Amino Acid Replacements Resulting from Suppression and Missense Reversion of a Chain-Terminator Mutation in Neurospora

The Neurospora crassa super-suppressor mutation, ssu-1, suppresses the auxotrophic phenotype of the mutant am(17) by inserting tyrosine at residue 313 of NADP-specific glutamate dehydrogenase, a position occupied in the wild type by glutamate. Two classes of am(17) revertants due to further mutation within the am gene have, respectively, tyrosine and leucine at residue 313. These replacements are consistent with a chain-terminating codon in am(17) of either the amber (UAG) or the ochre type (UAA), but are inconsistent with UGA. The Leu³¹³ and Tyr³¹³ variants of the enzyme have effective activity but are grossly different from the wild type in Michaelis constants (especially for ammonium) and heat stabilities at two different pH values. They show smaller but significant differences in these respects from each other.     

Glutamine up-regulates MAPK phosphatase-1 induction via activation of Ca2+→ ERK cascade pathway

The non-essential amino acid L-glutamine (Gln) displays potent anti-inflammatory activity by deactivating p38 mitogen activating protein kinase and cytosolic phospholipase A₂ via induction of MAPK phosphatase-1 (MKP-1) in an extracellular signal-regulated kinase (ERK)-dependent way. In this study, the mechanism of Gln-mediated ERK-dependency in MKP-1 induction was investigated. Gln increased ERK phosphorylation and activity, and phosphorylations of Ras, c-Raf, and MEK, located in the upstream pathway of ERK, in response to lipopolysaccharidein vitro and in vivo. Gln-induced dose-dependent transient increases in intracellular calcium ([Ca²⁺]_i) in MHS macrophage cells. Ionomycin increased [Ca²⁺]_i and activation of Ras → ERK pathway, and MKP-1 induction, in the presence, but not in the absence, of LPS. The Gln-induced pathways involving Ca²⁺→ MKP-1 induction were abrogated by a calcium blocker. Besides Gln, other amino acids including L-phenylalanine and l-cysteine (Cys) also induced Ca²⁺ response, activation of Ras → ERK, and MKP-1 induction, albeit to a lesser degree. Gln and Cys were comparable in suppression against 2, 4-dinitrofluorobenzene-induced contact dermatitis. Gln-mediated, but not Cys-mediated, suppression was abolished by MKP-1 small interfering RNA. These data indicate that Gln induces MKP-1 by activating Ca²⁺→ ERK pathway, which plays a key role in suppression of inflammatory reactions.     

Recombination in the lambda repressor gene: evidence that very short patch (VSP) mismatch correction restores a specific sequence

Summary The mutation am6 in the cI gene of bacteriophage is identified as a CT transition in a 5CC _T ^A GG sequence. In four-factor crosses of am6 with nearby mutations in cI, the frequencies of cI⁺ recombinants are much higher than expected from the physical distances. A very short patch (VSP) mismatch repair system is presumed to recognize am6/am ⁺ mispairs in the heteroduplexes that accompany recombination between the outside markers. Mutation am6 is corrected to am ⁺; correction of am ⁺ to am6 was not detected. Clear-plaque mutation 1-1 in cI is a TC transition in a 5CTTGG sequence, resulting in the sequence 5CC _T ^A GG. When 1-1 was crossed with nearby mutations in gene cI, there were no excess cI⁺ recombinants, which would result from repair of CCTGG (1-1) to CTTGG (cI⁺). However, in crosses of cI⁺ phages with mutation 1-1, there was an excess of cI^- recombinants, indicating that cI⁺ was repaired to 1-1. Preferential repair does not require adenine or cytosine methylation: when repairing a mismatch, the VSP repair system apparently identifies specific mispaired bases by sequence alone.     

