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PubMed2005

Bioactive hydroxyapatite coatings on polymer composites for orthopedic implants.

Hydroxyapatite [HA, Ca10(PO4)6(OH)2] coatings on polymer composite substrates were investigated for their bioactivity and their physicochemical and mechanical characteristics. HA holds key characteristics for use in orthopedic applications, such as for coating of the femoral stem in a hip replacement device. The plasma-spray technique was used to project HA onto a carbon fiber/polyamide 12 composite substrate. The resulting HA coatings exhibited mechanical adhesion as high as 23 MPa, depending on the surface treatment of the composite substrate. The purpose of this investigation was to evaluate the bioactivity of an HA-coated composite substrate. HA- coated samples have been immersed in simulated body fluid (SBF) and maintained within a shaker bath for periods of 1, 7, 14, 21, and 28 days at 37 degrees C. Scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction techniques were performed on the samples before and after immersion into SBF. SBF was analyzed using inductively coupled plasma atomic emission spectrometry for element concentration and evaluation of the solution's purity. SBF conditioning led to the deposition of crystalline HA onto the surface of the coatings. The calcium-to-phosphorous ratios of initial HA coating and of newly deposited HA were respectively 1.72 and 1.65, close to the HA theoretical calcium/phosphorous value of 1.67. Results demonstrated that bioactive HA coatings were produced by plasma spraying, because SBF conditioning induced newly formed HA with high crystallinity. Mechanical adhesion of the HA coatings was not significantly affected upon SBF conditioning.

Arthroplasty, Replacement, Hip↗

PubMed2006

Bioactive glass nanofiber-collagen nanocomposite as a novel bone regeneration matrix.

Nanoscale organized organic-inorganic nanocomposite systems have great potential in the development of biomaterials with advanced properties. Herein, we developed a novel nanocomposite biomaterial consisting of bioactive glass nanofiber (BGNF) and collagen reconstituted fibrous matrix for bone regenerative medicine. A sol-gel derived glass with a bioactive composition (58SiO(2). 38CaO.4P(2)O(5)) was electrospun to a nanoscale fiber with an average diameter of approximately 320 nm. The BGNF was subsequently hybridized with type I collagen, which is the main organic constituent of bone matrix. The BGNF and self-assembled collagen sol were combined in aqueous solution, and then crosslinked to produce a BGNF-collagen nanocomposite, in the form of either a thin membrane or a macroporous scaffold, by adopting appropriate processing conditions. The BGNF was observed to be distributed uniformly within the collagen reconstituted nanofibrous matrix. The nanocomposite matrices induced rapid formation of bone-like apatite minerals on their surfaces when incubated in a simulated body fluid, exhibiting excellent bioactivity in vitro. Osteoblastic cells showed favorable growth on the BGNF-collagen nanocomposite. In particular, the alkaline phosphatase activity of the cells on the nanocomposite was significantly higher than that on the collagen. This novel BGNF-collagen nanocomposite is believed to have significant potential in bone regeneration and tissue engineering applications.

Biocompatible Materials↗

PubMed2006

Diffusion of bioactive molecules through the walls of the medial tissue-engineered hybrid ePTFE grafts for applications in designs of vascular tissue regeneration.

