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Cleanrooms utilizing fan filter units (FFUs) have experienced rapid growth. Enabling this growth has been the flexibility to provide controls to clean environments, from minienvironments as small as workbenches to [...]

Normothermia during the first hour of life improves neonatal outcomes. Temperature is routinely assessed on admission to NICU, but the optimal time and method of assessing temperature after birth is not known.

The 1-propanediazonium ion, generated from N‘-nitro-N-nitroso-N-propylguanidine in aqueous solutions, was reacted with the purine nucleosides dGuo and dAdo or single-stranded or double-stranded DNA. After nucleobase liberation by acid hydrolysis, the percent yields of products were determined by LC/MS using either isotopically distinct internal standards in the case of the nucleoside reactions or an internal standard and the ratios of response factors of all other products that were separately determined in the case of the reactions with DNA. In the reactions of nucleosides, products of both n-propylation and iso-propylation at all of the heroatoms were observed. For these reactions, the yields of the three most abundant n-propyl adducts of Gua are in the order O⁶ > N7 > N², in the ratio of 9.0/6.4/1, while for Ade, the order of the yields of N-propyl products is N1 > N7 > N3 > N⁶ in the ratio 2.5/1.8/1.1/1. The ratios of n-propylated to iso-propylated products at each site, Pn/Pi, generally a measure of enhancement of SN2 displacement on the diazonium ion, vary with each heteroatom but by no more than a factor of 6 for Gua and a factor of 3 for Ade. In the reactions with duplex DNA, products of reactions at all sites could not be detected. In addition, much larger selectivities are observed, similar to what has been observed by others in the reactions with ethanediazonium ion. Thus, Pn/Pi = 30, 21, and 0.9 for N7, O⁶, and N² of Gua. Similarly, the values of Pn/Pi are 11 and 8 for N3 and N7 of Ade. Reactions with single-stranded DNA give values of Pn/Pi that are intermediate between the nucleoside reactions and the reactions of duplex DNA in most cases. The factors responsible for the relatively small atom site selectivities intrinsic to the nucleosides are analyzed, and reasons for enhanced SN2 nucleophilicity in duplex DNA are discussed.

The effects of N²-isopropylGua and N⁶-isopropylAde adducts in template DNA on polymerization by the human replicative DNA polymerase α (B-family) and the translesion synthesis DNA polymerases η and ι (Y-family) were investigated. A direct comparison between the accuracies of DNA synthesis using catalytic fragments of the human DNA polymerases η and ι is reported. We show that the N²-isopropylGua adduct is a powerful block to polymerization by DNA polymerase α. In contrast, the DNA polymerases η and ι synthesize DNA past the N²-isopropylGua lesion with efficiencies and accuracies opposite the lesion comparable to the unadducted Gua. All three DNA polymerases bypass the N⁶-isopropylAde adduct with only modest effects on efficiencies and accuracy. These results illustrate the dramatically different consequences to polymerization conferred by the position of the isopropyl adduct when catalyzed by DNA polymerase α and the lack of this effect on polymerization by the translesion synthesis DNA polymerases η and ι. A steady-state kinetic analysis of nucleotide insertion opposite the N²-isopropylGua and the N⁶-isopropylAde by the DNA polymerases η and ι was performed to measure the accuracy of DNA synthesis at these lesions. This analysis showed that the DNA polymerases η and ι preferably insert the correct nucleotide Cyt opposite the N²-isopropylGua lesion and the correct nucleotide Thy opposite the N⁶-isopropylAde with levels of accuracy similar to those detected opposite the unadducted nucleotides, thus, demonstrating minimal blocking and mutagenic potential by these lesions to the translesion synthesis polymerases. Similarly, a kinetic analysis of polymerization opposite the N⁶-isopropylAde by the DNA polymerase α showed comparable levels of insertion accuracy relative to the unadducted Ade. These results suggest that positioning of the isopropyl adduct on the purine ring to locate this group into the minor groove of the DNA is an important determinant to effect blocked replication by a replicative (B-family) polymerase, but not to affect replication by a translesion synthesis (Y-family) polymerase.

