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At least 973 records · Page 54Linked to original sources
PubMed1988

A new alcohol dehydrogenase, reactive towards methanol, from Bacillus stearothermophilus.

An NAD+-dependent alcohol dehydrogenase (ADH) was purified to homogeneity from an aerobic strain of Bacillus stearothermophilus, DSM 2334 (ADH 2334), and compared with the ADH from B. stearothermophilus NCA 1503 (ADH 1503). When an antibody raised against ADH 2334 was used, no cross-reactivity with ADH 1503 was observed on Western blots; by means of an enzyme-linked-immunoabsorbent-assay ('e.l.i.s.a.') procedure, it was found that ADH 1503 had less than 6% of the antigenic activity of ADH 2334. Amino acid analyses detected very small differences in composition, equivalent to about 40 sequence changes, between the two enzymes. The new enzyme has the same six-amino-acid N-terminal sequence as ADH 1503. ADH 2334, but not ADH 1503, is reactive towards methanol; both enzymes can oxidize ethanol, propan-1-ol, butan-1-ol and butan-2-ol. The new enzyme has a distinctive pH optimum at pH 5.5-6 and has significantly lower KEthanolm and kEthanolcat. values than those of ADH 1503. From steady-state kinetic parameters of the reaction with ethanol, propan-1-ol and butan-1-ol, it was shown that ADH 2334 has an ordered mechanism in both directions, with NAD+ being the compulsory first substrate in alcohol oxidation and NADH release being the rate-limiting step. ADH 1503 has an ordered addition of NAD+ and alcohol, but NADH release is not rate-limiting.

Alcohol Dehydrogenase↗
PubMed1995

Methional derived from 4-methylthio-2-oxobutanoate is a cellular mediator of apoptosis in BAF3 lymphoid cells.

4-Methylthio-2-oxobutanoic acid is the direct precursor of methional, which is a potent inducer of apoptosis in a BAF3 murine lymphoid cell line which is interleukin-3 (IL3)-dependent. Cultures treated for 8 h with methional in the presence of IL3 show extensive DNA double-strand breaks on flow cytometric analysis, increases in DNA fragmentation as measured by the amount of non-sedimentable DNA present in the 30,000 g supernatant of cell lysates and the typical laddering pattern of multiples of 180 bp seen upon agarose gel electrophoresis. No such features of apoptosis were found in cells treated with 4-methylthio 2-oxobutanoic acid or propanal, suggesting that the simultaneous presence of the methylthio group on the propanal moiety is essential for apoptosis to take place. Methional is further metabolized in cells by two reactions: oxidation via aldehyde dehydrogenase to (methylthio)propionic acid or beta-hydroxylation to malondialdehyde. The formation of malondialdehyde from methional in vitro by chemical hydroxylation under the conditions of the Fenton reaction provides a mechanism for the beta-hydroxylation which takes place in vivo. During apoptosis induced by IL3 deprivation, the ratio of 2,4-DNPH MDA to 2,4-DNPH methional is 0.94 in cells in IL3- medium compared with 0.54 in cells in IL3+ medium. These results support a role of cellular methional and malondialdehyde in apoptosis.

Aldehydes↗
PubMed1997

Quantification of malondialdehyde and 4-hydroxynonenal adducts to lysine residues in native and oxidized human low-density lipoprotein.

