The mechanism of the mutagenic action of hydroxylamine XIII. Reversion of phage MS2 amber mutants in the presence of hydroxylamines.

The replication of the phage MS2 in the presence of either hydroxylamine (HA) or O-methylhydroxylamine (OMHA) (mutagenesis in vivo) results in an increase in the reversion frequency of two amber mutations in the maturation protein. When acting on the extracellular phage (mutagenesis in vitro) the mutagens do not affect the reversion frequency. The most probable mode of mutagenic action of the hydroxylamines on the vegetative MS2 phage involves the enzymic formation of modified precursors and their incorporation into RNA.     

Comparative analysis of deletion and base-change mutabilities of Escherichia coli B strains differing in DNA repair capacity (wild-type, uvrA-, polA-, recA-) by various mutagens.

Dose-response curves were compared for deletions [ColBR (resistant to colicin B) mutations being more than 80% deletions] and base changes (reversion of argFam to prototrophy argplus) induced in the same set of E. coli strains (wild-type for DNA repair, uvrA-, polA- and recA-) by N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), hydroxylamine (HA), 4-nitroquinoline I-oxide (4NQO), mitomycin C (MTC, UV and X-rays. All these agents induced deletions as well as base changes in the wild-type strain. Thus chemical mutagenesis differed in E. coli and bacteriophages in vitro, for HA, NTG, EMS and perhaps UV produced only point mutations in phage Tr. The patterns of deletion and base-change mutability in E. coli were surprisingly similar. (I) The recombination less recA- strain was mutable by only three (NTG, EMS, HA) of the seven mutagens for either deletions or base changes. (2) The uvrA- strain, unable to excise pyrimidine dimers, was very highly mutable by 4NQO and UV but immutable by MTC for both deletions and base changes. (3) The polA- strain, defective in DNA polymerase I due to a non-suppressible mutation, was very highly mutable by HA and highly mutable by MTC and 4NQO for both deletions and base changes but was highly mutable only for deletions by UV and X-rays, remaining normally mutable by the other agents for both deletions and base changes despite its high sensitivity to their inactivating action. We conclude that errors in the recA-dependent repair of induced DNA damage (after 4NQO, MTC, UV and X-rays) or errors in replication enhanced by damage to the replication system or to the template strands (after NTG, EMS, and HA) give rise to deletions as well as to base changes. From a comparative analysis of 14 dose-response curves for deletions and base changes, we conclude that the order of mutagenic efficiency relative to killing is (EMS, NTG) greater than (UV, 4NQO) greater than HA greater than (X-rays, MTC), and that X-rays, 4NQO, HA and MTC induce more ColBR deletions than Argplus base changes, whereas UV and EMS induce ColBR deletions and Argplus base changes at nearly equal rates and the specificity of NTG is intermediate between these two types.     

Detection of mutagen-induced lesions in isolated DNA by marker rescue of Bacillus subtilis phage phi 105

A new mutagenesis assay is described which detects the induction of forward mutations in isolated DNA. The assay utilizes replicative from DNA of the temperate Bacillus subtilis phage φ105 and tests the ability of chemicals to induce lesions which inactivate phage genes involved in lysogen formation. There is a cluster of such genes tightly linked to the φ105 genetic marker Jsus11 which restricts the host range of the phage to cells capable of suppressing sus mutations. In the actual assay chemically treated DNA, from wild-type J⁺ phage, is added to competent cells which are infected with φ105Jsus11. Wild-type phage, capable of producing plaques on cells which are nonpermissive for φ105Jsus11, are produced by recombination between the added chemically-treated DNA and infecting φ105Jsus11 DNA. If the added DNA also carried mutagenic lesions in any of the genes controlling lysogeny, clear plaque mutants are produced which are readily distinguishable from the turbid plaquing wild-type phage. This report demonstrates the capacity of this marker rescue-based assay to detect as mutagens the following DNA-reactive chemicals: hydroxylamine (HA); N-methyl-N′-nitro-N-nitrosoguanidine (MNNG); chloroacetaldehyde (CAA); propylene oxide (PO) and N-acetyl-N-acetoxy-2-amino-fluorene (AAAF). The effect of using a host cell, defective for excision repair, on the sensitivity with which the assay detected the mutagenic activities of CAA, PO and AAAF also was examined.The new mutagenesis assay offers 2 advantages over several other previously described transformation-based assays: (1) in contrast to assays based on the induction of ribosome-associated drug resistances, the new assay can detect frameshift as well as base-substitution-type mutagens and (2) the mutants generated can be detected at high plating densities. The assay thus may be useful for general mutagen screening especially with highly bactericidal compounds which are not readily tested in other microbial assays.     

