Direct measurement of oxidative and nitrosative stress dynamics in Salmonella inside macrophages

Many significant bacterial pathogens have evolved virulence mechanisms to evade degradation and exposure to reactive oxygen (ROS) and reactive nitrogen species (RNS), allowing them to survive and replicate inside their hosts. Due to the highly reactive and short-lived nature of ROS and RNS, combined with limitations of conventional detection agents, the mechanisms underlying these evasion strategies remain poorly understood. In this study, we describe a system that uses redox-sensitive GFP to nondisruptively measure real-time fluctuations in the intrabacterial redox environment. Using this system coupled with high-throughput microscopy, we report the intrabacterial redox dynamics of Salmonella enterica Typhimurium (S. Typhimurium) residing inside macrophages. We found that the bacterial SPI-2 type III secretion system is required for ROS evasion strategies and this evasion relies on an intact Salmonella-containing vacuole (SCV) within which the bacteria reside during infection. Additionally, we found that cytosolic bacteria that escape the SCV experience increased redox stress in human and murine macrophages. These results highlight the existence of specialized evasion strategies used by intracellular pathogens that either reside inside a vacuole or “escape” into the cytosol. Taken together, the use of redox-sensitive GFP inside Salmonella significantly advances our understanding of ROS and RNS evasion strategies during infection. This technology can also be applied to measuring bacterial oxidative and nitrosative stress dynamics under different conditions in a wide variety of bacteria.A central mechanism of the innate immune response to defend against pathogens is the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) by specialized phagocytic immune cells (1). Macrophages and neutrophils generate ROS after detection of pathogen-associated molecular patterns (PAMPs) through the NADPH oxidase complex, whereas RNS is produced by inducible nitric oxide synthase (iNOS), which generates NO• through the conversion of l-arginine and oxygen (1–3).To survive, replicate, and disseminate throughout the body, bacterial pathogens—especially those that reside in intracellular niches—must overcome the antimicrobial oxidative and nitrosative burst (1). General ROS and RNS enzymes such as catalases, peroxidases, superoxide dismutases, and DNA repair enzymes are used by most bacteria to survive exposure to ROS and RNS (4). In addition to these general defenses, many intracellular pathogens have evolved specific evasion strategies that allow them to live inside their host cells. For example, the intracellular pathogens Shigella flexneri and Listeria monocytogenes, which cause shigellosis and listeriosis, respectively, “escape” the phagolysosome to proliferate within the cytosol of macrophages. In contrast, the Gram-negative pathogen Salmonella enterica subsp. Typhimurium (S. Typhimurium), which is a major cause of gastroenteritis and some systemic diseases, remains inside a specific Salmonella-containing vacuole (SCV), where it injects bacterial effector proteins directly into the host cell through a type III secretion system (T3SS). A specific set of type III effectors associated with a Salmonella pathogenicity island (SPI), known as SPI-2 effectors, have been implicated in ROS and RNS evasion strategies (5, 6); however, the relationship between SPI-2 and oxidative stress evasion is a contentious topic after a recent study concluded that the contribution of SPI-2 to Salmonella pathogenesis is unrelated to its interaction with oxidative stress (7). Inadequate analytic tools for directly measuring ROS/RNS and rapid fluctuations due to the short-lived nature of ROS/RNS have also contributed to poor reproducibility in the studies of ROS and RNS evasion strategies (1, 8, 9).Herein, we describe a previously unidentified use of a redox-sensitive GFP (roGFP2) that has been engineered to form a reversible disulfide bond upon oxidation or S-nitrosylation of specific cysteines. Formation of the disulfide bond leads to a slight shift in its conformation (Fig. S1A) resulting in two isoforms (roGFP2_ox and roGFP2_red) of roGFP2. These isoforms have distinct excitation spectra with specific fluorescence signals after excitation at 405 and 480 nm, respectively (10). Consequently, the 405/480-nm ratio can be used as a measure for the roGFP2_ox/roGFP2_red ratio, which corresponds with the intrabacterial redox potential (11). Because roGFP2 reports the redox potential by ratiometric analysis, this system excludes variations due to differences in roGFP2 concentrations. Until now, redox-sensitive GFP has almost exclusively been used in eukaryotes (12). In this study, we used roGFP2 to measure the intrabacterial redox potential in bacteria (Salmonella) after challenges with exogenous oxidative agents and during infection of HeLa cells (epithelial), THP-1 cells (monocytic), and bone marrow-derived macrophages (BMDMs) from mice. Finally, with the use of high-throughput microscopy, we demonstrated the involvement of the SPI-2 T3SS in ROS evasion strategies, as well as the requirement for an intact SCV to evade ROS and RNS inside macrophages.     

Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells

Ras/ERK signaling plays an important role in T cell activation and development. We recently reported that endothelial nitric oxide synthase (eNOS)-derived NO regulates T cell receptor (TCR)-dependent ERK activation by a cGMP-independent mechanism. Here, we explore the mechanisms through which eNOS exerts this regulation. We have found that eNOS-derived NO positively regulates Ras/ERK activation in T cells stimulated with antigen on antigen-presenting cells (APCs). Intracellular activation of N-, H-, and K-Ras was monitored with fluorescent probes in T cells stably transfected with eNOS-GFP or its G2A point mutant, which is defective in activity and cellular localization. Using this system, we demonstrate that eNOS selectively activates N-Ras but not K-Ras on the Golgi complex of T cells engaged with APC, even though Ras isoforms are activated in response to NO from donors. We further show that activation of N-Ras involves eNOS-dependent S-nitrosylation on Cys¹¹⁸, suggesting that upon TCR engagement, eNOS-derived NO directly activates N-Ras on the Golgi. Moreover, wild-type but not C118S N-Ras increased TCR-dependent apoptosis, suggesting that S-nitrosylation of Cys¹¹⁸ contributes to activation-induced T cell death. Our data define a signaling mechanism for the regulation of the Ras/ERK pathway based on the eNOS-dependent differential activation of N-Ras and K-Ras at specific cell compartments.     

The crucial role of polymorphonuclear leukocytes in resistance to Salmonella dublin infections in genetically susceptible and resistant mice

Macrophages are considered to be the mediators of resistance to extra-intestinal Salmonella infections. Nevertheless, the initial cellular response to Salmonella infections consists primarily of polymorphonuclear leukocytes (PMN). To determine whether PMN serve an important function for the infected host, we made mice neutropenic with the rat mAb to RB6–8C5 and infected them i.v. with ≈10³ Salmonella dublin or an isogenic derivative that lacks the virulence plasmid (LD842). We infected BALB/c mice, which have a point mutation in the macrophage-expressed gene Nramp1 that makes them susceptible to Salmonella, and BALB/c.D2 congenic mice, which have the wild-type Nramp1 gene that makes them resistant to Salmonella. Both mouse strains were resistant to LD842, and neutropenia made only the BALB/c strain susceptible to this infection. Neutropenic congenic mice, however, were susceptible only to wild-type S. dublin (plasmid+). These results show a complex interplay between plasmid-virulence genes in Salmonella, host macrophages, and PMN. Mice with normal macrophages need PMN to defend against nontyphoid Salmonella that carry a virulence plasmid but not against Salmonella without virulence plasmids. Mice with a mutant Nramp1 gene need PMN to defend against all Salmonella, even those that lack virulence plasmids. These results, plus the evidence that PMN kill Salmonella efficiently in vitro, suggest that Salmonella have adapted to grow inside macrophages where they are relatively sheltered from PMN. The adaptations that allow Salmonella to survive in macrophages do not protect them from PMN.     

A novel KCNQ1 variant (L203P) associated with torsades de pointes-related syncope in a Steinert syndrome patient

Background

A 43-year-old woman suffering from Steinert syndrome was admitted after experiencing multiple episodes of torsades de pointes-related syncope.

Objectives

To elucidate the pathophysiology of these arrhythmic events.

