Open timelike curves violate Heisenberg's uncertainty principle.

Toy models for quantum evolution in the presence of closed timelike curves have gained attention in the recent literature due to the strange effects they predict. The circuits that give rise to these effects appear quite abstract and contrived, as they require nontrivial interactions between the future and past that lead to infinitely recursive equations. We consider the special case in which there is no interaction inside the closed timelike curve, referred to as an open timelike curve (OTC), for which the only local effect is to increase the time elapsed by a clock carried by the system. Remarkably, circuits with access to OTCs are shown to violate Heisenberg's uncertainty principle, allowing perfect state discrimination and perfect cloning of coherent states. The model is extended to wave packets and smoothly recovers standard quantum mechanics in an appropriate physical limit. The analogy with general relativistic time dilation suggests that OTCs provide a novel alternative to existing proposals for the behavior of quantum systems under gravity.

Bacterial manganese reduction and growth with manganese oxide as the sole electron acceptor.

Microbes that couple growth to the reduction of manganese could play an important role in the biogeochemistry of certain anaerobic environments. Such a bacterium, Alteromonas putrefaciens MR-1, couples its growth to the reduction of manganese oxides only under anaerobic conditions. The characteristics of this reduction are consistent with a biological, and not an indirect chemical, reduction of manganese, which suggest that this bacterium uses manganic oxide as a terminal electron acceptor. It can also utilize a large number of other compounds as terminal electron acceptors; this versatility could provide a distinct advantage in environments where electron-acceptor concentrations may vary.

p38γ MAPK is required for inflammation-associated colon tumorigenesis.

Chronic inflammation has long been considered to causatively link to colon cancer development. However, signal transduction pathways involved remain largely unidentified. Here, we report that p38γ mitogen-activated protein kinase mediates inflammatory signaling to promote colon tumorigenesis. Inflammation activates p38γ in mouse colon tissues and intestinal epithelial cell-specific p38γ knockout (KO) attenuates colitis and inhibits pro-inflammatory cytokine expression. Significantly, p38γ KO inhibits tumorigenesis in a colitis-associated mouse model. The specific p38γ pharmacological inhibitor pirfenidone also suppresses pro-inflammatory cytokine expression and colon tumorigenesis. The tumor-promoting activity of epithelial p38γ was further demonstrated by xenograft studies. In addition, p38γ is required for ß-catenin/Wnt activities and p38γ stimulates Wnt transcription by phosphorylating ß-catenin at Ser605. These results show that p38γ activation links inflammation and colon tumorigenesis. Targeting p38γ may be a novel strategy for colon cancer prevention and treatment.

Outer membrane cytochromes of Shewanella putrefaciens MR-1: spectral analysis, and purification of the 83-kDa c-type cytochrome.

The metal-reducing bacterium Shewanella putrefaciens MR-1 is known to localize a majority of its membrane-bound cytochromes to its outer membrane when grown under anaerobic conditions. In this study, pyridine hemochrome spectra confirmed that these outer membrane cytochromes are c-type, and electrophoretic data demonstrated the presence of four distinct outer membrane cytochromes, with apparent molecular masses of 150, 83, 65, and 53 kDa. Fourth-order derivative analysis of 77 K spectra of the outer membrane revealed four spectrally distinct c-type hemes, with peaks at 545.4, 548.0, 550.6, and 552.6 nm. Outer membrane cytochromes in the reduced state were rapidly re-oxidized by oxidized iron and manganese, which have previously been shown to serve as electron acceptors for anaerobic respiration in this bacterium. The 83-kDa outer membrane cytochrome was purified and a specific polyclonal antibody was generated against this protein. Western blot analysis demonstrated that the vast majority of this protein was localized to the outer membrane and an intermediate density membrane fraction of similar composition. Its levels, but not its subcellular distribution, were somewhat influenced by the electron acceptor used to support anaerobic growth, with levels higher in fumarate-grown cells relative to iron(III)- or trimethylamine N-oxide-grown cells. Its specific content in cells grown under aerobic conditions was only 14% of that of fumarate-grown cells, suggesting that a switch to anaerobic conditions significantly increases the de novo synthesis of this outer membrane cytochrome.

Isolation and sequence of omcA, a gene encoding a decaheme outer membrane cytochrome c of Shewanella putrefaciens MR-1, and detection of omcA homologs in other strains of S. putrefaciens.