Malondialdehyde (MDA) and 4-hydroxynonenal (HNE) are major end-products of oxidation of polyunsaturated fatty acids, and are frequently measured as indicators of lipid peroxidation and oxidative stress in vivo. MDA forms Schiff-base adducts with lysine residues and cross-links proteins in vitro; HNE also reacts with lysines, primarily via a Michael addition reaction. We have developed methods using NaBH4 reduction to stabilize these adducts to conditions used for acid hydrolysis of protein, and have prepared reduced forms of lysine-MDA [3-(N epsilon-lysino)propan-1-ol (LM)], the lysine-MDA-lysine iminopropene cross-link [1,3-di(N epsilon-lysino)propane (LML)] and lysine-HNE [3-(N epsilon-lysino)-4-hydroxynonan-l-ol (LHNE)]. Gas chromatography/MS assays have been developed for quantification of the reduced compounds in protein. RNase incubated with MDA or HNE was used as a model for quantification of the adducts by gas chromatography/MS. There was excellent agreement between measurement of MDA bound to RNase as LM and LML, and as thiobarbituric acid-MDA adducts measured by HPLC; these adducts accounted for 70-80% of total lysine loss during the reaction with MDA. LM and LML (0.002-0.12 mmol/ mol of lysine) were also found in freshly isolated low-density lipoprotein (LDL) from healthy subjects. LHNE was measured in RNase treated with HNE, but was not detectable in native LDL. LM, LML and LHNE increased in concert with the formation of conjugated dienes during the copper-catalysed oxidation of LDL, but accounted for modification of < 1% of lysine residues in oxidized LDL. These results are the first report of direct chemical measurement of MDA and HNE adducts to lysine residues in LDL. LM, LML and LHNE should be useful as biomarkers of lipid peroxidative modification of protein and of oxidative stress in vitro and in vivo.

Aldehydes↗
PubMed1997

Glutathione S-transferases from the white-rot fungus, Phanerochaete chrysosporium.

A glutathione S-transferase (GST) was purified to homogeneity from the white-rot fungus, Phanerochaete chrysosporium, by affinity chromatography on glutathione-agarose followed by Mono-Q ion-exchange FPLC. This protein immunoblotted with antisera to rat Theta class GST 5-5 and also showed N-terminal sequence similarity to the Theta class, including the presence of a conserved serine residue that has been specifically implicated in catalysis in this class [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133-2143] and other residues conserved in plant sequences. Catalytic activity was found to be highly labile in the purified protein, although preliminary evidence for activity (approx. 120 m-units/mg) with 1,2-epoxy-3-(p-nitrophenoxy)propane was obtained in some preparations. The enzyme seems to be a dimer with a subunit molecular mass of 25 kDa by SDS/PAGE. The native molecular masses estimated by non-denaturing electrophoresis and by Superose-12 gel filtration were 58 and 45 kDa respectively. A second protein purified in this study also gave low level of activity with 1,2-epoxy-3-(p-nitrophenoxy)propane and had a subunit molecular mass of 28 kDa (native size 62-63 kDa), but did not immunoblot with any GST class and seemed to be N-terminally blocked.

Agaricales↗
PubMed1996

Development of the gametophyte of the fern Schizaea pusilla.

Schizaea pusilla is a pteridophyte with several unique developmental characteristics. In contrast to most other fern species, S. pusilla gametophytes remain filamentous throughout their development, and the gametophytes are associated with an endophytic fungus which appears to be mycorrhizal. In terms of tropistic responses, apical filament cells of young gametophytes are negatively phototropic compared with germ filaments of other ferns which exhibit positive phototropism. Cryofixation (propane jet freezing and high-pressure freezing) in conjunction with freeze substitution electron microscopy was used to study young gametophytes. The results demonstrate that apical filament cells have a distinctive structural polarity and that rhizoids also can be successfully frozen by these methods. The cytoskeleton and endomembrane system were particularly well preserved in cryofixed cells. In addition, Schizaea gametophytes were used as a test system to evaluate potential artifacts of propane jet freezing and high pressure freezing. There was little apparent difference in ultrastructure between cells cryofixed by either freezing method. These gametophytes will be useful in determining the effectiveness of cryofixation techniques and as a model system in tip growth studies.

Cryopreservation↗
PubMed1996

Solubilization of high-affinity, guanine nucleotide-sensitive mu-opioid receptors from rat brain membranes.

