Interaction between the nitrate respiratory system of Escherichia coli K12 and the nitrogen fixation genes of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{chl}$^? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from $\text{K. pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$_Kp⁺ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by $\text{chl}$ genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity.     

The effect of cyclic AMP on anaerobic growth of Escherichia coli

Adenosine 3′,5′-cyclic phosphate (cyclic AMP) stimulated a cyclic AMP-deficient mutant strain of $\text{Escherichia coli}$ to grow anaerobically on glucose in a minimal medium and in media supplemented with nitrate or casein hydrolysate. Cyclic AMP was found to stimulate the production of the formic hydrogenlyase system in this mutant strain, but had no effect on its ability to carry out anaerobic reductions of nitrate or nitrite. It was also observed that CO₂ stimulated the anaerobic growth of the mutant in the absence of cyclic AMP.     

Modulators of monoamine oxidase in plasma

Addition of small amounts of plasma activated the deamination of tryptamine by platelet monoamine oxidase (MAO). At higher concentrations, plasma inhibited the deamination instead. The inhibition was increased with increasing amounts of plasma added. The inhibition was uncompetitive in nature, partially reversed by prior ultrafiltration of the plasma through PM30 membranes and completely reversed by protein precipitation of plasma with perchloric acid. Addition of high amounts of plasma $\text{in}$$\text{vitro}$ also inhibited the activity of bovine striatal MAO. The inhibition of striatal deamination of tryptamine by plasma was noncompetitive in nature, completely reversed by ultrafiltration through PM30 membranes and partially reversed by perchloric acid treatment. The inhibition of striatal deamination of serotonin was noncompetitive in nature, not reversed by ultrafiltration but completely reversed by perchloric acid treatment. The pattern of inhibition of platelet or striatal MAO by plasma was different from that induced by addition of bovine serum albumin (BSA). Low concentrations of BSA added $\text{in}$$\text{vitro}$ activated the deamination of tryptamine or serotonin by platelet or striatal MAO by decreasing the K_m, while higher concentrations also decreased the V_max. The presence of protein, non-albumin circulating modulators of platelet or striatal MAO in plasma is discussed.     

Bioactivation of carbon tetrachloride, chloroform and bromotrichloromethane: role of cytochrome P-450.

In order to define the site of bioactivation of CCl₄, CHCl₃ and CBrCl₃ in the NADPH cytochrome $\text{c}$ reductase-cytochrome P-450 coupled systems of liver microsomes, the ¹⁴C-labeled hepatotoxins were incubated $\text{in}$$\text{vitro}$ with isolated rat liver microsomes and a NADPH-generating system. The covalent binding of radiolabel to microsomal protein was used as a measure of the conversion of the hepatotoxins to reactive intermediates. Omission of NADPH, incubation under CO:O₂ (8:2) and addition of a cytochrome $\text{c}$ reductase specific antisera mardedly reduced the covalent binding of all three compounds. When cytochrome P-450 was reduced to less than 25% of normal by pretreatment of rats with allylisopropylacetamide (AIA), but cytochrome $\text{c}$ reductase activity was unchanged, the covalent binding of CCl₄, CHCl₃, and CBrCl₃ was decreased by 63, 83, 70%, respectively. Incubation under an atmosphere of N₂ enhanced the binding of CCl₄, inhibited the binding of CHCl₃ and did not influence the binding of CBrCl₃. It is concluded that cytochrome P-450 is the site of bioactivation of these three compounds rather than NADPH cytochrome $\text{c}$ reductase and that CCl₄ bioactivation proceeds by cytochrome P-450 dependent reductive pathways, while CHCl₃ activation proceeds by cytochrome P-450 dependent oxidative pathways.     

II. Novel deaminated sulfadiazine metabolites in neonatal calf tissue,plasma, and urine following oral treatment with 14C-sulfadiazine

Demonstration of the efficacy of Tribrissen® [trimethoprim(TMP): sulfadiazine(SDZ), 1:5] for treatment of calf scours led to a study of the disposition of ¹⁴C-SDZ in the neonatal calf. Two healthy male calves (～40 kg) received orally one Tribrissen® bolus [¹⁴C-SDZ (1.0 g):TMP (0.2 g)] each for five consecutive days. The calves were killed on day 14 after the last dose. Mean total SDZ-related residues were 0.04 ppm (muscle), 0.16 ppm (kidney) and 0.31 ppm (liver) on day 14. Intact SDZ was undetectable on day 14 in all three tissues. Two-dimensional TLC autoradiograms of tissue, plasma and urine extracts revealed the presence of SDZ, N⁴-acetyl SDZ, 4-hydroxy-SDZ, N⁴-acetyl-4-hydroxy SDZ and two novel metabolites that were identified by NMR and mass spectral analyses to be 2-benzenesulfonamidopyrimidine and 2-benzenesulfonamido-4-hydroxypyrimidine. Most of the SDZ dose (87%) was recovered in the urine with SDZ and N⁴-acetyl SDZ accounting for 16 and 42% of the dose. The other four compounds accounted for <5% of the dose. The data from this study indicate that assays that rely on the presence of the N⁴-amino group, such as the Bratton-Marshall procedure, measure only part of the SDZ-related tissue residues in neonatal calves.     

