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Cloturin: effect on energy-producing processes in Ehrlich ascites and P388 murine leukaemia cells.

The main purpose of the present investigation was to study the effect of cloturin on aerobic glycolysis, endogenous and exogenous respiration and the level of ATP in both Ehrlich ascites carcinoma (EAC) and P388 murine leukaemia cells incubated in vitro. Also its effect on the level of total (T-SH) and non-protein (NP-SH) thiol groups was investigated. A significant inhibition of aerobic glycolysis was found only in P388 cells after 60 min of cloturin action. Cloturin inhibited both endogenous and exogenous respiration of EAC with succinate as substrate. Cloturin decreased the level of ATP after 2 h incubation in both types of tumour cell. The level of NP-SH was decreased more than that of T-SH in both types of cell.

Adenosine Triphosphate↗

Characterization of the O2-induced manganese-containing superoxide dismutase from Bacteroides fragilis.

A manganese-containing superoxide dismutase (MnSOD) has been isolated from extracts of O2-induced Bacteroides fragilis. The enzyme, Mr 43,000, was a dimer composed of noncovalently associated subunits of equal size. A preparation whose specific activity was 1760 U/mg had 1.1 g-atoms Mn, 0.3 g-atoms Fe, and 0.2 g-atoms Zn per mol dimer. Exposing the enzyme to 5 M guanidinium chloride, 20 mM 8-hydroxyquinoline abolished enzymatic activity. Dialysis of the denatured apoprotein in buffer containing either Fe (NH4)2(SO4)2 or MnCl2 restored O2-. scavenging activity. The iron-reconstituted enzyme was inhibited 89% by 2 mM NaN3, similar to other Fe-containing superoxide dismutases. The Mn-reconstituted and native MnSOD were inhibited approximately 50% by 20 mM NaN3. Addition of ZnSO4 to dialysis buffer containing either the iron or manganese salt inhibited restoration of enzymatic activity to the denatured apoprotein. MnSOD migrated as a single protein band coincident with a single superoxide dismutase activity band in 7.5 or 10% acrylamide gels. Isoelectric focusing resulted in a major isozymic form with pI 5.3 and a minor form at pI 5.0. Mixtures of the MnSOD and the iron-containing superoxide (FeSOD), isolated from anaerobically maintained B. fragilis [E. M. Gregory and C. H. Dapper (1983) Arch. Biochem. Biophys. 220, 293-300], migrated as a single band on acrylamide gels and isoelectrically focused to a major protein band (pI 5.3) and a minor band at pI 5.0. The amino acid composition of MnSOD was virtually identical to that of the FeSOD. The data are consistent with synthesis of a single superoxide dismutase apoprotein capable of accepting either Mn or Fe to form the holoenzyme.

Amino Acids↗

Simultaneous N7,O6-binding of guanine to two zinc centers and its possible biological significance.

The reaction of ZnCl(2) with 9-ethylguanine (9-EtGH) produced a novel dinuclear Zn(II) complex, [Zn(2)Cl(4)(H(2)O)(mu-9-EtGH-N7,O6)(9-EtGH-N7), 1. The X-ray structure analysis (monoclinic, P2(1) (No. 4), a = 11.0636(6) A, b = 6.6546(4) A, c = 15.9630(9) A, beta = 101.069(1) degrees, V = 1153.4(1) A(3), Z = 2) revealed that one of the tetrahedrally coordinated Zn(II) atoms binds to the N7 site of 9-EtGH and to the exocyclic O6 atom of another 9-EtGH molecule. The remaining Zn(II) atom binds to the N7 site of the second 9-EtGH moiety.

Binding Sites↗

Transformation and structural discrimination between the neutral {Fe(NO)2}10 dinitrosyliron complexes (DNICs) and the anionic/cationic {Fe(NO)2}9 DNICs.

