Gelsolin is a Ca2+-dependent actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility through modulation of the actin network. Gelsolin was also recently suggested to be involved in the regulation of apoptosis: human gelsolin (hGsn) has anti-apoptotic activity, whereas mouse gelsolin (mGsn) exerts either proapoptotic or anti-apoptotic activity depending on different cell types. Here, we studied the basis of anti-apoptotic activity of hGsn. We showed that both endogenous and overexpressed hGsn has anti-apoptotic activity, that depends on its C-terminal half. We also found that hGsn and its C-terminal half but not mGsn could prevent apoptotic mitochondrial changes such as Apsi loss and cytochrome c release in isolated mitochondria to a similar extent as Bcl-xL, indicating that hGsn targets the mitochondria to prevent apoptosis via its C-terminal half. In the same way as anti-apoptotic Bcl-xL, which we recently found to prevent apoptotic mitochondrial changes by binding and closing the voltage-dependent anion channel (VDAC), hGsn and its C-terminal half inhibited the activity of VDAC on liposomes through direct binding in a Ca2+-dependent manner. These results suggest that hGsn inhibits apoptosis by blocking mitochondrial VDAC activity.

The inhibitor of apoptosis, cIAP2, functions as a ubiquitin-protein ligase and promotes in vitro monoubiquitination of caspases 3 and 7.

The inhibitor of apoptosis, cIAP2, contains a putative Ring finger motif at the C terminus. Using in vitro ubiquitination assays, we found that the Ring finger of cIAP2 alone possesses intrinsic ubiquitin ligase activity and promotes substrate-independent ubiquitination. It also promotes ubiquitination of caspases 3 and 7 but not caspase-1. The Ring fingers of c-Cbl and Apc11 failed to promote caspase-7 ubiquitination, suggesting that the Ring finger of cIAP2 itself is involved in substrate recognition.

A sandwich enzyme immunoassay for pulmonary surfactant protein D and measurement of its blood levels in drowning victims.

A sensitive sandwich enzyme immunoassay for human pulmonary surfactant protein D (SP-D) was developed and used to examine the blood SP-D levels of drowning victims. Human SP-D was purified from amniotic fluid by chromatographic methods, and an antibody against human SP-D was prepared. A polystyrene ball coated with anti-SP-D IgG was incubated with purified human SP-D, and then with anti-SP-D Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as the hydrogen donor. The detection limit of human SP-D was 5.2 pg per assay tube. Examination of cross-reactions of this sandwich enzyme immunoassay with proteins from other human organs showed it to be highly specific for lung, and Northern blot analysis detected specific SP-D mRNA expression only in lung. The SP-D concentration of normal human serum was 6.4+/-2.7 (mean+/-S.D.) ng ml(-1) (n=20). The recovery rates of 0.52 ng and 5.2 ng SP-D added to 5 microl normal human serum were 93.6+/-2.7% and 93.6+/-6.1%, respectively. Blood SP-D levels of victims from the saltwater drowning group (n=14) revealed higher concentrations (105.8+/-53.7 ng ml(-1)), while freshwater drowning victims (n=12) were estimated to be 74.1+/-43.9 ng ml(-1). The SP-D levels of 15 subjects who died of hemorrhage (n=5), heart failure (n=8), traumatic shock (n=1), and electrocution (n=1) were lower (22.0+/-8.5 ng ml(-1)), and those of asphyxia victims (n=10) were slightly higher (36.2+/-17.1 ng ml(-1)) than those of other causes of death, except for drowning. These results suggest that in drowning victims, SP-D flowed into the systemic circulation by physiological and physical mechanisms, and the differences of blood SP-D levels between saltwater drowning and freshwater drowning victims are presumed to be influenced by the type of agony and/or the length of survival time in water.

Enhanced sonodynamic antitumor effect of ultrasound in the presence of nonsteroidal anti-inflammatory drugs.

The antitumor effects of non-steroidal anti-inflammatory drugs, tenoxicam and piroxicam, against sarcoma 180 cells cultured in 7-week-old male mice were examined in vitro under ultrasonic irradiation. The survival rate of tumor cells when tenoxicam or piroxicam was added to sarcoma 180 suspension under ultrasonic irradiation was significantly lower than that when ultrasound alone was applied. Furthermore, when L-histidine, a scavenger of singlet oxygen and hydroxyl radical, or D-mannitol, a scavenger of hydroxyl radical, was used concurrently, the survival rate of tumor cells was significantly higher with L-histidine. From the above findings, it is surmised that tenoxicam and piroxicam increase the antitumor effects of ultrasound by increasing the production of singlet oxygen and other active oxygen species.

Bis, a Bcl-2-binding protein that synergizes with Bcl-2 in preventing cell death.

Bcl-2 is the best characterized inhibitor of apoptosis, although the molecular basis of this action is not fully understood. Using a protein interaction cloning procedure, we identified a human gene designated as bis (mapped to chromosome 10q25) that encoded a novel Bcl-2-interacting protein. Bis protein showed no significant homology with Bcl-2 family proteins and had no prominent functional motif. Co-immunoprecipitation analysis confirmed that Bis interacted with Bcl-2 in vivo. DNA transfection experiments indicated that Bis itself exerted only weak anti-apoptotic activity, but was synergistic with Bcl-2 in preventing Bax-induced and Fas-mediated apoptosis. These results suggest that Bis is a novel modulator of cellular anti-apoptotic activity that functions through its interaction with Bcl-2.

