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Studies on hyperactivity in rats given tranylcypromine stereoisomers and reserpine.

Hyperactivity, salivation, piloerection, exophthalmia and ejaculation occurred in rats given reserpine (5 mg/kg i.p.) 2 hr after an oral dose (5 mg/kg) of trans-dl-2-phenylcyclopropylamine (tranylcypromine, dl-Tcp). The same syndrome occurred in rats given the d-isomer (d-Tcp) plus reserpine but not in rats given the l-isomer (l-Tcp) lus reserpine. Neither alpha methyl-p-tyrosine (2 X 150 mg/kg s.c.) nor parachlorophenylalanine (200 mg/kg s.c.) pretreatment prevented the hyperactivity syndrome. The results suggest that mainly MAO inhibition and release of monoamines are involved in the hyperactivity syndrome produced by dl-Tcp plus reserpine.

Animals↗

Phosducin-like protein regulates G-protein betagamma folding by interaction with tailless complex polypeptide-1alpha: dephosphorylation or splicing of PhLP turns the switch toward regulation of Gbetagamma folding.

Phosducin-like protein (PhLP) exists in two splice variants PhLP(LONG) (PhLP(L)) and PhLP(SHORT) (PhLP(S)). Whereas PhLP(L) directly inhibits Gbetagamma-stimulated signaling, the G betagamma-inhibitory mechanism of PhLP(S) is not understood. We report here that inhibition of Gbetagamma signaling in intact HEK cells by PhLP(S) was independent of direct Gbetagamma binding; however, PhLP(S) caused down-regulation of Gbeta and Ggamma proteins. The down-regulation was partially suppressed by lactacystine, indicating the involvement of proteasomal degradation. N-terminal fusion of Gbeta or Ggamma with a dye-labeling protein resulted in their stabilization against down-regulation by PhLP(S) but did not lead to a functional rescue. Moreover, in the presence of PhLP(S), stabilized Ggamma subunits did not coprecipitate with stabilized Gbeta subunits, suggesting that PhLP(S) might interfere with Gbetagamma folding. PhLP(S) and several truncated mutants of PhLP(S) interacted with the subunit tailless complex polypeptide-1alpha (TCP-1alpha) of the CCT chaperonin complex, which is involved in protein folding. Knock-down of TCP-1alpha in HEK cells by small interfering RNA also led to down-regulation of Gbetagamma. We therefore conclude that the strong inhibitory action of PhLP(S) on Gbetagamma signaling is the result of a previously unrecognized mechanism of Gbetagamma-regulation, inhibition of Gbetagamma-folding by interference with TCP-1alpha.

Amino Acid Sequence↗

Type D retrovirus Gag polyprotein interacts with the cytosolic chaperonin TRiC.

The carboxy terminus-encoding portion of the gag gene of Mason-Pfizer monkey virus (M-PMV), the prototype immunosuppressive primate type D retrovirus, encodes a 36-amino-acid, proline-rich protein domain that, in the mature virion, becomes the p4 capsid protein. The p4 domain has no known role in M-PMV replication. We found that two mutants with premature termination codons that remove half or all of the p4 domain produced lower levels of stable Gag protein and of self-assembled capsids. Interestingly, yeast two-hybrid screening revealed that p4 specifically interacted with TCP-1gamma, a subunit of the chaperonin TRiC (TCP-1 ring complex). TRiC is a cytosolic chaperonin that is known to be involved in both folding and subunit assembly of a variety of cellular proteins. TCP-1gamma also associated with high specificity with the M-PMV pp24/16-p12 domain and human immunodeficiency virus p6. Moreover, in cells, Gag polyprotein associated with the TRiC chaperonin complex and this association depended on ATP hydrolysis. In the p4 truncation mutants, the Gag-TRiC association was significantly reduced. These results strongly suggest that cytosolic chaperonin TRiC is involved in Gag folding and/or capsid assembly. We propose that TRiC associates transiently with nascent M-PMV Gag molecules to assist in their folding. Consequently, properly folded Gag molecules carry out the intermolecular interactions involved in self-assembly of the immature capsid.

Capsid↗

Kinetic evidence for pentachlorophenol-dependent growth of a dehalogenating population in a pentachlorophenol- and acetate-fed methanogenic culture.