The major metabolite of the cancer chemopreventive oltipraz (1), a pyrrolopyrazine thione, 4, has been shown to be a phase two enzyme inducer, an activity thought to be a key to the cancer chemopreventive action of the parent compound. To understand the possible mechanism by which the metabolite acts as an inducer, a study of its potential to generate free radicals was undertaken. Electron paramagnetic resonance (EPR) spin trapping studies using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) were performed with 7-methyl-6,8-bis-methyldisulfanyl-pyrrolo[1,2-a]pyrazine, 5, a synthetic precursor to the metabolite in aqueous and organic solvents. In the presence of GSH, which rapidly liberates the metabolite from the precursor, a 1:2:2:1 quartet spectrum with hyperfine coupling constants aN = aH = 14.9 G, characteristic of the hydroxyl radical adduct of DMPO, was observed in the presence of oxygen. No signal was seen under anaerobic conditions. This signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase. In aqueous dimethyl sulfoxide (80 vol % DMSO), the metabolite prescursor 5, GSH, and DMPO exhibited an EPR spectrum with the hyperfine values of aN = 12.7 G, aH1 = 10.3 G, and aH2 = 1.3 G, corresponding to the superoxide radical adduct of DMPO. The amount of superoxide radical adduct formed from the reaction of 5 and GSH increases with GSH concentration in phosphate buffer solution. Kinetic studies show that the formation of superoxide radical anion is first-order with respect to GSH. The formation of superoxide radical anion by the metabolite in the presence of GSH is linear at lower concentrations of 5 but becomes nonlinear at high concentrations. Overall, these studies suggest a mechanism in which GSH reduces the metabolite 4 to 4^•, presumably a radical anion, that in turn donates an electron to oxygen resulting in superoxide radical anion formation. This GSH stimulated redox cycle of the metabolite 4 suggests a possible mechanism by which the parent compound oltipraz might effect the cancer chemopreventve increase in the transcription of phase two enzymes that is mediated by transcription factor Nrf2.

Reactions have been carried out in which 1,3-diisopropyltriazene or N-isopropyl-N-(1-hydroxyethyl)nitrosamine has been decomposed in neutral, buffered aqueous media in the presence of (¹⁵N²)2‘-deoxyguanosine and (¹⁵N⁶)2‘-deoxyadenosine. The products of covalent attachment of the isopropyl cation, derived from the isopropyl diazonium ion, to the heteroatoms of the purines have been separated and quantified by HPLC/electrospray mass spectrometry by employing isotopically distinct synthetic standards. The results indicate that the two different precursors of the isopropyl cation result in the formation of different yields of products in the reactions at all of the heteroatoms of both purines, outside experimental error, except possibly in the case of the N3 position of dAdo. For the different alkylating agents, the ratios of yields at any two sites vary as well. This leads to the conclusion that isopropylation occurs by a preassociation mechanism in which the isopropyl cation intermediate reacts in the solvation shell in which it is generated from its precursors. The reaction of N-isopropyl-N-(1-hydroxyethyl)nitrosamine results in alkylation of 2‘-deoxyguanosine in preference to 2-deoxyadenosine, by a factor of 3−4. In this reaction, the yields for reaction at N1, N3, N⁶, and N7 of adenine vary over less than a factor of 2, whereas the yields for N², N3, O⁶, and N7 of guanine vary over less than a factor of 4. The N1 atom of guanine is disfavored over the major product, the O⁶ adduct, by a factor of <8. The reaction of 1,3-diisopropyltriazene shows a similar preference for alkylation of 2-deoxyguanosine, with a similar range of product distribution in the reactions at adenine heteroatoms and a somewhat larger range of distribution at guanine heteroatoms. In particular, the yield of 1-isopropylguanosine is 50-fold lower than that of O⁶-isopropylguanosine. The comparable yields of products of reaction at the “hard” and “soft” sites of the purines lead to the conclusion that nucleophilicity is unimportant in site selectivity of alkylation by the isopropyl cation. The noteworthy selectivities, above, are rationalized by:  differences in the association constants of the precursors of the cations with the two nucleosides; steric, statistical, and electrostatic effects that favor reaction of the O⁶ atom of guanine; and larger steric and/or desolvation requirements for association of the 1,3-diisopropyltriazenium cation with the N1 atom of guanine. The reaction of N-isopropyl-N-(1-hydroxyethyl)nitrosamine with double-stranded DNA has been similarly analyzed. The product distribution is remarkably similar in profile to that observed for the nucleosides in solution. In particular, exocyclic amino groups are competitive with the more traditional sites of diazonium ion-mediated alkylation. A comparison to earlier literature data on alkylation by methyl- and ethyl-diazonium ions illustrates some fundamental differences between the reaction of the diazonium ions and the isopropyl cation derived from the isopropyl diazonium ion.