Strategies of better vascular tissue engineering may require delivery of soluble bioactive signals in cell culture medium to the cells in tissue-regenerating constructs. We measured the diffusivity and permeability of model tissue-engineering bioactive molecules such as water and heparin through the walls of both a hybrid ePTFE graft and a porcine carotid artery, a model vascular tissue. While diffusivities of H(3)-water and H(3)-heparin were measured as 3.9 x 10(-) (6) and 1.6 x 10(-) (6) cm(2)/s in the artery, respectively, under diffusional circulation of cell culture medium through the lumens of the carotid arteries, their corresponding permeabilities were 4.7 x 10(-) (5) and 2.0 x 10(-) (5) cm/s. On the other hand, diffusivities of H(3)-water and H(3)-heparin were also measured as 5.1 x 10(-) (6) and 4.7 x 10(-) (6) cm(2)/s, respectively, in the tissue-engineered hybrid ePTFE grafts; their corresponding permeabilities were 5.1 x 10(-) (5) and 3.7 x 10(-) (5) cm/s. The hybrid graft tissues were engineered by replacing the biodegradable, porous poly(lactide-co-glycolide) layers coated on the ePTFE surfaces with smooth muscle cell-derived tissues for 6 weeks. We analyzed the morphologies of the artery and the engineered hybrid ePTFE tissues with scanning electron microscopy and H&E stains. While the artery had its typical structure properties with layers of intima, media and adventitia, the tissue-engineered ePTFE hybrid graft had two layers of engineered tissues on the inner and outer surfaces of the ePTFE. There were no significant differences among the luminal tissue morphologies of the test samples from the effects of diffusion flow applications, with minor changes on their luminal surfaces. The results of water and heparin diffusion experiments indicated that these bioactive molecules were well transported from the cell culture medium to the tissue-engineering cells, enough to support tissue regeneration. We hope that these transport results may elucidate the transport behaviors of soluble nutrient molecules and biological signals through the vascular constructs under tissue engineering processes.

Absorbable Implants↗

PubMed2007

A mathematical model for predicting controlled release of bioactive agents from composite fiber structures.

A mathematical model for predicting bioactive agent release profiles from core/shell fiber structures was developed and studied. These new composite fibers, which combine good mechanical properties with desired protein release profiles, are designed for use in tissue regeneration and other biomedical applications. These fibers are composed of an inner dense polymeric core surrounded by a porous bioresorbable shell, which encapsulates the bioactive agent molecules. The model is based on Fick's second law of diffusion, and on two major assumptions: (a) first-order degradation kinetics of the porous shell, and (b) a nonconstant diffusion coefficient for the bioactive agent, which increases with time because of degradation of the host polymer. Three factors are evaluated and included in this model: a porosity factor, a tortuosity factor, and a polymer concentration factor. Our study indicates that the model correlates well with in vitro release results, exhibiting a mean error of less than 2.2% for most studied cases. In this study, the model was used for predicting protein release profiles from fibers with shells of various initial molecular weights and for predicting the release of proteins with various molecular weights. This new model exhibits a potential for simulating fibrous systems for a wide variety of biomedical applications.

Biocompatible Materials↗

PubMed2003

Frontal sinus and skull bone defect obliteration with three synthetic bioactive materials. A comparative study.

Three synthetic bioactive materials were studied in an experimental model to compare their usability in a frontal sinus and a skull bone defect obliteration. Bioactive glass number 9 (BAG(1)), bioactive glass number 13 (BAG(2)), and hydroxyapatite (HA) granules were investigated. BAG(1) and HA granules have been previously tested clinically. The clinical usefulness of BAG(2) granules has not been tested. Upper bony walls of 45 Elco rabbits' frontal sinuses were drilled open from four separate holes with the use of a standard method. The skull bone defects and the sinuses in frontal bone were filled with BAG(1) or BAG(2) on one side, and with HA on the other side. Two parallel posterior defects were covered with a pedicled periosteum flap, and two anterior defects with a free flap. The resorption of materials, new bone, and fibrous-tissue formation were observed with a histomorphometric method at 1, 3, and 6 months postoperatively. Scanning-electron microscopy (SEM) and Fourier-transform infrared spectroscopy (FTIR) were done at 6 months. In histomorphometry, the new bone formation increased with all the investigated materials throughout the study (p < 0.001), but the results showed higher new bone formation in the defects filled with BAG(1) than in corresponding BAG(2)- or HA- filled defects. New bone formation and resorption of materials were faster in defects covered by pedicled than by free periosteum flaps (p < 0.001). Intimate contact between the used materials and new bone was confirmed by SEM. FTIR analysis of bone produced by BAG(1) and BAG(2) was of the same type as natural frontal bone. BAG(2) can be manufactured in various shapes, and thus, could possibly be used in clinical conditions requiring a special anatomical implant shape. However, more research is needed regarding this property of BAG(2).