Slow oxygen release on the first two flashes in chemically stressed Photosystem II membrane fragments results from hydrogen peroxide oxidation

Flash-induced amperometric signals were measured with a Joliot-type O₂ rate electrode in spinach Photosystem II (PS II) membrane fragments exposed to very low concentrations of added hydroxylamine or hydrogen peroxide. In both cases anomalous O₂ signals were observed on the first two flashes, and oscillating four-flash patterns were observed on subsequent flashes. The anomalous signals were eliminated in the presence of catalase but not EDTA. The rise times of the O₂-release kinetics associated with the anomalous signals were slow (ca. 20 ms with NH₂OH and ca. 120 ms with H₂O₂) compared to the kinetics of O₂ release on subsequent flashes and in control membranes (3–6 ms). It is proposed that when the intact PS II O₂-evolving complex is perturbed with small concentrations of added reductant, H₂O₂ can gain access and bind to the complex. Bound H₂O₂ can then reduce lower S states in some centers leading to anomalous O₂ signals on the first two flashes. A model is presented to explain both types of anomalous O₂ production. Oxygen observed on the third and subsequent flashes is due to the normal photosynthetic O₂-evolution process arising from the S₃-state. Anomalous O₂ production could be a protective mechanism in PS II centers subjected to stress conditions.Abbreviations DCIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - MES 4-morpholine-ethanesulfonic acid - OEC oxygen-evolving complex - PS II Photosystem II - Y_i O₂ flash yield on the i^th flash - Y_ss steady-state O₂ flash yield level in algae, chloroplasts, or thylakoids after flash-driven S-state oscillations have been damped Formerly, the Solar Energy Research Institute and operated by the Midwest Research Institute for the US Department of Energy under Contract DE-AC-02-83CH10093. Government and MRI retain non-exclusive, royalty-free license to publish or reproduce published articles, or allow others to do so for Government purposes.     

Chemical oxidation and reduction of the O2-evolution center in Photosystem II

Treatment of Photosystem II (PS II) with low concentrations of hydroxylamine is known to cause a two-flash delay in the O₂-evolution pattern, and in the formation of the S₂-state multiline EPR signal, due to the two-electron reduction of the S₁-state by hydroxylamine to form the S_-1-state. Past work has shown that these delays are not reversed by washing out the hydroxylamine nor by adding DCBQ or ferricyanide to oxidize the residual hydroxylamine, but are reversed by illumination with two saturating flashes followed by a 30-min dark incubation. We have examined the effects of treatments aimed at restoring the normal flash-induced O₂-evolution pattern and S₂-state multiline EPR signal after treatment of PS II with 40 M hydroxylamine. In agreement with past work, we find that the two-flash delay in O₂ evolution is not reversed when the hydroxylamine is removed by three cycles of centrifugation and resuspension in hydroxylamine-free buffer nor by adding ferricyanide or DCBQ to oxidize the unreacted hydroxylamine. However, the normal flash-induced O₂-evolution pattern is restored by illumination with two saturating flashes followed by a 30-min dark incubation (after the sample was first treated with 40 M hydroxylamine and the unreacted hydroxylamine was removed); illumination with one saturating flash followed by a 30-min dark incubation is only partially effective. These results show that ferricyanide and DCBQ are not effective at oxidizing the S_-1-state to the S₁-state. In contrast, adding hypochlorite (OCl^-) after treatment with hydroxylamine restored the normal flash-induced O₂-evolution pattern and also restored the formation of the S₂-state multiline EPR signal by illumination at 200 K. We conclude that hypochlorite is capable of oxidizing the S_-1-state to the S₁-state. This is the first example of a chemical treatment that advances the delayed flash-induced O₂ evolution pattern.Abbreviations DCBQ 2,5-dichloro-p-benzoquinone - OEC O₂-evolving center     

Optimization of the hydroxylamine cleavage of an expressed fusion protein to produce a recombinant antimicrobial peptide

Hydroxylamine was used to cleave the Asn-Gly peptide bond between the fusion partner and the antimicrobial peptide of interest, a magainin derivative (MSI-344). The efficiency of reaction depended on the hydroxylamine concentration, denaturant, pH, and the fused protein concentration. The optimal cleavage solution consisted of guanidine HCl as the denaturant, pH 8.1, and 6.7 mg ml^–1 of fused MSI-344. This optimized cleavage solution resulted in a high yield (95% ) of MSI-344 from a cultivation of E. coli. This result suggests potential applications for using hydroxylamine to cleave basic peptides produced from fusion proteins.     

