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At least 253 records · Page 14Linked to original sources
PubMed2007

Influence of the activator in an acrylic bone cement on an array of cement properties.

In all but one of the acrylic bone cement brands used in cemented arthroplasties, N,N-dimethyl-4-toluidine (DMPT) serves as the activator of the polymerization reaction. However, many concerns have been raised about this activator, all related to its toxicity. Thus, various workers have assessed a number of alternative activators, with two examples being N,N-dimethylamino-4-benzyl laurate (DMAL) and N,N-dimethylamino-4-benzyl oleate (DMAO). The results of limited characterization of cements that contain DMAL or DMAO have been reported in the literature. The present work is a comprehensive comparison of cements that contain one of these three activators, in which the values of a large array of their properties were determined. These properties range from the setting time and maximum exotherm temperature of the curing cement to the variation of the loss elastic modulus of the cured cement with frequency of the applied indenting force in dynamic nanoindentation tests. The present results, taken in conjunction with those presented in previous reports by the present authors and co-workers on other properties of these cements, indicate that both DMAL and DMPT are suitable alternatives to DMPT.

Acrylic Resins↗
PubMed1997

Oxygen activation by cytochrome P450BM-3: effects of mutating an active site acidic residue.

A highly conserved acid residue is found in the I-helix of most cytochrome P450s and has been suggested to play a critical function in oxygen activation and substrate hydroxylation in these monooxygenases. We have investigated this hypothesis for cytochrome P450BM-3 by replacing the naturally occuring glutamate at position 267 with a glutamine residue. In the case of P450BM-3, mutation of the glutamate to glutamine as position 267 drastically reduces the catalytic activity of the enzyme when palmitate is used as a substrate for hydroxylation. On the other hand, the activity change toward laurate hydroxylation is relatively small. The much slower catalytic turnover by the mutant enzyme in palmitate hydroxylation compared with wild type allows the observation of a new spectral intermediate in the hemoprotein. This intermediate is similar to that observed in the corresponding active site acid-to-amide replacement in cytochrome P450cam (N. C. Gerber and S. G. Sligar (1994) J. Biol. Chem. 269, 4260-4266). Also, in analogy with P450cam, this mutation does not lead to any side oxidation processes which produce hydrogen peroxide. Interestingly, however, the alteration in the active site structure which is implied by the change in regio specificity may also effect substrate packing thus leading to the uncoupling of the enzyme to produce additional water rather than a commitment to substrate oxidation. In addition, the distribution of hydroxylation products is altered by this mutation, suggesting some perturbation of the recognition property in P450BM-3.

Bacterial Proteins↗
PubMed2000

Human fatty acid omega-hydroxylase, CYP4A11: determination of complete genomic sequence and characterization of purified recombinant protein.

The gene of the human fatty acid omega-hydroxylase, CYP4A11, has been isolated from a human BAC library, and its complete genomic sequence has been determined. The CYP4A11 gene spanned 12,568 bp and contained 12 exons. The known PPAR recognition elements (PPRE), which were reported to be involved in the induction of CYP4A6 by clofibric acid, were not observed within the 5'-flanking region of the CYP4A11 gene. The recombinant CYP4A11 protein expressed in Escherichia coli using the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 6.4 nmol of P450/mg of protein. This P450 exhibited omega-hydroxylation activity toward laurate, with a turnover number of 14.7 nmol/min/nmol of P450. The apparent K(m) and V(max) values were 56.7 microM and 15.2 nmol/min/nmol of P450, respectively. It also showed omega-hydroxylation activity toward palmitate, with a turnover number of 0.78 nmol/min/nmol of P450. Although several reports from other groups described that CYP4A11 preparations catalyzed omega-hydroxylation of arachidonic acid, our purified recombinant protein exhibited no activity toward arachidonic acid nor prostaglandin A(1).

Amino Acid Sequence↗
PubMed2000

Expression and catalytic activity of mouse leukotriene B4 omega-hydroxylase, CYP4F14.