Identification of NRF2, a member of the NF-E2 family of transcription factors, as a substrate for caspase-3(-like) proteases.

Apoptosis is mediated by members of the interleukin-1beta converting enzyme (ICE) family of proteases (caspases), which are activated by diverse stimuli, although the downstream molecular targets of caspases are still poorly understood. Using the modified yeast two-hybrid system, which we recently established to clone genes for caspase substrates, we identified NRF2 as a novel caspase substrate. NRF2 is a member of the NF-E2 family of basic region leucine-zipper transcription factors and has been shown to induce phase II detoxifying enzymes through anti-oxidant response elements. NRF2 was cleaved at two sites by recombinant caspase-3 in vitro as well as in HeLa cells during TNFalpha-mediated apoptosis. Overexpression of the C-terminal cleavage fragment containing the DNA binding and leucine-zipper domains induced apoptosis in HeLa cells. These observations suggest that NRF2 might have some role in the induction of apoptosis after cleavage by caspases.

Cooperation of six and eya in activation of their target genes through nuclear translocation of Eya.

Drosophila sine oculis and eyes absent genes synergize in compound-eye formation. The murine homologues of these genes, Six and Eya, respectively, show overlapping expression patterns during development. We hypothesized that Six and Eya proteins cooperate to regulate their target genes. Cotransfection assays were performed with various combinations of Six and Eya to assess their effects on a potential natural target, myogenin promoter, and on a synthetic promoter, the thymidine kinase gene promoter fused to multimerized Six4 binding sites. A clear synergistic activation of these promoters was observed in certain combinations of Six and Eya. To investigate the molecular basis for the cooperation, we first examined the intracellular distribution of Six and Eya proteins in transfected COS7 cells. Coexpression of Six2, Six4, or Six5 induced nuclear translocation of Eya1, Eya2, and Eya3, which were otherwise distributed in the cytoplasm. In contrast, coexpression of Six3 did not result in nuclear localization of any Eya proteins. Six and Eya proteins were coimmunoprecipitated from nuclear extracts prepared from cotransfected COS7 cells and from rat liver. Six domain and homeodomain, two evolutionarily conserved domains among various Six proteins, were necessary and sufficient for the nuclear translocation of Eya. In contrast, the Eya domain, a conserved domain among Eya proteins, was not sufficient for the translocation. A specific interaction between the Six domain and homeodomain of Six4 and Eya2 was observed by yeast two-hybrid analysis. Our results suggest that transcription regulation of certain target genes by Six proteins requires cooperative interaction with Eya proteins: complex formation through direct interaction and nuclear translocation of Eya proteins. This implies that the synergistic action of Six and Eya is conserved in the mouse and is mediated through cooperative activation of their target genes.

The induction of smooth muscle alpha actin in a transformed rat cell line suppresses malignant properties in vitro and in vivo.

A normal rat fibroblast 3Y1 cell line expresses smooth muscle a actin and the expression of alpha actin is suppressed in the transformant (SR-3Y1-2) induced by a Raus sarcoma virus. Gene transfer with smooth muscle alpha actin into the SR-3Y1-2 cell line reduced growth and invasiveness in vitro, as well as tumor growth and experimental lung metastasis depending on the expression of the alpha actin. These results indicated that smooth muscle alpha actin is involved in the regulation of cell growth as well as cell motility and thus leads to the suppression of malignant phenotypes in transformed cells.

Characteristics of intracardiac potentials at successful ablation sites in concealed Wolff-Parkinson-White syndrome.

The authors investigated bipolar electrograms recorded from the catheter tip at the actual successful ablation sites in 22 consecutive patients with concealed Wolff-Parkinson-White syndrome to clarify the characteristics of the potentials indicating the optimal site for catheter ablation. In all patients the retrograde transaortic approach to their left-sided accessory pathways, and a temperature-controlled (60 degrees C) energy delivery, were performed. The authors assumed that a shorter dissociation time (time from energy delivery to ventriculoatrial conduction dissociation) indicated more accurate catheter mapping. A significant negative correlation (r = 0.527, p < 0.05) between the AV ratio (ratio of the amplitudes of the atrial to ventricular potentials) recorded at the ablation catheter tip and the dissociation time was observed. When the AV ratio and the dissociation time were compared among the groups classified according to the corresponding Npeak (the number of positive potential peaks in the electrogram obtained from the ablation catheter tip during right ventricular apical pacing) value, they differed significantly (p < 0.05 and p < 0.01, respectively), ie, a higher AV ratio and a shorter dissociation time related to a multipeak electrogram from the ablation catheter tip. The authors conclude that the atrial insertion site of the accessory pathway, exhibiting a multipeak complex electrogram that may represent nonuniform anisotropic characteristics, is an adequate ablation site.

Spatial coherence of bending magnet radiation and application limit of the van Cittert-Zernike theorem.

In this paper we discuss how the first-order spatial coherence of bending magnet radiation is determined. We present an analytical representation of the coherence and compare it with the numerical calculations based on the first principles. It is shown that if the electron-beam size is so large that some conditions are satisfied, the van Cittert-Zernike theorem can be used without any modification in the vertical and horizontal directions. The formalism presented will be useful in judging whether or not the electron-beam size in the storage ring can be estimated directly from the van Cittert-Zernike theorem using the synchrotron radiation interferometer.