Isolation and characterization of 5-carbamoylmethyluridine and 5-carbamoylmethyl-2-thiouridine from human urine

Two new modified uracil nucleosides, 5-carbamoylmethyuridine (ncm5U, I) and 5-carbamoylmethyl-2-thiouridine (ncm5s2U, II) were isolated from a 24 hr collection of a normal human urine. The structures were assigned on the basis of UV, NMR and mass spectral data and confirmed by comparison of the spectral data and HPLC mobilities with those of authentic samples. On the basis of experimental data it appears possible that 5-carbamoylmethyl-2-thio-uridine (ncm5s2U, II) may be a degradation product produced from a labile precursor by the chemical treatments during the isolation procedure. However, the other nucleoside (ncm5U,I) certainly appears to be of metabolic origin and was also found in the urines of one chronic myelogenous leukemia and one lung carcinoma patient. Abbreviations used are: tRNA-transfer ribonucleic acid, TMS-trimethylsilyl, RP-HPLC--reverse phase high performance liquid chromatography, EI--electron impact, cm5U-5-carboxymethyluridine, mcm5U-5-methoxycarbonylmethyluridine, cm5s2U-5-carboxymethyl-2-thiouridine, mcm5s2U-5-methoxycarbonylmethyl-2-thiouridine, t6A-9-beta-D-ribofuranosyl-[N(purin-6-yl)carbamoyl]-1-threonine, C-cytidine, acp3u-3-(3-amino-3-carboxypropyl)uridine, AICR-aminoimidazole carboxamide riboside, alpha-4-PCNR & beta-4-PCNR-9-alpha-D-(or beta-D)-ribofuranosyl-pyridin-4-one-3-carboxamide, H x 7R-7-beta-D-ribofuranosyl hypoxanthine, m3U-3-methyluridine, m1I-1-methylinosine, m1G-1-methylguanosine, DI-5'-deoxyinosine, dms5OA-5'-deoxy-5'-methylthioadenosine sulfoxide, m2(2)G-N2-dimethylguanosine, psi-psi-uridine, A-adenosine, I-inosine, CML-chronic myelogenous leukemia mam5s2U-5-methylaminomethyl-2-thiouridine, ncm5U-5-carbamoylmethyluridine, ncm5s2U-5-carbamoylmethyl-2-thiouridine, UV-ultraviolet, NMR-nuclear magnetic resonance, HPLC-high performance liquid chromatography, GC-MS-gas chromatography-mass spectrometry.     

The role of wobble uridine modifications in +1 translational frameshifting in eukaryotes

In Saccharomyces cerevisiae, 11 out of 42 tRNA species contain 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), 5-methoxycarbonylmethyluridine (mcm⁵U), 5-carbamoylmethyluridine (ncm⁵U) or 5-carbamoylmethyl-2′-O-methyluridine (ncm⁵Um) nucleosides in the anticodon at the wobble position (U₃₄). Earlier we showed that mutants unable to form the side chain at position 5 (ncm⁵ or mcm⁵) or lacking sulphur at position 2 (s²) of U₃₄ result in pleiotropic phenotypes, which are all suppressed by overexpression of hypomodified tRNAs. This observation suggests that the observed phenotypes are due to inefficient reading of cognate codons or an increased frameshifting. The latter may be caused by a ternary complex (aminoacyl-tRNA*eEF1A*GTP) with a modification deficient tRNA inefficiently being accepted to the ribosomal A-site and thereby allowing an increased peptidyl-tRNA slippage and thus a frameshift error. In this study, we have investigated the role of wobble uridine modifications in reading frame maintenance, using either the Renilla/Firefly luciferase bicistronic reporter system or a modified Ty1 frameshifting site in a HIS4A::lacZ reporter system. We here show that the presence of mcm⁵ and s² side groups at wobble uridines are important for reading frame maintenance and thus the aforementioned mutant phenotypes might partly be due to frameshift errors.     