Methods and Results

We obtained DNA from the patient and sequenced the coding region of KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. A single nucleotide change was identified in the KCNQ1 gene at position 608 (T608C), resulting in a substitution from leucine to proline at position 203 (L203P). CHO cells were used to express either wild-type KCNQ1, wild-type KCNQ1+L203P KCNQ1 (50:50), or L203P KCNQ1, along with KCNE1 to recapitulate the slow cardiac delayed rectifier potassium current (I_Ks). Patch-clamp experiments showed that the variant L203P causes a dominant negative effect on I_Ks. Coexpression of wild-type KCNQ1 and L203P KCNQ1 (50:50) caused a ~75% reduction in current amplitude when compared to wild-type KCNQ1 alone (131.40 ± 23.27 vs 567.25 ± 100.65 pA/pF, P < .001). Moreover, when compared with wild-type KCNQ1 alone, the coexpression of wild-type KCNQ1 and L203P KCNQ1 (50:50) caused a 7.5-mV positive shift of midpoints of activation (from 27.5 ± 2.4 to 35.1 ± 1.2 mV, P < .05). The wild-type KCNQ1 and L203P KCNQ1 (50:50) coexpression also caused alteration of I_Ks kinetics. The activation kinetics of the L203P variant (50:50) were slowed compared with wild-type KCNQ1, while the deactivation kinetics of L203P (50:50) were accelerated compared with wild type, all these further contributing to the “loss-of-function” phenotype of I_Ks associated with the variant L203P.

Conclusion

Torsades de pointes and episodes of syncope are very likely to be due to the KCNQ1 variant L203P found in this patient.     

Single nucleotide polymorphisms in the promoter region of the IL1B gene influence outcome in multiple myeloma patients treated with high-dose chemotherapy independently of relapse treatment with thalidomide and bortezomib

Little is known on the impact of polymorphisms in the IL1B gene on outcome in multiple myeloma. In a population-based study of 348 Danish myeloma patients treated with high-dose treatment (HDT), 146 patients treated with INF-?? maintenance treatment, and in 243 patients with relapse, we analysed the impact on outcome of HDT, INF-?? maintenance treatment, and treatment with thalidomide and bortezomib at relapse, in relation to the major identified functional polymorphisms in the promoter region of IL1B. The wild-type C-allele of IL1B C-3737T and non-carriage of the IL1B promoter haplotype TGT (?3737^T, ?1464^G and ?31^T), giving high IL1B promoter activity, were associated with longer time-to-treatment failure (TTF) (HR, 1.4 (1.0?C1.9) and 1.5 (1.1?C2.0)) and overall survival (HR, 1.8 (1.2?C2.6) and 1.6 (1.1?C2.3)) after HDT. Among INF-?? treated patients, a trend towards better TTF was found in patients carrying the wild-type C-allele of IL1B C-3737T (HR, 1.6 (1.1?C2.4)). Furthermore, among INF-?? treated patients, gene?Cgene interaction studies on IL1B C-3737T and NF??B1-94ins/del ATTG revealed a fourfold increase in TTF for homozygous carriers of wild-type alleles at both loci as compared to variant allele carriers at both loci. No relation to genotype and outcome was found for relapse patients treated with thalidomide or bortezomib. Our results indicate that a subpopulation of myeloma patients carrying the wild-type C-allele of IL1B C-3737T and non-carriers of the promoter haplotype TGT (?3737^T, ?1464^G and ?31^T) benefit from a better outcome of HDT and INF-?? treatment, an effect that may be related to the NF-??B pathway.     

Up-regulation of inducible nitric oxide synthase expression in cancer-prone p53 knockout mice

High concentrations of nitric oxide (NO) cause DNA damage and apoptosis in many cell types. Thus, regulation of NO synthase (NOS) activity is essential for minimizing effects of cytotoxic and genotoxic nitrogen oxide species. We have shown previously that NO-induced p53 protein accumulation down-regulates basal and cytokine-modulated inducible NOS (NOS2) expression in human cells in vitro. To further characterize the feedback loop between NOS2 and p53, we have investigated NO production, i.e., urinary nitrate plus nitrite excretion, and NOS2 expression in homozygous p53 knockout (KO) mice. We report here that untreated p53 KO mice excreted 70% more nitrite plus nitrate than mice with wild-type (wt) p53. NOS2 protein expression was constitutively detected in the spleen of untreated p53 KO mice, whereas it was undetectable in the spleen of wt p53 controls. Upon treatment with heat-inactivated Corynebacterium parvum, urinary nitrite plus nitrate excretion of p53 KO mice exceeded that of wt controls by approximately 200%. C. parvum treatment also induced p53 accumulation in the liver. Splenectomy reduced the NO output of C. parvum-treated p53 KO mice but not of wt p53 controls. Although NO production and NOS2 protein expression were increased similarly in KO and wt p53 mice 10 days after injection of C. parvum, NOS2 expression returned to baseline levels only in wt p53 controls while remaining up-regulated in p53 KO mice. These genetic and functional data indicate that p53 is an important transrepressor of NOS2 expression in vivo and attenuates excessive NO production in a regulatory negative feedback loop.     