On the action of hydroxylamine,hydrazine and their derivatives on the water-oxidizing complex

Photosynthetic water oxidation proceeds by a four-step sequence of one-electron oxidations which is formally described by the transitions S₀ S₁, S₁ S₂, S₂ S₃, S₃ (S₄) S₀. State S₁ is most stable in the dark. Oxygen is released during S₃ (S₄) S₀. Hydroxylamine and hydrazine interact with S₁. They cause a two-digit shift in the oxidation sequence as observed from the dark equilibrium, i.e. from S₁ S₂ : S₂ S₃ : S₃ (S₄) S₀ : S₀ S₁ :... in the absence of the agents, to S₁ ^* S₀ : S₀ S₁ : S₁ S₂ : S₂ S₃ :... in the presence of hydroxylamine or hydrazine.We measured the concentration dependence of this two-digit shift via the pattern of proton release which is associated with water oxidation. At saturating concentrations hydroxylamine and hydrazine shift the proton-release pattern from OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H(S₃ S₀) : 1H⁺(S₀ S₁) :... to 2H⁺(S₁ ^* S₀) : 1H⁺(S₀ S₁) : OH⁺(S₁ S₂) : 1H⁺(S₂ S₃) : 2H⁺(S₃ S₀) :... The 2H⁺ were released upon the first excitation with a half-rise time of 3.1 ms, both with hydroxylamine and withydrazine. The concentration dependence of the shift was rather steep with an apparent Hill coefficient at half saturation of 2.43 with hydroxylamien (Förster and Junge (1985) FEBS Lett. 186, 53–57) and 1.48 with hydrazine. The concentration dependence could be explained by cooperative binding of n3 molecules of hydroxylamine and of n2 molecules of hydrazine, respectively. Tentatively, we explain the interaction of hydroxylamine and hydrazine with the water-oxidizing complex (WOC) as follows: Two bridging ligands, possible Cl^- or OH^-, which normally connect two Mn nuclei, can be substituted by either 4 molecules of hydroxylamine or 2 molecules of hydrazine when the WOC resides in state S₁.Abbreviations DNP-INT dinitrophenylether of iodonitrothymol - FWHM full width at half maximum - NR neutral red (3-amino-7-dimethylamino-2-methylphenazine-HCI) - PS II photosystem II - WOC or (in formulas:) W water-oxidizing complex Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Electron transport chains and bioenergetics of respiratory nitrogen metabolism in Wolinella succinogenes and other Epsilonproteobacteria

Recent phylogenetic analyses have established that the Epsilonproteobacteria form a globally ubiquitous group of ecologically significant organisms that comprises a diverse range of free-living bacteria as well as host-associated organisms like Wolinella succinogenes and pathogenic Campylobacter and Helicobacter species. Many Epsilonproteobacteria reduce nitrate and nitrite and perform either respiratory nitrate ammonification or denitrification. The inventory of epsilonproteobacterial genomes from 21 different species was analysed with respect to key enzymes involved in respiratory nitrogen metabolism. Most ammonifying Epsilonproteobacteria employ two enzymic electron transport systems named Nap (periplasmic nitrate reductase) and Nrf (periplasmic cytochrome c nitrite reductase). The current knowledge on the architecture and function of the corresponding proton motive force-generating respiratory chains using low-potential electron donors are reviewed in this article and the role of membrane-bound quinone/quinol-reactive proteins (NapH and NrfH) that are representative of widespread bacterial electron transport modules is highlighted. Notably, all Epsilonproteobacteria lack a napC gene in their nap gene clusters. Possible roles of the Nap and Nrf systems in anabolism and nitrosative stress defence are also discussed. Free-living denitrifying Epsilonproteobacteria lack the Nrf system but encode cytochrome cd₁ nitrite reductase, at least one nitric oxide reductase and a characteristic cytochrome c nitrous oxide reductase system (cNosZ). Interestingly, cNosZ is also found in some ammonifying Epsilonproteobacteria and enables nitrous oxide respiration in W. succinogenes.     