Mammalian ALKBH8 Possesses tRNA Methyltransferase Activity Required for the Biogenesis of Multiple Wobble Uridine Modifications Implicated in Translational Decoding

Uridines in the wobble position of tRNA are almost invariably modified. Modifications can increase the efficiency of codon reading, but they also prevent mistranslation by limiting wobbling. In mammals, several tRNAs have 5-methoxycarbonylmethyluridine (mcm⁵U) or derivatives thereof in the wobble position. Through analysis of tRNA from Alkbh8^−/− mice, we show here that ALKBH8 is a tRNA methyltransferase required for the final step in the biogenesis of mcm⁵U. We also demonstrate that the interaction of ALKBH8 with a small accessory protein, TRM112, is required to form a functional tRNA methyltransferase. Furthermore, prior ALKBH8-mediated methylation is a prerequisite for the thiolation and 2′-O-ribose methylation that form 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U) and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm⁵Um), respectively. Despite the complete loss of all of these uridine modifications, Alkbh8^−/− mice appear normal. However, the selenocysteine-specific tRNA (tRNA^Sec) is aberrantly modified in the Alkbh8^−/− mice, and for the selenoprotein Gpx1, we indeed observed reduced recoding of the UGA stop codon to selenocysteine.tRNAs are frequently modified at the wobble uridine, a feature that is believed to either promote or restrict wobbling depending on the type of modification. In the case of eukaryotes, the functions of wobble uridine modifications have been studied in the greatest detail in Saccharomyces cerevisiae. Here, the modifications 5-methoxycarbonylmethyluridine (mcm⁵U), 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U), and 5-carbamoylmethyluridine (ncm⁵U) or its 2′-O-ribose-methylated form, ncm⁵Um, are found in 11 out of 13 wobble uridine-containing tRNAs (22). mcm⁵U and mcm⁵s²U are mostly found in “split” codon boxes, where the pyrimidine- and purine-ending codons encode different amino acids, while ncm⁵U is found in “family” codon boxes, where all four codons encode a single amino acid. Early reports based on in vitro experiments suggested that wobble nucleosides, such as mcm⁵U, ncm⁵U, and their derivatives, may restrict wobbling (17, 37, 45), but the results of a recent comprehensive study performed in vivo in S. cerevisiae show that such modifications can improve the reading both of the cognate, A-ending codons and of the wobble, G-ending codons (22). This may suggest that the primary role of these modified nucleosides is to improve translational efficiency rather than to restrict wobbling.The characterization of wobble uridine modifications in higher eukaryotes is very limited, and little is known about the enzymes that introduce them. In mammals, mcm⁵s²U has been found in the wobble position of tRNA^Glu(UUC), tRNA^Lys(UUU), and tRNA^Arg(UCU) (40). Unlike yeast, mammals possess a specialized tRNA that is responsible for recoding the UGA stop codon to insert the 21st amino acid, selenocysteine (Sec). The mammalian tRNA^Sec population consists of two subpopulations containing either mcm⁵U or the ribose-methylated derivative mcm⁵Um in the wobble position. Interestingly, ribose methylation of mcm⁵U in tRNA^Sec appears to have a role in regulating selenoprotein synthesis, as the expression of some selenoproteins, such as glutathione peroxidase 1 (Gpx1), appears to be promoted by mcm⁵Um-containing tRNA^Sec (5, 7, 9, 32).Some years ago, the Escherichia coli AlkB protein was found to be a 2-oxoglutarate- and iron-dependent dioxygenase capable of demethylating the lesions 1-methyladenosine and 3-methylcytosine in DNA (13, 42). Multicellular organisms generally possess several different AlkB homologues (ALKBH), and bioinformatics analysis has identified eight different mammalian ALKBH proteins, denoted ALKBH1 to ALKBH8 in humans and Alkbh1 to Alkbh8 in mice, as well as the somewhat-less-related, obesity-associated FTO protein (2, 16, 30). Among the ALKBH proteins of unknown function, ALKBH8 is the only one containing additional annotated protein domains. Here, the AlkB domain is localized between an N-terminal RNA recognition motif (RRM) and a C-terminal methyltransferase (MT) domain. Interestingly, the MT domain has sequence homology to the S. cerevisiae tRNA methyltransferase Trm9, which has been shown to catalyze the methyl esterification of modified wobble uridine (U34) residues of tRNA^Arg and tRNA^Glu, resulting in the formation of mcm⁵U and mcm⁵s²U, respectively (23, 43). Until recently, human ALKBH8 was incorrectly annotated in the protein sequence database, and another human protein, KIAA1456, has been designated the human Trm9 homologue (3, 23).We have generated for this study Alkbh8-targeted mice that lack exons critical for both the MT and AlkB activities of Alkbh8. The mice did not display any overt phenotype, but tRNA from these mice was completely devoid of mcm⁵U, mcm⁵s²U, and mcm⁵Um, and the relevant tRNA isoacceptors instead contained the acid form 5-carboxymethyluridine (cm⁵U) and/or the amide forms ncm⁵U/ncm⁵s²U. Furthermore, we show that recombinant ALKBH8 and TRM112 form a heterodimeric complex capable of catalyzing the methyl esterification of cm⁵U and cm⁵s²U to mcm⁵U and mcm⁵s²U, respectively. In agreement with the involvement of mcm⁵Um in selenoprotein synthesis, we observed a reduced level of Gpx1 in the Alkbh8^−/− mice, and tRNA^Sec from these mice showed a reduced ability to decode the UGA stop codon to Sec.     