The structural role of the zinc ion can be dispensable in prokaryotic zinc-finger domains

The recent characterization of the prokaryotic Cys₂His₂ zinc-finger domain, identified in Ros protein from Agrobacterium tumefaciens, has demonstrated that, although possessing a similar zinc coordination sphere, this domain is structurally very different from its eukaryotic counterpart. A search in the databases has identified ≈300 homologues with a high sequence identity to the Ros protein, including the amino acids that form the extensive hydrophobic core in Ros. Surprisingly, the Cys₂His₂ zinc coordination sphere is generally poorly conserved in the Ros homologues, raising the question of whether the zinc ion is always preserved in these proteins. Here, we present a functional and structural study of a point mutant of Ros protein, Ros_56–142C82D, in which the second coordinating cysteine is replaced by an aspartate, 5 previously-uncharacterized representative Ros homologues from Mesorhizobium loti, and 2 mutants of the homologues. Our results indicate that the prokaryotic zinc-finger domain, which in Ros protein tetrahedrally coordinates Zn(II) through the typical Cys₂His₂ coordination, in Ros homologues can either exploit a CysAspHis₂ coordination sphere, previously never described in DNA binding zinc finger domains to our knowledge, or lose the metal, while still preserving the DNA-binding activity. We demonstrate that this class of prokaryotic zinc-finger domains is structurally very adaptable, and surprisingly single mutations can transform a zinc-binding domain into a nonzinc-binding domain and vice versa, without affecting the DNA-binding ability. In light of our findings an evolutionary link between the prokaryotic and eukaryotic zinc-finger domains, based on bacteria-to-eukaryota horizontal gene transfer, is discussed.     

Single crystal spectroscopy and multiple structures from one crystal (MSOX) define catalysis in copper nitrite reductases

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br^2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1Cu-T2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no inter-Cu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins.