High-affinity mu-opioid receptors have been solubilized from rat brain membranes. In most experiments, rats were treated for 14 days with naltrexone to increase the density of opioid receptors in brain membranes. Occupancy of the membrane-associated receptors with morphine during solubilization in the detergent 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate appeared to stabilize the mu-opioid receptor. After removal of free morphine by Sephadex G50 chromatography and adjustment of the 3-[(3-cholamidopropyl)dimethyl]-1-propane sulfonate concentration to 3 mM, the solubilized opioid receptor bound [3H][D-Ala2,N-Me-Phe4,Gly-Dl5]-enkephalin([3H]DAMGO), a mu-selective opioid agonist, with high affinity (KD = 1.90 +/- 0.93 nM; Bmax = 629 +/- 162 fmol/mg of protein). Of the membrane-associated [3H]-DAMGO binding sites, 29 +/- 7% were recovered in the solubilized fraction. Specific [3H]DAMGO binding was completely abolished in the presence of 10 microM guanosine 5'-O-(3-thiotriphosphate). The solubilized receptor also bound [3H]diprenorphine, a nonselective opioid antagonist, with high affinity (KD = 1.4 +/- 0.39 nM, Bmax = 920 +/- 154 fmol/mg of protein). Guanosine 5'-O-(3-thiotriphosphate) did not diminish [3H]diprenorphine binding. DAMGO at concentrations between 1 nM and 1 microM competed with [3H]diprenorphine for the solubilized binding sites; in contrast, [D-Pen2, D-Pen5]-enkephalin, a delta-selective opioid agonist, and U50488H, a kappa-selective opioid agonist, failed to compete with [3H]diprenorphine for the solubilized binding sites at concentrations of < 1 microM. In the absence of guanine nucleotides, the DAMGO displacement curve for [3H]diprenorphine binding sites better fit a two-site than a one-site model with KDhigh = 2.17 +/- 1.5 nM, Bmax = 648 +/- 110 fmol/mg of protein and KDlow = 468 +/- 63 nM, Bmax = 253 +/- 84 fmol/mg of protein. In the presence of 10 microM guanosine 5'-O-(3-thiotriphosphate), the DAMGO displacement curve better fit a one- than a two-site model with KD = 815 +/- 33 nM, Bmax = 965 +/- 124 fmol/mg of protein.

Animals↗
PubMed2005

Self-association behaviour of protein:surfactant systems in alcohol/water mixtures.

The effect of the addition of short-chain monohydric alcohols (ethanol and propan-2-ol) to the protein:surfactant system lysozyme:sodium dodecyl sulfate (Lz:SDS) in aqueous solution was investigated using a conductometric technique. A second protein:surfactant system, bovine serum albumin:SDS (BSA:SDS) was also investigated so that the effect of a different protein conformation and composition could be compared. The critical aggregation concentration (CAC) of the protein forming the complex and the critical micelle concentration (CMC *) of SDS in the presence of protein, at different alcohol concentrations, were determined. It was found in both cases that the addition of alcohol does not produce a significant change in the CAC, whereas the CMC * displays variation with alcohol concentration that shows an inversion in the ranges 0.05-0.06 ethanol mole fraction and 0.02-0.03 propan-2-ol mole fraction. This suggests that, in contrast with the CAC behaviour, the major factor that drives SDS micellization in the presence of protein is the variation in water structure. Results also suggest that it occurs in the same way for both proteins, where electrostatic interactions are the main force in the formation of the complex. Conversely, hydrophobic interactions play the dominant role at the micellization stage, and only the extent of the interaction between protein:surfactant aggregates and surfactant species seems to depend on protein nature.

1-Propanol↗
PubMed2002

Preformulation approaches to improve the oral bioavailability of two novel piperidine renin inhibitors in dog.

