Observation of a charmoniumlike state produced in association with a J/psi in e+e- annihilation at sqrt[s] approximately equal to 10.6 GeV.

We report the first observation of a charmoniumlike state recoiling from the J/psi in the inclusive process e+e- -->J/psi+anything at a mass of (3.943+/-0.006+/-0.006) GeV/c{2}. We also observe the decay of this state into D*D[over ] and determine its intrinsic width to be less than 52 MeV/c{2} at the 90% C.L. These results are obtained from a 357 fb{-1} data sample collected with the Belle detector near the Upsilon(4S) resonance, at the KEKB asymmetric-energy e+e- collider.

Evidence for B-->eta'pi and improved measurements for B-->eta'K.

We report evidence for the exclusive two-body charmless hadronic B meson decay B-->eta'pi, and improved measurements of B-->eta'K. The results are obtained from a data sample of 386x10(6) BB pairs collected at the Upsilon(4S) resonance, with the Belle detector at the KEKB asymmetric-energy e+e- collider. We measure B(B+-->eta'pi+)=[1.76(-0.62)(+0.67)(stat)(-0.14)(+0.15)(syst)]x10(-6) and B(B0-->eta'pi0)=[2.79(-0.96)(+1.02)(stat)(-0.34)(+0.25)(syst)]x10(-6). We also find the ratio of B(B+-->eta'K+)/B(B0-->eta'K0)=1.17+/-0.08(stat)+/-0.03(syst) and measure the direct CP asymmetries for the charged modes.

Evidence for large direct CP violation in B+/- --> rho(770)0K+/- from analysis of three-body charmless B+/- --> K+/-pi+/-pi+/- decays.

We report results on a Dalitz analysis of three-body charmless B+/- --> K+/-pi+/-pi+/- decay including searches for direct CP violation. We report the first observation of the decay B+/- --> f2(1270)K+/- with a statistical significance above 6sigma. We also observe first evidence for large direct CP violation in the B+/- --> rho(770)0K+/- channel. The results are obtained with a data sample that contains 386 10(6) BB pairs collected at the Y(4s) resonance with the Belle detector at the KEKB asymmetric-energy e+e- collider.

Differential regulation of CXC ligand 1 transcription in melanoma cell lines by poly(ADP-ribose) polymerase-1.

The continuous production of the CXC ligand 1 (CXCL1) chemokine by melanoma cells is a major effector of tumor growth. We have previously shown that the constitutive expression of this chemokine is dependent upon transcription factors nuclear factor-kappa B (NF-kappaB), stimulating protein-1 (SP1), high-mobility group-I/Y (HMGI/Y), CAAT displacement protein (CDP) and poly(ADP-ribose) polymerase-1 (PARP-1). In this study, we demonstrate for the first time the mechanism of transcriptional regulation of CXCL1 through PARP-1 in melanoma cells. In its inactive state, PARP-1 binds to the CXCL1 promoter in a sequence-specific manner and prevents binding of NF-kappaB (p65/p50) to its element. However, activation of the PARP-1 enzymatic activity enhances CXCL1 expression, owing to the loss of PARP-1 binding to the CXCL1 promoter, accompanied by enhanced binding of p65 to the promoter. The delineation of the role of NF-kappaB-interacting factors in the putative CXCL1 enhanceosome will provide key information in developing strategies to block constitutive expression of this and other chemokines in cancer and to develop targeted therapy.

Observation of b-->dgamma and determination of /Vtd/Vts/.

We report the observation of the flavor-changing neutral current process b-->dgamma using a sample of 386 x 106 B meson pairs accumulated by the Belle detector at the KEKB e+e- collider. We measure branching fractions for the exclusive modes B--->rho-gamma, B0rho0gamma, and B0omegagamma. Assuming that these three modes are related by isospin, we find B(B-->(rho,gamma)gamma)=[Formula: See Text] with a significance of 5.1sigma. This result is used to determine the ratio of Cabibbo-Kobayashi-Maskawa matrix elements /Vtd/Vts/ to be [Formula: See Text].

Measurement of azimuthal asymmetries in inclusive production of hadron pairs in e(+)e(-) annihilation at Belle.

The Collins effect connects transverse quark spin with a measurable azimuthal dependence in the yield of hadronic fragments around the quark's momentum vector. Using two different reconstruction methods, we find evidence of statistically significant azimuthal asymmetries for charged pion pairs in e(+)e(-) annihilation at a center-of-mass energy of 10.52 GeV, which can be attributed to a transverse polarization of the primordial quarks. The measurement was performed using a sample of 79 x 10(6) hadronic events collected with the Belle detector.

Improved constraints on D0-D0 mixing in D0 --> K+ pi- decays from the Belle detector.