Redox reactions are an essential component in a wide range of biological systems, most notably in respiration (1) and photosynthesis (2). These critical reactions are often performed by metal-containing systems including metalloproteins that form a large portion of the protein kingdom. It is estimated that one-third of all proteins in nature require metals to perform their biological roles and nearly one-half of all enzymes must associate with a particular metal to function (3, 4). These metal ions can be either a single atom or combined with other atoms to form part of a cluster, playing a variety of life sustaining roles in the bacterial, plant, and animal kingdoms. They exploit the oxidation states of metals to perform redox cycling during catalysis and include metalloenzymes such as cytochrome c oxidase, hydrogenases, nitrogenases, and nitrite reductases where catalysis involves the controlled delivery of electrons and protons to the active site for the utilization of chemical substrates. Most of these systems utilize multiple redox centers among which these catalytic events are often coordinated, coupled, and orchestrated by structural signals that remain poorly understood. The redox-active metalloenzyme copper nitrite reductase (CuNiR) has become a good model system for studying these complex processes in biological systems with coupled redox centers due to their amenability to spectroscopic, fast kinetic, and advanced structural approaches capable of providing a movie of catalytic reaction activated by electron transfer (ET) in crystallo between the coupled centers and its utilization for the conversion of substrate providing information on reaction intermediates, some of which may be transitory (5–9).CuNiRs catalyze the conversion of nitrite to gaseous nitric oxide (NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O) in the first committed step of the denitrification pathway. This highly conserved family of enzymes is widespread in nature (10) and is of major importance in several pathways in the biogeochemical nitrogen cycle (11, 12). These homo-trimeric proteins contain two types of redox Cu center per monomer, as follows: an electron accepting type-1 Cu (T1Cu) site, which receives an electron from a physiological redox partner (cytochrome c or pseudoazurin/azurin), and a catalytic type-2 Cu (T2Cu) site. The T1Cu site is located near the top of the monomer and is responsible for giving rise to the color of the enzyme (blue or green) in its oxidized [Cu^II] state, depending on subtle differences in the immediate coordination chemistry of the site. The T2Cu is located within the interface of two adjacent monomers and is responsible for the binding of nitrite and its catalysis. The two redox Cu centers are coupled via the neighboring Cys-His residues that form a conserved hard-wired 12.6-Å ET bridge. When T2Cu is in the oxidized substrate-free [Cu^II] state, with a Cu^II-(His)₃-H₂O coordination, the difference between the reduction potentials of the two Cu sites are small or energetically unfavorable to allow ET from T1Cu to T2Cu. Displacement of the coordinated water by nitrite increases the reduction potential of T2Cu and promotes inter-Cu ET, an event heavily gated by the provision of protons from two conserved residues (His_CAT and Asp_CAT) in the T2Cu pocket to the substrate (5, 13). In the CuNiR of Achromobacter xylosoxidans (AxNiR), laser flash photolysis had shown the rate of inter-Cu ET to be the same as the rate of proton uptake, providing clear evidence for proton-coupled ET (PCET). Following the reduction of nitrite to nitric oxide (NO), the product dissociates from the T2Cu site and water rebinds to return to the resting T2Cu^II state. A delicate and ordered mechanism ensures a “dead-end” inactive species is not formed where the T2Cu redox center is in a reduced [Cu^I] state with the water ligand disociated, that is then unable to bind the substrate (14, 15). This mechanism for CuNiR is supported by spectroscopic, kinetic, mutagenesis, and structural studies primarily from the well-studied blue AxNiR and the green CuNiRs of Achromobacter cycloclastes (AcNiR) and Alcaligenes faecalis (AfNiR) (13–18).Biological systems containing redox centers, including metalloproteins such as CuNiR, are prone to reduction from X-ray sources during data collection (19, 20) due to solvated electrons within the crystal being produced from radiolysis of waters that can rapidly reduce their primary redox centers, e.g., T1Cu in CuNiRs is reduced from Cu^II to Cu^I redox state but the T2Cu remains oxidized (17). This phenomenon has been exploited to initiate enzyme turnover to generate reaction intermediates through redox driven catalysis (21). We have developed the MSOX (multiple structures from one crystal) approach that enables the collection of several structures from the same spot in a crystal (6–8). This approach has been applied successfully to AcNiR at several temperatures to provide structural movies of the catalytic reaction in nitrite-soaked crystals. However, these studies have not provided any information on the redox state of metal centers during catalysis in the crystals.A two-domain blue CuNiR from a Rhizobia species (Br^2DNiR) has recently been structurally characterized showing an unusual oxidized T2Cu^II-(His)₃-(H₂O)₂ coordination site, requiring the displacement of two water molecules by the substrate (22, 23) instead of a single water molecule in a prototypic CuNiR (15, 24). The recent availability of highly diffracting crystals of Br^2DNiR and the ability to achieve full occupancy of nitrite at the T2Cu site in nitrite-soaked crystals (22) prompted us to utilize the MSOX approach to probe structural changes during enzyme turnover. MSOX of as-isolated crystals of Br^2DNiR was combined with the recently commissioned on-line single crystal optical spectroscopy at SPring-8 BL26B1, enabling the redox status of the optically visible T1Cu to be monitored during serially recorded multiple structures and reporting on the structural changes at the catalytic T2Cu site for a CuNiR in the resting state. This has enabled us to provide a spectroscopically validated MSOX movie of an as-isolated CuNiR. We have combined detailed structural observations recorded in these MSOX movies and single crystal spectroscopy with solution measurements of reduction potentials and inter-Cu ET using laser-flash photolysis. We provide unambiguous evidence for a strong gating of ET between the coupled redox centers that is removed in the presence of the substrate. We demonstrate that the experimental approach (marrying single crystal spectroscopy/MSOX with solution data) utilized here is powerful in dissecting complex redox reactions and suggest the approach should be applicable to many complex redox systems in biology.     