人白介素-4与大肠杆菌二硫键异构酶DsbC在大肠杆菌中的融合表达及羟胺切割后共复性

目的:通过融合表达、羟胺切割、与二硫键异构酶共复性,获得高表达、高纯度、高生物活性的重组人白细胞介素-4(rhIL-4)。方法:将5端引入了羟胺切割位点的hIL-4基因克隆到大肠杆菌二硫键异构酶DsbC的原核表达载体pET-DsbC中,IPTG诱导表达,对包涵体进行纯化,然后在变性条件下经羟胺切割,利用DsbC的分子伴侣功能与hIL-4进行共复性,最后利用阳离子交换层析纯化获得rhIL-4蛋白。结果:融合蛋白DsbC-hIL-4的表达量占细菌总蛋白的40%以上,以包涵体形式存在;纯化后得到的rhIL-4的相对分子量为15×103,与预期一致,电泳纯度达95%;细胞学实验测定其具有良好的生物学活性。结论:通过融合表达的方法可以提高hIL-4的原核表达量;利用共复性的方式极大地提高了hIL-4的复性率和生物活性。     

Evidence for Nitric Oxide Mediated Effects on Islet Hormone Secretory Phospholipase C Signal Transduction Mechanisms

We have investigated the putative role of nitric oxide (NO) as a modular of islet hormone release, when stimulated by the muscarinic receptor agonist–phospholipase C activator, carbachol, with special regard to whether the IP₃-Ca²⁺ or the diacylglycerol-protein kinase C messenger systems might be involved. It was observed that the NO synthase (NOS) inhibitor N^G-nitro-L-arginine methylester (L-NAME) markedly potentiated insulin release and modestly inhibited glucagon release induced by carbachol. Similarly, insulin release induced by the phorbol ester TPA (protein kinase C activator) was markedly potentiated. Glucagon release, however, was unaffected. Dynamic perifusion experiments with ⁴⁵C²⁺-loaded islets revealed that the inhibitory action of L-NAME on carbachol-stimulated NO-production was reflected in a rapid and sustained increase in insulin secretion above carbachol controls, whereas the ⁴⁵Ca²⁺-efflux pattern was similar in both groups with the exception of a slight elevation of ⁴⁵C²⁺ in the L-NAME-carbachol group during the latter part of the perifusion. No difference in either insulin release or ⁴⁵Ca²⁺-efflux pattern between the carbachol group and L-NAME-carbachol group was seen in another series of experiments with identical design but performed in the absence of extracellular Ca²⁺ . However, it should be noted that in the absence of extracellular Ca²⁺ both ⁴⁵Ca²⁺-efflux and, especially, insulin release were greatly reduced in comparison with experiments in normal Ca²⁺. Further, in the presence of diazoxide, a potent K⁺ _ATP-channel opener, plus a depolarizing concentration of K⁺ the NOS-inhibitor L-NAME still markedly potentiated carbachol-induced insulin release and inhibited glucagon release. The enantiomer D-NAME, which is devoid of NOS-inhibitory properties, did not affect carbachol-induced hormone release. TPA-induced hormone release in depolarized islets was not affected by either L-NAME or D-NAME. The pharmacological intracellular NO donor hydroxylamine dose-dependently inhibited insulin release stimulated by TPA. Furthermore, a series of perifusion experiments revealed that hydroxylamine greatly inhibited carbachol-induced insulin release without affecting the ⁴⁵Ca²⁺-efflux pattern. In summary, our results suggest that the inhibitory effect of NO on carbachol-induced insulin release is not to any significant extent exerted on the IP₃-Ca²⁺ messenger system but rather through S-nitrosylation of critical thiol-residues in protein kinase C and/or other secretion-regulatory thiol groups. In contrast, the stimulating action of NO on carbachol-induced glucagon release was, at least partially, connected to the IP₃-Ca²⁺ messenger system. The main effects of NO on both insulin and glucagon release induced by carbachol were apparently exerted independently of membrane depolarization events.