The effects of N²-ethylGua, O⁶-ethylGua, and O⁶-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases α and η have been investigated. The N²-ethylGua adduct blocks polymerization by the replicative DNA polymerase α to a much greater extent than does the O⁶-ethyl- or the O⁶-methylGua adducts. The DNA polymerase η efficiently and accurately bypasses the N²-ethylGua lesion but like DNA polymerase α is similarly blocked by the O⁶-ethyl- or the O⁶-methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N²-ethylGua and the O⁶-ethylGua adducts by the DNA polymerases α and η and extension from 3‘-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N²-ethylGua adduct by DNA polymerase α is approximately 10⁴-fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N²-ethylGua:Cyt 3‘-terminus by DNA polymerase α is approximately 10³-fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N²-ethylGua lesion by the DNA polymerase η is about 370-fold more efficient than by the DNA polymerase α, and extension from the N²-ethylGua:Cyt 3‘-terminus by the DNA polymerase η is about 3-fold more efficient than by the DNA polymerase α. Furthermore, the DNA polymerase η preferably inserts the correct nucleotide Cyt opposite the N²-ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O⁶-ethylGua adduct by the DNA polymerases α and η. In reactions containing both DNA polymerases α and η, synthesis past the N²-ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N²-ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase α and lesion bypass catalyzed by DNA polymerase η.

Treatment for 48 h of murine Hepa 1c1c7 cells in culture with the cancer chemopreventive oltipraz (1) followed by addition of CD3I and immediate cell lysis yields, by LC/MS analysis, three isotopomers of the methylated pyrrolopyrazine (2), a known human metabolite of oltipraz. The major isotopomer (58%) is the one containing two CD3− groups attached to the pendant sulfur atoms of the pyrrolopyrazine ring, the others containing one CD3− and one CH3− group or two CH3− groups. It is concluded from this that the unmethylated pyrrolopyrazine (4) is the major metabolite of oltipraz. Prodrugs 5 and 6, which have been shown to rapidly generate 4 in the presence of GSH at physiological pH, induce the phase 2 enzyme NQO1 in Hepa 1c1c7 cells with potencies on par with oltipraz itself:  CDNQO1 = 14.4 ± 1.3, 20.1 ± 4.6, and 23.6 ± 1.6 μM for oltipraz, 5, and 6, respectively. Pretreatment of oltipraz, 5, and 6 in cell culture media with 1 mM GSH, which is shown to immediately convert 5 and 6 to 4, followed by incubation with Hepa 1c1c7 cells shows similar potencies for oltipraz and the (decomposed) produrgs, with CDNQO1 = 18.0 ± 4.4 μM for 5, 17.8 ± 0.2 μM for 6, and 13.5 ± 1.4 μM for oltipraz. Treatment with compound 6 of murine hepatoma cells containing a luciferase gene under the control of the antioxidant response element (ARE) from the mouse heme oxygenase (ho-1) gene elicits induction of luciferase activity, CD = 35.8 ± 2.8 μM, somewhat greater than the potency than oltipraz itself. Western blots of nuclear proteins isolated from Hepa 1c1c7 cells and probed with anti-Nrf2 indicate that as compared to vehicle DMSO, compound 6 stimulates nuclear translocation of Nrf2 from the cytosol. From this study, it is concluded that the major metabolite of the cancer chemopreventive oltipraz is a phase 2 enzyme inducer of comparable potency that activates the ARE and initiates nuclear translocation of transcription factor Nrf 2.

N-Nitroso-3-hydroperoxymorpholine was synthesized in 12 steps starting from a commercially available serine derivative and was subsequently characterized by elemental analysis, one-dimensional ¹H and ¹³C NMR spectroscopy, and homonuclear and heteronuclear two-dimensional methods. Treatment of the hydroperoxide with triarylphosphine or trialkylphosphine in CD2Cl2 or CD3CN yields N-nitroso-3-hydroxymorpholine, a putative metabolite of the carcinogen N-nitrosomorpholine, as indicated by ¹H and ¹³C NMR spectroscopy. A study of the decomposition of N-nitroso-3-hydroxymorpholine in aqueous buffer solution, ionic strength 1 M (KCl), at 25 °C is reported. The kinetics of decay obey a three term rate law, with terms for a hydrogen ion-dependent, pH-independent, and hydroxide ion-dependent reactions, similar to what has been reported for other α-hydroxynitrosamines. The stability of this compound is similar to that of α-hydroxynitrosopiperidine reported previously. The reaction of 3-hydroxymorpholine in aqueous solution, 25 °C, pH 6.6, yields (2-hydroxy-ethoxy)acetaldehyde (62%), acetaldehyde (26%), and glycolaldehyde (26%), based on analysis of the dintrophenylhydrazine derivatives. Both the stability and the products of decay of N-nitroso-3-hydroxymorpholine that are reported here contrast strongly with a previous description.