A method was developed to evaluate growth of a reductively dechlorinating bacterial population within a pentachlorophenol (PCP)- and acetate-fed, mixed, methanogenic culture. In 6- to 12-day experiments, a computer-monitored/feedback-controlled bioreactor was used to maintain constant pH, temperature, and acetate concentration, while transformation of multiple PCP additions was monitored. The potential at a platinum electrode, EPt, was not controlled externally, but was maintained constant at -0.25 +/- 0.002 V (vs. SHE) by iron sulfides in the medium and the activity of the culture. PCP was reductively dechlorinated at the ortho position, yielding 3, 4,5-trichlorophenol (3,4,5-TCP) via 2,3,4,5-tetrachlorophenol (2,3,4, 5-TeCP). Below an initial PCP concentration of 0.5 microM, PCP was transformed to 3,4,5-TCP within 3 to 6 h. Biomass concentration changes were small during this period, and PCP and 2,3,4,5-TeCP transformations were modeled as pseudo-first-order reactions. Increases in pseudo-first-order rate constants for PCP and 2,3,4, 5-TeCP were directly related to the amount of PCP transformed to 3,4, 5-TCP, suggesting enrichment of a PCP-catabolizing population. Moreover, rate constant increases were independent of the amount of acetate consumed, changes in the overall volatile suspended solids (VSS) concentration, and the experimental duration. When PCP was added to the reactor at increasingly shorter time intervals in an exponential pattern, pseudo-first-order rate constants increased exponentially. An average rate constant doubling time of 1.7 days (1. 4 to 2.3 d) was estimated. While the VSS concentration of the culture increased 60% in an 8-day period, pseudo-first-order rate constants increased by a factor of approximately 6. This large increase in transformation rate constants suggests growth of a bacterial population capable of using PCP and 2,3,4,5-TeCP as terminal electron acceptors.

Acetates↗

Enhanced healing of segmental tibial defects in sheep by a composite bone substitute composed of tricalcium phosphate cylinder, bone morphogenetic protein, and type IV collagen.

Diaphyseal segmental defects in the tibia of 18 sheep were used to evaluate the healing potential of a composite bone substitute device (CBS) composed of a tricalcium phosphate cylinder (TCP), naturally occurring sheep bone morphogenetic protein (sBMP), and type IV collagen. A total of 100 mg of sBMP and 20 mg of type IV collagen in the high-dose group (CBSH), and 13 mg of sBMP and 2.5 mg of type IV collagen in the low-dose group (CBSL) were adsorbed to TCP cylinders, respectively. TCP cylinders impregnated with type IV collagen alone (TCPC) were used as control. A significantly larger area and more highly integrated intensity of newly formed external callus between CBSH and CBSL or TCPC group were quantified by computerized image analyzer at both 3 and 6 weeks. A torsion test showed that the maximal torque capacity, maximal angular deformation, and bone stiffness of healed osteotomized tibia with implants recovered 117-125% in CBSH, 72-109% in CBSL, and 63-80% in TCPC, compared with the corresponding contralateral tibia at 16 weeks. A healing superiority of the segmental bone defects replaced by the implants was demonstrated in the CBSH group. Thus, the composite bone substitute device defined in this study was shown to possess osteoinductivity, osteoconductivity, and mechanical strength.

Animals↗

Mechanical properties of acrylic bone cement containing PMMA-SiO2 hybrid sol-gel material.

An organic-inorganic hybrid material, poly(methyl methacrylate) (PMMA)-SiO2 (SiO2 content of 72 wt%), was prepared by incorporating PMMA structure units covalently into an SiO2 glass network via the sol-gel approach. The hybrid sol-gel material PMMA-SiO2 was subsequently used as the solid powder component of bone cement and its mechanical properties were evaluated. The effects of the addition of tricalcium phosphate (TCP), hydroxyethyl methacrylate (HEMA), and ethylene glycol dimethacrylate (EGDMA) on the properties of the sol-gel hybrid bone cement were also investigated. The influence of these components on the temperature rise during polymerization was discussed. It was found that the new bone cement containing PMMA-SiO2 hybrid sol-gel material had higher modulus than that of Simplex-P bone cement. The addition of TCP in the new bone cement increased the Young's modulus and the polymerization time; the inverse was observed for the tensile, bending, and compressive strengths, and the polymerization temperature. The addition of HEMA and EGDMA in the new bone cement had the opposite effect of TCP. The comparison between the new sol-gel bone cement and the commercial Simplex P bone cement was discussed.

