Differential recognition of Staphylococcus aureus quorum-sensing signals depends on both extracellular loops 1 and 2 of the transmembrane sensor AgrC.

Virulence in Staphylococcus aureus is regulated via agr-dependent quorum sensing in which an autoinducing peptide (AIP) activates AgrC, a histidine protein kinase. AIPs are usually thiolactones containing seven to nine amino acid residues in which the thiol of the central cysteine is linked to the alpha-carboxyl of the C-terminal amino acid residue. The staphylococcal agr locus has diverged such that the AIPs of the four different S. aureus agr groups self-activate but cross-inhibit. Consequently, although the agr system is conserved among the staphylococci, it has undergone significant evolutionary divergence whereby to retain functionality, any changes in the AIP-encoding gene (agrD) that modifies AIP structure must be accompanied by corresponding changes in the AgrC receptor. Since AIP-1 and AIP-4 only differ by a single amino acid, we compared the transmembrane topology of AgrC1 and AgrC4 to identify amino acid residues involved in AIP recognition. As only two of the three predicted extracellular loops exhibited amino acid differences, site-specific mutagenesis was used to exchange the key AgrC1 and AgrC4 amino acid residues in each loop either singly or in combination. A novel lux-based agrP3 reporter gene fusion was constructed to evaluate the response of the mutated AgrC receptors. The data obtained revealed that while differential recognition of AIP-1 and AIP-4 depends primarily on three amino acid residues in loop 2, loop 1 is essential for receptor activation by the cognate AIP. Furthermore, a single mutation in the AgrC1 loop 2 resulted in conversion of (Ala5)AIP-1 from a potent antagonist to an activator, essentially resulting in the forced evolution of a new AIP group. Taken together, our data indicate that loop 2 constitutes the predicted hydrophobic pocket that binds the AIP thiolactone ring while the exocyclic amino acid tail interacts with loop 1 to facilitate receptor activation.

Diastereoselective formation of 2-aryl-3-arenesulfonyl 4-ethyl-1,3-oxazolidines: an X-ray crystallographic and 1H NMR study

N-Arylsulfonamides of (R)- and (S)-2-amino-1-butanol, on condensation with aromatic aldehydes produced diastereomerically pure 2-aryl-3-arenesulfonyl 4-ethyl-1,3-oxazolidines. The absolute configurations of one enantiomeric pair have been determined from two fully refined X-ray structures, supplemented by nmr data.

Synthesis, X-ray crystal structure and reactivity of the polymeric copper(II) dicarboxylic acid complex {Cu2(η1η1μ2-oda)2(py)4(H2O)2}n(odaH2 = octanedioic acid, PY = pyridine)

An aqueous solution of the α-ω-dicarboxylic acid octanedioic acid (odaH2) reacts with [Cu2(μ-O2CCH3)4(H2O)2] in the presence of an excess of pyridine (py) to give the crystalline copper(II) complex {Cu2(η1η1μ2-oda)2(py)4(H2O)2}n (1). structure of 1, as determined by X-ray crystallography, consists of polymeric chains in which bridging oda2− anions link two crystallographically identical copper atoms. The copper atoms are also ligated by two transoidal pyridine nitrogens and an oxygen atom from an apical water molecule, giving the metals an overall N2O3 square-pyramidal geometry. If the blue solid 1 is gently heated, or if it is left to stand in its mother liquor for prolonged periods, it loses one molecule of pyridine and half a molecule of water and the green complex {Cu (oda)(py)(H2O)0.5}n (2) is formed. Spectroscopic and magnetic data for both complexes are given, together with the electrochemical and thermogravimetric measurements for 1.

Synthesis and structure of the Mn II,II complex salt [Mn2(η1η1μ2-oda)(phen)4(H2O)2][Mn2][Mn2(η1η1μ2-oda)(phen)4(η1-oda)2].4H2O (odah2 = octanedioic acid): a catalyst for H2O2 disproportionation

The synthesis and X-ray crystal structure of the MnII,11 complex double salt [Mn2(η1η1µ2-oda)(phen)4(H2O)2][Mn2(η1η1µ2-oda(phen)4(η1-oda)2]·4H2O is reported, together with its catalytic activity towards the disproportionation of H2O2.

