FeoC from Klebsiella pneumoniae uses its iron sulfur cluster to regulate the GTPase activity of the ferrous iron channel.

Bacteria depend on the ferrous iron transport (Feo) system for the uptake of ferrous iron (Fe2+). The Feo system is crucial for colonization and virulence of pathogens. In γ-proteobacteria, the system consists of FeoA, FeoB, and FeoC. The function of FeoA remains unknown. FeoB likely forms the channel, whose regulation has been suggested to involve its GTPase domain (part of its NFeoB domain). FeoC from Klebsiella pneumonia was found to contain a [4Fe4S] cofactor, whose presence was speculated to enhance the GTPase activity of FeoB (Hsueh, K.-L., et al., J. Bacteriol. 2013 195(20): 4726-34). We present results here that support and extend that hypothesis. We monitored the GTPase activity of FeoB by NMR spectroscopy and found that the presence of 7% FeoC-[4Fe-4S]3+ (the highest level of cofactor achieved in vitro) increased the GTPase rate of NFeoB by 3.6-fold over NFeoB. The effect depends on the oxidation state of the cluster; with reduction of the cluster to [4Fe-4S]2+ the GTPase greatly decreased the GTPase rate. From the effects of point mutations in FeoC on GTPase rates, we conclude that Lys62 and Lys68 on FeoC each contribute to increased GTPase activity on NFeoB. Mutation of Thr37 of NFeoB to Ser nearly abolished the GTPase activity. The GTPase activity of the isolated K. pneumoniae NFeoB-FeoC complex (NFeoBC) was found to be higher in KCl than in NaCl solution. We solved the X-ray structure of the NFeoBC crystallized from KCl and compared it with a prior X-ray structure crystalized from NaCl. We propose a hypothesis, consistent with these results, to explain the factors that influence the GTPase activity. Bacteria may use the oxygen-sensitive cluster as a sensor to up-regulate the gate closing speed.

Repositioning of the Angiotensin II Receptor Antagonist Candesartan as an Anti-Inflammatory Agent With NLRP3 Inflammasome Inhibitory Activity.

Aberrant activation of the NLRP3 inflammasome promotes the pathogenesis of many inflammatory diseases. The development of the NLRP3 inflammasome inhibitors from existing drugs for new therapeutic purposes is becoming more important. Candesartan is an angiotensin II receptor antagonist widely used as a blood pressure-lowering drug; however, the inhibitory potential of candesartan on the NLRP3 inflammasome has not yet been investigated. We demonstrated that candesartan significantly inhibited the NLRP3 inflammasome and pyroptosis in macrophages. Mechanistic analysis revealed that candesartan inhibited the expression of NLRP3 and proIL-1ß by suppressing NF-κB activation and reducing the phosphorylation of ERK1/2 and JNK1/2. Candesartan reduced mitochondrial damage and inhibited the NLRP3 inflammasome assembly by suppressing NLRP3 binding to PKR, NEK7 and ASC. In addition, candesartan inhibited IL-1ß secretion partially through autophagy induction. Furthermore, oral administration of candesartan reduced peritoneal neutrophil influx, NLRP3 and ASC expression in peritoneal cells, and lavage fluid concentrations of active caspase-1, IL-1ß, IL-6 and MCP-1 in uric acid crystal-injected mice. These results indicated that candesartan has board anti-inflammatory effects and has the potential to be repositioned to ameliorate inflammatory diseases or NLRP3-associated complications.

The Effect of Hypoxia on Relative Biological Effectiveness and Oxygen Enhancement Ratio for Cells Irradiated with Grenz Rays.

Grenz-ray therapy (GT) is commonly used for dermatological radiotherapy and has a higher linear energy transfer, relative biological effectiveness (RBE) and oxygen enhancement ratio (OER). GT is a treatment option for lentigo maligna and lentigo maligna melanoma. This study aims to calculate the RBE for DNA double-strand break (DSB) induction and cell survival under hypoxic conditions for GT. The yield of DSBs induced by GT is calculated at the aerobic and hypoxic conditions, using a Monte Carlo damage simulation (MCDS) software. The RBE value for cell survival is calculated using the repair-misrepair-fixation (RMF) model. The RBE values for cell survival for cells irradiated by 15 kV, 10 kV and 10 kVp and titanium K-shell X-rays (4.55 kV) relative to 60Co γ-rays are 1.0-1.6 at the aerobic conditions and moderate hypoxia (2% O2), respectively, but increase to 1.2, 1.4 and 1.9 and 2.1 in conditions of severe hypoxia (0.1% O2). The OER values for DSB induction relative to 60Co γ-rays are about constant and ~2.4 for GT, but the OER for cell survival is 2.8-2.0 as photon energy decreases from 15 kV to 4.55 kV. The results indicate that GT results in more DSB induction and allows effective tumor control for superficial and hypoxic tumors.

Monte Carlo Simulation of Double-Strand Break Induction and Conversion after Ultrasoft X-rays Irradiation.

