ATP phosphoribosyl transferase (ATP-PRT) joins ATP and 5-phosphoribosyl-1-pyrophosphate (PRPP) in a highly regulated reaction that initiates histidine biosynthesis. The unusual hetero-octameric version of ATP-PRT includes four HisG S catalytic subunits based on the periplasmic binding protein fold and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. Here, we present the first structure of a PRPPbound ATP-PRT at 2.9 Å and provide a structural model for allosteric activation based on comparisons with other inhibited and activated ATPPRTs from both the hetero-octameric and hexameric families. The activated state of the octameric enzyme is characterized by an interstitial phosphate ion in the HisZ-HisG interface and new contacts between the HisZ motif 2 loop and the HisG S dimer interface. These contacts restructure the interface to recruit conserved residues to the active site, where they activate pyrophosphate to promote catalysis. Additionally, mutational analysis identifies the histidine binding sites within a region highly conserved between HisZ and the functional HisRS. Through the oligomerization and functional re-assignment of protein domains associated with aminoacylation and phosphate binding, the HisZ-HisG octameric ATP-PRT acquired the ability to initiate the synthesis of a key metabolic intermediate in an allosterically regulated fashion. Phosphoribosyl transferases (PRTs)5 catalyze the attack of a nitrogenous and/or aromatic base on 5-phosphoribosyl-1-pyrophosphate (PRPP), and thereby participate in essential reactions in the biosynthesis of nucleotides and the amino acids tryptophan and histidine (1, 2). The largest family (type I) of these structurally diverse enzymes includes many nucleotide salvage enzymes that share a five-stranded parallel ␤ sheet fold with a substrate binding hood domain (3, 4). The folds of type II (5) and type III enzymes (6) are distinct from the class I enzymes, and the type III enzymes resemble nucleoside phosphorylase. Among the PRTs with complex quaternary structures and sophisticated regulation are the glutamine PRPP amidotransferase, which catalyzes the first committed step of purine biosynthesis (7), and ATP phosphoribosyl transferase (ATP-PRT), which joins ATP and PRPP to initiate synthesis of histidine (8, 9). Glutamine PRPP amidotransferase and ATP-PRT both exhibit pathway end product inhibition, and regulation by cellular energy levels (7, 10). ATP-PRT is competitively inhibited by AMP and ADP (8, 11-13) and non-competitively inhibited by histidine (Fig.

Many transition metal complexes mediate DNA oxidation in the presence of oxidizing radiation, photosensitizers, or oxidants. The final DNA oxidation products vary depending on the nature of metal complexes and the structure of DNA. Here we propose a mechanism of oxidation of a nucleotide, deoxyguanosine 5'-monophosphate (dGMP) by trans-d,l-1,2-diaminocyclohexanetetrachloroplatinum (trans-Pt(d,l)(1,2-(NH(2))(2)C(6)H(10))Cl(4), [Pt(IV)Cl(4)(dach)]; dach = diaminocyclohexane) to produce 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) stoichiometrically. The reaction was studied by high-performance liquid chromatography (HPLC), (1)H and (31)P nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). The proposed mechanism involves Pt(IV) binding to N7 of dGMP followed by cyclization via nucleophilic attack of a phosphate oxygen at C8 of dGMP. The next step is an inner-sphere, two-electron transfer to produce a cyclic phosphodiester intermediate, 8-hydroxyguanosine cyclic 5',8-(hydrogen phosphate). This intermediate slowly converts to 8-oxo-dGMP by reacting with solvent H(2)O.

The role of gastroesophageal reflux and micro-aspiration as a trigger of airways hyperresponsiveness (AHR) in patients with asthma is controversial. The role of acid reflux and aspiration as a direct cause of AHR in normal subjects is also unclear. We speculated that aspiration of a weak acid with a pH (1.8) equivalent to the upper range of typical gastric contents would lead to AHR in naive mice. We further speculated that modest reductions in aspirate acidity to a level expected during gastric acid suppression therapy (pH 4.0) would impede aspiration-induced AHR. BALB/c female mice were briefly anesthetized with isoflurane and allowed to aspirate 75 microl of saline with HCl (pH 1.8, 4.0, or 7.4) or underwent sham aspiration. Mice were re-anesthetized 2 or 24 h later, underwent tracheostomy, and were coupled to a mechanical ventilator. Forced oscillations were used to periodically measure respiratory impedance (Zrs) following aerosol delivery of saline and increasing doses of methacholine to measure for AHR. Values for elastance (H), airways resistance (R(N)), and tissue damping (G) were derived from Zrs. Aspirate pH of 1.8 led to a significant overall increase in peak R(N), G, and H compared with pH 4.0 and 7.4 at 2 and 24 h. Differences between pH 7.4 and 4.0 were not significant. In mice aspirating pH 1.8 compared with controls, airway lavage fluid contained more neutrophils, higher protein, and demonstrated higher permeability. We conclude that acid aspiration triggers an acute AHR, driven principally by breakdown of epithelial barrier integrity within the airways.