Pre-steady-state kinetic study on the formation of Compound I and II of ligninase

The reaction between ligninase and hydrogen peroxide yielding Compound I has been investigated using a stopped-flow rapid-scan spectrophotometer. The optical absorption spectrum of Compound I appears different to that reported by Andrawis, A. et al. (1987) and Renganathan, V. and Gold, M.H. (1986), in that the Soret-maximum is at 401 nm rather than 408 nm. The second-order rate constant (4.2·10⁵ M⁻¹·s⁻¹) for the formation of Compound I was independent of pH (pH 3.0–6.0). In the absence of external electron donors, Compound I decayed to Compound II with a half-life of 5–10 s at pH 3.1. The rate of this reaction was not affected by the H₂O₂ concentration used. In the presence of either veratryl alcohol or ferrocyanide, Compound II was rapidly generated. With ferrocyanide, the second-order rate constant increased from 1.9·10⁴ M⁻¹·s⁻¹ to 6.8·10⁶ M⁻¹·s⁻¹ when the pH was lowered from 6.0 to 3.1. With veratryl alcohol as an electron donor, the second-order rate constant for the formation of Compound II increased from 7.0·10³ M⁻¹·s⁻¹ at pH 6.0 to 1.0·10⁵ M⁻¹·s⁻¹ at pH 4.5. At lower pH values the rate of Compound II formation no longer followed an exponential relationship and the steady-state spectral properties differed to those recorded in the presence of ferrocyanide. Our data support a model of enzyme catalysis in which veratryl alcohol is oxidized in one-electron steps and strengthen the view that veratryl alcohol oxidation involves a substrate-modified Compound II intermediate which is rapidly reduced to the native enzyme.     

A novel proton transfer salt of 2-amino-6-sulfamoylbenzothiazole and its metal complexes: the evaluation of their inhibition effects on human cytosolic carbonic anhydrases