Animals↗

PubMed2004

In vivo aging test for a bioactive bone cement consisting of glass bead filler and PMMA matrix.

The degradation of a new bioactive bone cement (GBC), comprised of an inorganic filler (bioactive MgO-CaO-SiO(2)-P(2)O(5)-CaF(2) glass beads) and an organic matrix [high-molecular-weight polymethyl methacrylate (PMMA)], was evaluated in an in vivo aging test. Hardened rectangular specimens (20 x 4 x 3 mm) were prepared from two GBC formulations (containing 50% w/w [GBC50] or 60% w/w [GBC60] bioactive beads) and a conventional PMMA bone cement control (CMW-1). Initial bending strengths were measured with the use of the three-point bending method. Specimens of all three cements were then implanted into the dorsal subcutaneous tissue of rats, removed after 3, 6, or 12 months, and tested for bending strength. The bending strengths (MPa) of GBC50 at baseline (0 months), 3, 6, and 12 months were 136 +/- 1, 119 +/- 3, 106 +/- 5 and 104 +/- 5, respectively. Corresponding values were 138 +/- 3, 120 +/- 3, 110 +/- 2 and 109 +/- 5 for GBC60, and 106 +/- 5, 97 +/- 5, 92 +/- 4 and 88 +/- 4 for CMW-1. Although the bending strengths of all three cements decreased significantly from 0 to 6 months, those of GBC50 and GBC60 did not change significantly thereafter, whereas that of CMW-1 declined significantly between 6 and 12 months. Thus, degradation of GBC50 and GBC60 does not appear to continue after 6 months, whereas CMW-1 degrades progressively over 12 months. Moreover, the bending strengths of GBC50 and GBC60 (especially GBC60) were significantly higher than that of CMW-1 throughout. It is believed that GBC60 is strong enough for use under weight-bearing conditions and that its mechanical strength is retained in vivo; however, its dynamic fatigue behavior will need assessment before application in the clinical setting.

Animals↗

PubMed2005

Dose- and time-dependent effect of bioactive gel-glass ionic-dissolution products on human fetal osteoblast-specific gene expression.

Bioactive glasses dissolve upon immersion in culture medium, and release their constitutive ions into solution. There has been some evidence suggesting that these ionic-dissolution products influence osteoblast-specific processes. Here, the effect of 58S sol-gel-derived bioactive glass (60% SiO(2), 36% CaO, 4% P(2)O(5), in molar percentage) on primary osteoblasts derived from human fetal long bone explant cultures is investigated, and it is hypothesized that critical concentrations of sol-gel-dissolution products (consisting of a combination of simple inorganic ions) can enhance osteoblast phenotype in vitro by affecting the expression of a number of genes associated with the differentiation and extracellular matrix deposition processes. Cells were exposed to a range of 58S dosages continuously for a period of 4-14 days in monolayer cultures. Quantitative real-time RT-PCR analysis of a panel of osteoblast-specific markers showed a varied gene expression pattern in response to the material. The highest concentration of Ca and Si tested (96 and 50 ppm, respectively) promoted upregulation of gene expression for most markers (including alkaline phosphatase, osteocalcin, and osteopontin) at the latest time point, compared to non-58S-treated control, although this observation was not statistically significant. The same 58S concentration produced higher ALP activity levels and increased proliferation throughout the culture period, compared to lower dosages tested; however, the results generated were again not statistically significant. The data overall suggest that no significant effect can be ascribed to the ionic products of 58S bioactive gel-glass dissolution tested here and their ability to stimulate osteoblastic marker gene expression.

Alkaline Phosphatase↗

PubMed2005

Mineralization of SaOS-2 cells on enzymatically (silicatein) modified bioactive osteoblast-stimulating surfaces.