Study of the oxidation of alkanes in their mixtures with oxygen and air under the action of a pulsed volume nanosecond discharge

The slow oxidation of alkanes (from methane to hexane) in their stoichiometric mixtures with oxygen or air under the action of nanosecond pulsed discharges was investigated. The discharges were excited in a tube of diameter 5 cm and length of 20 cm by 25-ns voltage pulses with an amplitude of 10 kV and a repetition rate of 40 Hz. The initial pressure in the mixture was varied in the range 0.76–10.1 torr. The current, the electric field strength, and the power deposited in a discharge were measured with a nanosecond time resolution. In time-resolved and time-integrated measurements, the intensities of the following bands were determined: CO ₂ ⁺ (B²Σ → X²Π, δv=0), CH(A²Δ, v′=0 → X²Π, v″=0), OH(A²Σ, v′=0 → X²Π, v″=0), CO(B¹Σ, v′=0 → A¹Π, v″=2), NO(A²Σ → X²Π, δv=3), N₂(C³Π, v′=1 → B³Π, v″=7), N₂(B³Π, v′=6 → A³Σ, v″=3), and N ₂ ⁺ (B²Σ, v′=0 → X²Σ, v″=2). The methane concentration was measured from the absorption of He-Ne laser radiation. Based on the results of optical measurements, the times of the complete oxidation of hydrocarbons were determined.     

Effect of hydrogen bonds on the energetics of macromolecules in the course of electron transfer

For a model system consisting of a bacteriochlorophyll dimer (P) and a primary quinone with the nearest environment (Q_A), which are the electron donor and acceptor in the recombination reaction in the Rhodobacter spheroides reaction center, the energies of states P⁺Q _A ^? and PQ_A have been calculated at several stable conformations of Q_A that differ in the positions of the proton involved in the hydrogen bond. It is shown that the position of the proton has a considerable influence on the energy of vertical transition P⁺Q _A ^? → PQ_A.     

Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence

The changes in intrinsic fluorescence parameters induced by thermal transitions in proteins are developed on the background of the common thermal fluorescence quenching due to an activation of collisions between the excited chromophores and neighbouring quenching groups. Two methods of separation of the thermai quenching and conformational change contributions to the temperature dependence of the fluorescence parameters are presented. One is based on the use of the linearity of the plots of the reciprocal fluorescence quantum yield, l/q, vs. the t/η ratio (T. temperature; η, solvent viscosity) for native proteins containing a single fluorescing chromophore (T.L. Bushueva, E.P. Busel and E.A. Burstein, Biochim. Biophys. Acta 534 (1978) 141). The other method is based on a consideration of the phase plots for the tryptophan fluorescence of proteins (fluorescence intensity at a fixed wavelength vs. intensity at any other fixed wavelength). The methods have been used for a study of the thermal transitions in Mg²⁺-loaded whiting parvalbumin (tryptophan fluorescence), Mg²⁺-loaded pike parvalbumins pI 4.2 (tyrosine fluorescence) and pI 5.0 (phenylalanine fluorescence), and Ca²⁺-loaded bovine α-lactalbumin (tryptophan fluorescence). The thermal denaturation curves for the parvalbumins show two-stepped character. The main change of the protein conformation occurs at the higher temperature step. Comparison of the fluorescence data with the microcalorimetry results shows that the maxima of the asymmetric heat sorption peaks for pike parvalbumins correlate with the mid-points of the higher temperature steps of the fluorimetric curves.     

Conformation of the common purine (β) ribosides in solution: further evidence for a correlation between N-S state of the ribose moiety and syn-anti equilibrium

The solution conformations of adenosine, guanosine and inosine in liquid ND₃ have been determined by NMR. Comparison of the Karplus analysis of the proton HR spectra of the ribose moiety obtained in this solvent with the data from aqueous solutions of A and I proves that the conformations of the nucleosides are very similar in both liquids. From the analysis of the vicinal coupling constants of the ring protons it has been deduced that the S state C(2′)-endo is slightly preferred. The mole fraction in S approximates 0.6 for all three nucleosides. C-13 relaxation measurements have been applied in the determination of the correlation times for rotational diffusion. Only at temperatures below −40‡ C is the pseudorotation of the furanoside ring slowed down sufficiently for it not to contribute to the measured relaxation rates. From NOE studies and T¹ measurements on the individual protons it is derived that the N, C(3′)-endo, form of the ribose is correlated with an anti conformation of the base (Y≈210‡ to 220‡) and the S, C(2′)-endo, form of the ribose with a syn conformation of the base (Y≈30‡ to 50‡). The glycosyl torsion angles derived for the two conformations of A, G, and I are equal within the limits of accuracy.     