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No abstract

Allen GB, Cloutier ME, Larrabee YC, Tetenev K, Smiley ST, Bates JH. Neither fibrin nor plasminogen activator inhibitor-1 deficiency protects lung function in a mouse model of acute lung injury. Am J Physiol Lung Cell Mol Physiol 296: L277-L285, 2009. First published December 5, 2008 doi:10.1152/ajplung.90475.2008.-Fibrin impairs surfactant function in vitro, and inhibition of fibrinolysis by plasminogen activator inhibitor (PAI-1) is thought to promote fibrin accumulation in acute lung injury (ALI). This has led to speculation that impaired PAI-1 and fibrin accumulation should protect lung function in ALI. We tested this hypothesis by investigating ALI severity in fibrinogen-deficient (FgnϪ/Ϫ) and PAI-1-deficient (PAI-1Ϫ/Ϫ) mice. PAI-1Ϫ/Ϫ, C57BL/6, FgnϪ/Ϫ, and Fgnϩ/Ϫ females were anesthetized and allowed to aspirate 4 l/g of hydrochloric acid (pH 1.0) and then reanesthetized and connected to a ventilator 48 h later. Naive C57BL/6 and Fgnϩ/Ϫ females served as controls. Following deep inflation (DI), forced oscillations were delivered periodically over 8 min to measure changes in elastance (H) as a surrogate of lung derecruitment, at positive end-expiratory pressures (PEEP) of 6, 3, and 1 cmH 2O. Increases in H following DI in acid-injured mice were greater than naive strain-matched controls. Increases in H were no different between injured PAI-1Ϫ/Ϫ and C57BL/6, or between injured FgnϪ/Ϫ and ϩ/Ϫ mice, at any PEEP. Pressure-volume curves were no different between injured groups. Total lung fibrin was lower in injured PAI-1Ϫ/Ϫ and FgnϪ/Ϫ mice relative to injured C57BL/6 and Fgnϩ/Ϫ mice, respectively, but indices of permeability were no different between strains. Unexpectedly, neither fibrin nor PAI-1 deficiency protects lung mechanical function in mice with acid-induced ALI. We speculate that in vivo lung function may be more closely tied to permeability and alveolar protein in general, rather than being linked specifically to fibrin. lung mechanics; respiratory impedance; acid aspiration; coagulation ACUTE LUNG INJURY (ALI) is a severe form of noncardiogenic pulmonary edema and hypoxemic respiratory failure stemming from numerous causes (60). Current treatment of ALI rests largely on supportive care with mechanical ventilation, and its prognosis remains poor with a mortality of 30 -40% in the general population, and higher in the elderly (49). The pathology of ALI typically progresses through an initial exudative phase characterized by neutrophil infiltration, edema, and accumulation of hyaline membranes, the latter consisting primarily of necrotic debris and fibrin (58). In this regard, the coagulation pathway and its end product fibrin have excited particular interest, in part due to the ability of fibrin to inhibit surfactant function in vitro (52,54), and the increasingly recognized interplay between coagulation and innate immunity (17,63). Fibrin formation and clearance in the lung are governed by the relative quantity and activity of fibrinolysis promoters such as plasminogen activators and fibrinolysis inh...

Approximately 20% of all negative results on SPT will have a positive ID test, more likely for indoor allergens. If a high suspicion for allergy exists in a patient with a negative SPT result, it may be useful to proceed with ID testing. However, the clinical significance of a positive ID test after negative SPT still needs to be elucidated.

This study suggests that common variants, rather than rare mutations, are the cause for association between IRF6 and nonsyndromic CL/P. rs2235371, but not rs642961, shows association with CL/P, suggesting a functional role for this polymorphism in our Honduran population. rs642961 has been previously reported to have an effect in other populations, suggesting that different populations may be affected by different polymorphisms.