There is a demand for novel bioactive supports in surgery, orthopedics, and tissue engineering. The availability of recombinant silica-synthesizing enzyme (silicatein) opens new possibilities for the synthesis of silica-containing bioactive surfaces under ambient conditions that do not damage biomolecules like proteins. Here it is shown that growth of human osteosarcoma SaOS-2 cells on cluster plates precoated with Type 1 collagen is not affected by additional coating of the plates with the recombinant silicatein and incubation with its enzymatic substrate, tetraethoxysilane (TEOS). However, the enzymatic modification of the plates by biosilica deposition on the protein-coated surface caused a marked increase in calcium phosphate formation of SaOS-2 cells as revealed by alizarin red-S staining to quantify calcium mineral content. The increased occurrence of calcium-phosphate nodules on the modified surface was also observed by scanning electron microscopy. These results suggest that by supporting calcium-phosphate deposition in vitro, biosilica (silicatein)-modified surfaces are potentially bioactive in vivo, by stimulating osteoblast mineralization function.

Biocompatible Materials↗

PubMed2006

Bioactive and mechanically strong Bioglass-poly(D,L-lactic acid) composite coatings on surgical sutures.

New coating processes have been investigated for degradable (Vicryl) and nondegradable (Mersilk) sutures with the aim to develop Bioglass coated polymer fibers for wound healing and tissue engineering scaffold applications. First, the aqueous phase of a Bioglass particle slurry was replaced with a poly(D,L-lactic acid) (PDLLA) polymer dissolved in solvent dimethyle carbonate (DMC) to act as third phase. SEM observations indicated that this alteration significantly improved the homogeneity of the coatings. Second, a new coating strategy involving two steps was developed: the sutures were first coated with a Bioglass-PDLLA composite film followed by a second PDLLA coating. This two-step process of coating has addressed the problem of poor adherence of Bioglass particles on suture surfaces. The coated sutures were knotted to determine qualitatively the mechanical integrity of the coatings. The results indicated that adhesion strength of coatings obtained by the two-step method was remarkably enhanced. A comparative assessment of the bioactivity of one-step and two-step produced coatings was carried out in vitro using acellular simulated body fluid (SBF) for up to 28 days. Coatings produced by the two-step process were found to have similar bioactivity as the one-step produced coatings. The novel Bioglass/PDLLA/Vicryl and Bioglass/PDLLA/Mersilk composite sutures are promising bioactive materials for wound healing and tissue engineering applications.

Apatites↗

PubMed2006

Manufacturing, mechanical characterization, and in vitro performance of bioactive glass 13-93 fibers.

Fibers were manufactured from the bioactive glass 13-93 by melt spinning. The fibers were further characterized by measuring their tensile and flexural strength, and their in vitro performance was characterized by immersing them in simulated body fluid, which analyzed changes in their mass, their flexural strength, and surface reactions. The strength of glass fibers is highly dependent on fiber diameter, test method, and possible surface flaws, for example, cracks due to abrasion. In this study, the thinnest fibers (diameter between 24 and 33 microm) possessed the highest average tensile strength of 861 MPa. The flexural strength was initially 1353.5 MPa and it remained at that level for 2 weeks. The Weibull modulus for both tensile and flexural strength values was initially about 2.1. The flexural strength started to decrease and was only approximately 20% of the initial strength after 5 weeks. During the weeks 5-40, only a slight decrease was detected. The flexural modulus decreased steadily from 68 to 40 GPa during this period. The weight of the samples initially decreased due to leaching of ions and further started to increase due to precipitation of calcium phosphate on the fiber surfaces. The mass change of the bioactive glass fibers was dependent on the surface area rather than initial weight of the sample. The compositional analysis of the fiber surface after 24 h and 5 weeks immersion did confirm the initial leaching of ions and later the precipitation of a calcium phosphate layer on the bioactive glass 13-93 fiber surface in vitro.

Biocompatible Materials↗

PubMed2006

Characterization of human fetal osteoblasts by microarray analysis following stimulation with 58S bioactive gel-glass ionic dissolution products.