Specific features of the kinetics of H2-O2-O2(a 1Δ g ) mixtures: I. formation and quenching of electronically and vibrationally excited (A′) molecules in H2-O2-O2(A 1Δ g ) mixtures at a temperature of 300 K

Available data on the kinetic processes in H₂-O₂-O₂(a ¹Δ _g ) mixtures are analyzed theoretically, and the ranges in which the rate constants of these processes can vary are determined. The processes of energy transformation in an O₂(a ¹Δ _g )-H₂-H-HO₂ system in the approximations of the fast and slow (in comparison with the vibrational relaxation time of the HO₂ radical) quenching of the electronically excited state are considered. The experiments on the quenching of singlet delta oxygen (SDO) molecules O₂(a ¹Δ _g ) excited in a microwave discharge at a temperature of 300 K and pressure of 6 Torr in a lean hydrogen-oxygen mixture are simulated (by a lean fuel mixture is meant a mixture in which the ratio of the fuel to the oxidizer mass fraction is less than the stoichiometric ratio, which is 2: 1 for a hydrogen-oxygen mixture). It is shown that, over the experimental observation times, the SDO quenching frequency depends on the concentration of molecular hydrogen, the residual odd oxygen fraction in the gas flow, and the ratio between the rate constants of kinetic processes involving HO₂ and HO₂* radicals. Simulations of the microwave discharge and the transport of excited oxygen along the drift tube make it possible to determine the residual odd oxygen concentration in the gas flow. Recommendations on the choice of the rate constants for the O₂(a ¹Δ _g ) + HO₂)A″, v₃″ = 0) ? O₂ + HO₂*(A′, v₃′ = 1), HO₂*(A′v₃′ ≤ 1) + O₂(a ¹Δ _g ) → HO₂*(A′,v₃′ ≥ 6) + O₂, and HO₂*(A′,v₃′ ≤ 1) + O₂(a ¹Δ _g ) → H + O₂ + O₂ processes are given. It is shown that, in the case of slow quenching in a H₂-O₂-O₂(a ¹Δ _g ) mixture at a low temperature, the intensity of hydrogen oxidation is enhanced due to the reaction + HO₂*(A′,v₃′ ≤ 1) + O₂(¹Δ) → H + O₂ + O₂.     

New Mutational Variants of Neurospora Nadp-Specific Glutamate Dehydrogenase

The am locus of Neurospora codes for NADP-dependent glutamate dehydrogenase (GDH). Four new am mutants that produced mutationally altered GDH have been characterized. Mutant am₁₁₉ is a CRM-negative, complementing mutant that maps between am₂ and am₁. The other three mutants are CRM formers that produce varieties of GDH that can be activated by glutamate or succinate. The GDH of am₁₃₀ and am₁₃₁ is similar in terms of activation properties to that of am₃. The GDH of am₁₂₂ requires very high concentrations of dicarboxylate for activity. The mutation in am₁₃₀ maps between am₁₄ and am₂ and resulted in a replacement at residue 75 of the GDH (pro → ser). The mutation in am₁₂₂ maps near am₁₁ and apparently resulted in the replacement of the tryptophan residue at position 389 with an unknown amino acid. The mutation in am₁₃₁ maps between am₂ and am₁.     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

