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Validation of 8-isoprostane and prostaglandin E(2) measurements in exhaled breath condensate.

OBJECTIVE: To qualitatively validate radioimmunoassays for 8-isoprostane and prostaglandin (PG) E(2) in exhaled breath condensate. SUBJECTS: Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection. METHODS: Samples were pooled together and purified by reverse phase high performance liquid chromatography (RP-HPLC). The eluted fractions were assayed for 8-isoprostane-like immunoreactivity and PGE(2)-like immunoreactivity by radioimmunoassays. In addition, simultaneous measurements of exhaled breath condensate unextracted samples with two anti-8-isoprostane and anti-PGE(2) sera with different cross-reactivity were performed. RESULTS: A single peak of 8-isoprostane-like immunoreactivity and PGE(2)-like immunoreactivity co-eluting with 8-isoprostane (retention time: 13 min) and PGE(2) (retention time: 21 min) standards, respectively, was identified by radioimmunoassays. Testing with two different antisera showed similar results for both 8-isoprostane-like immunoreactivity (limits of agreement = 4.5 pg/ml and - 4.1 pg/ml, n = 12) and PGE(2)-like immunoreactivity (limits of agreement = 6.1 pg/ ml and - 6.1 pg/ml, n = 12). CONCLUSION: This study provides evidence for the specificity of the radioimmunoassays for 8-isoprostane and PGE(2) in exhaled breath condensate. This is critical for proposing these markers as a non-invasive way for monitoring airway inflammation.

Breath Tests↗

Validation of leukotriene B4 measurements in exhaled breath condensate.

OBJECTIVE: To qualitatively validate an enzyme immunoassay to measure leukotriene B4 in exhaled breath condensate. Exhaled breath condensate is a new non-invasive method to monitor airway inflammation. SUBJECTS: Twenty-two subjects with different lung diseases attended the outpatient clinic on one occasion for exhaled breath condensate collection. METHODS: Samples were pooled together and purified by reverse-phase high-performance liquid chromatography. The fractions eluted were assayed for leukotriene B4 by enzyme immunoassay. RESULTS: A single peak of leukotriene B4-like immunoreactivity co-eluting with leukotriene B4 standard (retention time: 24 min) was identified by enzyme immunoassay. Reverse phase-high performance liquid chromatography peak of leukotriene B4 was clearly separated from those of 6-trans-leukotriene B4 (retention time: 14 min) and leukotriene B5 (retention time: 18 min) for which the antiserum used in the enzyme immunoassay had the highest cross-reactivity. Leukotriene B4 recovery was 64%. CONCLUSIONS: This study provides evidence for the presence of leukotriene B4 in the exhaled breath condensate and the specificity of the enzyme immunoassay used.

Breath Tests↗

Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation.

We have generated and characterized a novel site-specific antibody highly specific for the phosphorylated form of the amino-terminus of histone H3 (Ser10). In this study, we used this antibody to examine in detail the relationship between H3 phosphorylation and mitotic chromosome condensation in mammalian cells. Our results extend previous biochemical studies by demonstrating that mitotic phosphorylation of H3 initiates nonrandomly in pericentromeric heterochromatin in late G2 interphase cells. Following initiation, H3 phosphorylation appears to spread throughout the condensing chromatin and is complete in most cell lines just prior to the formation of prophase chromosomes, in which a phosphorylated, but nonmitotic, chromosomal organization is observed. In general, there is a precise spatial and temporal correlation between H3 phosphorylation and initial stages of chromatin condensation. Dephosphorylation of H3 begins in anaphase and is complete immediately prior to detectable chromosome decondensation in telophase cells. We propose that the singular phosphorylation of the amino-terminus of histone H3 may be involved in facilitating two key functions during mitosis: (1) regulate protein-protein interactions to promote binding of trans-acting factors that "drive" chromatin condensation as cells enter M-phase and (2) coordinate chromatin decondensation associated with M-phase.

Amino Acid Sequence↗

Involvement of Notch signaling in initiation of prechondrogenic condensation and nodule formation in limb bud micromass cultures.

Notch signaling is an evolutionarily conserved mechanism that plays a critical role in the determination of multiple cellular differentiation pathways and morphogenesis during embryogenesis. The limb bud high-density culture is an established model that recapitulates mesenchymal condensation and chondrocyte differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) showed that Notch and its related genes were expressed in such cultures on day 1 and reached a peak between day 3 and day 5, when cell condensation and nodule formation were initiated. Immunohistochemical experiments revealed that the expression of Notch1 was initially localized within the nodules and shifted to their peripheral region as the cell differentiation progressed. We disrupted Notch signaling by using a gamma-secretase inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), to analyze the function of Notch signaling in the culture system. The blocking of Notch signaling by DAPT apparently promoted the initiation of prechondrogenic condensation and fusion of the nodules, and such an effect was reversed by exogenous expression of the Notch cytoplasmic domain. DAPT treatment also induced chondrogenic markers and bone morphogenetic protein (BMP)-related molecules, including type II collagen, Sox9, GDF5, and Id1. These observations imply that the Notch signal may have an important role in chondrogenic differentiation by negatively regulating the initiation of prechondrogenic condensation and nodule formation.