Bioactive glasses dissolve upon immersion in culture medium, releasing their constitutive ions in solution. There is evidence suggesting that these ionic dissolution products influence osteoblast-specific processes. Here, we investigated the effect of 58S sol-gel-derived bioactive glass (60 mol % SiO2, 36 mol % CaO, 4 mol % P2O5) dissolution products on primary osteoblasts derived from human fetal long bone explant cultures (hFOBs). We used U133A human genome GeneChip oligonucleotide arrays to examine 22,283 transcripts and variants, which represent over 18,000 well-substantiated human genes. Hybridization of samples (biotinylated cRNA) derived from monolayer cultures of hFOBs on the arrays revealed that 10,571 transcripts were expressed by these cells, with high confidence. These included transcripts representing osteoblast-related genes coding for growth factors and their associated molecules or receptors, protein components of the extracellular matrix (ECM), enzymes involved in degradation of the ECM, transcription factors, and other important osteoblast-associated markers. A 24-h treatment with a single dosage of ionic products of sol-gel 58S dissolution induced the differential expression of a number of genes, including IL-6 signal transducer/gp130, ISGF-3/STAT1, HIF-1 responsive RTP801, ERK1 p44 MAPK (MAPK3), MAPKAPK2, IGF-I and IGFBP-5. The over 2-fold up-regulation of gp130 and MAPK3 and down-regulation of IGF-I were confirmed by real-time RT-PCR analysis. These data suggest that 58S ionic dissolution products possibly mediate the bioactive effect of 58S through components of the IGF system and MAPK signaling pathways.

Fetus↗

PubMed2007

Characteristic comparison of bioactive scaffolds based on polyhydroxyalkoanate/bioceramic hybrids.

In this study, bioactive hybrid scaffolds were prepared by combining poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV) with three kinds of bioceramics, which are sol-gel bioglass (SGBG), tricalcium phosphate (TCP), and hydroxyapatite. The bioactivity and biodegradation of various scaffolds were analyzed with simulated experiments in vitro by immersing the scaffolds into simulated body fluid to evaluate their biomineralizing ability. The changes of the surface components and structures were determined by FTIR, XRD, and SEM analyses. Cell cultures and observation were made in vitro to assess the proliferation of osteoblast on these scaffolds. The results show that there might be ion exchanges and biodegradation reactions occurring between the hybrids and simulated body fluid solution, which result in the weight loss of the scaffolds and the pH changes in solution. An obvious biomineralized deposit layer was formed on the surface of the PHBV/SGBG and PHBV/TCP scaffolds, which was determined to be the bonelike crystalline hydroxyl-carbonate-apatite. The experiments on cell culture show that osteoblast could grow and proliferate on the bioactive hybrid scaffolds.

Animals↗

PubMed1992

Synergistic interaction of bacterial lipopolysaccharide and the monocyte-macrophage colony-stimulating factor: potential quantitative and qualitative changes in macrophage-produced cytokine bioactivity.

In this brief definitive report, we show that over a 6-h period and under serum-free conditions, recombinant monocyte-macrophage colony-stimulating factor 1 (rCSF-1) and lipopolysaccharide (LPS) synergize and induce macrophages to express higher levels of mRNA for interleukin 1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor alpha (TNF-alpha), and IL-6 and to release more bioactivity than macrophages treated with LPS alone. This synergy was regulated by the amount of LPS in the culture medium. Paraformaldehyde-fixed macrophages like-wise showed augmentation of IL-1 activity, but whereas all of the bioactivity associated with the fixed macrophages could be neutralized by anti-IL-1 alpha antibody only approximately 40% of the supernate activity could be attributed to IL-1 alpha. Preliminary data suggest that the augmenting effect induced by CSF-1 cannot be explained solely on a quantitative basis because the addition of rIL-1 alpha to supernates of macrophages treated with LPS alone or with the combination of LPS and CSF-1 resulted in an increase in thymocyte mitogenic activity to a level that could not be explained on an additive basis. Although the supernates contained TNF and IL-6, antibody neutralization assays made it unlikely that these were directly responsible for the augmenting effect. These results suggest that CSF-1 not only enhances basic genetic responses induced by LPS alone but also may induce a mechanism that amplifies cytokine bioactivity.