Different experimental formulations based on aqueous and oily systems, water miscible solvents, and solid dispersions were investigated for their potential to increase the oral bioavailability (F) of two novel piperidine renin inhibitors (Ro-X1: (R)-1-methoxy-3-[(3S,4R,5R)-4-[4-[3-(2-methoxy-benzyloxy)-propoxy]-phenyl]- 5-(4-methoxy-naphthalen-2-ylmethoxy)-piperidin-3-yloxy]-propan-2-ol; Ro-X2: (R)-3-[(3S,4R,5R)-4-[4-[3-(2-methoxy-benzyloxy)-propoxy]-phenyl]-5-(4- methoxy-naphthalen-2-ylmethoxy)-piperidin-3-ylmethoxy]-propane-1,2-diol) in dogs compared to their administration as acidic aqueous solution. The compounds were characterized by a low solubility at pH 7 (Ro-X1: 3 micrograms/ml, Ro-X2: 24 micrograms/ml) and a high lipophilicity (Ro-X1: LogP = 5.7, Ro-X2: LogP = 3.7). For Ro-X1 oil-based vehicles resulted in an improvement in the oral bioavailability compared to the aqueous solution (F = 6 +/- 1.2%) with the best result being achieved with a solution in Capmul (F = 14.6 +/- 3.5%). By contrast, for Ro-X2 the highest bioavailability (F = 27.1 +/- 8.4%) was achieved using an aqueous solution. Computer simulations based on the physicochemical parameters of the compounds only predicted that the fraction of compound absorbed in man should be almost quantitative for Ro-X2 and only about 28% for Ro-X1. These results suggest that other factors such as extensive gut and/or hepatic metabolism as well as exclusion by intestinal transporters such as p-glycoprotein, rather than incomplete solubilization in the gut, are the major reasons for the limited oral bioavailability of both compounds.

Animals↗
PubMed2001

7-feruloylloganin: an iridoid glucoside from stems of Lonicera insularis.

A new iridoid glucoside, 7-feruloylloganin (1), was isolated from stems of Lonicera insularis, along with six known lignans including (-)-pinoresinol, 9alpha-hydroxypinoresinol, balanophonin, erythro-1-(4-hydroxy-3-methoxyphenyl)-2-[4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy]-propane-1,3-diol, threo-1-(4-hydroxy-3-methoxyphenyl)-2-[4-[2-formyl-(E)-vinyl]-2-methoxyphenoxy]-propane-1,3-diol and buddlenol A. The structure of 1 was determined by analyses of 2D NMR (1H-1H COSY, HMQC and HMBC) and HRFABMS.

Glucosides↗
PubMed1991

[Regeneration of the ciliary beat of human ciliated cells].

The influence of an isotonic, alkaline saline solution (diluted "Emser Sole" or brine from the spa of Bad Ems) on the ciliary beat of isolated cultured human ciliated cells of the upper respiratory tract was investigated. The ciliary beat was observed via an inverted phase contrast microscope (Zeiss Axiomat IDPC) and measured microphotometrically under physiological conditions and after the damaging influence of 1% propanal solution. Under physiological conditions the saline solution had a positive, although statistically not significant influence on the frequency of the ciliary beat. After damage of the cultivated cells by 1% propanal solution, the saline solution had a significant better influence on the regeneration of the cultured cells than a physiological sodium chloride solution. It is concluded that diluted brine from Bad Ems has a positive effect on the ciliary beat of the respiratory epithelium and accelerates its regeneration after damage by viral and bacterial infections, surgery or inhaled noxae.

Aldehydes↗
PubMed2003

Biofiltration of trichloroethylene-contaminated air: a pilot study.