A novel proton transfer compound (SMHABT)⁺(HDPC)^? (1) obtained from 2-amino-6-sulfamoylbenzothiazole (SMABT) and 2,6-pyridinedicarboxylic acid (H₂DPC) and its Fe(III), Co(II), Ni(II) complexes (2–4), and Fe(II) complex of SMABT (5) have been prepared and characterized by spectroscopic techniques. Additionally, single crystal X-ray diffraction techniques were applied to complexes (2–4). All complexes (2–4) have distorted octahedral conformations and the structure of 5 might be proposed as octahedral according to spectral and analytical results. All compounds, including acetazolamide (AAZ) as the control compound, were also evaluated for their in vitro inhibition effects on human hCA I and hCA II for their hydratase and esterase activities. The synthesized compounds have remarkable inhibitory activities on hCA I and hCA II. Especially, the inhibition potentials of the salt and the metal complexes (1–5) are comparable with AAZ. Inhibition data have been analyzed by using a one-way analysis of variance for multiple comparisons (p?相似文献   

Synthesis,characterization and in vitro inhibition of metal complexes of pyrazole based sulfonamide on human erythrocyte carbonic anhydrase isozymes I and II

Sulfonamides represent an important class of biologically active compounds. A sulfonamide possessing carbonic anhydrase (CA) inhibitory properties obtained from a pyrazole based sulfonamide, ethyl 1-(3-nitrophenyl)-5-phenyl-3-((5-sulfamoyl-1,3,4-thiadiazol-2-yl)carbamoyl)-1H-pyrazole-4-carboxylate (1), and its metal complexes with the Ni(II) for (2), Cu(II) for (3) and Zn(II) for (4) have been synthesized. The structures of metal complexes (2–4) were established on the basis of their elemental analysis, ¹H NMR, IR, UV–Vis and MS spectral data. The inhibition of two human carbonic anhydrase (hCA, EC 4.2.1.1) isoenzymes I and II, with 1 and synthesized complexes (2–4) and acetazolamide (AAZ) as a control compound was investigated in vitro by using the hydratase and esterase assays. The complexes 2, 3 and 4 showed inhibition constant in the range 0.1460–0.3930?µM for hCA-I and 0.0740–0.0980?µM for hCA-II, and they had effective more inhibitory activity on hCA-I and hCA-II than corresponding free ligand 1 and than AAZ.     

Elongator complex influences telomeric gene silencing and DNA damage response by its role in wobble uridine tRNA modification

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm⁵U₃₄), 5-methoxycarbonylmethyluridine (mcm⁵U₃₄), and 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA^Lys _{s² UUU}, tRNA^Gln _{s² UUG}, and tRNA^Glu _{s² UUC}, which in a wild-type background contain the mcm⁵s²U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U₃₄. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm⁵s²U nucleoside in tRNA^Lys _mcm⁵s²UUU, tRNA^Gln _mcm⁵s²UUG, and tRNA^Glu _mcm⁵s²UUC. These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U₃₄ are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.     

The reactivity of myeloperoxidase compound I formed with hypochlorous acid

Abstract

The reaction of human myeloperoxidase (MPO) with hypochlorous acid (HOCl) was investigated by conventional stopped-flow spectroscopy at pH 5, 7, and 9. In the reaction of MPO with HOCl, compound I is formed. Its formation is strongly dependent on pH. HOCl (rather than OCl^-) reacts with the unprotonated enzyme in its ferric state. Apparent second-order rate constants were determined to be 8.1×10⁷ M^-1s^-1 (pH 5), 2.0×10⁸ M^-1s^-1 (pH 7) and 2.0×10⁶ M^-1s^-1 (pH 9) at 15°C. Furthermore, the kinetics and spectra of the reactions of halides and thiocyanate and of physiologically relevant one-electron donors (ascorbate, nitrite, tyrosine and hydrogen peroxide) with this compound I were investigated using the sequential-mixing technique. The results show conclusively that the redox intermediates formed upon addition of either hydrogen peroxide or hypochlorous acid to native MPO exhibit the same spectral features and reactivities and thus are identical. In stopped-flow investigations, the MPO/HOCl system has some advantage since: (i) in contrast to H₂O₂, HOCl cannot function as a one-electron donor of compound I; and (ii) MPO can easily be prevented from cycling by addition of methionine as HOCl scavenger. As a consequence, the observed absorbance changes are bigger and errors in data analysis are smaller.     