This report summarizes an investigation of the reactions of biological and other thiols with the cancer chemopreventive oltipraz and other dithiolethiones. Analysis of the kinetics of reaction of 4-methyl-5-(pyrazin-2-yl)-1,2-dithiole-3-thione (oltipraz) 1 with monothiols and dithiols in the range of 0.75−20 mM in aqueous 15% ethanol, at pH 7.5 (0.1 M Tris buffer) and at 37 °C has been undertaken. A plot of kobsd against [thiol] shows that reactions of mono- and dithiols are first order in thiol concentration. The dependence on pH of these reactions shows that the active species is the thiolate anion. Specific second-order rate constants, k2 (M^-1 s^-1) for reaction of the thiolate anions with oltipraz have been determined to be cysteine, 0.040 ± 0.001; 2-mercaptoethanol, 2.0 ± 0.02; glutathione, 0.099 ± 0.001; mercaptoacetic acid anion, 4.0 ± 0.01; dithiothreitol, 1.33 ± 0.02; 1,3-propanedithiol, 10 ± 0.5; 1-mercaptopropane-3-ol, 6.5 ± 0.1; 1-mercaptopropane-2,3-diol, 1.26 ± 0.05. A plot of pKa against log k2 for monothiols shows a linear dependence of k2 on pKa, βnuc 1.1 ± 0.07, which accounts for most of the reportedly enhanced reactivity of dithiols over monothiols. The pseudo-first-order rate constant for the solvolysis of oltipraz has been measured as 2.2 (±0.2) × 10^-8 s^-1. The kinetics of reaction of three other dithiole-3-thiones with glutathione has also been studied for comparison with oltipraz. The specific second-order rate constants, k2 (M^-1 s^-1) are 5-phenyl-1,2-dithiole-3-thione, 4.7 × 10^-4; 5-(4-methoxyphenyl)-1,2-dithiole-3-thione, 4.1 × 10^-4; and 1,2-dithiole-3-thione 0.08. Important implications for the mode of biological action of these compounds and the nature of the putative biological targets of the compounds are discussed.

A study of the decomposition of α-hydroxydialkylnitrosamines in aqueous 9% acetonitrile, with an ionic strength of 1 M (NaClO4), at 25 °C is reported. Plots of the logarithm of the buffer-independent rate constant, ko, against pH are concave up and indicate a three-term rate law for the solvent reaction, including acid (kH^{+)-, base (kOH)-, and pH-independent (kHOH) terms. Secondary α-deuterium isotope effects for compound 1a, (N-nitrosomethylamino)phenylmethanol, are as follows:  kαH/kαD = 1.12 ± 0.03 and 1.19 ± 0.02 for kH+ and kOH, respectively. General acid (kHA) and general base (kA−) catalysis by more acidic carboxylic acid buffers is also observed. Structure reactivity and other parameters obtained in this study, and their changes with substrate and catalyst structure, permit the assignment of mechanisms for the kH+, kOH, kHA, and kA− processes.}

Diisopropyltriazene in aqueous 10% acetonitrile (pH 7.0 ± 0.4) yields N⁶-isopropyl-2‘-deoxyAdo as the predominant product and N²-isopropyl-2‘-deoxyGuo in yields comparable with the O⁶ adduct in reactions with 2‘-deoxyAdo and 2‘-deoxyGuo, respectively. These observations are inconsistent with what is expected on the basis of the regnant hypothesis concerning factors that determine atom site selectivity in diazonium ion-mediated alkylations. An alternative explanation based on the fleeting existence of the reactive intermediates involved is consistent with these observations.

The performance of cushions made from a number of fabrics and in-fills have been compared in small-scale fire experiments designed to simulate conditions in the fire at the Woolworths Store in Manchester on 8 May 1979.It is concluded that contrary to much comment at that time, other materials in addition to the polyurethane foam present made a significant contribution to the fire and that replacement of polyurethane by some other conventional in-fill materials would not have made a major difference to the rate of development of the fire or of smoke and toxic gases.Tests in the same configuration indicate the beneficial effects of composite design and of the major improvement obtained by the use of a polyurethane barrier foam composition.