Quinone reductase inhibitors block SAPK/JNK and NFkappaB pathways and potentiate apoptosis.

A variety of environmental stresses stimulate the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEKK) > stress-activated protein kinase (SAPK)-ERK kinase (SEK) > SAPK/c-Jun NH(2)-terminal kinase (JNK) stress-activated protein kinase cascade and coordinately activate the transcription factor NFkappaB. Mechanisms of stress activation upstream of MEKK1 have not been precisely determined. Redox mechanisms involving sulfhydryls are likely because N-acetyl-cysteine at millimolar concentrations blocks stress signals. Because intracellular sulfhydryl concentrations can be regulated through redox cycling involving reactive quinones (1), we tested the ability of quinone reductase inhibitors to alter stress signaling. Several quinone reductases are inhibited by dicoumarol, a coumarin derivative. Dicoumarol prevented SAPK activation in vivo by chemical cell stressors and also prevented SAPK activation induced by expression of the tumor necrosis factor alpha (TNFalpha) receptor-associated protein TRAF2 but not by expression of truncated active MEKK1. Other coumarin derivatives failed to block SAPK activation, but other inhibitors of quinone reductases, particularly menadione, similarly blocked SAPK activation. Cells deficient in a major quinone reductase, NQO1, displayed hypersensitivity to dicoumarol stress inhibition, whereas SAPK in cells reconstituted with the NQO1 gene displayed relative dicoumarol resistance. Consistent with the proposed role of overlapping upstream signaling cascades in activation of NFkappaB, dicoumarol also blocked NFkappaB activation in primary macrophages stimulated with either lipopolysaccharide or TNFalpha. In addition, dicoumarol strongly potentiated TNFalpha-induced apoptosis in HeLa cells, probably by blocking the anti-apoptotic effect of NFkappaB. The ability of dicoumarol to simultaneously inhibit SAPK and NFkappaB activation and to potentiate apoptotic cell death suggests that SAPK is not an obligate participant in apoptosis. Dicoumarol, currently in clinical use as an oral anticoagulant, represents a potential therapeutic inhibitor of the SAPK and NFkappaB response.

Quinone reductase (NQO1), a sensitive redox indicator, is increased in Alzheimer's disease.

NAD(P)H:quinone oxidoreductase 1 (NQO1), a redox-regulated flavoenzyme, plays a central role in monitoring cellular redox state. NQO1 acts to protect against oxidative stress induced by a variety of metabolic situations, including metabolism of quinones and other xenobiotics, by: (i) functioning as a two electron donor to provide a shunt that competes with the formation of reactive oxygen species; (ii) maintaining reduced coenzyme Q; and (iii) regulating the stress activated kinase pathway. In Alzheimer's disease, while there is abundant evidence for the involvement of oxidative stress, the cause or the consequences are largely unresolved. We suspected that increased NQO1 could signal a major shift in redox balance in Alzheimer's disease and, in this study, found that NQO1 is localized not only to neurofibrillary tangles but also the cytoplasm of hippocampal neurons. By marked contrast, there is very little NQO1 in the same neuronal populations in young and age-matched controls. This novel association of NQO1 further buttresses the nexus of oxidative stress, via free radicals, with selective neuronal vulnerability and also supports a fundamental abnormality in redox balance in Alzheimer's disease.

Fas-induced proteolytic activation and intracellular redistribution of the stress-signaling kinase MEKK1.

The stress-activated protein kinase (SAPK, alternatively JNK) is activated rapidly by cell stress stimuli such as inflammatory cytokines and oxidative stress, and more slowly by the initiation of the apoptotic cell death response by events such as ligation of the Fas protein. Mitogen-activated protein kinase/Erk kinase kinase-1 (MEKK1) is an activator of SAPK, serving as a SAPK-kinase-kinase through intermediate phosphorylation of the SAPK kinase SEK1. By sequencing proteolytic cleavage products of MEKK1, we found that the proapoptotic protease caspase 3 (CPP32) cleaves MEKK1 after residue D68 both in vivo and in vitro. Cleavage of MEKK1 after D68 is blocked by viral and chemical protease inhibitors. Cleavage of MEKK1 at D68 changes the intracellular distribution of the protein from a Triton-insoluble compartment to a Triton-soluble compartment, reflected in a redistribution from a particulate to a diffuse cytoplasmic staining seen by immunofluorescence. Activation of both SAPK and MEKK1 after Fas ligation is prevented by both viral and chemical caspase 3 inhibitors, which in contrast fail to block activation of SAPK by rapidly acting cell stresses. Stress factor-induced SAPK signaling is not dependent on caspase 3 function. We propose that two mechanisms of stress signaling through MEKK1 exist. One is rapid, independent of proteases, and occurs in the particulate Triton-insoluble compartment. The other is more slowly activated and involves liberation of particulate MEKK1 by proteolytic cleavage and activation by caspase 3.

High frequency intragenic recombination during macronuclear development in Tetrahymena thermophila restores the wild-type SerH1 gene.