The sequence of the omcA gene, which encodes a decaheme cytochrome c that is localized to the outer membrane (OM) of Shewanella putrefaciens MR-1, was determined. The 2202 bp nucleotide sequence of omcA encodes for 734 amino acids with a predicted molecular protein mass of 78.6 kDa. Comparison with the amino-terminal sequence of the mature protein suggests the presence of a hydrophobic leader sequence which is cleaved during translocation of the protein to the OM. This leader sequence has a lipoprotein consensus sequence for signal peptidase II at the cleavage site. The predicted mature protein is comprised of 708 amino acids with a predicted molecular mass of 75.8 kDa, but the addition of ten covalently attached heme c groups and covalent lipid modification to the amino-terminal cysteine increases the predicted mass to 82.7 kDa. This is consistent with its apparent mass of 83 kDa in SDS-PAGE gels. The predicted amino acid sequence for the OmcA protein shows no significant homology to known proteins. A RNA of approx. 2300 bases that hybridizes to the omcA gene was detected in anaerobically grown MR-1 cells. The size of this transcript is similar to the coding region of the omcA gene, suggesting that it is not part of a multicistronic operon. Similar to MR-1, four other strains of S. putrefaciens were all found to localize a majority of their membrane-bound cytochromes to the OM when grown under anaerobic conditions, and all contained an OM cytochrome of similar size to OmcA. In two of these strains, MR-4 and MR-8, a homolog of omcA was identified by RT-PCR and Southern blotting using primers and probes specific for omcA of MR-1. Western blot analysis using a polyclonal antibody to OmcA was similarly positive in strains MR-4 and MR-8. Partial nucleotide sequence analysis of these homologs demonstrated 74-77% predicted amino acid homology with OmcA of MR-1. In contrast, strains MR-30 and MR-42 tested negative for omcA homologs by Southern and Northern blots, RT-PCR, and Western blots.

The conserved Trp114 residue of thioredoxin reductase 1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death.

The selenoprotein thioredoxin reductase 1 (TrxR1) has several key roles in cellular redox systems and reductive pathways. Here we discovered that an evolutionarily conserved and surface-exposed tryptophan residue of the enzyme (Trp114) is excessively reactive to oxidation and exerts regulatory functions. The results indicate that it serves as an electron relay communicating with the FAD moiety of the enzyme, and, when oxidized, it facilitates oligomerization of TrxR1 into tetramers and higher multimers of dimers. A covalent link can also be formed between two oxidized Trp114 residues of two subunits from two separate TrxR1 dimers, as found both in cell extracts and in a crystal structure of tetrameric TrxR1. Formation of covalently linked TrxR1 subunits became exaggerated in cells on treatment with the pro-oxidant p53-reactivating anticancer compound RITA, in direct correlation with triggering of a cell death that could be prevented by antioxidant treatment. These results collectively suggest that Trp114 of TrxR1 serves a function reminiscent of an irreversible sensor for excessive oxidation, thereby presenting a previously unrecognized level of regulation of TrxR1 function in relation to cellular redox state and cell death induction.

Nitric oxide inhibits aldosterone synthesis by a guanylyl cyclase-independent effect.

To investigate the mechanism of nitric oxide (NO) inhibition of aldosterone release, this study compared the effects of type A natriuretic peptide and heat-stable enterotoxin to a nitric oxide donor, deta nonoate, on cGMP production and angiotensin II-stimulated aldosterone synthesis ill primary cultures of bovine adrenal zona glomerulosa cells. Type A natriuretic peptide (10(-10)-10(-6) M) and deta nonoate (10(-6)-10(-3) M) stimulated concentration-related increases in cGMP production. Heat-stable enterotoxin (10(-6) M) failed to stimulate cGMP synthesis in zona glomerulosa cells. Type A natriuretic peptide and deta nonoate attenuated angiotensin II-stimulated aldosterone production over the same concentration range that stimulated cGMP production. Heat-stable enterotoxin (10(-6) M) was without effect on aldosterone release. To further test the hypothesis that cGMP mediated the inhibition of aldosterone synthesis, the selective inhibitor of soluble guanylyl cyclase, 1H-(1,2,4)oxadiazolo [4,3-a]quinoxalin-1-one (ODQ) was used. ODQ pretreatment (10(-5) M) completely prevented deta nonoate-stimulated cGMP production without altering the inhibitory effect of deta nonoate on angiotensin II-stimulated steroidogenesis. Consistent with its selectivity for inhibiting soluble guanylyl cyclase, ODQ did not block type A natriuretic peptide-stimulated cGMP synthesis or type A natriuretic peptide inhibition of steroidogenesis. Deta nonoate completely blocked 25-hydroxycholesterol- and progesterone-stimulated aldosterone synthesis in zona glomerulosa cells and inhibited the conversion of 25-hydroxycholesterol to pregnenolone in mitochondrial fractions from bovine adrenal cortex. Deta nonoate-derived NO gave an absorbance maximum of the mitochondrial cytochrome P450 of 453 nm and inhibited the absorbance at 450 nm caused by carbon monoxide binding to the enzyme. These results suggest that deta nonoate reduces steroidogenesis independent of guanylyl cyclase activation and that NO has a direct effect to inhibit the activity of cytochrome P450, probably by binding to the heme groups of the cytochrome.