We have isolated a cDNA for a mouse leukotriene B4 omega-hydroxylase, CYP4F14. The cDNA encoded a protein with 524 amino acids, whose sequence similarity is 95% that of rat CYP4F1. The microsomes from yeast cells transfected with CYP4F14 expression vector showed 0.1 nmol P450/mg protein and catalyzed omega-hydroxylations of leukotriene B4, 6-trans-leukotriene B4, lipoxin A4, prostaglandin A1, and several hydroxyeicosatetraeonic acids (HETEs), with 8-HETE being the most active substrate. In contrast, no activity was detected toward lipoxin B4, laurate, and arachidonate. The mRNA for CYP4F14 had three different 5' untranslated sequences. Analysis of the CYP4F14 gene showed that two exon I sequences with different transcription start sites are located in the gene, and two splicing signals on the 3' end of intron I are alternatively used. The mRNA for this P450 was detected only in the liver by Northern blot analysis, whereas a small amount of the mRNA was detected in the brain using RT-PCR. Administration of clofibrate had no effect on microsomal 6-trans-leukotriene B4 omega-hydroxylase activity, but resulted in a marked reduction in the content of mRNA for this P450 in the liver. These findings indicate that CYP4F14 is very similar to CYP4F1 except for its expression in the brain and 5' untranslated sequences.

5' Untranslated Regions↗
PubMed2006

Enhanced transdermal delivery of pranoprofen from the bioadhesive gels.

Percutaneous delivery of NSAIDs has advantages of avoiding hepatic first pass effect and delivering the drug for extended period of time at a sustained, concentrated level at the inflammation site that mainly acts at the joint and the related regions. To develop the new topical formulations of pranoprofen that have suitable bioadhesion, the gel was formulated using hydroxypropyl methylcellulose (HPMC) and poloxamer 407. The effects of temperature on drug release was performed at 32 degrees C, 37 degrees C and 42 degrees C according to drug concentration of 0.04%, 0.08%, 0.12%, 0.16%, and 0.2% (w/w) using synthetic cellulose membrane at 37+/-0.5 degrees C. The increase of temperature showed the increased drug release. The activation energy (Ea), which were calculated from the slope of lop P versus 1000/T plots was 11.22 kcal/ mol for 0.04%, 10.79 kcal/mol for 0.08%, 10.41 kcal/mol for 0.12% and 8.88 kcal/mol for 0.16% loading dose from the pranoprofen gel. To increase the drug permeation, some kinds of penetration enhancers such as the ethylene glycols, the propylene glycols, the glycerides, the non-ionic surfactants and the fatty acids were incorporated in the gel formulation. Among the various enhancers used, propylene glycol mono laurate showed the highest enhancing effects with the enhancement factor of 2.74. The results of this study suggest that development of topical gel formulation of pranoprofen containing an enhancer is feasible.

Adhesives↗
PubMed1980

Influence of free fatty acid anion on the binding of warfarin to cytoplasmic proteins from rat liver.

In vitro binding studies have shown that warfarin binds strongly to both ligandins (Y) and Z protein obtained from rat liver cytosol with dissociation constants of 11.7 and 10.1 microM respectively. Increasing concentrations of oleate ion significantly increased the dissociation constant of warfarin with either protein, whereas laurate ion showed the same behavior only with Z protein. On the other hand, the binding of warfarin to liver cytoplasmic proteins in vivo was decreased in 72-h-pre-fasted rats, although such fasting failed to produce any increase in the in vivo levels of the cytoplasmic free fatty acids (FFA). However, based on the results of the in vitro binding study, it is suggested that changes in the composition of hepatic cytoplasmic free fatty acids as a result of fasting could reduce the in vivo binding of warfarin to Y and Z proteins and hence could lead to an increase of unbound warfarin in liver cytosol.

Animals↗
PubMedDate not supplied

Rates of H2O2 generation from peroxisomal beta-oxidation are sufficient to account for fatty acid-stimulated ethanol metabolism in perfused rat liver.

Fatty acids generate H2O2 via peroxisomal beta-oxidation and increase ethanol metabolism markedly in a system that involves catalase-H2O2. The present studies were conducted to understand why fatty acid-stimulated ethanol metabolism occurs much faster than rates of H2O2 generation reported previously in perfused rat liver. A new method was developed to measure rates of H2O2 generation based on the fact that methanol is oxidized only by catalase in rat liver. Rates of H2O2 generation were estimated from the time necessary for the steady-state level of catalase-H2O2 measured spectrophotometrically (660-640 nm) through a lobe of the liver to return to basal values following the addition of a known quantity of methanol in a closed perfusion system containing 4% bovine serum albumin. Under these conditions, basal rates of H2O2 production and rates of 4-methylpyrazole-insensitive ethanol oxidation were in a similar range (10 to 20 mumol/g/hr). Rates of H2O2 generation were increased up to 80 mumol/g/hr by addition of laurate, palmitate or oleate (1 mM); half-maximal increases in rates were observed with 0.6 mM oleate. Hexanoate, a short-chain fatty acid, did not stimulate H2O2 production or ethanol uptake. In these studies, rates of H2O2 generation compared well with rates of fatty acid-stimulated ethanol uptake measured in the presence of 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, with all fatty acids studied. It is concluded, therefore, that rates of H2O2 generation are sufficient to account for rates of fatty acid-stimulated ethanol metabolism via catalase-H2O2. In addition, these data indicate that catalase may contribute significantly to ethanol oxidation under physiological conditions in the presence of fatty acids.