This project demonstrated the biofiltration of a trichloroethylene (TCE)-contaminated airstream generated by air stripping groundwater obtained from several wells located at the Anniston Army Depot, Anniston, AL. The effects of several critical process variables were investigated to evaluate technical and economic feasibility, define operating limits and preferred operating conditions, and develop design information for a full-scale biofilter system. Long-term operation of the demonstration biofilter system was conducted to evaluate the performance and reliability of the system under variable weather conditions. Propane was used as the primary substrate necessary to induce the production of a nonspecific oxygenase. Results indicated that the process scheme used to introduce propane into the biofiltration system had a significant impact on the observed TCE removal efficiency. TCE degradation rates were dependent on the inlet contaminant concentration as well as on the loading rate. No microbial inhibition was observed at inlet TCE concentrations as high as 87 parts per million on a volume basis.

Air Pollution↗
PubMed2004

Evaluation of a multi-parameter biomarker set for oxidative damage in man: increased urinary excretion of lipid, protein and DNA oxidation products after one hour of exercise.

The objective of the present study was to evaluate a comprehensive set of urinary biomarkers for oxidative damage to lipids, proteins and DNA, in man. Eighteen moderately trained males (mean age 24.6+/-0.7) exercised 60min at 70% of maximal O2 uptake on a cycle ergometer. Urine fractions for 12 h were collected 1 day before, and for 3 consecutive days after exercise. As biomarkers of lipid peroxidation, 8 aldehydes (i.e. propanal, butanal, pentanal, hexanal, heptanal, octanal, nonanal and malondialdehyde-MDA)and acetone were analyzed in urines by gas chromatography with electron capture detection (GC-ECD). As a biomarker of protein oxidation, o,o'-dityrosine was analyzed in urine samples by a recently developed isotope dilution HPLC-atmospheric pressure chemical ionization (APCI)-tandem-mass spectrometry (HPLC-APCI-MS/MS) methodology. As a biomarker of oxidative DNA damage, urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was measured by an ELISA method. On the day of exercise, significant increases were observed in urinary excretions of acetone (p < 0.025, n = 18) and butanal (p < 0.01, n = 18) in the 12h daytime fractions compared to the daytime fraction before exercise. The urinary acetone excretion was also significantly (p < 0.05) increased on the 1st day after exercise. Octanal and nonanal were increased in the daytime urine fraction on the 2nd day after exercise. However, these increases were of borderline significance (p = 0.09 and p = 0.07, respectively). Significantly elevated urinary o,o'-dityrosine amounts were observed in the daytime fraction on the day of exercise (p < 0.025) and on the 1st day after exercise (p = 0.07) compared to the before exercise daytime fraction. Excretion of urinary 8-OHdG was statistically significantly increased in the daytime fractions on the day of exercise (p = 0.07) and on the 1st day after exercise (p < 0.025) compared to before exercise daytime fraction. Increases in urinary excretions of acetone, propanal, pentanal, MDA and 8-OHdG significantly correlated with training status (hours of exercise/week) of the volunteers, while o,o'-dityrosine did not. To our knowledge, the present study is the first to evaluate a multi-parameter non-invasive biomarker set for damage to three main cellular targets of ROS. It shows that 1 h of exercise may already induce oxidative damage in moderately trained individuals and that the chosen urinary biomarkers are sensitive enough to monitor such damage.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1984

An exploration of cryoprotective compounds for fowl spermatozoa.

Glycerol is an effective cryoprotective for fowl spermatozoa, but after thawing the frozen semen it must be reduced in concentration from the level adequate to protect spermatozoa during freezing, otherwise it has a contraceptive action. A series of alternative cryoprotective compounds were tested for their effect on fertility when fowl spermatozoa were inseminated fresh in their presence. Under these circumstances dimethylsulphoxide, dimethylacetamide, ethane-diol, propane-diol and methylpyrrolidone did not depress fertility when used in concentrations equivalent to that of glycerol or in amounts reported previously to be non-toxic and adequate to protect cells during freezing. Dimethylacetamide was compared with propane-diol for use in freezing fowl semen and the former enabled encouraging levels of fertility to be obtained.

Acetamides↗
PubMed1982

Biliary excretion of cadmium in rat. V. Effects of structurally related mercaptans on chelation of cadmium from metallothionein.

