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PubMed2002

Identification of a 34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing domain by site-directed mutagenesis.

The C-terminus of histone H1 is necessary for the folding of polynucleosomal arrays into higher-order structure(s) and contains octapeptide repeats each having DNA binding S/TPKK motifs. These repeat motifs were earlier shown to mimic the DNA/chromatin-condensing properties of the C-terminus of histone H1 (Khadake, J. R., and Rao, M. R. S. (1995) Biochemistry 36, 1041-1051). In the present study, we have generated a series of C-terminal mutants of rat histone H1d and studied their DNA-condensation properties. The single proline to alanine mutation in the S/TPKK motifs either singly or in combination resulted in only a 20% decrease in the DNA-condensation property of histone H1. Deletion of all the three S/TPKK motifs resulted in a 45% decrease in DNA condensation. When the three octapeptide repeats encompassing the S/TPKK motifs were deleted, there was again a 45% decrease in DNA condensation. On the other hand, when the entire 34 amino acid stretch (residue 145-178) was deleted, there was nearly a 90% decrease in DNA condensation brought about by histone H1d. Interestingly, deletion of the 10 amino acid spacer between the octapeptide repeats (residues 161-170) also reduced the DNA condensation by 70%. Deletion of the region (residues 115-141) immediately before the 34 amino acid stretch and after the globular domain and the region (residues 184-218) immediately after the 34 amino acid stretch had only a marginal effect on DNA condensation. The importance of the 34 amino acid stretch, including the 10 amino acid spacer, was also demonstrated with the recombinant histone H1d C-terminus. We have also determined the induced alpha-helicity of histone H1 and its various mutants in the presence of 60% trifluoroethanol, and the experimentally determined induced helical contents agree with the theoretical predictions of secondary structural elements in the C-terminus of histone H1d. Thus, we have identified a 34 amino acid stretch in the C-terminus of histone H1d as the DNA-condensing domain.

Amino Acid Sequence↗

PubMed1999

Metabolic stability of glutaraldehyde cross-linked peptide DNA condensates.

The stability of peptide DNA condensates was examined after introducing glutaraldehyde to cross-link surface amine groups. A 20 amino acid peptide (CWK(18)) was used to condense DNA into small (70 nm) condensates. The reaction between glutaraldehyde and peptide DNA condensates was indirectly monitored using a fluorescence-based assay to establish reaction completion in 4-5 h when using glutaraldehyde-to-peptide ratios of 1 to 4 mol equiv. Higher levels of glutaraldehyde cross-linking led to significant increases in particle size. The improved stability imparted by glutaraldehyde cross-linking was demonstrated by the increased resistance of DNA condensates to shear stress induced fragmentation. The cross-linked condensates were also significantly more resistant to in vitro metabolism by serum endonucleases. A decrease in the magnitude of transient gene expression was determined for cross-linked DNA condensates which also resulted in a 10-day steady-state expression when cross-linking with 4 mol equiv of glutaraldehyde. The results suggest that cross-linking DNA condensates may provide a means to alter the time course of transient gene expression by inhibiting DNA metabolism.

Chloroquine↗

PubMed1999

Formulation of highly soluble poly(ethylene glycol)-peptide DNA condensates.

Two poly(ethylene glycol) (PEG)-peptides were synthesized and tested for their ability to bind to plasmid DNA and form soluble DNA condensates with reduced spontaneous gene expression. PEG-vinyl sulfone or PEG-orthopyridyl disulfide were reacted with the sulfhydryl of Cys-Trp-Lys(18) (CWK(18)) resulting in the formation of nonreducible (PEG-VS-CWK(18)) and reducible (PEG-SS-CWK(18)) PEG-peptides. Both PEG-peptides were prepared on a micromole scale, purified by RP-HPLC in >80% yield, and characterized by (1)H NMR and MALDI-TOF. PEG-peptides bound to plasmid DNA with an apparent affinity that was equivalent to alkylated (Alk)CWK(18), resulting in DNA condensates with a mean diameter of 80-90 nm and zeta (zeta) potential of +10 mV. The particle size of PEG-peptide DNA condensates was constant throughout the DNA concentration range of 0. 05-2 mg/mL, indicating these to be approximately 20-fold more soluble than AlkCWK(18) DNA condensates. The spontaneous gene transfer to HepG2 cells mediated by PEG-VS-CWK(18) DNA condensates was over two orders of magnitude lower than PEG-SS-CWK(18) DNA condensates and three orders of magnitude lower than AlkCWK(18) DNA condensates. PEG-VS-CWK(18) efficiently blocked in vitro gene transfer by reducing cell uptake. The results indicate that a high loading density of PEG on DNA is necessary to achieve highly soluble DNA condensates that reduce spontaneous in vitro gene transfer by blocking nonspecific uptake by HepG2 cells. These two properties are important for developing targeted gene delivery systems to be used in vivo.