Animals↗

PubMed2000

Assessment of resorbable bioactive material for grafting of critical-size cancellous defects.

Bioactive glasses form a surface apatite layer in vivo that enhances the formation and attachment of bone. Sol-gel Bioglass graft material provides greater nanoscale porosity than bioactive glass (on the order of 50-200 A), greater particle surface area, and improved resorbability, while maintaining bioactivity. This study histologically and biomechanically evaluated, in a rabbit model, bone formed within critical-sized distal femoral cancellous bone defects filled with 45S5 Bioglass particulates, 77S sol-gel Bioglass, or 58S sol-gel Bioglass and compared the bone in these defects with normal, intact, untreated cancellous bone and with unfilled defects at 4, 8, and 12 weeks. All grafted defects had more bone within the area than did unfilled controls (p < 0.05). The percentage of bone within the defect was significantly greater for the 45S5 material than for the 58S or 77S material at 4 and 8 weeks (p < 0.05), yet by 12 weeks equivalent amounts of bone were observed for all materials. By 12 weeks, all grafted defects were equivalent to the normal untreated bone. The resorption of 77S and 58S particles was significantly greater than that of 45S5 particles (p < 0.05). Mechanically, the grafted defects had compressive stiffness equivalent to that of normal bone at 4 and 8 weeks. At 12 weeks, 45S5-grafted defects had significantly greater stiffness (p < 0.05). At 8 and 12 weeks, all grafted defects had significantly greater stiffness than unfilled control defects (p < 0.05). In general, the 45S5-filled defects exhibited greater early bone ingrowth than did those filled with 58S or 77S. However, by 12 weeks, the bone ingrowth in each defect was equivalent to each other and to normal bone. The 58S and 77S materials resorbed faster than the 45S5 materials. Mechanically, the compressive characteristics of all grafted defects were equivalent or greater than those of normal bone at all time points.

Animals↗

PubMed1983

Comparative study of immunoactivity and bioactivity of sodium insulin.

The bioactivity of equal doses (by radioimmunoassay) of regular insulin and sodium insulin were measured in six preconditioned female mongrel hound dogs by a previously described bioassay. The hypoglycemic nadir of blood glucose induced by intravenous injection of the drugs was compared with the nadirs induced by a known amount of the international standard insulin. The level of blood glucose was monitored-instantaneously and continuously using a nonthrombogenic continuous glucose-monitoring system. The hypoglycemic effect of subcutaneously injected sodium insulin was compared with the effect of subcutaneously injected regular insulin. The two drugs were injected at 1-2-week intervals to seven dogs. While the bioactivity of intravenously administered sodium insulin was equal to the bioactivity of intravenously administered regular insulin, the hypoglycemic effect of subcutaneously injected sodium insulin differed from the effect of subcutaneously injected regular insulin. The blood glucose nadir after subcutaneous injection of regular insulin occurred 101 +/- 21 min after the injection. The nadir after subcutaneous injection of sodium insulin occurred 163 +/- 33 min after the injection.

Animals↗

PubMed1995

2-Aminofluorene bioactivation by human term placental peroxidase.