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

5-Taurinomethyluridine (τm⁵U) and 5-taurinomethyl-2-thiouridine (τm⁵s²U) are located at the wobble position of human mitochondrial (hmt) tRNA^Leu(UUR) and tRNA^Lys, respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm⁵U and τm⁵s²U into 17-mers related to the sequence of the anticodon arms hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys, respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF₃) are compatible with the blockage of the canonical monomeric units. The synthesis of τm⁵s²U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.     

Synthesis and characterization of some metal complexes of a proton transfer salt,and their inhibition studies on carbonic anhydrase isozymes and the evaluation of the results by statistical analysis

Abstract

A novel proton transfer compound (HMeOABT)?⁺?(HDPC)^? (1) and its Fe(III), Co(II), Ni(II) and Cu(II) complexes (2–5) have been prepared and characterized by spectroscopic techniques. Complex 4 has distorted octahedral conformation revealed by single crystal X-ray diffraction method. Structures of the other complexes might be proposed as octahedral according to experimental data. All compounds were also evaluated for their in vitro inhibition effects on hCA I and II for their hydratase and esterase activities. Although there is no inhibition for hydratase activities, all compounds have inhibited the esterase activities of hCA I and II. Data have been analyzed by using a one-way analysis of variance. The comparison of the inhibition studies of 1–5 to parent compounds indicates that 1–5 have superior inhibitory effects. The inhibition effects of 2–5 are also compared to inhibitory properties of the metal complexes of MeOABT and H2DPC, revealing an improved transfection profile.     

Recruitment of divalent metal ions by incorporation of 4-thio-2′-deoxythymidine or 4-thio-2′-deoxyuridine into DNA

The modified nucleosides 4-thio-2-deoxyuridine (s⁴dU) and 4-thio-2-deoxythymidine (s⁴dT) are incorporated into dinucleosides, and s⁴dT is incorporated into a DNA hairpin loop to provide divalent metal ion binding sites. Binding of two different metal ions to these sites is studied, including Cd(II) as an NMR spectroscopy probe and Cu(II) as a reactive metal ion for DNA cleavage. Binding of Cd(II) to 4-thiouridine (s⁴U) and s⁴dT nucleosides, s⁴dU- and s⁴dT-containing dinucleosides, and a hairpin loop oligonucleotide containing s⁴dT is monitored by following the change in UV-vis absorbance of the thionucleosides at 340 nm and 21 °C in solutions containing 20.0–40 mM buffer, 1.00 M NaCl, and 15.0 mM BaCl₂. Cd(II) binds to the N(3) deprotonated form of s⁴dT with a binding constant (K=1.1×10⁴ M^–1) that is similar to that for Cd(II) binding to d(Tps⁴T) (K=9.2×10³ M^–1). Apparent binding constants (K_app) at pH 7.7 of Cd(II) to dinucleosides d(Gps⁴T), d(s⁴TpG), and d(Gps⁴U) are similar to those of their respective nucleosides s⁴U and s⁴dT, suggesting that neither the phosphate diester nor the second nucleoside has a major effect on Cd(II) binding. Binding of Cd(II) to s⁴U and d(Gps⁴U) is studied by use of ¹¹³Cd NMR and ¹H NMR spectroscopy, respectively. Binding strength and stoichiometry of the Cd(II) complex with d(Gps⁴U) as studied by ¹H NMR spectroscopy are similar to that obtained by UV-vis spectroscopy. Cd(II) binds strongly to s⁴dT in the loop portion of a DNA hairpin loop (K_app=2.7×10³ M^–1 at pH 7.7). However, the hairpin loop is moderately destabilized by Cd(II) binding, with a decrease in T_m of 14 °C in the presence of 10.0 mM Cd(II) as determined by optical melting experiments. Cu(II) oxidizes s⁴dT to form the disulfide of s⁴dT, limiting the usefulness of further studies with Cu(II).Electronic Supplementary Material Supplementary material is available in the online version of this article at .Abbreviations s⁴dU 4-thio-2-deoxyuridine - s⁴dT 4-thio-2-deoxythymidine - s⁴U 4-thiouridine     

Transfer RNA Bound to MnmH Protein Is Enriched with Geranylated tRNA – A Possible Intermediate in Its Selenation?