Biocompatible Materials↗

Improvement of the mechanical properties of new calcium phosphate bone cements in the CaHPO4-alpha-Ca3(PO4)2 system: compressive strength and microstructural development.

The hardening properties of calcium phosphate cements in the CaHPO4-alpha-Ca3(PO4)2 (DCP-alpha-TCP) system have been investigated with interest focused on the compressive strength and microstructure development. Previous studies have shown that the addition of CaCO3(CC) leads to a modification of the calcium-deficient apatite structure of the reaction product, which results in a material more similar to the apatite in bone mineral. The addition of 10% w/w of CC to the initial DCP-alpha-TCP powder mixture resulted, with time, in a retardation of the development of compressive strength. However, the optimum compressive strength reached values up to 40% higher than CC-free samples. This retarding effect also has been monitored as a function of the calcium to phosphorus (Ca/P) ratio of the DCP and alpha-TCP mixture, showing the importance of the final cement properties of the relative quantities of the reactants in the mixture.

Bone Cements↗

The cement setting reaction in the CaHPO4-alpha-Ca3(PO4)2 system: an X-ray diffraction study.

The setting reactions of calcium phosphate cements in the CaHPO4-alpha-Ca3(PO4)2 (DCP-alpha-TCP) system have been investigated. X-ray diffraction (XRD) analyses were performed on DCP-alpha-TCP cement samples of varying calcium to phosphorus (Ca/P) ratios after setting for 24 h in Ringer's solution at 37 degrees C. XRD measurements showed that the intensity of the DCP peaks decreased linearly as the Ca/P ratio of the mixture increased. However, the intensity of the peaks of a new calcium-deficient hydroxyapatite [CDHA; Ca9(HPO4)(PO4)5OH] precipitating phase increased linearly as the Ca/P ratio increased. Alpha-TCP was not detected after 24 h of setting in any sample. A two-phase mixture XRD model was applied to explain the results, and suitable fits were obtained between observed and expected values of the relevant peak heights. The method used for this study also can be applied to studies of the kinetic behavior of other cement systems.

Bone Cements↗

Adsorption and release of insulin-like growth factor-I on porous tricalcium phosphate implant.

In order to develop bone substitutes, the design of biomaterials like calcium phosphate ceramic loaded with bone growth factor are of great interest. However, it is necessary to control the amount of growth factor adsorbed onto ceramics and the kinetics of its release. Radiolabeling of insulin-like growth factor-I (IGF-I) with 125-iodine ([(125)I]-IGF-I) and its adsorption onto porous tricalcium phosphate (TCP) cylinders enabled us to establish the time-adsorption and time-release curves using various concentrations of IGF-I. The adsorption curve increased rapidly and then flattened out at 72 h; 90% of the maximum was already reached at 24 h; and 20% of the adsorbed IGF-I was released in water within 4 days. In human serum the release was faster at 82% within 4 days. In vivo evaluation on an animal model was then performed. Rabbits' bilateral femoral cylindrical bone defects were filled with the TCP cylinders, which were either carrying IGF-I or implanted alone as a control in each rabbit. Bone turnover and ceramic resorption were stimulated by IGF-I loaded TCP according to standard radiography, dual-energy X-ray absorptiometry, histology, and histomorphometry.

Adsorption↗

Comparison of MRI- and CT-based post-implant dosimetric analysis of transperineal interstitial permanent prostate brachytherapy.