HFE S65C Variant Is Not Associated with Increased Transferrin Saturation in Voluntary Blood Donors

ABSTRACT: Two amino acid variants in the HFE gene, C282Y and H63D, have been reported in most cases of hereditary hemochromatosis. A recently discovered novel amino acid variant of HFE, namely S65C, has been implicated to be responsible for a mild form of iron overload.We determined genotypes of the HFE S65C variant in 230 voluntary blood donors with a transferrin saturation >45%, who did not carry the HFE C282Y variant. The control group consisted of 248 first time blood donors who had a transferrin saturation < 45%. We also determined genotypes of the HFE H63D variant in the two groups.For the HFE S65C variant, the frequency of the HFE C65 allele was 1.7% and 2.2% in the high and low transferrin saturation groups, respectively (p = 0.65). In contrast, for the HFE H63D variant, the frequency of the HFE D63 allele was 24.8% and 14.7% in the high and low transferrin saturation groups, respectively (p = 0.0009).This study demonstrates no association of the HFE C65 allele with the phenotype of high transferrin saturation. The results do not support the use of DNA genotyping for the HFE S65C mutation in population screening studies for hemochromatosis.     

Identification of a urate transporter,ABCG2, with a common functional polymorphism causing gout

Genome-wide association studies (GWAS) have successfully identified common single nucleotide polymorphisms (SNPs) associated with a wide variety of complex diseases, but do not address gene function or establish causality of disease-associated SNPs. We recently used GWAS to identify SNPs in a genomic region on chromosome 4 that associate with serum urate levels and gout, a consequence of elevated urate levels. Here we show using functional assays that human ATP-binding cassette, subfamily G, 2 (ABCG2), encoded by the ABCG2 gene contained in this region, is a hitherto unknown urate efflux transporter. We further show that native ABCG2 is located in the brush border membrane of kidney proximal tubule cells, where it mediates renal urate secretion. Introduction of the mutation Q141K encoded by the common SNP rs2231142 by site-directed mutagenesis resulted in 53% reduced urate transport rates compared to wild-type ABCG2 (P < 0.001). Data from a population-based study of 14,783 individuals support rs2231142 as the causal variant in the region and show highly significant associations with urate levels [whites: P = 10⁻³⁰, minor allele frequency (MAF) 0.11; blacks P = 10⁻⁴, MAF 0.03] and gout (adjusted odds ratio 1.68 per risk allele, both races). Our data indicate that at least 10% of all gout cases in whites are attributable to this causal variant. With approximately 3 million US individuals suffering from often insufficiently treated gout, ABCG2 represents an attractive drug target. Our study completes the chain of evidence from association to causation and supports the common disease-common variant hypothesis in the etiology of gout.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:In vitro evolution of horse heart myoglobin to increase peroxidase activity

Random mutagenesis and screening for enzymatic activity has been used to engineer horse heart myoglobin to enhance its intrinsic peroxidase activity. A chemically synthesized gene encoding horse heart myoglobin was subjected to successive cycles of PCR random mutagenesis. The mutated myoglobin gene was expressed in Escherichia coli LE392, and the variants were screened for peroxidase activity with a plate assay. Four cycles of mutagenesis and screening produced a series of single, double, triple, and quadruple variants with enhanced peroxidase activity. Steady-state kinetics analysis demonstrated that the quadruple variant T39I/K45D/F46L/I107F exhibits peroxidase activity significantly greater than that of the wild-type protein with k₁ (for H₂O₂ oxidation of metmyoglobin) of 1.34 × 10⁴ M⁻¹ s⁻¹ (≈25-fold that of wild-type myoglobin) and k₃ [for reducing the substrate (2, 2′-azino-di-(3-ethyl)benzthiazoline-6-sulfonic acid] of 1.4 × 10⁶ M⁻¹ s⁻¹ (1.6-fold that of wild-type myoglobin). Thermal stability of these variants as measured with circular dichroism spectroscopy demonstrated that the T_m of the quadruple variant is decreased only slightly compared with wild-type (74.1°C vs. 76.5°C). The rate constants for binding of dioxygen exhibited by the quadruple variant are identical to the those observed for wild-type myoglobin (k_on, 22.2 × 10⁻⁶ M⁻¹ s⁻¹ vs. 22.3 × 10⁻⁶ M⁻¹ s⁻¹; k_off, 24.3 s⁻¹ vs. 24.2 s⁻¹; K_O2, 0.91 × 10⁻⁶ M⁻¹ vs. 0.92 × 10⁻⁶ M⁻¹). The affinity of the quadruple variant for CO is increased slightly (k_on, 0.90 × 10⁻⁶ M⁻¹s⁻¹ vs. 0.51 × 10⁻⁶ M⁻¹s⁻¹; k_off, 5.08 s⁻¹ vs. 3.51 s⁻¹; K_CO, 1.77 × 10⁻⁷ M⁻¹ vs. 1.45 × 10⁻⁷ M⁻¹). All four substitutions are in the heme pocket and within 5 Å of the heme group.     