The effects of several mono-, di-, and trithiols (400 mumol/kg) in mobilizing Cd from metallothionein were studied in rats 24 h after a single injection of 109CdCl2 (1 mg kg/Cd). BAL (2,3-dimercaptopropanol) and propanetrithiol (1,2,3-trimercaptopropane) were the most effective mercaptans in increasing the biliary excretion of Cd (3.1 and 5.5% of administered dose, respectively, compared to 0.04% in control injected with propylene glycol) in in situ experiments with a significant decrease in hepatic Cd. Propane-1-thiol was inactive and propanetrithiol was the most toxic of the compounds studied. A number of propane dithiols having sulfhydryl groups at different carbon positions with and without substituents (OH or SO-3) and dimercaptoethane were also tested for effectiveness in removing Cd. All the lipophilic compounds with two adjacent sulhydryl groups were effective in increasing the biliary excretion of Cd, but were less effective than BAL and propanetrithiol. The major form of Cd in liver cytosols of rats pretreated with CdCl2 and injected with mercaptans was metallothionein. A small amount of Cd in liver cytosol and a major portion of biliary Cd in propanetrithiol-injected rats were bound to high-molecular-weight proteins when fractionated on Sephadex G-75 columns. On the other hand, after injection of BAL, most of the Cd in the bile was associated with a fraction of molecular weight 10,000. Even though Cd was present mainly as metallothionein in livers of Cd-pretreated rats, the biliary forms of Cd after injection of BAL and propanetrithiol were different. Similar results were obtained when Cd was added in vitro to bile samples collected from control rats that were injected with these chelating agents alone. However, the Sephadex G-75 elution profile of Cd-BAL and Cd-propanetrithiol after direct addition to control rat bile showed Cd complexes of identical molecular weight (less than 6000). These results suggested that the Cd-binding ligands present in bile after injection of BAL and propanetrithiol were different from and had a higher molecular weight than the complexes in vitro with Cd and the respective chelating agents.

Animals↗
PubMed2004

Dipalmitoylphosphatidylcholine (DPPC) and DPPC/cholesterol liposomes as predictors of the cytotoxicity of bis-GMA related compounds.

In light of recent development, dental materials such as 2, 2-bis [4-2(-hydroxy-3-methacryloyloxypropoxy)phenyl] propane, ( bis-GMA); 2, 2-bis [4-(1-hydroxymethyl-2-methacryloxy)phenyl] propane, (iso-bis-GMA); and triethyleneglycol dimethacrylate, (TEGDMA) were investigated to determine whether their phase transition properties (phase transition temperature, temperature width, cooperativity) could be induced in samples of DPPC or DPPC/cholesterol (CHOL) liposomes using differential scanning calorimetry (DSC). The changes in phase transition properties of DPPC liposomes caused by addition of TEGDMA were greater than those caused by addition of bis-GMA or iso-bis-GMA, but the extent of changes in the properties of DPPC/CHOL (10:1 or 4:1) liposomes declined in the order of bis-GMA > iso-bis-GMA > TEGDMA. The degree of alteration was related to the cytotoxicity of these compounds. DPPC/CHOL liposomes were found to be better predictors of cytotoxicity than DPPC liposomes. Whether the computational approach to studying the molecular mechanism of alteration is applicable using descriptors such as reactivity of energy of the highest occupied molecular orbital (HOMO) and/or lowest unoccupied molecular orbital (LUMO) was investigated, and the data suggested that these descriptors are useful for studying the interactive roles of dental materials.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2006

Reproducibility and workability of the European test standard EN 12791 regarding the effectiveness of surgical hand antiseptics: a randomized, multicenter trial.