Reaction of Fe(CO)2(NO)2 and sparteine/tetramethylethylenediamine (TMEDA) in tetrahydrofuran afforded the electron paramagnetic resonance (EPR)-silent, neutral {Fe(NO)2}10 dinitrosyliron complexes (DNICs) [(sparteine)Fe(NO)2] (1) and [(TMEDA)Fe(NO)2] (2), respectively. The stable and isolable anionic {Fe(NO)2}9 DNIC [(S(CH2)3S)Fe(NO)2]- (4), with a bidentate alkylthiolate coordinated to a {Fe(NO)(2)} motif, was prepared by the reaction of [S(CH2)3S]2- and the cationic {Fe(NO)2}9 [(sparteine)Fe(NO)2]+ (3) obtained from the reaction of complex 1 and [NO][BF4] in CH(3)CN. Transformation from the neutral complex 1 to the anionic complex 4 was verified via the cationic complex 3. Here complex 3 acts as an {Fe(NO)2}-donor reagent in the presence of thiolates. The EPR spectra of complexes 3 and 4 exhibit an isotropic signal with g = 2.032 and 2.031 at 298 K, respectively, the characteristic g value of {Fe(NO)2}9 DNICs. On the basis of N-O/Fe-N(O) bond lengths of the single-crystal X-ray structures of the {Fe(NO)2}9/{Fe(NO)2}10 DNICs, the oxidation level of the {Fe(NO)2} core of DNICs can be unambiguously assigned. The mean N-O distances falling in the range of 1.214(6)-1.189(4) A and the Fe-N(O) bond distances in the range of 1.650(7)-1.638(3) A are assigned as the neutral {Fe(NO)(2)}(10) DNICs. In contrast, the mean N-O bond distances ranging from 1.178(3) to 1.160(6) A and the mean Fe-N(O) bond distances ranging from 1.695(3) to 1.661(4) A are assigned as the anionic/neutral/cationic {Fe(NO)2}9 DNICs. In addition, an EPR spectrum in combination with the IR nu(NO) (the relative position of the nu(NO) stretching frequencies and their difference Deltanu(NO)) spectrum may serve as an efficient tool for discrimination of the existence of the anionic/cationic/neutral {Fe(NO)2}9 DNICs and the neutral {Fe(NO)2}10 DNICs.

Anions↗

LR16, a compound with potent effects on the oxygen affinity of hemoglobin, on blood cholesterol, and on low density lipoprotein.

2-[4-(3,4-Dichlorophenylureido)phenoxy]-2-methylpropionic acid, LR16, combines with two symmetrically related sites in the central cavity of deoxyhemoglobin, 20 A away from the binding site of 2,3-bisphosphoglycerate, and acts as an allosteric effector synergistic with 2,3-bisphosphoglycerate. LR16 (1 mM) raises P50, the partial pressure of oxygen needed to achieve half-saturation with oxygen of a hemolysate of human hemoglobin, about 50 times more strongly than 1 mM 2,3-bisphosphoglycerate. Oral administration of LR16 (at small doses that produced no ill effects) to rats that were fed a diet rich in cholesterol caused substantial reductions of total serum cholesterol and low density lipoprotein-cholesterol, while high density lipoprotein-cholesterol remained unchanged.

Animals↗

Dynamics of protozoan and metazoan communities in a full scale wastewater treatment plant by rotating biological contactors.

Performance of a full-scale wastewater treatment plant by rotating biological contactors (RBC) system was monitored during a year by physico-chemical and microbial characterisation. Six points along wastewater treatment were selected in the plant: three points along the water line (influent, sedimentation tank and effluent) and three points along RBC system (RBC1, RBC2 and RBC3). Although a large seasonal change in the values of physico-chemical parameters was observed, operation of the plant was optimal during all year (90% of removal in BOD5 and SS influent content). Microbial characterisation was approached by determining the structure and dynamics of protozoan and metazoan communities. Protozoa were the most abundant in all stages in the plant, heterotrophic flagellates being the most representative group in the water line and ciliates in the RBC system. The same seasonal preference was only observed for heterotrophic flagellates in the water line and green flagellates in the RBC system, both groups having highest abundances in summer and spring, respectively. Identification of ciliated protozoa populations rendered 58 species of ciliates in the plant. Most of these species are typical of aerobic wastewater treatment systems except three of them, which are cited for the first time in this type of ecosystems: Chaenea stricta, Holosticha mancoidea and Oxytricha lanceolata. Along the water line 34 species were identified, and half of them only appeared occasionally (once in all the study), while along the RBC system biofilms 55 species were observed, and the majority appeared permanently in this system. Our results indicate that the type of habitat, rather than the physico-chemical water parameters, was the primary factor in determining the different distribution of protozoan and metazoan communities in the plant. In RBC biofilms, the structure of ciliate protozoa community was found to be quite sensitive to changes in physico-chemical parameters, mainly to organic loading (BOD5) variations.

Amoeba↗

Effect of local change in O2 saturation of hemoglobin on cerebral vasodilation from hypoxia and hypotension.