On the mechanism of bromouracil-induced mutagenesis

Bromouracil (BU)-induced mutagenesis of λC17 am o8 phage, in relation to the recombination systems of phage (red) or bacteria (rec), was studied. The mutations investigated were am → am⁺. For efficient BU-induced mutagenesis, red or recA genes as well as bacterial lex gene functions, known to be involved in UV-induced mutagenesis, were required. This suggests a common mechanism or some common step(s) in UV- and BU-induced mutagenesis. Moreover, a several-fold increase was observed in the number of mutants induced by BU in the excision-repair-deficient strain (uvrA), implying that incorporated BU induces some premutational lesions that are recognized and repaired by excision-repair enzymes. A hypothesis on the possible mechanism of BU-induced mutagenesis is proposed, which assumes a common mechanism for UV- and BU-induced mutagenesis, involving recombination repair processes. Incorporation of a tautomeric or ionized form of BU is considered only as a premutational change in DNA activating the dark-repair mechanisms in cells. The observation that BU enhances the frequency of recombination in λ phages also supports teh idea that recombination processes are involved in BU-induced mutagenesis.     

Complexes of hydroxamates. Part 1. Interaction of methioninehydroxamic acid with iron(III) in aqueous solution

The binding affinity of Fe(III) to methioninehydroxamate (MX) has been studied spectrophotometrically at I=0.15 M NaCl and T=25 °C. Equilibrium data have been assessed by the program SQUAD(II) in the wavelength range 400–550 nm and in the pH range 1.5–5.0. Five formation constants were determined for the species Fe(MX)(H)³⁺, Fe(MX)²⁺, Fe(MX)₂(H)₂³⁺, Fe(MX)₂(H)²⁺ and Fe₂(MX)₃³⁺. The stopped-flow kinetic data studied at 470 nm and in the pH range 1.0–3.0 is collectively expressed by the following rate equation at a given pH Rate=(A + BT_MX)T_MX where T_MX=the analytical connection of MX and the parameters A and B are both functions of pH in the range 1.7–3.0, but only A in the range 1.2– 1.7. A proposed mechanism was discussed, based on the equilibrium study, where the role of the chloro species of Fe(OH)²⁺ and Fe(OH)₂⁺ in the complex formation of Fe(III) with MX has been emphasized. Correlation of the results with pertinent systems has also been discussed.     

Formation and decay of radical-pair state P+I− in Chloroflexus aurantiacus reaction centers

We have examined the room temperature kinetics of the absorption changes associated with the formation of state P⁺I⁻ (P⁺BPh⁻) and its subsequent decay to state P⁺Q⁻_A in reaction centers from Chloroflexus aurantiacus. Our data, acquired using 30-ps excitation flashes, strongly suggest that formation of P⁺I⁻ (P⁺BPh⁻) takes longer in Chloroflexus than in reaction centers from Rhodopseudomonas sphaeroides. The reduction of the photoactive bacteriopheophytin (BPh) could take as long as 13 ps. Absorption changes different from those due to P⁺I⁻ are observed early in the excitation flash, but the detailed identity of the transient remains unclear. We also find that the kinetics observed subsequent to P⁺I⁻ formation differ with detection wavelength. The time constant measured in the anion band (I⁻) at 655 nm is 324 ± 20 ps and probably reflects the rate of electron transfer from I⁻ (BPh⁻) to Q_A. However, the kinetics measured in the BPh ground-state absorption bands are slightly longer: 365 ± 19 and 367 ± 21 ps at 538 and 760 nm, respectively. At 810 nm, a wavelength normally associated with the monomeric bacteriochlorophyll (BChl) in the Chloroflexus reaction center, a slightly faster (281 ± 19 ps) time constant is observed. This detection-wavelength dependence of the kinetics is similar to that observed recently in Rps. sphaeroides reaction centers. Comparison of these results suggests that the kinetics observed in the ground-state absorption bands of the BPhs and BChls in Chloroflexus may contain contributions from readjustments of the pigments and/or protein in response to the charge separation process.     