Animals↗
PubMed2006

Analysis of ligand binding curves on basis of mean intrinsic thermodynamic quantities.

The mean intrinsic thermodynamic quantity can be defined by considering the relative population of complex species in the solution and the value of intrinsic thermodynamic quantity corresponds to each step of ligation. In the present study a new method is introduced for analysis of experimental ligand binding data on basis of mean intrinsic thermodynamic quantities. In this regard, a deviation parameter was defined by comparing the non-interacting system with the cooperative interactive one. This parameter can be calculated just by estimation of the first binding constant. A set of relations between this deviation parameter and other binding characteristics, such as mean intrinsic Gibbs free energy of binding and mean Gibbs free energy of site-site interaction, have been developed. This model presents binding mechanism in a unified way that is simple, yet stringent, more straightforward, more reliable and informative. This analyzing method has been successfully applied for evaluation of various systems such as oxygen binding to hemoglobin, laurate and warfarin binding to human serum albumin, and reveals some new biological features of these binding systems.

Hemoglobins↗
PubMed2005

Identification of extracellular lipases/esterases produced by Thermus thermophilus HB27: partial purification and preliminary biochemical characterisation.

Thermus thermophilus HB27 produces important levels of extracellular lipolytic activity when grown for 30 h at 70 degrees C in a complex medium. A method to detect esterase activity in these samples after non-reducing SDS-polyacrylamide electrophoresis was developed. The method, that implies the renaturalisation of the enzymes in the SDS-gels by washing with Triton X-100 at high temperatures, allowed detecting three esterases with different molecular weights (108, 62 and 34 kDa, respectively). The electrophoretic mobility determined under different experimental conditions suggested that the 34- and 108 kDa-esterases might correspond with two oligomeric states of a sole enzyme (monomer and trimer). Dissociation of the trimer into the monomer started when the samples were heated at temperatures higher than 60 degrees C in the presence of sodium dodecyl sulphate. Evidences were found that indicated the independent nature of the 62 kDa-esterase. A method to purify these enzymes from postincubates of T. thermophilus HB27 was developed following three steps: sodium cholate treatment, ethanol/ether precipitation and hydrophobic chromatography. In this way, an enzyme solution was obtained that contained the identified esterases/lipases. The partially purified enzymes showed an optimum of activity for the hydrolysis of p-nitrophenyl laurate at alkaline pH values and 80 degrees C, a high thermal stability and were very stable in the presence of high concentrations of isopropanol.

2-Propanol↗
PubMed1976

Hydrolysis of an aliphatic monoester in emulsion by swine pancreas lipase: influence of interfacial bile salts molecules upon reaction rate.

The accumulation of four bile salts at the interface between water and n-hexyl laurate is studied. Gibb's interfacial excesses of these salts are calculated, starting from interfacial tension measurements. At the same time, emulsions of the ester are treated by lipase and reactions rates are plotted against the bile salt concentrations present in the water phase of the emulsions. Inhibition by conjugated bile salts appear before the critical micellar concentration is approached. There is no defined relationship between this inhibition and the fraction of interfacial area covered by detergent molecules. In accordance with recent publication, the discussion suggests that the energy level of interfaces is important for lipase action and that, when interfacial tension becomes too small, lipase does not attach to interfaces and appears as inactive.

Animals↗
PubMedDate not supplied

Lipoprotein lipase immobilization onto porous chitosan beads.

Lipoprotein lipase (LPL) was covalently immobilized onto the surface of porous chitosan beads (ChB) without or with spacers of different lengths. The relative activity (RA) of the immobilized LPL was found to be high toward a small ester substrate, p-nitrophenyl laurate (pNPL). LPL immobilized with spacer gave an almost constant activity, in marked contrast with the immobilized LPL without spacer, whose activity decreased with decreasing surface concentration. Further, the RA of LPL immobilized with spacer was higher than that of LPL immobilized without a spacer. The pH, thermal, and storage stabilities of the immobilized LPL were higher than those of the free one. LPL immobilized directly to the surface of ChB without any spacer gave a higher stability than that immobilized with spacer, in spite of the lower RA. The spacer effect on RA could be explained in terms of the mobility of the immobilized LPL molecule.