Cell Line↗

PubMed2005

Structural study of DNA condensation induced by novel phosphorylcholine-based copolymers for gene delivery and relevance to DNA protection.

Poly[2-(dimethylamino)ethyl methacrylate-b-2-methacryloyloxyethyl phosphorylcholine] (DMA-MPC) is currently under investigation as a new vector candidate for gene therapy. The DMA block has been previously demonstrated to condense DNA effectively. The MPC block contains a phosphorylcholine (PC) headgroup, which can be found naturally in the outside of the cell membrane. This PC-based polymer is extremely hydrophilic and acts as a biocompatible steric stabilizer. In this study, we assess in detail the morphologies of DNA complexes obtained using the diblock copolymer series DMA(x)MPC30 (where the mean degree of polymerization of the MPC block was fixed at 30 and the DMA block length was systematically varied) using transmission electron microscopy (TEM) and liquid atomic force microscopy (AFM). Both techniques indicate more compact complex morphologies (more efficient condensation) as the length of the cationic DMA block increases. However, the detailed morphologies of the DMA(x)MPC30-DNA complexes observed by TEM in vacuo and by AFM in aqueous medium are different. This phenomena is believed to be related to the highly hydrophilic nature of the MPC block. TEM studies revealed that the morphology of the complexes changes from loosely condensed structures to highly condensed rods, toroids, and oval-shaped particles as the DMA moiety increases. In contrast, morphological changes from plectonemic loops to flower-like and rectangular block-like structures, with an increase in highly condensed central regions, are observed by in situ AFM studies. The relative population of each structure is clearly dependent on the polymer molecular composition. Enzymatic degradation assays revealed that only the DMA homopolymer provided effective DNA protection against DNase I degradation, while other highly condensed copolymer complexes, as judged from TEM and gel electrophoresis, only partially protected the DNA. However, AFM images indicated that the same highly condensed complexes have less condensed regions, which we believe to be the initiation sites for enzymatic attack. This indicates that the open structures observed by AFM of the DNA complexation by the DMA(x)MPC30 copolymer series are closer to in vivo morphology when compared to TEM.

Biopolymers↗

PubMed2005

Temperature and gas-phase composition measurements in supersonic flows using tunable diode laser absorption spectroscopy: the effect of condensation on the boundary-layer thickness.

We used a tunable diode laser absorption spectrometer and a static-pressure probe to follow changes in temperature, vapor-phase concentration of D2O, and static pressure during condensation in a supersonic nozzle. Using the measured static-pressure ratio p/p0 and the mass fraction of the condensate g as inputs to the diabatic flow equations, we determined the area ratio (A/A*)wet and the corresponding centerline temperature of the flow during condensation. From (A/A*)wet we determined the boundary-layer displacement thickness during condensation (delta#)wet. We found that (delta#)wet first increases relative to the value of delta# in a dry expansion (delta#)Dry before becoming distinctly smaller than (delta#)Dry downstream of the condensation region. After correcting the temperature gradient across the boundary layers, the temperature determined from p/p0 and g agreed with the temperature determined by the laser-absorption measurements within our experimental error (+/-2 K), except when condensation occurred too close to the throat. The agreement between the two temperature measurements let us draw the following two conclusions. First, the differences in the temperature and mole fraction of D2O determined by the two experimental techniques, first observed in our previous study [P. Paci, Y. Zvinevich, S. Tanimura, B. E. Wyslouzil, M. Zahniser, J. Shorter, D. Nelson, and B. McManus, J. Chem. Phys. 121, 9964 (2004)], can be explained sufficiently by changes in delta# caused by the condensation of D2O, except when the phase transition occurs too close to the throat. Second, the extrapolation of the equation, which expresses the temperature dependence of the heat of vaporization of bulk D2O liquid, is a good estimate of the heat of condensation of supercooled D2O down to 210 K.

Journal Article↗

PubMed1984

The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. 1. Voluntary intake.