The purpose of this work was to investigate how a recently developed MRI-based post-implant dosimetric analysis technique for ultrasound guided transperineal interstitial permanent prostate brachytherapy (TIPPB) compared with the currently accepted CT-based technique. The study was based upon 3-mm MRI and CT scans of 15 patients who had received either 125I or 103Pd implantation. All images were acquired on post-operative day 1 and within 1 hr of each other. Prostate volumes were determined by the same physician. Sources were digitized and calculations performed using an in-house treatment planning system with a nearest neighbor seed sorting routine and AAPM TG43 formalism. Prostate volume, geometric source distribution spread (rcom), dose volume histogram (DVH), and tumor control probability (TCP) calculations were performed from both image sets. Differences in source localization were evaluated by comparing source spread and prescription isodose volumes. Differences in dosimetric analysis were evaluated through prostate-specific DVH and TCP comparisons. Prostate volume as determined from MRI was larger than that of CT by an average of +9.1% (R = 0.70). Calculated rcom was smaller by an average of -0.9 mm (R = 0.81). Isodose volumes at 80, 90, 100, and 150% of the prescription dose differed by an average of +2.5, +2.9, -2.9, and +4.8%, respectively (R = 0.97, 0.98, 0.98, and 0.91). Percentage volume of the prostate encompassed by 80, 100, and 150% of the prescription dose differed by an average of -0.9, -0.9, and -0.1%, respectively (R = 0.34, 0.35, and 0.35). TCP differed by an average of -0.8% (R = 0.37). The results of this study further support our initial findings that MRI may be used to reliably localize the implanted sources for TIPPB. This study also demonstrated that MRI-based post-implant dosimetric analysis is possible. However, it is evident that differences in prostate localization from MRI to CT can result in significantly different assessments of prostate volume coverage. There is clearly a need to further quantify the differences between these two imaging modalities in this application and address whether greater accuracy in describing the dose-volume relationship based on improvements in visualization of the prostate gland from MRI will translate into improved correlation with treatment outcome.

Brachytherapy↗

Structural study and stability of hydroxyapatite and beta-tricalcium phosphate: two important bioceramics.

The stability of hydroxyapatite (OHAP) and beta-tricalcium phosphate (beta-TCP) ceramics depends on the temperature of preparation and the partial pressure of water in the reaction atmosphere. In bioceramics used in the study of biomaterials, these two phosphates often coexist in distinct proportions; therefore, structural characterization of the individual phases is hindered. The structure of these two individual calcium phosphates have been studied using X-ray diffraction, and mainly by high-resolution electron microscopy. Whereas the X-ray diffraction profile of OHAP has been refined in the space group P6(3)/m, the HREM images oriented along the [001] direction at the edges of the crystal show a threefold symmetry. The X-ray diffraction pattern of beta-TCP has been refined in the space group R3c and the HREM image oriented along [0001] zone axis shows regular contrast. Both these calcium phosphate ceramics are unstable to the electron beam for a prolonged exposure time, and show areas of decomposition in HREM images of thin regions of the crystal. The regions of decomposition in the beta-TCP crystal show the presence of CaO as one of the major phases.

Calcium Phosphates↗

Low temperature formation of calcium-deficient hydroxyapatite-PLA/PLGA composites.

Hydroxyapatite-biodegradable polymer composites have been formed by a low temperature chemical route. Precomposite structures were prepared by combining alpha-Ca(3)(PO(4))(2) (alpha-tricalcium phosphate or alpha-TCP) with poly(L-lactic) acid and poly(DL-lactide-co-glycolide) copolymers. The final composite structure was achieved by in situ hydrolysis of alpha-TCP to Ca(9)(HPO(4))(PO(4))(5)OH (calcium deficient hydroxyapatite or CDHAp) either in solvent cast or pressed precomposites. Hydrolysis was performed at 56 degrees C-a temperature slightly above the glass transition of the polymers. The effects of polymer chemistry, composite formation technique, and porosity on hydrolysis kinetics and degree of transformation were examined with isothermal calorimetry, X-ray diffraction (XRD), Fourier transform infrared spectroscopy, and scanning electron microscopy. Calorimetric data and XRD analyses revealed that hydrolysis reactions were inhibited in the presence of the polymers. Isothermal calorimetry indicated the extent of the alpha-TCP to CDHAp transformation in 24 h to be 85% in the solvent cast composites containing PLGA (85:15) copolymer; however, XRD analyses suggested almost complete reaction. The CDHAp formation extent was 26% for the pressed composites containing the same polymer. In the presence of NaCl as a pore generator, 81% transformation was observed for the pressed composites. This transformation occurred without any chemical reaction between the polymer-inorganic components, as determined by Fourier transform infrared spectroscopy. Minimal transformation to CDHAp occurred in composites containing poly(L-lactic) acid.