The wobble nucleoside 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U) is present in bacterial tRNAs specific for Lys and Glu and 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U) in tRNA specific for Gln. The sulfur of (c)mnm⁵s²U may be exchanged by selenium (Se)–a reaction catalyzed by the selenophosphate-dependent tRNA 2-selenouridine synthase encoded by the mnmH (ybbB, selU, sufY) gene. The MnmH protein has a rhodanese domain containing one catalytic Cys (C97) and a P-loop domain containing a Walker A motif, which is a potential nucleotide binding site. We have earlier isolated a mutant of Salmonella enterica, serovar Typhimurium with an alteration in the rhodanese domain of the MnmH protein (G67E) mediating the formation of modified nucleosides having a geranyl (ge)-group (C₁₀H₁₇-fragment) attached to the s² group of mnm⁵s²U and of cmnm⁵s²U in tRNA. To further characterize the structural requirements to increase the geranylation activity, we here report the analysis of 39 independently isolated mutants catalyzing the formation of mnm⁵ges²U. All these mutants have amino acid substitutions in the rhodanese domain demonstrating that this domain is pivotal to increase the geranylation activity. The wild type form of MnmH⁺ also possesses geranyltransferase activity in vitro although only a small amount of the geranyl derivatives of (c)mnm⁵s²U is detected in vivo. The selenation activity in vivo has an absolute requirement for the catalytic Cys97 in the rhodanese domain whereas the geranylation activity does not. Clearly, MnmH has two distinct enzymatic activities for which the rhodanese domain is pivotal. An intact Walker motif in the P-loop domain is required for the geranylation activity implying that it is the binding site for geranylpyrophosphate (GePP), which is the donor molecule in vitro in the geranyltransfer reaction. Purified MnmH from wild type and from the MnmH(G67E) mutant have bound tRNA, which is enriched with geranylated tRNA. This in conjunction with earlier published data, suggests that this bound geranylated tRNA may be an intermediate in the selenation of the tRNA.     

State adaptations in the cyanobacterium Synechcoccus 6301 (PCC): Dependence on light intensity or spectral composition?

A profile of high light to intense self-shading conditions was constructed using a white light source and cultures of the cyanobacterium Synechococcus 6301; this profile approximates to a natural self-shading gradient of decreasing light intensity and PS II/PS I excitation ratio. Samples of S.6301 were placed along this profile and allowed to state adapt. To separate the effects of light intensity and wavelength on state adaptation, samples were also placed in a shade profile produced by a white light source and neutral density filters. After adaptation, samples were fixed in their resulting state by the addition of glutaraldehyde, and fluorescence measurements were made at 35° C or –160 °C. It is concluded:

1. Under conditions of deep shade (<5 μmol m^?2s^?1 PAR) and weak shade (>200 μmol m^?2s^?1 PAR), cells adapt to a low PS II fluorescence state (state 2); in moderate shade (20–60 μmol m^?2s^?1PAR) cells adapt to a high PS II fluorescence state (state 1). We suggest these findings provide evidence for the operation of different factors on the control of state adaptations in cyanobacteria; one set operates at low light and another at high light intensities.
2. Under conditions of self-shading, there is little evidence to support the contention that state adaptations in cyanobacteria are produced by wavelength-dependent changes in the PS II/PS I excitation ratio, instead, it appaers they are produced by changes in the intensity of incident irradiation.
3. The observed fluorescence changes do not appear to involve major changes in the phycobilisome sensitisation of PS II and PS I. Instead, it appears that these changes are effected by alterations in Φ^F of PS II (i.e. changes in PS II excitation density caused by alterations in the rate constants controlling spillover to PS I, photochemistry, fluorescence emission or thermal deactivation.