Post-embedding direct immunogold detection of a protein antigen in insect tissue.

An immunocytochemical study was performed to localize the site of hemoglobin synthesis in larvae and embryos of the insect Chironomus thummi. Heterologous antisera specific for C. thummi hemoglobins were prepared using a highly purified hemoglobin extract. Tissue samples were prepared by glutaraldehyde fixation of whole dissected larvae or whole embryos without osmium tetroxide postfixation. Epoxy resin-embedded thin sections were labeled with a direct immunogold conjugate. Immune label was localized in rough endoplasmic reticulum of fat body cells of larvae. Immune label was also present in embryos. The technique, which did not require chemical etching of the sections, proved very useful for demonstration of this intracellular protein antigen.

Antibodies to a synthetic peptide that react with flavin-containing monooxygenase (HLFMO3) in human hepatic microsomes.

Flavin-containing monooxygenases (FMOs) catalyze the oxidation of a diverse array of xenobiotic compounds. The purpose of this investigation was to develop a specific immunological probe to human hepatic flavin-containing monooxygenase (HLFMO3). An oligopetide corresponding to amino acid residues 257-270 of HLFMO3 was coupled to keyhole limpet hemocyanin (KLH) through the sulfhydryl group of a cysteine residue added to the amino-terminus of the peptide. This peptide-KLH conjugate was used to generate a polyclonal antibody. The resulting immunoglobulin showed specific Western blot reactivity with HLFMO3 protein in human hepatic microsomes, the same protein that is recognized by a polyclonal antibody directed against macaque liver FMO. These findings demonstrate that an antibody directed against a synthetic peptide derived from HLFMO3 can be easily produced in large quantities and used in studies for the immunodetection and immunoquantification of HLFMO3. This is also the first antipeptide antibody directed against an FMO of any species.

Cytochrome b(5) plays a key role in human microsomal chromium(VI) reduction.

The reduction of chromium(VI) to Cr(III) results in the formation of reactive intermediates that contribute to the cytotoxicity, genotoxicity, and carcinogenicity of Cr(VI)-containing compounds. Previous studies suggest that human microsomal Cr(VI) reduction likely proceeds through cytochrome b(5). In order to better understand Cr(VI) toxicity in humans, the role of cytochrome b(5) in combination with P450 reductase was examined in the reductive transformation of Cr(VI). Proteoliposomes containing human recombinant cytochrome b(5) and P450 reductase were constructed. The ability of P450 reductase to mediate efficient electron transfer from NADPH to cytochrome b(5) was confirmed by spectral analysis. The NADPH-dependent Cr(VI) reduction rate mediated by proteoliposomes was then compared to that of human microsomes. When these rates were normalized to equivalent cytochrome b(5) concentrations, the NADPH-dependent Cr(VI) reduction rates mediated by human microsomes were essentially identical to those for proteoliposomes containing cytochrome b(5) plus P450 reductase. Proteoliposomes containing only P450 reductase or cytochrome b(5) exhibited poor Cr(VI) reducing capabilities. Since it had been previously shown that trace amounts of iron (Fe) could dramatically stimulate microsomal Cr(VI) reduction, the ability of Fe to stimulate Cr(VI) reduction by proteoliposomes was examined. Both ferric chloride (FeCl(3)) and ferric adenosine-5'-diphosphate (FeADP) were shown to stimulate Cr(VI) reduction; this stimulation could be abolished by the addition of deferoxamine, a specific Fe(III) chelator. The NADPH-dependent reduction rates of various ferric complexes by proteoliposomes were sufficient to account for the increased Cr(VI) reduction rates seen with the addition of FeCl(3) or FeADP. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy as a transient intermediate formed during NADPH-dependent Cr(VI) reduction mediated by proteoliposomes containing cytochrome b(5) and P450 reductase. Overall, cytochrome b(5) in combination with P450 reductase can account for the majority of the NADPH-dependent Cr(VI) reduction seen with human microsomes.

Fracture in mode I using a conserved phase-field model.