Earlier investigations from our laboratory demonstrated that human term placental peroxidase (HTPP) is capable of metabolism of xenobiotics and endogenous compounds. In this study, purified HTPP was found to bioactivate 2-aminofluorene (2-AF) in the presence of H2O2. 2-AF oxidation was studied spectrophotometrically while radiometry was employed to assess the bioactivation. The rate of oxidation and covalent binding to protein and DNA was dependent upon the pH of the reaction medium and the concentration of 2-AF, the enzyme, and H2O2. To observe maximal enzyme velocity of oxidation, the presence of 16.5 microM H2O2, 100 microM 2-AF, 37 micrograms of the enzyme protein/ml, and pH 7.2 was required. Under optimal assay conditions, the range of specific activity between 130 and 165 nmol of 2-AF oxidized/min/mg HTPP was observed. Using similar assay conditions, the magnitude of covalent binding of [3H]-2-AF to protein (BSA) and calf thymus DNA was found to be about 508 pmol bound/min/mg HTPP/mg BSA and 84 pmol bound/min/mg HTPP/mg DNA, respectively. Potassium cyanide and sodium azide, the known inhibitors of different peroxidases, significantly blocked both the oxidation and covalent binding of 2-AF in a dose dependent manner. These results strongly suggest that peroxidase may be one of the important pathways responsible for the bioactivation of arylamines in human term placenta.

Azides↗

PubMed1998

Cloning and expression of CYP2F3, a cytochrome P450 that bioactivates the selective pneumotoxins 3-methylindole and naphthalene.

Members of the CYP2F gene subfamily are selectively expressed in lung tissues and have been implicated as important catalysts in the formation of reactive intermediates from several pneumotoxic chemicals. Human CYP2F1 bioactivates 3-methylindole (3MI), while mouse CYP2F2 bioactivates naphthalene. Although 3MI is a potent pneumotoxin in ruminants and rodents, the participation of cytochrome P450s from the 2F subfamily in 3MI bioactivation has not been fully defined. To test the hypothesis that a goat lung 2F homologue uniquely catalyzes the dehydrogenation of 3MI to the putative electrophile 3-methylene-indolenine, the CYP2F3 cDNA was cloned from a goat lung cDNA library and expressed in Escherichia coli. The predicted amino acid sequence of CYP2F3 possessed 82% identity to both human CYP2F1 and mouse CYP2F2. CYP2F3 was mutated at the 5' end, expressed in E. coli, and shown to have a molecular mass of 50 kDa. The reconstituted enzyme uniquely catalyzed only the dehydrogenation of 3MI to form 3-methylene-indolenine, an electrophilic intermediate, without detectable formation of other products, thus demonstrating highly unusual selectivity for dehydrogenation rather than hydroxylation of a substrate. Immunoinhibition studies demonstrated that about 20% of the production of the intermediate in goat lung microsomal samples was produced by CYP2F3. The CYP2F3 enzyme had a specific activity that was similar to that of human cDNA-expressed CYP2F1. CYP2F3 also stereoselectively catalyzed the formation of the 1R,2S-oxide from naphthalene; this stereoisomer is the putative pneumotoxin. The enzyme, however, lacked catalytic activity with other common P450 substrates including 7-ethoxycoumarin, a substrate for CYP2F1, indicating that the substrate selectivity of CYP2F3 appears to be high.

Amino Acid Sequence↗

PubMed1999

Identical primary sequence but different conformations of the bioactive regions of canine CCK-8 and CCK-58.

The C-terminal bioactive regions of CCK-8 and CCK-58 are identical (DY*MGWMDF-NH(2), Y* denotes a sulfated tyrosine residue), but these peptides have different patterns of bioactivity. Specifically, CCK-58 binds more avidly to the CCK(A) receptor while CCK-8 is more potent for stimulation of amylase secretion from pancreatic acini. We postulate that these seemingly contradictory observations reflect a stable conformational change in CCK-58 that enhances binding, but diminishes activation of second messenger. We used CD and NMR spectra to evaluate postulated structural differences between CCK-8 and the carboxy-terminus of synthetic CCK-58. The CD spectra indicate the presence of turns in CCK-8 but a mixture of helical and random coil structures for CCK-58. Comparisons of partial NMR chemical shift assignments of CCK-58 and full assignments for CCK-8 also indicate differences in the backbone conformations for these residues. The data support the hypothesis that these peptides have different, stable, carboxy-terminal structures that may influence bioactivity.

Animals↗