Voluntary intake was determined with vegetative Lotus pedunculatus cut and fed fresh to growing sheep of 42-46 kg live weight. Effects attributable to condensed tannins were assessed by growing the plant under high and low levels of soil fertility, inducing low and high concentrations of tannin (Expt 1), or by binding the tannins through spraying the herbage with polyethylene glycol (molecular weight 3350, PEG; Expt 2). Primary-growth lotus was used in Expt 1 and secondary-growth lotus in Expt 2. Concentrations of total and free condensed tannin were determined in fresh lotus, free tanning being defined as that not bound by mascerates of the plant. In Expt 1 the herbages fed contained respectively 46 and 106 g total condensed tannin/kg dry matter (DM) and 3 and 14 g free condensed tannin/kg DM. Mean metabolizable energy (ME) intakes were 0.89 and 0.77 MJ/kg live weight0 .75 per d (P less than 0.05) respectively. The lotus used in Expt 2 contained 63 and 5 g total reactive condensed tannin and free condensed tannin/kg DM respectively. After spraying with PEG at 2.4 g/g total condensed tannin, these values were reduced to 7 and 0.5 g/kg DM respectively. PEG addition increased apparent digestibility (proportion of each nutrient ingested) of cellulose, hemicellulose and nitrogen by 0.05, 0.08 and 0.26, and increased ME intake from 0.48 to 0.69 MJ/kg live weight0 .75 per d. It was concluded that high concentrations of condensed tannins depressed ME intake, due to depressions in both the voluntary intake and digestion of organic matter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗

PubMed1988

Comparative study of the condensation of chicken erythrocyte and calf thymus chromatins by di- and multivalent cations.

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidity, precipitation and electric dichroism measurements. For all the cations investigated (Mg2+, Tb3+, Co(NH3)6(3+), spermidine Spd2+ and spermine Sp4+) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg2+ and Tb3+. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a "precondensed" state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the "cross-linker" models. Tb3+ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that "30 nm fibers" were formed. Very little aggregation was produced by Tb3+. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.

Animals↗

PubMedDate not supplied

Exhaled breath condensate as matrix for toluene detection: a preliminary study.

The study was designed to investigate whether exhaled breath condensate, obtained by cooling exhaled air in spontaneous breathing, could be a suitable matrix for toluene quantitative analyses. Nine healthy subjects were exposed for a short period (20 min) to a known concentration of toluene. Exhaled breath condensate samples were collected before and at the end of the exposure, while the environmental concentration of toluene was continuously monitored. Toluene was analysed by head-space gas-chromatography mass spectrometry, and assay repeatability was also estimated in vitro. Baseline and post-exposure measurement of hippuric acid, the urinary toluene metabolite, was performed to assess current toluene exposure. Before the exposure toluene concentrations in the exhaled breath condensate were lower than the detectable limit in all subjects, while after the exposure toluene was detectable with a median value 0.35 microg l-1 (range 0.15-0.55 microg l-1) in all the exhaled breath condensate samples. As compared with the standard calibration in distilled water, the curves obtained by exhaled breath condensate were linear and comparable with the range examined in vivo for toluene. A significant correlation was found between the environmental toluene levels and toluene in the exhaled breath condensate at the end of exposure. Furthermore, a significant relationship between increased exhaled breath condensate toluene levels and urinary hippuric acid after the exposure was found. In conclusion, exhaled breath condensate is a promising matrix for toluene assessment, although its application in humans requires further investigations.

Adult↗

PubMed1994

Chromosome condensation and sister chromatid pairing in budding yeast.

We have developed a fluorescent in situ hybridization (FISH) method to examine the structure of both natural chromosomes and small artificial chromosomes during the mitotic cycle of budding yeast. Our results suggest that the pairing of sister chromatids: (a) occurs near the centromere and at multiple places along the chromosome arm as has been observed in other eukaryotic cells; (b) is maintained in the absence of catenation between sister DNA molecules; and (c) is independent of large blocks of repetitive DNA commonly associated with heterochromatin. Condensation of a unique region of chromosome XVI and the highly repetitive ribosomal DNA (rDNA) cluster from chromosome XII were also examined in budding yeast. Interphase chromosomes were condensed 80-fold relative to B form DNA, similar to what has been observed in other eukaryotes, suggesting that the structure of interphase chromosomes may be conserved among eukaryotes. While additional condensation of budding yeast chromosomes were observed during mitosis, the level of condensation was less than that observed for human mitotic chromosomes. At most stages of the cell cycle, both unique and repetitive sequences were either condensed or decondensed. However, in cells arrested in late mitosis (M) by a cdc15 mutation, the unique DNA appeared decondensed while the repetitive rDNA region appeared condensed, suggesting that the condensation state of separate regions of the genome may be regulated differently. The ability to monitor the pairing and condensation of sister chromatids in budding yeast should facilitate the molecular analysis of these processes as well as provide two new landmarks for evaluating the function of important cell cycle regulators like p34 kinases and cyclins. Finally our FISH method provides a new tool to analyze centromeres, telomeres, and gene expression in budding yeast.