Animals↗

Spectrophotometric assay for the condensing enzyme activity of the microsomal fatty acid chain elongation system.

A rapid and simple spectrophotometric method was developed to measure the activity of the condensing enzyme component of the microsomal fatty acid chain elongation system. The intermediate product of the condensation reaction is the beta-ketoacyl CoA which exists in two tautomeric forms, i.e., keto and enol. The addition of bovine serum albumin (BSA) to a cuvette cell containing a beta-ketoacyl CoA derivative resulted in the formation of a 303-nm absorbance peak, characteristic of enolate formation. The beta-ketoacyl CoAs with carbon chain length of 6 to 18 interacted with BSA to produce the 303-nm peak; acetoacetyl CoA was the only beta-keto compound tested which did not interact with BSA to produce the peak. Other compounds which were unaffected by BSA included CoA, free beta-keto acid, beta-hydroxyacyl CoA, acyl CoA, trans-2-enoyl CoA, and malonyl CoA. BSA could not be replaced by ovalbumin; furthermore, denatured (boiling) BSA could not induce the 303-nm peak. The specific activity of the condensing enzyme measured by the spectrophotometric method compares favorably with the activity obtained by the radioactive method. The apparent extinction coefficient (epsilon) for the absorbance peak generated by the beta-keto thioester varied from 5 to 30 mM-1 cm-1 depending on the beta-keto derivative. The spectrophotometric procedure can be used in the determination of the condensing enzyme activity in not only hepatic microsomes but also in kidney and brain microsomes both of which have significantly lower activity. The advantages of the novel method over the radioactive method are that (i) it does not involve the use of radioactive compounds, (ii) it is much less cumbersome and significantly less costly, and (iii) it is rapid and easy to perform.

Animals↗

The effect of fatty acids on the surface potential of phospholipid vesicles measured by condensed phase radioluminescence.

(1) The carboxyl group of fatty acids has a very low pK value, which is shifted into the physiological pH range when they are incorporated in a phospholipid membrane. As a result of a pH increase the surface charge and surface potential of the membrane increase. (2) The titration of the carboxyl group was observed with condensed phase radioluminescence. This technique uses the electron emitted by the tritiated membrane probe (oleic acid or cholesterol) to excite a fluorophore also incorporated in the bilayer. (3) The phase transition of dimyristoyl phosphatidylcholine vesicles labelled within 12-(9-anthroyloxy)stearic acid was measured by condensed phase radioluminescence at different pH values. (4) We related the condensed phase radioluminescence signal to the asymmetrical distribution of the fluorophore between the inner and outer layer of the lipid membrane which is induced by the repulsion of the negatively charged fatty acids. (5) We showed that the condensed phase radioluminescence signal is proportional to the protonation of the carboxyl group. On this basis, the broadening of the titration curve can be explained as an effect of the self-induced membrane potential calculated using the Gouy-Chapman theory. (6) Ca2+ drastically reduces the flip-flop rate of fatty acids across the membrane and also caused a decrease in the asymmetric distribution. (7) We concluded that a fatty acid can act as a membrane surface buffer. The pK value of 12-(9-anthroyloxy)stearic acid in a dimyristoyl phosphatidylcholine membrane is 7.0 +/- 0.3. (8) We discuss the results with respect to aggregation, fusion and clustering.

Cholesterol↗

Cholesterol condensation of alpha-linolenic and gamma-linolenic acid-containing phosphatidylcholine monolayers and bilayers.

Cholesterol is demonstrated to condense phosphatidylcholine (PC) monolayers and bilayers containing stearic acid in the sn-1 position and alpha-linolenic acid in the sn-2 position (18:0, alpha-18:3 PC) but has no effect when gamma-linolenic acid occupies the sn-2 position (18:0,gamma-18:3 PC). Cholesterol-induced condensation is measured by area/molecule determinations made on monolayers using a Langmuir trough, while condensation in bilayers is followed by the fluorescent dyes merocyanine (MC540) and dansyllysine. Permeability to erythritol is also demonstrated to be diminished by cholesterol for the condensable 18:0,alpha-18:3 PC bilayer membranes but not the 18:0,gamma-18:3 PC membranes. alpha- and gamma-linolenic acid are isomers containing 18 carbons and three unsaturations. Both fatty acids have unsaturations at positions 9 and 12 and differ only in the location of the third unsaturation, at either position 6 for gamma-linolenic acid (an omega-6 fatty acid) and at position 15 for alpha-linolenic acid (an omega-3 fatty acid). Here lipid-cholesterol interaction is used to distinguish the effect of position of unsaturation on membrane structure.