Macronuclear development in ciliates is characterized by extensive rearrangement of genetic material, including sequence elimination, chromosome fragmentation and telomere addition. Intragenic recombination is a relatively rare, but evolutionarily important phenomenon occurring in mitosis and meiosis in a wide variety of organisms. Here, we show that high frequency intragenic recombination, on the order of 30%, occurs in the developing amitotic macronucleus of the ciliate Tetrahymena thermophila. Such recombination, occurring between two nonsense transition mutations separated by 726 nucleotides, reproducibly restores wild-type expression of the SerH1 surface protein gene, thus mimicking complementation in trans heterozygotes. Recombination must be considered a potentially important aspect of macronuclear development, producing gene combinations not present in the germinal micronucleus.

Regulation of the activity of MEK kinase 1 (MEKK1) by autophosphorylation within the kinase activation domain.

MEK kinase 1 (MEKK1) shares sequence identity with the yeast kinases Ste11 and Byr2, and is capable of phosphorylation and activation of both mitogen-activated protein/extracellular signal-related protein kinase (MAP/ERK) kinase (MEK) and stress-activated protein kinase (SAPK)/ERK kinase (SEK) in vitro. In vivo, however, MEKK1 predominantly activates the SEK/SAPK kinase cascade. Mechanisms of activation of MEKK1 are unclear. We have identified a major site of autophosphorylation (Thr-575) within the 'activation loop' of MEKK1 between the kinase subdomains VII and VIII. Phosphatase treatment of a constitutively active MEKK1 decreased kinase activity by 59%. Dephosphorylated T575 was rapidly re-(auto)phosphorylated by MEKK1. Mutation of T575 to alanine decreased MEKK1 transphosphorylation activity with a SEK substrate to approx. 30% of wild-type. Mutation of a second threonine residue (Thr-587) to alanine eliminated the phosphorylation of MEK or SEK substrate but not autophosphorylation. MEKK1 autophosphorylation is an intramolecular reaction because active MEKK1 cannot transphosphorylate a kinase-inactive MEKK1. Inactive MEKK1 was not phosphorylated on Thr-575 within cells, suggesting that the phosphorylation of Thr-575 in vivo results from autophosphorylation rather than phosphorylation by an upstream kinase. Autoactivation of MEKK1 via autophosphorylation of Thr-575 might be an immediate response to initial kinase activation through non-phosphorylation mechanisms.

High-power picosecond mid-infrared optical parametric amplifier for infrared Raman spectroscopy.

A high-power (50-MW), kilohertz, picosecond, mid-IR optical parametric amplifier that is pumped by an amplified Ti:sapphire laser and also produces a fixed-frequency visible pulse is described. Mid-IR pulse energies of 40-55 microJ with 0.6-0.8-ps durations and 35-cm (-1) bandwidths are reported in the 3650- 2800-cm (-1) range. The combination of picosecond mid-IR and visible pulses is useful for two-color spectroscopies, which require simultaneous time and frequency resolution. To illustrate the above, we present vibrational relaxation data for the polyatomic molecule nitromethane, using time-resolved infrared Raman spectroscopy.

Sequence, codon usage and cysteine periodicity of the SerH1 gene and in the encoded surface protein of Tetrahymena thermophila.

The temperature-regulated SerH1 gene coding for an immunodominant surface glycoprotein (i-Ag H1) of Tetrahymena thermophila has been sequenced. The gene is reproducibly rearranged during macronuclear development and steady state mRNA levels are present at < 36 degrees C. The deduced i-Ag H1 amino acid (aa) sequence is rich in Ser, Thr and Cys, and contains three periods each consisting of 85 aa punctuated by eight Cys with the general formula, CX6CX17CX2CX18CX2CX11CX2CX19 (where X = any aa). Such Cys periodicity is common to ciliate i-Ag. Codon usage in Tt, Paramecium primaurelia and P. tetraurelia i-Ag encoding genes is similar, with approx. 80% A+T in the 3' position which is in marked contrast to the approx. 54% 3' A+T in other ciliate genes.

Activation of stress-activated protein kinase by MEKK1 phosphorylation of its activator SEK1.

A kinase distinct from the MEK activator Raf, termed MEK kinase-1 (MEKK), was originally identified by virtue of its homology to kinases involved in yeast mating signal cascades. Like Raf, MEKK is capable of activating MEK in vitro. High-level expression of MEKK in COS-7 cells or using vaccinia virus vectors also activates MEK and MAPK, indicating that MEKK and Raf provide alternative means of activating the MAPK signalling pathway. We have derived NIH3T3 cell sublines that can be induced to express active MEKK. Here we show that induction of MEKK does not result in the activation of MAPK, but instead stimulates the stress-activated protein kinases (SAPKs) which are identical to a Jun amino-terminal kinase. We find that MEKK regulates a new signalling cascade by phosphorylating an SAPK activator, SEK1 which in turn phosphorylates and activates SAPK.

Rate of phenotypic assortment in Tetrahymena thermophila.

During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf, the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published Rf values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of Rf. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with approximately 45 assorting units, as has been asserted.