We present a continuum phase-field model of crack propagation. It includes a phase-field that is proportional to the mass density and a displacement field that is governed by linear elastic theory. Generic macroscopic crack growth laws emerge naturally from this model. In contrast to classical continuum fracture mechanics simulations, our model avoids numerical front tracking. The added phase-field smooths the sharp interface, enabling us to use equations of motion for the material (grounded in basic physical principles) rather than for the interface (which often are deduced from complicated theories or empirical observations). The interface dynamics thus emerges naturally. In this paper, we look at stationary solutions of the model, mode I fracture, and also discuss numerical issues. We find that the Griffith's threshold underestimates the critical value at which our system fractures due to long wavelength modes excited by the fracture process.

Evaluating molar CYP1A level in fish hepatic microsomes by competitive ELISA using recombinant membrane-free CYP1A standard protein.

Fish cytochrome P4501A (CYP1A) is a widely accepted environmental biomarker, detecting biological effects of several xenobiotic groups present in aquatic environments, when evaluated in target tissues of a biosensor species. However, appropriate utilization of its protein level as a routine environmental diagnostic tool requires evaluation of properly normalized molar levels, mitigating comparison among different laboratories, during a multi-annual time scale and over a variety of tested populations of the biosensor species. A competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of CYP1A of the striped sea bream, Lithognathus mormyrus, using our previously described antibody raised to a trout CYP1A synthetic peptide, and a recombinant L. mormyrus CYP1A as a competitor. The L. mormyrus CYP1A-cDNA was cloned and modified by truncating its 5' hydrophobic membrane anchor, as well as by addition of 4x histidine tag, permitting its partial purification on a nickel-NTA column. The modified cDNA was ligated into the PCWOri+ vector and heterologously produced in Escherichia coli as a cytosolic, membrane-free protein, retaining its immuno-affinity with the anti-CYP1A antibody in the presence of the detergent Triton X-100. The detergent was added to the ELISA solution during the competitive step, rendering the microsomal CYP1A more accessible to the antibody. ELISA components, including coated levels of the modified standard CYP1A, and the concentrations of the Triton X-100, CYP1A-specific antibody, and the range of dissolved CYP1A standard protein, were optimized. Hypothesized immuno-affinity differences between the microsomal and the recombinant CYP1As, and among microsomal samples, as well as assay accuracy, were examined and discussed. This ELISA can serve for more efficient utilization of fish CYP1A as a pollution biomarker, and also as a model for establishing competitive ELISAs aimed at quantification of many different microsomal P450 proteins.

Satisfiability, sequence niches and molecular codes in cellular signalling.

Biological information processing as implemented by regulatory and signalling networks in living cells requires sufficient specificity of molecular interaction to distinguish signals from one another, but much of regulation and signalling involves somewhat fuzzy and promiscuous recognition of molecular sequences and structures, which can leave systems vulnerable to crosstalk. A simple model of biomolecular interactions that reveals both a sharp onset of crosstalk and a fragmentation of the neutral network of viable solutions is examined as more proteins compete for regions of sequence space, revealing intrinsic limits to reliable signalling in the face of promiscuity. These results suggest connections to both phase transitions in constraint satisfaction problems and coding theory bounds on the size of communication codes.

Optimal experimental design in an epidermal growth factor receptor signalling and down-regulation model.

We apply the methods of optimal experimental design to a differential equation model for epidermal growth factor receptor signalling, trafficking and down-regulation. The model incorporates the role of a recently discovered protein complex made up of the E3 ubiquitin ligase, Cbl, the guanine exchange factor (GEF), Cool-1 (beta -Pix) and the Rho family G protein Cdc42. The complex has been suggested to be important in disrupting receptor down-regulation. We demonstrate that the model interactions can accurately reproduce the experimental observations, that they can be used to make predictions with accompanying uncertainties, and that we can apply ideas of optimal experimental design to suggest new experiments that reduce the uncertainty on unmeasurable components of the system.

Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of menaquinone.

Shewanella putrefaciens MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome c. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the cymA gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a cymA knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the cymA transcript and CymA protein was also restored, by complementation of MR1-CYMA with the cymA gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1.

Cell-cycle-specific oscillation in the composition of chromatophore membrane in Rhodospirillum rubrum.

Synchrony in phototrophic cultures of Rhodospirillum rubrum was induced by stationary-phase cycling or by alterations in light intensity. Intracytoplasmic chromatophore membranes were prepared by differential centrifugation. Analysis of the composition of chromatophores obtained from cells at different times indicated that the protein/bacteriochlorophyll a ratio was constant throughout the cell cycle but that the protein/phospholipid ratio oscillated. This cell-cycle-dependent fluctuation in chromatophore membrane composition was reflected in the buoyant densities of the isolated chromatophores.