Synthesis of the μ-PH hexaosmium clusters [Os6H2(CO)20L(μ3-PH)](L = CO or MeCN) and their reactions with base: X-ray crystal structures of [Os6H2(CO)20(MeCN)(μ3-PH)] and [Os6H3(CO)19(CO2Me)(μ3-PH)]

Mild heating of the phosphidotriosmium cluster [Os3H(CO)10(µ2-PH2)](1) with [Os3(CO)12 –n(MeCN)n](n= 1 or 2) gives high yields of the (µ3-PH) bridged hexaosmium clusters (2) and (3); reactions of (2) and (3) with bases and X-ray structure analyses of (3) and of (6), which was obtained from (3) and MeO– followed by acid treatment are described.

Low-level Laser Therapy Improves Skeletal Muscle Performance, Decreases Skeletal Muscle Damage and Modulates mRNA Expression of COX-1 and COX-2 in a Dose-dependent Manner

We tested if modulation in mRNA expression of cyclooxygenase isoforms (COX-1 and COX-2) can be related to protective effects of phototherapy in skeletal muscle. Thirty male Wistar rats were divided into five groups receiving either one of four laser doses (0.1, 0.3, 1.0 and 3.0 J) or a no-treatment control group. Laser irradiation (904 nm, 15 mW average power) was performed immediately before the first contraction for treated groups. Electrical stimulation was used to induce six tetanic tibial anterior muscle contractions. Immediately after sixth contraction, blood samples were collected to evaluate creatine kinase activity and muscles were dissected and frozen in liquid nitrogen to evaluate mRNA expression of COX-1 and COX-2. The 1.0 and 3.0 J groups showed significant enhancement (P < 0.01) in total work performed in six tetanic contractions compared with control group. All laser groups, except the 3.0 J group, presented significantly lower post-exercise CK activity than control group. Additionally, 1.0 J group showed increased COX-1 and decreased COX-2 mRNA expression compared with control group and 0.1, 0.3 and 3.0 J laser groups (P < 0.01). We conclude that pre-exercise infrared laser irradiation with dose of 1.0 J enhances skeletal muscle performance and decreases post-exercise skeletal muscle damage and inflammation.

Extracellular Signal-Regulated Kinase 1/2 Activation, via Downregulation of Mitogen-Activated Protein Kinase Phosphatase 1, Mediates Sex Differences in Desoxycorticosterone Acetate-Salt Hypertension Vascular Reactivity

Extracellular signal-regulated kinase (ERK) 1/2 has been reported to play a role in vascular dysfunction associated with mineralocorticoid hypertension. We hypothesized that, compared with female rats, an upregulation of ERK1/2 signaling in the vasculature of male rats contributes to augmented contractile responses in mineralocorticoid hypertension. Uninephrectomized male and female Sprague-Dawley rats received desoxycorticosterone acetate (DOCA) pellets (200 mg per animal) and saline to drink for 3 weeks. Control uninephrectomized rats received tap water to drink. Blood pressure, measured by telemetry, was significantly higher in male DOCA rats (191 +/- 3 mm Hg) compared with female DOCA rats (172 +/- 7 mm Hg; n=5). DOCA treatment resulted in augmented contractile responses to phenylephrine in aorta (22 +/- 3 mN; n=6) and small mesenteric arteries (13 +/- 2 mN; n=6) from male DOCA rats versus uninephrectomized male rats (16 +/- 3 and 10 +/- 2 mN, respectively; P<0.05) and female DOCA rats (15 +/- 1 and 11 +/- 1 mN, respectively). ERK1/2 inhibition with PD-98059 (10 mu mol/L) abrogated increased contraction to phenylephrine in aorta (14 +/- 2 mN) and small mesenteric arteries (10 +/- 2 mN) from male DOCA rats, without any effects in arteries from male uninephrectomized or female animals. Compared with the other groups, phosphorylated ERK1/2 levels were increased in the aorta from male DOCA rats, whereas mitogen-activated protein kinase phosphatase 1 expression was decreased. Interleukin-10 plasma levels, which positively regulate mitogen-activated protein kinase phosphatase 1 activity, were reduced in male DOCA-salt rats. We speculate that augmented vascular reactivity in male hypertensive rats is mediated via activation of the ERK1/2 pathway. In addition, mitogen-activated protein kinase phosphatase 1 and interleukin 10 play regulatory roles in this process. (Hypertension. 2010; 55: 172-179.)