Germ Line Variants of Human N-Methylpurine DNA Glycosylase Show Impaired DNA Repair Activity and Facilitate 1,N6-Ethenoadenine-induced Mutations

Human N-methylpurine DNA glycosylase (hMPG) initiates base excision repair of a number of structurally diverse purine bases including 1,N⁶-ethenoadenine, hypoxanthine, and alkylation adducts in DNA. Genetic studies discovered at least eight validated non-synonymous single nucleotide polymorphisms (nsSNPs) of the hMPG gene in human populations that result in specific single amino acid substitutions. In this study, we tested the functional consequences of these nsSNPs of hMPG. Our results showed that two specific arginine residues, Arg-141 and Arg-120, are important for the activity of hMPG as the germ line variants R120C and R141Q had reduced enzymatic activity in vitro as well as in mammalian cells. Expression of these two variants in mammalian cells lacking endogenous MPG also showed an increase in mutations and sensitivity to an alkylating agent compared with the WT hMPG. Real time binding experiments by surface plasmon resonance spectroscopy suggested that these variants have substantial reduction in the equilibrium dissociation constant of binding (K_D) of hMPG toward 1,N⁶-ethenoadenine-containing oligonucleotide (ϵA-DNA). Pre-steady-state kinetic studies showed that the substitutions at arginine residues affected the turnover of the enzyme significantly under multiple turnover condition. Surface plasmon resonance spectroscopy further showed that both variants had significantly decreased nonspecific (undamaged) DNA binding. Molecular modeling suggested that R141Q substitution may have resulted in a direct loss of the salt bridge between ϵA-DNA and hMPG, whereas R120C substitution redistributed, at a distance, the interactions among residues in the catalytic pocket. Together our results suggest that individuals carrying R120C and R141Q MPG variants may be at risk for genomic instability and associated diseases as a consequence.     

Activation of Asparaginyl Endopeptidase Leads to Tau Hyperphosphorylation in Alzheimer Disease

Neurofibrillary pathology of abnormally hyperphosphorylated Tau is a key lesion of Alzheimer disease and other tauopathies, and its density in the brain directly correlates with dementia. The phosphorylation of Tau is regulated by protein phosphatase 2A, which in turn is regulated by inhibitor 2, I₂^PP2A. In acidic conditions such as generated by brain ischemia and hypoxia, especially in association with hyperglycemia as in diabetes, I₂^PP2A is cleaved by asparaginyl endopeptidase at Asn-175 into the N-terminal fragment (I_2NTF) and the C-terminal fragment (I_2CTF). Both I_2NTF and I_2CTF are known to bind to the catalytic subunit of protein phosphatase 2A and inhibit its activity. Here we show that the level of activated asparaginyl endopeptidase is significantly increased, and this enzyme and I₂^PP2A translocate, respectively, from neuronal lysosomes and nucleus to the cytoplasm where they interact and are associated with hyperphosphorylated Tau in Alzheimer disease brain. Asparaginyl endopeptidase from Alzheimer disease brain could cleave GST-I₂^PP2A, except when I₂^PP2A was mutated at the cleavage site Asn-175 to Gln. Finally, an induction of acidosis by treatment with kainic acid or pH 6.0 medium activated asparaginyl endopeptidase and consequently produced the cleavage of I₂^PP2A, inhibition of protein phosphatase 2A, and hyperphosphorylation of Tau, and the knockdown of asparaginyl endopeptidase with siRNA abolished this pathway in SH-SY5Y cells. These findings suggest the involvement of brain acidosis in the etiopathogenesis of Alzheimer disease, and asparaginyl endopeptidase-I₂^PP2A-protein phosphatase 2A-Tau hyperphosphorylation pathway as a therapeutic target.     

The properties and reactions of haemoglobin F(1) and their bearing on the dissociation equilibrium of haemoglobin

1. Hb-F_I, with the previously proposed structure α^A₂γ^Fγ^AcF, would be an exception to the general hypothesis that haemoglobins are in mobile equilibrium with their sub-units at all pH values. However, studies presented in this paper suggest that this is not so. 2. Gel-filtration and sedimentation analyses show that Hb-F_I dissociates at acid pH and gives only the usual types of hybrids in recombination experiments. When self-hybridized, Hb-F_I is the main haemoglobin species re-formed, although small but increasing amounts of Hb-F_II appear on prolonged exposure to acid. 3. Exchange experiments with isotopically labelled Hb-F_II and unlabelled Hb-F_I show no exchange of sub-units at neutral pH or after brief exposure to acid pH. Under equilibrium conditions at acid pH non-α^A-chains do not exchange, although α^A-chains equilibrate completely between the two species. 4. These results indicate that Hb-F_I does not contain γ^F-chains and its possible structure is discussed on this basis. Since the dissociation properties of Hb-F_I are not markedly different from those of Hb-A or Hb-F_II it is concluded that Hb-F_I, like other haemoglobins, is an equilibrium system.     