Chitin↗
PubMed2001

Effect of the cationic detergent CTAB on the involvement of ADP/ATP antiporter and aspartate/glutamate antiporter in fatty acid-induced uncoupling of liver mitochondria.

The influence of the positively charged amphiphilic compound cetyltrimethyl ammonium bromide (CTAB) on palmitate- and laurate-induced uncoupling and on carboxyatractylate and glutamate recoupling effects in liver mitochondria have been studied. CTAB (40 microM) in the presence of 3 mM MgCl2 had little (if any) effect on the palmitic acid-stimulated respiration of mitochondria; the glutamate recoupling effect increased, and the carboxyatractylate recoupling effect decreased to the same degree with the combined effect (about 80%) remaining unchanged. Thus, CTAB decreases the ADP/ATP antiporter involvement and increases to the same extent the aspartate/glutamate antiporter involvement in the fatty acid-induced uncoupling. The carboxyatractylate and glutamate recoupling effects were less pH dependent in the presence of CTAB than in its absence. These data could be interpreted with the assumption that fatty acid anions are more accessible to the ADP/ATP antiporter and their neutral forms are more accessible to the aspartate/glutamate antiporter, and that CTAB changes the relative anion carrier involvement in the fatty acid-induced uncoupling as it forms neutral complexes with fatty acid anions.

Amino Acid Transport Systems, Acidic↗
PubMed2003

Temperature dependence of rat liver mitochondrial respiration with uncoupling of oxidative phosphorylation by fatty acids. Influence of inorganic phosphate.

The respiration rate of liver mitochondria in the course of succinate oxidation depends on temperature in the presence of palmitate more strongly than in its absence (in state 4). In the Arrhenius plot, the temperature dependence of the palmitate-induced stimulation of respiration has a bend at 22 degrees C which is characterized by transition of the activation energy from 120 to 60 kJ/mol. However, a similar dependence of respiration in state 4 is linear over the whole temperature range and corresponds to the activation energy of 17 kJ/mol. Phosphate partially inhibits the uncoupling effect of palmitate. This effect of phosphate is increased on decrease in temperature. In the presence of phosphate the temperature dependence in the Arrhenius plot also has a bend at 22 degrees C, and the activation energy increases from 128 to 208 kJ/mol in the range from 13 to 22 degrees C and from 56 to 67 kJ/mol in the range from 22 to 37 degrees C. Mersalyl (10 nmol/mg protein), an inhibitor of the phosphate carrier, similarly to phosphate, suppresses the uncoupling effect of laurate, and the effects of mersalyl and phosphate are not additive. The recoupling effects of phosphate and mersalyl seem to show involvement of the phosphate carrier in the uncoupling effect of fatty acids in liver mitochondria. Possible mechanisms of involvement of the phosphate carrier in the uncoupling effect of fatty acids are discussed.

2,4-Dinitrophenol↗
PubMed1995

Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants.

Amino Acid Sequence↗
PubMed1993

Replacement of Thr-303 of P450 2E1 with serine modifies the regioselectivity of its fatty acid hydroxylase activity.

Threonine-303 of rabbit P450 2E1, which is putatively located at the distal heme surface, was replaced by serine and valine via site-directed mutagenesis. In the oxidized state, the Ser-mutated P450 exhibited a low- and high-spin mixed-type (low > high) absorption spectrum, whereas the Val-mutated P450, like the wild-type P450, exhibited a nearly high-spin type spectrum. The reduced CO complexes of the Ser- and Val-mutated P450s, as well as that of the wild-type P450, showed a Soret absorption maximum at 452 nm. Both mutated P450s were active in the hydroxylation of C10 to C18 fatty acids at somewhat lower rates than the wild-type P450. The Val-mutated P450 gave the same two products (the major one is probably the omega-1 hydroxy analog) as the wild-type P450, while additional products were formed on incubation with C11 to C17 fatty acids as substrates of the Ser-mutated P450; a total of four products was detected for each of the C12 to C15 fatty acids, and three for each of the C11, C16, and C17 homologues. The metabolites of laurate were determined by GC-MS analysis to be the omega-1, omega-2, omega-3, and omega-4 hydroxy counterparts. The Ser-mutated P450 hydroxylated drug substrates at almost the same rates as the wild-type P450, while the mutation to valine significantly lowered the drug hydroxylase activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Cytochrome P-450-dependent fatty acid hydroxylase system in Japanese house musk shrew, Suncus murinus.