Chromatids↗

PubMed1976

Control of chromosome behavior in amphibian oocytes. II. The effect of inhibitors of RNA and protein synthesis on the induction of chromosome condensation in transplanted brain nuclei by oocyte cytoplasm.

We studied the effects of actinomycin D, alpha-amanitin, puromycin, and cycloheximide on the cytoplasmic activity of maturing Rana pipiens oocytes that induces chromosome condensation in transplanted brain nuclei. Treatment of oocytes with each inhibitor suppressed the chromosome condensation induced by metaphase oocytes to varying degrees depending upon the dose of inhibitor, despite the fact that untreated metaphase I oocytes already possessed chromosome condensation activity (CCA). Treatment of brain nuclei before injection completely suppressed condensation at all doses used. Chromosome condensation induced by metaphase II oocyte cytoplasm, however, was insensitive to all the inhibitors, even when the brain nuclei were pretreated. Oocytes treated with alpha-amanitin throughout maturation induced chromosome condensation when tested at metaphase II. Removal of the oocyte chromosomes after the germinal vesicle (GV) broke down did not prevent the development of CCA, whereas removal of the entire GV before initiation of maturation deprived oocytes of CCA. The results suggest that metaphase I oocyte cytoplasm stimulates synthesis of brain nuclear RNAs that are translated into proteins necessary for chromosome condensation, whereas metaphase II oocytes possess all the factors for chromosome condensation. In both cases, GV nucleoplasm appears indispensable for the development of CCA, whereas immediate activity of the oocyte genome is not required.

Amanitins↗

PubMed1994

Separate metabolic pathways leading to DNA fragmentation and apoptotic chromatin condensation.

Apoptosis is the predominant form of cell death observed in a variety of physiological and pathological conditions such as cancer involution, insect metamorphosis, the development of the immune and nervous systems, and embryogenesis. The typical nuclear changes taking place in apoptotic cells include extensive condensation of chromatin and internucleosomal DNA fragmentation into units of 200 base pairs. However, the mechanisms responsible for both chromatin condensation and DNA fragmentation have yet to be elucidated. In this study, micrococcal nuclease and the divalent cations, Ca2+ and Mg2+, were applied to isolated nuclei in an attempt to reconstitute in vitro the digestion of genomic DNA associated with apoptosis. Micrococcal nuclease was found to induce a typical pattern of DNA fragmentation, but did not give rise to chromatin condensation, whereas Ca2+/Mg2+ induced both chromatin condensation and DNA fragmentation in isolated mouse liver nuclei. When the endonuclease inhibitor ZnCl2 was used, the DNA fragmentation induced by Ca2+/Mg2+ in nuclei could be completely inhibited, but chromatin condensation still occurred. For comparison, intact liver cells were treated with valinomycin, a potassium ionophore, which gave rise to an atypical cell death, with chromatin condensation appearing without DNA fragmentation. Our results suggest that endonuclease activation in apoptosis is neither necessary nor sufficient to induce chromatin condensation, and that DNA fragmentation and chromatin condensation may be triggered through separate pathways during apoptosis.

Animals↗

PubMed2005

Long-term in vivo gene expression via delivery of PEI-DNA condensates from porous polymer scaffolds.

Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding beta-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed beta-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for beta-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55-60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of beta-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2- week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI-condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.

Animals↗

PubMed2004

Short-term in vitro and in vivo analyses for assessing the tumor-promoting potentials of cigarette smoke condensates.