Cholesterol↗

Complementary recognition in condensed DNA: accelerated DNA renaturation.

The functional consequences of DNA condensation are investigated. The recognition of complementary strands is profoundly modified by this critical phenomenon. (1) Condensation of denatured DNA greatly accelerates the kinetics of DNA renaturation. We propose a unifying explanation for the effects of several accelerating solvents studied here including polymers, di- and multivalent cations, as well as effects seen with the phenol emulsions and single-stranded nucleic acid binding proteins. Optimal conditions for renaturation at or above the calculated three dimensional diffusion limit are theoretically consistent with a limited search space in the condensed phases. (2) In addition to these effects on association of two single strands, similar condensation acceleration effects can be seen in strand exchange experiments with double stranded DNA without proteins. These may model a mechanism of recombinational protein function.

Bacteriophage lambda↗

The influence of condensation method on porosity and shade of body porcelain.

Condensation of porcelain is an important laboratory variable that can affect a variety of physical properties in metal ceramic restorations. This study examined four methods of condensation (brush with vibration, ultrasonic vibration, spatulation, and noncondensation) to evaluate their effects on porosity and color. Standardized 12 x 12 mm acetate plastic squares were invested and cast with Olympia alloy. After oxidation, the metal substructures received one of the following opaque and body porcelains: Ceramco II G Series, Will-Ceram V-Series, Vita VMK-68, or Jelenko. The examination of each sample consisted of three parts: (1) apparent specific gravity analysis (porosity); (2) tristimulus colorimeter analysis; and (3) subjective observer color analysis. No statistical differences (p less than or equal to 0.05) in apparent specific gravity were found between methods of condensation within any of the four porcelains. Statistical differences (p less than or equal to 0.05) in color were noted between methods of condensation within several of the porcelains by both the tristimulus colorimeter and the subjective observers.

Analysis of Variance↗

Factors affecting mutagenic activity of cigarette smoke condensate in Salmonella typhimurium TA 1538.

Smoke condensates from Burley tobacco, bright-type tobacco and various brands of commercial cigarettes were tested for mutagenicity by using a microsomal test system with Salmonella typhimurium TA 1538. Smoke condensate from Burley tobacco had much higher mutagenic activity than that from bright-type tobacco. Increased mutagenic activity was observed with smoke condensates from Burley tobacco grown with increasing amounts of nitrogen fertilizer, and from commercial cigarettes blended with Burley tobacco. There was a significant correlation between nitrate content of cigarette and mutagenic activity of the resulting smoke condensate. The results suggest that nitrate in cigarettes may influence the formation of potential mutagens during the burning of a cigarette.

Histidine↗

Periapical condensing osteitis and endodontic treatment.

Forty-nine roots of thirty-six teeth with the diagnosis of condensing osteitis were followed up by means of radiographs and patient files after endodontic treatment. The mean observation period was 4.3 years. Prior to endodontic treatment the periodontal membrane was widened on thirty-eight roots but on follow-up only on twelve. The periapical condensing osteitis showed total regression on thirty-six of the forty-nine roots. No condensing osteitis showed progress. The results show rebuilding of bone structures to normal appearance after endodontic treatment of periapical condensing osteitis.

Adolescent↗

Photoelastic stress comparison of warm (Endotec) versus cold lateral condensation techniques.

Stress generated during gutta-percha obturation of root canals can create microcracks and ultimately lead to failure. A new thermosoftening device, the Endotec, which develops a warm lateral condensation technique, was tested against the traditional lateral condensation procedure. The results indicated that the warm lateral condensation technique created less stress during obturation than did cold lateral condensation.

Cold Temperature↗

Effects of increased lifespan on chromatin condensation in the adult male housefly.

Analysis of the chromatin condensation pattern in optic lobe nuclei from the adult male housefly has shown significant differences in males from a long lived, low activity (LA) group as compared to a short lived, high activity (HA) group. The rate of chromatin condensation in the LA group was very much less than that observed in the HA group, leaving the nuclei from the former group in a permanently lowered condensation state. Moreover, a detailed comparison of the amount and spatial distribution of a single high density chromatin component (HDC) suggests that the low activity conditions not only lowers the rate of chromatin condensation but also alters the normal program controlling this process. Linear discriminant analysis showed that a consistently higher percentage of nuclei from the LA group, as compared to the HA group, could be classified with a 1-day-old model derived from the HA group. These results are discussed with respect to environmental influences on lifespan and are compared to the response of lipofuscin accumulation in low activity male houseflies.