The substrate specificity and other properties of a fatty acid hydroxylase system in liver microsomes of the Japanese house musk shrew, Suncus murinus, were examined. Shrew liver microsomes catalyzed the hydroxylation of various saturated fatty acids (C8-C18) to the corresponding omega- and (omega-1)-hydroxy derivatives. The relative activities of the hydroxylase with these substrates were as follows: C12 (100, actual conversion: 7.05 nmol/mg of microsomal protein/min), C10 (90), C14 (87), C16 (23), C8 (19), and C18 (11). The specific activity of the fatty acid hydroxylase in shrew liver was much higher than that in other species. The omega/omega-1-hydroxylation ratio decreased with increasing chain length of fatty acid substrates (C10-C18), but it was 0 for the C8 fatty acid. Both NADPH and O2 were required for hydroxylase activity, and NADH had little effect. The apparent Km value for laurate was 1.6 X 10(-5) M. The hydroxylase activity was 92% inhibited by CO at a CO-O2 ratio of 9. p-Chloromercuribenzoate (0.1 mM) inhibited hydroxylation by 94% whereas iodoacetate (0.1 mM) inhibited it by only 8%. SKF 525-A (1 mM) and menadione (0.01 mM), respectively, caused 41% and 29% inhibition of the activity. It is concluded that the hydroxylase catalyzing fatty acid hydroxylation in shrew liver microsomes is a typical cytochrome P-450-linked monooxygenase.

Animals↗
PubMed1991

Fatty acid oxidation and the beta-oxidation complex in Mycobacterium leprae and two axenically cultivable mycobacteria that are pathogens.

Intact, non-growing Mycobacterium leprae, M. avium and M. microti oxidized a wide range of 1-14C-labelled fatty acids (C8 to C24) to 14CO2. Laurate (C12) was oxidized most rapidly, and its oxidation by M. leprae was inhibited by the antileprosy agents Dapsone, clofazamine and rifampicin. Key enzymes of beta-oxidation were detected in extracts from all three mycobacteria. All these activities (both in intact mycobacteria and the enzymes) were stimulated in M. avium grown in Dubos medium plus palmitate but activities in M. microti or M. avium grown either in Dubos medium with added liposomes or triolein, or in vivo were similar to those detected in the same strain grown in Dubos medium alone. M. avium could be grown in medium in which 95% of its fatty acyl elongase activity is acetyl-CoA dependent. In this medium growing M. avium organisms oxidized [1-14C]palmitate to 14CO2 but simultaneously elongated palmitate to C24 acids and even longer. Acetyl-CoA-dependent elongase activity is similar but clearly not identical to reversed beta-oxidation, but the exact point(s) of difference have not yet been identified.

Acetyl Coenzyme A↗
PubMed2007

Effect of high hydrostatic pressure on four genotypes of F-specific RNA bacteriophages.

AIMS: The pressure responses of four genotypes of F-specific RNA bacteriophages, f2, GA, Qbeta and SP, were evaluated with respect to pressure magnitude, treatment temperature and suspending medium. METHOD AND RESULTS: The pressure responses were studied with respect to pressure magnitude (350 to 600 MPa), treatment temperature (-10 to 50 degrees C) and suspending media. Phages f2 and GA had much higher pressure resistances than Qbeta and SP. Pressure resistances of Qbeta and SP were enhanced with increase in salt concentrations in the range of 350 to 600 MPa from -10 to 50 degrees C in PBS. Qbeta and SP had greater pressure resistances when suspended in phosphate-buffered saline (PBS) with added glucose (5%, w/w), UHT whole milk and Dulbecco's Modified Eagle's Medium plus 10% fetal bovine sera than they did in PBS. Two surfactants, sucrose laurate and monolaurin, and one chelating agent, ethylenediamine tetraacetic acid (EDTA), increased the pressure resistance of Qbeta and SP, but had modest effect on either f2 or GA. CONCLUSIONS: Four representative F-specific RNA bacteriophages, f2 (serotype I), GA (serotype II), Qbeta (serotype III) and SP (serotype IV) showed different resistances to hydrostatic pressure in the range of 350-600 MPa. SIGNIFICANCE AND IMPACT OF THE STUDY: This study screened for practical surrogates of HAV for validation of commercial high hydrostatic pressure processing.

Chelating Agents↗