Previous studies found that repeated application of smoke condensate from tobacco-burning reference cigarettes to chemically initiated SENCAR mouse skin promoted the development of tumors in a statistically significant and dose-dependent manner, while condensate from prototype cigarettes that primarily heat tobacco promoted statistically fewer tumors. Based on the recognized correlation between sustained, potentiated epidermal hyperplasia and tumor promotion, we conducted tests to examine the utility of selected short-term analyses for discriminating between condensates exhibiting significantly different promotion activities. In vitro analyses assessing the potential for inducing cytotoxicity (ATP bioluminescence) or free radical production (cytochrome c reduction, salicylate trapping) demonstrated significant reductions when comparing condensate collected from prototype cigarettes to reference condensate. Short-term in vivo analyses conducted within the context of a mouse skin, tumor-promotion protocol (i.e., comparative measures of epidermal thickness, proliferative index, myeloperoxidase activity, leukocyte invasion, mutation of Ha-ras, and formation of modified DNA bases) provided similar results. Reference condensate induced statistically significant and dose-dependent increases (relative to vehicle control) for nearly all indices examined, while prototype condensate possessed a significantly reduced potential for inducing changes that we regarded as consistent with sustained epidermal hyperplasia and/or inflammation. Collectively, these data support the contention that selected short-term analyses associated with sustained hyperplasia and/or inflammation are capable of discriminating between smoke condensates with dissimilar tumor-promotion potentials. Moreover, our results suggest that comparative measures of proliferative index and myeloperoxidase activity, both possessing favorable correlation coefficients relative to tumor formation (i.e., > or = 0.95 after 8 or 12 weeks of promotion), may constitute reasonable end points for further investigation.

Adenosine Triphosphate↗

PubMed2002

Nephron induction revisited: from caps to condensates.

Conversion of mesenchyme to epithelium in the metanephric kidney is clearly a multimolecular, multistep and partly redundant process. The present short review focuses on a neglected morphological aspect of kidney differentiation: the development of two transitory mesenchymal condensations that precede epithelial differentiation of nephrons. The first appearing condensate covers the tips of the collecting ducts and is termed a cap condensate. In the early kidney rudiment this structure has been referred to as a primary or early condensate. A few cells of the cap condensate (maybe only four to six cells), situated at the lateral edge of the cap, start proliferating rapidly and form a pretubular aggregate (or pretubular condensate), which converts to secretory nephron epithelia and finally segregates to different tubule segments. Throughout nephrogenesis, the cap condensates and pretubular aggregates are clearly distinguishable structures that show only partly overlapping gene expression profiles. Apart from being the source for the pretubular aggregates, the role of the cap condensate is unknown. It is now proposed that the cap regulates ureteric branching morphogenesis.

Animals↗

PubMed1997

When rDNA transcription is arrested during mitosis, UBF is still associated with non-condensed rDNA.

The mechanisms that control inactivation of ribosomal gene (rDNA) transcription during mitosis is still an open question. To investigate this fundamental question, the precise timing of mitotic arrest was established. In PtK1 cells, rDNA transcription was still active in prophase, stopped in prometaphase until early anaphase, and activated in late anaphase. Because rDNA transcription can still occur in prophase and late anaphase chromosomes, the kinetics of rDNA condensation during mitosis was questioned. The conformation of the rDNA was analyzed by electron microscopy from the G2/M transition to late anaphase in the secondary constriction, the chromosome regions where the rDNAs are clustered. Whether at transcribing or non-transcribing stages, non-condensed rDNA was observed in addition to axial condensed rDNA. Thus, the persistence of this non-condensed rDNA during inactive transcription argues in favor of the fact that mitotic inactivation is not the consequence of rDNA condensation. Analysis of the three-dimensional distribution of the rDNA transcription factor, UBF, revealed that it was similar at each stage of mitosis in the secondary constriction. In addition, the colocalization of UBF with non-condensed rDNA was demonstrated. This is the first visual evidence of the association of UBF with non-condensed rDNA. As we previously reported that the rDNA transcription machinery remained assembled during mitosis, the colocalization of rDNA fibers with UBF argues in favor of the association of the transcription machinery with certain rDNA copies even in the absence of transcription. If this hypothesis is correct, it can be assumed that condensation of rDNA as well as dissociation of the transcription machinery from rDNA cannot explain the arrest of rDNA transcription during mitosis. It is proposed that modifications of the transcription machinery occurring in prometaphase could explain the arrest of transcription, while reverse modifications in late anaphase could explain activation.

Animals↗

PubMedDate not supplied

Anaerobic treatment of condensates: trial at a kraft pulp and paper mill.