We tested the effect of certain newly synthesized allosteric modifiers of hemoglobin on the dilation induced by arterial hypoxia, arterial hypotension, and arterial hypercapnia in cerebral arterioles of anesthetized cats equipped with cranial windows for the observation of the cerebral microcirculation. The allosteric modifiers of hemoglobin are isomers of 2-(aryloxy)-2-methylpropionic acid. They shift the oxygen dissociation of hemoglobin to the right, thereby facilitating the local release of oxygen. When these compounds were applied topically by superfusion at a rate of 1 ml/min in a concentration of 0.1 mM, they had no significant effect on baseline arteriolar diameter but reduced significantly the vasodilation from arterial hypoxia and arterial hypotension. They did not influence the vasodilation from arterial hypercapnia. Spectrophotometric measurements of optical densities from pial veins 50-80 microns in diameter indicated that the superfusion with the allosteric compounds reduced hemoglobin oxygen saturation both during room air breathing and during hypoxia. We conclude that the vasodilations from arterial hypoxia and arterial hypotension are mediated by local oxygen-dependent mechanisms. The allosteric modifiers of hemoglobin may be useful as tools in investigating oxygen-dependent mechanisms.

Animals↗

A study of the reactions of H3O+, NO+ and O2+ ions with nine alkoxy alcohols.

Following a selected ion flow tube mass spectrometry (SIFT-MS), analysis of the headspace of a commercial available nail polish remover pad in which gamma-butyrolactone (GBL) and 2-butoxy-1-ethanol were found to be the major volatiles, a study of the reactions of H3O+, NO+ and O2+* ions with nine alkoxy alcohols (R1-O-R2OH) was carried out using selected ion flow tube (SIFT) at a carrier gas (helium) pressure of 9. 3 x 10(1) Pa. Experiments were also performed at various carrier gas pressures (4 x 10(1)-1.1 x 10(2) Pa) for some reactions and under moist air condition. The number and distribution of the hydrates for the product ions were used to identify their structures and to investigate reaction mechanisms. The H3O+ reactions proceed via nascent ion-molecule complex (H3O+ .M)*, then produce R1-O-R2OH. H+, ( R1-O-R2)+, HOR2OH. H+ and R2=O. H+ ions through various channels. Similarly, via a nascent complex (NO+.M)*, NO+. M, (M-H)+ and (M-ROH)+ ions were produced in the NO+ reactions. The collisions between the nascent complexes (H3O+ .M)* and (NO+.M)* with a third body (He, N2 and O2 etc) have influence on the product ion distributions. The O2+ reactions produce mainly dissociative product ions and it is uncertain whether they proceed via the nascent ion-molecule complex (O2+ . M)*. This study provides rate coefficients and product ions in the SIFT-MS database for the analysis of these compounds in normal and humid air samples. It will be further utilised into addiction and drug abuse, respiratory medicine and other research areas using SIFT-MS.

Air Pollutants↗

Emerging awareness of the critical roles of S-phosphocysteine and selenophosphate in biological systems.

S-Phosphocysteine residues in proteins are formed as key intermediates in certain enzyme-catalyzed reactions. In phosphoenolpyruvate (PEP)-dependent carbohydrate transport processes, the phosphoryl group of PEP is transferred sequentially to histidine residues of two cytoplasmic proteins, Enzyme I and HPr, common to all of the PEP-dependent carbohydrate transport systems. The phosphoryl group of HPr then is transferred to an essential histidine and an essential cysteine residue located in the cytoplasmic domains of certain substrate-specific, membrane-bound proteins, the actual transporters. Both the N-phosphohistidine and the S-phosphocysteine residues in these Enzyme II domains are transient catalytic intermediates in the final steps that lead to phosphorylation of the bound carbohydrate substrate. In a different type of biological system, numerous proteins involved in signal transduction pathways are phosphorylated on specific tyrosine residues by various kinases and the effects of these modifications are modulated by a family of protein tyrosine phosphate-specific phosphatases. The mechanism of action of these phosphatases involves the transfer of the phosphoryl group from a phosphotyrosine residue in the protein substrate to an essential ionized cysteine residue in the phosphatase polypeptide forming an S-phosphocysteine residue. Reaction of the latter with water and release of orthophosphate complete the phosphatase-catalyzed reaction. Selenophosphate, formed by phosphorylation of a selenol, is a key selenium donor compound in prokaryotes. This extremely oxygen-labile compound is synthesized from ATP and selenide by selenophosphate synthetase. This novel reactive selenium compound is the selenium donor for selenocysteyl-tRNA biosynthesis and for conversion of 2-thiouridine residues in tRNAs to 2-selenouridine.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

Oxygen radical generation by Maillard compounds.