Deamination of 5-methoxytryptamine, serotonin and phenylethylamine by rat MAO in vitro and in vivo.

Pretreatment of rats with clorgyline, a selective inhibitor of MAO-A, significantly inhibited the $\text{in vivo}$ deamination of intraventricularly administered serotonin (5-HT) and 5-methoxytryptamine (5-MT), but not phenylethylamine (PEA). Pretreatment with d, l-deprenyl, a selective inhibitor of MAO-B, significantly inhibited the $\text{in vivo}$ deamination of all three substrates. Brain and liver homogenates from rats pretreated with clorgyline showed a decreased ability to deaminate ($\text{in vitro}$) 5-MT and 5-HT, but not PEA. Homogenates from animals pretreated with d,l-deprenyl showed a decreased capacity to deaminate PEA, but not 5-MT or 5-HT. Clorgyline, when added to brain and liver homogenates, selectively blocked the deamination of 5-MT and 5-HT, but not PEA, whereas, d,l-deprenyl blocked the deamination of PEA without affecting that of 5-MT or 5-HT. In addition, 5-MT was found to be 100 X more potent than PEA at inhibiting the $\text{in vitro}$ deamination of 5-HT. These findings suggest that 5-MT and 5-HT are favored substrates for MAO-A $\text{in vitro}$ and $\text{in vivo}$. However, $\text{in vivo}$, significant amounts of 5-MT and 5-HT can also be deaminated by MAO-B.     

Charge distribution in electron transport components: cytochrome c and cytochrome c oxidase mixtures

Mixtures of cytochrome $\text{c}$ oxidase and cytochrome $\text{c}$ have been titrated by coulometrically generated reductant, methyl viologen radical cation, and physiological oxidant, O₂. Charge distribution among the heme components in mixtures of these two redox enzymes has been evaluated by monitoring the absorbance changes at 605 and 550 nm. Differences in the pathway of the electron transfer process during a reduction cycle as compared to an oxidation cycle are indicated by variations found in the absorbance behavior of the heme components during successive reductive and oxidative titrations. It is apparent that the potential of the cytochrome $\text{a}$ heme is dependent upon whether oxidation or reduction is occurring.     

OKY-1581: A selective inhibitor of thromboxane synthesis invivo and invitro

OKY-1581 is an effective inhibitor of thromboxane synthesis $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$. The generation of thromboxane B₂ (TxB₂), prostaglandin E (PGE) and prostaglandin F (PGF) was measured following clotting and during platelet aggregation induced by collagen. The presence of OKY 1581 either $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$ caused a reduction in TxB₂ generation during clotting and platelet aggregation with a concomitant increase in PGE and PGF. The effect could be observed two hours after oral or subcutaneous administration of 5 to 100 mg per rabbit and lasted for 24 to 48 hours. The reduction in TxB₂ was not accompanied by an inhibition of clotting or platelet aggregation. OKY-1581 appears to be a suitable agent for studying the role of TxB₂ in atherosclerosis.     

Studies on a non-pyridine nucleotide dependent, membrane-bound L-(+)-glutamate oxidoreductase in Azotobacter vinelandii