This paper estimates the yields of DNA double-strand breaks (DSBs) induced by ultrasoft X-rays and uses the DSB yields and the repair outcomes to evaluate the relative biological effectiveness (RBE) of ultrasoft X-rays. We simulated the yields of DSB induction and predicted them in the presence and absence of oxygen, using a Monte Carlo damage simulation (MCDS) software, to calculate the RBE. Monte Carlo excision repair (MCER) simulations were also performed to calculate the repair outcomes (correct repairs, mutations, and DSB conversions). Compared to 60Co γ-rays, the RBE values for ultrasoft X-rays (titanium K-shell, aluminum K-shell, copper L-shell, and carbon K-shell) for DSB induction were respectively 1.3, 1.9, 2.3, and 2.6 under aerobic conditions and 1.3, 2.1, 2.5, and 2.9 under a hypoxic condition (2% O2). The RBE values for enzymatic DSBs were 1.6, 2.1, 2.3, and 2.4, respectively, indicating that the enzymatic DSB yields are comparable to the yields of DSB induction. The synergistic effects of DSB induction and enzymatic DSB formation further facilitate cell killing and the advantage in cancer treatment.

Impact of Hypoxia on Relative Biological Effectiveness and Oxygen Enhancement Ratio for a 62-MeV Therapeutic Proton Beam.

This study uses the yields of double-strand breaks (DSBs) to determine the relative biological effectiveness (RBE) of proton beams, using cell survival as a biological endpoint. DSB induction is determined when cells locate at different depths (6 positions) along the track of 62 MeV proton beams. The DNA damage yields are estimated using Monte Carlo Damage Simulation (MCDS) software. The repair outcomes are estimated using Monte Carlo excision repair (MCER) simulations. The RBE for cell survival at different oxygen concentrations is calculated using the repair-misrepair-fixation (RMF) model. Using 60Co γ-rays (linear energy transfer (LET) = 2.4 keV/µm) as the reference radiation, the RBE for DSB induction and enzymatic DSB under aerobic condition (21% O2) are in the range 1.0-1.5 and 1.0-1.6 along the track depth, respectively. In accord with RBE obtained from experimental data, RMF model-derived RBE values for cell survival are in the range of 1.0-3.0. The oxygen enhancement ratio (OER) for cell survival (10%) decreases from 3.0 to 2.5 as LET increases from 1.1 to 22.6 keV/µm. The RBE values for severe hypoxia (0.1% O2) are in the range of 1.1-4.4 as LET increases, indicating greater contributions of direct effects for protons. Compared with photon therapy, the overall effect of 62 MeV proton beams results in greater cell death and is further intensified under hypoxic conditions.

The Effects of Dimethylsulfoxide and Oxygen on DNA Damage Induction and Repair Outcomes for Cells Irradiated by 62 MeV Proton and 3.31 MeV Helium Ions.

Reactive oxygen species (ROS) play an essential role in radiation-induced indirect actions. In terms of DNA damage, double strand breaks (DSBs) have the greatest effects on the repair of DNA damage, cell survival and transformation. This study evaluated the biological effects of the presence of ROS and oxygen on DSB induction and mutation frequency. The relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) of 62 MeV therapeutic proton beams and 3.31 MeV helium ions were calculated using Monte Carlo damage simulation (MCDS) software. Monte Carlo excision repair (MCER) simulations were used to calculate the repair outcomes (mutation frequency). The RBE values of proton beams decreased to 0.75 in the presence of 0.4 M dimethylsulfoxide (DMSO) and then increases to 0.9 in the presence of 2 M DMSO while the RBE values of 3.31 MeV helium ions increased from 2.9 to 5.7 (0-2 M). The mutation frequency of proton beams also decreased from 0.008-0.065 to 0.004-0.034 per cell per Gy by the addition of 2 M DMSO, indicating that ROS affects both DSB induction and repair outcomes. These results show that the combined use of DMSO in normal tissues and an increased dose in tumor regions increases treatment efficiency.

Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB inductions and conversions induced by therapeutic proton beams.

Purpose: This study evaluated the DNA double strand breaks (DSBs) induced by indirect actions and its misrepairs to estimate the relative biological effectiveness (RBE) of proton beams.Materials and methods: From experimental data, DSB induction was evaluated in cells irradiated by 62 MeV proton beams in the presence of dimethylsulphoxide (DMSO) and under hypoxic conditions. The DNA damage yields for calculating the RBE were estimated using Monte Carlo Damage Simulation (MCDS) software. The repair outcomes (correct repairs, mutations and DSB conversions) were estimated using Monte Carlo Excision Repair (MCER) simulations.Results: The values for RBE of 62 MeV protons (LET = 1.051 keV/µm) for DSB induction and enzymatic DSB under aerobic condition (21% O2) was 1.02 and 0.94, respectively, as comparing to 60Co γ-rays (LET = 2.4 keV/µm). DMSO mitigated the inference of indirect action and reduced DSB induction to a greater extent when damaged by protons rather than γ-rays, resulting in a decreased RBE of 0.86. DMSO also efficiently prevented enzymatic DSB yields triggered by proton irradiation and reduced the RBE to 0.83. However, hypoxia (2% O2) produced a similar level of DSB induction with respect to the protons and γ-rays, with a comparable RBE of 1.02.Conclusions: The RBE values of proton beams estimated from DSB induction and enzymatic DSB decreased by 16% and 12%, respectively, in the presence of DMSO. Our findings indicate that the overall effects of DSB induction and enzymatic DSB could intensify the tumor killing, while alleviate normal tissue damage when indirect actions are effectively interrupted.