Biocompatible Materials↗

Replacement of segmental bone defects using porous bioceramic cylinders: a biomechanical and X-ray diffraction study.

A porous ceramic material [hydroxyapatitetricalcium phosphate (HA-TCP)] was implanted in the femora of 30 dogs to investigate the possibility of using this material to repair segmental bone defects. A bone segment, 1.5 cm in length, was removed from the diaphysis of one femur in each dog to create the defect. Cylinders of corresponding size were inserted into the defects. The animals were divided into three groups with recovery times of 2 months, 4 months, and 6 months, respectively. The implants were harvested and subjected to biomechanic tests (bending strength) and X-ray diffraction analysis. The bending strengths of the implant construct increased gradually over time postoperatively. The values of strength for the three different time groups had significant variations (p < 0.05). The X-ray diffraction analysis indicated that the peaks of the TCP included in the cylinders decreased in intensity after implantation and tended to be similar to those of natural bone by 6 months after operation. Conversely, the peaks for the HA had fewer changes compared with preimplantation values. Based on the results of this experiment it was concluded that the porous HA-TCP ceramic cylinders have potential for repair of segmental bone defects if assisted by adequate stabilizing fixtures during the early postoperative period.

Animals↗

Adsorption characteristics of proteins on calcium phosphates using liquid chromatography.

The adsorption characteristics of various proteins on beta-Ca(3)(PO(4))(2) (beta-TCP), CaHPO(4). 2H(2)O (DCPD) and CaHPO(4) (DCPA) have been investigated using liquid chromatography. Acidic proteins were eluted from these calcium phosphate surfaces by potassium phosphate buffer (KP) but not by KCl and MgCl(2), whereas basic proteins were eluted by all three eluents. Such elution behavior can be interpreted by considering two kinds of adsorbing sites on the calcium phosphates, namely positively charged sites (Ca sites) which adsorb acidic proteins, and negatively charged sites (P sites) which adsorb basic proteins. A similar behavior has also been observed on Ca(10)(PO(4))(6)(OH)(2) (HAp) and Ca(8)H(2)(PO(4))(6). 5H(2)O (OCP). The order of the ratios of Ca-to-P sites was estimated to be DCPD > OCP > HAp >> DCPA >> beta-TCP, which is in agreement with the order of the surface zeta potentials. In addition, the (0k0) surface of DCPD is suggested to consist mainly of Ca sites. The total number of Ca sites on beta-TCP is thought to be significantly smaller than the other calcium phosphates.

Adsorption↗

Composites made of rapidly resorbable ceramics and poly(lactide) show adequate mechanical properties for use as bone substitute materials.

Porous composite materials made of poly(L, DL-lactide) and a ceramic component, alpha-tricalcium phosphate (alpha-TCP) or one of the rapidly resorbable glass ceramics, GB9N or GB14N, respectively, were developed to be used as bone substitutes. The present article describes the mechanical properties and the in vitro degradation characteristic of the different composite materials. The yield strength, the elastic modulus, and the molecular weight were measured after in vitro degradation up to 78 weeks. The initial strengths of the alpha-TCP composite (12.5 +/- 0.7 MPa) was higher than that of the GB9N and GB14N composites (8.3 +/- 0.2 MPa and 10.9 +/- 0.2 MPa, respectively). The initial elastic moduli of the composites were between 450 and 650 MPa. The mechanical properties remained constant until a degradation period of 26 weeks. Then they decreased continuously until they were completely lost at week 52. The molecular weight (M(w)) decreased steadily from 91,000 D in the case of the alpha-TCP composite and 78,000 D and 85,000 D in the case of the GB9N or GB14N composites, respectively, to about 10,000 D at week 78. It was concluded that the composites show adequate mechanical properties in the range of cancellous bone and a suitable degradation characteristic to be used as bone substitute materials.

Animals↗

Proliferation and differentiation rates of a human osteoblast-like cell line (SaOS-2) in contact with different bone substitute materials.