Amino sugars such as galactosamine are hepatotoxic. It has been verified that toxic hepatitis induced by galactosamine is similar to that of CCl4 poisoning, and that both were inhibited by O2* scavengers. Fructosamine results from the union of glucose with the epsilon-amine of lysine. A test for fructosamine quantification is based on nitroblue tetrazolium (NBT) reduction, in which O2- is involved, the reduction being inhibited in the presence of superoxide dismutase (SOD). Given these facts, we attempted to elucidate if galactosamine and glucosamine reduce NBT and if that reduction is inhibited by SOD. This was confirmed. Subsequently, we incubated aminoacids (glycine, lysine, alanine) with glucose and galactose for 7 days and studied the action of the incubation products on NBT, using amino acids and sugars as controls. We found that NBT reduction increases proportionally to the length of incubation time of glucose/galactose with lysine, but not with other amino acids. Reduction of NBT by the Amadori compounds formed is inhibited by SOD. We suggest that oxygen radical generation by Amadori compounds must be taken into consideration as one cause of damage in diabetes of long duration.

Amino Sugars↗

Anthracycline-induced oxygen consumption and oxidative damage in rat liver microsomes are not necessarily coupled. A study with 8 structurally related anthracyclines.

The stimulative effect of 8 anthracyclines (the parent compounds daunorubicin and doxorubicin and 6 structurally closely related anthracyclines) on the production of thiobarbituric acid (TBA)-reactive material was investigated in liver microsomes. Except for daunorubicinone and doxorubicinone, all derivatives stimulated NADPH-dependent production of TBA-reactive material. Doxorubicinone had no effect, daunorubicinone inhibited TBA-reactivity at concentrations up to 50 microM. However, the latter two compounds stimulated oxygen consumption in the presence of EDTA to a degree comparable to that induced by the parent compounds. Since the oxygen uptake under these circumstances represents redox cycling of the drugs, apparently redox cycling and production of TBA-reactive material were not coupled for these compounds. Spectral measurements showed no decisive role for interaction with free iron (Fe3+) ions in the non-coupling of redox cycling and production of TBA reactive material. Evidence for a role of bound iron ions was not obtained. It is discussed that for the aglycones oxygen consumption and production of TBA reactive material might be non-coupled through their different interaction with microsomal RNA.

Animals↗

Deactivation of singlet molecular oxygen by organo-selenium compounds exhibiting glutathione peroxidase activity and by sulfur-containing homologs.

The bimolecular rate constants (k) of quenching of molecular singlet oxygen 1O2 (1 delta g) by organo-selenium compounds exhibiting glutathione peroxidase activity and by sulfur analogs have been determined by time resolved phosphorescence detection of 1O2 in CD3OD and C6D6, with no solvent effect. The rate constants of quenching by the Se-containing compounds were found to be approximately one order of magnitude higher than those of the S-containing homologs. A linear correlation was observed between log k and the Hammett constant omega ortho with p = -0.89, the rate constant being higher for molecules with an electron-donating substituent and lower for those with an electron-withdrawing substituent. This observation is consistent with the involvement of a charge transfer complex in the deactivation of singlet oxygen.

Antioxidants↗

Photoreactivity of biologically active compounds, XIV: influence of oxygen on light induced reactions of primaquine.

The influence of molecular oxygen and oxygen radicals on the photoreactivity of the antimalarial drug primaquine (PQ) has been investigated. Oxygen is directly involved in photodecomposition of the drug. Flushing with helium gas prior to and during irradiation to suppress the oxygen level of the medium, retards the degradation rate of PQ (followed by HPLC) and leads to the formation of only two degradation products (identified by MS) compared to eight main- and several minor products under normal atmospheric conditions. Flushing with oxygen gas prior to and during irradiation to increase the oxygen content of the medium accelerates the degradation rate of PQ. PQ produces oxygen radicals (hydroxyl and superoxide) during photolysis, while the photoproducts of PQ seem likely to induce singlet oxygen formation (detected by addition of radical scavengers). Sensitization reactions involving singlet oxygen lead to decomposition of PQ (followed by HPLC). On the basis of our results, photochemical reaction mechanisms of PQ are postulated and discussed. At physiological conditions (aqueous, neutral pH, oxygen rich) PQ has a large potential to decompose after light absorption. The photoreaction seems to be initiated at the quinoline nitrogen. The ability to form an intramolecular hydrogen bond seems to be essential for the luminescence properties of the drug. Phosphorescence lifetime of PQ is about 5 microseconds. Fast chemical reactions may occur from the short-lived triplet state of the drug, but the excited compound can diffuse only a limited distance prior to deexcitation. This can be important concerning light-induced adverse effects which may appear after medication with PQ.

Antimalarials↗