We report the results of a search for D0-D0 mixing in D0 --> K+ pi- decays based on 400 fb(-1) of data accumulated by the Belle detector at KEKB. Both assuming CP conservation and allowing for CP violation, we fit the decay-time distribution for the mixing parameters x and y, as well as for the parameter R(D), the ratio of doubly Cabibbo-suppressed decays to Cabibbo-favored decays. The 95% confidence level region in the (x'2,y') plane is obtained using a frequentist method. Assuming CP conservation, we find x'2 < 0.72 x 10(-3) and -9.9 x 10(-3) < y' < 6.8 x 10(-3) at the 95% confidence level; these are the most stringent constraints on the mixing parameters to date. The no-mixing point (0,0) has a confidence level of 3.9%. Assuming no mixing, we measure R(D) = (0.377 +/- 0.008 +/- 0.005)%.

Observation of a chi(c2)' candidate in gamma gamma --> DD production at belle.

We report on a search for new resonant states in the process gamma gamma --> DD. A candidate C-even charmonium state is observed in the vicinity of 3.93 GeV/c2. The production rate and the angular distribution in the gamma gamma center-of-mass frame suggest that this state is the previously unobserved chi(c2)', the 2(3)P2 charmonium state.

Production of manganese peroxidase by pellet culture of the lignin-degrading basidiomycete, Pleurotus ostreatus.

Pleurotus ostreatus No. 42 produced the ligninolytic enzymes, manganese peroxidase (MnP) and laccase, in agitation culture in glucose/peptone/wheat-bran medium. Formation of mycelial pellets 1-2 mm in diameter was essential for the production of MnP; and the concentration of dissolved oxygen in the culture medium greatly influenced the production of MnP, a concentration over 5 ppm being necessary for MnP production. The maximal activity of MnP was obtained on days 7-9 of culture, after the consumption of nutrient glucose. Introduction of oxygen from the start of the cultivation caused large pellet formation, which resulted in a low MnP activity level. P. ostreatus No. 42 produced two MnP isozymes in agitation culture. The major isozyme, F-2, was 36.4 kDa and had a pI of 3.95. The MnP characteristics, Km values, dependence on Mn2+ and optimum pH showed the similarity between this isozyme and MnP 3, which was produced under different culture conditions. Analysis of the N-terminal amino acid sequence indicated the close similarity of F-2 to MnP 3.

Poly(ADP-ribose) polymerase-1 is required for efficient HIV-1 integration.

Poly(ADP-ribose) polymerase-1 (PARP-1; EC ) is an abundant nuclear enzyme, activated by DNA strand breaks to attach up to 200 ADP-ribose groups to nuclear proteins. As retroviral infection requires integrase-catalyzed DNA strand breaks, we examined infection of pseudotyped HIV type I in fibroblasts from mice with a targeted deletion of PARP-1. Viral infection is almost totally abolished in PARP-1 knockout fibroblasts. This protection from infection reflects prevention of viral integration into the host genome. These findings suggest a potential for PARP inhibitors in therapy of HIV type I infection.

FKBP12, the 12-kDa FK506-binding protein, is a physiologic regulator of the cell cycle.

FKBP12, the 12-kDa FK506-binding protein, is a ubiquitous abundant protein that acts as a receptor for the immunosuppressant drug FK506, binds tightly to intracellular calcium release channels and to the transforming growth factor beta (TGF-beta) type I receptor. We now demonstrate that cells from FKBP12-deficient (FKBP12(-/-)) mice manifest cell cycle arrest in G(1) phase and that these cells can be rescued by FKBP12 transfection. This arrest is mediated by marked augmentation of p21(WAF1/CIP1) levels, which cannot be further augmented by TGF-beta1. The p21 up-regulation and cell cycle arrest derive from the overactivity of TGF-beta receptor signaling, which is normally inhibited by FKBP12. Cell cycle arrest is prevented by transfection with a dominant-negative TGF-beta receptor construct. TGF-beta receptor signaling to gene expression can be mediated by SMAD, p38, and ERK/MAP kinase (extracellular signal-regulated kinase/mitogen-activated protein kinase) pathways. SMAD signaling is down-regulated in FKBP12(-/-) cells. Inhibition of ERK/MAP kinase fails to affect p21 up-regulation. By contrast, activated phosphorylated p38 is markedly augmented in FKBP12(-/-) cells and the p21 up-regulation is prevented by an inhibitor of p38. Thus, FKBP12 is a physiologic regulator of cell cycle acting by normally down-regulating TGF-beta receptor signaling.

Reactive oxygen species are critical for the growth and differentiation of medullary thyroid carcinoma cells.