Comparison of signal-to-cutoff values in first, second, and third generation enzyme immunoassays for the diagnosis of HTLV-1/2 infection in ""at-risk"" individuals from Sao Paulo, Brazil

Data obtained during routine diagnosis of human T-cell lymphotropic virus type 1 (HTLV-1) and 2 (HTLV-2) in ""at-risk"" individuals from Sao Paulo, Brazil using signal-to-cutoff (S/C) values obtained by first, second, and third generation enzyme immunoassay (EIA) kits, were compared. The highest S/C values were obtained with third generation EIA kits, but no correlation was detected between these values and specific antibody reactivity to HTLV-1, HTLV-2, or untyped HTLV (p = 0.302). In addition, use of these third generation kits resulted in HTLV-1/2 false-positive samples. In contrast, first and second generation EIA kits showed high specificity, and the second generation EIA kits showed the highest efficiency, despite lower S/C values. Using first and second generation EIA kits, significant differences in specific antibody detection of HTLV-1, relative to HTLV-2 (p = 0.019 for first generation and p < 0.001 for second generation EIA kits) and relative to untyped HTLV (p = 0.025 for first generation EIA kits), were observed. These results were explained by the composition and format of the assays. In addition, using receiver operating characteristics (ROC) analysis, a slight adjustment in cutoff values for third generation EIA kits improved their specificities and should be used when HTLV ""at-risk"" populations from this geographic area are to be evaluated. (C) 2009 Elsevier B.V. All rights reserved.

Upper limit on the cosmic-ray photon flux above 10(19) eV using the surface detector of the Pierre Auger Observatory

A method is developed to search for air showers initiated by photons using data recorded by the surface detector of the Auger Observatory. The approach is based on observables sensitive to the longitudinal shower development, the signal risetime and the curvature of the shower front. Applying this method to the data, tipper limits on the flux of photons of 3.8 x 10(-3), 2.5 x 10(-3), and 2.2 x 10(-3) km(-2) sr(-1) yr(-1) above 10(19) eV, 2 x 10(19) eV, and 4 x 10(19) eV are derived, with corresponding limits on the fraction of photons being 2.0%, 5.1%, and 31% (all limits at 95% c.l.). These photon limits disfavor certain exotic models of sources of cosmic rays. The results also show that the approach adopted by the Auger Observatory to calibrate the shower energy is not strongly biased by a contamination from photons. (C) 2008 Elsevier B.V. All rights reserved.

Structural role of fluoride in the ion-conducting glass system B(2)O(3)-PbO-LiF studied by single- and double-resonance NMR

The local structure of an ion-conducting glass with nominal composition 50B(2)O(3)-10PbO-40LiF has been investigated by complementary (7)Li, (11)B, (19)F, and (207)Pb single- and double-resonance experiments. The results give insight into the structural role of the lithium fluoride additive in borate glasses: (1) LiF is seen to actively participate in the network transformation process contributing to the conversion of three- into four-coordinate boron units, as shown by (11)B single-resonance as well as by (11)B{(19)F} and (19)F{(11)B} double-resonance experiments. (2) (19)F signal quantification experiments suggest substantial fluoride loss, presumably caused by formation of volatile BF(3). A part of the fluoride remains in the dopant role, possibly in the form of small LiF-like cluster domains, which serve as a mobile ion supply. (3) The extent of lithium-fluorine and lead-fluorine interactions has been characterized by (7)Li{(19)F} and (207)Pb{(19)F} REDOR and SEDOR experiments. On the basis of these results, a quantitative structural description of this system has been developed.