The aim of our study was to investigate the influence of four bone substitutes on the growth behavior of a human osteoblast-like cell line (SaOS-2) culture: pure alpha tricalcium phosphate (alpha-TCP = BIOBASE), a bioactive glass (bioglass), a neutralized glass-ceramic (GB9N), and solvent dehydrated bone. We established an in vitro cell culture model with three-dimensional scaffolds (cubes of 0.7 x 0.7 x 1.0 cm) of porous bone substitutes to investigate proliferation and differentiation rates of SaOS-2 cells. The cultures were analyzed for individual cell morphology after 5 days of growing using scanning electron microscopy. Fracture preparations of the cubes showed that cells could infiltrate the porous structures, but the cell shapes varied from individual round-shaped cells to wide spread cells and cell clusters, depending on the material. Also, the differentiation of the seeded cells was dissimilar after a 5-day incubation. The specific alkaline phosphatase (ALP) enzyme activity (ALP/DNA) measured in the supernatants of alpha-TCP-grown cells was nine times higher than the lowest activity, as observed by cells incubated on GB9N. Early (Collagen1, ALP) and late marker (osteocalcin, bone sialoprotein) of osteoblastic differentiation were proofed by reverse transcriptase-polymerase chain reaction analysis. Cells grown on bone substitutes and bioglass seem to be less differentiated than alpha-TCP-grown cells, because of noticeably less amounts of osteocalcin and bone sialoprotein. The cultivation on GB9N seems to dedifferentiate the cells, because even the ALP expression was reduced as well. Our results indicate that distinct bone substitutes influence proliferation and differentiation of osteoblastic cells in different manners. These results might influence the selection of an adequate bone substitute for clinical use as well, part from degradative and biomechanical properties.

Bone Substitutes↗

Direct separation of tranylcypromine enantiomers and their profile in an atypical depressive patient.

An isocratic and simple high-performance liquid chromatographic method is developed for the direct resolution of the tranylcypromine (TCP) enantiomers. The method involves the use of an S-18-crown-6-ether chiral stationary phase known as the Crownpak CR (+) column. The stereochemical separation factor (alpha) obtained was 1.30 and the stereochemical resolution factor (Rs) was 0.69 when using a mobile phase consisting of 0.1 N perchloric acid containing 12% methanol at 23 degrees C. The method has been used to monitor and identify, qualitatively, the profile of enantiomers of TCP in urine of an atypical depressive patient. It was found that the (-)-S-TCP concentration is significantly higher than the (+)-R-enantiomer.

Chromatography, Liquid↗

New homoleptic organometallic derivatives of vanadium(III) and vanadium(IV): synthesis, characterization, and study of their electrochemical behaviour.

The arylation of [VCl3(thf)3] with LiR(Cl), where R(Cl) is a polychlorinated phenyl group [C6Cl5, 2,4,6-trichlorophenyl(tcp), or 2,6-dichlorophenyl (dcp)] gives four-coordinate, homoleptic organovanadium(III) derivatives with the formula [Li(thf)(4)][V(III)(R(Cl))(4)] (R(Cl) = C(6)Cl(5) (1), tcp (2), dcp (3)). The anion [V(III)(C6Cl5)4]- has an almost tetrahedral geometry, as observed in the solid-state structure of [NBu4][V(C6Cl5)4] (1') (X-ray diffraction). Compounds 1-3 are electrochemically related to the neutral organovanadium(IV) species [V(IV)(R(Cl))4] (R(Cl) = C6Cl5 (4), tcp (5), dcp (6)). The redox potentials of the V(IV)/V(III) semisystems in CH2Cl2 decrease with decreasing chlorination of the phenyl ring (E(1/2) = 0.84 (4/1), 0.42 (5/2), 0.25 V (6/3)). All the [V(IV)(R(Cl))4] derivatives involved in these redox couples could also be prepared and isolated by chemical methods. The arylation of [VCl(3)(thf)(3)] with LiC6F5 also gives a homoleptic organovanadium(III) compound, but with a different stoichiometry: [NBu4]2[V(III)(C6F5)5] (7). In this five-coordinate species, the C6F5 groups define a trigonal bipyramidal environment for the vanadium atom (X-ray diffraction). EPR spectra for the new organovanadium compounds 1-6 are also given and analysed in terms of an elongated tetrahedral structure with C(2v) local symmetry. It is suggested that the R(Cl) groups exert a protective effect towards the vanadium centre.

Journal Article↗