Synthesis and Pharmacological Evaluation of Triazolopyrimidinone Derivatives as Noncompetitive, Intracellular Antagonists for CC Chemokine Receptors 2 and 5.

CC chemokine receptors 2 (CCR2) and 5 (CCR5) are involved in many inflammatory diseases; however, most CCR2 and CCR5 clinical candidates have been unsuccessful. (Pre)clinical evidence suggests that dual CCR2/CCR5 inhibition might be more effective in the treatment of such multifactorial diseases. In this regard, the highly conserved intracellular binding site in chemokine receptors provides a new avenue for the design of multitarget ligands. In this study, we synthesized and evaluated the biological activity of a series of triazolopyrimidinone derivatives in CCR2 and CCR5. Radioligand binding assays first showed that they bind to the intracellular site of CCR2, and in combination with functional assays on CCR5, we explored structure-affinity/activity relationships in both receptors. Although most compounds were CCR2-selective, 39 and 43 inhibited ß-arrestin recruitment in CCR5 with high potency. Moreover, these compounds displayed an insurmountable mechanism of inhibition in both receptors, which holds promise for improved efficacy in inflammatory diseases.

Effects of indirect actions and oxygen on relative biological effectiveness: estimate of DSB induction and conversion induced by gamma rays and helium ions.

Clustered DNA damage other than double-strand breaks (DSBs) can be detrimental to cells and can lead to mutagenesis or cell death. In addition to DSBs induced by ionizing radiation, misrepair of non-DSB clustered damage contributes extra DSBs converted from DNA misrepair via pathways for base excision repair and nucleotide excision repair. This study aimed to quantify the relative biological effectiveness (RBE) when DSB induction and conversion from non-DSB clustered damage misrepair were used as biological endpoints. The results showed that both linear energy transfer (LET) and indirect action had a strong impact on the yields for DSB induction and conversion. RBE values for DSB induction and maximum DSB conversion of helium ions (LET = 120 keV/µm) to (60)Co gamma rays were 3.0 and 3.2, respectively. These RBE values increased to 5.8 and 5.6 in the absence of interference of indirect action initiated by addition of 2-M dimethylsulfoxide. DSB conversion was ∼1-4% of the total non-DSB damage due to gamma rays, which was lower than the 10% estimate by experimental measurement. Five to twenty percent of total non-DSB damage due to helium ions was converted into DSBs. Hence, it may be possible to increase the yields of DSBs in cancerous cells through DNA repair pathways, ultimately enhancing cell killing.

Fast Monte Carlo simulation of DNA damage induction by Auger-electron emission.

PURPOSE: A local damage model (LDM) was developed to estimate the biological efficiency of Auger-electron-emitting radionuclides. MATERIALS AND METHODS: The LDM required information on the local dose distribution, local energy spectrum, and clustered DNA damage yields in the cell nucleus. To apply the model, the nucleus was divided into concentric shells where each shell contributed its own local dose, energy spectrum, and damage yield. The local doses and energy spectra were computed using the PENELOPE (PENetration and Energy LOss of Positrons and Electrons) code. The DNA damage yields were estimated using the MCDS (Monte Carlo damage simulation) code. RESULTS: For a typical 4-µm radius mammalian cell nucleus, the absorbed doses to the cell nucleus per unit cumulated activity, equal to 0.0065, 0.00418, 0.0028, 0.0027 and 0.0015 Gy Bq(-1) s(-1) for (125)I, (119)Sb, (123)I, (111)In and (99m)Tc, were within 6% difference with the MIRD (Medical Internal Radiation Dose) published data. The simulated total (simple and complex) single-strand break (SSB) and double-strand break (DSB) yields were in the same order, i.e., (125)I > (119)Sb > (123)I > (111)In > (99m)Tc. The agreement between present results and literature data for the DNA damage yields was generally good. More than 75% of the total SSB and DSB yields were contributed from regions within 2.5 µm of the nucleus center. CONCLUSIONS: The proposed methodology was computationally efficient and could be applied to other irradiation geometries such as cell clusters.

Residues involved in the catalysis, base specificity, and cytotoxicity of ribonuclease from Rana catesbeiana based upon mutagenesis and X-ray crystallography.

The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by site-directed mutagenesis, k(cat)/K(m) analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase.d(ACGA) complex was determined at 1.80 A resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P(1) site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B(1) base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B(2) site and determine B(2) base specificity.