A new type of membrane-bound oxidoreductase is described that carries out an oxidative deamination reaction that specifically involves L-glutamate. This enzyme is found in a subcellular fraction of $\text{Azotobacter}$$\text{vinelandii}$ strain 0. It can oxidize L?(+)-glutamate using molecular oxygen and produces α-ketoglutarate and NH₃ as end products. Neither NAD⁺ nor NADP⁺ are involved in this oxidation. The reaction is carried out by the membranous “R₃” fraction which is obtained from sonically ruptured resting cells by differential centrifugation. In addition to O₂, the electron acceptors that allowed for L-glutamate oxidation were phenazine methosulfate (PMS), K₃Fe(CN)₆, and 2, 6-dichloroindophenol (DCIP). This oxidation appears to be an integral part of the $\text{Azotobacter}$ electron transport system as the L-glutamate oxidase rate is also highly sensitive to known electron transport inhibitors, $\text{i.e.}$, 2-n-hydroxy-4-quinoline-N-oxide, cyanide, and thenoyltrifluoroacetone. Spectral absorption studies on the $\text{Azotobacter}$ R₃ electron transport fraction revealed that the cytochrome and flavoprotein (non-heme iron) components also could be reduced completely upon the addition of L-glutamate. Preliminary results suggest that this is a new type of L-glutamate oxidoreductase that does not as yet have an Enzyme Commission number and appears to be (a) a specific flavoprotein enzyme that is not a type of L-amino acid oxidase, (b) tightly bound (and functionally attached) to the $\text{Azotobacter}$ electron transport system, and (c) capable of carrying out specifically the oxidative deamination of L-glutamate in the absence of pyridine nucleotides.     

On the purification of nitrite reductase from Thiobacillus denitrificans and its reaction with nitrite under reducing conditions.

Nitrite reductase (cytochrome $\text{cd}$) from $\text{T. denitrificans}$ has been crystallized in high yield in three simple and rapid steps. The spectral absorption ratio at 408 to 280 nm was 1.52. Light absorption spectra in the oxidized and reduced states were virtually identical to those of nitrite reductase from $\text{P. aeruginosa}$. EPR spectroscopy of nitrite reductase at 12° showed a low-spin ferric heme resonance with g-values at 2.52, 2.45 and 1.73 assigned to the d-heme. Reaction of nitrite reductase with nitrite in the presence of the reducing systems [(ascorbate + PMS) or sulfide] resulted in the formation of nitric oxide (confirmed by gas chromatography) which reacted with both $\text{c}$- and $\text{d}$-hemes of nitrite reductase yielding an EPR-detectable enzyme-NO complex with g-values at 2.07, 2.04 and 1.99 and a ¹⁴N hyperfine splitting constant of 22.5 gauss. The amount of nitric oxide produced enzymatically with sulfide as electron donor was only 5% of that found when ascorbate plus PMS served as reductant.To our knowledge the detection of the unique enzyme-NO complex is the first definitive EPR evidence for the mandatory liganding of nitric oxide with pure nitrite reductase during nitrite reduction.     

Trinitrotoluene (TNT) as a sole nitrogen source for a sulfate-reducing bacteriumDesulfovibrio sp. (B strain) isolated from an anaerobic digester

A sulfate-reducing bacterium (SRB),Desulfovibrio sp. (B strain), isolated from a continuous anaerobic digester (Boopathy and Daniels, Current Microbiology, 23:327–332, 1991) was found to use 2,4,6-trinitrotoluene (TNT) as sole nitrogen source. This bacterium also used nitrate, nitrite, and ammonium as nitrogen source. A long lag period was noticed when TNT or nitrite was used as nitrogen source. Nitrate, nitrite and TNT also served as electron acceptor in the absence of sulfate for this bacterium. Under nitrogen-limiting condition, 100% removal of TNT was observed within 8 days of incubation. The main intermediate observed was diaminonitrotoluene, which was further converted to toluene via triaminotoluene by reductive deamination process. Under nitrogen-rich conditions (presence of ammonium), TNT was converted to diaminonitrotoluene, and toluene was not produced. This isolate did not degrade TNT all the way to CO₂. This study demonstrated the possibility of using this isolated to decontaminate the soil and water contaiminated with TNT under anaerobic conditions.     

Intraperitoneal injection of zymosan in mice induces pain,inflammation and the synthesis of peptidoleukotrienes and prostaglandin E2

Intraperitoneal injection of zymosan in mice induced rapid extravasation and accumulation of plasma proteins in the peritoneal cavity. Neutrophils began to appear in the peritoneal cavity after a lag period of approximately 3 hours. The injected mice exhibited a pain response (writhing) during the first 30 minutes after injection, but writhing ceased before protein or cell accumulation had reached maximum levels. The injection of zymosan induced synthesis of PGE₂ (measured by RIA) which reached maximum levels of 30 minutes, then declined slowly. Peptido-leukotriene levels (detected by bioassay, RIA and HPLC) increased rapidly after injection, reached a peak within an hour of injection and declined to undetectable levels within 4 hours. The early peptido-LT was predominantly LTC₄, while later, LTE₄ was the major component. LTD₄ levels remained low throughout and no LTB₄ was detected at any time. Indomethacin treatment elevated levels of peptido-LTs, recued PGE₂ levels and inhibited writhing. Phenidone reduced peptido-LT levels. $\text{In}$$\text{vitro}$ studies demonstrated that zymosan stimulates LTC4 synthesis by peritoneal cells whereas LTE₄, LTD₄, LTB₄ or monoHETES were not detectable (using HPLC methods). The source of enzymes responsible for the $\text{in}$$\text{vivo}$ metabolism of LTC₄ to LTD₄ and LTE₄ could not be identified.     