Formation of the parental replicative form DNA of bacteriophage phi-X174 and initial events in its replication

Intracellular φX174 DNA was studied under a variety of conditions that prevent the replication of the parental replicative form DNA. These conditions included treatment with 150 μg of chloramphenicol per ml., the use of the rep₃⁻ mutation of the host cell, amber mutation (am 8) in the viral gene responsible for RF replication (gene A) ^† and combinations thereof. In all cases the majority of the parental RF was in the covalently closed form (RFI). The relative amount of RF with a discontinuity in one strand (RFII) in these cases was between 2 and 10% of the total RF and independent of the multiplicity of infection. The only exception was seen in infections of rep₃ cells with φX am 3 (a mutant in the lysis gene, gene E, used as a wild-type representative). In this case a fairly constant absolute amount of RFII (1 to 4 per cell), independent of the multiplicity of infection, was formed, consisting almost exclusively of a closed complementary and an open parental viral strand. Since the formation of this type of RFII was dependent on protein synthesis and the presence of the product of φX gene A, it is concluded that the discontinuity in the parental viral strand represents the result of the action of the gene A product on the DNA. Possible mechanisms for the mode of action of the gene A product are discussed.     

Theoretical studies on the conformations of methyl glycopyranosides

The σ-charges on various atoms of methyl glycosides have been computed by using the MO-LCAO method of Del Re. The potential and free energies of methyl aldohexopyranosides and methyl aldopentopyranosides in their C1(d) and 1C(d) conformations have been calculated. Minimization of the energies of these conformations has been studied by suitably tilting the axial C-C and C-O bonds. Considerable release of strain is achieved when tilts of 4.5 and 3° are given to the axial hydroxymethyl and hydroxyl groups, respectively, that are involved in Hassel-Ottar effect. A tilt of 3° is also found necessary for the axial OMe group involved in the Hassel-Ottar effect. The calculated free-energy values are in accord with experimental ones, after adding a value of 0.8 kcal.mole^?1 for the anomeric effect of -OMe group. These studies predict that all of the methyl aldohexopyranosides, except methyl α-d- and methyl β-d-idopyranosides, favour the C1 conformation. On the other hand, the energy calculations also predict that, of the eight methyl aldopentopyranosides studied, only methyl α-d- and methyl β-d-xylopyranosides and methyl α-d -ribopyranoside favour the C1(d) conformation; for the other pentopyranosides, considerable amounts of both C1(d) and 1C(d) conformations are present in the equilibrium mixture. The calculated values of the percentage of α-anomer present in the equilibrium mixture agree fairly well with those obtained experimentally.     

The uncatalysed and the copper(II) promoted hydrolysis of 4-nitrophenyl L-leucinate

The uncatalysed hydrolysis of 4-nitrophenyl L-leucinate has been studied in detail over a range of pH and temperature at I=0.1 M (KNO₃). Base hydrolysis of the ester is strongly promoted by copper(II) ions. Rate constants have been obtained for the following reactions (where EH⁺ is the N- protonated ester and E is the free base form) EH⁺ + OH⁻ → products E + OH⁻ → products E + H₂O → products CuE²⁺ + OH⁻ → products Base hydrolysis of the copper(II) complex CuE²⁺ is 3.8 × 10⁵ times faster than that of E and 75 times faster than that of EH⁺ at 25 °C and I=0.1 M. Activation parameters for these reactions have been determined and possible mechanisms are considered.