Animals↗

Chromosome assembly in vitro: topoisomerase II is required for condensation.

The role of topoisomerase II (topo II) in chromosome condensation was studied in a mitotic extract derived from Xenopus eggs by specific immunodepletion. HeLa nuclei, which have a high complement of endogenous topo II, are converted to mitotic chromosomes in the topo II-depleted extract equally well as in the control. Chicken erythrocyte nuclei, however, which have a very low content of topo II, do not convert to condensed chromosomes in the depleted extract, although their condensation is normal upon addition of purified topo II. Dosage experiments support the possible notion of a structural involvement of topo II in chromosome condensation. In the topo II-depleted extract the erythrocyte nuclei progress to precondensation chromosomes, which lack the nuclear membrane-lamina complex and consist of a cluster of swollen chromatids.

Animals↗

Separation of glutaraldehyde and some of its aldol condensation products by hydroxyl-aldehyde group affinity chromatography.

Glutaraldehyde exists in aqueous solution in equilibrium with monomers and polymers of cyclic and open-chain hemialdals and hydrates. At alkaline pH oligomeric and polymeric alpha,beta-unsaturated aldehyde derivatives are formed from primarily produced aldol condensation products. This communication reports a method for separation of such aldol condensates by means of a new high performance liquid chromatography technique based on the affinity of aldehyde groups for hydroxyl groups of a hydroxylated polyether matrix (Bio-Gel SEC-10). Five peaks corresponding to different aldol condensates of glutaraldehyde were obtained from the affinity column. They have been distinguished by 1H-NMR and UV spectroscopy. Kinetic measurements yielded formation rates for the different aldol condensates.

Alcohols↗

Mutagenicity of marijuana and Transkei tobacco smoke condensates in the Salmonella/microsome assay.

Extracts and smoke condensates of marijuana, Transkei home-grown tobacco and also commercial cigarette tobaccos were assayed for their mutagenic activity to Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, both with and without metabolic activation. No mutagenic activity was detected in dichloromethane extracts of marijuana and tobacco per se, but all the smoke condensates exhibited mutagenicity with metabolic activation. The only strain not mutated by any of the pyrolyzates was TA1535. Transkei tobacco pyrolyzate proved to be the most mutagenic, followed by marijuana, pipe and cigarette tobacco. Mutagenicity was positively associated with the nitrogen content of the various products. The potent mutagenic action of marijuana smoke condensate, coupled with a condensate yield of more than 50% higher than that of cigarette and pipe tobacco, indicates a high carcinogenic risk associated with marijuana smoking.

Cannabis↗

Karyotyping chromosomes by electron microscopy. Condensation-inhibition of G bands in human and Chinese hamster chromosomes by a BrdU-Hoechst 33258 treatment.

A BrdU-Hoechst 33258 treatment of living cells, which selectively induced condensation-inhibition of G-band chromatin in human and Chinese hamster chromosomes, is presented. As a consequence mitotic chromosomes showed high resolution R-banding patterns when examined by light and electron microscopy. Besides each whole chromosome identification, this procedure also permitted the electron microscopic study of specific structures, such as satellites, secondary constrictions, telomeres, centromeres, as well as G and R bands, some of them no visible by light microscopy. We have also observed that the chromatin of G and R bands behave as blocks of chromatin condensation and that G-band chromatin develops condensation along G2. Under the BrdU-Hoechst 33258 treatment, chromatin fibers seem to invert their spontaneous pattern of condensation within the chromosomes.

Animals↗

An electro-optical study of the mechanisms of DNA condensation induced by spermine.

Condensation of DNA by spermine has been studied by electric dichroism, electric birefringence and rotational relaxation times at 1 mM ionic strength. Using Manning's theory, we found that condensation occurs for a fraction of neutralized phosphate charges (r) equal to 0.90, in good agreement with previous studies using spermidine, synthetic polyamines and trivalent cations (e.g. Co(NH3)36 +, Tb3 +). Our results are compatible with the presence in solution of torus-shaped condensed structures in a narrow range of spermine concentration; further addition of the polyamine produced precipitation due to the self-aggregation of several toroids. For spermine concentrations lower than that required for collapse, important changes of the orientation mechanism in the electric field and of DNA stiffness were observed. Whereas free DNA was mainly oriented by a fast-induced polarizability mechanism, DNA-spermine complexes displayed an important permanent dipole component, in the spermine concentration range where extension of the DNA molecules was present. The birefringence relaxation times suggested that, in the first step, the stiffness of the DNA molecules increased, and then, at higher spermine concentration, bending of the DNA molecules occurred so that condensation into toroidal particles became possible.

Birefringence↗