     

Metal-based carboxamide-derived compounds endowed with antibacterial and antifungal activity

Abstract

A series of three bioactive thiourea (carboxamide) derivatives, N-(dipropylcarbamothioyl)-thiophene-2-carboxamide (L¹), N-(dipropylcarbamothioyl)-5-methylthiophene-2-carboxamide (L²) and 5-bromo-N-(dipropylcarbamothioyl)furan-2-carboxamide (L³) and their cobalt(II), copper(II), nickel(II) and zinc(II) complexes (1)–(12) have been synthesized and characterized by their IR,¹H-NMR spectroscopy, mass spectrometry and elemental analysis data. The Crystal structure of one of the ligand, N-(dipropylcarbamothioyl)thiophene-2-carboxamide (L¹) and its nickel(II) and copper(II) complexes were determined from single crystal X-ray diffraction data. All the ligands and metal(II) complexes have been subjected to in vitro antibacterial and antifungal activity against six bacterial species (Escherichia coli. Shigella flexneri. Pseudomonas aeruginosa. Salmonella typhi. Staphylococcus aureus and Bacillus subtilis) and for antifungal activity against six fungal strains (Trichophyton longifusus. Candida albicans. Aspergillus flavus. Microsporum canis. Fusarium solani and Candida glabrata). The in vitro antibacterial and antifungal bioactivity data showed the metal(II) complexes to be more potent than the parent ligands against one or more bacterial and fungal strains.     

Isolation and Partial Characterization of Four Host-specific Toxins of Helminthosporium maydis (Race T)

Helminthosporium maydis, race T, produces four host-specific toxins in culture. These have been designated toxins I, II, III, and IV. A method for isolation and purification of the four toxins is presented, and the criteria of purity of preparations of toxins I, II, and III are given. Toxins I and II are chemically similar and yield the same molecular ion when subjected to mass spectrometry, while toxin III appears to be a glycoside of a compound related to toxins I and II. Toxins I, II, and III can be biologically derived from ¹⁴C-mevalonic acid or ¹⁴C-acetate, permitting preparation of ¹⁴C-labeled toxins. Some chemical, spectral, and chromatographic properties of toxins I, II, and III are presented, and these data are discussed relative to the possible structure of the three compounds. In addition, four host-specific toxins have been isolated from corn infected with H. maydis (race T). These toxins are recovered in the same fractions as toxins I, II, III, and IV using the isolation procedure described here. Three of the toxins isolated from infected corn cannot be distinguished from toxins I, II, and III on the basis of infrared spectra or chromatographic mobility.     

Synthesis and characterization of a proton transfer salt between 2,6-pyridinedicarboxylic acid and 2-aminobenzothiazole,and its complexes and their inhibition studies on carbonic anhydrase isoenzymes

Abstract

A novel proton transfer compound (HABT)⁺(Hdipic)^? (1) obtained from ABT and H₂dipic and its metal complexes (2–5) have been prepared and characterized by spectroscopic techniques. Single crystal X-ray diffraction method has also been applied to 2 and 5. While complex 2 has a distorted octahedral conformation, 5 exhibits a distorted square pyramidal structure. The structures of 3 and 4 might be proposed as octahedral according to experimental data. All compounds were also evaluated for their in vitro inhibition effects on hCA I and II for their hydratase and esterase activities. Although there is no inhibition for hydratase activities, all compounds have inhibited the esterase activities of hCA I and II. The comparison of the inhibition studies of 1–5 to parent compounds indicates that 1–5 have superior inhibitory effects. The inhibition effects of 2–5 are also compared to inhibitory properties of the metal complexes of ABT and H₂dipic, revealing an improved transfection profile.     