Enzymatic reduction of chromium(VI) by human hepatic microsomes.

The reduction of chromium(VI) by human hepatic microsomes was investigated. The reduction rates were proportional to the amount of microsomes added and reduction was mediated by an NADPH-dependent enzymatic system which exhibited a Km for chromate of 1.04 +/- 0.18 microM and a Vmax of 5.03 +/- 0.49 nmol/min/mg protein. Relative to incubation under 0% O2, 21% O2 inhibited microsomal Cr(VI) reduction in three individuals by 53, 36 and 37%. Cr(VI) reduction was not inhibited by metyrapone, carbon monoxide, aminopyrine, piperonyl butoxide or chloroform, suggesting that cytochrome P450s did not play a major role. Thallium trichloride (0.13 and 0.26 mM), a known flavoprotein inhibitor, caused a complete inhibition of both Cr(VI) reduction and NADPH:cytochrome P450 (c) reductase activity. A partial inhibition of Cr(VI) reduction was seen in the presence of n-octylamine, which may suggest a possible role for flavin-containing monooxygenase (FMO). Overall, human microsomal Cr(VI) reduction is very different from the P450-mediated microsomal reduction observed in rodents. Specifically, the human system is much less oxygen-sensitive, has a much greater affinity for chromate and is apparently mediated by flavoproteins.

Cloning and sequence of cymA, a gene encoding a tetraheme cytochrome c required for reduction of iron(III), fumarate, and nitrate by Shewanella putrefaciens MR-1.

The cymA gene, which encodes a tetraheme cytochrome c, was cloned from Shewanella putrefaciens MR-1. This gene complemented a mutant which had a TnphoA insertion in cymA and which was deficient in the respiratory reduction of iron(III), nitrate, fumarate, and manganese(IV). The 561-bp nucleotide sequence of cymA encodes a protein of 187 amino acids with a predicted molecular mass of 20.8 kDa. No N-terminal signal sequence was readily apparent; consistent with this, a cytochrome with a size of 21 kDa was detected in the wild type but was absent in the insertional mutant. The cymA gene is transcribed into an mRNA; the major transcript was approximately 790 bases, suggesting that it is not part of a multicistronic operon. This RNA transcript was not detected in the cymA mutant. The CymA protein was found in the cytoplasmic membrane and soluble fraction of MR-1, and it shares partial amino acid sequence homology with multiheme c-type cytochromes from other bacteria. These cytochromes are ostensibly involved in the transfer of electrons from the cytoplasmic membrane to acceptors in the periplasm. The localization of the fumarate and iron(III) reductases to the periplasm and outer membrane of MR-1, respectively, suggests the possibility of a similar electron transfer role for CymA.

Ferric iron reduction-linked growth yields of Shewanella putrefaciens MR-1.

The anaerobic reduction of ferric citrate by Shewanella putrefaciens MR-1 cells was inhibited markedly by p-chloromercuriphenylsulphonate, moderately by potassium cyanide, and to a small extent by 2-heptyl-4-hydroxyquinolone-N-oxide. Iron reduction was accompanied by increases in total cellular protein, with values of 0.33-7.54 g cell protein produced per mol Fe(III) reduced. The growth yields were dependent upon the growth conditions of the inoculum and the initial concentration of Fe(III) citrate in the medium. Specifically, maximum growth yields were obtained when the inoculum was pregrown anaerobically and when the initial Fe(III) citrate concentrations were 5-10 mmol l-1. Lower growth yields were obtained with initial Fe(III) citrate concentrations of 20-30 mmol l-1, suggesting that cell growth was partially inhibited by higher concentrations of Fe(III) or Fe(II). Maximal growth yields were also observed early (6-24 h), after which continued increases in cell protein were minimal.

Isolation and characterization of a transposon mutant of Shewanella putrefaciens MR-1 deficient in fumarate reductase.

A transposon mutant, designated CMTn-3, of Shewanella putrefaciens MR-1 that was deficient in fumarate reduction was isolated and characterized. In contrast to the wild-type, CMTn-3 could not grow anaerobically with fumarate as the electron acceptor, and it lacked benzyl viologen-linked fumarate reductase activity. Consistent with this, CMTn-3 lacked a 65 kDa c-type cytochrome, which is the same size as the fumarate reductase enzyme. CMTn-3 retained the wild-type ability to use nitrate, iron(III), manganese(IV) and trimethylamine N-oxide (TMAO) as terminal electron acceptors. The results indicate that the loss of the fumarate reductase enzyme does not affect other anaerobic electron transport systems in this bacterium.