OBJECTIVES: To evaluate the reproducibility and workability of the in vivo test model of the European test standard EN 12791 regarding the effectiveness of surgical hand antiseptics and, as a secondary objective, to evaluate the power of the model to discriminate between the effectiveness of various formulations of surgical hand antiseptics. DESIGN: Prospective, randomized, multicenter study with a Latin square design. SETTING: Five laboratories at 2 universities, 2 disinfectant manufacturers, and 1 private testing institution. PARTICIPANTS: Twenty healthy adults in each laboratory. INTERVENTION: Surgical hand antisepsis was performed by scrubbing with chlorhexidine gluconate 4% detergent (CHG) or by rubbing the hands with propan-2-OL (70% by volume; Iso 70) or ethanol 85% (E 85); rubbing the hands and forearms for 3 minutes with propan-1-OL (N 60) was used as the reference disinfection procedure. We deliberately chose to use these antiseptics at the given concentrations because they were intended to cover the range of typical antiseptics submitted for approval according to EN 12791. METHODS: In once-weekly tests, the immediate effects of the 4 antiseptics were established according to the method laid down in EN 12791 by assessing the release of skin flora from the fingertips as viable bacteria counts per milliliter of sampling fluids before treatment and viable bacteria counts immediately after treatment, separately for both hands, such that after 4 weeks each volunteer had used every formulation once. RESULTS: The mean log reduction factor (RF) for the release of bacterial skin flora (the log RF was calculated as the log count before treatment minus the log count after treatment) and corresponding standard deviations for the 4 hand antisepsis formulations were as follows: for CHG, 1.1+/-0.3 colony-forming units (cfu) per milliliter of sampled fluid; for Iso 70, 1.7+/-0.3 cfu/mL; for E 85, 2.1+/-0.3 cfu/mL; and for N 60, 2.4+/-0.4 cfu/mL. The differences between these values proved significant (P<.001) by analysis of variance and in Tukey's "honestly significantly different" (HSD) post hoc test. Although, with regard to their immediate antibacterial activity, the same ranking of these antiseptics was found at all laboratories, the levels of efficacy were significantly different across laboratories (P<.001); no statistical difference was found between left and right hands (P>.01). Relating the log RF values of the other 3 formulations to those of the reference formulation (N 60) abolished differences between laboratories (P=.16); in addition, the interclass correlation coefficient decreased from 9.1% to 4.5%. With 20 volunteers, a minimum difference of 0.47 log between the mean log RFs of the reference formulation and an inferior test formulation will be detected as significant at an alpha of .05 (1-sided) and a 1- beta value of .8. CONCLUSION: The test method described in EN 12791 yielded the same conclusion on the effectiveness of the tested formulations in every laboratory and proved, therefore, reproducible and workable.

2-Propanol↗
PubMed2004

Alcohol-mediated error-prone PCR.

The effect of urea, isopropanol, propan-1-ol, and butan-1-ol on PCR using three different DNA polymerases was investigated. In the presence of these agents, polymerases were active as expected up to a critical concentration where they became progressively inhibited. Critical concentrations of alcohols generally increased with thermoresistance of the polymerases and decreased with the hydrophobicity of the alcohols. These results indicate that an important aspect of the inhibition involved conformational loosening due to a decrease in the hydrophobic effect. A mutagenic effect occurred with Vent(r) (exo-) DNA polymerase in the presence of 7.0 to 8.0% v/v propan-1-ol, affording mutation frequencies of up to 9.8 x 10(-3) mutation/bp/PCR. Under these conditions the preferential replacement of Gs and Cs was observed, in opposition to standard error-prone PCR that favors replacement of As and Ts. Comparison of various PCR conditions indicates that propanol and MnCl2 have different modes of action, and that the decrease in fidelity promoted by propanol is due to a finely tuned partial destabilization of the polymerase. The PCR conditions developed in this study provide a useful alternative for targeting different sequence space for directed evolution experiments.

1-Propanol↗
PubMed1998

Inhibition of chromosomal separation provides insights into cleavage furrow stimulation in cultured epithelial cells.

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinoderm embryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3, 5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.

Actins↗