Role of cis-cis muconic acid in the catalysis of Pseudomonas putida chlorocatechol 1,2-dioxygenase

Chlorocatechol 1,2-dioxygenase (1,2-CCD) is a non-heme iron protein involved in the intradiol cleavage of aromatic compounds that are recalcitrant to biodegradation. In particular, 1,2-CCD catalyzes the conversion of catechol and its halogenated derivatives to cis-cis muconic acid. In this study we describe a series of experiments concerning the interaction of chlorocatechol 1,2-dioxygenase from Pseudomonas putida (Pp1,2-CCD) with cis-cis muconic acid. We used single-injection ITC to show that the reaction product inhibits enzyme kinetics. DSC and EPR measurements probed whether this was accomplished by a direct binding of the product to the enzyme active site. DSC shows that cis-cis muconic acid affects the thermal unfolding of the protein and allowed us to estimate a binding constant. Furthermore, EPR spectra of the Fe(III) center demonstrate that, upon product binding, a significant decrease in resonance intensity is observed, indicating that cis-cis muconic acid binds directly to the active site. Based on the increasing interest for understanding dioxygenases mechanism of action and, moreover, how to control such process, our data indicate that the product of the reaction does play a relevant role in the catalysis and should therefore be taken into account when one thinks about ways of regulating enzyme activity. (C) 2010 Elsevier B.V. All rights reserved.

Structure and thermal reactivity of Zn(II) salts of isocinchomeronic acid (2,5-pyridinedicarboxylic acid)

The synthesis, an improved refined crystal and molecular structure re-determination, and the thermal decomposition behavior of two Zn(II) derivatives of isocinchomeronic acid (2,5-pyridinedicarboxylic acid or H(2)2,5-pydc) are presented. [Zn(2,5-pydc)(H(2)O)(3)Zn(2,5-pydc)(H(2)O)(2)](2) (1) crystallizes in the triclinic P-1 space group with a = 7.106(2), b = 11.450(2), c = 11.869(1) angstrom, alpha = 107.29(1), beta = 104.08(1), gamma = 90.32(2)degrees, and Z = 2. [Zn(2,5-pydc)(H(2)O)(2)] center dot H(2)O (2) is orthorhombic (P2(1)2(1)2(1) space group), with a = 7.342(1), b = 9.430(1), c = 13.834(2) angstrom, and Z = 4. The structures were refined to agreement R(1)-factors of 0.0315 (1) and 0.0336 (2). Complex (1) is arranged as molecular Zn(4)(2,5-pydc)(4)(H(2)O)(10) tetramers, the cages of which define channels that remain unblocked by anions. Compound (2) is polymeric with Zn(2,5-pydc)(H(2)O)(2) and Zn(2,5-pydc)(H(2)O)(3) units linked through bridging ligands. Both compounds were synthesized under mild conditions in aqueous media, without need to resort to hydrothermal media. Changing the pH from 4.51 to 5.75 suffices to direct the chemical processes toward the orthorhombic compound rather than to the triclinic one.

Examples of Self-Iterating Lie Algebras, 2

We study properties of self-iterating Lie algebras in positive characteristic. Let R = K[t(i)vertical bar i is an element of N]/(t(i)(p)vertical bar i is an element of N) be the truncated polynomial ring. Let partial derivative(i) = partial derivative/partial derivative t(i), i is an element of N, denote the respective derivations. Consider the operators v(1) = partial derivative(1) + t(0)(partial derivative(2) + t(1)(partial derivative(3) + t(2)(partial derivative(4) + t(3)(partial derivative(5) + t(4)(partial derivative(6) + ...))))); v(2) = partial derivative(2) + t(1)(partial derivative(3) + t(2)(partial derivative(4) + t(3)(partial derivative(5) + t(4)(partial derivative(6) + ...)))). Let L = Lie(p)(v(1), v(2)) subset of Der R be the restricted Lie algebra generated by these derivations. We establish the following properties of this algebra in case p = 2, 3. a) L has a polynomial growth with Gelfand-Kirillov dimension lnp/ln((1+root 5)/2). b) the associative envelope A = Alg(v(1), v(2)) of L has Gelfand-Kirillov dimension 2 lnp/ln((1+root 5)/2). c) L has a nil-p-mapping. d) L, A and the augmentation ideal of the restricted enveloping algebra u = u(0)(L) are direct sums of two locally nilpotent subalgebras. The question whether u is a nil-algebra remains open. e) the restricted enveloping algebra u(L) is of intermediate growth. These properties resemble those of Grigorchuk and Gupta-Sidki groups.