Reactive oxygen species have recently been demonstrated to play a role in numerous cellular signal transduction pathways. Here we investigate the involvement of H2O2 in Raf-1-mediated differentiation in the human medullary thyroid carcinoma (MTC) cell line TT:deltaRaf-1:ER. Catalase, but not Cu/Zn superoxide dismutase, completely inhibited Raf-1-induced differentiation of beta-estradiol-treated TT: deltaRaf-1:ER. In addition, catalase treatment down-regulated RET expression at both the mRNA and protein levels and induced apoptosis in the parental TT cell line and uninduced TT:deltaRaf-1:ER human MTC cells. These results implicate H2O2 as a downstream mediator of c-Raf-1-induced differentiation and as a survival factor in MTC cells.

Poly(ADP-ribose) polymerase-1 in the nervous system.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme, activated by DNA strand breaks to participate in DNA repair. Overactivation of PARP by cellular insults depletes its substrate NAD(+) and then ATP, leading to a major energy deficit and cell death. This mechanism appears to be prominent in vascular stroke and other neurodegenerative processes in which PARP gene deletion and PARP-inhibiting drugs provide major protection. Cell death associated with PARP-1 overactivation appears to be predominantly necrotic while apoptosis is associated with PARP-1 cleavage, which may conserve energy needed for the apoptotic process. Novel forms of PARP derived from distinct genes and lacking classic DNA-binding domains may have nonnuclear functions, perhaps linked to cellular energy dynamics.

Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion.

Apoptotic and necrotic cell death are well characterized and are influenced by intracellular ATP levels. Poly(ADP-ribose) polymerase (PARP), a nuclear enzyme activated by DNA strand breaks, physiologically participates in DNA repair. Overactivation of PARP after cellular insults can lead to cell death caused by depletion of the enzyme's substrate beta-nicotinamide adenine dinucleotide and of ATP. In this study, we have differentially elicited apoptosis or necrosis in mouse fibroblasts. Fibroblasts from PARP-deficient (PARP(-/-)) mice are protected from necrotic cell death and ATP depletion but not from apoptotic death. These findings, together with cell death patterns in PARP(-/-) animals receiving other types of insults, indicate that PARP activation is an active trigger of necrosis, whereas other mechanisms mediate apoptosis.

1-(N-alkylamino)-11-(N-ethylamino)-4,8-diazaundecanes: simple synthetic polyamine analogues that differentially alter tubulin polymerization.

Polyamine analogues such as bis(ethyl)norspermine and N1-(cyclopropylmethyl)-N11-ethyl-4,8-diazaundecane (CPENSpm) act as potent modulators of cellular polyamine metabolism in vitro and possess impressive antitumor activity against a number of cell lines. Some of these polyamine analogues appear to produce their cell-type-specific cytotoxic activity through the superinduction of spermidine/spermine N1-acetyltransferase (SSAT). However, there are several analogues (e.g., N1-(cycloheptylmethyl)-N11-ethyl-4, 8-diazaundecane (CHENSpm)) which are effective cytotoxic agents but do not superinduce SSAT. We have previously demonstrated that CPENSpm and CHENSpm both initiate the cell death program, although by different mechanisms, and that CHENSpm (but not CPENSpm) induces a G2/M cell cycle arrest. We now report that one potential mechanism by which some polyamine analogues can retard growth and ultimately produce cytotoxicity is through interference with normal tubulin polymerization. In these studies, we compare the effects of the polyamine analogues CHENSpm, CPENSpm, and (S)-N1-(2-methyl-1-butyl)-N11-ethyl-4,8-diazaundecane (IPENSpm) on in vitro tubulin polymerization. These spermine analogues behave very differently from spermine and from each other in terms of tubulin polymerization rate, equilibrium levels, and time of polymerization initiation. These results demonstrate that structurally similar polyamine analogues with potent antitumor effects can produce significantly different cellular effects. The discovery of polyamine analogues that can alter tubulin polymerization provides a series of promising lead compounds that may have a similar spectrum of activity to more difficult to synthesize compounds typified by paclitaxel.

The natural polyamine spermine functions directly as a free radical scavenger.

The polyamines are small organic cations that are absolutely required for eukaryotic cell growth. Although their growth requirements are well established, the molecular functions of the polyamines are ill-defined. Oxidative damage to DNA by reactive oxygen species is a continual problem that cells must guard against to survive. The polyamine spermine, which is normally found in millimolar concentrations in the nucleus, is shown here to function directly as a free radical scavenger, and adducts formed as a result of this function are identified. These data suggest that spermine is a major natural intracellular compound capable of protecting DNA from free radical attack.

Unsymmetrically substituted polyamine analogue induces caspase-independent programmed cell death in Bcl-2-overexpressing cells.