Genetic basis of multidrug resistance in Salmonella enterica serovars Enteritidis and Typhimurium isolated from diarrheic calves in Egypt

Up to this date, nothing is known about the molecular basis of antimicrobial resistance in Salmonella isolated from animals in Africa. Therefore, this study was carried out to screen the incidence of multidrug-resistant (MDR) strains of Salmonella from neonatal calf diarrhea in Egypt and also to characterize the molecular basis of this resistance. Nine unique Salmonella isolates were obtained from 220 fecal samples, and six of these showed multidrug resistance phenotypes and harbored at least two antimicrobial resistance genes. Four were Salmonellaenterica serovar Typhimurium and two were S.enterica serovar Enteritidis. Class 1 integrons were identified in all MDR Salmonella isolates. The identified gene cassettes within class 1 integrons were as follows; aminoglycoside adenyltransferase type A (aadA1, aadA2 and aadA5), which confer resistance to streptomycin and spectinomycin, and dihydrofolate reductase gene cassettes (dfrA1, dfrA15 and dfrA15), which confer resistance to trimethoprim. A class 2 integron containing dfrA1-sat2-aadA1 gene cassettes was identified in only one isolate of S. enterica serovar Enteritidis. The β-lactamase-encoding gene, bla_TEM-1, was identified in five isolates and the extended-spectrum β-lactamase-encoding genes, bla_CMY-2 and bla_SHV-12, were identified in S. enterica serovar Typhimurium. Furthermore, the plasmid-mediated quinolone resistance genes, qnrB, qnrS and aac(6′)-Ib-cr, were also identified. To the best of our knowledge, this is the first report of qnrS in S. enterica serovar Enteritidis, qnrB in S. enterica serovar Typhimurium, and aac(6′)-Ib-cr in Salmonella of animal origin. Also, this is the first report of the molecular characterization of antimicrobial resistance in Salmonella isolated from animals in Africa.     

Gly482Ser polymorphism in the peroxisome proliferator-activated receptor γ coactivator-1α gene is associated with oxidative stress and abdominal obesity

The objective of the study was to o investigate the relationship of the Gly482Ser (G482S) polymorphism in the peroxisome proliferator-activated receptor γ coactivator-1α (PPARGC1A) gene and type 2 diabetes mellitus (T2DM), obesity, and oxidative status in Chinese adults. We enrolled 276 T2DM patients and 1049 nondiabetic subjects aged at least 35 years. The G482S variant was detected using polymerase chain reaction and restriction enzyme digestion. The levels of thiobarbituric acid reactive substance, an indicator of lipid peroxidation, were measured in plasma samples. The homeostasis model assessment-estimated insulin resistance (HOMA-IR) index was determined for nondiabetic subjects. P values were adjusted for age, sex, and body mass index by using a generalized linear model. In this series, there was no association between G482S polymorphism and T2DM and obesity (body mass index >25 kg/m²). However, the plasma fasting insulin levels and HOMA-IR indices were significantly higher in nondiabetic subjects harboring the variant (S/S) genotype than in those with the heterozygous (G/S) genotype. With regard to the effect of the different genotypes on body fat distribution, overweight nondiabetic subjects harboring the S/S or G/S genotype had a significantly higher waist-to-hip ratio than those with the wild-type (G/G) genotype. Furthermore, subjects with the S/S genotype had significantly higher serum thiobarbituric acid reactive substance levels than those with the G/G genotype; the diabetic group mainly contributed to this significant association (P < .001). In overweight, nondiabetic Chinese adults, G482S polymorphism in the PPARGC1A gene is associated with hyperinsulinemia, HOMA-IR indices, and abdominal obesity. Furthermore, in hyperglycemia, the S482 allele is related to increased oxidative stress.     

Two-component kinase-like activity of nm23 correlates with its motility-suppressing activity

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1^S12OG and nm23-H1^P96S. Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1^P96S or nm23-H1^S120G, and another was phosphorylated much more slowly by nm23-H1^P96S and by nm23-H1^S120G than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATP-citrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1^S120G and nm23-H1^P96S were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1^S120 was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1^P96S was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.