[(13)C(2)]- Acetaldehyde Promotes Unequivocal Formation of 1,N(2)-Propano-2 `-deoxyguanosine in Human Cells

Acetaldehyde is an environmentally widespread genotoxic aldehyde present in tobacco smoke, vehicle exhaust and several food products. Endogenously, acetaldehyde is produced by the metabolic oxidation of ethanol by hepatic NAD-dependent alcohol dehydrogenase and during threonine catabolism. The formation of DNA adducts has been regarded as a critical factor in the mechanisms of acetaldehyde mutagenicity and carcinogenesis. Acetaldehyde reacts with 2`-deoxyguanosine in DNA to form primarily N(2)-ethylidene-2`-deoxyguanosine. The subsequent reaction of N(2)-ethylidenedGuo with another molecule of acetaldehyde gives rise to 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo), an adduct also found as a product of the crotonaldehyde reaction with dGuo. However, adducts resulting from the reaction of more than one molecule of acetaldehyde in vivo are still controversial. In this study, the unequivocal formation of 1,N(2)-propanodGuo by acetaldehyde was assessed in human cells via treatment with [(13)C(2)]-acetaldehyde. Detection of labeled 1,N(2)-propanodGuo was performed by HPLC/MS/MS. Upon acetaldehyde exposure (703 mu M), increased levels of both 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo), which is produced from alpha,beta-unsaturated aldehydes formed during the lipid peroxidation process, and 1,N(2)-propanodGuo were observed. The unequivocal formation of 1,N(2)-propanodGuo in cells exposed to this aldehyde can be used to elucidate the mechanisms associated with acetaldehyde exposure and cancer risk.

Ultrasensitive Simultaneous Quantification of 1,N(2)-Etheno-2 `-deoxyguanosine and 1,N(2-)Propano-2 `-deoxyguanosine in DNA by an Online Liquid Chromatography-Electrospray Tandem Mass Spectrometry Assay

Exocyclic DNA adducts produced by exogenous and endogenous compounds are emerging as potential tools to study a variety of human diseases and air pollution exposure. A highly sensitive method involving online reverse-phase high performance liquid chromatography with electrospray tandem mass spectrometry detection in the multiple reaction monitoring mode and employing stable isotope-labeled internal standards was developed for the simultaneous quantification of 1,N(2)-etheno-2`-deoxyguanosine (1,N(2)-epsilon dGuo) and 1,N(2)-propano-2`-deoxyguanosine (1,N(2)-propanodGuo) in DNA. This methodology permits direct online quantification of 2`-deoxyguanosine and ca. 500 amol of adducts in 100 mu g of hydrolyzed DNA M the same analysis. Using the newly developed technique, accurate determinations of 1,N(2)-etheno-2`-deoxyguanosine and 1,N2-propano-2`-deoxyguanosine levels in DNA extracts of human cultured cells (4.01 +/- 0.32 1,N(2)-epsilon dGuo/10(8) dGuo and 3.43 +/- 0.33 1,N(2)-propanodGuo/10(8) dGuo) and rat tissue (liver, 2.47 +/- 0.61 1,N(2)-epsilon dGuo/10(8) dGuo and 4.61 +/- 0.69 1,N(2)-propanodGuo/108 dGuo; brain, 2.96 +/- 1.43,N(2)-epsilon dGuo/10(8) dGuo and 5.66 +/- 3.70 1,N(2)-propanoclGuo/10(8) dGuo; and lung, 0,87 +/- 0.34 1,N(2)-edGuo/ 10(8) dGuo and 2.25 +/- 1.72 1,N(2)-propanodGuo/10(8) dGuo) were performed. The method described herein can be used to study the biological significance of exocyclic DNA adducts through the quantification of different adducts in humans and experimental an with pathological conditions and after air pollution exposure.