The Domtar Papers pulp and paper mill in Windsor, Quebec, Canada, investigated the potential for anaerobic treatment of contaminated kraft mill condensates. The objectives of this project were to assess the technical feasibility of replacing the steam stripper with anaerobic treatment, to provide basic information for the design of an anaerobic treatment process for condensates, and to provide information on treated condensate quality for eventual reuse. The project involved extensive chemical characterization of condensates, followed by treatability tests. The tests included laboratory bench-scale tests and on-site pilot testing using direct feed from the process. Characterization showed that the organic content of the condensates was essentially methanol, as expected, but that foul evaporator condensates had high sulfide contents. It was found that undiluted foul condensates at the Windsor mill are toxic to the anaerobic biomass because of these high concentrations of sulfides. Treatment of combined condensates is possible at an approximate volumetric loading of 10 to 12 g/L.d chemical oxygen demand (COD) with good production of biogas (0.35 L/g of COD removed) and excellent methanol removal (better than 95%). The biogas produced is of excellent fuel quality with close to 90% methane, but with a high sulfide content (close to 4%).

Bacteria, Anaerobic↗

PubMed2000

Reaction mechanism of recombinant 3-oxoacyl-(acyl-carrier-protein) synthase III from Cuphea wrightii embryo, a fatty acid synthase type II condensing enzyme.

A unique feature of fatty acid synthase (FAS) type II of higher plants and bacteria is 3-oxoacyl-[acyl-carrier-protein (ACP)] synthase III (KAS III), which catalyses the committing condensing reaction. Working with KAS IIIs from Cuphea seeds we obtained kinetic evidence that KAS III catalysis follows a Ping-Pong mechanism and that these enzymes have substrate-binding sites for acetyl-CoA and malonyl-ACP. It was the aim of the present study to identify these binding sites and to elucidate the catalytic mechanism of recombinant Cuphea wrightii KAS III, which we expressed in Escherichia coli. We engineered mutants, which allowed us to dissect the condensing reaction into three stages, i.e. formation of acyl-enzyme, decarboxylation of malonyl-ACP, and final Claisen condensation. Incubation of recombinant enzyme with [1-(14)C]acetyl-CoA-labelled Cys(111), and the replacement of this residue by Ala and Ser resulted in loss of overall condensing activity. The Cys(111)Ser mutant, however, still was able to bind acetyl-CoA and to catalyse subsequent binding and decarboxylation of malonyl-ACP to acetyl-ACP. We replaced His(261) with Ala and Arg and found that the former lost activity, whereas the latter retained overall condensing activity, which indicated a general-base action of His(261). Double mutants Cys(111)Ser/His(261)Ala and Cys(111)Ser/His(261)Arg were not able to catalyse overall condensation, but the double mutant containing Arg induced decarboxylation of [2-(14)C]malonyl-ACP, a reaction indicating the role of His(261) in general-acid catalysis. Finally, alanine scanning revealed the involvement of Arg(150) and Arg(306) in KAS III catalysis. The results offer for the first time a detailed mechanism for a condensing reaction catalysed by a FAS type II condensing enzyme.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗

PubMed2002

Isolation of compounds from bleached kraft mill recovery condensates associated with reduced levels of testosterone in mummichog (Fundulus heteroclitus).

Previous studies have identified chemical recovery condensates as a primary source of hormonally active substances within a Canadian bleached kraft pulp mill. Although reverse osmosis treatment of condensates raises the exposure threshold that alters circulating levels of testosterone in mummichog (Fundulus heteroclitus), the responsible chemicals have not been characterized. In this study, a solid-phase extraction (SPE) method was developed to isolate chemical recovery condensate extractives for evaluation of hormonal activity. Condensates generated during softwood and hardwood pulp production were investigated for their relative potential to affect both circulating and gonadal production of sex steroids in mummichog. Mummichog were exposed to whole condensates, extracts from suspended particulates (> 1 microm), two fractions from SPE, and residual condensates after SPE. The distribution of bioactivity among condensate fractions was similar for both wood furnishes. In both sexes, significant depressions in circulating testosterone and in in vitro gonadal testosterone production were associated with exposure to whole condensates, particulate extracts, one SPE fraction, and residual material after SPE. An optimized SPE method subsequently demonstrated complete recovery of polar, bioavailable chemicals that reduced testosterone levels in both sexes. Characterizations of active fractions by gas chromatography-mass spectrometry (GC-MS) showed the presence of extractives with molecular masses > or = 240 amu possessing functionalities consistent with lignin degradation products. This study provides the first isolation of chemicals derived from pulp production associated with impaired reproductive performance in fish.

Animals↗