The polyamine analogue, N1-ethyl-N11-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm)-induced programmed cell death in NCI H157 cells is accompanied by cytochrome c release, the loss of mitochondrial membrane potential, activation of caspase-3, caspase-mediated poly(ADP-ribose) polymerase cleavage, G2-M arrest, and DNA and nuclear fragmentation. Overexpression of Bcl-2 completely inhibits CHENSpm-induced cytochrome c release, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. However, Bcl-2 does not abrogate CHENSpm-induced programmed cell death. These results suggest that although cytochrome c release and activation of the caspase-3 protease cascade contribute to the rapid and efficient execution of apoptosis, a caspase cascade-independent pathway also exists and can be activated by CHENSpm treatment.

Structural specificity of polyamines and polyamine analogues in the protection of DNA from strand breaks induced by reactive oxygen species.

Reactive oxygen species are known to induce strand breaks and/or base modifications in DNA. DNA strand breaks are associated with many pathologies and programmed cell death. We have examined the ability of the polyamines and their analogues to protect phi X-174 plasmid DNA from strand breakage induced by a oxygen-radical generating system. Spermine and several unsymmetrically substituted polyamine analogues reduced the amount of strand breakage at a physiologically relevant concentration of 1 mM. However, putrescine, spermidine, N1-acetylspermine, N1-acetylspermidine and symmetrically alkylated polyamine analogues were not able to reduce strand breakage at the same concentration. Thus, the unsymmetrically alkylated polyamine analogues and natural spermine can protect DNA against strand breakage induced by Cu(II)/H2O2 generated ROS similar to other more classical antioxidants.

The role of polyamine catabolism in polyamine analogue-induced programmed cell death.

N1-ethyl-N11-[(cyclopropyl)methyl]-4,8,-diazaundecane (CPENSpm) is a polyamine analogue that represents a new class of antitumor agents that demonstrate phenotype-specific cytotoxic activity. However, the precise mechanism of its selective cytotoxic activity is not known. CPENSpm treatment results in the superinduction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT) in sensitive cell types and has been demonstrated to induce programmed cell death (PCD). The catalysis of polyamines by the SSAT/polyamine oxidase (PAO) pathway produces H2O2 as one product, suggesting that PCD produced by CPENSpm may be, in part, due to oxidative stress as a result of H2O2 production. In the sensitive human nonsmall cell line H157, the coaddition of catalase significantly reduces high molecular weight (HMW) DNA (>/=50 kb) and nuclear fragmentation. Important to note, specific inhibition of PAO by N,N'-bis(2, 3-butadienyl)-1,4-butane-diamine results in a significant reduction of the formation of HMW DNA and nuclear fragmentation. In contrast, the coaddition of catalase or PAO inhibitor has no effect on reducing HMW DNA fragmentation induced by N1-ethyl-N11-[(cycloheptyl)methyl]-4,8,-diazaundecane, which does not induce SSAT and does not deplete intracellular polyamines. These results strongly suggest that H2O2 production by PAO has a role in CPENSpm cytotoxicity in sensitive cells via PCD and demonstrate a potential basis for differential sensitivity to this promising new class of antineoplastic agents. Furthermore, the data suggest a general mechanism by which, under certain stimuli, cells can commit suicide through catabolism of the ubiquitous intracellular polyamines.

Isolation of a polyamine transport deficient cell line from the human non-small cell lung carcinoma line NCI H157.

In an effort to study the mechanism underlying the observed phenotype-specific response of human lung cancer cell lines to a polyamine analogue, N1,N12-bis(ethyl)spermine(BESpm), we have isolated a BESpm resistant cell line from the BESpm-sensitive large cell lung carcinoma line NCI H157. The mutant line exhibits identical growth rates in the presence or absence of the analogue. However, the overall growth of mutant cells reaches stationary phase earlier than that of the parental cells. In contrast to the parental cells, where a superinduction of spermidine/spermine N1-acetyltransferase (SSAT) is associated with BESpm toxicity, treatment of this resistant line with BESpm did not induce SSAT mRNA or enzyme activity. BESpm treatment was not effective in depleting the intracellular polyamine pools and very low intracellular BESpm levels were detected. This BESpm resistance is not mediated by multidrug resistance (MDR) protein, since these cells maintain their sensitivity to the antineoplastic agent adriamycin. Treatment of these cells with methylglyoxal bis(guanylhydrazone) (MGBG), an AdoMetDC inhibitor which enters cell using polyamine transport system, shows no inhibition of cell growth. Our data suggest that these mutant cells are deficient in polyamine transport. Consistent with this hypothesis, exogenous polyamines did not prevent difluoromethylornithine (DFMO) induced growth inhibition in the mutant cells.