Oxygen-18 studies on the oxidative deamination mechanism of alicyclic primary amines in rabbit liver microsomes

The mechanism of microsomal oxidative deamination of alicyclic primary amines: cyclopentylamine, cyclohexylamine, cycloheptylamine, 1- and 2-aminoindan, 1- and 2-aminotetralin, was studied under an atmosphere of ¹⁸O₂ or in a medium containing H₂¹⁸O. The oxygen-18 contents of the products determined by gas-liquid chromatography/mass spectrometry revealed that almost all (75–100 atom%) of the oxygen of oximes was derived from molecular oxygen, whereas a part (4–25 atom% ) of the oxygen of ketones. The studies on the hydrolysis of oximes and the oxygen exchange reaction of ketones proved that the latter proceeded at a considerable rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.5–336}\text{min}$) and the former made a minor contribution, to explain why the major portion (75–96 atom%) of the oxygen in ketones was derived from water. The results support the mechanism that microsomal deamination proceeds mainly through a carbinolamine intermediate, which is initially hydroxylated at the α carbon to the amino group, partially equilibrating with the imine, and then rearranges to form a ketone and ammonia.     

Effect of Blood Nitrite and Nitrate Levels on Murine Platelet Function

Nitric oxide (NO) appears to play an important role in the regulation of thrombosis and hemostasis by inhibiting platelet function. The discovery of NO generation by reduction of nitrite (NO₂ ⁻) and nitrate (NO₃ ⁻) in mammals has led to increased attention to these anions with respect to potential beneficial effects in cardiovascular diseases. We have previously shown that nitrite anions at 0.1 µM inhibit aggregation and activation of human platelet preparations in vitro in the presence of red blood cells and this effect was enhanced by deoxygenation, an effect likely due to NO generation. In the present study, we hypothesized that nitrite and nitrate derived from the diet could also alter platelet function upon their conversion to NO in vivo. To manipulate the levels of nitrite and nitrate in mouse blood, we used antibiotics, NOS inhibitors, low nitrite/nitrate (NOx) diets, endothelial NOS knock-out mice and also supplementation with high levels of nitrite or nitrate in the drinking water. We found that all of these perturbations affected nitrite and nitrate levels but that the lowest whole blood values were obtained by dietary restriction. Platelet aggregation and ATP release were measured in whole blood and the results show an inverse correlation between nitrite/nitrate levels and platelet activity in aggregation and ATP release. Furthermore, we demonstrated that nitrite-supplemented group has a prolonged bleeding time compared with control or low NOx diet group. These results show that diet restriction contributes greatly to blood nitrite and nitrate levels and that platelet reactivity can be significantly affected by these manipulations. Our study suggests that endogenous levels of nitrite and nitrate may be used as a biomarker for predicting platelet function and that dietary manipulation may affect thrombotic processes.     

The effect of oxygen on nitrate and nitrite assimilation in leaves of Zea mays L. under dark conditions

The assimilation of nitrate under dark-N₂ and dark-O₂ conditions in Zea mays leaf tissue was investigated using colourimetric and ¹⁵N techniques for the determination of organic and inorganic nitrogen. Studies using ¹⁵N indicated that nitrate was assimilated under dark conditions. However, the rate of nitrate assimilation in the dark was only 28% of the rate under non-saturating light conditions. No nitrite accumulated under dark aerobiosis, even though nitrate reduction occurred under these conditions. The pattern of nitrite accumulation in leaf tissue in response to dark-N₂ conditions consisted of three phases: an initial lag phase, followed by a period of rapid nitrite accumulation and finally a phase during which the rate of nitrite accumulation declined. After a 1-h period of dark-anaerobiosis, both nitrate reduction and nitrite accumulation declined considerably. However, when O₂ was supplied, nitrate reduction was stimulated and the accumulated nitrite was rapidly reduced. Anaerobic conditions stimulated nitrate reduction in leaf tissue after a period of dark-aerobic pretreatment.     