Oxidation of tryptophan by redox intermediates of myeloperoxidase and inhibition of hypochlorous acid production

Abstract

The neutrophil enzyme myeloperoxidase catalyzes the oxidation of tyrosine to tyrosyl radicals, which cross-link to proteins and initiate lipid peroxidation. Tryptophan is present in plasma at about the same concentration as tyrosine and has a similar one-electron reduction potential. In this investigation, we have determined the ability of myeloperoxidase to catalyze the oxidation of tryptophan to assess whether or not this reaction may contribute to oxidative stress at sites of inflammation. We show that tryptophan is a poor substrate for myeloperoxidase because, even though it reacts rapidly with compound I (k_I 2.1×10⁶ M^-1s^-1), it reacts sluggishly with compound II (k_II 7 M^-1s^-1). Tryptophan reversibly inhibited production of hypochlorous acid by purified myeloperoxidase by converting the enzyme to a mixture of compound II and compound III. It gave 50% inhibition (I₅₀) at a concentration of 2 µM. In contrast, it was an ineffective inhibitor of hypochlorous acid production by human neutrophils (I₅₀ 80 µM) unless superoxide dismutase was present (I₅₀ 5 µM). We propose that compound I of myeloperoxidase will oxidize tryptophan at sites of inflammation. Enzyme turnover will result from the reaction of superoxide or tyrosine with compound II. Thus, tryptophan radicals are potential candidates for exacerbating oxidative stress during inflammation.     

Assay of both activities of the bifunctional tRNA-modifying enzyme MnmC reveals a kinetic basis for selective full modification of cmnm5s2U to mnm5s2U

Transfer RNA (tRNA) contains a number of complex ‘hypermodified’ nucleosides that are essential for a number of genetic processes. Intermediate forms of these nucleosides are rarely found in tRNA despite the fact that modification is not generally a complete process. We propose that the modification machinery is tuned into an efficient ‘assembly line’ that performs the modification steps at similar, or sequentially increasing, rates to avoid build-up of possibly deleterious intermediates. To investigate this concept, we measured steady-state kinetics for the final two steps of the biosynthesis of the mnm⁵s²U nucleoside in Escherichia coli tRNA^Glu, which are both catalysed by the bifunctional MnmC enzyme. High-performance liquid chromatography-based assays using selectively under-modified tRNA substrates gave a K_m value of 600 nM and k_cat 0.34 s⁻¹ for the first step, and K_m 70 nM and k_cat 0.31 s⁻¹ for the second step. These values show that the second reaction occurs faster than the first reaction, or at a similar rate at very high substrate concentrations. This result indicates that the enzyme is kinetically tuned to produce fully modified mnm⁵(s²)U while avoiding build-up of the nm⁵(s²)U intermediate. The assay method developed here represents a general approach for the comparative analysis of tRNA-modifying enzymes.     

Metal based drugs: design,synthesis and in-vitro antimicrobial screening of Co(II), Ni(II), Cu(II) and Zn(II) complexes with some new carboxamide derived compounds: crystal structures of N-[ethyl(propan-2-yl)carbamothioyl]thiophene-2-carboxamide and its copper(II) complex

A new series of compounds derived from thiophene-2-carboxamide were synthesized and characterized by IR, ¹H-NMR and ¹³C-NMR, mass spectrometry and elemental analysis. These compounds were further used to prepare their Co(II), Ni(II), Cu(II) and Zn(II) metal complexes. All metal(II) complexes were air and moisture stable. Physical, spectral and analytical data have shown the Ni(II) and Cu(II) complexes to exhibit distorted square-planar and Co(II) and Zn(II) complexes tetrahedral geometries. The ligand (L¹) and its Cu(II) complex were characterized by the single-crystal X-ray diffraction method. All the ligands and their metal(II) complexes were screened for their in-vitro antimicrobial activity. The antibacterial and antifungal bioactivity data showed that the metal(II) complexes were found to be more potent than the parent ligands against one or more bacterial and fungal strains.