Babesia major in Britain: blood-induced infections in splenectomized and intact calves

A strain of Babesia major, originally isolated from field collections of Haemaphysalis punctata in Kent, England was maintained in splenectomized calves by the intravenous inoculation of infected blood. Rapid passage from carrier calves, that had recovered from a tick-induced infection, resulted in a marked increase in virulence; 4 out of 6 calves of the second passage underwent fatal infections and the others suffered severe reactions.Five splenectomized and 5 intact calves of the same breed and age were infected with the same number of infected erythrocytes (RBC). The intact calves reacted mildly with maximum parasite counts ranging from < $\text{1}\text{1000}$ RBC to $\text{5}\text{1000}$ RBC; haemoglobin levels and packed cell volume values, however, fell sharply but recovered swiftly. The group of splenectomized calves exhibited one fatal case, 2 severe reactions and 2 mild infections; maximum parasitaemias varied from $\text{7}\text{1000}$ RBC to $\text{322}\text{1000}$ RBC. Packed cell volumes and haemoglobin concentrations declined to low levels and took several weeks to return to normal.It is concluded that B. major should be regarded as a potential pathogen of British cattle.     

Hg2+-stimulated NADH-oxidase activity of Ascaris muscle microsomal lipoamide dehydrogenase.

In the presence of Hg²⁺$\text{Ascaris}$ lipoamide dehydrogenase stimulated the reduction of oxygen, ferricyanide, and 2,6-dichlorophenolindophenol with NADH, which was inhibited by lipoic acid. On the other hand, Cu²⁺ stimulated the reduction of the artificial dyes, but only a little the reduction of oxygen. Hg²⁺ changed the visible absorption spectrum of the lipoamide dehydrogenase, but did not change the fluorescence curve. Lipoic acid decreased the fluorescence, but did not change the visible absorption spectrum. The $\text{Ascaris}$ lipoamide dehydrogenase have two SH groups per one subunit and 5–6 moles of HgCl₂ and 3–4 moles of CuSO₄ per one subunit were required for the maximal activity.     

Crystal data, molecular dimensions and molecular symmetry in cytochrome oxidase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa cytochrome oxidase (nitrite reductase, cytochrome cd) has been crystallized in space group P2₁2₁2 with cell dimensions $\text{a = 122.8}\text{A}\text{?}\text{, b = 87.2}\text{A}\text{?}\text{, c = 73.4}\text{A}\text{?}$. Density measurements suggest that the asymmetric unit contains one 63,000 molecular weight subunit of the dimeric molecule. Crystal data agree well with electron microscopy of single molecules. The X-ray pattern extends beyond 2.5 Å resolution, and structure analysis is in progress.     

Control of nitrate and nitrite assimilation by carbohydrate reserves,adenosine nucleotides and pyridine nucleotides in leaves of Zea mays L. under dark conditions

The rate of in-vivo nitrate reduction by leaf segments of Zea mays L. was found to decline during the second hour of dark anaerobic treatment. On transfer to oxygen the capacity to reduce nitrate under dark conditions was restored. These observations led to the proposal that nitrate reductase is a regulatory enzyme with ADP acting as a negative effector. The effect of ADP on the invitro activity of nitrate reductase and the changes in the in-vivo adenylate pool under dark-N₂ and dark-O₂ were investigated. It was found that ADP inhibited the activity of partially purified nitrate reductase. Similarly, the in-vivo anaerobic inhibition of nitrate reduction was associated with a build-up of ADP in the leaf tissue. Under anaerobic conditions nitrite accumulated and on transfer to oxygen the accumulated nitrite was reduced. To explain this phenomenon the following hypothesis was proposed and tested. Under anaerobic conditions the supply of reducing equivalents for nitrite reduction in the plastid becomes restricted and nitrite accumulates as a consequence. On transfer to oxygen this restriction is removed and nitrite disappears. This capacity to reduce accumulated nitrite was found to be dependent on the carbohydrate status of the leaf tissue.     

Electron transport-linked nitrous oxide synthesis and reduction by Paracoccusdenitrificans monitored with an electrode

The nitrous oxide reductase activity of $\text{Paracoccus}\text{denitrificans}$ can be conveniently measured using an electrochemical method for determining N₂O. Introduction of this procedure has shown that (i) N₂O reductase activity is reversibly inhibited by oxygen; (ii) antimycin strongly inhibits electron flow to N₂O and that the inhibition is bypassed by tetramethyl-p-phenylenediamine; (iii) ascorbate plus tetramethyl-p-phenylenediamine, presumably by donating electrons to cytochrome c, is an effective reductant for nitrous oxide reductase; (iv) in the presence of the nitrous oxide reductase inhibitor, acetylene, N₂O is promptly produced from nitrite, consistent with the product of nitrite reductase being N₂O.